VITAMIN E ISOFORM γ-TOCOTRIENOL ALLEVIATES ASTHMA AND COPD
PEH HONG YONG (B.Sc. (Hons.), NUS)
A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE
Supervisor: Associate Professor Wong Wai-Shiu Fred Examiners: Professor Lim Tow Keang Assistant Professor Thai Tran Professor Clive Page, King’s College London
I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously.
Peh Hong Yong Matric No.: A0035464H 17 July 2017
ACKNOWLEDGEMENTS First and foremost, I would like to extend my deepest appreciation to my PhD supervisor, A/Prof Fred Wong Wai-Shiu, for his guidance, encouragement and invaluable advice throughout these five years. I would also like to thank my thesis advisor Prof Ong Choon Nam and Dr Fong Chee Wai for their endless support, recommendation and enriching research collaboration. I sincerely thank the both of them for their support in my research endeavors and the constant inspiration to excel in my PhD journey. I also wish to acknowledge the President Graduate Fellowship and the National University of Singapore Industry Relevant Project (IRP) research scholarship with Davos LifeSciences, which provided research, educational and financial support for my PhD education. I am also truly honored to be the recipient of National University of Singapore Overseas Postdoctoral Fellowship, for the opportunity to begin my next milestone at Harvard Medical School. I wish to express my appreciation to Prof Benny Tan, Dr Deron Herr and Dr Judy Sng for their guidance, motivation and support. I would like to thank my mentors Eugene Ho and Cheng Chang. I am truly grateful for all the help they have provided and the skills they have imparted to me, making my experience a painless and pleasant one. I would also like to express my gratitude to other members of various laboratories (FW: Yong Loo Lin School of Medicine Pharmacology, OCN: Saw Swee Hock School of Public Health, and FCW: Davos LifeSciences) – Chan Tze Khee, Daniel Tan Wan Shun, Alan Koh Hock Meng, Lee Bee Lan, Su Jin, Winston Liao Wupeng, Lee Suet Hoay, Dong Jin Rui, Zhou Shuo, Jonathan Lim, Fera Goh, Guan Shou Ping, Lah Lin Chin, Khaing Nwe Win, Genevieve Seow, Pow Chen Wei, Amy Yong and Eunice Peh; for providing me with listening ears. I will always remember the good times we shared. I wish to also thank the Yong Loo Lin School of Medicine, Department of Pharmacology staff – Ho Lai Har, Alan Koh Hock Meng, Khoo Yok Moi, Ratnasari Bte Mohamed Basri, Cheong Yoke Ping, Fan Lu, Ho Woon Fei, Tan Yen Ling and Ting Wee Lee, for their kind assistance in making my lectures to life sciences students such a pleasant and fulfilling experience. I further thank all professors from the National University of Singapore whom I have encountered. I truly learnt a lot during my four years of PhD education. Last but not least, I would like to thank my life partner, family and friends for their boundless support and companionship through the harsh and good times. It would not have been possible without their countless encouragement and motivation.
TABLE OF CONTENTS ACKNOWLEGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
HONORS AND AWARDS
LIST OF PUBLICATIONS
CHAPTER 1: INTRODUCTION 1.1 MOTIVATION AND GOAL ............................................................................................ 1 1.2 SCOPE ..................................................................................................................... 2 1.3 VITAMIN E – TOCOPHEROLS AND TOCOTRIENOLS ....................................................... 5 1.3.1 Introduction ...................................................................................................... 5 22.214.171.124
History .................................................................................................. 5
Biochemical and Physical Properties of Vitamin E Isoforms .................. 6
Sources of Vitamin E ............................................................................ 9
1.3.2 Bioavailability and Pharmacokinetics of Vitamin E.......................................... 12 1.3.3 Vitamin E as an Antioxidant ........................................................................... 15 1.3.4 Vitamin E Therapy in Inflammatory Diseases ................................................. 17 1.3.5 Summary and Outlook of Vitamin E ............................................................... 20 1.4 ASTHMA ................................................................................................................. 25
1.4.1 Epidemiology of Asthma................................................................................. 27 1.4.2 Development of Asthma ................................................................................. 29 1.4.3 Immunopathology of Allergic Asthma ............................................................. 30 126.96.36.199
Classical Hallmarks of Asthma ............................................................ 33
Eosinophils ......................................................................................... 35
1.4.4 Oxidative Stress ............................................................................................. 39 1.4.5 Oxidative Stress in Asthma ............................................................................ 41 188.8.131.52
Impaired Antioxidant Defenses in Asthma ........................................... 44
184.108.40.206.1 Superoxide Dismutase (SOD) ............................................................... 45 220.127.116.11.2 Catalase (CAT) ........................................................................................ 45 18.104.22.168.3 Glutathione Peroxidase (GPx) .............................................................. 47 22.214.171.124.4 Nuclear Erythroid 2-Related Factor 2 (Nrf2) ....................................... 47 126.96.36.199 Increased Oxidative Damage in Asthma ................................................ 48 188.8.131.52.1 Lipid Peroxidation (8-Isoprostane) ....................................................... 48 184.108.40.206.2 Protein Nitration (3-Nitrotyrosine) ......................................................... 50 220.127.116.11.3 Oxidative DNA Damage (8-OHdG) ...................................................... 51 18.104.22.168.4 Relief of Asthma by targeting Oxidative Stress in the Lungs ........... 52 1.4.6 Current Therapies of Asthma ......................................................................... 52 1.4.7 Corticosteroids in Asthma: Prednisolone ........................................................ 57 1.4.7 Vitamin E as a Potential Therapy in Asthma................................................... 60 1.4.8 Hypotheses & Objectives ............................................................................... 61 1.5 CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) ........................................... 64 1.5.1 Epidemiology of COPD .................................................................................. 67 1.5.2 Etiology of COPD ........................................................................................... 68
1.5.3 Inflammation in COPD ................................................................................... 70 22.214.171.124
Macrophages ...................................................................................... 75
STAT3 and NF-κB............................................................................... 76
1.5.4 Oxidative Stress in COPD .............................................................................. 79 126.96.36.199
Impaired Antioxidants in COPD........................................................... 82
188.8.131.52.1 Superoxide Dismutase (SOD) ............................................................... 83 184.108.40.206.2 Catalase ................................................................................................... 83 220.127.116.11.3 Glutathione Peroxidase (GPx) .............................................................. 84 18.104.22.168.4 Nuclear Erythroid 2-Related Factor 2 (Nrf2) ....................................... 85 22.214.171.124
Heightened Oxidative Damage in COPD ............................................ 86
126.96.36.199.1 Lipid Peroxidation (8-Isoprostane) ....................................................... 87 188.8.131.52.2 Protein Nitration (3-Nitrotyrosine) ......................................................... 88 184.108.40.206.3 Protein Oxidation (AOPP) ...................................................................... 89 220.127.116.11.4 DNA Oxidative Damage (8-OHdG) ...................................................... 90 18.104.22.168.5 Targeting Oxidative Stress with Antioxidants in COPD .................... 90 1.5.5 Protease-Antiprotease Imbalance in COPD ................................................... 91 1.5.6 Emphysema in COPD .................................................................................... 94 1.5.7 Current Therapies in COPD ........................................................................... 95 1.5.8 Prednisolone and Vitamin E Isoform γ-Tocotrienol ....................................... 100 1.5.9 Hypotheses and Objectives .......................................................................... 101 1.6 CONCLUSIONS...................................................................................................... 102 CHAPTER 2: METHODS 2.1 REAGENTS ........................................................................................................... 105 2.2 MURINE ASTHMA MODEL ...................................................................................... 106
2.2.1 Mice ............................................................................................................. 106 2.2.2 House dust mite (HDM) Airway Challenge ................................................... 106 2.2.3 Treatment Groups for HDM-development .................................................... 107 2.2.4 Treatment Groups for screening of vitamin E isoforms ................................. 108 2.2.5 Treatment Groups for vitamin E isoform γ-tocotrienol in HDM-induced asthma mouse model ............................................................................................... 110 2.3 MURINE COPD MODEL ......................................................................................... 112 2.3.1 Mice ............................................................................................................. 112 2.3.2 Cigarette smoke (CS) exposure ................................................................... 113 2.3.3 Treatment groups for vitamin E isoform γ-tocotrienol in acute 2-weeks CSinduced COPD mouse model ....................................................................... 115 2.3.4 Treatment groups for vitamin E isoform γ-tocotrienol in chronic 2-months CSinduced COPD mouse model ....................................................................... 117 2.4 BRONCHOALVEOLAR LAVAGE ................................................................................ 118 2.5 TOTAL CELL COUNT ............................................................................................. 119 2.6 DIFFERENTIAL CELL COUNT OF BAL FLUID ............................................................ 119 2.7 2,7-DICHLORODIHYDROFLUORESCIN DIACETATE (DCFH-DA) ASSAY ....................... 120 2.8 HISTOLOGICAL EXAMINATION ................................................................................ 121 2.8.1 Hematoxylin and Eosin (H&E) Staining .......................................................... 121 2.8.2 Periodic Acid-Fluorescence Schiff (PAFS) Staining ....................................... 123 2.9 FREEZE DRY OF LUNG TISSUE .............................................................................. 124 2.10 ENZYMATIC ASSAY ............................................................................................... 125 2.10.1 Total Antioxidant Capacity (TAC) Assay....................................................... 125 2.10.2 Superoxide Dismutase (SOD) Assay............................................................ 125 2.10.3 Catalase (CAT) Assay .................................................................................. 126 2.10.4 Glutathione Peroxidase (GPx) Assay ........................................................... 127
2.11 ENZYME IMMUNOASSAY (EIA) ............................................................................... 127 2.11.1 Multiplex ....................................................................................................... 128 2.11.2 8-Isoprostane and 8-hydroxy-2-deoxyguanosine (8-OHdG) EIA................... 129 2.11.3 3-Nitrotyrosine (3-NT) EIA ............................................................................ 129 2.11.4 Advanced Oxidation of Protein Products (AOPP) EIA .................................. 130 2.12 AIRWAY HYPERRESPONSIVENESS (AHR) ............................................................... 131 2.13 PULMONARY FUNCTION TEST (PFT) ...................................................................... 131 2.14 IMMUNOBLOTTING (W ESTERN BLOTTING) ............................................................... 132 2.15 NF-ΚB, NRF2 AND STAT3 TRANSACTIVATION ASSAY ............................................. 133 2.16 REAL-TIME POLYMERASE CHAIN REACTION ........................................................... 134 2.16.1 RNA Isolation ............................................................................................... 134 2.16.2 Reverse Transcription .................................................................................. 136 2.16.3 Real-time Polymerase Chain Reaction ......................................................... 136 2.17 PHARMACOKINETICS OF γ-TOCOTRIENOL ............................................................... 139 2.18 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH) ASSAY ................................................. 140 2.19 STATISTICAL ANALYSIS ......................................................................................... 140 CHAPTER 3: TIME COURSE DEVELOPMENT OF INFLAMMATION AND OXIDATIVE STRESS IN HOUSE DUST MITE-INDUCED ASTHMA, AND SELECTION OF VITAMIN E ISOFORM 3.1 ABSTRACT ........................................................................................................... 141 3.2 INTRODUCTION ..................................................................................................... 143 3.3 MOUSE MODELS AND STATISTICAL ANALYSIS .......................................................... 145 3.4 RESULTS ............................................................................................................. 146 3.4.1 Increased Inflammation and Mucus Hypersecretion in Asthma .................... 146 3.4.2 Augmented Oxidative Stress in Asthma ....................................................... 154
3.4.3 Upregulated NF-κB and Downregulated Nrf2 Levels Contribute to Inflammation and Oxidative Stress in Asthma ................................................................... 156 3.4.4 γ-Tocotrienol is Selected Amongst the Vitamin E Isoforms ........................... 158 3.5 DISCUSSION ......................................................................................................... 165 3.6 SUPPLEMENTARY ................................................................................................. 172 CHAPTER 4: VITAMIN E ISOFORM γ-TOCOTRIENOL DOWN REGULATES HOUSE DUST MITE-INDUCED ASTHMA 4.1 ABSTRACT ........................................................................................................... 173 4.2 INTRODUCTION ..................................................................................................... 175 4.3 RESULTS ............................................................................................................. 178 4.3.1 γ-Tocotrienol Attenuates HDM-Induced Airway Inflammation ....................... 178 4.3.2 γ-Tocotrienol Ameliorates HDM-Induced Oxidative Stress in the Airways ..... 182 4.3.3 γ-Tocotrienol Abrogates HDM-Induced AHR ................................................ 186 4.3.4 γ-Tocotrienol Reduces Nuclear NF-κB and Promotes Nuclear Nrf2 .............. 188 4.4 DISCUSSION ......................................................................................................... 190 4.5 SUPPLEMENTARY ................................................................................................. 196 CHAPTER 5: VITAMIN E ISOFORM γ-TOCOTRIENOL PROTECTS AGAINST EMPHYSEMA IN CIGARETTE SMOKE-INDUCED COPD 5.1 ABSTRACT ........................................................................................................... 197 5.2 INTRODUCTION ..................................................................................................... 199 5.3 RESULTS ............................................................................................................. 202 5.3.1 γ-Tocotrienol Attenuates CS-Induced Airway Inflammation .......................... 202 5.3.2 γ-Tocotrienol Abates Inflammation Via Downregulation of Nuclear STAT3 and NF-κB........................................................................................................... 206 5.3.3 γ-Tocotrienol Suppresses CS-Induced Oxidative Stress in the Airways ......... 208 5.3.4 γ-Tocotrienol Strengthens Antioxidant Defence Promoting Nuclear Nrf2 ........ 211
γ-Tocotrienol Ameliorates 2-month CS-Induced Emphysema ...................... 214
5.3.6 γ-Tocotrienol Abrogates 2-month CS-Induced Lung Function Impairment ..... 216 5.4 DISCUSSION ......................................................................................................... 218 CHAPTER 6: SUMMARY, LIMITATIONS AND FUTURE WORK 6.1 SUMMARY AND CONCLUSION ................................................................................. 225 6.2 LIMITATIONS AND FUTURE WORK........................................................................... 231 6.2.1 Modifications to Improve Bioavailability ........................................................ 231 6.2.2 In Vitro Analyses .......................................................................................... 232 6.2.3 Clinical Testing ............................................................................................. 232 6.2.4 Complement Corticosteroids and/or Steroid-Resensitization ........................ 233 REFERENCES ................................................................................................................... 235
Ph.D. Thesis Vitamin E isoform γ-tocotrienol alleviates asthma and COPD By Hong Yong Peh Department of Pharmacology Yong Loo Lin School of Medicine National University of Singapore
Summary The discovery of vitamin E began in 1922 as a vital component required in reproduction. Today, there are eight naturally occurring vitamin E isoforms, namely α-, β-, γ- and δ-tocopherol and α-, β-, γ- and δ-tocotrienol. Vitamin E are potent antioxidants, capable of neutralizing free radicals directly by donating hydrogen. While α-Tocopherol had been regarded as the dominant form in vitamin E, recent findings reveal that tocotrienols possess superior antioxidant and anti-inflammatory properties over α-tocopherol. Asthma is a chronic inflammatory disease, affecting over 300 million people worldwide, where eosinophils are the main inflammatory cells involved. Although corticosteroids are the first-line treatment for asthma, a subset of patients is steroid-resistant, and chronic corticosteroid use causes side effects. In my first project, I employed a house dust mite (HDM)-induced asthma mouse model to study the time course development of inflammation and oxidative stress in asthma. I observed increased inflammation with each HDM challenge in the airways, which impairs antioxidant defenses, resulting in oxidative damage in the lungs. This led to mucus hypersecretion and airway hyperresponsiveness after ix
11 days post-HDM challenge. As vitamin E possesses both anti-oxidative and antiinflammatory properties, we screened vitamin E isoforms as potential therapeutic candidates in HDM experimental asthma. γ-Tocotrienol was the selected isoform, as it displayed better free-radical neutralizing activity in vitro and inhibition of bronchoalveolar lavage (BAL) fluid total, eosinophil and neutrophil counts in HDM mouse asthma in vivo, as compared to other vitamin E isoforms. For my second project, I focused on using γ-tocotrienol to attenuate HDM-induced asthma, with efficacies compared against corticosteroid prednisolone. The study showed that γtocotrienol abated HDM-induced elevation of BAL fluid cytokine and chemokine levels, total reactive oxygen species and oxidative damage biomarker levels, and serum IgE levels, but promoted lung antioxidant activities. Mechanistically, γtocotrienol was found to block nuclear NF-κB level and enhance nuclear Nrf2 levels in lung lysates to greater extents than prednisolone. More importantly, γtocotrienol suppressed airway hyperresponsiveness in asthma. Corticosteroids are known to be ineffective against neutrophilic inflammation, where we observed that γ-tocotrienol was more effective than prednisolone at ameliorating neutrophil infiltration in this study. We needed a neutrophilic disease to ascertain if γtocotrienol is indeed more potent than corticosteroid. Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide, where patients develop emphysema and reduced lung function. Neutrophils are the main inflammatory cells involved, and cigarette smoke (CS) inhalation is the leading risk factor for COPD. Thus, for my third project, I focused on using γ-tocotrienol in abating CS-induced COPD mouse models in comparison with prednisolone.
BALB/c mice were exposed to CS daily for 2 weeks or 2 months. γ-Tocotrienol reduced CS-induced BAL fluid neutrophil count and levels of cytokines, chemokines and oxidative damage biomarkers, and pulmonary pro-inflammatory and pro-oxidant gene expression, but restored lung endogenous antioxidant activities. γ-Tocotrienol acted by inhibiting nuclear translocation of STAT3 and NFκB, and up-regulating Nrf2 activation in the lungs. In mice exposed to 2-month cigarette smoke, γ-tocotrienol ameliorated bronchial epithelium thickening and destruction of alveolar sacs in lungs, and improved lung functions. In comparison with prednisolone, γ-tocotrienol demonstrated better anti-oxidative efficacy, and protection against emphysema and lung function in COPD. This study suggests a novel role for γ-tocotrienol in protecting the lungs from emphysematous destructions of alveolar sacs. Taken together, this thesis revealed for the first time the efficacies of γ-tocotrienol in both asthma and COPD. γ-Tocotrienol was more effective than prednisolone in abrogating oxidative damage in both diseases, and superior in attenuating neutrophilic-induced inflammation. γ-Tocotrienol may have therapeutic potential for the treatment of asthma and COPD.
Thesis supervisor: W.S. Fred Wong, Ph.D. Head & Associate Professor, Department of Pharmacology Yong Loo Lin School of Medicine National University of Singapore
LIST OF TABLES
CHAPTER 1 Table 1A| Natural sources of tocopherols and tocotrienols
Table 1B| Molecular targets modulated by tocopherols and tocotrienols
Table 1C| Current therapies in Asthma
CHAPTER 2 Table 2A| Sources of chemicals and reagents
Table 2B| Treatment groups for HDM-development asthma study
Table 2C| Treatment groups for screening of vitamin E isoforms study
Table 2D| Treatment groups for γ-tocotrienol in HDM-induced asthma study
Table 2E| Treatment groups for 2-weeks CS-induced COPD model
Table 2F| Treatment groups for 2-months CS-induced COPD model
Table 2G| Primer sequences for various targets
CHAPTER 3 Table 3A| Complete blood count and chemical analysis of serum in mice
CHAPTER 4 Table 4A| Complete blood count and chemical analysis of serum in mice
LIST OF FIGURES CHAPTER 1 Figure 1.01| Chemical structures of tocopherol and tocotrienol
Figure 1.02| Anatomy of an asthmatic airways
Figure 1.03| The global prevalence of asthma
Figure 1.04| Dendritic cell presentation in asthma
Figure 1.05| Immunology of asthma
Figure 1.06| Immunological pathways leading to inflammation of the airway
Figure 1.07| Eosinophils effector functions in asthma
Figure 1.08| Brief Overview of Oxidative Stress
Figure 1.09| Oxidative stress and oxidative damage in asthma
Figure 1.10| Superoxide anion presence results in decreased activity of catalase
Figure 1.11| Self-propagating mechanism of lipid peroxidation
Figure 1.12| Production of 8-hydroxy-2-deoxyguanosine
Figure 1.13| Proposed guidelines for asthma treatment
Figure 1.14| Chemical structure of prednisolone
Figure 1.15| Mechanism of action of glucocorticoid
Figure 1.16| GOLD classification of COPD
Figure 1.17| Differences between asthma and COPD
Figure 1.18| Effector functions of inflammatory cells in COPD
Figure 1.19| Role of STAT3 in COPD
Figure 1.20| Role of NF-κB in COPD
Figure 1.21| Pathophysiology of oxidative stress in COPD
Figure 1.22| Oxidative damage biomarkers in COPD
Figure 1.23| Treatment ladder for each stage of COPD
CHAPTER 2 Figure 2.01| Murine asthma model design
Figure 2.02| Set-up of two peristaltic pumps to deliver 4% cigarette smoke into the ventilated chambers
Figure 2.03| Acute 2-weeks CS-induced COPD model design
Figure 2.04| Chronic 2-months CS-induced COPD model design
CHAPTER 3 Figure 3.01| Recruitment of inflammatory cells into the airways in HDM-induced asthma lungs
Figure 3.02| Elevated airway inflammation and leukocytes infiltration in HDMchallenged lungs
Figure 3.03| Mucus hypersecretion in lungs of asthmatic mice
Figure 3.04| Inflammatory cytokines and chemokines heightened in HDM-induced asthma
Figure 3.05| Increased levels of free radicals and oxidative damage in HDMchallenged lungs
Figure 3.06| Antioxidants expression decreased with repeated exposure to HDM
Figure 3.07| Nuclear levels of NF-κB and Nrf2 in asthma
Figure 3.08| Comparison of free radical neutralizing effects and anti-inflammatory actions of vitamin E isoforms
Figure 3.09| γ-tocotrienol is superior over α-tocopherol in modulating nuclear levels of NF-kB and Nrf2 in HDM-challenged lungs
Figure 3.10| Pharmacokinetics analysis of oral γ-tocotrienol in mice
CHAPTER 4 Figure 4.01| Protective effects of γ-tocotrienol on HDM-induced allergic airway inflammation
Figure 4.02| Inhibitory effects γ-tocotrienol on HDM-induced lung cytokines and chemokines
Figure 4.03| Protective effects of γ-tocotrienol on HDM-induced oxidative stress in the lungs
Figure 4.04| γ-Tocotrienol strengthens antioxidant defense in HDM-challenged lungs
Figure 4.05| γ-Tocotrienol prevents HDM-induced airway hyperresponsiveness (AHR) to increasing concentrations of methacholine
Figure 4.06| Protective effects of γ-tocotrienol are mediated through regulation of nuclear levels of NF-kB and Nrf2 in HDM-challenged lungs
CHAPTER 5 Figure 5.01| Therapeutic effects of γ-tocotrienol on CS-induced airway inflammation 203 Figure 5.02| Inhibitory effects γ-tocotrienol on CS-induced lung cytokines, chemokines, mucin and proteases
Figure 5.03| Anti-inflammatory effects of γ-tocotrienol are mediated through regulation of nuclear levels of STAT3 and NF-kB in CS-challenged lungs
Figure 5.04| Efficacies of γ-tocotrienol on CS-induced oxidative stress in the lungs
Figure 5.05| γ-Tocotrienol strengthens antioxidant defense in CS-challenged lungs
Figure 5.06| γ-Tocotrienol strengthens antioxidant defense in CS-challenged lungs
Figure 5.07| Protective effects of γ-tocotrienol against hallmark features of COPD in 8 weeks chronic CS exposed mice
Figure 5.08| γ-Tocotrienol protects mice from impaired lung function in 8 weeks chronic CS exposure
LIST OF ABBREVIATIONS 3-NT
α-Tocopherol transport protein
Animal Holding Unit, NUS
One-Way Analysis of Variance
Advanced oxidation of protein products
Antioxidant response element
Airway smooth muscle
Bronchoalveolar lavage fluid
Bovine serum albumin
CCAAT-enhancer binding protein
Cyclic adenosine monophosphate
Complementary deoxyribonucleic acid
Chronic obstructive pulmonary disease
Cyclic adenosine monophosphate response element binding protein
CuZnSOD Cytosolic copper zinc SOD (SOD type 1) DALY
Disability-adjusted life year
Extracellular SOD (SOD type 3)
Enzyme-linked immunosorbent assay
High affinity IgE receptor
Forced expiratory volume
Functional residual capacity
Forced vital capacity
Granulocyte colony-stimulating factor
Granulocyte macrophage colony-stimulating factor
Global initiative for chronic obstructive lung disease
Glutathione (reduced state)
Glutathione (oxidized state)
Hematoxylin and Eosin
Histone deacetylase 2
House dust mite
Institutional Animal Care and Use Committee
Intercellular cell adhesion molecule 1
Inducible nitric oxide synthase
Long-acting β2-adrenergic receptor agonist
Long-acting muscarinic receptor antagonist
Leukemia inhibitory factor
Leukotriene receptor antagonist
Major basic protein
Macrophage inflammatory protein
Mitochondrial manganese SOD (SOD type 2)
Nicotinamide adenine dinucleotide phosphate
Nuclear factor-kappa B
No Observed Adverse Effects Level
Nitric oxide synthase
NADPH:quinone oxidoreductase 1
Nuclear erythroid 2-related factor 2
Periodic acid-fluorescence schiff
Polymerase chain reaction
Pulmonary function test
Protein kinase C
Pattern recognition receptor
Reactive nitrogen species
Reactive oxygen and nitrogen species
Reactive oxygen species
Revolutions per minute
Short-acting β2-adrenergic receptor agonist
Short-acting muscarinic receptor antagonist
Standard error mean
Secretory leukoprotease inhibitor
Steroid receptor coactivator-1
Signal transducer and activator of transcription 3
Tissue inhibitor of MMP
Total lung capacity
Tumor necrosis factor-α
Tocotrienol rich fraction
Vascular cell adhesion molecule 1
HONOURS & AWARDS 1. NUS President Graduate Fellowship (Nov 2014 – Present) 2. NUS-Industry Research Programme Scholarship (Aug 2012 – Oct 2014) 3. Lee Foundation International Conference Travel Award, to attend American Thoracic Society Conference 2016 (May 2016) 4. Best Oral Presenter Award, 1st place, Yong Loo Lin School of Medicine 6th Annual Graduate Scientific Congress (Mar 2016) 5. Global Young Scientist Summit Travel Fellowship, organized by National Research Foundation Singapore, with nomination from Nobel Laureate Sir Richard Roberts (Jan 2016) 6. Best Young Scientist Poster Award, IUPHAR World Conference on the Pharmacology of Natural and Traditional Medicine (Jul 2015) 7. HOPE Meeting with Nobel Laureates Travel Fellowship, organized by Japan Society for the Promotion of Science (Mar 2015) 8. Society for Redox Biology & Medicine Travel Award, to attend conference organized by Society for Redox Biology and Medicine (Nov 2014) 9. Best Oral Presenter Award, 1st place, Yong Loo Lin School of Medicine 4th Annual Graduate Scientific Congress (Mar 2014) 10. Invited Manuscript Reviewer: •
International Journal of Chronic Obstructive Pulmonary Disease (2017Present)
Pulmonary Medicine and Respiratory Research (2017-Present)
British Journal of Pharmacology (2014-Present)
PLoS One (2013-Present)
LIST OF PUBLICATIONS
1. Peh HY, Tan WSD, Chan TK, Pow CW, Foster PS, et al. (2017) Vitamin E isoform γ-tocotrienol protects against emphysema in cigarette smoke-induced COPD. Free Radical Biology and Medicine. 110:332-344. 2. Peh HY, Tan WSD, Liao WP and Wong WSF. (2016) Vitamin E therapy beyond cancer: tocopherol versus tocotrienol. Pharmacology and Therapeutics. 162:152-169. 3. Peh HY, Ho WE, Chan TK, Seow ACG, Cheng C, et al. (2015) Vitamin E isoform γ-Tocotrienol down regulates house dust mite-induced asthma. Journal of Immunology. 195: 437-444. [selected for Cover Page of 195(2) issue] 4. Chan TK, Tan WSD, Peh HY and Wong WSF. (2017) Aeroallergens induce reactive oxygen species production and DNA damage and dampen antioxidant responses in bronchial epithelial cells. Journal of Immunology. 199: doi:10.4049/jimmunol.1600657 5. Dong JR, Liao WP, Peh HY, Chan TK, Tan WSD, et al. (2017) Ribosomal protein S3 gene silencing protects against experimental allergic asthma. British Journal of Pharmacology. 174:540-552. 6. Liao WP, Dong JR, Peh HY, Tan LH, Lim KS, et al. (2017) Oligonucleotide therapy for obstructive and restrictive respiratory diseases. Molecules. 22:139. 7. Chan JHS, Chua YL, Peh HY, Jovanovic V, Gascoigne NRJ, et al. (2017) Molecular engineering of a therapeutic antibody for Blo t 5-induced allergic asthma. Journal of Allergy and Clinical Immunology. 139:1705-1708. 8. Chua YL, Liong KH, Huang CH, Wong HS, Zhou Q, Ler SS, Tang YF, Low CP, Koh HY, Kuo IC, Zhang YL, Wong WSF, Peh HY, et al. (2016) Blomia tropicalis–specific TCR transgenic Th2 cells induce inducible BALT and severe asthma in mice by an IL-4/IL-13–dependent mechanism. Journal of Immunology. 197: 3771-3781. 9. Chan TK, Loh XY, Peh HY, et al. (2016) House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs. Journal of Allergy and Clinical Immunology. 138:84-96. 10. Tan WSD, Peh HY, Liao WP, Pang CH, Chan TK, et al. (2016) Cigarette Smoke-Induced Lung Disease Predisposes to More Severe Infection with Nontypeable Haemophilus influenzae: Protective Effects of Andrographolide. Journal of Natural Products. 79:1308-1315. 11. Footitt J, Mallia P, Durham AL, Ho WE, Trujillo M, Telcian AG, Rosario AD, Cheng C, Peh HY, et al. (2015) Oxidative and nitrosative stress and histone xxii
deacetylase-2 activity in exacerbations of Chronic Obstructive Pulmonary Disease. Chest. 149:62-73. 12. Ho WE, Xu Y-J, Xu FG, Cheng C, Peh HY, et al. (2015) Anti-malarial drug artesunate restores metabolic changes in experimental allergic asthma. Metabolomics. 11:380-390. 13. Ho WE, Peh HY, Chan TK and Wong WSF. (2014) Artemisinins: pharmacological actions beyond anti-malarial. Pharmacology and Therapeutics. 142:126-139. 14. Ho WE, Xu YJ, Cheng C, Peh HY, Tannenbaum SR, et al. (2014) Metabolomics reveals inflammatory-linked pulmonary metabolic alterations in a murine model of house dust mite-induced allergic asthma. Journal of Proteome Research. 13:3771-3782. 15. Guan SP, Tee W, Ng DSW, Chan TK, Peh HY, et al. (2013) Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity. British Journal of Pharmacology. 168:1707-1718. 16. Ho WE, Xu YJ, Xu F, Cheng C, Peh HY, et al. (2013) Metabolomics reveals altered metabolic pathways in experimental asthma. American Journal of Respiratory Cell and Molecular Biology. 48:204-211. 17. Ho WE, Cheng C, Peh HY, Xu F, Tannenbaum SR, et al. (2012) Anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma. Free Radical Biology and Medicine. 53:498-507.
MANUSCRIPT IN PROGRESS 1) Peh HY, Ho WE, Tan WSD, Cheng C, Chan TK, et al. Time course development of inflammation and oxidative stress in house dust mite-induced asthma. 2017; manuscript in preparation 2) Tan WSD, Liao WP, Peh HY, Pang CH, Chan TK, et al. Andrographolide promotes autophagy in bronchial epithelial cells exposed to cigarette smoke extract. 2017; manuscript in review
CONFERENCE ABSTRACTS 1. Peh HY, et al. Efficacy of Vitamin E γ-Tocotrienol in Asthma and COPD. Oral presentation delivered at Hong Yong Peh’s research seminar in Harvard Medical School, Boston, USA, May, 2016. 2. Peh HY, et al. Vitamin E Isoform γ-Tocotrienol Attenuates Cigarette Smokeinduced Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine. 193: A3166. Thematic poster presentation at the American Thoracic Society meeting, San Francisco, USA, May, 2016. 3. Peh HY, et al. Vitamin E Isoform y-Tocotrienol Alleviates Cigarette SmokeInduced Chronic Obstructive Pulmonary Disease. Free Radical Biology and Medicine Abstracts. 87: S139-S140. Poster presentation delivered at Society of Redox Biology and Medicine meeting, Boston, USA, November, 2015. 4. Peh HY, et al. Vitamin E Isoform γ-Tocotrienol Alleviates Cigarette Smokeinduced Chronic Obstructive Pulmonary Disease. Chinese Journal of Pharmacology and Toxicology. 2015-S1: 51. Poster presentation delivered at the IUPHAR World Conference on the Pharmacology of Natural and Traditional Medicine, Singapore, July, 2015. 5. Peh HY, et al. Vitamin E γ-Tocotrienol Ameliorates Experimental House Dust Mite Asthma. Oral and Poster presentation delivered at the 7th HOPE Meeting, Tokyo, Japan, March, 2015. 6. Peh HY, et al. Vitamin E γ-Tocotrienol Ameliorates Experimental House Dust Mite Asthma. Free Radical Biology and Medicine Abstracts. 76: S88. Poster presentation delivered at the Society of Redox Biology and Medicine meeting, Seattle, USA, November, 2014. 7. Peh HY, et al. Vitamin E Isomer γ-Tocotrienol Abrogates Lung Inflammation & Oxidative Stress In Experimental Allergic Asthma. Oral presentation delivered at Yong Loo Lin School of Medicine Graduate Scientific congress, Singapore, March, 2014. 8. Peh HY, et al. Vitamin E Isomer γ-Tocotrienol Abrogates Lung Inflammation & Oxidative Stress In Experimental Allergic Asthma. Poster presentation delivered at International Conference on Pharmacology and Drug Development, Singapore, December 2013.
Motivation and Goal
Vitamin E tocotrienols are gaining traction in recent decade over tocopherols, with its success in attenuating inflammation and oxidative stress in various diseases (bone, cardiovascular, eye, neurological and kidney diseases, cancer, lipid disorder and radiation damage). However, there were no studies that evaluated efficacy of tocotrienols in respiratory diseases in both human and animal models. Our laboratory is specialized in investigating pharmacology of potential therapeutics in respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and fibrosis. Asthma affects over 300 million patients worldwide and with its increasing prevalence, it is the most common disease in the lungs. There are numerous patients (approximately 20 million or more) who are resistant to corticosteroids, thus creating a huge unmet medical need. Asthma is also a chronic inflammatory disease with heightened oxidative damage in the lungs. With all these factors, we collaborated with Davos LifeSciences (who kindly provided vitamin E isoforms for our research) to elucidate the efficacy of tocotrienol in asthma, compare the effectiveness against tocopherol, and deduce the isoform with the most therapeutic potential. Upon completing the asthma study, it sparked the interest of the subsequent study which investigated the efficacy of γ-tocotrienol in COPD. Although the major inflammatory cell responsible in asthma is eosinophil, there were some data which suggested the potential of tocotrienol in
ameliorating neutrophils. As COPD is primarily a destructive disease of the airways caused by neutrophils, and there were no other studies conducted which evaluated tocotrienols in COPD, it was the perfect opportunity for the next study. COPD affects over 200 million patients worldwide, and accounts for approximately 5% of deaths globally in year 2004. It is estimated to be the third leading cause of death worldwide by year 2020. There are only two FDA-approved medications for COPD, namely corticosteroids and roflumilast (phosphodiesterase 4 inhibitor), which have low therapeutic index and many unwanted side effects. Overall, this research endeavor would not only further our knowledge of the pathology of both asthma and COPD, but more importantly, create a potential new class of drug (a naturally occurring vitamin with negligible side effects) in both diseases.
There are 8 isoforms of vitamin E, while majority of the population only knows of α-tocopherol. They are naturally occurring antioxidants with reported anti-inflammatory properties. In Chapter 1, I will elaborate on vitamin E and its isoforms, pharmacokinetics, antioxidant capacity, reported targets that it can modulate and its uses in various diseases. I will provide a brief overview of comparison between tocopherols and tocotrienols and outlook of vitamin E. I will also discuss the molecular mechanisms and immunology that drive the development of both asthma and COPD, to better understand how various inflammatory cells 2
orchestrate the inflammation in both diseases. The antioxidant-oxidant imbalance in asthma and COPD that contributes to oxidative stress and chronic inflammation will be described. The free radicals generated in asthma and COPD resulted in oxidative damage on key biomolecules such as lipids, proteins and DNA will be discussed as well. This should provide an insight on our hypotheses that vitamin E tocotrienol can protect against asthma and emphysema in COPD.
In Chapter 3, the time course development of inflammation and oxidative stress will be studied in a 3-weeks mouse model, with exposure to house dust mite (HDM) intratracheally once weekly. The mouse will be sacrificed at various time-points throughout the model, to investigate the status of inflammation
transcription factors analyses), and deduce if inflammation or oxidative stress occurs first. With the data, I will conduct a screening of vitamin E isoforms to measure their antioxidant capacity, anti-inflammatory and antioxidative potentials, as well as modulation of key transcription factors (NFκB and Nrf2). This will answer if tocotrienols or tocopherols are superior in experimental asthma, and allow the selection of the vitamin E isoform of interest for pharmacokinetics and subsequent studies.
In Chapter 4, with the selected vitamin E isoform (γ-tocotrienol) from previous study, I will examine the efficacies of γ-tocotrienol (three doses) against clinically prescribed corticosteroid, prednisolone, in HDM-induced 3
asthma. I employed oral gavage to administer the various treatment groups, which is the same route of administration for humans, so as to provide more clinical relevant data. The ability to attenuate inflammation, oxidative stress/damage, mucus hypersecretion and airway hyperresponsiveness, as well as mechanism of actions of γ-tocotrienol will be measured in this study.
In the final study of the thesis, Chapter 5, the effectiveness of γ-tocotrienol (three doses) will be compared against corticosteroid, prednisolone, in a 2weeks cigarette smoke (CS)-induced mouse model. Similarly, the route of administration to the mice was kept similar to human patients, via oral gavage. I will measure the modulation of inflammatory and oxidative stress biomarkers, and mechanism of actions of γ-tocotrienol in COPD. In order to capture emphysema in experimental COPD, it requires a chronic exposure to CS. Therefore, using a 2-months CS-induced COPD mouse model, I will quantify the protection against emphysema and impairment of lung function in COPD for both γ-tocotrienol and prednisolone treated mice. Lastly, in Chapter 6, I will summarize the key findings from each study, and also discuss the limitations faced and potential future works.
Vitamin E – Tocopherols and Tocotrienols
1.3.1 Introduction 22.214.171.124
Vitamin E is one of the most widely consumed vitamins known for its antioxidant capacity and multiple health benefits. In 1912, Casimir Funk coined the term “vitamine” (obtained from vital and amine, meaning amine of life) as it was hypothesized that vitamine was chemical amines present in micronutrient food factors which prevented beriberi and other dietarydeficiency diseases (Funk, 1912). It was validated subsequently that not all vitamine, such as vitamin C and vitamin E, contained amine group, thus it was shortened to “vitamin” in English. The discovery of vitamin E began in 1922 by Herbert Evans and Katherine Bishop when they isolated an uncharacterized fat-soluble compound from green leafy vegetables which is required for reproduction (Evans and Bishop, 1922). Upon identifying the compound in 1924, it was termed as tocopherol (obtained from Greek words tokos and phero, meaning to bear children), also known as αtocopherol presently. The search beyond α-tocopherol isoform started in 1947 by Stern et al, and it was in 1964 when tocotrienols were first discovered (Sen et al., 2007a). Despite its discovery in 1964, it was only until 1980s where tocotrienol research took off in various diseases, which triggered the debate of supremacy between tocopherols and tocotrienols. α-Tocopherol was the main focus of vitamin E research and regarded as the major isoform with the most potent antioxidant and biological activity. However, recent studies have shown otherwise, where tocotrienols may 5
triumph with its superior anti-cholesterolemic (unique to tocotrienols only), antioxidant,
neuroprotective properties. Despite the steady growth in publications of tocotrienols since 1966, it comprises merely 3% of vitamin E research papers when obtained from PubMed. Currently, there are eight naturally occurring isoforms in the vitamin E family.
Biochemical and Physical Properties of Vitamin E Isoforms
Vitamin E can be classified into tocopherols and tocotrienols, resulting in a total of eight isoforms: namely α-, β-, γ-, and δ-tocopherol, and α-, β-, γ-, and δ-tocotrienol (Figure 1.01). Vitamin E in its pure form is a yellow viscous oil/liquid that oxidizes readily when exposed to light, oxygen and transition metal ions. They are not water-soluble but dissolves in alcohol, organic solvents and vegetable oils (Bramley et al., 2000). They have the same basic chemical structure where a C16 isoprenoid side chain is attached at the C-2 of a chromane ring. Tocotrienols differ from tocopherols with an unsaturated farnesyl isoprenoid side chain at C-3’, C-7’ and C-11’ over a saturated phytyl isoprenoid side chain (Fig. 2). The greek letter prefixes of both tocopherols and tocotrienols depend on the number and position of methyl groups on the chromanol ring, whereby α-isoform is 2,5,7,8-tetramethyl; β-isoform is 2,5,8-trimethyl; γ-isoform is 2,7,8-trimethyl; and δ-isoform is 2,8-dimethyl. Therefore, using the α-isoform, the structural terminology
trimethyltridecyl)-6-chromanol while α-tocotrienol is 2,5,7,8-tetramethyl-26
amphipathic molecules with a polar chromanol ring and lipophilic isoprenoid side chain. The unsaturated isoprenoid side chain of tocotrienols potentially enhances its penetration into fatty tissues, such as brain and liver, and better distribution on the cell membranes (Suzuki et al., 1993). Vitamin E in nature consists of the dextrorotatory enantiomers only with a single stereoisomer. Tocopherols contain three chiral stereocenters at C-2, C-4’ and C-8’ (e.g.: d-RRR-α-tocopherol), while tocotrienols contain only one chiral stereocenter at C-2 as the other two chiral stereocenters are not possible with C=C unsaturation in the isoprenoid tail (Colombo, 2010).
Figure 1.01| Chemical structures of tocopherol and tocotrienol. Numbering for the chromanol ring in green and numeration for the isoprenoid side chain in red. Blue R groups are listed for each isoform in the table with molecular weight. Tocopherol and tocotrienol are largely similar, with the exception of three C=C double bonds in the isoprenoid side chain for tocotrienol. Naturally occurring tocopherol chiral stereocenters are 2’R,4’R,8’R, while tocotrienol chiral stereocenter is 2’R only due to the C=C unsaturation in the isoprenoid tail.
Sources of Vitamin E
In nature, vitamin E can be found in vegetables, plants and plant oils. The distribution of tocopherols in the plant kingdom is primarily α-tocopherol in green leafy plants and γ-tocopherol in non-green plant parts such as fruits and seeds (Zielinski, 2008). Common food sources of α-tocopherol can be found in almonds, avocados, hazelnuts, peanuts and sunflower seeds; βtocopherol in oregano and poppy seeds; γ-tocopherol in pecans, pistachios, sesame seeds and walnuts; δ-tocopherol in edamame and raspberries (obtained from United States Department of Agriculture nutrient database, http://ndb.nal.usda.gov/). Common food oils including corn, peanut and soybean oil contains largely α-tocopherol and γtocopherol. In contrast, tocotrienols are relatively rarer in food sources and plant kingdom. It was suggested that tocotrienols are bioconverted to tocopherols and may function as intermediates to biosynthesize αtocopherol in plants (Pennock, 1983). In plants, tocotrienols can be found in the seeds of monocotyledonous plants, fruits of dicotyledonous plants [list of plants can be found in (Horvath et al., 2006; Zielinski, 2008)] and latex of rubber trees (Hevea brasiliensis) (Horvath et al., 2006). Tocotrienols levels thrive in abundance in plant foods, namely rice bran oil, palm oil and annatto seeds (ratio of tocopherol to tocotrienol in plant oils – 45:55, 30:70 and 0:100 respectively). The extraction of crude palm oil from oil palm (Elaeis guineensis) fruits can reach up to 800 mg/kg of tocotrienols, consisting mainly of α-tocotrienol and γ-tocotrienol (Theriault et al., 1999). Annatto seeds are unique as unlike rice bran and palm oil, it 9
contain only tocotrienol (mostly δ-tocotrienol), but no tocopherols (Frega et al., 1998). Tocotrienols can also be acquired in cereal grains like barley, oat, rice, rye and wheat. They are characterized with higher content of tocotrienols to tocopherols within the kernels (ratio of tocopherol to tocotrienol – 21:79, 23:77, 33:67, 49:51 and 45:55 respectively). Table 1A lists the various sources of vitamin E and its respective components of tocopherols and tocotrienols.
Synthetic forms of vitamin E consist mainly of α-tocopherol, and it was first synthesized in 1938 (Karrer et al., 1938). Unlike naturally occurring dRRR-α-tocopherol, synthesized α-tocopherol consists of a racemic mixture of eight stereoisomers [all-rac-α-tocopherol (RRR-, RRS-, RSS-, RSR-, SRS-, SSR-, SRR- and SSS-α-tocopherol)] (Kiyose et al., 1997). Various attempts to synthesize tocotrienols were not as successful with tocopherols, as it only produced low to moderate yields and purity (Netscher, 2007).
Table 1A| Natural sources of tocopherols and tocotrienols
Plants and Fruits Species
Aesculus hippocastanum Hort. b Bixa orellana L. a Hevea brasiliensis Müll.Arg. a Rosmarinus officinalis L. a Vitis vinifera L. a Litchi chinensis Sonn. a Delphinium ajacis L. b
Horse chestnuts Lipstick tree Rubber tree Rosemary Grape Litchi (lychee) Larkspur
seed seed latex seed seed seed seed
Tocopherol (TP, µg/g tissue) α β γ δ 195 1758 5.0 2883 18.0 124.1 120
528.1 1.10 435.2 9.2 78
88 606.5 241.6 30.8 1.9 59.2 83
174.7 506.3 17.4 -
Tocotrienol (T3, µg/g tissue) α β γ δ 97 18.7 522.4 560.5 5.0 566
1.84 300.3 153
626 534.7 196.7 109.4 25.0 3.37 -
% of T3
336 977.9 1870 488.0 -
78.9 53.9 77.6 22.5 60.1 70.1 71.9
Plant Oils and Cereals
Family Elaeis guineensis L. c Oryza sativa L. c Oryza sativa L. e Cocos nucifera L. c Zea mays L. d Hordeum vulgare L. d Avena sativa L. a Amaranthus ssp. e Triticum sp. a Secale cereal f Arachis hypogaea L. c Carthamus tinctorius L. c Glycine max L. c
Palm oil Rice bran oil Black rice Brown rice White rice Coconut oil Corn Barley Oat Sorghum Wheat Rye Peanut oil Safflower oil Soybean oil
890 860 93.7 26.4 7.4 36 66.9 76.1 15.8 17.9 45 27.87 367 774 958
Tocopherol (TP, µg/g) α β γ δ 152 324 12.4 4.2 0.7 5 3.7 8.6 5.4 1.4 15.4 11.46 130 387 101
18 0.2 0.9 5.0 2.28 -
53 2.5 1.2 0.1 45.0 5.6 3.2 13.3 216 387 593
38.6 6 1.0 0.7 0.2 21 264
Tocotrienol (T3, µg/g) α β γ δ 205 116 13.8 5.6 0.8 5 5.3 40.3 4.2 2.1 5.0 5.94 -
1 8.7 19.6 8.19 -
439 349 26.4 15.4 5.8 19 11.3 10.4 2.1 0.8 -
% of T3
94 0.4 0.9 0.9 0.1 -
82.9 54.1 42.9 79.5 89.2 69.4 25.4 79.2 45.6 16.8 54.7 50.7 -
Tocols: tocopherols and tocotrienols. Reference: a (Horvath et al., 2006), b (Matthaus et al., 2003), c (http://tocotrienol.org/), d (Panfili et al., 2003), e (Choi et al., 2007), f (Zielinski et al., 2001).
1.3.2 Bioavailability and Pharmacokinetics of Vitamin E Studies in the past two decades have shown that both α-tocopherol transport protein (αTTP) and ω-hydroxylase are vital components for the absorption of vitamin E, α-tocopherol in particular (Hosomi et al., 1997; Sontag and Parker, 2002). αTTP in the liver facilitates the packing of vitamin E into lipoproteins and transportation to other tissues via the bloodstream. While αTTP has a high affinity to α-tocopherol (100%), it has lower affinity for other vitamin E isoforms: approximately 50% for βtocopherol, 10-30% for γ-tocopherol and 1% for δ-tocopherol (BrigeliusFlohe and Traber, 1999; Traber, 2007). A study has also shown that αTTP has 8.5 fold lower affinity to bind to α-tocotrienol than α-tocopherol (Hosomi et al., 1997). In the liver, unbounded isoforms of vitamin E to αTTP will be susceptible to catabolization via cytochrome P450 (CYP4F2)initiated ω-hydroxylation and oxidation by ω-hydroxylase (Jiang, 2014). Due to these two antagonizing interactions, α-tocopherol is regarded as the predominant isoform to be accumulated in tissues where other vitamin E
hydroxycarboxychromanol and carboxyethyl-hydroxychroman derivatives (for tocotrienols) (Birringer et al., 2002; Lodge et al., 2001). With that in mind, it led to a huge debate for a decade if orally administered tocotrienols can reach vital organs in the body, which dampened the research on tocotrienols in the 1990s. Despite the fact that αTTP has a lower affinity towards tocotrienols, it is not clear, or to what extent, the transport of orally administered tocotrienols to vital organs depends on 12
αTTP. In one study, female αTTP knockout mice remained deficient in αtocopherol despite dietary supplementation with α-tocopherol (Jishage et al., 2001). Interestingly, oral supplementation of α-tocotrienol to female mice restored fertility in these αTTP knockout mice under α-tocopherol deficiency (Khanna et al., 2005). This suggests other transport machinery or mechanisms for the absorption and transport of tocotrienols beyond the αTTP.
Following up on the debate of tocotrienol viability, pharmacokinetics studies on tocotrienols were performed. A study in 2003 determined the pharmacokinetics
intraperitoneal, intravenous and oral routes in rats (Yap et al., 2003). The absorption
intraperitoneal routes was negligible and should be avoided. Tocotrienols have incomplete absorption and limited bioavailability in rats, where the bioavailability of α-tocotrienol was approximately 28%, and 9% for both γand δ-tocotrienols (Yap et al., 2003). The time required to reach peak plasma concentration for α-, γ-, and δ-tocotrienol were 3.3, 3.0 and 2.8 hours respectively, while the half-life was approximately 3 hours for αtocotrienol and 2 hours for γ- and δ-tocotrienols in rats (Yap et al., 2003). In humans, the half-life of α-, γ-, and δ-tocotrienol in plasma was estimated to be 2.3, 4.4 and 4.3 hours, respectively (Yap et al., 2001), while the halflife of α-tocopherol and γ-tocopherol were 57 and 13 hours, respectively (Leonard et al., 2005). In a separate double-blind placebo-controlled study, 13
human subjects took tocotrienol supplements at a dose of 250 mg/day for 8 weeks, where the mean plasma levels of α-, γ-, and δ-tocotrienol were 0.8 µM, 0.54 µM and 0.09 µM (O’Byrne et al., 2000). As tocotrienols are oil-based compounds, it faces limited bioavailability. As emulsions are known to increase absorption of fat-soluble compounds, based on selfemulsifying drug delivery systems technology (Gao and Morozowich, 2005), self-emulsifying formulations of tocotrienols were produced, such as Tocovid™ SupraBio™ and Naturale³. The supplementation of Tocovid SupraBio™ elevated peak plasma concentration of α-tocotrienol in humans to approximately 3 µM, a threefold increase as compared to 0.8 µM in previous study without self-emulsification (Khosla et al., 2006).
Although tocotrienols are not well-absorbed in the liver, they appear to be similarly absorbed as tocopherols with dietary fat and are secreted into chylomicron particles. The chylomicron-bound vitamin E isoforms were reported to be transferred to peripheral tissues such as adipose tissues, bones, brain, lung, muscle and skin, via the lymphatic system (Ikeda et al., 2001; Uchida et al., 2012). This might explain the accumulation of non-αtocopherol isoforms of vitamin E in peripheral tissues, despite the fact of low uptake in the liver αTTP. A study involving Caco2 human epithelial colorectal cells elucidated that absorption of γ-tocotrienol supersedes that of α-tocopherol (Tsuzuki et al., 2007). The rapid epithelial transport of tocotrienols over tocopherols in Caco2 cells were attributed to the difference in saturation of the isoprenoid side chains, where unsaturated 14
tocotrienols were more lipophilic. The toxicological aspect of tocotrienol is relatively safe, where human subjects fed with 250 mg/day for 8 weeks (O’Byrne et al., 2000), and female rats fed with 130 mg/kg daily for 13 weeks to one year displayed no side effects (Nakamura et al., 2001; Tasaki et al., 2008).
1.3.3 Vitamin E as an Antioxidant Vitamin E is widely accepted as one of the most potent antioxidants. The antioxidant property is attributed to the hydroxyl group from the aromatic ring of tocochromanols, which donates hydrogen to neutralize free radicals or reactive oxygen species (ROS). In homogenous solutions and in vitro assays, the reaction rates between the vitamin E isoforms largely depends on the number of methyl groups on the chromanol ring. The antioxidant activity of α-, β- and γ-isoforms of both tocopherol and tocotrienol are similar, except the δ-isoform which has weaker activity, when tested in pyrogallolsulfonphthalein and 2,7-dichlorodihydrofluorescein diacetate assays (Peh et al., 2015; Yoshida et al., 2007).
However, the distinction of antioxidant activities in biological systems between tocopherols and tocotrienols is clear. Free radicals attack on cell membrane results in peroxyl radicals and lipid peroxidation which is responsible for hypercholesterolemia and cardiovascular diseases. Studies have displayed the superiority of α-tocotrienol over α-tocopherol in neutralizing the peroxyl radicals and lipid peroxidation in rat liver and 15
liposomal membranes (Ghafoorunissa et al., 2004; Serbinova et al., 1991; Suzuki et al., 1993). In HUVEC (human umbilical vein endothelial cell) cells, palm tocotrienol rich fraction (TRF) was more efficient than αtocopherol in attenuating thiobarbituric reactive substances (TBARS) (Ghafoorunissa et al., 2004). In rat brain mitochondria, γ-tocotrienol elicited stronger protective effect against oxidative damage (Kamat and Devasagayam,
administration of tocotrienols abated protein carbonylation and extended mean life span, where α-tocopherol supplementation had no effect (Adachi and Ishii, 2000). This could be explained with a few possible mechanisms. Firstly, tocotrienols are more uniformly distributed in the cell membrane bilayer (Palozza et al., 2006). Secondly, tocotrienols have a stronger disordering effect on phospholipids due to its unsaturated isoprenoid side chain which results in its “arc” conformation over the chromanol ring, thus rendering a more effective interaction to lipid radicals (Serbinova et al., 1991; Simone et al., 2008). Lastly, tocotrienols have a higher recycling efficiency from its chromanoxyl radicals over tocopherols (Packer et al., 2001; Theriault et al., 1999).
The antioxidant efficacy of vitamins E on reactive nitrogen species (RNS) is gaining more attention recently. RNS includes nitric oxide (NO), nitrogen dioxide (NO2) and peroxynitrite (ONOO-). The reaction of α-tocopherol with NO2 yields a nitrosating agent, while γ-tocopherol reduces NO2 to NO without generating nitrosating species (Cooney et al., 1995). It was 16
hypothesized that possessing an unsubstituted 5-position on the chromanol ring, γ- and δ-isoforms, but not α- and β-isoforms of vitamin E, renders the ability to neutralize RNS (Christen et al., 1997). The scavenging of NO2 and ONOO- by γ-tocopherol is superior to that of αtocopherol, which resulted in the formation of 5-nitro-γ-tocopherol. This was proven in a study where 5-nitro-γ-tocopherol was detected in the plasma of zymosan-induced peritonitis in rats (Christen et al., 2002). Similarly in radiological threat, nitric oxide synthase (NOS) is stimulated and produced RNS, where only prophylactic administration of γ-tocotrienol among the vitamin E isoforms protected mice from radiation damage (Berbée et al., 2009; Ghosh et al., 2009).
1.3.4 Vitamin E Therapy in Inflammatory Diseases In lipopolysaccharide-induced RAW264.7 macrophage and IL-1β-induced lung epithelial cell models, vitamin E isoforms could attenuate PGD2 and PGE2, respectively, with varying potencies: γ-tocotrienol ≈ δ-tocopherol > γ-tocopherol >> α-/β-tocopherol (Jiang et al., 2008). Likewise, the production of LTB4 and LTC4 in HL-60 cells and human neutrophils when stimulated with ionophore (A23187) was abrogated by γ-, δ-tocopherol and γ-tocotrienol. α-Tocopherol was much less potent in reducing leukotrienes with IC50 of 40-60 µM as compared to γ-tocotrienol with IC50 of approximately 5 µM (Jiang et al., 2008). It has been shown that γtocotrienol was more potent than all tocopherol isoforms in ameliorating lipopolysaccharide-induced RAW246.7 macrophage production of IL-6 and 17
G-CSF, probably via the downregulation of NF-κB activation and suppression of CCAAT-enhancer binding protein (C/EBP) (Wang and Jiang, 2013).
As for allergic dermatitis, in a randomized, double-blind, placebocontrolled trial, patients were fed 400 mg/day α-tocopherol for 60 days, and beneficial effects were observed (Javanbakht et al., 2010; Kosari et al., 2010). In a murine picryl chloride-induced dermatitis model, mice developed scratching behavior, dermal thickening, mast cell degranulation and heightened histamine levels in serum (Tsuduki et al., 2013). Oral 1 mg/day of rice bran tocotrienol (97.5% of tocotrienol – 3.5% α-tocotrienol, 89.9% γ-tocotrienol and 4.1% δ-tocotrienol) reversed the disease outcome via the inhibition of protein kinase C (PKC) activity.
Patients suffering from inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, were found to have lower levels of vitamin E than normal healthy subjects (Bousvaros et al., 1998). In experimental colitis induced by acetic acid in rats, oxidative damage and both macroscopic and microscopic structural damages in colon were observed. Oral administration of 100 mg/kg/day of α-tocopherol abated oxidative and structural damage on the acetic acid-challenged colon (Bitiren et al., 2010). In a randomized controlled trial of stable but oxidatively stressed Crohn’s disease patients, supplementation of 533 mg/day α-tocopherol for four weeks significantly reduced oxidative stress (Aghdassi et al., 2003). The 18
development of bowel stenosis in Crohn’s disease involves extracellular matrix deposition. Human intestinal fibroblasts isolated from colonic or ileal tissue of Crohn’s disease and ulcerative colitis, when treated with TRF, reduced cell proliferation and enhanced programmed cell death via apoptosis and autophagy of the intestinal fibroblasts (Luna et al., 2011b). In a follow-up study, it was observed that TRF-mediated decrease in extracellular matrix production and fibrogenesis of human intestinal fibroblasts was due to inhibition of TGF-β1 (Luna et al., 2011a).
In a transgenic KRN/NOD mouse model of human rheumatoid arthritis, treatment with 0.134 mg/day α-tocopherol for six weeks decreased IL-1β, reduced white blood cell counts to those of the control, abated oxidative stress and prevented joint destruction. However, it was unable to modify the date of onset of disease or alter the disease intensity (Bandt et al., 2002). In a carrageenan-induced air pouch inflammation model, combined γ-tocopherol and aspirin provided better anti-inflammatory action than combined α-tocopherol and aspirin or aspirin alone. In a recent study using 4 mg/kg of collagen in complete Freund’s adjuvant rheumatoid arthritis rat model, the collagen-induced arthritic rats had increased body weight, paw edema, CRP, TNF-α; and reduced glutathione and superoxide dismutase level. Oral treatment with 5 mg/kg/day γ-tocotrienol for 25 days significantly restored all targets back to levels found in control rats. Arthritic rats had synovial fibrosis, congestion and hyperplasia in histopathological analysis of joint morphology, where treatment with γ19
tocotrienol exhibited mild synovial hyperplasia (Radhakrishnan et al., 2014). In a similar model where arthritis in rats was induced by intradermal injection of collagen type II emulsified in complete Freund’s adjuvant, arthritic rats developed paw edema, bodyweight gain, histopathological changes in synovial hyperplasia and inflammation, CRP level and collagen-specific T-cells (Haleagrahara et al., 2014). Collagen-induced arthritis rats orally treated with 10 mg/kg δ-tocotrienol for 25 days, had reduced paw edema, bodyweight restoration, suppression of collagenspecific T-cells and CRP level. δ-Tocotrienol treatment was more effective than glucosamine in reduction of paw edema. The significant reversal of collagen-induced arthritis by δ-tocotrienol may be a viable therapy for rheumatoid arthritis. These findings suggest that tocotrienols might be a better candidate than tocopherols as anti-inflammatory agents.
1.3.5 Summary and Outlook of Vitamin E Vitamin E research is nearly a century long in a couple of years’ time. The masses know vitamin E (α-tocopherol) as a fertility factor and a key component in cosmetic products. However, the public is unaware that there are a total of 8 naturally occurring forms within the vitamin E family, and an additional seven relatively physiologically inactive forms created in synthetic dl-rac-α-tocopherol (Burton et al., 1998; Traber and Arai, 1999). Tocotrienols, consisting of one half of vitamin E family, has been the subject of approximately 3% of the studies published when obtained from 20
PubMed. The scientific community recognizes that vitamin E is one of the most
oxidative/nitrosative damage (Cooney et al., 1995; van Acker et al., 1993). In addition to vitamin E ability to neutralize free radicals as an antioxidant, it has also been shown to modulate signaling pathways including PPAR, C/EBP, STAT6, NF-κB and Nrf2, as well as signaling/inflammatory molecules such as apoptotic regulators (Bcl-2 and caspase-3), cytokines (IL-1β, IL-4, IL-5, IL-6, IL-13, TNF-α, TGF-β and G-CSF), kinases (c-Src, ERK, MAPK, PI3K, PK6 and PKC) and enzymes (AP, Cat, GPx, SOD, eNOS, GTPase, HMG-CoA reductase, HO-1, COX-2, 5-LOX, 12-LOX and PLA2) (Table 1B).
Table 1B| Molecular targets modulated by tocopherols and tocotrienols Molecular Targets Modulated
↑ ↑ ↔ ↓ ↓ ↓ ↓ ↓
Bcl-2 Caspase-3 IL-1β IL-4, IL-5 IL-6 IL-13 G-CSF
↓ ↔ ↑ ↑ ↓ ↓ ↓ ↔
T3 ↑ ↓ ↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
↓↓ ↑ ↑ ↑↑ ↓ ↓ ↓ ↓
↓ ↓ ↓ ↑ ↔ ↔ ↓ ↔ ↔ ↔ ↔ ↔ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓
12-LOX, PLA2 c-Src, ERK MAPKα, Src MAPKβ PI3K PK6 PKC C/EBP NF-κB Nrf2 PPARα STAT6 Akt Apolipoprotein A1 Apolipoprotein B C-reactive protein
↓↓ ↓↓ ↓↓ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑↑ ↓ ↓ ↓↓ ↓ ↓ ↓
G-CSF (radiation) Alkaline phosphatase Catalase, GPx, SOD COX-2, 5-LOX eNOS GTPase
Collagen type I α1 Histamine LTB4, LTC4, LTD4
References (Lekli et al., 2010; Mukherjee et al., 2008) (Kuhad and Chopra, 2009b; Miyoshi et al., 2005) (Devaraj et al., 2008; Kuhad and Chopra, 2009b) (Peh et al., 2015; Wagner et al., 2008) (Chin and Ima-Nirwana, 2014; Wang and Jiang, 2013) (Peh et al., 2015; Wang et al., 2012) (Peh et al., 2015; Wang and Jiang, 2013) (Chin and Ima-Nirwana, 2014; Devaraj et al., 2008; Qureshi et al., 2010) (Jiang et al., 2011; Kuemmerle et al., 1997; Kuhad and Chopra, 2009b) (Li et al., 2013; Satyamitra et al., 2011; Singh et al., 2014b) (Norazlina et al., 2000) (Matough et al., 2014; Musalmah et al., 2002; Peh et al., 2015; Venditti et al., 2011) (Jiang, 2014; Jiang et al., 2008) (Das et al., 2008; Freedman et al., 2000; Mukherjee et al., 2008) (de Diego-Otero et al., 2008; Mo et al., 2012) (Berbée et al., 2009; Deng et al., 2014; Parker et al., 1993; Qureshi et al., 2002) (Das et al., 2008; Jenkins et al., 2001; Mukherjee et al., 2008; Niess et al., 2000) (Khanna et al., 2010) (Sen et al., 2000) (Das et al., 2008; Mukherjee et al., 2008) (Das et al., 2008; Mukherjee et al., 2008) (Nakaso et al., 2014) (Li et al., 2013) (Freedman et al., 2000; Tsuduki et al., 2013) (Wang and Jiang, 2013) (Peh et al., 2015; Wang and Jiang, 2013) (Li et al., 2012; Peh et al., 2015) (Fang et al., 2010) (Wang et al., 2012) (Lekli et al., 2010; Mukherjee et al., 2008) (Cloarec et al., 1987; Daud et al., 2013; Nicod and Parker, 2013; Qureshi et al., 1991) (Devaraj and Jialal, 2000; Haghighat et al., 2014) (Chin and Ima-Nirwana, 2014; Chojkier et al., 1998; Jiang et al., 2011; Norazlina et al., 2000) (Gueck et al., 2002; Tsuduki et al., 2013) (Jiang, 2014; Jiang et al., 2008) (Chin and Ima-Nirwana, 2014; Jenkins et al., 2001; Norazlina et al., 2000) (Gueck et al., 2002; Jiang, 2014; Jiang et al., 2008)
TP: tocopherol; T3: tocotrienol; ↓: moderate decrease; ↓↓: substantial decrease ↑: moderate increase; ↑↑: substantial increase; ↔: no effect; ↓↑: contradicting results where both ↓ and ↑ were reported.
α-Tocopherol had been regarded as the main isoform of vitamin E after the discovery of αTTP in the liver which selectively binds to α-tocopherol to prevent its degradation (Min et al., 2003), thus explaining its much longer half-life in the plasma over the other seven isoforms (Leonard et al., 2005). Despite that, γ-tocotrienol was observed to be absorbed significantly faster than α-tocopherol in the intestines (Tsuzuki et al., 2007). Aside from bioavailability issue, tocotrienols were shown to have comparable or superior efficacies in various diseases. In particular, tocopherols do not have cholesterol-lowering properties via the inhibition of HMG-CoA reductase
Supplementation of tocotrienols has potential to prevent atherosclerosis (Daud et al., 2013), and tocotrienols were shown to have better therapeutic benefits over tocopherols in platelet thrombosis (Qureshi et al., 2011). Tocotrienols anti-inflammatory actions are superior to α-tocopherol, possibly due to the ability to inhibit activation of STAT6 (Wang et al., 2012). Both γ-tocopherol and γ-tocotrienol, but not α-tocopherol, abrogate COX and 5-LOX-mediated eicosanoids in biological system to reduce inflammation (Jiang, 2014). Nonetheless, despite vitamin E (tocopherols) has an established safety record, it is necessary to ascertain the safety of tocotrienols in chronic studies and determine the maximum tolerated dose since tocotrienols differ quite substantially from tocopherols.
In light of this, it is highly recommended that future research claims on vitamin E to specify the specific form employed to prevent any confusion. 23
For instance, vitamin E was reported to perform below expectations in a clinical trial in preventing cardiovascular diseases, and to actually increase all-cause of mortality in test subjects (Miller et al., 2005). It is scientifically inaccurate to discount vitamin E on the whole when the study only adopted the use of α-tocopherol. In recent decade, vitamin E took quite a hit when clinical trials yielded disappointing or conflicting results (CookMills et al., 2013; Greenberg, 2005; Lee et al., 2005; Lonn et al., 2005; Riccioni et al., 2006). The isoform of vitamin E applied in most clinical trials then was α-tocopherol. As shown in clinical trials registered and papers published, tocotrienols holds barely 3% foothold in the entire vitamin E collection. With tocotrienols success in various diseases in preclinical models, one frontier for tocotrienol research in the next decade shall be the translation from bench to bedside.
Asthma is a chronic inflammatory disease of the airways, with four classical hallmarks – inflammation, mucus hypersecretion, airway hyperresponsiveness (AHR) and airway remodeling (Figure 1.02) (Fanta, 2009). This chronic inflammatory disorder in the lungs is orchestrated by various inflammatory cells and cellular elements to mount a combination of immune and allergic responses in the host. Bronchial biopsies conducted in asthmatic patients proved the presence of inflammation (Busse and Lemanske, 2004). Inflammatory responses observed in asthma include mast cell degranulation and infiltration of eosinophils and neutrophils. Transcription factors like NF-κB are activated to express pro-inflammatory genes, where the accumulation of all these factors initiate, worsen and sustain asthma (Barnes, 1996). Airway inflammation can initiate oxidative stress in asthma where it is believed to contribute to the recruitment of inflammatory cells into the airway and pathophysiology of asthma (MacNee, 2001). Among the inflammatory leukocytes, eosinophils and neutrophils are reported to produce the most amounts of reactive oxygen and nitrogen species. Under inflammatory conditions, antioxidant enzymes activities are observed to be reduced (Giustarini et al., 2009). The inability to defend against these oxidants results in oxidative damage on important biomolecules such as lipids, proteins and DNA present in the cells or lung tissue. Oxidative damage to the cells, if left untreated, will impede its normal physiological functions, thus supporting the pathology of asthma (Andreadis et al., 2003). The accumulated inflammation and oxidative 25
damage results in AHR that leads to four clinical hallmarks – recurrent episodes of coughing, shortness of breath, chest tightness and wheezing (Asher and Pearce, 2014). The failure to adhere to treatments, developing resistance to current therapeutics, or worsening to chronic bronchitis can eventually lead to death (Masoli et al., 2004).
Current treatments for asthma include inhaled/oral corticosteroids and β2adrenoreceptor
corticosteroids have many undesirable adverse effects like easy bruising, poor wound healing, obesity, increased susceptibility to infection and thinning of skin (Rang et al., 2011). A group of asthmatic patients displayed insensitivity to steroidal treatment from the very first dose, while others are prone to develop resistance against corticosteroids therapy with time (Sher et al., 1994). Therefore, there is a need for a new therapeutic agent with increased efficacies and reduced or negligible side effects to treat asthma.
Figure 1.02| Anatomy of an asthmatic airways. Figure obtained from National Heart, Lung, and Blood Institute – NIH, USA.
1.4.1 Epidemiology of Asthma Asthma is becoming common and rising globally, creating an increased stress and burden socially in developed and developing countries (Barnes, 2008b). Asthma is estimated to have affected over 300 million people worldwide, with an estimated increase of 100 million more people by year 2025 (Masoli et al., 2004; Peters et al., 2006). Its prevalence increases 50% every decade (Braman, 2006), thus becoming a major public-health concern (Devereux, 2006). Facing approximately 250,000 deaths annually, the direct and indirect costs surmounted by asthma exceeds that of HIV/AIDS and tuberculosis combined (Braman, 2006; Martinez and Vercelli, 2013). It is currently the 14th most important disease worldwide, due to the extent and duration of disability, and its huge economic burden
from increased pharmaceutical costs and reduced productivity (Croisant, 2014; Nieto et al., 2001).
The incidence of asthma is higher in developed countries – Australia (14.7%), Canada (14.1%), New Zealand (15%), United Kingdom (>15%) and United States of America (10.9%) (Figure 1.03) (Braman, 2006). In Singapore, as obtained from the Singapore Health Promotion Board 2015, approximately 20% of children and 5% of adult suffer from asthma, with estimation of prevalence to increase another 25% in the next 15 years. In USA alone, the total annual healthcare cost of asthma is approximately US$56 billion dollars (Barnett and Nurmagambetov, 2011). In Singapore, the cost is estimated to be US$33.93 million per annum (Chew et al., 1999). This was made up of US$17.22 million in direct costs (medication and hospitalization) and US$16.71 million in indirect costs (loss of productivity and absence from work). The growing prevalence, mortality and economical costs of asthma encourage the need to better understand asthma and discover new novel cost-effective therapeutic agents.
Figure 1.03| The global prevalence of asthma. Figure adapted from (Devereux, 2006).
1.4.2 Development of Asthma There are many causes of asthma, one of such is inhaled allergens (house dust mite, pollen, animal dander) or isocyanates, which form haptens and mount an immunological response, leading to inflammation of the airway (Wisnewski et al., 1999). It is classified under environmental factors, where its persistence is the likely cause of pathogenesis (Busse and Rosenwasser, 2003). The genetics aspect of asthma is yet to be ascertained, as the underlying cause and inheritance pattern of the genome is complex (Lemanske and Busse, 2003). A study using DNA microarray analysis suggests that arginase, the final enzyme in the urea cycle, is one of the causes of asthma pathogenesis (Zimmermann et al., 2003). Among all the causes, the interaction between genetics and 29
environmental factors generally lead to the inflammation and pathology of the airway (Cohn et al., 2004). In the past two decades, asthma therapy was mainly focused on bronchodilation and anti-inflammation of the airways (Halayko et al., 2006).
1.4.3 Immunopathology of Allergic Asthma Allergic asthma is commonly initiated by an inappropriate immune response towards inhaled/exposed allergens that are normally innocuous (Martinez and Vercelli, 2013). Allergens are mostly protein-based, such as HDM, spores, pollens, animal dander or certain food (e.g.: peanut, eggs, etc) (Galli et al., 2008). The major allergen is HDM, with two major strains – Dermatophagoides pteronyssinus and Dermatophagoides farina, which can activate receptors on the airway epithelium such as Toll-like receptor (TLR) and pattern recognition receptor (PRR) to initiate allergic responses (Hammad and Lambrecht, 2008). This results in the capture and processing of these antigens by dendritic cells in the basement membrane of the airway epithelium, where they mature and migrate to lymph nodes to present the processed antigen to naïve CD4+ T cells (Busse and Lemanske, 2001; Larché et al., 2003). The presence of IL-4 cytokines drive the differentiation of naïve CD4+ T cells into CD4 T-helper 2 cells (Th2 cells). The generation of various cytokines like IL-4, IL-5, IL-9 and IL13 by Th2 cells contribute to the inflammation of the airway (Figure 1.04).
Figure 1.04| Dendritic cell presentation in asthma. Figure obtained from (Lambrecht and Hammad, 2015).
IL-4 and IL-13 induce B cells to undergo isotype class switching from IgM to IgE (Corry and Kheradmand, 1999). The interaction between Th2 cells and B cells (CD40 – CD40L), and the cytokines activates B cells to produce IgE antibodies into the bloodstream, which binds to IgE receptors (FcεRI) present on mast cells (Kabesch et al., 2006). The cross-linking of IgE and FcεRI on mast cells surface activates mast cell degranulation, releasing pro-inflammatory mediators like histamine and prostaglandins (Figure 1.05). This brings about the early phase reactions (1 to 4 hours after onset) in asthma, consisting constriction of airway smooth muscles and bronchoconstriction (Janeway et al., 2008).
Figure 1.05| Immunology of asthma. Figure obtained from (Holgate and Polosa, 2008).
IL-5 regulates the maturation and migration of eosinophils into the blood circulation. IL-5 too mediates the survival of eosinophils (Epstein and Rothenberg, 1998). The entry of eosinophils into the lungs, combined with macrophages and lymphocytes which secrete pro-inflammatory mediators, cytokines and chemokines, results in the late phase reactions (6 to 18 hours after onset). These reactions include bronchospasm, damage to airway epithelial cells and mucus hypersecretion (Figure 1.06) (Janeway et al., 2008; Roquet et al., 1997).
Figure 1.06| Immunological pathways leading to inflammation of the airway. Figure obtained from (Devereux, 2006).
Classical Hallmarks of Asthma
The first hallmark is airway inflammation, whose degree is correlated to the severity of the asthmatic condition of the patient and may determine the responsiveness towards treatment (Lemanske and Busse, 2003). Bronchial biopsies conducted in asthmatic patients proved the presence of inflammation (Busse and Lemanske, 2004). Inflammatory responses observed in asthma include presence of IgE antibodies, mast cell degranulation and infiltration of eosinophils and neutrophils. Transcription factors like NF-κB are activated to express proinflammatory genes (Barnes, 1996), where the accumulation of all these factors initiate, worsen and sustain asthma. Airway inflammation can initiate oxidative stress in asthma (MacNee, 2001; Nadeem et al., 2003).
The second hallmark is mucus hypersecretion. In healthy individuals, mucus in the lungs protects the epithelium from injury and assists the
removal of bacteria and debris from the lungs. However, overproduction of mucus in the airways will narrow the airway lumen, and in severe asthma, form plugs that can block the airway (Lemanske and Busse, 2003). The sources of mucin glycoprotein (MUC) are goblet cells and mucus glands. There are 13 known genes of MUC (MUC1-4, MUC5AC, MUC5B, MUC6-9 and MUC11-13) in the human airways (Seong et al., 2002). Current literature states that IL-4, IL-5, IL-13 and IL-17 are required for goblet cell hyperplasia and MUC5AC production (Zhao et al., 2009). Muc5ac gene expression hinges on the transcriptional activity of NF-κB, where the downregulation of NF-κB activation attenuated mucus secretion (Fujisawa et al., 2009). Clinically, MUC5AC is the predominant gel-forming mucin expressed in asthmatic patients sputum and is released during exerciseinduced bronchoconstriction (Hallstrand et al., 2007).
The third hallmark is airway remodeling, where the smooth muscles in the airway thickens, along with collagen deposits (Lemanske and Busse, 2003). The accumulation of airway inflammation, mucus hypersecretion and airway remodeling results in the final hallmark, airway hyperresponsiveness (AHR), which causes breathlessness and wheezing. Clinical management of asthma relies largely on spirometry to monitor the lung function in patients (Fanta, 2009). AHR is defined as an augmented bronchoconstrictor response to a stimulus. Inflammatory mediators like IL4, IL-5, IL-13 and IL-17 are revealed to potentiate AHR in asthma. IL-5 recruits eosinophils into the airways, leading to the release of major basic 34
protein, eosinophil peroxidase and cysteinyl-leukotrienes, which are associated with AHR (Mould et al., 2000). IL-13 involvement in AHR is essential, as IL-13-/- mice failed to develop allergen-induced AHR and administration of recombinant IL-13 restored AHR responses (Walter et al., 2001). IgE-mediated mast cell degranulation is necessary for AHR also by producing numerous inflammatory cytokines and mediators (Kobayashi et al., 2000). It is gaining acceptance that oxidative stress can mediate AHR too. Pre-treatment of catalase in sheep suppressed AHR (Sugiura and Ichinose, 2008). It was also observed that ONOO˙ overproduction can induce AHR in guinea pigs (Sadeghi-Hashjin et al., 1996). Accumulation of H2O2 causes airway smooth muscle cell contraction, which in turn induces AHR in mice (Lee et al., 2006).
The infiltration of inflammatory cells into the airway, particularly eosinophils, is the key characteristic feature and central effector in the inflamed asthmatic airway (Figure 1.07) (Kelly et al., 2007; Sampson, 2000). Transmigration of eosinophils into the airway involves a cascade of signaling pathways, where the priming and activation of CD4 Th2 cells releases cytokines such as IL-3 and IL-5. In combination with chemokines like eotaxin and adhesion molecules such as ICAM-1, VCAM-1, Eselection and VLA-4; eosinophilia in the airways occurs (Hamelmann and Gelfand, 2001; Hogan et al., 2008; Jia et al., 1999).
Eosinophils when activated, secretes cytotoxic granule proteins like major basic protein (MBP) and eosinophil peroxidase (EPO), where the former induces the release of histamine from mast cells and basophils, resulting in the damage of airway epithelial cells (Gleich, 1990, 2000). EPO consists approximately 25% of the total granular proteins secreted. It is involved in the catalysis of free oxygen radicals and nitric oxide, which leads to the formation of reactive oxygen and nitrogen species, inducing oxidative damage to cells present in the lung tissues (MacPherson et al., 2001; Rothenberg and Hogan, 2006).
Besides eliciting damage to the airways directly from its production of granular proteins, studies have shown that eosinophils are the source of leukotriene (LT) – C4 (Cowburn et al., 1998; Gleich and Adolphson, 1986). LTC4 can be converted to metabolites via enzymatic modifications, such as LTD4 and LTE4, which are involved in the airway hyperresponsiveness, mucus hypersecretion and bronchoconstriction in asthma (Csoma et al., 2002; Kariyawasam and Robinson, 2007). The multiple involvements of eosinophils in asthma have led to the eosinophil hypothesis in asthma.
In a study conducted by Foster et al (1996), it was shown that a deficiency in IL-5 reduces eosinophils trafficking into the airways, where eosinophilia in the BALF was observed to be reduced by over 90%. The reduction of eosinophils
hyperresponsiveness and lung damage in the mouse asthma model (Foster et al., 1996). In the sputum of asthmatic patients, the decrease in levels of MBP (generated by eosinophils) with treatment, leads to the improvement in airway function (Gleich, 1990). These suggest the potential of targeting eosinophils in the treatment of asthma.
Figure 1.07| Eosinophils effector functions in asthma. Figure obtained from (Walford and Doherty, 2014).
Besides eosinophils, neutrophils are also observed to play a role in the pathogenesis of asthma (Fujisawa et al., 1993; Lamblin et al., 1998). Although the direct relationship between neutrophils and asthma has yet 37
to be ascertained (Kay and Corrigan, 1992), the presence of neutrophils can help indicate the severity of the asthmatic condition. The infiltration of neutrophils is observed to be minimally present in the airways of patients with mild-to-moderate asthma, but it is noticeable in the airways of patients with severe asthma and acute asthma exacerbations (Jatakanon et al., 1999; Sampson, 2000). Therefore, neutrophilia could possibly be one of the biomarkers for chronic or severe asthma.
Neutrophils possess many granular secretory vesicles which contain myeloperoxidase (MPO), thromboxane A, matrix metalloproteinase-9 (MMP-9), cathepsins and serine proteases, that can result in inflammation and oxidative damage upon its release (Fahy, 2009; Sampson, 2000). Neutrophils can attract eosinophils through chemoattractant IL-8 (or KC in mouse), and induce eosinophils degranulation by secreting neutrophilic lactoferrin and elastase (Monteseirin, 2009). MMP-9 is primarily produced by neutrophils, which also promotes the migration of eosinophils into the airways. MMP-9 is also involved in the remodeling of the airways (Atkinson and Senior, 2003). Dendritic cells presentation requires MMP-9 for antigen uptake in the airway, therefore, MMP-9 knockout mice resulted in the reduction in allergic airway inflammation (Vermaelen and Pauwels, 2003). Neutrophils are also major mediators in the production of free radicals, such as superoxide anion and hydrogen peroxide (Hanazawa et al., 2000b). During inflammation, MPO in neutrophils can react with hydrogen peroxide to form hypochlorous acid (HOCl). Interestingly, it was 38
also shown that neutrophils obtained from asthmatic lungs were producing higher levels of free radicals as compared to neutrophils from healthy lungs (Monteseirín et al., 2002). This results in increased oxidative damage in the lungs.
Although corticosteroids are effective at abating eosinophilia in the airways, it has been consistently shown that they are ineffective against neutrophils (Ito et al., 2008b; Maneechotesuwan et al., 2007). It is gaining traction that neutrophilia is correlated to usage of corticosteroids. It was hypothesized that neutrophilia in severe asthmatics is attributable to the broad suppression of inflammatory cells in the airways, except neutrophils. Chronic usage of corticosteroids may lead to elevated neutrophils in the lungs, which eventually develops into the most severe form of asthma, known as steroid-resistant asthma (Jatakanon et al., 1999; McKinley et al., 2008; Sur et al., 1993). Therefore, it would be beneficial to discover novel therapeutics which can attenuate neutrophils in asthma, so as to complement corticosteroid therapy.
1.4.4 Oxidative Stress The consensus definition of oxidative stress by Helmut Sies in 1985 would be the imbalance between oxidants and antioxidants, where oxidants are favored (Sies et al., 1985). Under normal conditions, the electron leakage of mitochondrial electron carriers and enzymes during respiration leads to
the formation of free oxygen radicals or oxidants. Phagocytic inflammatory cells like macrophages do secrete oxidants too (Kehrer, 1993). Oxidants are produced constantly in the body and are regulated by antioxidants enzymes, thus making it challenging to maintain this balance (Giustarini et al., 2009). Reactive oxygen or nitrogen species are molecules that contain oxygen or nitrogen which generate free radicals. Under pathophysiological conditions, generation of both reactive oxygen and nitrogen species are significantly increased by various mechanisms like inflammation and parasites invasion, resulting in the shift in balance (Halliwell and Cross, 1994). The defenses by antioxidants are not able to sustain the hike in oxidants, and this occurrence is termed oxidative stress, which will lead to oxidative damage in the host (Sies, 1991). Key antioxidant enzymes are superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) (Sies, 1997).
Oxidative stress involves two distinct observations (Giustarini et al., 2009): (1) increased levels of oxidants (highlighted in yellow in Figure 1.08) probably due to the reduced activities of antioxidants (enzymes circled in blue in Figure 1.08); and (2) the presence of oxidative damage markers to lipids, proteins and DNA (molecules circled in red in Figure 1.08).
O2 . -
Figure 1.08| Brief Overview of Oxidative Stress. Figure adapted from (Kirkham and Rahman, 2006). Molecules highlighted in yellow represents the oxidants; enzymes circled in blue represents the antioxidant enzymes; items circled in red represents oxidative damage biomarkers.
1.4.5 Oxidative Stress in Asthma The classical hallmarks of asthma are listed as airway inflammation, airway hyperresponsiveness, airway remodeling and mucus hypersecretion (Devereux, 2006; Fanta, 2009). Oxidative stress in asthma is believed to contribute to these classical hallmarks, recruitment of inflammatory cells into the airway, pathophysiology of asthma, and even possibly insensitivity to steroidal treatments (MacNee, 2001; Wood et al., 2000). Among the inflammatory leukocytes, eosinophils and neutrophils are reported to produce the most amounts of reactive oxygen and nitrogen species (Dozor, 2010; Giustarini et al., 2009). Some examples of reactive oxygen species include hydrogen peroxide (H2O2), hydroxyl radicals (OH˙), superoxide anion (O2˙−) and nitric oxide (NO), while reactive nitrogen 41
species include peroxynitrite (ONOO-) (Caramori and Papi, 2004; Dworski, 2000). Normally, the host can tolerate low levels of oxidants by the protection of antioxidants present in the lungs. However, under inflammatory conditions, antioxidant enzymes activities are observed to be reduced (Comhair, 2005; Nadeem et al., 2003; Sies, 1997), resulting in high levels of oxidative stress which in turn promotes and supports the ongoing inflammation. The inability to defend against these oxidants results in oxidative damage on important biomolecules such as lipids, proteins and DNA present in the cells or lung tissue (Cho et al., 2004a; Montuschi et al., 1999). Oxidative damage to the cells, if left untreated, will impede its normal physiological functions, thus supporting the pathology of asthma (Andreadis et al., 2003; Wood et al., 2003).
The first evidence of oxidative stress in asthma includes the observed reductions of antioxidants activities from human asthmatic patients (Ghosh et al., 2006; Hasselmark et al., 1990; Perisic et al., 2007; Powell et al., 1994). This study will measure the activity and changes in expression of each key antioxidant enzyme pertaining to asthma, namely superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) (Sies, 1997). The second evidence includes the detection of increased levels of oxidants and oxidative damage biomarkers. Clinically used biomarkers of oxidative damage to the lipids, proteins and DNA are namely 8isoprostane,
(Louhelainen et al., 2008; Spencer et al., 1995). The biomarkers were 42
bronchoalveolar lavage fluid of human asthma patients (Mak and ChanYeung, 2006; Montuschi and Barnes, 2002; Nadeem et al., 2003; Sugiura and Ichinose, 2008; Takao, 2005). NADPH oxidase (NOX), a pro-oxidant, is a major producer of superoxide anion. Clinically, gene expressions of NOX were observed to be increased in asthmatic patients when compared to healthy individuals (Ökrös et al., 2012). NOX was also studied to play a pertinent role in the regulation and trafficking of both eosinophils and neutrophils into the lungs (Abdala-Valencia et al., 2007; Boldogh et al., 2005). Inducible nitric oxide synthase (iNOS) is another pro-oxidant responsible for the formation of NO, whereby its product fuels the production of ONOO˙ to attack tyrosine residues, resulting in 3-NT (Nadeem et al., 2007). Levels of iNOS were found to be heightened in asthmatic patients (Hansel et al., 2003).
Recent studies have revealed that oxidative stress promotes corticosteroid resistance in asthmatic patients (Adcock and Barnes, 2008; Barnes, 2004b). In steroid-resistant animal models, strategies to reverse steroid resistance focus on using antioxidants, Nrf2 activators, theophylline, PI3Kδ inhibitors and HDAC2 activators (Adcock and Ito, 2004; Barnes, 2004b, 2013a). Therefore, there is an urgent need to discover novel therapeutics to complement corticosteroids, which can ameliorate oxidative stress to reverse the development of steroid resistance in patients.
Impaired Antioxidant Defenses in Asthma
The levels of oxidants and antioxidants are kept at homeostasis. The downregulation of antioxidants will result in imbalance and oxidative stress. Studies have only shown that antioxidants activities are reduced from the exhaled breath condensate of human asthmatic patients (Sugiura and Ichinose, 2008). Key antioxidant enzymes include SOD, catalase, GPx and heme oxygenase (HO) (Figure 1.09), while key non-enzymatic antioxidants include glutathione (GSH) and antioxidants obtained from food such as vitamin C, vitamin E and flavonoid (Comhair and Erzurum, 2009).
Figure 1.09| Oxidative stress and oxidative damage in asthma. Molecules highlighted in yellow represents the oxidants; enzymes circled in blue represents the antioxidant enzymes; items circled in red represents oxidative damage biomarkers; green boxes represent the key biomolecules in the cell. 44
Superoxide Dismutase (SOD)
Superoxide dismutases (SODs) are metalloenzymes and also the only ones that dismutate superoxide anion (O2•-) into hydrogen peroxide (H2O2) (Figure 1.09). There are three subtypes of SOD, namely copper-zinc (CuZnSOD), manganese (MnSOD) and extracellular (ECSOD) SODs, respectively in order of their numerical subtypes (Kinnula and Crapo, 2003). The location of each SOD isotype differs. SOD1 is located in the cytoplasm, SOD2 in the mitochondria and SOD3 in the extracellular spaces of the cell. In severe asthma, impaired SOD activity was associated with AHR, airflow obstruction and airway remodeling (Chung and Marwick, 2010). On top of that, studies that measured the levels of oxidants and antioxidants in asthmatic patients revealed that the total SOD activity is reduced (Comhair, 2005; Kinnula and Crapo, 2003; Smith et al., 1997).
Catalase is a well-studied enzyme for many decades since the 1930s (Morton et al., 2008). It is present in most aerobic cells and its catalytic activity lies in the conversion of toxic hydrogen peroxide (H2O2) to water and molecular oxygen (McMurry, 2008) (Figure 1.09). Hydrogen peroxide is formed under normal respiration and pathogenic reactive oxygen species production (Loukides et al., 2002). The reduced activity of catalase was shown to be related to increased levels of oxidative stress (Nadeem et al., 2005). Under pathological conditions in asthma where 45
levels of oxidants are heightened due to the released reactive oxygen and nitrogen species by inflammatory cells (Emelyanov et al., 2001), the production of superoxide anion would result in the conversion into hydrogen peroxide by SOD. Hydrogen peroxide is toxic and is targeted by catalase for degradation, where catalase will be converted to compound I via the oxidation of its heme group from Fe(III) to Fe(V) (Kono and Fridovich, 1982). In the presence of superoxide anion, compound I catalase will be converted to compound II via the reduction from Fe(V) to Fe(IV), in which catalase is now inactivated (Figure 1.10). Catalase functional catalytic activity will be restored by reacting with a second superoxide anion. These additional steps results in the decreased activity of catalase.
Fe(III) + H2O2 2 H20 + O2 + Fe(V) Catalase
Fe(V) + O2- O2 + Fe(IV) Compound I
Fe(IV) + O2- O2 + Fe(III) Compound II
Figure 1.10| Superoxide anion presence results in decreased activity of catalase. Equations obtained from (Kono and Fridovich, 1982).
A recent study by Ghosh et al, (2006) revealed that oxidation of a specific cysteine (Cys377) results in the inactivation of catalase. In the BAL fluid and serum of asthmatic patients, catalase activity was reduced up to 50% when compared to healthy individuals (Ghosh et al., 2006; Novák et al.,
1991). The reduction of catalase activity resulted in the accumulation of hydrogen peroxide, which is toxic and will lead to oxidative damage in the lungs (Dworski, 2000; Ganas et al., 2001; Horvath et al., 1998).
Glutathione Peroxidase (GPx)
The role of glutathione peroxidase (GPx) is to convert hydrogen peroxide to water and oxygen, with the help of reduced glutathione (GSH) (Ho et al., 1997). GSH will be converted to oxidized glutathione (GSSG) in the process, which will be recycled by glutathione reductase (GR) (Figure 1.09). Several studies had shown that under severe asthmatic conditions, GPx activity is observed to be decreased significantly when compared to healthy individuals (Misso et al., 1996; Nadeem et al., 2003; Powell et al., 1994).
Nuclear Erythroid 2-Related Factor 2 (Nrf2)
Nrf2 is a redox-sensitive transcription factor involved in binding to the antioxidant response element (ARE), a promoter of many antioxidant genes that is essential for their inducible activation (Sussan and Biswal, 2014). Disruption of Nrf2 in mice were detected to be more susceptible to allergen-mediated airway inflammation (Rangasamy et al., 2005). Activation of Nrf2 in airway smooth muscle cells extracted from airway biopsies
(Michaeloudes et al., 2011a). Steroid resistance account up to 10% of the
asthmatics with critical Th17 responses, and Nrf2 mediators were hypothesized to be alternative drugs to such patients (Barnes, 2013a).
Increased Oxidative Damage in Asthma
The second common observation of oxidative stress is the increased levels of oxidative damage to the cells (Giustarini et al., 2009). As oxidants such as superoxide anion, peroxynitrite and hydrogen peroxide are too transient with very short half-lives, most studies adopt the measurements of oxidative damage biomarkers instead (Louhelainen et al., 2008; Wood et al., 2003). Key biomarkers of oxidative damage for the important biomolecules of the cell (lipids, proteins and DNA) are circled red in Figure 1.09.
Lipid Peroxidation (8-Isoprostane)
Lipids in the cell membrane and cytoplasm are present in large quantities, thus making it a common target of free radicals attack. The process of attack is known as lipid peroxidation, where it propagates continuously once initiated and can only be stopped in the presence of antioxidants (Figure 1.11). Products of lipid peroxidation are reactive and can lead to oxidative damage to the cells by modifying important biomolecules (Montuschi, 2004).
Initiation: R• + LH RH + L• Propagation: L• + O2 LOO• LOO• + LH LOOH + L• Termination: L• + AH LH + A• A• + LOO• LOO-A
Figure 1.11| Self-propagating mechanism of lipid peroxidation. Equations obtained from (Montuschi, 2004). R•, free radicals; L, lipids; A, antioxidants.
8-Isoprostane, also known as 8-iso Prostaglandin F2α, were found to be formed in human in vivo only in year 1990 and is the best characterized compound among the F2-isoprostanes (Morrow et al., 1990). Since year 2000, F2-isoprostanes emerged as one of the most representative biomarkers of oxidative stress (Wood et al., 2003). Several studies conducted clinically observed the increased production of 8-Isoprostane in exhaled breath condensates of asthmatic patients, whereby increase in 8Isoprostane levels were directly proportional with increasing severity of asthma (Baraldi et al., 2003a; Van Hoydonck et al., 2004; Zanconato et al., 2004). The levels of 8-Isoprostane in mild asthmatic patients were significantly higher than normal healthy individuals (Baraldi et al., 2003b; Montuschi et al., 1999; Shahid et al., 2005; Wood et al., 2000). Interestingly, a study performed by Montuschi et al, (1999) revealed that oxidative stress was not reduced in patients with severe asthma even when treated with corticosteroids, indicating that potent anti-inflammatory corticosteroids were not effective in the relief of lipid peroxidation to the lungs.
Protein Nitration (3-Nitrotyrosine)
An established biomarker of oxidative damage to proteins where proteins are nitrated, is 3-nitrotyrosine (3-NT) (Andreadis et al., 2003). In the presence of nitric oxide synthases (NOS) and arginine residues, nitric oxide (NO) is produced. The combination of NO with superoxide anion (O2•-) forms peroxynitrite (ONOO-), which attacks tyrosine residues to form 3-NT (Dedon and Tannenbaum, 2004; Ghosh et al., 2006) (Figure 1.09). There are many mechanisms to invoke the nitration of proteins, resulting in the gain or loss of that protein functionality and activity (Schopfer et al., 2003). A study by (Radi, 2004) showed that nitration of tyrosine residues on MnSOD results in the loss of activity of the antioxidant enzyme. Interestingly, peroxynitrite and 3-NT have shown to display dual roles in the cells; where 1) its increased levels indicate the presence of oxidative stress and oxidative damage, and 2) on its own, it is involved in cell signaling pathways (Szabo et al., 2007). A study had shown that peroxynitrite is involved in the activation of phosphopnositide3-kinase (PI3K)/Akt signaling pathway (Delgado-Esteban et al., 2007).
In asthma, levels of 3-NT measured from cellular fluids or exhaled breath condensate was observed to be increased in asthmatic patients when compared to healthy individuals (Hanazawa et al., 2000a; Kaminsky et al., 1999; Sugiura and Ichinose, 2008). Similarly in mouse asthma model, studies have shown that 3-NT levels were increased under asthmatic conditions (Cho et al., 2004a; Ito et al., 2008a; Okamoto et al., 2006). 50
When asthmatic patients or mice were treated with antioxidants, 3-NT levels were reduced, which corresponded to relief in inflammation and oxidative stress in the airways (Cho et al., 2004b; Kirkham and Rahman, 2006).
Oxidative DNA Damage (8-OHdG)
The classical biomarker for oxidative damage to DNA is 8-hydroxy-2deoxyguanosine (8-OHdG), where it is produced by reactive oxygen and nitrogen species attack on deoxyguanosine in DNA (Giustarini et al., 2009) (Figure 1.12). A study conducted by (Fitzpatrick et al., 2011), presented that 8-OHdG levels are increased under asthmatic conditions. A recent study had shown that free radicals in asthma will attack the DNA, resulting in modifications on both purine and pyrimidine bases such as crosslinking damage and DNA single/double-strand breaks (Chan et al., 2016). 8OHdG can be repaired by DNA base excision repair mechanisms, where DNA repair capacity were shown to be inhibited in asthma. It was suggested to adopt DNA repair drugs as potential therapy in asthma.
Figure 1.12| Production of 8-hydroxy-2-deoxyguanosine. Figure adapted from Osato Research Institute. 51
Relief of Asthma by targeting Oxidative Stress in the Lungs
Many studies have shown that by relieving oxidative stress in the lungs, the pathology of asthma will be reduced (Bowler, 2004; MacNee, 2001; Nel et al., 2001). A study which used α–lipoic acid, an antioxidant, on asthma mice model showed substantial reductions in oxidative damage to proteins and airway inflammation (Cho et al., 2004a). The restoration of antioxidants in the host to reduce oxidative stress was shown to be a sound therapeutic strategy in the treatment of asthma (Kirkham and Rahman, 2006).
1.4.6 Current Therapies of Asthma Various treatments for asthma are developed to target the inflammation and physiological remodeling of the airway, such as oral/inhaled corticosteroids,
antagonists and anti-IgE monoclonal antibody (Table 1C) (Fanta, 2009; Halayko et al., 2006). Current therapies serve primarily to control the disease, but not cure it. There are two main strategies to treat asthma, 1) to control the airway inflammation, and 2) to prevent and/or relieve bronchoconstriction (Blakey et al., 2013).
Table 1C| Current therapies in Asthma β2-agonists
Leukotriene receptor antagonists
Tachycardia, Tremor, Asthma Exacerbation
Rash, Arm/leg pain, Nausea
Quick Relief Long-term Control
Anti-IgE monoclonal antibody
Throat irritation, Osteoporosis, Dysphonia
Corticosteroids are the first-line of treatment and gold standard for treating asthma, where it relief asthma by reducing inflammation-associated leukocytes infiltration to the airway and suppress airway inflammation. It inhibits
transcription factors like NF-κB and activator-protein 1. It can upregulate β2-adrenoreceptor
bronchospasm through the dilation of bronchioles, airway smooth muscle relaxation and increasing airflow into the lungs (Fanta, 2009; Sovijärvi et al., 2003). Corticosteroids suppress but not cure airway inflammation, thus along with its undesirable side effects (water retention, lipids and cortisol metabolism dysfunction, cataracts, osteoporosis, and increased risks of opportunistic infections) over prolonged treatment is not ideal (Fanta, 2009; Sovijärvi et al., 2003). Another major setback is that some patients eventually become resistant to corticosteroids, which necessitates the need for higher doses, converting the use of inhaled corticosteroids to oral corticosteroids for systemic suppression of uncontrolled inflammation. 53
Corticosteroids were reported to be ineffective against neutrophilic-based inflammation in asthma as well. Moreover, there are approximately 10% of the asthmatic population who responds poorly or does not respond to corticosteroids from the start, and account for about 50% of the total healthcare cost in managing asthma (Barnes, 2012). In the recent decade, inhaled corticosteroids are combined with long-acting β2-adrenergic agonists (ICS+LABA) for quick relief of the airways, as well as long term control of the ongoing inflammation (Lalloo, 2002; NHLB-NIH, 2007; Rosenhall et al., 2002). It is well-tolerated in patients and proven to be effective clinically in mild-severe asthma.
β2-agonists coupled to Gs proteins activate adenylate cyclase to convert ATP to cAMP, which catalyzes the activation of PKA to phosphorylate MLCK
bronchodilation (Rang et al., 2007). Despite their potent capabilities for bronchodilation, β2-agonists are unable to suppress the ongoing inflammation in the airways. Short-acting β2-agonists (SABA) provide temporary short-term relief (onset in 5 mins, duration of action 4-6 hours) of the airway, while long-acting β2-agonists (LABA) deliver a long-term (more than 12 hours) bronchodilation (Fanta, 2009). SABA, a standalone therapy, is recommended for use only when needed for quick relief or before exposure to known asthma triggers, as the duration of action may shorten with regular use via β2-adrenergic receptor desensitization and downregulation (Nishikawa et al., 1994). On the otherhand, LABA is 54
prescribed as a combined therapy with inhaled corticosteroids (Fanta, 2009). The usage of LABA alone was reported to increase risk of asthmatic exacerbations (Beasley et al., 2010). The reduction of AHR by LABA without abating the airway inflammation, leads to false perception of controlling the disease, and result in uncontrolled progression of the inflammatory process.
Leukotriene receptor antagonists bind to cysteinyl leukotriene 1 receptors expressed on the airway smooth muscles and block the action of leukotriene C4, D4 and E4 ligands, resulting in bronchodilation and reduction of circulating eosinophils in the blood (Reiss et al., 1998). While leukotriene receptor antagonists displayed clinical improvements in asthmatic symptoms and lung function, they are less effective than inhaled corticosteroids. However, they are still being utilized as they are effective orally, lesser unwanted side effects, and provide an alternative treatment for patients who are resistant to corticosteroids (Barnes, 2011).
Omalizumab is the only novel anti-IgE monoclonal antibody therapy that has been approved for the treatment of severe asthma (Barnes, 2012). It is usually indicated for treatment when inhaled corticosteroids, LABA and leukotriene modifiers do not provide adequate control or cannot be used because of intolerable adverse effects. Other than binding to the portion of IgE that recognizes its high affinity receptor (FcεRI) on the surface of mast
cells and basophils, omalizumab can down-regulate expression of FcεRI, neutralizing IgE-mediated responses in asthma (Fanta, 2009).
The proposed guidelines for asthma treatment in increasing severity of asthma – 1) SABA only as needed; 2) SABA + low-dose inhaled corticosteroid (ICS); 3) low-dose ICS+LABA, or low-dose ICS + leukotriene receptor antagonist (LTRA); 4) medium/high-dose ICS+LABA or high-dose ICS+LTRA; 5) high-dose ICS+LABA and anti-IgE or oral corticosteroids (Figure 1.13). This excludes ~10% of the asthmatics who are resistant to current therapies.
Figure 1.13| Proposed guidelines for asthma treatment. Figure obtained from (Reddel et al., 2015).
In spite of decades of research into asthmatic treatments, approximately 5-10% of asthma patients remain resistant to corticosteroids. Current therapies can relief the symptoms but are unable to cure it, not neglecting the side effects of each drug. Moreover, current approved therapies only focused on attenuating inflammation, which neglected the oxidative stress and oxidative damage in the lungs. Therefore, the search of novel antiinflammatory drugs that function differently from corticosteroids, or drugs with higher therapeutic index that can ameliorate oxidative stress and complement corticosteroids (or re-sensitize steroid receptors) is required.
1.4.7 Corticosteroids in Asthma: Prednisolone Corticosteroids, or glucocorticoids, are endogenously produced or synthetic steroidal hormones which control multiple anti-inflammatory actions in the body. While dexamethasone is the most potent synthetic derivative available, the most commonly prescribed derivative in asthma is prednisolone (Figure 1.14) (Bentley et al., 1996). This is due to the balance between potency of anti-inflammatory actions against adverse effects profile. Dexamethasone and prednisolone are approximately 30 times and five times more potent than endogenous cortisol, respectively (Rang et al., 2011). Oral prednisolone is typically prescribed at a dose of 25-40 mg/day daily for five days, with a half-life of 18-36 hours in humans (Shefrin and Goldman, 2009). Currently, prednisolone is recommended for moderate to severe asthmatics and asthmatic exacerbations.
Figure 1.14| Chemical structure of prednisolone.
Anti-inflammatory potential of corticosteroids are facilitated by upregulating transcription of various anti-inflammatory mediators and inhibiting various NF-κB-induced pro-inflammatory genes (Sorrells and Sapolsky, 2007). Prednisolone binds to the glucocorticoid receptor (GR) which is activated upon ligand binding. The ligand-receptor complex then translocates to the nucleus, homodimerize, and bind to glucocorticoid response element (GRE) in the promoter region of steroid-sensitive genes (Figure 1.15). This switches off the transcription of multiple mediators such as inflammatory cytokines, chemokines, adhesion molecules, enzymes and receptors (Barnes, 2011). It also inhibit the production of leukotrienes and prostaglandins via the inhibition of phospholipase A2 and cyclooxygenase (COX-1
Corticosteroids also bind to coactivator molecules such as steroid receptor coactivator-1
element binding protein (CREB) and p300/CBP-associated factor (PCAF). The binding to coactivator molecules causes acetylation of lysine on histone-4, which activates genes encoding anti-inflammatory proteins,
such as secretory leukoprotease inhibitor (SLPI) (Barnes and Adcock, 2003). The activation of GR by corticosteroids can bind to coactivator molecules to inhibit histone acetyltransferase (HAT) activity directly. They can also promote activity of histone deacetylase (HDAC), which leads to suppression of pro-inflammatory genes.
Figure 1.15| Mechanism of action of glucocorticoid. Figure obtained from (Lowe et al., 2008).
Though highly effective, glucocorticoids have many undesirable adverse effects like easy bruising, poor wound healing, obesity, increased susceptibility to infection and thinning of skin (Rang et al., 2007). A group of asthmatic patients displayed insensitivity to steroidal treatment from the very first dose, while others are prone to develop resistance against glucocorticoids therapy with time (Corrigan et al., 1996; Sher et al., 1994).
While corticosteroids are effective at abating eosinophilia in the airways, it has been consistently shown that they are ineffective against neutrophils (Ito et al., 2008b; Maneechotesuwan et al., 2007). Although eosinophilia is dominant in allergic asthma, prominent neutrophilic inflammation was reported to be involved during asthmatic exacerbations (Fahy et al., 1995), and in cases of sudden-onset fatal asthma (Sur et al., 1993). It was also revealed in severe asthma that high-doses or chronic usage of corticosteroids treatment contribute to neutrophilia, as corticosteroids can increase apoptosis of eosinophils but decrease apoptosis of neutrophils (Cox, 1995; Meagher et al., 1996). Also, oxidative stress is a major component in asthma, where corticosteroids are ineffective in attenuating oxidative stress. High oxidants burden is hypothesized to be involved in steroid resistance as well (Marwick et al., 2007). Therefore, it is necessary to discover new therapies that can replace or complement corticosteroids.
1.4.7 Vitamin E as a Potential Therapy in Asthma The detailed background (history, biochemical and physical properties, sources and antioxidant capacity) and therapeutic uses of vitamin E was covered in Section 1.3 of this PhD thesis.
We were interested to employ vitamin E in asthma, as not only was vitamin E reported to have anti-inflammatory properties and proven effective in several inflammatory diseases (such as allergic dermatitis, inflammatory bowel diseases and rheumatoid arthritis). It is a naturally 60
occurring antioxidant to counter oxidative stress/damage in various diseases (such as cardiovascular diseases, osteoporosis, nephropathy and radiation damage) (Aggarwal et al., 2010; Peh et al., 2016). In particular, γ-tocotrienol has anti-cancer properties by inhibiting NF-κB pathway through the downregulation of receptor-interacting protein and TAK1, which leads to suppression of anti-apoptotic gene products and potentiates apoptosis. Persistent NF-κB activation was reported in both patients and animal models of asthma (Liu et al., 2010; Pantano et al., 2008; Yick et al., 2013). Various strategies to target NF-κB signalling pathway, namely receptor-based inhibition of NF-κB, IKK inhibition, inhibition of IκB degradation, and inhibition of NF-κB nuclear translocation, have shown to be effective in asthma (Gilmore and Garbati, 2011; Tak and Firestein, 2001). On top of that, tocotrienols were shown to decrease IL13/STAT6-activated eotaxin secretion in an in vitro study, where eotaxin is a major chemokine involved in asthma to promote eosinophils infiltrations into the airways in asthma (Wang et al., 2012). This evidence supports the great promise of using vitamin E in asthma.
1.4.8 Hypotheses & Objectives Inflammation and oxidative stress were reported to be involved in the pathogenesis of asthma. Using an established house dust mite-induced mouse model, I would like to validate if the mouse model is clinically relevant and if inflammation and oxidative stress do take place in
experimental asthma. For this study (Chapter 3), I hypothesize that inflammation and oxidative stress occurs in experimental asthma. The objectives for this study are: 1. To investigate the roles and interactions of inflammation and oxidative stress in asthma. 2. To observe the time course development of inflammation and oxidative stress, to deduce which takes place first. 3. To fill the gaps of knowledge in asthma development, and also ascertain current literature findings of asthma pathophysiology.
With the same experimental asthma mouse model, I will perform screening of various vitamin E isoforms to select the most potent isoform, and also validate if tocotrienols are superior over tocopherol in asthma. In this study (Chapter 3), I hypothesize that tocotrienol has more therapeutic potential than tocopherol in asthma mouse model. The objectives for this study are: 1. To examine if vitamin E isoforms can attenuate inflammation and oxidative stress in asthma. 2. To ascertain if tocotrienols are indeed superior over tocopherols in asthma. 3. To analyze the toxicity profile of vitamin E isoforms administered near the maximum tolerated dose over two weeks in experimental asthma. 4. To deduce which vitamin E isoform has the most therapeutic potential in asthma for the subsequent study. 62
5. To perform pharmacokinetics study on the selected vitamin E isoform to ensure that oral feeding of vitamin E will be distributed to the lungs, and optimize dosing regimen in the subsequent study.
With the selected vitamin E isoform, I will conduct a more detailed analysis of its efficacy in asthma with various doses, and also compare effectiveness in asthma against clinically prescribed drug, prednisolone. In this study (Chapter 4), I hypothesize that the selected vitamin E isoform can attenuate house dust mite-induced allergic asthma, through the amelioration of both inflammation and oxidative stress. The objectives for this study are: 1. To conduct a dose-dependent study for the selected vitamin E isoform, with detailed modulations of inflammation and oxidative stress in house dust mite-induced asthma. 2. To compare therapeutic potentials of selected vitamin E isoform against prednisolone, and deduce if the selected vitamin E isoform can complement with prednisolone clinically. 3. To investigate the mechanism of actions of selected vitamin E isoform.
Chronic Obstructive Pulmonary Disease (COPD)
Chronic obstructive pulmonary disease (COPD) is a progressive lifethreatening lung disease that is characterized by the irreversible development of airflow limitation and deterioration of pulmonary function, leading to breathlessness and predisposes to exacerbations and other serious illness (Vogelmeier et al., 2017). This is in contrast to asthma where the airflow obstruction is usually reversible with treatment. The airflow limitation in COPD is normally associated with chronic inflammation to noxious gases and particles (such as cigarette smoke and industrial fumes) in the peripheral airways and lung parenchyma, which results in narrowing and obstruction of the airways (Agusti and Soriano, 2008). COPD often manifests itself in four anatomical lesion – chronic bronchitis, emphysema, pulmonary hypertension and small airway remodeling (Wright et al., 2008), along with five common respiratory symptoms – chronic cough, sputum production, wheezing, dyspnea and chest tightness (Vogelmeier et al., 2017).
Currently, COPD is under-diagnosed due to its slow onset and usually only becomes apparent after 40-50 years of age in humans. The primary method of diagnosing COPD today is spirometry to measure the lung function of patients (Voelkel et al., 2011). Parameters of spirometry for COPD patients include forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), which represents the volume of air forcibly exhaled in the first second and volume of air forcibly exhaled after 64
maximal inspiration, respectively. The minimum requirement to be diagnosed for COPD is when the FEV1/FVC ratio is less than 0.7 (Vogelmeier et al., 2017). According to the Global Initiate for Chronic Obstructive Lung Disease (GOLD) criteria, the patients are categorized into four stages based on FEV1 – GOLD1) mild, GOLD2) moderate, GOLD3) severe, GOLD4) very severe (Figure 1.16) (Vogelmeier et al., 2017). In mild GOLD1, patients have chronic cough and sputum production. This stage is usually unnoticeable and not detected, as the patients are unaware of the abnormality in their lung function. In moderate GOLD2, patients develop dyspnea due to the decline in airflow. This is the stage that patients start seeking medical attention as they experience chronic respiratory symptoms or an episode of exacerbation. In severe GOLD3, the airflow limitation and lung function in patients further worsen, where they encounter greater shortness of breath, fatigue, reduced exercise capacity and repeated exacerbations that reduce their quality of life. In most severe GOLD4, patients have chronic respiratory failure with limited quality of life, and episodes of exacerbations may be life threatening (Vogelmeier et al., 2017). The disease severity is irreversible and persists even after cessation to exposure of noxious gases or particles that cause COPD. Current treatments include corticosteroids and phosphodiesterase-4 inhibitor to attenuate the inflammation in COPD (Barnes, 2013a; Grootendorst et al., 2007). Besides, there is a significant portion of COPD patients (>10%) who are resistant to corticosteroids and are in need of novel therapies. 65
Figure 1.16| GOLD classification of COPD. Figure adapted from (Vogelmeier et al., 2017).
The impairment of lung function in COPD is commonly associated with chronic bronchitis and emphysema, in which both conditions co-exist (Chung and Adcock, 2008). Bronchitis is defined as the inflammation of the bronchi, and results in remodeling of the airways due to peribronchiolar fibrosis (narrowing of the small airways) and mucus hypersecretion (luminal obstruction in the lungs) (Ekberg-Aronsson et al., 2005). Emphysema is the major cause of death in COPD, where the inner walls of alveolar sacs rupture, resulting in larger air spaces with reduced surface area for sufficient gas exchange in the lungs (Voelkel et al., 2011). The heightened activity and levels of proteases not only destroy the lung structures, but reduce lung elasticity. In general, the increase in
inflammation and inflammatory cell infiltrations, elevated oxidative stress and oxidative damage, mucus hypersecretion and augmented proteases activities are involved in the progression of COPD (Atkinson and Senior, 2003; Barnes, 2008b; Stratev et al., 2013).
1.5.1 Epidemiology of COPD The prevalence of COPD is increasing, with a global burden of over 600 million people, where more than 210 million people have moderate to severe COPD. (Sin and Vestbo, 2009). The primary risk factor in COPD is cigarette smoke, and other risk factors include – indoor air pollution (solid fuel for cooking), outdoor air pollution, dusts and chemicals (vapors and fumes). Initially, COPD was more common in men due to tobacco smoking in high-income countries, and higher risk for women in low-income countries due to indoor air pollution (Mannino and Buist, 2007). It affects men and women almost equally today. According to the World Health Organization report November 2016 (http://www.who.int/respiratory/copd), it is estimated that approximately three million deaths were caused by COPD in year 2015, which accounts for about 5% of all deaths globally in that year. Among the death tolls, more than 90% of deaths occur in low and middle-income countries. Currently, COPD is the fourth leading cause of death worldwide and is predicted to be the third leading cause of death by the year 2020 (Soriano and Rodríguez-Roisin, 2011), and responsible for ~7.8% of all deaths by year 2030 (Mathers and Loncar, 2006). COPD may account for more than 10% of lost disability-adjusted life years 67
(DALYs) worldwide and is ranked 10th for the most frequent cause of disability in the world (Speizer et al., 2006). Almost half of all COPD patients reported that COPD affects their quality of life, ability to work and sleeping pattern. This will incur a huge amount on indirect healthcare costs. The economic burden of COPD is immense, and estimated to exceed those of asthma by over three folds (Skrepnek and Skrepnek, 2004). In the European Union, the total healthcare budget is estimated to be €38.6 billion. In United States of America, the direct and indirect healthcare costs is estimated to be US$32.1 billion (Skrepnek and Skrepnek, 2004). With the rising prevalence, mortality and economic burden of COPD, there is a huge unmet medical need for the discovery of novel cost-effective therapies that can cure or delay the disease progression.
1.5.2 Etiology of COPD Cigarette smoke remains as the most important risk factor for COPD, where 73% of COPD mortality is smoking related (Mannino and Buist, 2007). The other risk factors include indoor air pollution (solid fuel for cooking), outdoor air pollution (forest fire, vehicle exhaust fumes and haze), occupational hazards (dusts in coal mines and fumes in factories), and history of childhood respiratory infections (Mannino and Buist, 2007; Vogelmeier et al., 2017). Although smoking vastly increases the chances to get COPD, the susceptibility to tobacco smoke varies greatly among individuals (Castaldi et al., 2011). There are over 1.1 billion tobacco 68
smokers worldwide, where it was estimated that some 47% of men and 12% of women smoke on the global scale (Fagerström, 2002).
Within the cigarette smoke, it contains/produces nicotine, methanol, carbon monoxide, harmful free radicals and ammonia when inhaled (Park et al., 1998; Renaud et al., 1984). Studies had revealed that carbon monoxide and nicotine inhalation can affect platelet functions and mount an inflammatory response in the lungs, as well as infiltration of macrophages and neutrophils in the airways (Barnes, 2008b). As a result of inflammation, levels of activated NF-kB are upregulated and mucus hypersecretion can be found on the bronchial epithelium (Simone et al., 2011). Each puff of CS contains 1015 free radicals, including nitric oxide (NO), nitrogen dioxide (NO2), peroxynitrite (ONOO˙), superoxide anion (O2˙–), hydrogen peroxide (H2O2) and hydroxyl radical (OH˙) (Pryor and Stone, 1993). Similarly, inflammatory cells infiltrated into the airways can produce hypochlorous acid (HOCl), O2˙−, OH˙, NO and H2O2 (Aktan, 2004; Drost et al., 2005; Kirkham and Barnes, 2013). The antioxidants capacities are inhibited in COPD, tipping the balance between oxidants and antioxidants in favor of oxidants, resulting in oxidative damage in the lungs (Kirkham and Rahman, 2006). Clinically, oxidative stress levels were observed to be drastically increased in severe exacerbations in COPD (Drost et al., 2005). The ablation of Nrf2 in cigarette smoke exposed mice enhances cigarette smoke-induced emphysema and disease outcome in the lungs (Rangasamy et al., 2004). In COPD, The balance between 69
matrix metalloproteinases (MMPs) and their inhibitors is dysregulated and they play a big role in the destruction of alveolar walls, leading to development of emphysema eventually (Mocchegiani et al., 2011). The increase in inflammation, along with increase in oxidative stress and MMPs, results in airway remodeling and irreversible damage to the lungs. It would beneficial to discover novel drugs that can not only attenuate inflammation in COPD, but ameliorate oxidative stress as well.
1.5.3 Inflammation in COPD Despite the similarities between asthma and COPD on how both are chronic obstructive inflammatory disease of the airways, there are marked differences in the pattern of inflammation in the respiratory tract and involvement of inflammatory cells (Barnes, 2008b). Inflammation in asthma occurs mainly in the larger airways, while inflammation in COPD predominantly occurs in the smaller airways and lung parenchyma (Jeffery, 2000). The difference in distribution of inflammation may be attributable to the inhaled agents, such as house dust mites in asthma and cigarette smoke in COPD (Figure 1.17). Interestingly, although cigarette smoke is majorly responsible for inflammation in COPD, the inflammation does not cease even after the patients stop smoking (Lapperre et al., 2006). While asthma is predominantly driven by Th2 inflammatory responses (Kaiko and Foster, 2011; Peh et al., 2015), it is not as distinct for COPD although COPD is generally perceived to be mediated by Th1 immune responses (Chung and Adcock, 2008; Majori et al., 1999). While eosinophils are the 70
major inflammatory cell involved in asthma, elevated numbers of macrophages and neutrophils were observed in both BAL fluid and sputum of COPD patients (Molet et al., 2005; Pesci et al., 1998; Vernooy et al., 2004). Contrary to asthma, eosinophils are not usually involved in COPD except during episodes of exacerbation in severe patients or patients who have concomitant asthma (Singh et al., 2014a; Siva et al., 2007). Moreover, cigarette smoke has the capability to inhibit normal lung repair responses by blocking the regulatory responses that normally downregulate inflammation (such as Treg and IL-10), which promotes inflammatory cells infiltrations and potentiation of damages in the lungs (Rennard, 2002).
Figure 1.17| Differences between asthma and COPD. Figure obtained from (Barnes, 2004c)
Besides inflammatory cells, there are many mediators such as cytokines/chemokines and proteases involved in the pathology of COPD (Barnes, 2008b). Mechanistically, persistent STAT3 and NF-κB activation 71
have been detected in COPD (Di Stefano et al., 2002; Qu et al., 2009). Inflammation is unlikely to be to sole contributor to the development of COPD, judging from the plethora of biomarkers and disease outcomes. It is likely to also include the involvement of oxidative stress and proteaseantiprotease imbalance in COPD, which ultimately results in emphysema.
Neutrophilia is a characteristic feature of COPD. Neutrophil levels were found to be increased in the sputum and BAL fluid of COPD patients and the levels correlate with disease severity (Keatings et al., 1996; Quint and Wedzicha, 2007). It was also found to be augmented in the bronchial epithelium and lamina propria in smokers compared to healthy controls (Pesci et al., 1998). As mentioned in Section 1.5.3, neutrophilia was found to be elevated more in the small airways than larger airways (Battaglia et al., 2007). Similarly in bronchial biopsies from GOLD3 and GOLD4 patients with COPD, heightened neutrophil counts were revealed in the airway submucosa (Di Stefano et al., 2009). Neutrophils infiltrations into the airways are due to the increase in CXC-chemokines, such as CXCL1 and CXCL8 (also known as IL-8), which bind and activate CXCR2 that is expressed mainly on neutrophils surface (Barnes, 2008b). In sputum obtained from COPD patients, IL-8 levels was found to correlate with neutrophil counts (Keatings et al., 1996). Cigarette smoke on its own can activate macrophages in the airways to release CXCL1 and CXCL8 to attract neutrophils into the lungs (Figure 1.18). 72
Neutrophils are a rich source of inflammatory mediators, free radicals and proteases, which contribute to airway obstruction and lung parenchymal destruction (Stockley, 2002). Neutrophils also possess many granular secretory vesicles which contain neutrophil elastase, myeloperoxidase (MPO), thromboxane A, matrix metalloproteinase-9 (MMP-9), cathepsins and serine proteases, that can result in inflammation and oxidative damage upon its release and contribute to emphysema (Fahy, 2009; Sampson, 2000). Under normal conditions, neutrophils are recruited to defend against foreign microorganisms by activating the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) present on the plasma membrane to generate large amounts of free radicals (superoxide anion, hydrogen peroxide and peroxynitrite), also known as respiratory bursting (Dahlgren and Karlsson, 1999). During inflammation and extended periods of neutrophilia, neutrophils deal immense damage at the site of recruitment and can be harmful to healthy tissue (Quint and Wedzicha, 2007). Being a potent cell of both inflammation and oxidative damage, apoptotic neutrophils are cleared by phagocytic macrophages. However,
phagocytosis of neutrophils by macrophages via the inhibition of actin rearrangement (Minematsu et al., 2011). MMP-9 is primarily produced by neutrophils and also involved in the remodeling of the airways (Atkinson and Senior, 2003). MMP-9 levels were upregulated in COPD patients’ lungs, and are responsible for mucus overproduction in the airways
(Barnes, 2008b; Mercer et al., 2005). The accumulated neutrophils in the airways result in the clinical abnormalities of COPD.
Despite that corticosteroids are the first-line of therapy in COPD, they have been consistently shown to be ineffective against neutrophils (Ito et al., 2008b; Maneechotesuwan et al., 2007). As there are more COPD patients who are resistant to corticosteroids than asthma patients (Barnes, 2013a), this creates an urgent need to discover alternative class of drugs with similar or better anti-inflammatory actions than corticosteroids, or complement corticosteroids to combat against steroid resistance in COPD.
Figure 1.18| Effector functions of inflammatory cells in COPD. Figure obtained from (Barnes, 2008b). 74
Besides neutrophilia, macrophages also play a critical role in COPD. Macrophage numbers were increased in the lungs of COPD patients (Molet et al., 2005). It was reported that the disease severity correlated to the number of macrophages in the airways (Di Stefano et al., 1998). In cigarette smoke exposure, chemoattractants such as CCL2 (also known as monocyte chemotactic protein-1, MCP-1) and CXCL1 promotes the infiltration of macrophages into the airways (Traves et al., 2004). The increase in number of macrophages is partly due to the prolonged survival of macrophages in the lungs as well. Besides CXCL1 and CXCL8 secreted by macrophages to attract neutrophils into the airways, macrophages can release macrophage inflammatory protein-1α (MIP1α/CCL3), another chemoattractant for neutrophils.
In cigarette smoke exposure, epithelial cells secrete cytokine IL-1β, which is a potent activator of alveolar macrophages in COPD (Russell et al., 2002b), to secrete inflammatory cytokines/chemokines and free radicals (Keatings et al., 1996). This is validated clinically when levels of IL-1β was observed to increase in both sputum and BAL fluid in smokers (Ryder et al., 2002). As it is more harmful than beneficial to eliminate macrophages in COPD, there is an ongoing clinical trial which is testing the therapeutic potential of canakinumab (anti-IL-1β monoclonal antibody) in COPD by reducing activation of macrophages in COPD patients (Rogliani et al., 2015). It is gaining traction that macrophages orchestrate inflammation in 75
COPD via the release of chemokines to attract neutrophils, and the secretion of proteases such as MMP-9 and MMP-12 (Barnes, 2004a; Molet et al., 2005).
STAT3 and NF-κB
Cigarette smoke and IL-1β in the lungs can trigger inflammatory and epithelial cells to produce IL-6 and LIF, which activate their cognate cytokine receptors resulting in STAT3 nuclear translocation (Qu et al., 2009; West et al., 2015). Both STAT3 and phospho-STAT3 levels were revealed to be significantly increased in the lungs of COPD patients when compared to healthy controls (Yew-Booth et al., 2015). STAT3 is also essential for the differentiation of Th17 cells, to produce IL-17 responsible for inflammation and steroid resistance in COPD (Figure 1.19) (Barnes, 2013a; Yang et al., 2007). In a recent study, STAT3-deficient mice had reduced airway inflammation and proteases activity, however, it still developed emphysema (Ruwanpura et al., 2012). This possibly suggests that while STAT3 plays a critical role in inflammation of COPD, it is less involved in the pathogenesis of emphysema at the later stages of COPD.
Figure 1.19| Role of STAT3 in COPD. Figure obtained from (Barnes, 2008b).
Levels of IL-1β, IL-8, TNF-α, RANTES, nitric oxide synthase 2 (NOS2), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were found to be upregulated in the lower airways, along with augmented MUC5AC and neutrophil elastase levels in bronchiolar epithelium of COPD patients (Caramori et al., 2015). All of these targets are inducible by NF-κB, thus suggesting the involvement of NF-κB in the development of COPD (Figure 1.20). Inflammatory cytokines and free radicals can activate NF-κB, a master transcription factor of inflammation in COPD (Chen et al., 2011; Edwards et al., 2009). Macrophages obtained from the sputum of COPD patients revealed elevated NF-κB activation (Caramori et al., 2003). It is also highly expressed in bronchial biopsies from COPD patients (Di Stefano et al., 2002). In that study, there is a positive correlation between the number of
p65-positive epithelial cells and the degree of airflow limitation. There are growing evidences to target against NF-κB in COPD. The pharmacological inhibition of IKK-β/IKK-2 using PHA-408 attenuated neutrophilia and inflammatory
inflammation (Rajendrasozhan et al., 2010). In another study, lung inflammation and respiratory dysfunction induced by cigarette smoke were attenuated when treated with NF-κB decoy oligodeoxynucleotides (Li et al., 2009). Therefore, new therapies that can inhibit the activation of STAT3 and NF-κB may be promising in the treatment of COPD.
Figure 1.20| Role of NF-κB in COPD. Figure adapted from (Sekine et al., 2012).
1.5.4 Oxidative Stress in COPD Oxidative stress was introduced earlier in Sections 1.4.4 and 1.4.5. COPD patients suffer shortness of breath (dyspnea) due to the underlying chronic bronchitis and emphysema in the airways, which are contributed by oxidative stress and oxidative damage in the lungs. Cigarette smoking is the main etiological factor for the development of COPD. Each puff of cigarette smoke contains 1015 free radicals, including nitric oxide (NO), nitrogen dioxide (NO2), superoxide anion (O2˙–), hydrogen peroxide (H2O2) and hydroxyl radical (OH˙) (Pryor and Stone, 1993). Neutrophils and activated macrophages that infiltrated the airways will also produce free radicals (Figure 1.21) (Mak, 2008; Quint and Wedzicha, 2007). Some examples of free radicals released from inflammatory cells include hydrogen peroxide, hydroxyl radicals, superoxide anion, nitric oxide, peroxynitrite (ONOO-) and hypochlorous acid (HOCl). Normally, the host can tolerate low levels of oxidants by the protection of antioxidants present in the lungs. However, under repeated insults from oxidants in cigarette smoke and inflammatory conditions, antioxidant enzymes activities are observed to be reduced in COPD (Kirkham and Barnes, 2013; Stratev et al., 2013), resulting in high levels of oxidative stress which in turn promotes and supports the ongoing inflammation. The inability to defend against these oxidants results in oxidative damage on important biomolecules such as lipids, proteins and DNA present in the cells or lung tissue (ben Anes et al., 2014; Kinnula et al., 2007). The pulmonary levels of oxidative damage biomarkers and degree of pulmonary oxidant79
antioxidant imbalance were found to correlate with the severity of COPD (Drost et al., 2005; Louhelainen et al., 2008). Oxidative damage to the cells, if left untreated, will impede its normal physiological functions, thus supporting the pathology of COPD (Chung and Marwick, 2010).
Figure 1.21| Pathophysiology of oxidative stress in COPD. Figure obtained from (Kirkham and Barnes, 2013).
Oxidative stress is also responsible for supporting the ongoing inflammation and recruitment of inflammatory cells into the airways, as well as insensitivity to corticosteroids treatments (Ahmad et al., 2013; Barnes, 2013a; Chung and Marwick, 2010; Drost et al., 2005). Oxidants from cigarette smoke can activate the redox-sensitive transcription factor
NF-κB to mount an inflammatory response (Bowie and O’Neill, 2000), as well as activate airway epithelial cells and alveolar macrophages to secrete chemoattractants to recruit neutrophils into the lungs (Louhelainen et al., 2008). As a consequence, this will perpetuate the oxidative stress in the lungs and result in a vicious cycle of persistent inflammation and oxidative stress/damage. Hypochlorous acid produced by neutrophils can react with plasma proteins to form advanced oxidation protein products (AOPP), where AOPP elevation has been detected in Tunisian COPD patients (ben Anes et al., 2014), and was also linked to smokers with depression (Vargas et al., 2013). NADPH oxidase (NOX) is a cytoplasmic enzyme that produces O2˙−, with increased levels of NOX2 and NOX4 observed in COPD (Griffith et al., 2009). Although O2˙− are relatively weak oxidizing agents, they can form more reactive radicals when reacted with NO to produce ONOO˙, or be dismutated by SOD into H2O2. It has been shown that NOX-produced oxidants can initiate redox-sensitive NF-κB activation, linking oxidative stress to inflammation in the lungs once again (Lee and Yang, 2012).
Recent studies have revealed that oxidative stress promotes corticosteroid resistance in COPD patients (Adcock and Barnes, 2008; Barnes, 2013a). In cigarette smoke challenged mice or steroid-resistant animal models, the use of antioxidants and Nrf2 activators showed promise to attenuate COPD symptoms, as well as potentially reversing steroid resistance (Barnes, 2004b; Boutten et al., 2011; Kirkham and Rahman, 2006). As 81
such, it would be beneficial to develop novel therapies that can inhibit oxidative stress/damage, and complement the use of corticosteroids in COPD.
Impaired Antioxidants in COPD
Antioxidants are the first-line of defense against oxidants or free radicals in the body, to convert the latter into less toxic molecules or compounds. The balance between antioxidants and oxidants is kept at homeostasis under normal conditions, where tipping either sides will result in harmful effects in the host (Sies, 1986). The total antioxidant capacity in living organism is rather complex, which takes into account endogenous and exogenous, enzymatic and non-enzymatic antioxidants. Key antioxidant enzymes include SOD, catalase, GPx and heme oxygenase (HO) (Figure 1.09), while key non-enzymatic antioxidants include glutathione (GSH) and bilirubin, and antioxidants obtained from food such as vitamin C, vitamin E and flavonoid (Comhair and Erzurum, 2009). The total antioxidant capacity in COPD patients is lower than that of healthy individuals, and the deterioration of lung function correlates to how impaired the antioxidant capacity is (Rahman et al., 2000). It was also observed that the antioxidant potential in GOLD4 patients was the lowest compared to the other less severe COPD patients (Pirabbasi et al., 2013). There is potential in antioxidant supplementation in COPD to suppress oxidative stress.
Superoxide Dismutase (SOD)
Superoxide dismutases (SODs) are metalloenzymes and also the only ones that dismutate superoxide anion (O2•-) into hydrogen peroxide (H2O2) (Figure 1.09). There are three subtypes of SOD, namely copper-zinc (CuZnSOD), manganese (MnSOD) and extracellular (ECSOD) SODs, respectively in order of their numerical subtypes (Kinnula and Crapo, 2003). The location of each SOD isotype differs. SOD1 is located in the cytoplasm, SOD2 in the mitochondria and SOD3 in the extracellular spaces of the cell. Clinically, the SOD activity of healthy smokers, nonsmoking COPD patients and smoking COPD patients were decreased when compared to healthy non-smokers (Gavali et al., 2013). In the same study, SOD activity was positively correlated with FEV1 in COPD patients. The polymorphism of ECSOD such as R213G mutation which increases the level of circulating ECSOD in the body, can protect the smoker against the development of COPD (Oberley-Deegan et al., 2009). The pharmacological augmentation of ECSOD in mice had reduced oxidative stress in response to cigarette smoke, and were protected against emphysema (Yao et al., 2010).
Catalase is present in most aerobic cells and its catalytic activity lies in the conversion of toxic hydrogen peroxide (H2O2) to water and molecular oxygen (McMurry, 2008) (Figure 1.09). Hydrogen peroxide is formed under normal respiration and pathogenic reactive oxygen species 83
production (Loukides et al., 2002). The chemical equations and description on how oxidants can inhibit the activity of catalase is mentioned in Section 126.96.36.199.2. Catalase expression was reduced in both smokers without COPD and COPD patients, when compared to non-smoking healthy individuals (Altuntaş et al., 2002). Even when comparing smokers with COPD against non-smoking COPD patients, catalase expression was lowered in bronchiolar epithelium cells of smokers (Betsuyaku et al., 2013). In C57BL/6 mice exposed to cigarette smoke daily for three months, catalase levels were temporarily upregulated after one day of cigarette smoke exposure, and was not restored thereafter, even after a month of withdrawal from cigarette smoke once emphysema developed (Betsuyaku et al., 2013).
Glutathione Peroxidase (GPx)
The role of glutathione peroxidase (GPx) is to convert hydrogen peroxide to water and oxygen, with the help of reduced glutathione (GSH) (Ho et al., 1997). GSH will be converted to oxidized glutathione (GSSG) in the process, which will be recycled by glutathione reductase (GR) (Figure 1.09). In response to acute cigarette smoke exposure, glutathione levels are upregulated to counteract the initial barrage of oxidants, but are eventually overwhelmed with time. Cigarette smoke is also responsible for downregulating GPx and glutathione reductase (Harju et al., 2008). Clinically, GPx activity was lowest in very severe COPD patients when comparing patients from different GOLD stages. The severity of COPD 84
was also negatively correlated to GPx activity (Kluchova et al., 2007; Tkacova et al., 2005). Experimentally, glutathione peroxidase mimetics were found to be potentially effective in the treatment of COPD (Kirkham and Barnes, 2013).
Nuclear Erythroid 2-Related Factor 2 (Nrf2)
Nrf2 is a redox-sensitive transcription factor involved in binding to the antioxidant response element (ARE), a promoter of many antioxidant genes that is essential for their inducible activation (Sussan and Biswal, 2014). Nrf2 directly regulates expressions of GPx, GR, heme oxygenase-1 (HO-1) and NADPH: quinone oxidoreductase 1 (NQ01) (Adair-Kirk et al., 2008), and is able to indirectly regulate the other antioxidants. Nrf2 activation was found down-regulated in COPD patients (Ma, 2013; Mercado et al., 2011). Nrf2-regulated HO-1 levels in the lungs were also found to be decreased in COPD patients when compared to healthy individuals (Malhotra et al., 2008). The knocking out of Nrf2 not only hastened, but worsened the development of emphysema in mice (Rangasamy et al., 2004). Compounds that promote Nrf2 activation afforded protection against experimental COPD (Harvey et al., 2011; Singh et al., 2009; Sussan et al., 2009). The discovery of novel therapeutics that can booster the antioxidant defenses will serve to be beneficial in the treatment of COPD.
Heightened Oxidative Damage in COPD
The second component of oxidative stress is the increased levels of oxidative damage to the cells (Giustarini et al., 2009). As oxidants such as superoxide anion, peroxynitrite and hydrogen peroxide are too transient with very short half-lives, most studies adopt the measurements of oxidative damage biomarkers instead (Louhelainen et al., 2008). Key biomarkers of oxidative damage for the important biomolecules of the cell (lipids, proteins and DNA) are circled red in Figure 1.09, and Figure 1.22.
Figure 1.22| Oxidative damage biomarkers in COPD. Red arrows demarcate changes in COPD conditions; yellow highlights represent the oxidants involved; blue words are for antioxidant enzymes; and purple words are for oxidative damage biomarkers.
Lipid Peroxidation (8-Isoprostane)
Lipids in the cell membrane and cytoplasm are present in large quantities, thus making it a common target of free radicals attack. The process of attack is known as lipid peroxidation, where it propagates continuously once initiated and can only be stopped in the presence of antioxidants (Figure 1.11). Extensive lipid peroxidation in cell membrane can lead to loss of fluidity, drops in membrane potential, increase in ions permeability and eventual cell rupture, which may contribute to the pathogenesis of various tissue injuries and diseases (Gutteridge, 1995).
8-Isoprostane, a lipid peroxidation product, is a prostaglandin-F2α isomer which is formed in vivo by free radical-catalyzed peroxidation of arachidonic acid (Montuschi et al., 2000). It has been used extensively as the oxidative damage biomarker for lipids in numerous studies (DalleDonne et al., 2006). Several clinical studies conducted had observed an increase in the production of 8-Isoprostane in either exhaled breath condensates or sputum from smokers and COPD patients, where increase in 8-Isoprostane levels correlate with the severity of lung injury or COPD (Biernacki et al., 2003; Kinnula et al., 2007; Montuschi et al., 2000). These studies show that 8-Isoprostane is an effective biomarker to detect oxidative stress in smokers and COPD patients, therefore, 8-Isoprostane levels were measured in this study.
Protein Nitration (3-Nitrotyrosine)
An established biomarker of oxidative damage to proteins where proteins are nitrated, is 3-nitrotyrosine (3-NT) (Dalle-Donne et al., 2006). In the presence of nitric oxide synthases (NOS) and arginine residues, nitric oxide (NO) is produced. The combination of NO with superoxide anion (O2•-) forms peroxynitrite (ONOO-), which attacks tyrosine residues to form 3-NT (Dedon and Tannenbaum, 2004) (Figure 1.09 and 1.22). There are many mechanisms to invoke the nitration of proteins, resulting in the gain or loss of that protein functionality and activity (Schopfer et al., 2003). A study showed that nitration of tyrosine residues on MnSOD results in the loss of activity of the antioxidant enzyme (Radi, 2004). Interestingly, peroxynitrite and 3-NT have shown to display dual roles in the cells; where 1) its increased levels indicate the presence of oxidative stress and oxidative damage, and 2) on its own, it is involved in cell signaling pathways (Szabo et al., 2007). A study had shown that peroxynitrite is involved in the activation of phosphopnositide-3-kinase (PI3K)/Akt signaling pathway (Delgado-Esteban et al., 2007).
Clinically, 3-NT levels measured from plasma and exhaled breath condensate in smokers and COPD patients were significantly higher than healthy individuals (Lee et al., 2014; Petruzzelli et al., 1997). The levels of 3-NT were negatively correlated to FEV1 in COPD patients as well (Sugiura et al., 2004). Interestingly, the use of xanthine oxidase inhibitor allopurinol was able to ameliorate 3-NT formation in COPD patients 88
(Ichinose et al., 2003). In order to combat against corticosteroids resistance in COPD, there was a study that observed superior efficacies of theophylline in attenuating nitrative stress (3-NT levels) and neutrophilia than inhaled corticosteroid (Hirano et al., 2006).
Protein Oxidation (AOPP)
Advanced Oxidation Protein Products (AOPP) are created through the reaction of plasma proteins with chlorinated oxidants such as chloramines or hypochlorous acid (HOCl) (Witko-Sarsat et al., 1996) (Figure 1.22). One of the major sources of chlorinated oxidants is neutrophil, which contains heme enzyme myeloperoxidase (MPO) that catalyzes the reaction of hydrogen peroxide (H2O2) with chloride ion (Cl-) to generate large amounts of hypochlorous acid (Iwao et al., 2006). As COPD is primarily a neutrophilic disease of the airways, it would be useful to measure levels of AOPP in this study. AOPP is used as a protein oxidative damage biomarker in various diseases such as uremia, hemodialysis patients, acute coronary syndrome and type 2 diabetes mellitus (Piwowar et al., 2007; Škvařilová et al., 2005; Witko-Sarsat et al., 1996; WitkoSarsat et al., 2003). Likewise in COPD, AOPP levels were found to correlate with the severity of COPD (Stanojkovic et al., 2011). AOPP elevation has been detected in Tunisian COPD patients (ben Anes et al., 2014), and was also linked to smokers with depression (Vargas et al., 2013). In experimental COPD model, rats exposed to cigarette smoke had heightened levels of AOPP in the lungs as well (Lau et al., 2012). 89
DNA Oxidative Damage (8-OHdG)
The classical biomarker for oxidative damage to DNA is 8-hydroxy-2deoxyguanosine (8-OHdG), where it is produced by reactive oxygen species (most commonly hydroxyl radical, OH-) attack at the C8 position of deoxyguanosine in DNA (Dalle-Donne et al., 2006; Giustarini et al., 2009) (Figure 1.12). 8-OHdG is excised during the repair of oxidative damage to deoxyguanosine sites in DNA. Clinically, 8-OHdG was used to estimate extensiveness of DNA damage in humans after cigarette smoke exposure (Valavanidis et al., 2009). In another clinical study, COPD patients had more 8-OHdG levels in the lungs when compared to non-COPD smokers and non-smokers (Neofytou et al., 2012). DNA double-strand breaks (DSBs) were positively correlated to levels of 8-OHdG in lung tissue samples obtained from COPD patients, asymptomatic smokers and healthy individuals. It was revealed that DSBs was primarily caused by oxidative stress, which contributed to the pathogenesis of COPD by inducing apoptosis and pro-inflammatory responses (Aoshiba et al., 2012).
Targeting Oxidative Stress with Antioxidants in COPD
As oxidative stress/damage is a major contributor to the pathogenesis of COPD, it is highly beneficial to complement the use of antioxidants or discover novel drugs that can subdue oxidative stress in the treatment of COPD (Boutten et al., 2011; Kirkham and Rahman, 2006). It was shown in many studies that by relieving oxidative stress in the lungs, the pathophysiology of COPD will be reduced (Boutten et al., 2011; Kirkham 90
and Rahman, 2006; Singh et al., 2009). The targeting of Nrf2 with triterpenoid CDDO-imidazolide in cigarette smoke-induced emphysema in mice managed to downregulate the etiopathogenesis of emphysema and oxidative stress in the lungs (Sussan et al., 2009). The most commonly used antioxidant in experimental COPD is N-acetylcysteine (NAC), which is a precursor of glutathione with established safety record (Dekhuijzen and van Beurden, 2006). While there were some clinical studies that observed the beneficial effects of NAC in COPD (Hansen et al., 1994; Stav and Raz, 2009), the largest trial of an antioxidant in COPD named BRONCHUS (bronchitis randomized on NAC cost-utility study) study failed to show any effect of NAC on slowing down disease progression or exacerbation frequency (Kirkham and Barnes, 2013). All hopes are not lost, as a subsequent study named PANTHEON (placebo-controlled study on efficacy and safety of N-acetylcysteine high dose in exacerbations of COPD) which administered higher dose of NAC to Chinese patients with moderate-to-severe COPD over a period of 1 year, observed reduced COPD exacerbations and patients had better symptom relief (Zheng et al.). Newer studies are adopting the use of more potent antioxidants in COPD.
1.5.5 Protease-Antiprotease Imbalance in COPD The protease-antiprotease imbalance hypothesis in COPD started in the early 1960s, which was believed to be involved in the pathogenesis of emphysema. An initial observation that the administration of papain, a 91
plant elastolytic proteinase, into hamsters’ lungs resulted in emphysema (Gross et al., 1965). This introduced the notion that the secretion of unchecked proteases by inflammatory cells led to the destruction of the parenchymal matrix, and eventually the development of emphysema (Abboud and Vimalanathan, 2008). Although earlier studies of COPD focused on the effects of proteases-induced lung inflammation/injury, it is still believed to play a critical role in the development of COPD.
Among the proteases, neutrophil elastase (NE) is one of the more potent serine protease that hydrolyzes many proteins. Neutrophil elastase is stored in azurophilic granules in neutrophils and necessary in healthy host defence, as shown in mice with NE knockout were more susceptible to bacterial-induced sepsis (Belaaouaj, 2002). However, the overproduction of NE would degrade the extracellular matrix and is shown to be implicated in various destructive diseases including COPD (Shapiro, 2002a). The administration of elastase into the airways resulted in neutrophil infiltrations, mucus hypersecretion, pulmonary edema and hemorrhage (Wright et al., 2008). These changes led to impairment of lung function, which is consistent with clinical findings. As elastase alone can rapidly induce emphysema in mice, many early experiment COPD models adopted the use of elastase to discover novel therapies. However, the inflammation induced by elastase is short-lived and resolves within a week of administration, which does not reflect the slow progressive nature
of COPD. Thus, elastase-induced emphysema mouse models gradually faded out (Stevenson and Birrell, 2011).
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases responsible for the degradation of extracellular matrix components. Alveolar macrophages express MMPs when exposed to cigarette smoke (Lemaître and D'Armiento, 2006). Among the MMPs, MMP-12 is critically involved in COPD, as MMP-12 knockout mice were protected against emphysema induced by cigarette smoke (Hautamaki et al., 1997; Zheng et al., 2000). Besides inducing tissue destruction, MMP12 can also cleave pro-TNF-α to mature TNF-α to promote inflammation (Black et al., 1997). Interestingly, while MMP-9 is a major contributor to emphysema in humans (Mercer et al., 2005), the genetic knockout of MMP-9 in mice did not prevent the development of emphysema (Lanone et al., 2002). MMPs are regulated by four tissue inhibitor of MMP (TIMP14), where TIMPs can be secreted as soluble proteins (TIMP-1, TIMP-2 and TIMP-4) or bounded to extracellular matrix (TIMP-3) (Gomez et al., 1997). TIMPs form non-covalent complexes with MMPs, by binding to the zinc binding catalytic site present in MMPs (Mocchegiani et al., 2011). TIMP-1 levels were observed to be decreased in COPD patients, which is normally upregulated from alveolar macrophages in response to inflammation in the airways in healthy individuals, and thus support elastolysis and tissue degradation (Russell et al., 2002a).
Both NE and MMPs are in-sync with each other, as they inhibit their counterpart inhibitors. NE inhibit TIMPs (regulators of MMPs), while MMPs degrade α1-antitrypsin (regulator of NE) (Shapiro, 2002b). Both proteases cleave the extracellular matrix to produce elastin fragments or collagenderived peptides, such as N-acetyl proline-glycine-proline (PGP), which are chemoattractants for monocytes and neutrophils (Weathington et al., 2006). The imbalance of protease-antiprotease in COPD is mainly attributed to oxidative stress, as oxidants itself can induce proteases release, and cigarette smoke exposure was shown to inhibit endogenous antiproteases activities (Cavarra et al., 2001).
1.5.6 Emphysema in COPD Emphysema is pathologically defined as an abnormal permanent enlargement of the airspaces distal to the terminal bronchioles, together with destruction of the alveolar walls without obvious fibrosis (Voelkel et al., 2011). The inflammation within the alveoli upregulates proteases that damage the alveolar surfaces (Mocchegiani et al., 2011). Neutrophil degranulation also leads to destruction of alveolar walls and affects mucociliary function, thus impeding mucociliary clearance (Quint and Wedzicha, 2007). The obliteration of alveolar walls results in fewer and larger floppy air sacs instead of many tiny ones. This reduces the surface area for gas-exchange in the lung and decreases the elasticity of the lung. Consequently, the smaller airways have difficulty in supporting breathing due to the reduced elastic recoil of alveolar septum, and are more prone to 94
collapse during breathing (Mitzner, 2011). Ultimately, the collapsing bronchioles cause further limitation of airflow. Mechanistically, IL-1β, MMP12 and NF-κB have been shown to contribute to the development of emphysema in COPD (Di Stefano et al., 2002; Lappalainen et al., 2005; Taraseviciene-Stewart and Voelkel, 2008). Neutrophil elastase and MMP12 released by macrophages enzymatically breakdown the scaffold of the alveolar sacs, resulting in emphysema (Taraseviciene-Stewart and Voelkel, 2008). Treatment with MMP9/MMP12 inhibitor ameliorated emphysema and airway remodeling in cigarette smoke-induced COPD in guinea pigs (Churg et al., 2007). Besides inflammation, oxidative stress is also essential in emphysema and COPD. Genetic deletion of glutathione S-transferase increased susceptibility to develop COPD (Cheng et al., 2004), while polymorphism in SOD3 is associated with reduced lung function in COPD patients (Dahl et al., 2008). In experimental COPD, Nrf2-deficient mice are more susceptible to develop emphysema when exposed to cigarette smoke (Iizuka et al., 2005).
1.5.7 Current Therapies in COPD Current FDA-approved treatments of COPD include bronchodilators, corticosteroids and phosphodiesterase 4 (PDE4) inhibitors. They are only able to relieve symptoms, improve quality of life and relatively slowdown disease progression, rather than curing the underlying condition (Mushtaq, 2014). Given that COPD is a chronic inflammatory disease, effective antiinflammatory agents would likely be effective and reduce the risk of COPD 95
bronchodilators – short-acting β2-adrenergic receptor agonist, salbutamol; long-acting β2-adrenergic receptor agonist, salmeterol and formoterol; short-acting muscarinic receptor antagonist (anticholinergic), ipratropium; long-acting anticholinergic, tiotropium. Although bronchodilators are able to provide bronchodilation, improve airflow limitation and reduce the time to first exacerbation when compared to placebo (Tashkin and Cooper, 2004), they lack anti-inflammatory properties and hence, fail to inhibit the progression of COPD (Barnes, 2013b). Thus, the main therapies of COPD today still rely on anti-inflammatory corticosteroids and PDE4 inhibitors (such as roflumilast).
Corticosteroids relieve COPD by reducing inflammation-associated leukocytes infiltration to the airway and suppress airway inflammation. It inhibits
transcription factors like NF-κB and activator-protein 1 (Alsaeedi et al., 2002). Corticosteroids were observed to reduce exacerbation rates in COPD in several clinical studies (Alsaeedi et al., 2002; Calverley et al., 2007a). Corticosteroids suppress but not cure airway inflammation, thus along with its undesirable side effects (water retention, lipids and cortisol metabolism dysfunction, cataracts, osteoporosis, and increased risks of opportunistic infections) over prolonged treatment is not ideal (Caplan et al.,
Poetker and Reh,
2010). On another note, inhaled
corticosteroids were not able to alter the rate of decline of lung function or 96
improve survival rate in COPD (Calverley et al., 2007a; Vestbo et al., 1999). This poor clinical response to corticosteroids is mainly due to steroid resistance in COPD patients (Culpitt et al., 2003). The reduction in histone deacetylase 2 (HDAC2) expressions and activity in response to oxidative stress from cigarette smoke and COPD, results in acetylation of the glucocorticoid receptor and preventing its inhibition of NF-κB driven inflammation. As COPD has more oxidative stress than asthma, there are potentially even more COPD patients who are resistant to corticosteroids than asthmatics (Barnes, 2013a).
Roflumilast is a selective PDE4 inhibitor, which accumulates intracellular levels of cyclic adenosine monophosphate (cAMP) by inhibiting the hydrolysis of cAMP to AMP (Martorana et al., 2005). As a PDE4 inhibitor, its anti-inflammatory properties include decreased release of inflammatory mediators and cytokines, reduced expression of cell surface markers and diminished apoptosis. Roflumilast was able to prevent emphysema in cigarette smoke exposed mice and attenuated neutrophilia in COPD patients (Grootendorst et al., 2007; Martorana et al., 2005). Severe COPD patients were given roflumilast for one year, and it was shown to slightly improve lung function without changing the exacerbation rates (Calverley et al., 2007b). In a more recent study, severe COPD patients who are prone to frequent exacerbations were given roflumilast for one year. Roflumilast treatment was able to reduce exacerbations and hospital admissions when compared to placebo group (Martinez et al., 2015). 97
While roflumilast was newly approved by FDA for the treatment of COPD, patients suffer from dose-limiting major side effects such as severe nausea and vomiting, headache and weight loss (Page and Spina, 2012).
There is a proposed treatment ladder for COPD patients with increasing severity by GOLD. In GOLD1 mild COPD patients, they are prescribed with short-acting bronchodilators such as β2-adrenergic receptor agonists (SABA)
bronchodilation and improve airflow obstruction (Figure 1.23). In GOLD2 moderate
bronchodilators such as β2-adrenergic receptor agonists (LABA) or muscarinic receptor antagonists (LAMA) to provide longer periods of bronchodilation. In GOLD3 severe COPD patients, they are also given inhaled corticosteroids if they have repeated episodes of exacerbations, in attempt to curb the inflammation in the lungs. Lastly, in GOLD4 very severe COPD patients, they will be given oral corticosteroids for attenuation of systemic inflammation. In very severe COPD patients who do not respond to corticosteroids with deteriorating lung function, they would be administered with long-term oxygen or surgical treatments (Barnes, 2013b; Vogelmeier et al., 2017).
Figure 1.23| Treatment ladder for each stage of COPD. Figure obtained from www.goldcopd.com.
In light of this, with the lack of effective treatments and escalating prevalence and economic burden of COPD, it is impetuous to discover novel therapeutics which can attenuate inflammation and oxidative stress, as well as protect against the development of emphysema. It would be beneficial to complement existing corticosteroids or assist in resensitizing steroid resistance too.
1.5.8 Prednisolone and Vitamin E Isoform γ-Tocotrienol The background, mechanism of actions and corticosteroid resistance of prednisolone were covered in Section 1.4.7 in details. As mentioned in Section 1.5.7, oral corticosteroid such as prednisolone (30-40 mg/day for 7 days) is only administered in severe and very severe COPD patients, who require systemic suppression of inflammation.
The detailed background (history, biochemical and physical properties, sources and antioxidant capacity) and therapeutic uses of vitamin E was covered in Section 1.3 of this PhD thesis. We were interested to employ vitamin E isoform γ-tocotrienol in COPD for its anti-inflammatory and anti-oxidative properties. From Section 1.5 of this PhD thesis, it was shown that transcription factors STAT3, NF-κB and Nrf2 are modulated in COPD. Besides neutrophil as the major inflammatory cell involved in COPD, both inflammation and oxidative stress are key components of the development of COPD. We have recently published the first report on the anti-inflammatory and antioxidative effects of γ-tocotrienol in experimental asthma (Peh et al., 2015). Although γ-tocotrienol is able to mitigate eosinophilic inflammation in asthma and showed some indications to be effective against neutrophilia, we were unsure for certain if it is effective in disease with predominant neutrophilic inflammation such as COPD. A study had observed the ability of γ-tocotrienol in downregulating the activation of STAT3 (Kannappan et al., 2010). In the previous asthma study, we had revealed that γ-tocotrienol 100
was capable of upregulating Nrf2 activation and inhibiting NF-κB activation in the lungs. As such, we believe that γ-tocotrienol may be effective in the treatment of cigarette smoke-induced COPD. Currently, there are no approved drugs that can effectively delay/stop the progression of COPD, so it would be interesting to observe if γ-tocotrienol is capable of protecting the lungs from airway remodeling and emphysematous destruction.
1.5.9 Hypotheses and Objectives Following on from a previous study (Chapter 4), there were some preliminary results that indicated γ-tocotrienol efficacy in attenuating neutrophils. As covered in Section 1.5, COPD is the perfect disease to evaluate the modulation of neutrophilia in the lungs. Using γ-tocotrienol, I will conduct a detailed analysis of its efficacy in cigarette smoke-induced COPD with various doses, and also compare effectiveness in COPD against clinically prescribed drug, prednisolone. There are no current therapies that can protect against emphysema or delay the progression of emphysema in COPD, thus I will evaluate the potential of γ-tocotrienol against emphysema. In this study, I hypothesize that vitamin E isoform γtocotrienol can alleviate cigarette smoke-induced COPD, via the attenuation of both inflammation and oxidative stress. I also hypothesize that γ-tocotrienol can protect against emphysema in COPD. The objectives for this study are: 1. To examine the roles of inflammation and oxidative stress in 2-weeks cigarette smoke-induced COPD. 101
2. To conduct a dose-dependent study for vitamin E isoform γ-tocotrienol, with detailed modulations of inflammation and oxidative stress in cigarette smoke-induced COPD. 3. To compare therapeutic potentials of vitamin E isoform γ-tocotrienol against prednisolone. 4. To develop mouse emphysema and detect lung function impairment using a 2-months cigarette smoke-induced COPD model. 5. To investigate the modulation of emphysema and lung function by vitamin E isoform γ-tocotrienol against prednisolone.
Although asthma and COPD are lung diseases that are intensively studied worldwide, there is still a huge unmet medical need to discover novel drugs with higher therapeutic index. Vitamin E tocotrienol isoforms are naturally occurring antioxidants made in nature, with negligible side effects if consumed within the safety limits. The respective three studies hypotheses and objectives can be referred at Sections 1.4.8 and 1.5.9 in the introduction. For this thesis, I hypothesize that vitamin E tocotrienol can attenuate asthma and COPD.
In Chapter 3, I developed an in vivo house dust mite-induced asthma mouse model and examined the time course development of inflammation and oxidative stress in asthma. The airway inflammation (inflammatory cell counts, lung histology, cytokines and chemokines profile), mucus 102
hypersecretion (lung histology and mucin expression), oxidative stress (antioxidants expression, free radicals levels and oxidative damage biomarkers) and transcription factor activation (NF-κB and Nrf2) were investigated. I found that it is likely for inflammation to occur first, which led to respiratory burst and initiation of oxidative stress subsequently. As the experimental asthma model displayed both inflammation and oxidative stress, and vitamin E tocotrienol isoforms were not used in any lung diseases before, I screened the efficacies of various vitamin E isoforms. The antioxidant capacity of each isoforms, ability to attenuate HDMinduced airways inflammation and neutralization of free radicals of each vitamin E isoforms were measured. The summation of all the data led to the selection of γ-tocotrienol for subsequent studies.
In Chapter 4, I observed for the first time that γ-tocotrienol can abate airway inflammation and oxidative lung damage in experimental murine asthma, with therapeutic effects similar or better than prednisolone, a corticosteroid used as mainstream anti-inflammatory drug for asthma. Treatment
production, and leukocytes infiltration to the lungs. Anti-oxidative lung damage therapy by γ-tocotrienol was mediated by upregulating Nrf2 activation and restoration of antioxidants activities, as well as decreasing pro-oxidants
inflammation and oxidative stress led to the amelioration of airway hyperresponsiveness.
In Chapter 5, I report for the first time that oral γ-tocotrienol could abrogate acute cigarette smoke-induced pulmonary neutrophilic infiltration, cytokine production and oxidative damage in a 2-weeks cigarette smoke model. It also abated airway remodeling, emphysematous destruction of alveolar sacs and impaired lung function in an 8-weeks cigarette smoke (chronic) model. The protective effects of γ-tocotrienol are likely mediated by inhibiting the activations of STAT3 and NF-κB, and augmenting Nrf2 nuclear translocation in cigarette smoke-challenged lungs. γ-Tocotrienol was more effective than prednisolone in protecting the lungs from developing emphysema in cigarette smoke-induced COPD model.
2.1 Reagents Table 2A| Sources of chemicals and reagents
Chemicals and Reagents
1st BASE, Singapore
Tris base, tris buffered saline (TBS), tris-acetate-EDTA (TAE), sodium dodecyl sulfate (SDS), phosphate buffered saline (PBS) 10x
Advansta Inc., Menlo Park, CA, USA Ambion, Austin, TX, USA Comparative Medicine, NUS
WesternBright Enhanced chemiluminescent (ECL), horseradish peroxidase (HRP) substrate RNA later Anesthetic mixture (10 ml/g ketamine: medetomidine: H2O = 3:4:33), isoflurane
Applied Biosystems, Foster City, CA, USA
SYBR Green Polymerase chain reaction (PCR) Master Mix
Bio-Rad Laboratories, Hercules, CA, USA
Agarose, blotting paper, polyvinylidene difluoride (PVDF) membrane, tetramethylethylenediamine (TEMED), 3,3’,5,5’tetramethylbenzidine (TMB) substrate kit
DAKO, Carpinteria, CA, USA
Mayer Hematoxylin, Schiff reagent, Gill Hematoxylin
Davos LifeScienes Pte Ltd
Vitamin E isoforms, Olive oil
Greer Laboratories, NC, USA
House dust mite (Dermatophagoides pteronyssinus)
Hyclone, Logan, UT, USA
Fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 culture medium
Invitrogen, Grand Island, NY, USA Merck, Darmstadt, Germany
Trizol, trypan blue, 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA), Diethylpyrocarbonate (DEPC)-treated water Absolute ethanol, isopropanol, methanol
National Diagnostics, Atlanta, GA, USA
Quanta Biosciences, Gaithersburg, MD, USA
Quanta qScript cDNA Super Mix
Sigma Aldrich, St. Louis, MO, USA
HEPES buffer, Harris Hematoxylin, eosin Y, 10% neutral buffered formalin, bovine serum albumin (BSA), chloroform, Tween 20, Tween 80, Span 20, ammonium chloride, methyl green, methacholine, non-fat skim milk, 1,1-diphenyl-2-picrylhydrazyl, resveratrol, trolox, prednisolone
Thermo Scientific Inc., Waltham, MA, USA
Bicinchoninic acid (BCA) protein assay kit, trypan blue
University of Kentucky CTRP, Lexington, KY, USA
3R4F research cigarettes
Assay kits reagents were obtained from Cayman Chemical (USA) and Millipore (Massachusetts, USA).
Murine Asthma Model
2.2.1 Mice Female BALB/c mice 6 to 8 weeks of age, weighing 18-25 g (Animal Resources Centre, Canning Vale, WA, Australia) were purchased from InVivos Private Limited of National University of Singapore (NUS). The mice were housed in plastic cages of five mice/cage, in a temperaturecontrolled room (±22°C and relative humidity of ±54%, 12-hour day/night cycle) maintained by Animal Holding Unit (AHU), NUS. Beddings were changed on alternate days. Standard laboratory feed and water was provided ad libitum. Newly arrive mice were allowed to acclimatized for at least 4 days to adapt to their new environment. All procedures followed strictly the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of NUS, under protocol number 109/12 and 035/10.
2.2.2 House dust mite (HDM) Airway Challenge Purified HDM protein extracts (Dermatophagoides pteronyssinus) were purchased from Greer Laboratories (NC, USA), diluted to a stock concentration of 10 mg/ml in sterile saline and kept frozen in the -20oC freezer. HDM working solution was freshly prepared before each use, where the stock solution is further diluted to a concentration of 100 µg
extract in 40µl sterile saline. Mice were sensitized on Day 0 and 7, and challenged on Day 14 via intratracheal administration with 40 µl of 100 µg HDM extract (Figure 2.01) (Hammad et al., 2009).
Figure 2.01| Murine asthma model design.
2.2.3 Treatment Groups for HDM-development In order to deduce if both inflammation and oxidative stress are involved in the development of HDM-induced asthma, mice were sensitized and challenged with saline/HDM a total of three times. They were sacrificed at various time points in the 17-days animal model. This will be summarized in Table 2B.
Table 2B| Treatment groups for HDM-development asthma study Group (n=8) 0 1 Saline 1 HDM 4 Saline 4 HDM 7 Saline 7 HDM 8 Saline 8 HDM 11 Saline 11 HDM 14 Saline 14 HDM 15 Saline 15 HDM 17 Saline 17 HDM
Sacrifice Saline Sacrifice HDM Sacrifice Sacrifice Saline HDM Sacrifice Sacrifice Saline Sacrifice HDM Saline Saline Sacrifice HDM Sacrifice HDM Saline Sacrifice Saline Sacrifice HDM HDM Sacrifice Saline Saline Sacrifice HDM HDM Saline Sacrifice Saline Saline HDM Sacrifice HDM HDM Sacrifice Saline Saline Saline HDM Sacrifice HDM HDM -
2.2.4 Treatment Groups for screening of vitamin E isoforms Vitamin E isoforms were kindly provided by Davos Life Science Pte Ltd, with ≥97% purity as measured by HPLC. As the no observed adverse effect level (NOAEL) for tocotrienols (T3) was reported at 260 mg/kg daily in mice for 13 weeks or up to one year (Nakamura et al., 2001; ReaganShaw et al., 2008; Tasaki et al., 2008), the highest oral dose of vitamin E isoforms in this study was capped at 250 mg/kg. On days 7, 8, 9, 14, 15 and 16, α-Tocopherol (250 mg/kg), α-tocotrienol (250 mg/kg), δ-tocotrienol (250 mg/kg), γ-tocotrienol (250 mg/kg), or vehicle (oil emulsifier) were emulsified in 0.2 ml of sterile water and fed orally to the mice.
The mice were divided into 7 treatment groups as shown in Table 2C. All mice were subjected to oral gavage treatment of water or respective drugs, to account for oral gavage-induced stress as a confounder. Group 1 (Saline) mice were saline-sensitized, saline-challenged and oral gavaged with 0.2 ml of water, serving as a negative model control. Group 2 (HDM) mice were HDM-sensitized, HDM-challenged and fed orally with 0.2 ml of water, which was the positive asthma model control. Group 3 (HDM/Veh) mice were HDM-sensitized, HDM-challenged and fed orally 0.2 ml of 3% oil emulsifier. This group served as a vehicular control and a negative control for the drugs.
Group 4 (HDM/αTP) mice were HDM-sensitized, HDM-challenged, and oral gavage with 0.2 ml of α-tocopherol, which served as vitamin E isoform screening group 1. Group 5 (HDM/αT3) mice were HDM-sensitized, HDMchallenged and fed orally 0.2 ml of α-tocotrienol, serving as vitamin E isoform screening group 2. Group 6 (HDM/γT3) mice were HDM-sensitized, HDM-challenged and orally gavage with 0.2 ml of γ-tocotrienol, serving as vitamin E isoform screening group 3. Group 7 (HDM/δT3) mice were HDMsensitized, HDM-challenged and fed orally with 0.2ml of δ-tocotrienol, serving as vitamin E isoform screening group 4. All mice were sacrificed on day 17.
Table 2C| Treatment groups for screening of vitamin E isoforms study
2.2.5 Treatment Groups for vitamin E isoform γ-tocotrienol in HDMinduced asthma mouse model γ-Tocotrienol, one of eight isoform of vitamin E, was kindly provided by Davos Life Science Pte Ltd, with ≥97% purity as measured by HPLC. As the no observed adverse effect level (NOAEL) for tocotrienols was reported at 260 mg/kg daily in mice for 13 weeks or up to one year (Nakamura et al., 2001; Reagan-Shaw et al., 2008; Tasaki et al., 2008), the highest oral dose of γ-tocotrienol in this study was capped at 250 mg/kg. On days 7, 8, 9, 14, 15 and 16, γ-tocotrienol (γ-T3; 30, 100 and 250 mg/kg), or vehicle (oil emulsifier) were emulsified in 0.2 ml of sterile water and fed orally to the mice.
Prednisolone, a corticosteroid, was purchased from Sigma Aldrich (St. Louis, MO, USA) and stored at 4oC. On days 7, 8, 9, 14, 15 and 16,
prednisolone is freshly dissolved in sterile water before use to a working concentration of 10 mg/kg in 0.2 ml water and fed orally to the mice.
The mice were divided into 7 treatment groups as shown in Table 2D. All mice were subjected to oral gavage treatment of water or respective drugs, to account for oral gavage-induced stress as a confounder. Group 1 (Naïve) mice were neither sensitized nor challenged, but oral gavaged with 0.2 ml of water, serving as a background control. Group 2 (Naïve-γT3) mice were neither sensitized nor challenged, but subjected to 0.2 ml oral gavage of 250 mg/kg γ-tocotrienol once daily, for six days; serving as a background drug control to observe if γ-tocotrienol would have any cytotoxic effects on naïve mice. Group 3 (Saline) mice that were salinesensitized, saline-challenged and fed with 0.2 ml of water, served as a negative model control.
Group 4 (HDM) mice were HDM-sensitized, HDM-challenged and fed with 0.2 ml of water, which was the positive asthma model control. Group 5 (HDM/Veh) mice were HDM-sensitized, HDM-challenged and fed orally 0.2 ml of 3% oil emulsifier. This group served as a vehicular control and a negative control for the drugs.
Group 6 (HDM/γT3) mice were HDM-sensitized, HDM-challenged and orally gavage with 0.2 ml of γ-tocotrienol at three concentrations (30, 100 and 250 mg/kg), which serves as the drug of interest. Group 7 (HDM/Pred) 111
mice were HDM-sensitized, HDM-challenged and fed orally with 0.2ml of 10 mg/kg prednisolone, serving as a positive drug control. All mice were sacrificed on day 17. Table 2D| Treatment groups for γ-tocotrienol in HDM-induced asthma study
Murine COPD Model
2.3.1 Mice Female BALB/c mice 7 to 8 weeks of age, weighing 18-25 g (Animal Resources Centre, Canning Vale, WA, Australia) were purchased from InVivos Private Limited of National University of Singapore (NUS). The mice were housed in plastic cages of five mice/cage, in a temperaturecontrolled room (±22°C and relative humidity of ±54%, 12-hour day/night cycle) maintained by Animal Holding Unit (AHU), NUS. Beddings were changed on alternate days. Standard laboratory feed and water was provided ad libitum. Newly arrive mice were allowed to acclimatized for at least 4 days to adapt to their new environment. All procedures followed
strictly the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of NUS, under protocol number 109/12.
2.3.2 Cigarette smoke (CS) exposure Cigarette smoke (3R4F research cigarettes) was delivered to the ventilated chambers via two peristaltic pumps (Masterflex L/S, ColeParmer Instrument Co., Niles, IL, USA) at a constant rate of 1 L/min. One peristaltic pump is connected to the lighted cigarettes and delivers cigarette smoke at a rate of 40 mL/min, while the other peristaltic pump is extracting and delivering room air at a rate of 960 mL/min – to achieve 4% CS delivery to the ventilated chambers (Figure 2.02). Sham air control group was simultaneously placed in another ventilated chamber and exposed only to room air at a constant rate of 1 L/min.
Peristaltic pumps 40 mL/min
Cigarettes Room air extraction Ventilated chambers
Figure 2.02| Set-up of two peristaltic pumps to deliver 4% cigarette smoke into the ventilated chambers.
For the 2-week acute CS-induced COPD model, mice were exposed to 4% CS from nine 3R4F reference cigarettes (University of Kentucky, Lexington, KY, USA) daily at a frequency of 3 sticks every 2 hours on days 1-5 and 8-11 (Figure 2.03). Sham air mice were placed in separate ventilated chamber and exposed to room air. Mice were sacrificed 24 hours after the last CS or sham air exposure on day 12, with blood, BAL fluid and lung tissues collected for various analyses.
Figure 2.03| Acute 2-weeks CS-induced COPD model design
For the 2-month chronic CS-induced COPD model, mice were exposed to the same CS exposure regimen daily for 8 weeks at a frequency of 5 consecutive days per week (Figure 2.04). Sham air mice were placed in separate ventilated chamber and exposed to room air. Mice were subjected to pulmonary function test 24 hours after the last CS or sham air exposure on day 54, and sacrificed thereafter where both blood and lung tissues were collected for various analyses. 114
Figure 2.04| Chronic 2-months CS-induced COPD model design
2.3.3 Treatment groups for vitamin E isoform γ-tocotrienol in acute 2-weeks CS-induced COPD mouse model γ-Tocotrienol, an isoform of vitamin E, was kindly provided by Davos Life Science Pte Ltd, with ≥97% purity as measured by HPLC. As the no observed adverse effect level (NOAEL) for tocotrienols was reported at 260 mg/kg daily in mice for 13 weeks or up to one year (Nakamura et al., 2001; Reagan-Shaw et al., 2008; Tasaki et al., 2008), the highest oral dose of γ-tocotrienol in this study was capped at 250 mg/kg. On days 9 – 11, γ-tocotrienol (γ-T3; 30, 100 and 250 mg/kg), or vehicle (oil emulsifier) were emulsified in 0.2 ml of sterile water and fed orally to the mice.
Prednisolone, a corticosteroid, was purchased from Sigma Aldrich and stored at 4oC. On days 9 – 11, prednisolone was freshly dissolved in
sterile water before use to a working concentration of 10 mg/kg in 0.2 ml water and fed orally to the mice.
The mice were divided into 7 treatment groups as shown in Table 2E. All mice were subjected to oral gavage treatment of water or respective drugs, to account for oral gavage-induced stress as a confounder. Group 1 (Naïve) mice were exposed to neither sham air nor cigarette smoke, and oral gavaged with 0.2 ml of water, serving as a background control. Group 2 (Naïve-γT3) mice were also exposed to sham air, but subjected to 0.2 ml oral gavage of 250 mg/kg γ-tocotrienol once daily, for three days; serving as a background drug control to observe if γ-tocotrienol would have any cytotoxic effects on naïve mice. Group 3 (Sham) mice were exposed to sham air and fed with 0.2 ml of water, served as a negative model control.
Group 4 (CS) mice were exposed to CS and fed with 0.2 ml of water, which was the positive COPD model control. Group 5 (CS/Veh) mice were exposed to CS and fed orally 0.2 ml of 3% oil emulsifier. This group served as a vehicular control and a negative control for γ-tocotrienol.
Group 6 (CS/γT3) mice were exposed to CS and orally gavage with 0.2 ml of γ-tocotrienol at three concentrations (30, 100 and 250 mg/kg), which serves as the drug of interest. Group 7 (CS/Pred) mice were exposed to CS and fed orally with 0.2ml of 10 mg/kg prednisolone, serving as a positive drug control. All mice were sacrificed on day 12. 116
Table 2E| Treatment groups for 2-weeks CS-induced COPD model
2.3.4 Treatment groups for vitamin E isoform γ-tocotrienol in chronic 2-months CS-induced COPD mouse model γ-Tocotrienol (250 mg/kg), or prednisolone (10 mg/kg) in 0.2 mL of water was given via oral gavage 1 hour before the CS exposure on days 38–40, 43–47, and 50–53 to the mice.
The mice were divided into 5 treatment groups as shown in Table 2F. All mice were subjected to oral gavage treatment of water or respective drugs, to account for oral gavage-induced stress as a confounder. Group 1 (Sham) mice were exposed to sham air and fed with 0.2 ml of water, served as a negative model control. Group 2 (CS) mice were exposed to CS and fed with 0.2 ml of water, which was the positive COPD model control. Group 3 (CS/Veh) mice were exposed to CS and fed orally 0.2 ml of 3% oil emulsifier. This group served as a vehicular control and a negative control for γ-tocotrienol. Group 4 (CS/γT3) mice were exposed to 117
CS and orally gavage with 0.2 ml of γ-tocotrienol at a concentration of 250 mg/kg, which serves as the drug of interest. Group 5 (CS/Pred) mice were exposed to CS and fed orally with 0.2ml of 10 mg/kg prednisolone, serving as a positive drug control. All mice were sacrificed on day 54.
Table 2F| Treatment groups for 2-months CS-induced COPD model
Bronchoalveolar lavage (BAL) and cardiac puncture were performed on day 17 for the HDM-induced asthma model, and on day 12 for the CSinduced COPD model. Mice were anaesthetized via 0.3 ml intraperitoneal (i.p.) injections of anesthetic mixture, where operations begun 5 min after anesthetization to ensure that the mice were fully unconscious. Blood was first extracted from the heart of the mice by piercing the left ventricle with a 25G needle connected to a sterile 1 ml syringe. This process was done first to minimize contamination of the BAL fluid with erythrocytes. Tracheotomy was performed and a small transverse incision was made on the exposed trachea. A 20G cannula connected to a 1 ml sterile syringe was inserted into the trachea through this incision and 0.5 ml of ice-cold 118
phosphate-buffered saline (PBS, 4°C) was infused into the lungs. This process was repeated twice more to ensure flushing out most of the contents in the lung, where a final volume of 1.4 - 1.5 ml of BAL fluid was collected and used for the subsequent experiments.
Total Cell Count
BAL fluid collected from the lungs of the mouse was centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was collected and stored at -80°C. The pellet was dissolved in 200 µl of 8.5 mg/ml NH4Cl and incubated for 5 min at room temperature to allow the lysis of red blood cells. The cell suspension was then centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was discarded and the pellet was re-suspended in 200 µl of RPMI with 10 mg/ml BSA. The suspension of cells contained inflammatory cells that had infiltrated the airway. 10 µl of 0.4% trypan blue solution was mixed with 10 µl of cell suspension and the total number of viable cells was enumerated using a haemocytometer, under an inverted light microscope (Nikon Instruments, Tokyo, Japan) at 200× magnification. The count was performed in a single blinded manner to eliminate bias.
Differential Cell Count of BAL Fluid
Following the enumeration of the total cell count, dilutions were performed on the BAL fluid collected using the RPMI/BSA solution to achieve an average total cell count of about 1 × 105 cells per 150 µl of RPMI/BSA. For
cytological examination, cytocentrifugation was carried out. Smears of the diluted cell suspension were made on glass slides using a Cytospin centrifuge (Thermo Shandon, Pittsburgh, USA) at 600g for 10 min.
Slides were stained using Liu staining (modified Wright staining), where 800 µl of Liu A was pipetted onto the smears on the slides for 30 sec followed by 1600 µl of Liu B for 90 sec. Slides were left to dry overnight and glass cover slips were mounted onto the stain with histomount. Differential cell count was then performed on at least 500 cells at 1000× magnification under the light microscope (Leica Microsystems, Wetzler, Germany) for each cytospin slide. Four types of inflammatory cells namely macrophages, eosinophils, lymphocytes and neutrophils were identified and enumerated. The count was performed in a single blinded manner to eliminate bias.
2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) Assay
For DCFH-DA assay, BAL fluid was incubated with 10 mM DCFH-DA (Invitrogen, Grand Island, NY, USA) for 20 mins at 37oC, with its supernatant discarded after centrifuged at 3,000 rpm for 5 minutes at 4oC. The pellet was resuspended in RPMI and fluorescence measured with a spectrofluorometer of excitation 492 nm and emission 525 nm.
Lung samples from the mice were removed from the thoracic cavity by careful dissection 24 hours after the final respective challenges and stored in 10% neutral buffered formalin solution for at least 48 hours before it was sent to process in a tissue processor (Leica Microsystems, Wetzler, Germany). Briefly, lung samples were dehydrated by immersing them in increasing concentrations of ethanol (70% 80% 90% 100%) for 30 min each and two hours for 100%. The samples were then placed in xylene, a clearing agent, for 10.5 hours. Samples were emplaced in hot molten paraffin wax for 3 hours. Tissues were then further embedded in paraffin wax, fixed and sectioned into 4.5 µm pieces using a rotary microtome (Leica Microsystems, Wetzler, Germany). The sectioned tissues were then placed on a glass slide to be stained.
2.8.1 Hematoxylin and Eosin (H&E) Staining Lung sections were stained with H&E to investigate cell infiltration in the airways. Briefly, the sectioned tissues were deparaffinized in Histoclear for 5 min twice, then rehydrated in a series of descending concentrations of ethanol (100% 100% 90% 70%) for 1 min and 3 min for 100%, and finally in deionized water for 1 min. The slides were submerged in Mayer Hematoxylin (DAKO, Copenhagen, Denmark) for 2 min and washed in deionized water before differentiating with 0.1% acid alcohol for 30 sec. Slides were washed under running tap water for 30 sec before counterstaining with eosin for 30 sec and washed in deionized water. 121
Samples were dehydrated in ascending concentrations of ethanol (70% 90% 100% 100%) for 30 sec each. Lastly, the slides were incubated in Histoclear for 5 min before mounting with cover slips and Histomount. Both photo-micrograph of bronchioles and alveolars in the stained lung tissue were captured using light microscope camera (Nikon DSRi1, Japan) at 200X magnification.
Quantification of inflammation score was performed single-blinded to eliminate bias and scored based on a 5-point system as previously described (Myou et al., 2003). The inflammation scoring was: 0, no inflammatory cells; 1, occasional cuffing with few inflammatory cells; 2, most bronchi or vessels surrounded by a ring of cells one cell layer deep; 3, a thick layer of cells (2-4 cells layer deep); 4, a ring of cells more than 4 cells layer deep. Scoring was performed on at least 3 photo-micrographs taken per lung section, with at least 4 mice per treatment group.
Quantification of bronchial epithelium thickness was performed by taking photo-micrographs of bronchi at 200X magnification and then analyzed using ImageScope software (Leica Biosystems, Wetzlar, Germany) by measuring the epithelium thickness 12 times around each bronchus. The measurement was performed single-blinded on two bronchi per mice, with 4 mice per treatment group.
Quantification of alveolar diameter was performed by taking photomicrographs using light microscope camera (Nikon DSRi1) at 100X magnification that contain only alveolar and overlaid with a 12-horizontal line template, where alveolar diameter is calculated by dividing the length of 12 lines over number of intercepts (alveolar walls with lines), as previously described (Beckett et al., 2013). The counting and calculation was performed single-blinded on 3 photo-micrographs per mice, with 4 mice per treatment group.
2.8.2 Periodic Acid-Fluorescence Schiff (PAFS) Staining PAFS staining is used to determine mucus secretion. Similarly, slides were deparaffinized with Histoclear for 10 mins and rehydrated in decreasing concentrations of ethanol (100% 100% 90% 70%) for 1 min each. The slides were immersed in periodic acid for 5 min, deionized water for 5 min, and schiff’s reagent for 15 min. Next, the lung sections were washed under running tap water for 5 min and stained in Gill’s Hematoxylin for 90 sec. The slides were then dehydrated in ascending concentrations of ethanol (70% 90% 100% 100%) for 30 sec each and cleaned with Histoclear for 5 min. Relative intensity analyses of PAFS stain were performed single-blinded, where the ratio of red over green stains was normalized to saline control, using the ImageJ software (National Institutes of Health, Bethesda, USA), as previously described (Goh et al., 2013). The analyses were performed on at least 4 bronchi/bronchioles per mice, with 4 mice per treatment group. 123
Freeze Dry of Lung Tissue
Precise dissection at the mice thoracic cavity was performed to obtain undamaged lung samples on day 17 for HDM-induced asthma model and on day 12 for CS-induced COPD model, washed in ice-cold PBS to remove excess blood then kept in 2.0 ml eppendorf tubes. Labeled tubes containing the lungs, with the caps left open, were sent to the Freeze Dry machine (Labconco Corporation, Kansas City, USA) overnight. The vacuum pump in the machine kept the tissues sterile and extracted all the moisture. Tissue pestles (Krackeler Scientific Inc., New York, USA) were used to grind the dried lung tissues till powder-like form, and then stored in -80oC. This allowed accurate weighing of required lung tissues in subsequent analysis.
Homogenization of Freeze Dried Lung Tissue Powdered lungs were scooped out from stock tubes into freshly labeled ones and weighed. HEPES buffer (25 mM) was added into each tube accordingly, so as to obtain 1 mg/0.1ml concentration, followed by approximately 5 Precellys® 1.4mm ceramic beads (Cayman Chemical, USA). Thereafter, the tubes were placed in the Precellys® tissue homogenizer (Precellys, MD, USA) to fully lyse the lung fragments. The tubes were then centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was extracted and used in subsequent assay kits.
Enzymatic activity of respective enzymes in lung homogenates from Section 2.9 was measured using enzymatic assays. Assay kits and reagents were obtained from Cayman Chemical (USA), where the protocols of each assay were adapted from the manufacturer’s manual provided.
2.10.1 Total Antioxidant Capacity (TAC) Assay Principle of TAC Assay: The total antioxidant capacity present in the lungs, including both aqueous- and lipid-soluble antioxidants (such as vitamins, proteins, lipids, glutathione, enzymes, etc), are assessed with detection limits of 0.044 mM/mg of lungs. The antioxidants present in the sample will inhibit the oxidation of 2,2’-Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) to ABTS.+ radicals by metmyoglobin. Known concentrations of trolox standards, samples, metmyoglobin and ABTS reagents are added to each well, where the reactions are initiated by adding hydrogen peroxide
spectrophotometer at 750 nm. The TAC is inversely proportionate to the standard curve and derived as mM of antioxidant capacity per mg of lungs.
2.10.2 Superoxide Dismutase (SOD) Assay Principle of SOD Assay: It is to measure the activity of SOD with detection limits of 1.25 units/g of lungs, where one unit is the amount of SOD
required dismutating 50% of the superoxide anion. Tetrazolium salt solution (radicals detector) was added to separate wells containing samples and standards in the 96-well plate. Xanthine oxidase which catalyzes the formation of superoxide anions from oxygen was then added to start the reaction and timed for 20 min, before readings were computed in the spectrophotometer at 450 nm. The readings were directly proportional to SOD activity and standard curves were plotted to obtain the precise units of activity.
2.10.3 Catalase (CAT) Assay Principle of CAT Assay: CAT activity was quantified in this assay, with a detection limit of 20 nmol/min/g of lungs. Methanol and hydrogen peroxide can be converted to formaldehyde by CAT, which is the key mechanism in this assay. The chromogen Purpald (4-amino-3-hydrazino-5-mercapto1,2,4-triazole) is oxidized after interaction with form-aldehyde, resulting in the change in color from colorless to purple. Known concentrations of methanol and sample were added to the wells, with various concentrations of formaldehyde as the standard. Addition of hydrogen peroxide started the reaction, where potassium hydroxide was added 20 min later to stop the reaction. Purpald was added for 10 min before the addition of potassium periodate for 5 min. The intensity is directly proportional to the activity of CAT and was measured spectrophotometrically at 540 nm.
Standard curves were plotted to calculate the activity of CAT in nmol/min/g of lung.
2.10.4 Glutathione Peroxidase (GPx) Assay Principle of GPx Assay: It was employed to quantify the activity of GPx, with a detection limit of 5.0 nmol/min/g of lungs. Through the reduction of hydrogen peroxide, GSH is converted to oxidized glutathione (GSSG), where GSSG is recycled back to GSH by glutathione reductase (GR) and NADPH. The oxidation of NADPH to NADP+ results in a decrease in absorbance at 340 nm, which is directly proportional to the activity of GPx. Assay buffer and co-substrate mixture (containing NADPH, GSH and GR) were added to standards or samples wells. The reaction was initiated when cumene hydroperoxide was added to the wells and absorbance was read at 340 nm every minute, for 7 min. Standard curves and change in absorbance graphs were plotted to determine the activity of GPx.
Enzyme Immunoassay (EIA)
EIA, also known as Enzyme-Linked Immunosorbent Assay (ELISA), is employed to detect a specific antigen in the sample (Lequin, 2005). BAL fluid obtained from Section 2.4 and lung homogenate from Section 2.9 were used for EIA accordingly. Assay kit and reagents for multiplex (20-24 cytokines and chemokines) were obtained from Millipore (MA, USA). Assay
deoxyguanosine were obtained from Cayman Chemical (USA), while 3nitrotyrosine and Advanced Oxidation of Protein Products assay reagents and kit was obtained from Cell Biolabs (San Diego, CA, USA). The protocols of each assay were adapted from the manufacturer’s instructional manual provided.
2.11.1 Multiplex BAL fluid was thawed in ice prior to usage. Principle of Multiplex assay: The list of cytokines and chemokines for this multiplex are: G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-6, IL-9, IL-10, IL-12 (p40), IL-13, IL-17, KC, LIF, LIX, MIP-1α, MIP-1β, RANTES, TNFα, and VEGF. The 96-well plate is soaked with wash buffer for 10 mins before decant and adding standards and samples into each well accordingly. The premixed antibody beads were sonicated for 30 sec and then vortexed for 1 min, then added into each well subsequently. The plate is sealed and incubated overnight on a plate shaker at 500 rpm in the 4oC cold room. The plate is washed twice with a magnet to hold onto the magnetic beads, before adding secondary detection antibody and incubated at room temperature for 1 hour on a plate shaker at 500 rpm. Streptavidinphycoerythrin were added into the plate and further incubated for 30 mins at room temperature on the plate shaker at 500 rpm. The plate was washed with a magnet and sheath fluid was added into each well before using the MAGPIX instrument to read the plate. Standard curves were plotted to derive the concentrations of respective cytokines/ chemokines. 128
2.11.2 8-Isoprostane and 8-hydroxy-2-deoxyguanosine (8-OHdG) EIA BAL fluid was thawed in ice prior to usage in both assays. Principle of 8-Isoprostane & 8-OHdG Assay: It was performed to quantitatively measure the levels of 8-Isoprostane and 8-OHdG in the BAL fluid, with a detection limit of 2.7 ρg/ml and 33 ρg/ml respectively. The competitive binding between 8-Isoprostane/8-OHdG and its tracer (8Isoprostane-acetlycholinesterase/8-OHdG-acetylcholinesterase)
basis of this assay. A known concentration of tracer and specific antiserum to 8-Isoprostane/8-OHdG were added into the pre-coated 96-well plate (mouse anti-rabbit IgG), along with the standards and samples into respective wells for 18 hours at 4oC. The pre-coated antibody binds to the antiserum, which binds to 8-Isoprostane/8-OHdG and the tracer competitively. After incubation, the plate was washed five times before Ellman’s Reagent (substrate to acetylcholinesterase) was added to each well. The product of this enzymatic reaction was yellow in color and the intensity was measured spectrophotometrically at 412 nm after 60 min. The resulting intensity was inversely proportional to the amount of 8Isoprostane/8-OHdG in the BAL fluid, where standard curves were plotted to determine the exact amount.
2.11.3 3-Nitrotyrosine (3-NT) EIA Lung homogenate was used as samples in this assay.
Principle of 3-NT Assay: The quantification of levels of 3-NT in the lungs was the aim of this assay. Samples and standards were added accordingly in the nitrotyrosine coated EIA plate (Cell Biolabs) for 10 mins, before adding anti-nitrotyrosine antibody for 1 hour. Plates were washed and incubated with secondary antibody-enzyme conjugate for 1 hour, washed subsequently before substrate solution was added for 10 min and adding stop solution at the final step. The absorbance was measured with a spectrophotometer at 450 nm and standard curves were plotted to calculate the amount of 3-NT.
2.11.4 Advanced Oxidation of Protein Products (AOPP) EIA Lung homogenate was used as samples in this assay. Principle of AOPP Assay: Advanced oxidation protein products (AOPP) are produced during oxidative stress by chlorinated oxidants such as chloramines and hypochlorous acid (mainly produced by neutrophils). This serves as an indirect indicator of neutrophils-induced oxidative damage in the lungs. Samples and standards are added into each well accordingly and the reaction is initiated by the addition of chloramine reaction initiator for 5 min. The reaction is ceased by adding stop solution and absorbance measured using a spectrophotometer at 340 nm. Standard curves were plotted to calculate the amount of AOPP in the samples.
Airway Hyperresponsiveness (AHR)
AHR was measured using airway resistance (Rl) and dynamic compliance (Cdyn) in response to methacholine (0.5 – 8 mg/ml; Sigma-Aldrich) recorded using a wholebody plethysmograph chamber (Buxco, CT, USA). Rl is defined as the ratio of driving pressure over the rate of airflow, and Cdyn is defined as the lungs ability to stretch during a change in volume relative to an applied change in pressure. On sacrifice day, mice were anesthetized and tracheotomy was performed where a small transverse incision was made on the exposed trachea. The cannula is tightly secured in the trachea and connected to the whole body plethysmograph chamber. The chamber was connected to the pneumotach, ventilator and nebulizer. The machine was adjusted at a tidal volume of 200 µl/breath and a breathing rate of 130/min. Methacholine is freshly prepared before each use. The machine records the data and the mice were challenged with aerosolized 10 µl PBS first, followed by 10 µl of increasing doses of methacholine, with a time interval of 5 min between each nebulization. The results are presented as fold change percentage of the values in response to PBS.
Pulmonary Function Test (PFT)
Mice were anesthetized on day 53 of the 2-months CS-induced COPD mouse model, and tracheotomy was performed as described in section 2.12 with a cannula attached to the trachea, and placed in a
bodyplethysmograph connected to a computer controlled ventilator (Forced
Wilmington, NC, USA) (Vanoirbeek et al., 2010). This set-up specifically designed for mouse has a dead space of 0.70 ml and measures three maneuvers: Boyle’s Law FRC, quasistatic pressure volume and fast flow volume maneuver. The anesthetized mouse was allowed to acclimatize until a regular breathing pattern was obtained. Total lung capacity (TLC), functional residual capacity (FRC), static compliance (Cchord), forced expiratory volume at 100 ms over forced vital capacity (FEV100/FVC) and airway resistance (Rl) were recorded using the FinePointeTM data acquisition and analysis software (Buxco). Work of breathing was calculated using the area under the pressure-volume graph. Maneuvers for all three tests were repeated four times each to obtain a reliable mean for all parameters.
Immunoblotting (Western blotting)
Lung samples were harvested on respective sacrifice days of various mouse models, and stored at -80oC. The lung lobes were cut into small pieces and homogenized using Precellys tissue homogenizer. Both nuclear and cytosolic protein extracts were collected from the lung lysates using the nuclear extraction kit (Cayman Chemicals, USA) according to the manufacturer’s protocol. The BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to determine the protein
concentrations in both extracts. Loading the same amount of protein (25 µg) per lane in the gel, they were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membrane with a wet electrophoretic transfer system (Bio-Rad). Immunoblots were then blocked with 5% nonfat milk (Sigma Aldrich) in TBS with 0.05% Tween 20 at room temperature for 2 hours, and probed with various primary antibodies (NF-κB, Nrf2 and STAT3 from Cell Signaling Technology) at 4oC overnight. The membrane was washed with TTBS buffer and probed with corresponding secondary antibody for 2 hours, and subsequently developed on hyperfilms using chemiluminescence ECL reagent (HRP) (Advansta Inc., CA, USA). TATAbinding protein (TBP) was used as control to normalize nuclear extract proteins. Band intensity was quantitated using ImageJ software (National Institutes of Health, USA) for relative densitometry quantifications.
NF-κB, Nrf2 and STAT3 Transactivation Assay
Briefly, the pre-coated 96-well plate contains immobilized oligonucleotide with the respective consensus sequence (NF-κB: 5'-GGGACTTTCC-3', Nrf2:
TTCCCGGAA-3’). Nuclear extract proteins (20 µg), obtained as described in section 2.14, from the mouse samples are loaded into the wells and bind specifically to the immobilized oligonucleotide. After incubation of 1 hour, primary antibodies (anti-p65, anti-Nrf2, anti-STAT3) were added into
the wells for 1 hour, followed by HRP-conjugated secondary antibodies for another 1 hour. The plates were washed and added with developing substrate solution, and stopped with the stopping solution. The absorbance was measured at 450 nm with a spectrophotometer with a reference wavelength of 655 nm.
Real-Time Polymerase Chain Reaction
Lung samples from the mice were extracted from the thoracic cavity by careful dissection on sacrifice day (respective mouse model) and stored in RNAlater (Ambion, USA), an aqueous tissue storage reagent that rapidly permeated the lung tissues to stabilize and protect the RNA. The samples were first incubated at 4°C overnight to allow the RNAlater to permeate the tissues and were stored at -80°C.
2.16.1 RNA Isolation RNA isolation was performed using a single-step method as previously described (Chomczynski and Sacchi, 2006). Homogenization: Lung tissues stored previously at -80°C in RNAlater were taken out and 1 ml of TRIzol reagent (Invitrogen, USA) was added to a 1.5ml tube. A homogenizer was used to disrupt the lung tissues in an ice bath (to help reduce degradation of RNA). During sample homogenization, TRIzol reagent helped to maintain the integrity of the RNA while disrupting cells and dissolving cell components. The homogenate was then
centrifuged at 12,000 rpm for 10 min at 4°C to spin down the unwanted membranes and cell components. The cleared supernatant was transferred to another 1.5ml tube for the next step. Phase separation: The supernatant was left to incubate for 5 minutes at room temperature before 0.2 ml chloroform was added. The tubes were shaken vigorously for 15 sec and incubated at room temperature for 3 min. The resulting pinkish-white mixture was then centrifuged at 12,000 rpm for 15 min at 4°C, to separate the mixture into three distinct phases – a colorless upper aqueous phase containing RNA, a middle whitish organic layer containing proteins and a bottom reddish organic layer containing DNA. About 500 µl of the top aqueous layer was carefully transferred to another tube for further processing. RNA precipitation: 0.5 ml of isopropanol was added to the clear aqueous solution and the tubes were shaken vigorously for 30 sec, incubated at room temperature for 10 min and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was discarded and 1 ml of 75% ethanol was added to the RNA pellet. Centrifugation at 8500 rpm for 5 min at 4°C was then performed to wash the pellet. Redissolving RNA: The supernatant was discarded and the washed RNA pellet was exposed to air at room temperature for 10 min to allow the pellet to dry. The RNA pellet was dissolved in 100 µl of ribonuclease-free DEPC water by passing through a filter tip and incubated at 55°C for 10 min.
Quantification of RNA: A nano-drop ND-1000 spectrophotometer was used to quantify the amount of RNA present in the sample. 1 µl of RNA was added to the quantifying spot in the spectrophotometer using DEPC water as the blank and the concentration of the RNA was recorded. In addition, the A260, A280 and A260/A280 ratios were recorded as an indication of the purity of the RNA extracted. An A260/A280 ratio of 1.8 to 2.0 meant an acceptable level of purity of the RNA extracted.
2.16.2 Reverse Transcription 1 µg of total RNA, with oligo dT primer and AMV reverse transcriptase was used to reverse transcribe into cDNA by a thermal cycler (GeneAmp PCR system 2700, Applied Biosystems, USA).
2.16.3 Real-time Polymerase Chain Reaction The 20 µl real time PCR mixture contained 10 µl of 2x QuantiFast SYBR Green PCR Master Mix (Applied Biosystems, USA), 2 µl of 10x QuantiTect Primer Assay (Qiagen, USA), 1 µl template cDNA, and RNase-free water. The PCR primers used were obtained from Integrated DNA Technologies (Coraville, IA, USA) and are listed in Table 2G. Each sample was run in triplicate. Amplification and detection was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, USA) using the following settings: 2 min at 95oC, 40 cycles of 10 sec at 95oC and 30 sec at 60oC. 18s was used as an endogenous control to normalize each target sample.
All data were analyzed using the Sequence Detection System SDS software package, version 188.8.131.52 (Applied Biosystems, USA). A relative Quantification plate document was created for analysis. The mRNA expression levels for all samples were normalized to housekeeping gene β-actin. Table 2G| Primer sequences for various targets
Gene CAT Cu,ZnSOD E-Selectin EC-SOD GPx-1 GR HO-1 ICAM-1 iNOS IL-13 IL-17 IL-33 MMP9
Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Primer Sequences (5' 3') 5’ –ACCAGGGCATCAAAAACTTG-3’ 5’-CACCTTGAGCATGTAAGGCATAGG-3’ 5’-ACCAGTGCAGGACCTCATTTTAA-3’ 5’-TCTCCAACATGCCTCTCTTCATC-3’ 5’-AACGCCAGAACAACAATTCC-3’ 5’-TGAATTGCCACCAGATGTGT-3’ 5’- GTGTCCCAAGACAATC-3’ 5’- GTGCTATGGGGACAGG-3’ 5’-GGTTCGAGCCCAATTTTACA-3’ 5’-GCCCTGAAGCTTTTTGTCAG-3’ 5′-GCGTGAATGTTGGATGTGTACC-3′ 5′-GTTGCATAGCCGTGGATAATTTC-3′ 5’-CCTCACTGGCAGGAAATCATC-3’ 5’-CCTCGTGGAGACGCTTTACATA-3’ 5'-CCAGCACTCCGTGAAGATCC-3' 5'-CTGGTCTGGGAAGGCATACA-3' 5'-GTTCTCAGCCCAACAATACAAGA-3' 5'-GTGGACGGGTCGATGTCAC-3'
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5'-CAGAGGCCATGCAATATCCTC-3' 5'-CAGCATGGTATGGAGTGTGGA-3' 5'-GACCAGGATCTCTTGCTGGA-3' 5'-GAACTCTCCACCGCAATGA-3' 5'-CGGATCCACTTCACTTTTAACACAGTC-3' 5'-GAGATCTTTAGATTTTCGAGAGCTTA-3' 5′-CGCTCATGTACCCGCTGTAT-3′ 5′-TGTCTGCCGGACTCAAAGAC-3′
MMP12 Mn-SOD MUC5ac MUC5b NOX1 NOX2 NOX3 NOX4 NQ01 p22phox p67phox Periostin RANTES TGF-β TNF-α VCAM-1
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5′- CATGAAGCGTGAGGATGTAGAC-3′ 5′-TGGGCTAGTGTACCACCTTTG-3′ 5’-CACATTAACGCGCAGATCATG-3’ 5’-CCAGAGCCTCGTGGTACTTCTC-3’ 5'-GGACTTCAATATCCAGCTACGC-3' 5'-CAGCTCAACAACTAGGCCATC-3' 5'- TCCCTAGCATGAGCGCCTTA-3' 5'- CCACGACGCAGTTGGATGTT-3' 5’-AGGTCGTGATTACCAAGGTTGTC-3’ 5’-AAGCCTCGCTTCCTCATCTG-3’ 5’-AGCTATGAGGTGGTGATGTTAGTGG-3’ 5’-CACAATATTTGTACCAGACAGACTTGAG-3’ 5’-GCTGGCTGCACTTTCCAAAC-3’ 5’-AAGGTGCGGACTGGATTGAG-3’ 5’-CCCAAGTTCCAAGCTCATTTCC-3’ 5’-TGGTGACAGGTTTGTTGCTCCT-3’ 5'-AGAGAGTGCTCGTAGCAGGAT-3' 5'-GTGGTGATAGAAAGCAAGGTCTT-3' 5’-CGTGGCTACTGCTGGACGTT-3’ 5’-GCACACCTGCAGCGATAGAG-3’ 5’-CTTCGGATTCACCCTCAGTC-3’ 5’-CACCTTGAGCATGTAAGGCATAGG-3’
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5'-TAGCCCAATTAGGCTTGGCATCC-3' 5'-TAAGAAGGCGTTGGTCCATGCT-3' 5'-TTTGCCTACCTCTCCCTCG-3' 5'-CGACTGCAAGATTGGAGCACT-3' 5′-CTCCCGTGGCTTCTAGTGC-3′ 5′-GCCTTAGTTTGGACAGGATCTG-3′ 5'-CCAGTGTGGGAAGCTGTCTT-3' 5'-AAGCAAAAGAGGAGGCAACA-3' 5'-CCAGCACTCCGTGAAGATCC-3' 5'-CTGGTCTGGGAAGGCATACA-3'
Pharmacokinetics of γ-Tocotrienol
In order to obtain the levels of γ-tocotrienol in mice against time, serum of mice administered with γ-tocotrienol had to be obtained at various timepoints. Naïve mice were given oral γ-tocotrienol (250 mg/kg) and anesthetized prior to blood collection at different time intervals over a 12hour period. The blood collected was left to stand at room temperature for 4 hours. This allowed the clotted erythrocytes to settle, before centrifuged at 3000 rpm for 5 min at 4oC. The supernatant, containing the serum, was aliquot to 60 µl per tube and stored at -80oC.
Standards were prepared by adding 50 µl of naïve mice serum (not administered with γ-tocotrienol) to 200 µl of methanol and 1 µl of varying concentrations of γ-tocotrienol (2.5, 5, 10, 50, 100 and 200 ng/ml). As for the samples, 50 µl of serum from respective time-points was added to 200 µl of methanol. Both standards and samples were subjected to heavy vortex for 10 min before centrifuged at 13,200 rpm at 4oC for 10 min. 180 µl of the supernatant was removed and transferred to a new set of eppendorf tubes. The standards and samples were centrifuged at 13,200 rpm at 4oC for 10 min again to ensure the removal of red blood cells debris, where 140 µl of the supernatant was extracted to 2.0 ml glass vials. Serum levels of γ-tocotrienol were measured using the Waters Alliance 2695 HPLC System (Waters Corporation, Milford, MA, USA) as described (Lee et al., 2003). Data were pooled and analysed using the naïve pooled modelling approach (Burtin et al., 1996). Data were analysed using the 139
average concentration at each time point. The noncompartmental analysis was performed using WinNonlin professional version 6.3 software (Pharsight, Mountain View, CA).
1,1-diphenyl-2-picrylhydrazyl (DPPH) Assay
The DPPH assay was employed to determine if the drug of interest possess any radical scavenging activity. Different concentrations (1 – 300 µg/ml methanol) of a-tocopherol, tocotrienols (Davos Life Science), prednisolone, resveratrol, or trolox (Sigma-Aldrich) were incubated with 0.1 mM 1,1-diphenyl-2-picrylhydrazyl dissolved in methanol (DPPH; Sigma-Aldrich) for various time intervals (5 min to 4 hours). Timedependent and concentration-dependent free radical scavenging activities were measured colorimetrically at 517 nm post incubation at 37oC. Each sample was loaded in triplicates and experiments were performed at least three times.
Data are presented as mean ± SEM. Comparisons between different groups were conducted using one-way analysis of variance (ANOVA) and Dunnett’s test. The critical level for statistical significance was set at (p < 0.05) when SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for evaluating statistical values. *: P-value < 0.05, **: P-value < 0.01.
Chapter 3: Time Course Development of Inflammation and Oxidative Stress in House Dust Mite-Induced Asthma, and Selection of vitamin E isoform
Background: Vitamin E isoforms were reported to possess both antioxidative and anti-inflammatory properties. Recent reports revealed that tocotrienols
inflammation and oxidative damage in various diseases. Thus we needed a disease model with both inflammation and oxidative stress to investigate therapeutic potentials of vitamin E isoforms. Asthma is a chronic inflammatory disease with observed oxidative damage in patients.
Objective: We aimed to deduce if both inflammation and oxidative stress is involved in house dust mite (HDM) experimental asthma model. If so, we aim to examine if vitamin E isoforms do possess anti-inflammatory and anti-oxidative properties, and select the isoform with most therapeutic potential for further studies.
Methods: BALB/c mice were sensitized and challenged with HDM. Bronchoalveolar lavage (BAL) fluid was assessed for total and differential cell counts, oxidative damage biomarkers, and cytokine levels. Lungs were examined for cell infiltration and mucus hypersecretion, and the
expression of antioxidants and pro-inflammatory biomarkers, as well as transcription factors. Sera were assayed for γ-tocotrienol levels.
Results: HDM challenge elevated BAL fluid cytokine and chemokine levels, total reactive oxygen species and oxidative damage biomarker levels. Mucus hypersecretion was observed in the lungs of HDMchallenged mice. Both inflammation and oxidative stress transcription factors, NF-κB and Nrf2 respectively, were observed to be modulated in asthma. While screening among vitamin E isoforms, γ-Tocotrienol displayed better free-radical neutralizing activity in vitro and inhibition of BAL fluid total, eosinophil and neutrophil counts in HDM mouse asthma in vivo, as compared to other vitamin E isoforms including α-tocopherol. Also, γ-tocotrienol was found to block nuclear NF-κB level and enhance nuclear Nrf2 levels in lung lysates to greater extents than α-tocopherol.
Conclusion: Both inflammation and oxidative stress contribute to the pathogenesis of asthma. We have shown for the first time that γtocotrienol is more potent than α-tocopherol in experimental asthma.
3.2 Introduction One of the classical hallmarks of asthma is airway inflammation. Inflammatory
degranulation and infiltration of eosinophils and neutrophils. The secretion of inflammatory cytokines such as IL-4, IL-5, and IL-13 by Th2 cells contribute to the inflammation of the airway. Airway inflammation can initiate oxidative stress in asthma which contributes to the recruitment of inflammatory cells into the airway and pathophysiology of asthma (MacNee, 2001; Nadeem et al., 2003). Among the inflammatory leukocytes, eosinophils and neutrophils are reported to produce the most amounts of reactive oxygen and nitrogen species. Clinically, antioxidants activities such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) from asthmatic patients were observed to be reduced (Ghosh et al., 2006; Hasselmark et al., 1990; Perisic et al., 2007; Powell et al., 1994). Levels of oxidants and oxidative damage biomarkers, namely 8isoprostane, 3-nitrotyrosine (3-NT) and 8-hydroxy-2-deoxyguanosine (8OHdG),
bronchoalveolar lavage fluid of patients (Louhelainen et al., 2008; Mak and Chan-Yeung, 2006; Montuschi and Barnes, 2002; Takao, 2005). The inability to defend against these oxidants results in oxidative damage on important biomolecules in the cells or lung tissue (Andreadis et al., 2003). Transcription factors like Nuclear Factor Kappa B (NF-κB) and nuclear factor erythroid-2-related factor 2 (Nrf2) are modulated to express proinflammatory
accumulation of all these factors initiate, worsen and sustain asthma (Barnes, 1996). The increased inflammation and levels of oxidants can attack the airway epithelial cells to hypersecrete mucus (Bast et al., 1991; Wood et al., 2003). Common allergens and triggers of asthma include house dust mites, pollens and cigarette smoke (Sporik et al., 1992).
Vitamin E is a lipid-soluble natural supplement with antioxidant property, and it exists in eight distinct isomers, consisting of α, β, γ and δ isoforms of both tocopherols and tocotrienols (Aggarwal et al., 2010). The isomeric forms of vitamin E vary by both the number and location of methyl groups found on the chromanol ring: α is 5,7,8-trimethyl; β is 5,8-dimethyl; γ is 7,8-dimethyl; and δ is 8-monomethyl. Sources of tocopherols include corn, wheat and soybeans, whereas tocotrienols occur mainly in barley, oats, palm oil, and rice bran (Sen et al., 2006). Amidst its multiple biological functions, vitamin E is primarily known for its antioxidant functions (Bell, 1987; Burton and Traber, 1990). Besides acting as an antioxidant on its own, the antioxidant activities of tocotrienols are also mediated through induction of antioxidant enzymes such as SOD, catalase and GPx, which quench free radicals such as superoxide anion and hydrogen peroxide (Aggarwal et al., 2010; Burton et al., 1983). Apart from its anti-oxidative roles, tocotrienols had demonstrated a variety of anti-inflammatory properties. Tocotrienols had also been found to abrogate the level and activity of cytokines (TNF-α, IL-1β, IL-6 and IL-8), adhesion molecules
(VCAM-1), signaling molecules (STAT-3 and NF-κB) and enzymes (cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)) (Aggarwal et al., 2010; Bachawal et al., 2010; Kuhad and Chopra, 2009a; Matsunaga et al., 2012; Nakagawa et al., 2010). It also demonstrated anticancer, cardioprotective, and cholesterol-lowering activities (Aggarwal et al., 2010; Colombo, 2010; Wong et al., 2012). Although there are relatively few studies on the anti-inflammatory properties of tocotrienol, the results obtained were consistent and promising that tocotrienols can attenuate inflammation via the inhibition of NF-κB signaling pathway.
The efficacies of each vitamin E isoforms in ameliorating house dust mine (HDM)-induced murine model of allergic airway inflammation were examined in this study, where γ-tocotrienol was selected due to its potency and abundance among the isomers.
Mouse models and statistical analysis
As two different animal models were used in this study, for more details, it can be referred to sections 2.2.3 and 2.2.4, respectively. The statistical analyses for both models were performed with ANOVA, as mentioned in section 2.19. *p