益气活血方对心肌梗死大鼠心肌细胞凋亡影响的研究

目的观察益气活血方对心肌梗死大鼠心肌细胞凋亡和B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-Associated X protein,Bax)、活化的含半胱氨酸天冬氨酸蛋白水解酶-3(cleaved caspase 3,C-caspase 3)表达情况的影响,探讨益气活血方影响心肌细胞凋亡的作用机制。方法结扎左冠状动脉前降支构建心肌梗死大鼠模型,并随机分为模型组、益气活血组、培哚普利组,假手术组(即对照组,只穿线不结扎),每组大鼠10只。术后第二天开始灌胃给药,以28天作为观察时间点。小动物超声检测各组大鼠心脏结构和功能的变化,HE染色观察心肌组织病理形态变化,TdT介导的dUTP缺口末端标记(TUNEL)染色法检测大鼠心肌细胞凋亡指数,Western blot技术检测心肌梗死边缘区Bax、Bcl-2、C-caspase 3蛋白表达水平。结果与假手术组比较,模型组大鼠左心室射血分数(left venteicular ejection fraction,LVEF)、左室短轴缩短率(left ventricular shortening fraction,LVFS)均显著降低(P<0.01),左心室收缩末期内径(left ventricular end-systolic dimension,LVESD)、左心室舒张末期内径(left ventricular end-diastolic dimension,LVEDD)均升高(P<0.01);两用药组LVEF、LVFS相较于模型组均显著升高(P<0.01),LVESD、LVEDD均降低(P<0.01)。与假手术组相比,模型组心肌细胞凋亡指数明显上升(P<0.01);与模型组比较,益气活血组与培哚普利组大鼠心肌细胞凋亡指数明显降低(P<0.01)。Western blot结果显示,模型组大鼠Bax、C-caspase 3表达与假手术组相比均有明显升高(P<0.01),Bcl-2表达及Bcl-2/Bax相较于假手术组有所降低(P<0.01)。与模型组比较,益气活血组大鼠Bax蛋白表达有所下降(P<0.01),Bcl-2蛋白表达与Bcl-2/Bax显著升高(P<0.05,P<0.01),C-caspase 3蛋白表达有所降低,但差异无统计学意义(P>0.05);相较于模型组,培哚普利组大鼠Bax表达降低(P<0.01),Bcl-2/Bax比值上升(P<0.05),Bcl-2呈升高趋势,C-caspase 3有下降趋势,但差异无统计学意义(P>0.05)。结论益气活血方能够提高心肌梗死大鼠心功能,下调促凋亡蛋白Bax的表达,上调抗凋亡蛋白Bcl-2表达及Bcl-2/Bax比值,从而减轻心肌凋亡损伤以保护心脏。

Effects of Yiqi Huoxue formula on apoptosis of myocardial cells in rats with myocardial infarction

Objective To observe the effect of Yiqi Huoxue formula on apoptosis of myocardial cells and expression of B-cell lymphoma-2(Bcl-2),Bcl-2-Associated X protein(Bax)and cleaved-caspase 3(c-caspase 3).And to explore the mechanism of Yiqi Huoxue formula on apoptosis of myocardial cells.Methods The rat model of myocardial infarction was established by ligating the anterior descending branch of the left coronary artery,and the rats were randomly divided into the model group,the Yiqi Huoxue group,and the perindopril group,sham operation group(that is the control group,only threading without ligation),with 10 rats in each group.Administration by gavage began on the second day after the operation,and the observation time point was 28 days.The changes of heart’s structure and function in each group were detected by ultrasound.The pathological changes of myocardial tissue were observed by HE staining.The apoptosis index of myocardial cells in rats was detected by Tdt-mediated dUTP notch terminal marker(TUNEL)staining.The protein expression of Bax,Bcl-2 and c-caspase 3 in the marginal area of myocardial infarction were detected by western blot.Results Compared with the sham operation group,the left ventricular ejection fraction(LVEF)and left ventricular shortening(LVFS)of the model group rats decreased significantly(P<0.01),and the left ventricular end-systolic dimension(LVESD)and left ventricular end-diastolic dimension(LVEDD)increased(P<0.01).Compared with the model group,LVEF and LVFS in the two treatment groups increased significantly(P<0.01),and LVESD and LVEDD decreased(P<0.01).Compared with the sham operation group,the apoptosis index of myocardial cells in the model group increased significantly(P<0.01).Compared with the model group,the apoptosis index of myocardial cells in two treatment group significantly decreased(P<0.01).Western blot results showed that the expression of Bax and C-caspase 3 in the model group were significantly higher than those in the sham operation group(P<0.01),and the expression of Bcl-2 expression and Bcl-2/Bax decreased than those in the sham operation group(P<0.01).Compared with the model group,the expression of Bax protein in the Yiqi Huoxue group decreased(P<0.01),the expression of Bcl-2 protein and Bcl-2/Bax increased significantly(P<0.05,P<0.01),protein expression of c-caspase 3 decreased,but the difference was not statistically significant(P>0.05).Compared with the model group,expression of Bax in the perindopril group decreased(P<0.01),and the ration of Bcl-2/Bax increased(P<0.05).Bcl-2 showed an upward trend,and c-caspase 3 showed a downward trend,but there was no significant difference(P>0.05).Conclusion Yiqi Huoxue formula can improve cardiac function of rats with myocardial infarction,down-regulating the expression of pro-apoptotic protein Bax,up-regulating the expression of anti-apoptotic protein Bcl-2 and the ration of Bcl-2/Bax,so as to reduce the apoptosis damage of myocardial cells to protect the heart.