从线粒体膜稳定作用探讨线粒体K(MITO-KATP)通道开放剂改善老年冠心病大鼠心肌缺血再灌注损伤的机制

The mechanism of mitochondrial K (MITO-KATP) channel opening agent in improving myocardial ischemia reperfusion injury in aged CHD rats from the stable effect of mitochondrial membrane

目的从线粒体膜稳定作用探讨线粒体K+ATP(MITO-KATP)通道开放剂尼可地尔(Nicorandil,Nic)改善老年冠心病大鼠心肌缺血再灌注损伤的机制。方法选取60只健康雄性SD老年大鼠(18~20个月),体重400~600g,随机分为3组:假手术(Sham)组、模型(Model)组和尼可地尔(Nic)组,每组10只。建立老年冠心病大鼠心肌缺血再灌注(MI/R)模型,尼可地尔(Nic)组在再灌注之前,股静脉注射尼可地尔5mg/kg,假手术组和模型组注射等量的生理盐水。3组大鼠在术后24h进行腹主动脉取血,分别检测大鼠血清中,肌酸磷酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)的水平变化;采用流式细胞仪测定大鼠心肌细胞线粒体膜电位的变化情况;采用WesternBlot法测定心肌细胞线粒体中p-Cx43蛋白变化情况,心肌组织中Bcl-2和Bax蛋白表达情况的变化。结果与Sham组相比,模型组大鼠血清中CK-MB和LDH的含量增加(P<0.05),说明心肌缺血再灌注会导致心肌细胞损伤;与模型组相比,尼可地尔组大鼠血清中CK-MB和LDH的含量下降(P<0.05),说明尼可地尔有抗心肌缺血的作用。采用阳离子绿色荧光染料罗丹明123对线粒体的膜电位进行检测,与Sham组相比,模型组大鼠心肌线粒体膜电位降低(P<0.05),而尼可地尔组大鼠心肌线粒体膜电位高于模型组(P<0.05),说明心肌细胞凋亡时会导致线粒体膜电位降低以及功能变化,而线粒体K+ATP通道开放剂尼可地尔可以缓解线粒体的损伤情况。WesternBlot结果表明,与Sham组相比,模型组大鼠心肌线粒体p-Cx43的蛋白表达下调,而尼可地尔组大鼠心肌线粒体p-Cx43的蛋白表达高于模型组,说明尼可地尔能够上调心肌线粒体p-Cx43的蛋白表达,维持线粒体的稳定性。与Sham组相比,模型组大鼠心肌组织中Bcl-2的蛋白表达下调,Bax蛋白表达上调;而尼可地尔组大鼠心肌组织中Bcl-2的蛋白表达上调,Bax蛋白表达下调,说明线粒体K+ATP通道开放剂尼可地尔可有效抑制心肌缺血再灌注导致的心肌细胞凋亡。结论线粒体K+ATP通道开放剂尼可地尔对心肌缺血再灌注损伤有保护作用,其机制可能是通过降低大鼠血清中CK-MB和LDH的含量,维持线粒体膜稳定性,抑制心肌细胞的凋亡。

Objective In this study,the mechanism of mitochondrial K (MITO-KATP) channel opening agent in improving myocardial ischemia reperfusion injury in elderly coronary heart disease (CHD) rats were investigated from the stable effect of mitochondrial membrane. Methods Sixty healthy male SD rats (18-20 months) weighing 400-600 g were randomly divided into 3 groups:sham group,model group and nicorandil (Nic) Group,10 in each group. The model of myocardial ischemia reperfusion (MI/R) in elderly CHD rats was established. Before reperfusion,the Nic group was injected 5 mg/kg into the femoral vein. The sham group and model group were given the same amount of saline. The levels of CK-MB and LDH in serum of the three groups of rats were detected by abdominal aorta blood extraction 24 h after surgery. The changes of mitochondrial membrane potential in rat myocardial cells were measured by flow cytometry. The changes of p-Cx43 protein in the mitochondria of myocardial cells were determined by Western Blot. The protein expressions of Bcl-2 and Bax in myocardial tissue were determined by Western Blot. Results Compared with Sham group,the contents of CK-MB and LDH in serum of rats in the model group were increased (P<0.05),which indicated that myocardial ischemia reperfusion could lead to myocardial cell injury. Compared with the model group,the contents of CK-MB and LDH in serum of rats in Nic group were decreased (P<0.05),which indicated that nicodiltir had an antimyocardial ischemia effect. Cationic green fluorescent dye rhodamine 123 was used to detect the membrane potential of mitochondria. Compared with Sham group,the myocardial mitochondrial membrane potential of rats in the model group was decreased (P<0.05). However,the myocardial mitochondrial membrane potential was higher in the Nic group than in the model group (P<0.05). This indicated that apoptosis of cardiac cells would lead to the decrease of mitochondrial membrane potential and functional changes,while the mitochondrial K channel opening agent nicodiline could alleviate the mitochondrial damage. Western Blot results showed that compared with sham group,the protein expression of p-Cx43 in the myocardium of rats in the model group was decreased. The protein expression of p-Cx43 in myocardial mitochondria was higher in the Nic group than in the model group,which indicated that nicodiline could up-regulate the protein expression of cardiac mitochondria p-Cx43 and maintain the stability of mitochondria. Compared with Sham group,the protein expression of Bcl-2 in the myocardium tissue of the model group was down-regulated,and the protein expression of Bax was up-regulated. However,the protein expression of Bcl-2 was up-regulated and the protein expression of Bax was down-regulated in myocardial tissues of rats in Nic group,which indicated that mitochondrial K channel opening agent nicodiline could effectively inhibit myocardial apoptosis induced by myocardial ischemia reperfusion. Conclusion Mitochondrial K (MITO-KATP) channel opening agent nicodiline had a protective effect on myocardial ischemia reperfusion injury. The mechanism might be by reducing the contents of serum LDH and CK-MB, maintaining the stability of mitochondrial membrane,and inhibiting the apoptosis of myocardial cells.