Rho激酶信号通路参与ET-1诱导的人气道平滑肌细胞迁移和细胞骨架的变化

Rho-kinase signaling pathway participates in endothelin-1-induced human airway smooth muscle cell migration and cytoskeletal reorganization

目的研究Rho激酶信号通路在内皮素-1(ET-1)诱导的人气道平滑肌细胞(ASMCs)迁移及细胞骨架变化中的作用。方法组织块贴壁法培养人ASMCs,改良的Boyden小室检测ASMCs的迁移能力;激光共聚焦扫描显微镜观察细胞骨架的变化;Western blotting检测ET-1刺激不同时间后Rho激酶底物肌球蛋白磷酸酶目标亚单位1(MYPT1)的磷酸化水平。结果ET-1在0.1、1、10、100nmol/L浓度具有趋化ASMCs的迁移能力,10nmol/L的ET-1趋化ASMCs迁移的能力最强(与对照组相比,P<0.01)。Rho激酶抑制剂Y-27632可浓度依赖性地抑制ET-1趋化的ASMCs跨膜迁移,与ET-1组相比,10μmol/L的Y-27632显著抑制了ASMCs迁移(P<0.01);ET-1(10nmol/L)刺激后ASMCs细胞骨架和形态改变,伪足生成,应力纤维形成增多,Y-27632可显著抑制ET-1诱导的上述改变;ET-1刺激15、30min后p-MYPT1表达显著增高(与对照组相比,P<0.01),随着刺激时间的延长,p-MYPT1表达下降(P>0.05)。结论Rho激酶信号通路在ET-1诱导的ASMCs迁移和细胞骨架变化中发挥重要的作用。

Objective To investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell （ASMCs） migration and cytoskeletal reorganization induced by endothelin- 1 （ET-1）. Methods Primary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-I treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation ofmyosin-phosphatase target 1 （p-MYPTI） was examined using Western blot analysis. Results At the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect （P〈0.01）. Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 μmol/L significantly blocked ASMC migration （P〈0.01）. ET-1 （10 nmoi/L） exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPTI protein expression after ET-1 exposure for 15 and 30 min （P〈0.01）, but prolonged exposure failed to result in the expression enhancement （P〉0.05）. Conclusion Rho-kinase signaling pathway may play an important role in ET-l-induced ASMC migration and reorganization ofactin cytoskeleton.