| ObjectiveRheumatoid arthritis(RA) is a kind of systemic autoimmune diseases characterized by synovial hyperplasia and chronic articular synovial inflammation. It has been investigated that the abnormal proliferation and reduced apoptosis of RA synovial fibroblasts are involved in the pathogenesis of synovial hyperplasia. Studies have reported that calreticulin(CRT) may be associated with the pathogenesis of RA. In the early stage of the research, our group also found the high expression of CRT in RA serum, synovial membrane and synovial fluid, and the serum CRT had a positive correlation with RA disease activity DAS28, furthermore,CRT could promote the formation of pannus in RA via NO signal pathway. It has been reported that CRT could participate in the pathogenesis of RA by inhibiting Fas-Fas L-mediated T cell apoptosis. This present study is proposed to discuss whether CRT could promote the pathogenesis of RA synovial inflammation by regulating the anti-apoptotic protein c-FLIP in the basis of previous work and studies. We aim to provide some new theoretical and experimental basis for clinical therapeutic target of RA synovial inflammation.Methods1.Specimens for this research were collected form patients under knee arthroscopic surgery or arthroplasty in January 2014 to May 2015. Synovial fluid samples include: 32 cases of RA patients, 37 patients with OA. Synovial tissue samples including: 14 RA patients, 19 patients with OA.2.Synovial fluid levels of CRT were measured by ELISA.3.The semi-quantitative analysis and localization of CRT and c-FLIP in RA and OA synovium was detected by immunohistochemistry. Correlations of synovial CRT expression with c-FLIP were analyzed.4.The synovial fibroblasts were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and OA patients and cultured in vitro. The growth characteristics of RA and OA synovial fibroblasts were observed and analyzed while the cells were cultured.5.The proliferation of RA synovial fibroblasts after stimulated with CRT was examined by CCK-8 assay.6.The effect of CRT on inhibiting TRAIL-mediated RA synovial fibroblasts apoptosis was assessed by flow cytometry.7.The expression of CRT and c-FLIP in RA and OA synovial fibroblasts was detected by cellular immunofluorescence.8.Western blot and q RT-PCR were performed to detect the expression of c-FLIP in RA and OA synovial fibroblasts both in protein and m RNA levels. Also, the effect of different concentrations of recombinant human CRT on the expression of c-FLIP and the regulation of NF-κB signaling pathway were detected by q RT-PCR and Western Blot.9.Data were processed with SPSS software 20.0. Data were expressed as mean ± SD. Student’s t-test,Student-Newman-Keuls test and Pearson correlation analysis were performed for the statistical analysis. P < 0.05 was considered statistically significant.Results1.CRT levels in synovial fluid from RA [(7.2±3.0) ng/ml]patients were significantly higher than OA [(3.8±0.6) ng/ml](t=6.724, P<0.01).2.Immunohistochemistry results showed high expression of CRT and c-FLIP in the lining and sublining, inflammatory cells and perivascular areas of RA synovium. While, weak staining of CRT and c-FLIP was observed in the lining and perivascular areas of OA synovium. There was a signigficant positive correlation between synovial CRT expression and c-FLIP.3.Adherent primary FLS from both groups were initially round, and then stretched as spindle shape or fusiform. Primary FLS from RA group began to adhere at 12 to 24 hours after plating and spread to the bottom of culture flask at 4 to 5 days. Primary cells from OA group grew slowly, which began to adhere at 24 to 48 hours after plating and achieved cell confluency till 7 to 8 days. During subculture, the interval between adjacent passages was approximately 2 to 3 days in young group but 3 to 4 days in old group.4.CCK-8 results showed that CRT could significantly promote the proliferation of RAFLS.5.CRT had showed a suppressive effect on TRAIL-mediated RA FLS apoptosis refer to the flow cytometry results.6.q RT-PCR, Western Blot and Cell Immunofluorescence Test results showed that, compared to the OA synovial fibroblasts, CRT and c-FLIP in RA synovial fibroblasts were significantly higher in both m RNA and protein levels.7.After treated with CRT, the expression of c-FLIP in RA FLS was increased both in m RNA and protein levels.8.Increased levels of p-NF-κB were observed following CRT stimulation, while total NF-κB were not significantly changed. Treatment with NF-κB inhibitor inhibited CRT-mediated c-FLIP upregulation in FLS from RA patients.Conclusions1.c-FLIP showed higher expression in RA synovial tissue compared to OA, and the CRT and c-FLIP expression in RA synovial tissue were positively correlated.2.Both CRT and c-FLIP revealed increased expression in RA FLS. CRT could increase the expression of c-FLIP in RA FLS in a dose-dependent manner.3.CRT may regulate the phosphorylation level of NF-κB by up-regulating the expression of c-FLIP in RA FLS, which may participate in the process of synovial hyperplasia and continuous inflammation of RA. |
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