ABSTRACT
Objective To clarify the mechanisms that the response of fibroblast-like synovial (FLS) cellsto methotrexate (MTX) in rheumatoid arthritis (RA) and to provide theory basis for the drug treatment of RA.Methods Synovial fibroblasts were isolated from synovial tissue specimens obtained from patients with RA andexposed to MTX.Cell viability was measured using a MTT assay and cell apoptosis was valued by flow cytometry.Western blotting analysis of LC3 and immunocytochemistry were used to analyze the induction of autophagy in RA-FLS after treating with MTX.Transfection of siRNA was used to interfere the expression of Beclin1 to down-regulate the autophagy,cell apoptosis was valued by flow cytometry and western blot analysis was used to test the PARPp85 with or without the presence of MTX.Statistical product and service solutions (SPSS) 18.0 statistical software was used for statistical analysis of all experimental data.Independent sample t test was used according to data distribution status,homogeneity of variance,and normal distribution.GraphPad Prism 5.0 was used to draw statistical graphs.Results MTX induced apoptosis was increased in RA-FLS.MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation.In RA-FLS,transfection with Beclin1 siRNA inhibited autophagy and increased the susceptibility to MTX,which induced cell death.Conclusion Autophagy of RA-FLS contributes to the resistance to apoptosis induced by methotrexate.
ABSTRACT
Objective To study the relationship between E2F2 and the pathological features of RA and the mechanism of E2F2 expression.Methods The interference efficiency was detected by RT-PCR after RA FLs transfected with E2F2 siRNA.The cell proliferation was analyzed by MTS and the expression of numerous genes was tested by ELISA.To analyze the influence of E2F2 in the immune response by RT-PCR,RA FLs were separately treated with TNF-t and PDTC.The way of regulating E2F2 experssion was tested by Chip.Results Compared with negative control groups,the level of E2F2 mRNA were decreased in the cells transfected with E2F2 siRNA (P ＜ 0.01).Silencing E2F2 suppresses the proliferation of RA FLs (P ＜ 0.05).TNF-α regulates the expression of E2F2 in RA FLs via the NF-κB pathway.Conclusion Up-regulation of E2F2 is associated with the process of RA and TNF-α regulates the expression of E2F2 in RA FLs via the NF-κB pathway.Thus E2F2 might be a potential target for use in the development of new therapeutic approches of RA.
ABSTRACT
OBJECTIVE:To investigate the effects of triptolide(TP)on the proliferation of fibroblast-like synoviocytes(FLS) from patients with rheumatoid arthritis(RA)in vitro. METHODS:5 RA patients received knee arthroplasty or synovectomy to ob-tain synovial tissue. FLS was isolated,cultured and identified,and then incubated in the presence of TP [0 (blank control),50, 100 and 200 nmol/ml] for 24,48 and 72 h. The effects of TP on FLS was evaluated by MTT,and then proliferation inhibitory rate was calculated;flow cytometry was used to detect the apoptosis and cell cycle of FLS. RESULTS:The inhibitory rates of TP(50, 100 and 200 nmol/ml)on the proliferation of FLS were 17.46%-52.56%,which was positively correlated with drug concentration. Compared with blank control group,100 and 200 nmol/ml TP could increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in S phase,with statistical significance(P<0.05);200 nmol/ml TP could induce cell apoptosis. CONCLU-SIONS:TP could inhibit the proliferation and also could induce the apoptosis of FLS in RA patients in vitro,which may be one of its mechanism for treating RA.