基于RhoA/ROCK通路探讨桔梗提取物对类风湿关节炎成纤维样滑膜细胞增殖及转移的影响

Effects of Platycodon grandiflorum extract on proliferation and metastasis of rheumatoid arthritis fibroblast-like synovial cells based on RhoA/ROCK pathway

目的探讨桔梗提取物(PGE)对类风湿关节炎(RA)成纤维样滑膜细胞增殖和转移的影响及其作用机制。方法将成纤维样滑膜细胞MH7A细胞分为对照组、PGE-L组(25 mg·L^(-1)PGE)、PGE-M组(50 mg·L^(-1)PGE)、PGE-H组(100 mg·L^(-1)PGE)、PGE+血管紧张素(Ang)Ⅱ组[100 mg·L^(-1)PGE+100 nmol·L^(-1)Ras同源基因家族成员A(RhoA)/Rho激酶(ROCK)通路激活剂AngⅡ],给予相应的处理。采用CCK-8法、流式细胞术、Transwell实验分别检测细胞增殖、凋亡、迁移与侵袭情况,Western blot法检测相关蛋白表达水平,GST-pull down法检测细胞中RhoA活性。结果与对照组比较,PGE-L组、PGE-M组、PGE-H组的吸光度值(PGE处理24、48、72h)、迁移及侵袭细胞数目、细胞周期蛋白D1(cyclin D1)、基质金属蛋白酶(MMP)-2、MMP-9、ROCK1、ROCK2蛋白表达及RhoA活性降低,p21蛋白表达及细胞凋亡率升高(P<0.05),且PGE的浓度越高,对应的趋势越明显(P<0.05)。与PGE-H组比较,PGE+AngⅡ组的吸光度值(PGE处理24、48、72 h)、迁移及侵袭细胞数目、cyclin D1、MMP-2、MMP-9、ROCK1、ROCK2蛋白表达及RhoA活性升高,p21蛋白表达及细胞凋亡率降低(P<0.05)。结论PGE可抑制MH7A细胞增殖与转移,该机制可能与抑制RhoA/ROCK通路有关。

AIM To investigate the effects of Platycodon grandiflorum extract(PGE)on the proliferation and metastasis of fibroblast-like synovial cells in rheumatoid arthritis(RA)and its mechanism of action.METHODS Fibroblastlike synovial cells MH7A were divided into control group,PGE-L group(25 mg·L^(-1)PGE),PGE-M group(50 mg·L^(-1)PGE),PCE-H group(100 mg·L^(-1)PGE),PGE+angiotensin(Ang)Ⅱgroup(100 mg·L^(-1)PGE+100 nmol·L^(-1)Ras homologous gene family member A(RhoA)/Rho kinase(ROCK)pathway activator Ang I),and treated accordingly.Cell proliferation,apoptosis,migration and invasion were detected by CCK-8 method,flow cytometry and Transwell,respectively.The expression levels of related proteins were detected by Western blot,and the RhoA activity in cells was detected by GSTpull down method.RESULTS Compared with the control group,absorbance value(24,48 and 72 h after PGE treatment),the number of migrating and invading cells,the expression of cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,ROCK1 and ROCK2 proteins,and the activity of RhoA were significantly reduced,and p21 protein expression and the apoptosis rate were increased in PGE-L group.PGE-M group,and PGE-H group,and the higher the concentration of PGE.the more obvious the corresponding trend was(P<0.05).Compared with the PGE-H group,the absorbance value(24,48,72 h after PGE treatment),the number of migratory and invasive cells,the expression of cyclin D1,MMP-2,MMP-9,ROCK1,ROCK2 protein and RhoA activity were increased,amd p21 protein expression and apoptosis rate decreased in PGE+Ang I group(P<0.05).CONCLUSION PGE can inhibit the proliferation and metastasis of MH7A cells,which may be related to the inhibition of RhoA/ROCK pathway.