剪切力对成纤维细胞中p38和CREB活性影响和作用机制

The effects of shear stress on the activity of p38 and CREB in fibroblast cells and its mechanisms

目的探讨剪切力对成纤维细胞中p38丝裂原活化蛋白激酶(p38MAPK)和环磷腺苷效应元件结合蛋白(CREB)活性影响及其调控机制。方法将成纤维细胞传代于培养皿中,细胞贴壁后采用不同作用时间和强度的剪切力(Shear stress)作用于成纤维细胞。为了探索剪切力作用的机制,分别加入活性氧(ROS)抑制剂过氧化氢酶和钙离子螯合剂BAPTA预处理成纤维细胞,剪切力作用30min后收集细胞。采用免疫印迹方法分别检测细胞中p38、CREB的活性。结果剪切力呈时间和强度依赖性方式促进成纤维细胞中p38、CREB磷酸化升高;在5dyn/cm2强度下,p38和CREB活性在10～30min时段逐步升高;在剪切力5dyn/cm2和7dyn/cm2作用30min后,p38和CREB的磷酸化水平均出现明显升高。ROS抑制剂Catalase抑制剪切力作用下成纤维细胞p38和CREB磷酸化,而钙离子螯合剂BAPTA增强p38和CREB磷酸化。结论剪切力通过ROS途径促进成纤维细胞中p38和CREB的磷酸化。

Objective To explore the effects of shear stress on the activity of p38 and CREB in the fibroblast cells andits mechansims. Methods The shear stress of different persistent time and intensity was applied to the fibroblast cellsafter the cells were transferred onto culture dish and adhered to dish wall. In order to explore the mechanisms of shearstress,the cells were pretreated with reactive oxygen species （ROS）inhibitor hydrogen peroxide enzyme （Catalase）andcalcium ion echelating agent BAPTA,respectively,before exerting shear stress and then collected 30 minutes later aftershear stress applied. The activity of p38 and CREB in the cells were detected by western blotting. Results The shearstress promoted phosphorylation of p38 and CREB in fibroblasts witha time- and intensity-dependent pattern. Under thestrength of 5 dyn/cm2,p38 and CREB activities increased gradually during 10 min and 30min.The phosphorylation levelsof p38 and CREB significantly increased at 5 dyn/cm2and 7 dyn/cm230 min later. ROS inhibitor catalase inhibitedphosphorylation of P38 and CREB in fibroblasts by the effect of shear stress. Meanwhile, BAPTA increasedphosphorylation of P38 and CREB. Conclusion Shear stress may promote the activity of p38 and CREB in fibroblasts bythe way of ROS.