Rho激酶在乳鼠心肌细胞模拟缺血再灌注损伤细胞凋亡中的作用

摘要：目的观察Rho激酶在培养乳鼠心肌细胞缺血再灌注损伤细胞凋亡中的作用，及Rho激酶抑制剂盐酸法舒地尔（fasudil,F）对缺血再灌注损伤细胞凋亡的影响。方法原代培养出生1～2d的SD乳鼠心肌细胞，并行肌钙蛋白抗体免疫荧光染色鉴定，建立缺血再灌注损伤（I/R）模型。实验分3组：① 正常对照组；② I/R组;③ I/R+F组：建立I/R模型前20min 加入法舒地尔，使其终浓度分别为10μmol/L（F1组）、30μmol/L（F2组）、50μmol/L（F3组）。Western blot分别检测缺血2h,再灌注3h肌球蛋白磷酸酶目标亚单位1（myosin phosphatase target subunit 1，MYPT1）磷酸化水平，作为Rho激酶功能活化的标志。应用流式细胞仪Annexin-V/PI双色法检测再灌注3、6h时点心肌细胞的凋亡率。结果再灌注3h后MYPT1的磷酸化水平明显增加，I/R组为正常对照组的 5.7倍(P＜0.01)，F1、F2、F3组法舒地尔干预后较I/R组分别减少24.6%、40.1%、60.1%（P＜0.05）；法舒地尔组再灌注3、6h心肌细胞的凋亡率较I/R组同时间点显著下降，随给药浓度的增加，细胞凋亡率呈下降趋势。结论Rho激酶在缺血再灌注心肌细胞中有促凋亡作用，法舒地尔可减少缺血再灌注心肌细胞凋亡的发生，从而发挥良好的心肌保护作用。

Effects of Rho-kinase on cardiac apoptosis induced by ischemia/reperfusion injury in neonatal rat cardiomyocytes

To investigate the effects of Rho kinase on ischemia/reperfusion induced cardiac apoptosis and the effects of the Rho kinase inhibitor, fasudil on cardiac apoptosis in cultured neonatal rat cardiomyocytes. MethodsNeonatal rat ventricular myocytes were prepared from hearts of 1 2 day old Sprague Dawley rat pups, and then subjected to ischmemia and reperfusion injury in vitro. Cultured neonatal cardiomyocytes were visualized with immunofluorescence staining. Cultured cardiomyocytes were randomly divided into three groups: the control group, the I/R group, and the I/R+fasudil group(treated with three concentrations of 10?μmol/L, 30?μmol/L and 50?μmol/L). The extent of phosphorylation of myosin phosphatase target subunit 1(MYPT1) was quantified by Western blot analysis, and the activity of the Rho kinase. Cardiac myocyte apoptosis ratio was determined by flow cytometry with Annexin V and propidium iodide（PI）staining. ResultsCardiomyocyte ischemia reperfusion resulted in a 5.7 fold increase in the amount of phosphorylated MYPT1 compared with the control group(P＜0.01 ). In contrast, treatment with fasudil at 10μmol/L， 30μmol/L and 50μmol/L attenuated the amount of phosphorylated MYPT1 by 24.6%, 40.1% and 60.1%, respectively (P＜0.05).　The cardiac myocyte apoptosis ratio were significantly decreased when myocytes was subjected to 3?h and 6?h reperfusion in the fasudil group in a dose dependent fashion compared with the I/R group. ConclusionsRho kinase induces cardiomyocyte apoptosis, however, the Rho kinase inhibitor, fasudil can decrease cardiomyocyte apoptosis and protect cardiac cells.