الفهرس 
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Abstract
Rheumatoid arthritis (RA) is a progressive inflammatory autoimmune disease with an estimated global incidence of 1%. The exact cause is unknown, but genetic and environmental factors are contributory. RA is characterized by synovial inflammation and hyperplasia, autoantibody production, cartilage and bone destruction and systemic complications leading to substantial physical, psychological and economical effects. In addition, long term patient prognosis is poor. No treatment cures RA; therefore, the therapeutic goals are reduction of pain, protection of articular structures, prevention of loss of joint function, control of systemic complications and improvement of the quality of life. Although non steroidal anti-inflammatory drugs (NSAIDs) provide rapid anti-inflammatory and analgesic effects, they do not prevent or slow joint destruction. Therefore, disease modifying antirheumatic drugs (DMARDs) therapy should be initiated for all patients diagnosed with RA. Traditional DMARDs have demonstrated the ability to slow disease progression and prevent further destruction of the joints and involved tissues. However, they have the potential to cause serious toxicities and their onset of action is slow. The biologic response modifiers (BRMs), the newest class of DMARDs, target the proinflammatory and joint-damaging effects of mediators including tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), T-cells and B-cells but they are expensive, associated with serious side effects and have less data detailing their long-term effects.
Leflunomide is considered one of traditional DMARDs. Its main action is inhibiting denovo synthesis of pyrimidine leading to inhibition of T-cell proliferation. In addition, other mechanisms of leflunomide have been reported in the treatment of RA including anti-inflammatory effect and reduction of matrix metalloproteinase production. Quercetin is categorized as a flavonol, one of the six subclasses of flavonoid compounds. It is found in a variety of fruits and vegetables, such as onions, apples and tea. Reports show that many health beneficial effects, including hepatoprotection, anticancer effect, antioxidant and anti-inflammatory activity are associated with quercetin.
In view of such considerations, the design of the current work was set to evaluate and compare the efficacy of leflunomide, alone and in combination with quercetin, on oxidative stress, some inflammatory and arthritic markers, as well as hepatic indicators in adjuvant-induced arthritic (AIA) rat model, in an attempt to achieve better therapeutic management of RA accompanied with less hepatotoxicity.
In the present study, 40 adult male Sprague-Dawley rats were used. Rats were divided into 5 groups (n=8). Eight rats served as normal controls. AIA was induced in rats with intradermal injection of 0.1 ml suspension of heat-killed Mycobacterium butyricum, (12 mg/ml) in incomplete freund’s adjuvant at the base of the tail. chronic inflammation was allowed to progress in all animals for 15 days. Rats were then divided into 4 groups:
Group 1: Untreated AIA rats receiving the vehicle 1% sodium carboxy methyl cellulose (CMC-Na) daily via an oral gavage syringe.
Group 2: AIA rats treated with leflunomide (10 mg / kg), 5 times a week via an oral gavage syringe.
Group 3: AIA rats treated with quercetin (200mg/kg), every other day via an oral gavage syringe.
Group 4: AIA rats treated with a combination of leflunomide and quercetin as in groups 2 &3.
Both leflunomide and quercetin were suspended in 1% CMC-Na. Tested drugs were administered for 19 days, from day 15 till the end of the study. The hind paw diameter was measured every other day from day 0 till the end of the experiment to assess the swelling of the joint. Arthrogram score was also assessed to determine the severity of arthritis on days 15 (first day of therapy), 24 and 34 (end of the experiment). At the end of the experiment and after an overnight fast, rats of all groups were sacrificed by cervical dislocation. Blood samples were collected from posterior vena cava and serum was separated, kept under -80 °C and used for estimation of the following parameters:
1- Malondialdehyde (MDA).
2- Total antioxidant capacity (TAC).
3- Tumor necrosis factor alpha (TNF-α).
4- Cycloxygenase-2 (COX-2).
5- Matrix metalloproteinase-1 (MMP-1).
6- Alanine aminotransferase (ALT).
7- Aspartate aminotransferase (AST).
Livers and hind paws of all groups were excised and preserved in 10% formalin for histopathological examination by routine H&E stain.
Fifteen days after the induction of arthritis in rats, the untreated AIA showed an increase in the hind paw diameter and arthrogram score in comparison to normal controls, while the treatments given to AIA rats succeeded to decrease the hind paw diameter and arthrogram score as compared to the untreated AIA rats. Leflunomide treatment expressed more significant decrease in the swelling of the hind paw than quercetin treatment. Paradoxically, the combination of leflunomide and quercetin illustrated no significant reduction in the hind paw diameter and arthrogram score when compared to leflunomide treatment alone.
As regards histopathological examination of the ankle joint of rats, the microscopic picture was in line with the results of the hind paw diameter and arthrogram score. The joint of normal control rats revealed thin synovial layer covering the intact normal cartilage and bone while, the untreated AIA rat exhibited synovial proliferation with angiogenesis, invasion of cartilage and bone destruction. Quercetin treatment decreased the synovial proliferation and cartilage as well as bone erosion when compared to the untreated AIA; however its effect was the least in comparison to other treatment groups. Treatment with either leflunomide alone or in combination with quercetin presented with a nearly similar degree in the reduction of synovial proliferation. However, the cartilage and bone in the combination treated group were near normal.
The TNF-α, COX-2 and MMP-1 play an important role in inflammation development and cartilage and bone destruction. In untreated AIA rats, a significant rise in these parameters was observed in comparison to normal controls. There was a reduction in the level of TNF-α, COX-2 and MMP-1 in the monotherapy-treated groups. The highest significant decrease in the levels of TNF-α, COX-2 and MMP-1 in the current study has occurred in the combination group, thus demonstrating a potent anti-arthritic effect.
Estimation of serum level of both malondialdehyde and total antioxidant capacity was used for the assessment of oxidative status which is considered one of the pathways leading to arthritis development and progression. The MDA and TAC levels have been significantly changed in comparison to normal controls as the level of MDA increased while TAC level decreased in the diseased rat model. Treatment of the AIA rats with either leflunomide or quercetin, individually, decreased the MDA level and elevated the TAC level. A more pronounced effect was expressed by quercetin monotherapy regarding TAC level in comparison to leflunomide monotherapy. The combination treatment succeeded to induce the highest significant antioxidant activity by reducing the MDA level and elevating the TAC to a level which exceeded the normal values (normal control animals) and this effect is presumably due to the antioxidant effect of quercetin.
Hepatotoxicity is one of the major concerns with the use of leflunomide. Safety profile of drugs was assessed by measuring ALT and AST level in rat sera and histopathological examination of the liver tissue of rats. It was observed that both liver enzymes increased significantly in untreated AIA rats, which is considered one of the extra-articular manifestations of the rheumatoid arthritis. Noteworthy, leflunomide expressed the highest elevation of liver enzymes in the present study compared to untreated AIA rats indicating the hepatotoxic side effect of the drug. Liver enzymes showed significant reduction in both quercetin and combination therapy in comparison to untreated AIA and leflunomide treated rats respectively, thus reflecting the hepato-protective effect of quercetin.
Consistent with the results of liver enzymes, the histopathological examination of liver tissue in untreated AIA rats expressed moderate inflammatory cell infiltration with mild hepatic necrosis. Leflunomide treated rats revealed the highest inflammatory cell infiltrate forming granuloma with hepatic necrosis. When the rats were treated with either quercetin alone or combination therapy, the liver tissue presented with minimal inflammatory cell infiltration and no hepatic necrosis with a more pronounced effect in quercetin treated rats. Histopathological data confirmed the hepato-protective effect of quercetin.
Collectively , the current study has shown that the combination of leflunomide and quercetin significantly inhibited the progression of rheumatoid arthritis as regards oxidative stress, MMP-1 and inflammatory mediators including TNF-α and COX-2 when compared to each probed drug alone. However, the combination therapy was not significantly different from treatment with leflunomide alone concerning the hind paw diameter of the rat, arthrogram score and histopathological examination of rat hind paw joint. This may be due to the short treatment period adopted in the present work, thus long-term and large-scale studies are recommended to verify whether or not the suppressive effects of such combination will improve prognosis in the future.
It is, therefore, possible that by combining both leflunomide and quercetin, a window of opportunity exists in the disease process not only for suppressing the disease action but also for limiting inflammation and toxicity.