Chapter 7

EFFECT OF VITAMINS C AND E ON ANTI-INFLAMMATORY AND ANTIOXIDANT STATUS IN COLLAGEN INDUCED ARTHRITIS 7.1. INTRODUCTION Rheumatoid Arthritis is a well known inflammatory disease. In acute stage, there is usually a sterile joint effusion. Later the polymorphs diminish in the synovial fluid and lymphocytes become prominent. The joints get inflamed and become hot, swollen and tender. In chronic state, the synovium and the cartilage in the joints are damaged. Early clinical symptoms of the disease are fatigue, weakness and stiffness of joints with mild pain. As the disease progresses joints become swollen (oedematous) and deformities appear soon. Collagen induced arthritis (CIA) is an excellent model for Rheumatoid Arthritis as the causes, symptoms and biochemical events in both these pathologies are similar. Role of Oxygen Free Radicals in the inflammatory process is well established. Free radicals are highly reactive molecules capable of damaging cellular components. They contribute to the pathogenesis of a number of diseases including Rheumatoid Arthritis (RA) in which the synovium is destroyed. This leads to the inflammation at joints and subsequent damage to articular surfaces and bases. Free Oxygen Radicals which are the byproducts of normal activity of cells have been found in excess in rheumatoid arthritic joints

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and are believed to contribute to the joint damage. Thus, the normal antioxidant defensive mechanism is disrupted in RA. Increased oxidative stress and decreased levels of antioxidants have been reported in active RA [247]. The singlet molecular oxygen is involved in the breakdown of collagen which initiates inflammatory process. Some vitamins and minerals act as antioxidants and help protect cells against damage from free radicals. Ascorbic acid (Vitamin C) and α - tocopherol (Vitamin E) were found to reduce the oxidative stress by scavenging the Superoxide and other oxidant radicals which in turn could reduce the lipid peroxides in blood and other tissues. Their anti-inflammatory effect is mediated through the antioxidant activity. Inadequate nutrient intake of vitamins has been correlated to decreased levels of plasma antioxidants and decreased activity of scavenging enzymes in inflammatory diseases [258].

Also, it has been reported that the levels of

antioxidant markers in the blood of RA patients are lower than those in their controls. This was explained due to the excessive need for existing antioxidants during inflammatory conditions. Vitamin E has been reported to uncouple joint inflammation and destruction in transgenic mouse models of RA [270]. Natural antioxidants like β-carotene, ascorbic acid and α-tocopherol were shown to enhance immunosuppressant effect of certain anti inflammatory drugs [38]. A recent strategy of antioxidant therapy for the prevention and treatment of RA is fast progressing. The role of vitamins C and E as antioxidants in RA has been investigated by taking different tissues like blood/serum, liver, heart, kidney, testis and brain. The antioxidant effect of both these vitamins have been studied and discussed in detail.

7.2. MATERIALS AND METHODS As described elsewhere in Chapter 3.

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Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

7.3. RESULTS Fourteen days after the booster injection of type II collagen, the rats developed oedema in the paw. Inflamed paws were reddish in appearance. The thickness of the paw was measured using Vernier calipers and the readings were tabulated.

7.3.1. Effect on Paw Oedema The thickness of paw measured for control, arthritic and treated rats at intervals of 7 days are given in Table 7.1. The percentage variation from control value is given in parenthesis and graphically represented in Figure 7.1. Table 7.1. Efficacy of Vit. C and Vit. E in reducing Paw Oedema in Collagen Induced Arthritic rats Thickness of paw oedema (in mm) Group

Control

Arthritis

Vit. C

Vit. E

st

th

th

nd

th

1 day

8 day

15 day

22 day

30 day

3.090 a ± 0.035 (100) 4.506 b ± 0.055 (145.83) 4.510 b ± 0.034 (145.95) 4.500 b ± 0.042 (145.63)

3.128 p ± 0.025 (101.23) 4.678 q ± 0.046 (149.55) 4.670 q ± 0.042 (149.3) 4.610 q ± 0.042 (147.38)

3.176 a ± 0.024 (101.53) 4.838 c ± 0.051 (152.33) 4.492 b ± 0.034 (141.44) 4.522 b ± 0.055 (142.38)

3.256 p ± 0.023 (102.52) 4.886 s ± 0.034 (150.06) 4.324 q r ± 0.056 (132.8) 4.362 q r ± 0.065 (133.97)

3.320 a ± 0.046 (101.97) 4.912 d ± 0.048 (147.95) 4.062 b ± 0.032 (122.35) 4.214 c ± 0.07 (126.93)

Levels of Significance Levene’s t Paired t (2-tailed)* (2-tailed) $ P < 0.01

------

P < 0.01

P < 0.001

P < 0.001

P < 0.001

P < 0.05

P < 0.001

Values are mean ± SEM of 5 animals in each group. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

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The thickness of paw in arthritic rats increased significantly when compared to that of normal animals. A further increase in paw thickness was noticed for arthritic as well as the vitamin treated rats on the 8th day. But on the 15th day, the vitamin treated groups recorded a decrease in paw thickness whereas the arthritic group still experienced an increase. On the 22nd and 30th days too, the paw oedema was reduced towards normal in both vitamins C and E treated rats. But the untreated arthritic rats showed a further increase in paw thickness. However, the normal rats recorded minimal increase in paw thickness towards 30th day which was not significant statistically. Fig. 7.1. Anti-inflammatory effect of vitamins C and E on Paw Oedema in CIA rats

The increase in thickness of paw towards the 30th day in both normal and arthritic rats was significant (P < 0.01). The reductions in paw oedema brought about by vitamin E was significant (P < 0.05). But, the oedema suppression

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by vitamin C was highly significant (P < 0.001). The arthritic group differed significantly (P < 0.001) from normal. Both the treatments reduced the oedema very significantly (P < 0.001).

7.3.2. Effect on Catalase activity The activity of catalase in various tissues of normal, arthritic and treated rats are furnished in Table 7.2. The percentage variation of activities from the normal value is given in parenthesis in the same table and represented graphically in Figure 7.2. Table 7.2. Effects of Vit. C and Vit. E on Catalase activity in tissues of CIA rats Activity of Catalase in tissues ( µmoles / min / mg protein )

GROUP

Control

RA

Vit. C

Vit. E

SERUM

LIVER

HEART

a

p

a

0.211 ± 0.002 (100) 0.049 c ± 0.005 (23.22) 0.174 b ± 0.005 (82.46) 0.196 a ± 0.004 (92.89)

0.179 ± 0.009 (100) 0.051 r ± 0.006 (28.49) 0.126 q ± 0.003 (70.39) 0.158 p ± 0.003 (88.27)

1.250 ± 0.042 (100) 0.797 b ± 0.053 (63.76) 0.810 b ± 0.057 (64.80) 0.828 b ± 0.035 (66.24)

KIDNEY p

2.834 ± 0.135 (100) 0.901 r ± 0.090 (31.79) 1.698 q ± 0.078 (59.92) 1.763 q ± 0.072 (62.21)

TESTIS

BRAIN

c

0.271 p ± 0.012 (100) 0.174 q ± 0.013 (64.21) 0.210 q ± 0.013 (77.49) 0.223 q ± 0.007 (82.29)

0.444 ± 0.023 (100) 0.796 a ± 0.034 (179.30) 0.548 b ± 0.022 (123.40) 0.435 * c ± 0.012 (97.97)

Levels of Significance Paired t $ (2-tailed) ---

P < 0.01

P < 0.05

P < 0.05

* Values below the limit. Values are mean ± SEM of 5 animals. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

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There was significant decrease in the activity of this enzyme in all the tissues of CIA rats except testis. Catalase activity in the testis was elevated significantly. Treatment with vitamin C significantly regulated the catalase activity in serum, liver, kidney and testis. Levels were not affected in heart and brain. Vitamin E could normalize the catalase activity in serum, liver and testis of CIA rats. Kidney experienced a regulatory effect of vitamin E. In heart and brain, the levels were not affected significantly by the treatment.

Percentage

Fig. 7.2. Variations in Catalase activity in tissues of CIA rats treated with vitamins C and E

Catalase activity was decreased significantly (P < 0.01) in CIA rats compared to normal. Supplementation with both vitamins C and E showed significant elevation in activity (P < 0.05).

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Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

7.3.3. Effect on Superoxide dismutase activity Superoxide dimutase (SOD) activity in different tissues of normal, arthritic and treated rats are presented in Table 7.3. The percentage variation in activity from normal value is given in parenthesis and depicted in Figure 7.3. Table 7.3. Effects of Vit. C and Vit. E on Superoxide dismutase (SOD) activity in tissues of CIA rats Activity of SOD in tissues (Unit / mg Hb for Blood or mg protein for other tissues)

GROUP

Control

RA

Vit. C

Vit. E

BLOOD

LIVER

HEART

KIDNEY

TESTIS

BRAIN

0.244 a ± 0.010 (100)

0.685 p ± 0.092 (100)

3.654 a ± 0.457 (100)

0.890 p ± 0.145 (100)

1.047 a ± 0.055 (100)

1.087 p ± 0.079 (100)

0.027 c ± 0.005 (11.07)

0.045 r ± 0.010 (6.57)

0.789 c ± 0.071 (21.59)

0.237 q ± 0.064 (26.63)

0.625 b ± 0.078 (59.69)

0.332 r ± 0.071 (30.54)

0.084 b ± 0.008 (34.43)

0.336 q ± 0.047 (49.05)

1.449 b ± 0.068 (39.66)

0.655 p ± 0.023 (73.60)

0.734 b ± 0.040 (70.11)

0.472 r ± 0.026 (43.42)

0.083 b ± 0.014 (34.02)

0.561 p ± 0.019 (81.90)

1.796 b ± 0.090 (49.15)

0.554 q ± 0.042 (62.25)

1.028 a ± 0.021 (98.19)

0.789 q ± 0.046 (72.59)

Levels of Significance Paired t $ (2-tailed) ---

P < 0.001

P < 0.001

P < 0.001

Values are mean ± SEM of 5 animals. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

The levels of SOD activity were reduced significantly in all the tissues of CIA rats. Vitamin C treated CIA rats showed significant increase in SOD activity in their blood, liver and heart. SOD activity was raised in kidney which

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did not differ significantly from normal level. No significant effect was shown by this vitamin on SOD activity in testis and brain. Treatment of CIA rats with vitamin E also produced varying effects on SOD activity in tissues. Levels were increased significantly in blood, heart and brain. Activity was normalized in liver and testis. No significant effect was caused by vitamin E on kidney SOD activity. Fig. 7.3. Variations in Superoxide dismutase activity in tissues of CIA rats treated with vitamins C and E

The reduction in SOD activity in arthritic group was highly significant (P < 0.001). Treatments when compared to arthritic group revealed that both the vitamins C and E enhanced the SOD activity very significantly (P < 0.001).

7.3.4. Effect on Glutathione Level The GSH content of various tissues in normal, arthritic and treated rats are recorded in Table 7.4. The percentage variation in concentration from normal value is presented in the table in parenthesis with a depiction in Figure 7.4.

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Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

Table 7.4. Effects of Vit. C and Vit. E on reduced Glutathione (GSH) levels in tissues of CIA rats Levels of GSH in tissues ( µg / mg protein )

GROUP

Control

SERUM

LIVER

HEART

KIDNEY

TESTIS

BRAIN

6.828 a

31.820 p

7.430 a

6.268 p

43.647 a

7.926 p

±

±

±

±

±

±

0.264

2.298

0.219

0.299

1.092

0.779

(100)

(100)

3.615 RA

Vit. C

Vit. E

c

13.397

(100) r

1.328

(100) d

2.473

28.324

---

(100)

(100) r

Levels of Significance Paired t $ (2-tailed)

b

4.117 q

±

±

±

±

±

±

0.527

1.296

0.132

0.293

2.605

0.228

(52.94)

(42.10)

(17.87)

(39.45)

(64.89)

(51.94)

6.387 a

22.664 q

5.060 b

6.098 p

29.287 b

5.246 q

±

±

±

±

±

±

0.149

0.916

0.092

0.096

0.881

0.081

(93.54)

(71.23)

(68.10)

(97.29)

(67.10)

(66.19)

6.085 a

25.198 p q

2.193 c

4.947 q

37.074 a

4.565 q

±

±

±

±

±

±

0.122

0.704

0.113

0.111

1.391

0.084

(89.12)

(79.19)

(29.52)

(78.92)

(84.94)

(57.60)

P < 0.001

P < 0.001

P < 0.001

Values are mean ± SEM of 5 animals. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

GSH level was reduced significantly in all the tissues of arthritic rats. Vitamin C treatment increased the GSH content to normal level in serum and kidney of arthritic rats. GSH content was regulated significantly towards normal in liver and heart. Treated testis and brain did not record any significant elevation in GSH levels compared to arthritic. Vitamin E had significant effect on GSH levels of serum and testis. The level was brought to normal in these two tissues. The GSH content was raised significantly towards normal in treated liver, heart and kidney. Brain did not respond to vitamin E with regard to GSH content.

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Fig. 7.4. Variations in Glutathione level in tissues of CIA rats treated with vitamins C and E

The decline in the levels of GSH in arthritic group was highly significant (P < 0.001). The increases in GSH level brought by vitamin C and vitamin E were also highly significant (P < 0.001).

7.3.5. Effect on Conjugated Dienes Level The levels of Conjugated Dienes in different tissues of normal, arthritic and treated rats are given in Table 7.5. The percentage variation in level from normal value is given in presented in the same table in parenthesis with a graphical representation in Figure 7.5. The concentration of Conjugated Dienes was found to be increased significantly in all the tissues of CIA rats. Treatment with vitamin C decreased the levels significantly towards normal. Levels were normalized in blood, liver, kidney and brain. Conjugated Diene concentration was reduced significantly towards normal in treated heart and testis. Vitamin E also brought the levels of Conjugated Dienes to normal in all the tissues of treated rats except heart. In heart, the level was regulated significantly towards normal.

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Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

Table 7.5. Effects of Vit. C and Vit. E on the level of Conjugated Dienes (CD) in tissues of CIA rats Levels of Conjugated Dienes in tissues (µmoles / ml blood or µmoles / 100 mg wet weight tissue))

GROUP

Control

BLOOD

LIVER

HEART

KIDNEY

TESTIS

BRAIN

1.719 a

5.740 p

2.698 a

6.042 p

3.294 a

7.135 p

±

±

±

±

±

±

0.120

0.214

0.279

0.478

0.379

0.225

(100)

(100)

(100) b

2.363 RA

Vit. E

6.926

(100) c

8.920

(100) q

5.842

8.839 q

±

±

±

±

±

0.099

0.278

0.480

0.234

0.281

0.300

(137.46)

(142.77)

(256.82)

(177.35)

(123.88)

a

5.996

p

4.683

b

(147.63) 6.884

p

4.558

---

(100) c

±

1.951 Vit. C

8.195

q

Levels of Significance Paired t $ (2-tailed)

b

P < 0.001

7.837 p

±

±

±

±

±

±

0.023

0.267

0.135

0.110

0.073

0.137

(113.50)

(104.46)

(173.57)

(113.94)

(138.37)

(109.84)

1.820 a

6.207 p

4.934 b

6.178 p

3.894 a

7.261 p

±

±

±

±

±

±

0.022

0.174

0.147

0.110

0.073

0.111

(105.88)

(108.14)

(182.88)

(102.25)

(118.21)

(101.77)

P < 0.001

P < 0.001

Values are mean ± SEM of 5 animals. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

Arthritic group showed highly significant elevation (P < 0.001) in the level of Conjugated Dienes. The reductions in the level by both the vitamins C and E were also highly significant ( P < 0.001).

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Fig. 7.5. Variations in the level of Conjugated Dienes in tissues of CIA rats treated with vitamins C and E

7.3.6. Effect on Malondialdehyde Level The concentration of MDA in various tissues of normal, arthritic and treated rats are given in Table 7.6. The percentage variation in the level from normal value is presented in parenthesis followed by a graphical representation in Figure 7.6. The level of MDA was significantly increased in all the tissues of CIA rats. Vitamin C treatment was found to normalize the MDA levels in blood, liver, heart and testis. The level was regulated towards normal in kidney and brain. Vitamin E also expressed an effect similar to that of vitamin C. The levels were normalized in blood, liver, heart and testis and and reduced towards normal in kidney and brain.

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Table 7.6. Effects of Vit. C and Vit. E on the level of Malondialdehyde (MDA) in tissues of CIA rats Levels of MDA in tissues (µmoles / ml blood or µmoles / 100 mg wet weight tissue)

GROUP

Control

BLOOD

LIVER

HEART

KIDNEY

TESTIS

BRAIN

0.121 a

0.381 p

0.894 a

0.507 p

0.391 a

3.407 p

±

±

±

±

±

±

0.006

0.023

0.052

0.014

0.024

0.075

(100)

(100)

(100) b

0.168 RA

Vit. E

1.195

(100) b

1.461

(100) r

0.593

5.399 r

±

±

±

±

±

0.005

0.031

0.025

0.081

0.012

0.190

(138.94)

(183.02)

(133.72)

a

0.428

p

0.987

a

(288.01) 0.981

q

(151.87) 0.433

---

(100) b

±

0.132 Vit. C

0.696

q

Levels of Significance Paired t $ (2-tailed)

a

P < 0.001

(158.47) 4.153 q

±

±

±

±

±

±

0.004

0.009

0.024

0.014

0.012

0.127

(109.00)

(112.51)

(110.41)

(193.34)

(110.89)

(121.90)

0.133 a

0.426 p

0.917 a

0.989 q

0.392 a

4.380 q

±

±

±

±

±

±

0.004

0.010

0.006

0.016

0.011

0.106

(109.55)

(111.84)

(102.58)

(194.87)

(100.35)

(128.56)

P < 0.001

P < 0.001

Values are mean ± SEM of 5 animals. Percentage variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P < 0.01. $ Significant difference between Control and RA as well as RA and each Treatment.

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Fig. 7.6. Variations in the level of Malondialdehyde in tissues of CIA rats treated with vitamins C and E

Increase in the MDA level in arthritic group was highly significant (P < 0.001). Reductions brought by vitamin C and vitamin E treatments were also highly significant (P < 0.001).

7.4. DISCUSSION Reactive Oxygen Intermediates (ROI) together with Prostaglandins, leukotrenes and proteases are believed to be involved in the pathogenesis of inflammatory diseases like RA. Treatment of RA and alleviation of the painful symptoms associated with the disease involves use of drugs that show antioxidant activity. This part of the study was confined to the estimation of the antioxidant status of blood and other tissues in Collagen Induced Arthritis (CIA) by measuring the activity of catalase, superoxide dismutase and the levels of GSH, CD and MDA, the latter two being the indicators of lipid peroxidation. The modulatory effects of the vitamins C and E on these

Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

209

parameters were examined. The anti-inflammtory effect of these agents were investigated by assessing the reduction in paw oedema. The paw thickness was increased 145.83% of the normal in CIA rats at the beginning of the treatment. This was raised to 147.95% on 30 th day in untreated rats. Vitamin C treated CIA rats recorded 122.35% and vitamin E treated animals showed 126.93% of normal thickness at the end of the experiment. It was evident that both the vitamins produced marked change in the inflammatory status of CIA rat. Vitamin C was a little more effective than vitamin E in reducing the paw oedema within 30 days. The anti-inflammatory role of vitamin E had been explained by many workers [267,269,270,277]. Clinical symptoms of inflammation was reported to improve by vitamin E supplementation [272]. The mechanism of action of vitamin E was explained to be due to the reduction in the formation of major molecules during inflammation [273]. A much more effective role played by vitamin C in alleviating the symptoms of RA is to be investigated in detail. The activity of catalase and SOD were found to be decreased significantly in the serum as well as other tissues of CIA rats. Level of the metabolic antioxidant, GSH (reduced glutathione) was also found to be reduced significantly in all the tissues of CIA rats. The concentrations of lipid peroxidation products CD and MDA were elevated significantly in all the tissues analyzed. This observation shows that the reactive oxygen scavenging system is impaired in arthritic condition. A defective antioxidant status had been demonstrated in patients with RA by Karatas et al. [244]. They described lowered antioxidant levels and elevated MDA level in RA patients. Various workers have reported that the activities of catalase, SOD and GPx in blood are lowered during RA [244, 245]. Findings in

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this study are also in agreement with these reports. Humad et al. [88] had reported an increased lipid peroxidation in RA patients and had suggested that the pentane level of exhaled air of patients can be accepted as a marker of the disease. Pushendran et al. [338] had reported that lipid peroxidation as assessed by TBARS and CDs was maximum in brain followed by other organs like liver, kidney and heart. The higher susceptibility of brain and liver was explained by substrate availability and to a lesser extent, the level of antioxidant. This reflected the degenerative diseases of these organs. A similar observation in this study also hints the degenerative changes of organs in RA. Increased levels of CD and MDA in various tissues of CIA rats explain a similar mechanism of impaired lipid peroxidation. It is reiterated that Free Radicals/Reactive Oxygen Species are involved in the etiology of arthritis. The decreased levels of catalase in CIA rats were elevated significantly in all the tissues treated with vitamins C and E except for heart and brain. The levels were regulated towards normal by vitamin C in serum, liver, kidney and testis. Catalase activity was restored to normal by vitamin E in serum, liver and testis. Vitamin E was thus found to be more effective than vitamin C with regard to catalase activity. SOD activity in testis and brain of CIA rats was not at all influenced by vitamin C. The activity was normalized in kidney and regulated in blood, liver and heart by this vitamin. Vitamin E on the other hand, did not affect SOD activity of arthritic kidney, but regulated the activity in blood, heart and brain. SOD activity was brought to normalcy by vitamin E in liver and testis and regulated towards normal in Kidney. Again, vitamin E has been proven to be more effective on CIA rat tissues in rectifying anomalies in SOD activity.

Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

211

The GSH contents of arthritic testis and brain were not influenced by vitamin C. It could normalize the GSH level in serum and kidney and regulate the same in liver and heart. Though vitamin E could not affect GSH level in brain of CIA rats, it elevated the levels to normalcy in serum, liver and testis. GSH content was regulated by vitamin E in heart and kidney. Vitamin E was thus found to be more effective in this case also. Lipid peroxidation was increased in CIA as evidenced by the higher concentration of CD and MDA in various tissues of inflammed rats. Treatment with vitamins C and E could reduce the levels either to normalcy or towards it. Both vitamins C and E normalized the CD levels in blood, liver, kidney and brain. Lipid peroxidation was regulated in heart tissue of CIA rats by both these vitamins. In testis, the CD level was regulated by vitamin C whereas it was normalized by vitamin E. Vitamin E thus seemed to be more effective than vitamin C in controlling CD levels in arthritic tissues. Level of MDA was highly influenced by vitamins C and E. Both these vitamins acted similarly in such a way that the MDA levels were brought down to normalcy in blood, liver, heart and testis. Lipid peroxidation was regulated by both vitamins C and E in heart and brain of CIA rats as evidenced by reduction in MDA levels. In brain the CD level was normalized by vitamin treatments, but the MDA level was only regulated towards normal. Vitamin C and vitamin E are almost equally effective in controlling the lipid peroxidation in CIA rats. Ramanathan and Balakumar [241] had observed increased levels of lipid peroxidation and decreased levels of non enzymatic antioxidants and activities of enzymatic antioxidants in arsenic induced oxidative stress. They also reported that Ascorbic acid (vitamin C) and α-tocopherol (vitamin E) treatment could

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reduce lipid peroxidation and enhance the levels of GSH and activities of SOD, catalase, glutathione peroxidase and glutathione reductase. Bae et al., 2003 [258] had also shown that the level of plasma antioxidants and the activity of antioxidant enzymes are decreased in RA patients and the MDA concentration slightly increased. They correlated the low levels of vitamin A and β-carotene and vitamin E in the plasma of RA patients to reduced activities of SOD and GPx and enhanced levels of MDA in them. Arajuo et al., 1998 [262] had reported that low level of vitamin E intake caused juvenile RA in children. Many workers have cautioned that low level of vitamin E in serum is a possible risk factor of RA [261, 269, 272, 273].

More recently, vitamin E has been reported to be a

potential antioxidant with anti-inflammatory role in experimentally induced arthritis [266, 267, 270]. Its use in antioxidant therapy for the prevention and treatment of RA has been proposed by many researchers [258, 259, 268, 270]. Jaswal et al. have recommended co-administration of Vitamin E with conventional drugs to RA patients to reduce the level of MDA [268]. In the present study, vitamin C and vitamin E have shown to reduce the paw oedema in arthritic rats. Vitamin C expressed a better effect. Both these vitamins could also raise the decreased levels of catalase, SOD and GSH towards normal in almost all the arthritic tissues analyzed. Vitamin E was found to be a little more effective than vitamin C in rectifying the anomalies that occurred in CIA rats. The enhanced lipid peroxidation in arthritic state was lowered by both these vitamins. CD levels were regulated more successfully by vitamin E compared to vitamin C. However, both these vitamins were almost equally effective in regulating or normalizing the MDA levels in various tissues studied. This observation supports the earlier findings that Ascorbic acid and αtocopherol are effective antioxidants against RA.

Effect of Vitamins C and E on Anti-inflammatory and Antioxidant status in Collagen Induced Arthritis

213

7.5. CONCLUSION Treatments with Vitamins C and E had profound effects on antioxidant system of arthritic rats. The pathological impact was either reduced or removed completely by these vitamins. The efficacy of the two vitamins depend on the tissue involved and the parameter considered. Vitamin E is the most effective antioxidant (non enzymatic and nutrient antioxidant) which could restore the normal enzymatic antioxidant defense mechanism in liver and testis as well as the normal lipid peroxidation in blood, liver, heart, kidney and testis of CIA rats. Vitamin C, on the other hand, expressed better anti-inflammatory effect than vitamin E as evidenced by a greater reduction in paw oedema. Their antiinflammatory effect is supposed to be mediated by antioxidative mechanism as revealed by the results of this work.