SIRT1 Through The NF-?B Pathway Inhibition Of Rheumatoid Arthritis Synovial Fibroblasts Migrate And Invade The Mechanism Research

Background:Rheumatoid arthritis is a chronic systemic autoimmune and inflammatory disease in which synovial fibroblasts play a crucial role.Sirtuins belong to class III histone deacetylases which use NAD+ as a co?substrate for their enzymatic activities to regulate the acetylation level and activity of substrates including participating in gene silencing,DNA repair,cell apoptosis,material synthesis or decomposition,aging,inflammation and many other life processes in the body.In recent years,sirtuins have attracted much attention in chronic diseases,such as obesity,metabolic syndrome,coronary heart disease and arthritis,among which SIRT1 plays an important role in reducing inflammation.SIRT1 can regulate many signaling molecules and inflammatory factors,including IL-6,IL-1,and TNF-?,by regulating NF-?B,thus reducing inflammation and protecting cells.The role of SIRT 1 remains controversial in tumors and inflammatory diseases,especially arthritis.SIRT 1 can promote cell growth and apoptosis resistance in human synovium.However,the absence of SIRT1 aggravated inflammatory arthritis in mice and increased the production of pro-inflammatory cells,and the increased expression of SIRT1 inhibited the aggressiveness of RA-FLS.Currently,the role of sirtuin(SIRT)1 in RA(Rheumatoid Arthritis)is uncertain,It is worth noting that SIRT1 has been shown to protect joints in many mouse experiments,but its role and mechanism in human RA joint tissues still need to be further demonstrated by experiments.Objective:The role of SIRT1 in inhibiting the invasion of fibroblast synovial cells in rheumatoid arthritis through NF-? B pathway was proposed to search for new targets for the treatment of rheumatoid arthritis.Methods:1.Preparation of human synovial tissues and FLS:Synovial tissue samples were obtained from 10 patients with RA during joint replacement or synovectomy or at Northern Jiangsu Hospital.Non-autoimmune diseases synovial tissues from ten traumatic knee patients were used as normal controls.Synovial tissue samples were got during the surgery.FLS were isolated by digestion with 2.5g/l trypsin for 4h at37?C with gentle agitation.2.SIRT1 transfection RA-FLS:RA-FLS cells transfected with pcdna3.1-SIRT1 and interleukin PCDNA3.1 were used in cell proliferation,apoptosis,migration and invasion experiments.3.Analysis the effect of SIRT1 on the proliferation and apoptosis of RA-FLS:The proliferation of RA-FLS was measured with cck-8 Kit(Cell Counting kit-8)and compared.Annexin V-FITC / PI apoptosis kit and FACS were used to analyze and calculate the apoptosis rate of RA-FLS treated with SIRT1,and compared with RA-FLS not transfected with SIRT1.4.Analysis the effects of SIRT1 on migration and invasion of RA-FLS:4.1 The migration ability of pcdna3.1-SIRT1 and PCDNA3.1 was detected by scratch test.4.2The number of cells invaded by pcdna3.1-SIRT1 and PCDNA3.1 Transwell cells was detected by Transwell compartment invasion experiment.4.3 The effects of pcdna3.1-SIRT1 and PCDNA3.1 on the migration and invasion of RA-FLS were compared.5.To investigate whether SIRT1 could reduce the production of pro-inflammatory cytokines in RA-FLS and the expression of NF-? B related proteins:5.1 The concentrations of TNF-?,IL-6,IL-8 and IL-1 were determined by enzyme-linked immunosorbent assay(ELISA).5.2 Western blot: Western blot was used to determine the expression of protein expression of NF-? B that includ antibodies against NF-? B p65(ab16502),phospho-NF-?B p65(Ser536)(ab76302),acetyl-NF-? B p65(Lys310)(ab19870),SIRT1(ab110304),and GAPDH(ab8245).5.3 After NF-?B p65 was knocked down,cell migration and invasion experiments were carried out again.6.Statistical analysis: Student's t test was used to analyze the differences between the two groups.One-way ANOVA was used to One-way analysis of variance followed by a post hoc Tukey's test was used to compare the results of more than two groups.Differences with a P-value of less than 0.05 were considered statistically significant.All statistical analyses were performed using the SPSS19 software package.Result:1.The SIRT1 overexpression inhibited RA-FLS proliferation in RA2.The SIRT1 overexpression suppressed migration and invasion of RA-FLS3.Overexpression of SIRT1 decreased the production of pro-inflammatory cytokines in RA-FLS and the expression of NF-?B related proteins.4.Knockdown of p65 expression can partially reverse the inhibition of SIRT1 on the migration and invasion of RA-FLS.Conclusion:SIRT1 can inhibit the migration and invasion of RA-FLS through NF-KB pathway.