磷酸肌酸对糖尿病大鼠心肌缺血再灌注时细胞凋亡的影响

Effects of phosphocreatine on apoptosis following myocardial ischemia-reperfusion in diabetic melllitus rats

目的探讨磷酸肌酸对糖尿病大鼠心肌缺血再灌注时细胞凋亡的影响。方法雄性SD大鼠，体重150—170g，采用高脂饲养联合腹腔注射链脲佐菌素的方法制备糖尿病模型，造模成功的27只大鼠饲养2周后，采用随机数字表法，将其随机分为3组（n=9）：假手术组（S组）、缺血再灌注组（I／R组）和磷酸肌酸组（PP组）。I／R组和PP组采用结扎左冠状动脉前降支30min再灌注2h的方法制备心肌缺血再灌注模型，PP组于缺血前30min腹腔注射磷酸肌酸1g／kg，I／R组给予等容量生理盐水。再灌注2h时采集静脉血样，测定血浆肌钙蛋白T（cTnT）的浓度，然后处死大鼠，取心肌组织，采用免疫组化法测定Bcl-2、Bax和Caspase-3表达的水平，并计算Bcl-2与Bax表达的比值（Bcl-2／Bax比），采用TUNEL染色法检测细胞凋亡情况，计算凋亡指数（AI）；电镜下观察心肌细胞超微结构。结果与S组比较，I／R组血浆cTnT浓度升高，心肌组织Bcl-2、Bax和Caspase-3表达上调，Bcl-2／Bax比降低，AI升高（P〈0．05或0．01）；与I／R组比较，PP组血浆cTnT浓度降低，心肌组织Bcl-2表达上调，Bax和Caspase-3表达下调，Bcl-2／Bax比升高，AI降低（P〈0．01）。PP组心肌病理学损伤程度轻于I／R组。结论磷酸肌酸可抑制细胞凋亡，从而减轻糖尿病大鼠心肌缺血再灌注损伤，其机制与上调Bcl-2表达、下调Bax和Caspase-3的表达有关。

Objective To investigate the effects of phosphocreatine on apoptosis following myocardial ischemia-reperfusion （I/R） in diabetic rats. Methods Male SD rats weighing 150-170 g were used in this study. Diabetes niellitus was induced by high fat diet and intraperitoneal streptozotocin. Twenty-seven rats in which diabetes mellitus was successfully induced were randomly divided into 3 groups （ n = 9 each） ： sham operation group （group S）;myocardial I/R group（group I/R ）and phosphocreatine group （group PP）. Myocardial I/R was induced by 30 min occlusion of left anterior descending branch of coronary artery followed by 2 h reperfusion in I/R and PP groups. In group PP phosphocreatine 1 g/kg was given intraperitoneally 30 rain before myocardial I/R. Blood samples were collected at the end of 2 h reperfusion for determination of plasma concentration of calcium troponin T （cTnT）. The animals were then sacrificed and ischemic myocardial specimens were isolated. The expression of Bcl-2, Bax and Caspase-3 in ischemic myocardium was determined and Bcl-2/Bax ratio was calculated. Myocardial apoptosis was detected by TUNEL and apoptotic index was calculated. The uhrastructnre of cardiomyocytes was examined with electron microscope. Results Myocardial I/R significantly increased plasma cTnT concentration and Bcl-2, Bax and Caspase-3 expression in myocardium and apoptosis index and decreased Bcl-2/Bax ratio. Phosphocreatine significantly attenuated I/R-induced abeve-mentioned changes and myocardial damage. Conclusion Phosphocreatine can reduce myocardial I/R injury in diabetic mellitus rats by reducing myocardial apoptosis through up-regulation of Bel-2 expression and down-regulation of Bax and Caspase-3 expression.