促红细胞生成素抑制caspase-3蛋白表达对缺血再灌注大鼠的心脏保护作用

Cardio-protection of erythropoietin on the rats with ischemia-reperfusion injury through inhibiting caspase-3 protein expression

目的研究促红细胞生成素(EPO)对大鼠缺血再灌注(I/R)心肌缺血及梗死面积、心肌细胞凋亡和caspase-3蛋白表达的影响。方法采用在体大鼠心肌I/R模型,将24只健康SD大鼠按随机数字表法随机分成3组:(1)假手术组,以6-0号丝线在冠状动脉左前降支下穿过,不予以结扎;(2)I/R组,以6-0号丝线在冠状动脉左前降支下穿过,并结扎,在结扎线中埋置松解线,45 min后,松解结扎线,实现再灌注24 h;(3)EPO组,手术过程同I/R组,在再灌注开始即刻,给大鼠腹腔内注射重组人EPO(rhEPO),5 000 U/kg。再灌注24 h后用伊文蓝和氯化四唑(TTC)进行心肌染色,测量心肌缺血范围和梗死范围;采用原位缺口末端标记(TUNEL)检测心肌细胞凋亡;采用W estern印迹法检测心肌caspase-3蛋白p17亚基的表达。结果经过45 min缺血和24 h再灌注:心肌染色显示:假手术组心肌无缺血区和梗死区;与假手术组比较,I/R组和EPO组心肌有明显缺血区和梗死区,其缺血范围和梗死范围均显著增大(P<0.01);与I/R组比较,EPO组缺血范围略为缩小,其差异没有显著性(P>0.05),但梗死范围显著缩小(P<0.01)。TUNEL结果显示:与假手术组比较,I/R组和EPO组心肌细胞凋亡显著增加(P<0.01);与I/R组比较,EPO组心肌细胞凋亡显著减少(P<0.01);W estern印迹法结果显示:与假手术组比较,I/R组和EPO组caspase-3蛋白p17亚基表达水平显著升高(P<0.01);与I/R组比较,EPO组caspase-3蛋白p17亚基表达水平显著下降(P<0.01)。结论 EPO显著缩小I/R大鼠心肌缺血和梗死范围,减少心肌细胞凋亡,对I/R心肌具有明显的保护作用,其机制可能与下调caspase-3蛋白表达相关。

Objective To study the protective effects of erythropoietin（EPO） on the myocardium and its influence on the expression of caspase-3 protein in rats during ischemia-reperfusion（I/R） injury.Methods 24 healthy male SD rats were randomly divided into： sham-operated,I/R and EPO groups.In sham-operated group,the left anterior descending（LAD） coronary artery was exposed without further procedures.In I/R group,the LAD coronary artery was reperfused for 24 hours following 45 minutes occlusion.In EPO group,the rats were administered by intraperitoneal injection of recombinant human erythropoietin（rhEPO） 5 000 U/kg,at the beginning of reperfusion.The hemodynamic parameters were determined by multiple channel electrophysiolograph after 24 hour reperfusion.The ischemia and infarct size of hearts were measured by double staining with TTC-Evan′s blue dye.The apoptotic index of cardiomyocytes was investigated by TUNEL method.The expression of caspase-3 protein of cardiac tissues in rats was determined by Western blot assay.Results After 45-minute ischemia and 24-hour reperfusion,there were no differences in systolic pressure（SP）,diastolic pressure（DP） and left ventricular systolic pressure（LVSP） among three groups（P0.05）.Compared with sham-operated group,left ventricular diastolic pressure（LVDP） and left ventricular end diastolic pressure（LVEDP） were significantly increased while maximal rate of increase of ventricular pressure（dp/dtmax） and maximal rate of decrease of ventricular pressure（dp/dtmin） reduced remarkably in I/R group（P0.05）.LVDP and LVEDP were decreased significantly but dp/dtmax and dp/dtmin increased remarkably in EPO group when compared with I/R group.The myocardial infarct size was reduced in EPO group compared with I/R group（P0.05）.The TUNEL positive cardiomyocytes were increased dramatically after I/R injury but compared with I/R group,they were reduced significantly in EPO group（P0.05）.The expression of caspase-3 protein was higher in I/R group than that in sham-operated group but it was significantly reduced in EPO group when compared with I/R group.Conclusions EPO can provide an obvious cardioprotective effect on I/R myocardium.EPO can promote the recovery of myocardial function during I/R,reduce myocardial infarct size and inhibit the apoptosis of cardiomyocytes induced by myocardial I/R injury through downregulation of caspase-3 protein expression.