胰岛素对大鼠缺血后再灌注心肌细胞凋亡、Bad表达影响及机制研究

Role of Bad in anti-apoptotic mechanism of insulin during post-ischemia reperfusion myocardium injury in rats

目的:观察胰岛素对大鼠缺血后再灌注心肌细胞凋亡及促凋亡蛋白Bad磷酸化表达的影响,以进一步探讨胰岛素在缺血再灌注损伤中的抗凋亡机制。方法:46只SD雄性大鼠随机分为四组:缺血再灌注对照组(I/R,n=12),实施30min缺血/180min再灌注;胰岛素组(INS,n=12),于再灌注期前5min颈静脉内输注胰岛素(60U/L,每小时4ml/kg)至灌注期结束;胰岛素+LY294002组(INS+LY,n=12),再灌注前10min静脉注射LY294002(0.3mg/kg),5min后再静脉输注胰岛素+LY294002(每小时30μg/kg);假手术组(SHAM,n=10):仅给予冠脉穿线,不实施结扎。实验结束后,用原位末端标记法(TUNEL)和DNA梯带法(DNA Ladder)标测细胞凋亡,用免疫组化和Western-blotting法检测磷酸化Bad(pBad)表达。结果:①与SHAM组相比,I/R组凋亡指数(AI)明显增加(P<0.01),pBad表达降低(P<0.05);②与I/R组相比,INS组AI值明显减少(P<0.01),pBad表达升高(P<0.01);③与INS组相比,INS+LY组AI值明显增加(P<0.01),pBad表达降低(P<0.05)。结论:①缺血再灌注可导致心肌组织pBad低表达;②胰岛素对大鼠在体缺血再灌注心肌有明显抗凋亡作用;③PI3K/Akt/Bad途径可能为胰岛素抗缺血再灌注诱导心肌凋亡的重要信号转导机制,其机制可能与胰岛素通过PI3K/Akt途径减少pBad表达有关。

Objective：To observe anti-apoptotic effect of insulin,and explore the role of Bad in anti-apoptotic mechanism of insulin during post-ischemia reperfusion injury in myocardium of rats. Methods： Forty-six male Sprague-Dawley rats were randomly divided into 4 groups： ①I/R group was subjected to ischemia for 30 min and reperfusion for 180 min; ②INS group was subjected to intravenous infusion of Insulin （60 U/L, 4 ml/kg, h） at 5 min before 3 h of reperfusion;③INS＋LY group was subjected to intravenous infusion of LY294002 （0.3 mg/kg） at 10 rain before 3 h of reperfusion and intravenous infusion of insulin and LY294002 （30 μg·kg^-1h^-1） at 5 min before 3 h of reperfusion;④SHAM group was subjected to placing a silk around the left anterior descending coronary artery without a slipknot. At the end of 4 h of reperfusion, apoptosis of cardiomyocytes were assessed through TUNEL and DNA Ladder;Immunochemistry and Western-blotting were used to analyse the expression of phosphorylated Bad in all groups. Results： ①Compared with I/R group, apoptosis index （AI） were significantly decreased,and expression of phosphorylated Bad was prominently higher in INS group. ②There were a remarkably increase in AI and lower expression of phosphorylated Bad in INS＋LY group than that in INS group. Conclusion： ①Ischemia and rcperfusion resulted in lower expression of phosphorylated Bad in myocardium of rats; ②Insulin exerted an anti-apoptotic effect in myocardium of rats during ischemia and reperfusion; ③its mechanism may be involved in higher expression of phosphorylated Bad via P13-K/Akt pathway.