Abstract
In the present study, the biochemical effects of vitamin C, and aqueous extract of green tea (GTE) 
INTRODUCTION
Rheumatoid arthritis (RA) is a polyarticular disease affecting about 1 % of the population of the world 1 . It is an autoimmune disease characterized by chronic inflammation, progressive joint destruction, physical impairment, work disability and early mortality 2 . The process of disease progression is characterized by hyperplasia of synoviocytes, mainly of synovial fibroblasts, resulting in bone and joint destruction 3,4 . However, the proliferation of synovial cells is not limitless and spontaneous suppression of synovial proliferation has been observed 5 . Immunization of mice with collagen II (CII) leads to the development of arthritis "the collagen-induced arthritis model for RA". CII-specific activation of both T and B cells is critical for the development of arthritis and the transfer of both rodent and human serum with CII-specific antibodies induces arthritis in mice 6,7
INTRODUCTION
. Inflammation is known to result in increased production of nitric oxide (NO) and prostaglandins 8 . NO is an important mediator of diverse physiologic and pathologic processes, including arthritis 9 . Joint inflammation in autoimmune adjuvant-induced arthritis is dependent on the enhanced production of NO.
INTRODUCTION
McCartney-Francis et al. 10 (1999) reported that NO is ideally suited as a potent inflammatory mediator because of its strong reactivity with oxygen, superoxide, and ironcontaining compounds.
INTRODUCTION
Several lines of evidence suggest that oxidative stress has a role in the pathology of RA. This oxidative stress, associated with the generation of free radicals, is a major contributor to joint damage in RA
11
. The insufficiency of antioxidant defense systems and the acceleration of the oxidative reactions can result from the pro-oxidant/antioxidant imbalance in RA 12 . It was demonstrated that the level of free radical-induced damage to proteins in the synovial fluid was twice as high in RA 13 . Moreover, it was found that individuals with initially low levels of protecting antioxidants in their plasma, such as vitamins C and selenium, are at greater risk of developing RA
14
. The two most often suggested mechanisms for the increased incidence and activity of free radicals in RA joints are: (i) the production of various free radicals, such as 
Aim of work:
The present study was aimed to investigate the effect of antioxidants as vitamin C and GTE on rat model of RA. So, the plasma levels of lipid peroxides, NO, glutathione (GSH), CP, Cu and Zn were determined.
MATERIAL & METHODS

Chemicals:
L-Ascorbic acid, thiobarbituric acid, reduced glutathione, naphthylenediamine dihydrochloride, sulphanilamide, sodium nitrite, sodium azide, 5,5-dithio bis (2-nitrobenzoic acid), epinephrine and pphenylene diamine dihydrochloride, complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were fine grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade.
Animal treatment
Forty healthy male albino rats (Rattus norvegicus) their body weight 150-170 gm were included in the present study. All animals were conditioned at room temperature at natural photoperiod for 1 week before the start of the experiment. A commercial balanced diet and tap water were provided. The duration of experiment was 45 days. They were randomly divided into 4 groups (10 rats each) as the following:- (1998) 36 , by soaking 15 g. of instant green tea powder in 1 L of boiling distilled water for 5 minutes. The solution was filtered to make 1.5% GTE. This solution was provided to rats as their sole source of drinking water.
Animal treatment
All the tested antioxidants (vitamin C and GTE) were administrated daily for 45 days (experiment duration). The animals of different groups were sacrificed under light anesthesia 1 day after the end of the treatment. The blood samples from all groups were collected from the orbital vein in heparinized tubes and were centrifuged at 5000 rpm for 10 min for plasma separation. Plasma samples were divided into aliquots and kept at -40 o C until biochemical analysis.
Biochemical analysis
The plasma levels of lipid peroxides (LPO) were measured as thiobarbituric acid reactivity (TBARS). The product of the reaction between malondialdehyde and thiobarbituric acid was measured as described elsewhere
37
. The plasma levels of nitric oxide (NO) was determined as total nitrite after deproteinization with ZnSO 4 (30%), nitrate reduction with cadmium (activated by 2 % HCl) and color developed by the reaction with Griess reagent (1% sulfanilamide/ 0.1% naphthylethylene diamine diHCl, w/v in 2.5% H 3 PO 4 ) was recorded at 550nm against reagent blank using sodium nitrite as standard
38
. The plasma GSH levels were determined chemically as described by Ellman (1959)
39
. The plasma CP activity was determined using a paraphenylenediamine dihydrochloride method
40
. The plasma levels of zinc and copper were determined by employing flame atomic absorption spectrometry. The specific cathode lamps were used. Three determinations were made for each solution. The accuracy and precision of the analytical methods were tested with standard reference materials.
Statistical analysis
The results were expressed as mean ± standard error (SE). Differences between groups were assessed by one-way analysis of variance (ANOVA) using the Prism version 4 software packages for Windows. The level of significance was accepted with P <0.05. Table ( 1) shows the measured bioindices in different treated rat groups compared with control group. Fig. (1) shows the plasma levels of: (A) Copper, (B) Ceruloplasmin in different rat groups. Rat group with rheumatoid arthritis: In the C II group, the levels of LPO, NO, CP and Cu were significantly higher than controls. Contrarily, the levels of, GSH and Zn were significantly lower than controls. Vitamins C effect: In the C II + Vit. C group, the levels of LPO, NO, CP and Cu were significantly increased but the levels of Zn were significantly decreased in comparison with controls. GSH levels showed non significant increase when compared with that of controls.
RESULTS
In comparison with C II -treated group, the levels of GSH and Zn were significantly increased but the levels of NO, Cu and CP were significantly decreased in the C II + Vit.C group. Also, the levels of LPO were insignificantly decreased in vitamin C treated groups in comparison with the C II-treated group. GTE effect: In the C II + GTE group, the levels of LPO, NO, and Cu were significantly increased but the levels of GSH, CP and Zn did not show significantly changes in comparison with controls.
RESULTS
In comparison with C II -treated group, the levels of, LPO, NO, CP and Cu were significantly decreased but the levels of GSH, and Zn were significantly increased. Values are means ± SE for 10 rats (N= 10 for each group). The level of significance was accepted with P <0.05. 
Bull

DISCUSSION
Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints and tissue around the joints with infiltration of macrophages and activated T cells
41
. The pathogenesis of that disease is linked predominantly with the formation of free radicals at the site of inflammation. Previously, Heliovaara et al.,
41
14 reported that a low antioxidant level is a risk factor for RA.
41
The present study was performed to evaluate the effect of antioxidants on RA rats and to assess oxidative stress markers in the blood. Recent investigations have consistently indicated that the inflammatory process produces oxygen radicals and decreased antioxidant levels, which may worsen the symptoms of the RA (Darlington and Stone
42
). Lipid peroxidation has been implicated in the pathogenesis of degenerative diseases and inflammatory arthritis
43
. During lipid peroxidation, polyunsaturated fatty acids are oxidized to produce lipid peroxyl radicals that in turn lead to further oxidation of polyunsaturated fatty acid in a perpetuating chain reaction that can lead to cell membrane damage. Matrix degradation arising from cytokinestimulated chondrocytes was shown to be primarily due to lipid peroxidation and to be preventable by vitamin E, the primary antioxidant for lipids 44 . Oxidative injury and inflammatory status in various rheumatic diseases was reported 45 . In the current work, the levels of LPO, and NO in RA rat group were significantly higher than controls but NO levels were significantly reduced in all antioxidants (Vit C -, and GTE -) -treated groups. Moreover, levels of LPO were significantly reduced in GTE-treated groups. Similarly, the levels of plasma LPO were found to be significantly higher in RA than controls in many previous studies noticed that the green tea inhibits production of NO in human chondrocytes. Furthermore, green tea is shown to reduce inflammation in arthritis and is rich in antioxidants which may be useful in the prevention of onset and severity of RA (Tariq et al., 51 and Adcoks et al.,
54
). Moreover, the reduction of NO and PGE 2 by GTE in RA were also shown in some studies
53, 55, 56
.
Haqqi et al.,
57 found that the potential disease-modifying effect of green tea on arthritis came to light when it was shown that collagen type II-induced RA (C II RA) in mice, was ameliorated by prophylactic administration of green tea polyphenols in drinking water. The reduced C II RA incidence and severity was reflected in a marked inhibition of the inflammatory mediators COX2, interferon-gama and tumor necrosis factor -alpha (TNF-α) in arthritic joints of green tea-fed mice. Since increasing PGE 2 can cause inflammatory reactions, such as local congestion, edema, and pain in rheumatoid arthritis, the significant decrease of PGE 2 in rheumatoid rats may be due to inhibition of COX2 activity following the administration of GTE
58, 59
. An increase in the in vivo generation of oxidants and lipid peroxidation products in the plasma of RA was found to be negatively correlated with the antioxidant levels
60
. Ceruloplasmin (CP) is considered the principal plasma and synovial antioxidant in RA, being responsible for up to 70% of the protective capacity against superoxide free radicals
61
, which have been shown to be directly related to the pathogenesis of the inflamed joint in RA and the related increases in lipid peroxidation, ascorbate depletion and hyaluronate degradation
62
. 
63
. This activity as ferroxidase is increased during inflammation, infections, and other conditions, and these observations seem to suggest that there is a possibility that CP acts both as an antioxidant and an acute-phase reactant 63 . In the present study, the levels of CP and Cu were significantly higher in C II group than control group. The levels of CP and Cu were significantly reduced in all groups treated with antioxidants Vit C-and GTE-groups.
63
Similarly; many investigators found that the plasma levels of CP were significantly higher in RA than in controls 46, 47 . Moreover, Amancio et al. 64 found the significant increase of plasma Cu in RA. However, the increase in the antioxidant capacity produced by CP seems unable to cope with the RAinduced oxidative stress, and thus the induced lipid peroxidation is not fully prevented (as indicated by the increase in LPO values). The finding of raised Cu levels in the plasma is to be expected because of a concomitant rise of CP, which is an acute phase reactant 65 . Increased levels of CP observed in the present study may be related to its scavenging action of superoxide radicals that are generated during the inflammatory process of RA.
63
Acute or chronic inflammatory processes cause an accumulation of copper in many organs, particularly in the inflamed areas
66
. Additionally, a number of biologically active extracellular polypeptides, including cytokines and angiogenic factors, which participate in the pathogenesis and development of inflammatory processes, are known to be involved in trace metal metabolism. Copper plays an important role in development and maintenance of the immune system 66 . Zoli et al.
65
revealed that IL-1β and TNF-α levels significantly correlate with serum copper concentrations. In the recent study, in vivo, copper chelation with tetrathiomolybdate strongly repressed acute inflammation and onset of RA model through inhibition of mononuclear cell infiltration, and pannus formation 67 . Also, Brewer
68
reported that anticopper therapy such as penicillamine has efficacy in RA.
68
Zinc is a metal antioxidant and it is required in over 200 enzymes and so deficiency is likely to affect a number of different systems. Zinc, like copper, is a component of CuZnsuperoxide dismutase, an important antioxidant enzyme.
68
Zinc has a stabilising effect on membranes possibly by displacing bound transition metal ions and thereby preventing peroxidation of membrane lipids 69 . In the present study, the levels of Zn and GSH were significantly lower in C II group than control group. Moreover, the levels of Zn and GSH were significantly elevated in all groups treated with antioxidants Vitamin C-and GTE-groups in comparison with C II groups. Previously, Tuncer et al., 70 found plasma zinc levels were decreased significantly in RA. The authors suggested that low plasma Zn level in RA is one of the nonspecific features of inflammation. It has been postulated that low serum zinc may be caused by elevated IL-1 associated with RA
19
. With acute inflammation, the acute phase response may move Zn into the liver and the reduced plasma concentration may not be indicative of overall deficiency 71 . It is unclear whether chronic cytokine release, as is seen in RA, causes a shift of Zn from one compartment to another or if there is a true Zn depletion.
19
GSH plays an important role in the protection of cells and tissue structures.
19
Its role includes detoxification of xenibiotics, free radicals, peroxides and regulation of immune function 72 . The authors reported that low levels of GSH are implicated in RA. In addition, it is found that Zn-deficient rats have lowered GSH concentrations
73
. This finding may explain the reduction of plasma level of GSH in RA in our study. Moreover, Miesel and Zuber
74
suggested participation of xanthin oxidase in the depletion of serum GSH in RA. Also, Hassan et al.
75
found that RA was associated with significant depletion in GSH levels.
75
In conclusion, the current study suggests proper antioxidant intake may reduce free radical generation and improve antioxidant status in RA. GTE and vitamin C may effectively normalize in different degrees the impaired oxidant/ antioxidant system and may be useful in delaying the complication of RA. These antioxidants display considerable potency in anti-inflammatory action. 

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