氢吗啡酮后处理对大鼠心肌保护效应的机制:PI3K/Akt信号通路介导的自噬

Mechanism of protective effect of hydromorphone postconditioning on myocardium in rats:PI3K/Akt signaling pathway-mediated autophagy

目的评价氢吗啡酮后处理对大鼠心肌保护效应的机制与磷脂酰肌醇3-激酶/丝氨酸-苏氨酸蛋白激酶(PI3K/Akt)信号通路介导的自噬的关系。方法健康雄性SD大鼠40只,体重220~250 g,采用随机数字表法分为5组(n=8):假手术组(Sham组)、缺血再灌注组(IR组)、氢吗啡酮后处理组(HP组)、PI3K抑制剂组(W组)、氢吗啡酮后处理+PI3K抑制剂组(HP+W组)。采用左冠状动脉前降支结扎30 min再灌注120 min的方法建立心肌缺血再灌注损伤模型。HP组于再灌注前5 min经股静脉注射氢吗啡酮0.1 mg/kg。HP+W组于再灌注前5 min经股静脉注射氢吗啡酮0.1 mg/kg及PI3K抑制剂渥曼青霉素15μg/kg。W组于再灌注前5 min经股静脉注射渥曼青霉素15μg/kg。再灌注结束时,采用TTC染色法确定心肌梗死面积(IS),采用比色法检测血清乳酸脱氢酶(LDH)活性,电镜观察心肌超微结构变化,采用Western blot法检测磷酸化Akt(p-Akt)、微管相关蛋白1轻链3(LC3)表达水平,计算LC3-Ⅱ/Ⅰ比值。结果与Sham组比较,IR组IS、血清LDH活性增加,心肌组织p-Akt表达上调,LC3-Ⅱ/Ⅰ比值升高(P<0.05),心肌细胞自噬空泡增多,细胞超微结构损伤明显;与IR组比较,HP组IS、血清LDH活性降低,心肌组织p-Akt表达上调,LC3-Ⅱ/Ⅰ比值降低(P<0.05),心肌细胞自噬空泡减少,超微结构损伤减轻;与HP组比较,HP+W组IS、血清LDH活性增加,心肌组织p-Akt表达下调,LC3-Ⅱ/Ⅰ比值增加(P<0.05),心肌细胞自噬空泡增多,细胞超微结构损伤加重。结论氢吗啡酮后处理对大鼠心肌保护效应的机制与激活PI3K/Akt信号通路,抑制自噬有关。

Objective To evaluate the relationship between the mechanism of protective effect of hydromorphone postconditioning on myocardium and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway-mediated autophagy in rats.Methods Forty healthy male Sprague-Dawley rats,weighing 220-250 g,were divided into 5 groups(n=8 each)using a random number table method:sham operation group(Sham group),ischemia-reperfusion(I/R)group(group IR),hydromorphone postconditioning group(group HP),PI3K inhibitor group(group W)and hydromorphone postconditioning+PI3K inhibitor group(group HP+W).Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.In group HP,hydromorphone 0.1 mg/kg was injected via femoral vein at 5 min before reperfusion in group HP.In group HP+W,hydromorphone 0.1 mg/kg and wortmannin(PI3K inhibitor)15μg/kg were injected via femoral vein at 5 min before reperfusion.In group W,wortmannin 15μg/kg was injected via femoral vein at 5 min before reperfusion.At the end of reperfusion,the myocardial infarct size(IS)was determined by TTC staining,the activities of serum lactate dehydrogenase(LDH)was detected by colorimetry,myocardial specimens were collected for microscopic examination of the ultrastructure(with a electron microscope),the expression of phosphorylated Akt(p-Akt)and microtubule-associated protein 1 light chain 3(LC3)was determined by Western blot and the ratio of LC3-Ⅱ/Ⅰwas calculated.Results Compared with Sham group,IS and the activities of serum of LDH were significantly increased,p-Akt expression in myocardial tissues was up-regulated,the ratio of LC3-Ⅱ/Ⅰwas increased(P<0.05),autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was obvious in group IR.Compared with group IR,IS and the activities of serum of LDH were significantly decreased,p-Akt expression in myocardial tissues was up-regulated,the ratio of LC3-Ⅱ/Ⅰwas decreased(P<0.05),autophagic vacuoles were decreased and the damage of ultrastructure of cardiomyocytes was attenuated in group HR.Compared with group HR,IS and the activities of serum of LDH were significantly increased,p-Akt expression in myocardial tissues was down-regulated,the ratio of LC3-Ⅱ/Ⅰwas increased(P<0.05),autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was aggravated in group HR+W.Conclusion The mechanism of protective effect of hydromorphone postconditioning on myocardium is related to activation of PI3K/Akt signaling pathway and inhibition of autophagy in rats.