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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 the effects of insulin on myocardial cell apoptosis and the expression of pro-apoptotic protein Bad after ischemia in rats, so as to further explore the anti-apoptotic mechanism of insulin in ischemia-reperfusion injury. Methods: Forty-four SD male rats were randomly divided into four groups: ischemia / reperfusion control group (I / R, n = 12), ischemia 30 min Insulin + LY294002 group (INS + LY, n = 12) received intravenous injection of LY294002 (0.3 min before reperfusion) at 5 min before reperfusion mg / kg). Insulin + LY294002 (30μg / kg / h) was infused intravenously 5 minutes later. Sham operation group (SHAM, n = 10) was given coronary artery only. After the experiment, apoptosis was detected by TUNEL and DNA Ladder, and the expression of Bad (pBad) was detected by immunohistochemistry and Western-blotting. Results: ①Apoptosis index (AI) in I / R group was significantly increased (P <0.01) and pBad expression was decreased compared with SHAM group (P <0.05); ② Compared with I / R group, AI in INS group was significantly (P <0.01). (3) Compared with INS group, AI in INS + LY group increased significantly (P <0.01) and pBad expression decreased (P <0.05). Conclusion: ischemia-reperfusion can lead to low expression of pBad in myocardium; ② insulin has obvious anti-apoptotic effect on ischemia-reperfusion myocardium in rats; ③ PI3K / Akt / Bad pathway may be induced by insulin resistance to ischemia-reperfusion The mechanism of apoptosis, an important signal transduction mechanism, may be related to the decrease of pBad expression by insulin through the PI3K / Akt pathway.