参附注射液对缺血/再灌注损伤诱导大鼠心肌细胞凋亡的实验研究

The effect of Shenfu on ischemia-reperfusion induced cardiomyocyte apoptosis in rats

目的:通过观察参附注射液对缺血/再灌注损伤时,心肌细胞超微结构及心肌细胞凋亡参数的影响,探讨参附注射液拮抗心肌缺血/再灌注损伤的作用机制。方法:建立心肌缺血/再灌注损伤模型,结扎大鼠左冠状动脉前降支(LAD)30min,再灌注10min。将46只大鼠随机分为5组,(Ⅰ)假手术组;(Ⅱ)对照组心肌缺血30min+生理盐水;(Ⅲ)对照组心肌缺血30min/再灌注10min+生理盐水;(Ⅳ)给药组心肌缺血30min+参附注射液;(Ⅴ)给药组心肌缺血30min/再灌注10min+参附注射液。以上各组分别采集相同部位的心室肌组织;应用透射电镜观察心肌细胞超微结构,通过CIMAS多功能真彩色病理图像分析CIMAS系统对心肌细胞线粒体进行体视学分析。原位标记TUNEL法凋亡的心肌细胞,免疫组化方法检测心肌细胞Bcl-2、Bax蛋白的表达,病理图像分析系统进行凋亡心肌细胞、Bcl-2及Bax蛋白的表达分析。结果:与假手术组比较,给药组(Ⅳ、Ⅴ)心肌细胞的Bcl-2蛋白表达显著增多(P<0.05),Bax蛋白表达略增加(P>0.05)和显著降低(P<0.05),心肌细胞凋亡明显减少(P>0.05);心肌细胞组织结构损害明显减轻;对照组与假手术组比较线粒体有显著变化(P<0.01),给药组(Ⅳ、Ⅴ)与假手术组比较线粒体变化程度差异无统计学意义(P>0.05)。结论:参附注射液能明显抑制心肌缺血/再灌注损伤引起的心肌细胞凋亡,其作用机制可能与上调Bcl-2蛋白表达,下调Bax蛋白表达和保护线粒体相关。

Objective：To observe the effect of Shenfu injection on the ultrastructure and apoptosis of myocytes in rats with myocardial ischemia/reperfusion injuries,and to explore the protective mechanisms of Shenfu injection on myocytes.Methods：The myocardial ischemia /reperfusion models is established by ligating the left anterior descending coronary arteries（LAD） of rats for 30 min,and then reperfused for 10 min.A total of 46 rats were divided into 5 groups,（Ⅰ）Sham-operated group;（Ⅱ） myocardial ischemia 30 min ＋ saline;（Ⅲ） myocardial ischemia 30 min/reperfusion 10 min ＋ saline;（Ⅳ） treatment group myocardial ischemia 30 min ＋ Shenfu injection;（Ⅴ） treatment group myocardial ischemia 30 min/reperfusion 10 min ＋ Shenfu injection.Each group was collected the ventricular muscle tissue from the same part of the heart.Transmission electron microscopy was applied to observe the changes in myocardial ultrastructure,at 25,000-fold magnification,the changes of myocardial mitochondrial ultrastructure were analyzed by CIMAS image system.Myocardial cell apoptosis was determined by TUNEL method,the expression of Bcl-2,Bax protein in myocardial cells was detected with immunohistochemistry technique.The expression of Bcl-2,Bax protein and cellapoptosis were ana-lyzed by pathological image analysis system.Results：Compared to control group（Ⅰ）,in Shenfu-treated group（Ⅳ、Ⅴ）,the expression of Bcl-2 protein was increased significantly（P 0.01）,and a slight increase of Bax protein expression in Ⅳand significantly reduced（P 0.05,P 0.05）.Shenfu-treated group significantly reduced myocardial apoptosis（P 0.05）;Myocardial cell ultrastructure shows inⅤtreatment group had significantly reduced the damage;Myocardial ultrastructure stereologic analysis showed that the control group compared with the sham-operated group had a significant change in mitochondria（P 0.01）,Shenfu-treated group（Ⅳ、 Ⅴ） compared with the sham-operated group of mitochondrial degree of change was no significant difference（P 0.05）.Conclusion：Shenfu injection could inhibit myocardial cell apoptosis in rats with myocardial ischemia/reperfusion and its mechanism may be through up-regulating Bcl-2 protein expression and down-regulating Bax protein expression.