Effect Of Adiponectin On Proliferation And Apoptosis Of Synovial Fibroblasts In Rheumatoid Arthritis

Part1Promotional effect of adiponectin on proliferation of synovial fibroblasts from rheumatoid arthritis patientsObjective:During the past nine years, we have shown that adiponectin (AD) is highly expressed in the inflammatory joint of rheumatoid arthritis (RA) patients and correlates closely with progressive bone erosion, but the mechanism remained largely unclear. Rheumatoid arthritis synovial fibroblasts (RASFs) are proliferative and are known to participate in synovial hyerplasia, synovitis and bone erosion. In this study, we will test the role of AD on proliferation of RASFs.Methods:RASFs were treated with AD (0.1,1or10μg/ml) or not in vitro. After72hours, CCK-8and direct microscopic count were used to test the number of RASFs exposed to AD or not.Results:CCK-8showed that the number of cells with10μg/ml of AD treatment in vitro was significantly increased, compared with those without AD treatment (OD:0.78±0.06vs0.59±0.067, p<0.01). And the results of direct microscopic count were same as CCK-8(53.75±2.13×104vs33.25±2.46×104,p<0.05).Conclusions:Our results have demonstrated that AD cloud promote the proliferation of RASFs in a concentration-dependent manner. Part2Inhibitory effect of adiponectin on apoptosis in synovial fibroblasts from rheumatoid arthritis patientsObjective:Base on our previous studies, AD has been demonstrated to promote proliferation of RASFs. But, whether AD could only exert effect on proliferation or both apoptosis and proliferation of RASFs, is still unknown. In this study, we will test the role of AD on apoptosis in RASFs.Methods:RASFs were cultured in vitro and became to apoptosis when treated with PBS in the absence and presence of AD (0.1,1or10μg/ml). After24hours, the frequencies of apoptosis cells were measured by flow cytometry.Results:The frequencies of apoptosis cells were significantly decreased when the cells were exposed to10μg/ml of AD in vitro, compared with those without AD treatment (11.33±3.65%vs23.34±5.21%,p<0.01).Conclusions:Our results have identified an inhibitory function of AD on apoptosis in RASFs in a concentration-dependent manner. Part3Modulatory effect of adiponectin on apoptosis and cell cycle related genes expression in synovial fibroblasts from rheumatoid arthritis patientsObjective:Base on our previous studies, AD has been demonstrated to exert effect on both apoptosis and proliferation of RASFs, resulting in abnormal synovial hyperplasia, but the mechanism behind is still unknown. In this study, we will test the role of AD on apoptosis and cell cycle related genes expression in synovial fibroblasts from rheumatoid arthritis patients.Methods:RASFs were treated with AD (0.1,1or10μg/ml) or not in vitro. After72hours, Real-time PCR was used to test the expression of Bcl-2, Bax, p53, CDK4and PCNA mRNA, and western blot was used to test the protein expression of Bcl-2and Bax.Results:A markedly increased level of Bcl-2mRNA (p<0.01) and protein and a great decreased level of Bax-2mRNA (p<0.05) and protein were also observed in RASFs in the presence of10μg/ml of AD, compared with those without it. AD treatment with10μg/ml could significantly inhibit p53mRNA (p<0.05) and elevate Bcl-2/Bax (p<0.05), CDK4mRNA (p<0.05) and PCNA mRNA expression (p<0.05).Conclusions:Our results have demonstrated that AD cloud increase Bcl-2, CDK4and PCNA expression, decrease Bax and p53expression and elevate Bcl-2/Bax. Part4Up-Regulation of adiponectin on the expression of IL-6, IL-8and MMP-3mRNA in synovial fibroblasts from rheumatoid arthritis patientsObjective:To investigate the effect of AD on synovitis and bone erosion, here, we will test the role of AD on the expression of IL-6, IL-8and MMP-3mRNA in RASFs.Methods:RASFs were treated with AD (0.1,1or10μg/ml) or not in vitro. After72hours, real-time PCR was used to test the IL-6, IL-8and MMP-3mRNA expression in RASFs.Results:AD treatment with10μg/ml could significantly elevate IL-6mRNA (8.97±3.77folds,p<0.05), IL-8mRNA (12.71±2.05folds, p<0.001) and MMP-3mRNA (4.99±1.12folds,p<0.001) expression in RASFs, compared with those not exposed to AD.Conclusions:AD could exacerbate arthritis symptoms and bone erosion by promoting IL-6, IL-8and MMP-3expression in RASFs.