Rho激酶在急性心肌梗死大鼠中的表达及其对心肌细胞凋亡的影响

Expression of Rho-kinase in rats with acute myocardial infarction and its relationship to apoptosis

目的:分析心肌梗死(MI)后大鼠心肌组织中Rho激酶表达的变化,探讨Rho激酶信号通路与心肌细胞凋亡的关系及其选择性抑制剂法舒地尔对MI后心肌的保护作用。方法:选取雄性Wistar大鼠,结扎其左前降支建立急性心肌梗死(AMI)模型。将术后24 h存活的24只大鼠随机分为治疗组(n=12)和AMI组(n=12),另随机选取10只大鼠作为假手术组,只在其左前降支下穿线不结扎。治疗组腹腔注射法舒地尔5 mg/kg,每日两次;AMI组和假手术组给予等量的生理盐水。4周后,用Evens蓝及四唑氮蓝试验(NBT)双染确定缺血及梗死面积;用RT-PCR法测定Rho激酶mRNA的表达;DNA片段分析观察心肌细胞凋亡的情况;用免疫组化染色法测定凋亡相关蛋白bcl-2及bax表达的变化。结果:与AMI组相比,治疗组的梗死面积显著减小(P<0.05)。AMI大鼠缺血心肌组织中DNA片段分析显示,有DNA梯状条带(ladder)形成,假手术组大鼠却没有。AMI组大鼠中Rho激酶mRNA及凋亡相关蛋白bax的表达明显增加(P<0.01,P<0.01);bcl-2的表达明显减少(P<0.01)。以法舒地尔治疗4周后,Rho激酶mRNA及bax的表达均显著减少,bcl-2的表达显著增加(均P<0.01)。结论:MI大鼠心肌组织中Rho激酶的表达升高,心肌细胞凋亡增加。法舒地尔能够减少Rho激酶的表达,减少心肌细胞凋亡,发挥对心肌的保护作用。

AIM： To analyze the changes of Rho-kinase expression in rats with acute myocardial infarction（AMI） and to study the relationship between cardiac myocyte apoptosis and Rho/Rock signaling pathway and the effect of fasudil,a Rho kinase selective inhibitor,on cardiac protection after AMI.METHODS： Male Wistar rats were subjected to left anterior descending coronary artery（LAD） ligation for 24 h and the surviving rats were randomly divided into two groups： treatment group（n=12） and AMI group（n=12）.Another ten rats were randomly selected as the sham-operated group（n=10）,with no ligation of LAD.Fasudil（5 mg/kg） was given to rats of the treatment group twice a day by IP injection.Rats of the AMI group and sham-operated group during the same period were given an equivalent normal of saline.Rats were sacrificed 4 weeks after and the infarct size and ischemia size were determined by Evans-blue and NBT double staining.The Rho kinase mRNA expression in the ischemia region was measured by RT-PCR and the apoptotic cardiocytes in the ischemia region were determined by DNA ladder analysis.Bcl-2 and Bax protein expressions were measured by immunohistochemical staining.RESULTS： Infarct size was obviously reduced in treatment group compared with AMI group（P〈0.05） and no significant difference was observed in the ischemic area.The expression of Rho kinase increased significantly in AMI group compared with sham group.Expression of apoptosis-related protein and bax increased whereas bcl-2 decreased（P〈0.01）.Compared with those in AMI group,the expression of Rho kinase significantly attenuated,bax decreased and bcl-2 increased in the treatment group（P〈0.01）.DNA agarose gel electrophoresis of both AMI group and treatment group formed ladders,but no ladders were observed in sham group.CONCLUSION： In the myocardium of rats with AMI,the expression of Rho kinase and the apoptosis of myocardial cells are increased.Fasudil,a Rho kinase inhibitor,attenuates the expression of Rho kinase and apoptosis of myocardial cells and decreases infarct size,therefore playing an important role in cardiac protection after AMI.