Smooth Muscle Relaxation by the Herbal Medicine Ssanghwatang associated with Nitric Oxide Synthase Activation and Nitric Oxide Production

2006. Vol. 27. No. 4. 74-83 Korean Journal of Oriental Medicine Original Article Smooth Muscle Relaxation by the Herbal Medicine Ssanghwatang associ...
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2006. Vol. 27. No. 4. 74-83 Korean Journal of Oriental Medicine

Original Article

Smooth Muscle Relaxation by the Herbal Medicine Ssanghwatang associated with Nitric Oxide Synthase Activation and Nitric Oxide Production Joong-Kil Kim, Ha-Na Shim, Seung-HeeLee Kwan-Suk Yoo, Bong-Keun Song1) Department of Oriental Internal Medicine, College of Oriental Medicine, Wonkwang University Department of Constitutional Medicine, College of Oriental Medicine, Wonkwang University1) Ssanghwatang (SHT) has been known to prove effective in the treatment for erectile dysfunction (ED), and its modified formula is widely used in clinical practice. However, its fundamental mechanism of action is not clearly known. It is well known that endothelial cells can achieve the relaxation of vascular smooth muscles by the release of nitric oxide (NO). NO is synthesized by the enzyme NO synthase (NOS) from L-arginine and oxygen. It is widely accepted that NO plays an important role in the relaxation of corpus cavernous smooth muscle and vasculature. In addition, in terms of the penile erection, the NO/cGMP pathway is more potent than the PGE1 /cAMP pathway. The main purpose of the present study was to investigate the mechanism of the erectile effects of SHT by focusing on its direct effects on corpus cavernous smooth muscle cells. We investigated the NOS activity, nitrite concentration and cGMP levels in rat corpus cavernous smooth muscle cell lines activated by SHT extracts. Furthermore, we evaluated the effect of SHT extracts on penile smooth muscle relaxation following oral administration of SHT extract powder to rats by the dosage of 1 g/kg over fifteen days. As a result, we found that SHT stimulated NO release. NOS activity and cGMP levels were increased by SHT respectively. Furthermore, SHT relaxed the corpus cavernous smooth muscle. These results are consistent with the concept that penile erection by SHT is carried out through the NO/cGMP pathway. In conclusion, the present study shows that SHT increases the NOS activity, synthesizes NO and augments the cGMP, which mediates penile erection. Further determination of the SHT mechanism related with the NO/cGMP pathway strongly indicates that SHT can be used as a remedy for erectile impotence. Key Words : Ssanghwatang, smooth muscle relaxation, nitric oxide, penile erection, cGMP

Introduction Ssanghwatang (SHT), a natural product and traditional herbal medicine, has been used for thousands of years as a therapeutic formula in Received 1 September 2006;received in revised form 14 September 2006;accepted 30 September 2006 Corresponding author:Bong-Keun Song, Department of Internal Medicine, Wonkwang University Kwangju Medical Center, 543-8 Juwol Dong, Nam Gu, Kwangju, 503-310, Korea. Tel:82-63-850-2102 / FAX:82-63-841-0033 E-mail:[email protected]

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Korea, China and Japan and is still widely so used. SHT has been known to be effective against erectile dysfunction (ED), and its modified formula is widely used in clinical practice. However, its fundamental mechanism of action is not clearly known. In the present study, we have investigated the mechanism of its erectile action by determining its direct action on penile corpus cavernous muscle and cultured cells. Penile erection is the end result of smooth muscle relaxation in the penis. It is well established

Smooth Muscle Relaxation by the Herbal Medicine Ssanghwatang associated with Nitric Oxide Synthase Activation and Nitric Oxide Production (827)

that the balance between contraction and relaxant factors controls the degree of tone of the penile vasculature and the smooth muscle of the corpus cavernosum, and determines the functional state of the penis: flaccidity or erection. ED is defined as the "inability to achieve or maintain an erection adequate for sexual satisfaction"1). ED may be due to inability of penile smooth muscle to relax. This inability can have multiple causes, including nerve damage, endothelial damage, alteration in receptor expression/function, or in the transduction pathways that are implicated in the relaxation and contraction of the smooth muscle. It is well known that endothelial cells can achieve relaxation of vascular smooth by release of nitric oxide (NO). NO is synthesized by the enzyme NO synthase (NOS) from L-arginine and oxygen. An important role for NO in the relaxation of corpus cavernous smooth muscle and vasculature is widely accepted2,3). Also, in terms of the penile erection, the NO/cGMP pathway is more potent 4) than the PGE1/cAMP pathway . The aim of the present study was to estimate four hypotheses: 1) NO is involved in the mechanism of action of SHT; 2) SHT increases NOS activity; 3) SHT increases the cGMP level; 4) SHT has a direct effect on the corpus cavernous smooth muscle cell to relax. As a result, we found that SHT stimulated NO release, and NOS activity and cGMP level were increased by SHT. Furthermore, SHT relaxed the corpus cavernous smooth muscle. These results are consistent with the concept that penile erection by SHT is carried out through the NO/cGMP pathway.

Materials and methods 1. Materials 1) Herbal composition SHT is a mixture of herbal drugs. The com-

Table 1. Composition of SHT

Scientific name Paeonia Radix Rehmanniae Radix Astragali Radix Angelicae gigantis Radix Cassiae Cortex Glycyrrhizae Radix Paeonia Radix Cnidii Rhizoma Zingiberis Rhizoma Zizyphi inermis Fructus Total

Weight (g) 10 4 4 4 3 3 3 4 4 4 43

position of the mixture is as follows (Table 1): Paeonia Radix (10g), Rehmanniae Radix (4g), Astragali Radix (4g), Angelicae gigantis Radix (4g), Cnidii Rhizoma (4g), Cassiae Cortex (3g), Glycyrrhizae Radix (3g), Zingiberis Rhizoma (4g), Zizyphi inermis Fructus (4g). All the herbal materials were purchased from Wonkwang University Kwangju Oriental Medical Center and identified by herbal experts. 2) Reagents Dulbecco's modified Eagle's medium (DMEM), sodium citrate, L-arginine, ethylene diamine tetra sodium salt (EDTA) and lipopolysaccharide (LPS) were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was supplied by the HyClone (Logan, UT). AntieNOS antibodies were from BD Biosciences (San Jose, CA). 3) Animals Eight-week-old male Sprague-Dawley white rats (250-300g) were provided from Damul Science (Daejeon, Korea) and housed under conditions of constant temperature and controlled illumination for 2 weeks. Food and water were available ad libitum.

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(828) Korean J of Oriental Med 2006;27(4)

Fig 1. Effect of SHT on NOS. Cells were stimulated with LPS (100µg/ml) in the absence or presence of water extracts of SHT at 100mg/ml. After 24 hr incubation, NOS was measured. Data are mean ± SE. ++: significant difference between control and LPS group. *: significant difference between LPS and SHT group.

2. Methods 1) Preparation of extract and administration For water extraction, SHT (400 g) was mixed with 3,000 mL of distilled water and extracted under reflux for 3 hr by boiling the formula. The extracts were filtered with a Whitman paper filter. The filtrate was concentrated with a rotary evaporator at 50℃ under vacuum and freezedried to dryness. The total amount of extract powder was 91.38 g. The extract powder was dissolve in 0.9% saline (2 mL) and administered orally to rats by the dosage of 1 g/kg for fifteen days.

2) Culture of rat corpus cavernous smooth 5,6) muscle cells Rat corpus cavernous smooth muscle (CCSM) cells were obtained as sterile surgical specimens, the tissue was washed and cut into 1 to 2 mm pieces and placed into culture dishes with DMEM containing 20% FBS, penicillin (100 ㎍ /mL), streptomycin (100 ㎍/mL) and 2 mM glutamine. After explants attached to the culture dish, usually 1 to 2 days, DMEM supplement with 10% FBS, penicillin, streptomycin, and glutamine were added. Smooth muscle cells

Fig 2. Effect of water extracts of SHT on NO production by LPS primed rat penile smooth muscle cells. Cells were activated with LPS (10ng/ml) in the absence or presence of SHT at indicated doses. After 48hr incubation, nitrite concentration was measured by Griess method. Each value represents the mean ± SE. * p

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