AMPK/p38 MAPK/Nrf2信号通路在糖尿病大鼠心肌缺血再灌注损伤中的作用

Role of AMPK/p38 MAPK/Nrf2 signaling pathway in myocardial ischemia-reperfusion injury in diabetic rats

目的评价一磷酸腺苷依赖的蛋白激酶/p38丝裂原活化蛋白激酶/核因子E2相关因子2(AMPK/p38 MAPK/Nrf2)信号通路在糖尿病大鼠心肌缺血再灌注损伤中的作用。方法清洁级健康SD雄性大鼠,2~3月龄,体重220~280 g,高脂饲料喂养,腹腔注射1%链脲佐菌素50 mg/kg,连续4 d,制备糖尿病模型。取糖尿病模型制备成功的大鼠30只,按随机数字表法分为3组(n=10):假手术组(Sham组)、心肌缺血再灌注组(I/R组)和AMPK抑制剂Compound C+心肌缺血再灌注组(C+I/R组)。采用结扎左冠状动脉前降支30 min、再灌注120 min的方法制备心肌缺血再灌注损伤模型。C+I/R组于缺血前30 min时尾静脉注射Compound C 0.5 mg/kg,Sham组和I/R组尾静脉注射等量生理盐水。再灌注120 min时计算心肌梗死面积百分比,采用ELISA法测定血清CK-MB、LDH浓度,采用ELISA法测定心肌组织谷胱甘肽(GSH)、SOD、ROS水平,采用Western blot法测定心肌组织AMPK、磷酸化AMPK(p-AMPK)、p38 MAPK、磷酸化p38 MAPK(p-p38 MAPK)、Nrf2、血红素氧合酶1(HO-1)的表达。结果与Sham组比较,I/R组心肌梗死面积百分比、血清CK-MB、LDH水平升高,心肌组织GSH和SOD水平降低,ROS水平增加,AMPK、p-AMPK、p-p38 MAPK、Nrf2、HO-1表达上调(P<0.05);与I/R组比较,C+I/R组心肌梗死面积百分比、血清CK-MB、LDH水平升高,心肌组织GSH和SOD水平降低,ROS水平增加,AMPK、Nrf2、HO-1表达下调(P<0.05)。结论AMPK/p38 MAPK/Nrf2信号通路参与了糖尿病大鼠心肌缺血再灌注时内源性抗氧化应激机制。

Objective To evaluate the role of adenosine monophosphate-dependent protein kinase/p38 mitogen-activated protein kinase/nuclear factor E2-associated factor 2(AMPK/p38 MAPK/Nrf2)pathway in myocardial ischemia-reperfusion(I/R)injury in diabetic rats.Methods Clean-grade healthy Sprague-Dawley male rats,aged 2-3 months,weighing 220-280 g,were fed with a high fat diet,and 1%streptozotocin 50 mg/kg was intraperitoneally injected for 4 consecutive days to develop the model of diabetes mellitus.Thirty diabetic rats were divided into 3 groups(n=10 each)using the random number table method:sham operation group(sham group),myocardial I/R group(I/R group),and AMPK inhibitor compound C+myocardial I/R group(C+I/R group).The model of myocardial I/R injury was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion.Compound C 0.5 mg/kg was injected via the caudal vein at 30 min before ischemia in C+I/R group,while the equal volume of normal saline was given instead in Sham group and I/R group.At 120 min of reperfusion,the percentage of myocardial infarct size was calculated,the serum concentrations of creatine kinase isoenzymes(CK-MB)and lactic dehydrogenase(LDH)were determined by enzyme-linked immunosorbent assay,the levels of glutathione(GSH),superoxide dismutase(SOD)and reactive oxygen species(ROS)in myocardial tissues were measured by enzyme-linked immunosorbent assay,and the expression of AMPK,phosphorylated AMPK(p-AMPK),phosphorylated p38 MAPK(p-p38 MAPK),Nrf2 and heme oxygenase-1(HO-1)in myocardium was determined by Western blot.Results Compared with Sham group,the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased,the levels of GSH and SOD in myocardial tissues were decreased,ROS level was increased,and the expression of AMPK,p-AMPK,p-p38 MAPK,Nrf2 and HO-1 was up-regulated in I/R group(P<0.05).Compared with I/R group,the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased,the levels of GSH and SOD in myocardial tissues were decreased,ROS level was increased,and the expression of AMPK,Nrf2 and HO-1 was down-regulated in C+I/R group(P<0.05).Conclusions AMPK/p38 MAPK/Nrf2 signaling pathway is involved in the mechanism of endogenous antioxidant stress during myocardial I/R in diabetic rats.