丹酚酸B对人类风湿关节炎滑膜成纤维细胞增殖与凋亡的影响及其作用机制

Effect and mechanism of salvianolic acid B on proliferation and apoptosis of human rheumatoid arthritis synovial fibroblasts

目的探讨丹酚酸B对人类风湿关节炎(RA)滑膜成纤维细胞增殖和凋亡的影响及其作用机制。方法以不同浓度的丹酚酸B(0、1、10、20、30、40和50μmol/L)处理人RA滑膜成纤维细胞MH7A,采用MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,筛选丹酚酸B的最适作用浓度。取MH7A细胞,设置对照组(正常培养)、丹酚酸B组(加入丹酚酸B培养)与丹酚酸B+3-MA组(加入丹酚酸B和5 mmol/L自噬抑制剂3-MA共培养)。采用MTT法和流式细胞仪检测细胞增殖和凋亡情况;qRT-PCR检测caspase-3 mRNA相对表达水平;Western blotting检测caspase-3、LC3-Ⅰ、LC3-Ⅱ、Beclin-1和p62蛋白相对表达水平。结果与0μmol/L丹酚酸B组比较,30、40和50μmol/L丹酚酸B处理的MH7A细胞增殖率明显降低(P<0.05),凋亡率明显升高(P<0.05),据此选择50μmol/L丹酚酸B进行后续实验。与对照组比较,丹酚酸B组细胞增殖率、p62蛋白相对表达水平明显降低,细胞凋亡率、caspase-3 mRNA和蛋白以及LC3-Ⅱ/LC3-Ⅰ、Beclin-1蛋白相对表达水平明显升高(P<0.05);与丹酚酸B组比较,丹酚酸B+3-MA组细胞增殖率(63.47%±1.94%vs.51.33%±4.67%)、p62蛋白相对表达水平(0.97±0.06 vs.0.20±0.02)明显升高,细胞凋亡率(21.4%±1.8%vs.30.4%±0.6%)、caspase-3 mRNA(0.83±0.06 vs.1.27±0.15)和蛋白(0.64±0.05 vs.1.10±0.09)、LC3-Ⅱ/LC3-Ⅰ(0.29±0.07 vs.1.23±0.21)、Beclin-1(0.36±0.05 vs.1.05±0.08)蛋白相对表达水平明显降低(P<0.05)。结论丹酚酸B可能通过诱导自噬抑制人RA滑膜成纤维细胞的增殖并促进其凋亡。

Objective To investigate the effect and mechanism of salvianolic acid B(SB)on proliferation and apoptosis of human rheumatoid arthritis(RA)synovial fibroblasts.Methods Human RA synovial fibroblasts MH7A cells were treated with different concentrations of SB(0,1,10,20,30,40 and 50μmol/L).MTT method was used to detect cell proliferation and flow cytometry was used to detect cell apoptosis,and the optimal concentration of SB was selected.MH7A cells were set as control group(normal cultured cells),SB group(cells cultured with 50μmol/L SB)and SB+3-MA group(cells cultured with 50μmol/L SB and 5 mmol/L autophagy inhibitor 3-MA).MTT and flow cytometry were used to detect cell proliferation and apoptosis,qRT-PCR was used to measure the relative expression level of caspase-3 mRNA.Western blotting was used to detect the relative expression levels of caspase-3,LC3-Ⅰ,LC3-Ⅱ,Beclin-1 and p62 protein.Results Compared with 0μmol/L SB group,the proliferation rate decreased obviously(P<0.05)and the apoptosis rate increased significantly(P<0.05)of MH7A cells treated with 30,40 and 50μmol/L SB and 50μmol/L SB was used for follow-up experiments.Compared with control group,the cell proliferation rate and the relative expression level of p62 protein in SB group decreased significantly,while the apoptosis rate,the relative expression levels of caspase-3 mRNA and protein,as well as the relative expression levels of LC3-Ⅱ/LC3-Ⅰand Beclin-1 protein increased(P<0.05).Compared with SB group,the proliferation rate and the relative expression level of p62 protein increased obviously(63.47%±1.94%vs.51.33%±4.67%;0.97±0.06 vs.0.20±0.02)in SB+3-mA group,and the apoptosis rate(21.4%±1.8%vs.30.4%±0.6%),the relative expression levels of caspase-3 mRNA(0.83±0.06 vs.1.27±0.15)and protein(0.64±0.05 vs.1.10±0.09),as well as the relative expression levels of LC3-Ⅱ/LC3-Ⅰ(0.29±0.07 vs.1.23±0.21)and Beclin-1 protein(0.36±0.05 vs.1.05±0.08)decreased(P<0.05).Conclusion SB might inhibit the proliferation and promote apoptosis of human RA synovial fibroblast by inducing autophagy.