吡格列酮对缺血再灌注心肌细胞凋亡影响的实验研究

The effect of pioglitazone on apoptotic cardiomyocytes for ischemia reperfusion

目的观察吡格列酮对大鼠在体心肌缺血再灌注时心肌细胞凋亡的影响。方法实验动物随机分为2组,一为缺血30min再灌注30min组,进一步分为假手术组(n=5)、模型组(即溶剂对照组,n=6)和吡格列酮组(3mg/kg,n=7),测定心肌梗死面积;另一为缺血30min再灌注2h组,然后进一步分为假手术组(n=5)、模型组(n=6)及吡格列酮0·3mg/kg组(n=6)、1mg/kg组(n=7)和3mg/kg组(n=6),各用药组于缺血前30min静脉注射给药。然后,取心脏标本,石蜡包埋后切片,免疫组化检测凋亡蛋白Bax、Bcl-2、Caspase-3、PPARγ蛋白质表达,原位杂交方法检测PPARγmRNA表达。TUNEL法和DNA凝胶电泳观察心肌细胞凋亡。结果(1)与模型组比较,吡格列酮组梗死面积与缺血区面积之比减少28%(P<0·01),梗死面积与左室面积之比减少32%(P<0·01);(2)免疫组化和原位杂交结果示:吡格列酮0·3、1、3mg/kg可呈剂量依赖性减少Bax、Caspase-3,增加Bcl-2、PPARγ蛋白质以及PPARγmRNA表达;(3)TUNEL法检测吡格列酮可减少心肌细胞凋亡指数,3组作用均显著(P<0·05),但DNA凝胶电泳模型组、吡格列酮0·3、1mg/kg可见到DNA梯带,假手术组和吡格列酮3mg/kg则无DNA梯带。结论吡格列酮预处理可通过减少心肌细胞凋亡和梗死面积起到抗缺血再灌注损伤的作用。

Objective This study was to investigate the effect of pioglitazone on apoptotic cardiomyocytes with the model of ischemia-reperfusion at rat heart in vivo. Methods Sprague-Dawley rats were randomly divided into two groups. One was 30 min reperfusion group, which was subdivided into sham （ n = 5 ） , model （ vehicle, n = 6 ） and pioglitazone 3 mg/kg （ n = 7 ） with 30 min ischemia followed by 30 min reperfusion to detect the area of myocardial infarction （MI）. Another was 2 h reperfusion group, which was further subdivided into sham （n = 5 ） , model （vehicle, n = 6 ） , and pioglitazone 0. 3 mg/kg （n = 6）, 1 mg/kg （n = 7） and 3 mg/kg （n = 6）. Apart from the sham, pioglitazone and vehicle were administered intravenously 30 min before occlusion. Then hearts were excised, paraffined and cut into 4μm thick. Immunohistochemistry, in situ hybridization, TUNEL and DNA agarose gel electrophoresis were performed to detect the expression of Bax, Bcl-2, Caspase-3 and PPARy protein and PPARy mRNA. Results （1） Compared with model, nec/aar of pioglitazone decreased by 28% （ P 〈 0. 01 ） . The nec/lv ratio reduced by 32% （P 〈 0. 01 ）. （2）In a dose-dependent manner, the expressions of Bax and Caspase-3 were depressed, while the expression of Bcl-2, PPARy protein and PPARy mRNA were enhanced by pioglitazone. （3）The apoptotic index of subgroups injected pioglitazone reduced significantly by TUNEL compared with model （ P 〈 0. 05 ）. Agarose gel electrophoresis demonstrated that DNA ladder existed in model, pioglitazone 0. 3 mg/kg and pioglitazone 1 mg/kg , but not pioglitazone 3 mg/kg. Conclusions Pioglitazone could protect the heart from I/R injury evidenced by the improvement in the expression of PPARy at the levels of protein and mRNA after pioglitazone administrated, and by the decrease in the apoptotic cardiomyocytes.