稳心颗粒调节心肌梗死大鼠心肌缝隙连接蛋白43表达及自噬的研究

The Effect of Wenxin Granules on the Expression of Connexin 43 and Autophagy in Myocardial Infarction Rats

目的:探讨稳心颗粒对心肌梗死大鼠心肌缝隙连接蛋白43表达及自噬水平变化的影响。方法:通过心脏左冠状动脉前降支结扎法构建心肌梗死大鼠模型后,随机分为模型组、稳心颗粒低剂量组、稳心颗粒高剂量组及美托洛尔组,连续给药4周,另设正常组作为空白对照,每组8只大鼠。超声心动图检测各组大鼠左室短轴收缩末期前壁厚度(LVAWs)、左室舒张末期前壁厚度(LVAWd)、左室收缩末期内径(LVIDs)、左室舒张末期内径(LVIDd)以及左室射血分数(LVEF);心脏生理电刺激检测各组大鼠的心室颤动阈值;苏木精-伊红(HE)染色观察心脏组织病理学改变;采用免疫组织化学染色及蛋白免疫印迹法检测心肌缝隙连接蛋白43(CX43)、自噬相关蛋白微管相关蛋白1轻链3(LC3)、酵母自噬相关基因6哺乳动物同源体(Beclin1)、P62蛋白的表达。结果:与正常组比较,模型组大鼠LVAWs、LVAWd、LVEF降低,LVIDs、LVIDd升高(P<0.01);与模型组比较,稳心颗粒低剂量组、稳心颗粒高剂量组、美托洛尔组LVAWs、LVAWd、LVEF升高,LVIDs、LVIDd降低(P<0.05或P<0.01)。模型组大鼠心室颤动阈值明显低于正常组(P<0.01);与模型组比较,稳心颗粒低剂量组、稳心颗粒高剂量组、美托洛尔组心室颤动阈值有所增高(P<0.01)。与正常组比较,模型组LC3、Becli1蛋白平均光密度值明显增加,CX43蛋白平均光密度明显降低(P<0.01);与模型组比较,稳心颗粒高剂量组、美托洛尔组自噬正相关蛋白LC3、Beclin1平均光密度值均明显降低,CX43蛋白平均光密度值增加(P<0.01)。与正常组比较,模型组自噬正相关蛋白LC3Ⅱ/LC3Ⅰ、Beclin1表达增加,自噬负相关蛋白P62表达以及CX43蛋白表达降低(P<0.05);与模型组比较,稳心颗粒低剂量组Beclin1蛋白表达明显减少,P62蛋白表达明显升高(P<0.05),稳心颗粒高剂量组及美托洛尔组LC3Ⅱ、Beclin1蛋白表达明显降低,P62及CX43蛋白表达明显升高(P<0.05)。结论:稳心颗粒可改善心肌梗死大鼠的心功能并预防心律失常,其机制与调节心脏自噬水平和缝隙连接蛋白43表达有关。

Objective:To investigate the effects of Wenxin Granules on the expression of connexin and autophagy in rats with myocardial infarction.Methods:After the rat model of myocardial infarction was established by ligation of the anterior descending branch of the left coronary artery,the rats were randomly divided into model group,Wenxin Granules low-dose group,Wenxin Granules high-dose group,and metoprolol group,which were administered continuously for 4 weeks.The normal group was also set as blank control.Eight rats in each group.The left ventricular end-systolic anterior wall thickness(LVAWs),left ventricular end-diastolic anterior wall thickness(LVAWd),left ventricular end-systolic internal diameter(LVIDs),left ventricular end-diastolic internal diameter(LVIDd),and left ventricular ejection fraction(LVEF)were measured by echocardiography in each group.Cardiac electrophysiological stimulation was used to detect the ventricular fibrillation threshold of each group.Cardiac histopathological changes were observed by hematoxylin-eosin(HE)staining.The expressions of myocardial connexin 43(CX43),autophagy associated protein protein 1 light chain 3(LC3),yeast autophagy associated gene 6 mammillary homologous(Beclin1)and P62 were detected by immunohistochemical staining and Western Blot.Results:Compared with normal group,LVAWs,LVAWd and LVEF in model group were decreased,while LVIDs and LVIDd were increased(P<0.01).Compared with model group,LVAWs,LVAWd and LVEF were increased in Wenxin Granule low-dose group,Wenxin Granule high-dose group,and metoprolol group,while LVIDs and LVIDd were decreased(P<0.05 or P<0.01).The ventricular fibrillation threshold in model group was significantly lower than that in normal group(P<0.01).Compared with model group,the ventricular fibrillation threshold of Wenxin Granule low-dose group,Wenxin Granule high-dose group and metoprolol group was increased(P<0.05).Compared with normal group,the average optical density of LC3 and Becli1 protein in model group was significantly increased,while the average optical density of CX43 protein was significantly decreased(P<0.01).Compared with model group,the average optical density values of autophagy positive correlated proteins LC3 and Beclin1 in Wenxin Granule high-dose group and metoprolol group were significantly decreased,while the average optical density values of CX43 protein were increased(P<0.05).Compared with normal group,positive autophagy correlation protein LC3Ⅱ/LC3Ⅰand Beclin1 expression were increased in model group,while negative autophagy correlation protein P62 expression and CX43 protein expression were decreased(P<0.05).Compared with model group,Beclin1 protein expression was significantly decreased and P62 protein expression was significantly increased in Wenxin Granule low-dose group(P<0.05),LC3Ⅱand Beclin1 protein expressions were significantly decreased in Wenxin Granule high-dose group and metoprolol group,while P62 and CX43 protein expressions were significantly increased(P<0.05).Conclusion:Wenxin Granules can improve cardiac function and prevent arrhythmia in rats with myocardial infarction,and its mechanism is related to regulating cardiac autophagy level and connexin expression.