RhoA/ROCK信号通路对TLR-2配体诱导的类风湿关节炎患者成纤维滑膜细胞分泌趋化因子的调控作用

目的 研究RhoA/Rho激酶(ROCK)信号通路对Toll样受体2(TLR-2)配体诱导的类风湿关节炎(RA)成纤维样滑膜细胞(FLS)分泌趋化因子的影响.方法 RA FLS来源于活动性RA患者滑膜组织;肽聚糖被用于TLR-2配体;RhoA活性检测采用pull down方法;ROCK活性用磷酸化肌球蛋白结合亚单位1(MYPT1)蛋白表达来表示,磷酸化MYPT1蛋白检测采用免疫印迹法;细胞活性检测采用四甲基偶氮唑蓝比色(MTT)法,趋化因子水平采用酶联免疫吸附测定法(ELISA)检测.结果 肽聚糖(5 mg/L)刺激使体外培养的RA FLS分泌白细胞介素8(IL-8)、单核细胞趋化蛋白-2(MCP-2)和受激活调节正常T细胞表达和分泌因子(RANTES)明显增高,对RhoA和ROCK活化也有显著的刺激作用,肽聚糖诱导的上述作用能被TLR-2单克隆抗体所阻抑.RhoA抑制剂C3转化酶和ROCK抑制剂Y27632对PG诱导的IL-8、MCP-2和RANTES等趋化因子分泌有显著的抑制作用.结论 RhoA/ROCK信号通路对TLR2介导的RA FLS分泌趋化因子具有调控作用,通过抑制该通路活化可能有利于RA的治疗.

Abstract：

Objective To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway,a small Rho GTPase that is considered as an important modulator in inflammatory responses,on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS)from rheumatoid arthritis (RA) patients.Methods The RhoA activity was measured by a pull-down assay.And the ROCK activity was assessed by Western blot.The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay).MTT test was used to detect the cellular viability.Results The stimulation of peptidoglycan(PG,5 mg/L) increased the levels of IL-8 (interleukin-8),RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2)and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS.And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody.Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8,RANTES and MCP-2 in PG-induced RA FLS.Conclusion The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS.It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.

Modulation of RhoA/ROCK pathway on TLR-2 ligand-induced chemokine secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis

Objective To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway,a small Rho GTPase that is considered as an important modulator in inflammatory responses,on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS)from rheumatoid arthritis (RA) patients.Methods The RhoA activity was measured by a pull-down assay.And the ROCK activity was assessed by Western blot.The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay).MTT test was used to detect the cellular viability.Results The stimulation of peptidoglycan(PG,5 mg/L) increased the levels of IL-8 (interleukin-8),RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2)and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS.And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody.Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8,RANTES and MCP-2 in PG-induced RA FLS.Conclusion The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS.It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.