法舒地尔减轻大鼠缺血/再灌注心肌线粒体损伤和细胞凋亡

Fasudil attenuates mitochondrial injury and apoptosis in rat model of myocardial ischemia/reperfusion injury

目的观察线粒体融合蛋白2(Mfn2)和发动蛋白相关蛋白1(Drp1)在法舒地尔抑制Rho激酶对抗心肌缺血/再灌注(I/R)损伤中的变化,并分析其意义。方法离体大鼠心脏,结扎冠状动脉左前降支缺血0.5 h,持续再灌注2 h模拟心肌I/R损伤模型。实验分假手术组、I/R组、法舒地尔组。测定心室动力学变化和再灌注期间冠脉流出液中乳酸脱氢酶(LDH)含量,透射电镜观察心肌超微结构改变,免疫组织化学染色检测Rho激酶下游磷酸化的蛋白磷酸酶1调节亚基12A(p-PPP1R12A/p-MYPT1)表达,Western blot法检测Mfn2、Drp1和裂解型胱天蛋白酶3(c-caspase-3)蛋白水平。结果与假手术组相比,各组再灌注不同时间点左心室收缩和舒张功能均降低,LDH释出增加;与I/R组相比,法舒地尔组左心室收缩和舒张功能得到改善,LDH释出减少;透射电镜结果显示I/R组心肌明显肌丝断裂、肌原纤维结构不完整、线粒体损伤,免疫组织化学染色显示p-MYPT1蛋白表达上调;与I/R组比,法舒地尔组心肌肌原纤维和线粒体损伤减轻,p-MYPT1蛋白表达下调;Western blot结果显示,与假手术组相比,I/R组Mfn2蛋白表达减少,Drp1和c-caspase-3蛋白水平增加,与I/R组相比,法舒地尔组Mfn2蛋白水平无明显变化,Drp1和c-caspase-3蛋白水平减少。结论法舒地尔抑制Rho激酶的抗心肌I/R损伤作用与Mfn2蛋白表达无明显关联,可能与降低Drp1蛋白表达减少线粒体损伤,进而减少细胞凋亡有关。

Objective To observe the changes of mitochondria fusion protein 2（ Mfn2） and dynamin-related protein 1（ Drp1） in the cardioprotection of fasudil,and analyze the significance. Methods Hearts isolated from male Sprague-Dawley rats were subjected to ischemia for 30 minutes（ occlusion of left anterior descending artery）,and continuously perfusion for120 minutes to establish myocardial ischemia/reperfusion（ I/R） injury model. The rats were divided into 3 groups： sham group,I/R group and fasudil group. The left ventricular hemodynamics were continuously recorded; lactate dehydrogenase（ LDH） content was measured during reperfusion; myocardial ultrastructure was observed by electron microscopy; the protein expression of phosphorylated protein phosphatase 1 regulatory subunit 12A（ p-PPP1R12A/p-MYPT1） was detected by immunohistochemistry; and the protein expressions of Mfn2,Drp1 and cleaved caspase-3（ c-caspase-3） were detected by Western blot analysis. Results Compared with the sham group,the left ventricular systolic and diastolic function was weakened,and LDH release was promoted in the other two groups during reperfusion. Compared with the I/R group,fasudil improved left ventricular systolic and diastolic function and reduced LDH release. Electron microscopy and immunohistochemical results showed that myofibril and mitochondria were damaged obviously,and p-MYPT1 protein expression was enhanced in the I/R group. Compared with the I/R group,fasudil attenuated the damage of myofibril and mitochondria,and decreased p-MYPT1 protein expression. Western blotting showed that,compared with the sham group,Mfn2 protein expression decreased,Drp1 and c-caspase-3 protein expressions increased in the I/R group. Compared with I/R group,there was no obvious change in Mfn2 protein expression,while Drp1 and c-caspase 3 protein expressions decreased in the fasudil group.Conclusion Fasudil can protect against myocardial I/R damage through inhibiting Rho kinase,which has no clear correlation with Mfn2 protein expression,but may be related with decreasing Drp1 protein expression and reducing mitochondrial injury,thereby inhibiting apoptosis.