Effects Of The Active Metabolite Of Leflunomide, A771726, On The Immune Function Of Peritoneal Macrophages From Collagen-induced Arthritis Rats And Its Partly Mechanisms Of Action

ObjectiveTo study the effects of the active metabolite leflunomide, A771726, on the secondary inflammation and immune reactions on collagen-induced arthritis(CIA),In this article,we focus on the effect of A771726 on the immune function of peritoneal macrophages(PMФ) in collagen-induced arthritis (CIA) and its potential mechanism.MethodsChicken typeⅡcollagen (CⅡ) was used to induce CIA in rats; The changes of secondary paw-swelling were observed and scored;Pathological changes in synovium of joint were examined by Hematoxylineosin(HE) stain method.Anti-II collagen antibody and tumor necrosis factor-α(TNF-α)in blood was detected by enzyme-linked immunosorbent assay (ELISA).T,B lymphocytes Proliferation was determined by 3-(4,5-dimethylthiazal-2y1)2,5-diphenyltetrazoli-umbromide (MTT) assay ,Phagocytosis was surveyed by neutral red assay. interleukin-I(IL-1)activity production release from PMФwas measured by thymocyte proliferation assay. TNF-α, IL-10 , IL-6, TGF-β1 produced by PMФwas determined by ELISA assay. Oxidative stress were assessed by Chemical methods. The expression of TNF-αmRNA,IL-6 mRNA,IL-10 mRNA,TGF-β1 mRNA,iNOS mRNA,TLR4 mRNA of PMФfrom CIA rats were detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. The expression of protein of TLR4 of PMФfrom CIA rats was measured by western blot analysis.Results1.Leflunomide could alleviate the secondary inflammation in joint,regulate the function of immunity,and have apparently treatment effect to CIA rats. CIA rats occurrenced red swelling of the paws on d12 after immunity, which accompanied with paw swelling, polyarthritis , ear and tail Inflammatory nodule and erythema,decrease of body lesions. Crest-time of secondary inflammation was on d22～d26,and the inflammation began abatement on d26. Compare to the normal rat, in weight and the development of inflammatory CIA rats, the histological morphological of rats joints examination showed hyperplastic synovium, synovial lining hyperplasia, inflammatory cells infiltration, pannus formation and destruction of the cartilage, and faty changes in the synovial cells. A771726 (3,6,12 mg/kg,d12～d34)ig could mitigate the inflammation of joint at different degree and suppress the excessive T and B lymphocytes proliferation and decrease the elevated anti-II collagen antibody and TNF-αin blood.2.The effect of A771726 on the fuction of PMФfrom normal rats and CIA rats in vitro studiesA771726 effectively reduced the content of TNF-α, IL-6, NO, IL-1 in supernatant of PMФfrom CIA rats, but increased significantly the content of IL-10,TGF-β1;A771726 effectively reduced the content of NO and MDA,and the reduction of SOD content were increased in the supernatant of PMФfrom CIA rats.These results showed that A771726 (1,10μmol/L)significantly inhibited the secretion function of PMФfrom CIA rats.A771726 (1,10μmol/L) markedly ameliorate oxidative stress of PMΦfrom CIA rats.3. The effect of A771726 on the mRNA expression of TNF-α, IL-6 , iNOS, IL-10 and TGF-β1 of PMФfrom CIA rats in vitro studiesA771726 effectively inhibited the mRNA expression of TNF-α, IL-6,iNOS of PMФ, increased the mRNA expression of IL-10 and TGF-β1 of PMФ. These results suggested A771726 could suppress the expression inflammatory cytokine and relieve inflammatory reaction in CIA rats .4. The effect of A771726 on the expression of TLR4 mRNA of PMФfrom CIA rats in vitro studiesCompared with normal group, the expression of TLR4 protein and mRNA were obviously increased in model group. Compared with model group, the expression of TLR4 protein and mRNA of PMФfrom CIA rats were decreased significantly in A771726 (1, 10μmol/L) group. These results suggested the therapeutic effects of A771726 on CIA might be associated with down-regulating TLR4 expression.Conclusion1 leflunomide has an improvement effects on polyarthritis, paw swelling, and articular histopathological changes in rats with CIA, which is related to its modulating immune functions.2 A771726 maybe regulate the abnormal function of immunity in CIA rats through adjusting the function of PMФ.3 A771726 (1,10μmol/L)regulate the secretion function and oxidative stress, and advanced investigation for its mechanism whether or not be related to inhibitory LPS-TLR4 signal pathway of PMФfrom CIA rats would be performed in future.