目的 探讨自噬在法舒地尔(Fasudil)抑制Rho激酶心肌保护中的变化,并分析其作用及可能机制.方法 离体大鼠心脏Langendorff装置灌流,结扎冠状动脉左前降支30 min模拟局部心肌缺血,松开结扎线恢复灌流120 min复制心肌缺血/再灌注(ischemia/reperfusion,I/R)模型.实验分3组:I/R组、法舒地尔(Fasudil)组、和Fasudil+自噬抑制剂渥曼青霉素(Wort)组.连续记录左心室动力学变化,收集再灌注5、10 min冠脉流出液测乳酸脱氢酶(lactate dehydrogenase,LDH)含量;RT-PCR检测自噬相关基因Atg5、Beclin1和凋亡相关基因Bax、Bcl-2 mRNA表达的变化,Western blotting检测caspase3的表达变化.结果 与I/R组比,Fasudil使左心室发展压、左心室内压最大上升及下降速率、左心室做功明显得到改善,降低复灌期冠脉流出液中LDH的释放,Atg5、Beclin1 mRNA表达增加,Bcl-2/Bax mRNA升高,caspase 3蛋白表达降低.自噬抑制剂Wort减弱了法舒地尔的保护作用,抑制了心室动力学指标的恢复,LDH释放增多,Atg5和Beclin1 mRNA表达降低,Bcl-2/Bax降低,caspase 3蛋白表达升高.结论 抑制Rho激酶心肌保护作用中诱导了自噬的发生,且自噬起到保护心肌的作用,可能与减少细胞凋亡有关.%Objective To investigate the changes of autophagy in ischemic myocardium of rats treated with fasudil for inhibiting Rho kinase.Methods The hearts isolated from male Sprague-Dawley rats were subjected to 30 min of occlusion of the left anterior descending artery followed by 120 min of reperfusion with or without treatment with fasudil or fasudil+Wort.The left ventricular hemodynamics were continuously recorded,and the coronary effluent was collected during the reperfusion to determine lactate dehydrogenase (LDH) levels.The mRNA expressions of autophagy-related genes Atg5 and Beclin1 and apoptosis-related genes bax and bcl-2 were detected by RT-PCR,and the protein expression of caspase-3 was detected by Western blotting.Results Compared with I/R group,fasudil significantly improved the left ventricular developed pressure,maximal rise/fall rate of left ventricular pressure and rate pressure product,reduced LDH release during reperfusion,increased Atg5 and Beclin1 mRNA expression and the ratio of Bcl-2/Bax,and lowered caspase 3 protein expression.The autophagy inhibitor Wort significantly attenuated the effect of fasudil in the rat hearts.Conclusion Fasudil treatment for inhibiting Rho kinase promoted autophagy in ex vivo rat heart to protect against myocardial ischima-reperfusion injury possibly by reducing apoptosis of the cardiac myocytes.
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