脂联素及其受体在T2DM大鼠心肌I/R损伤中的作用及其机制

Role and mechanism of adiponectin and its receptor in myocardial I/R injury of T2DM rats

目的检测脂联素(adiponectin,APN)及其受体1(AdipoR 1)的表达水平,探讨脂联素受体激动剂ADP355对二型糖尿病(type 2 diabetes mellitus,T2DM)大鼠心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤的保护作用及其机制。方法60只SD大鼠经高脂高糖喂养,注射链脲佐菌素(streptozotocin,STZ)建立T2DM模型,随机分为3组(每组20只):糖尿病假手术组(sham组)、糖尿病心肌损伤组(I/R组)、ADP355+I/R组(ADP355预处理的心肌I/R损伤)。ADP355+I/R组每周腹腔注射2 ml ADP355(1 mg/kg),I/R组和sham组注射同等体积PBS,连续4周后,I/R组与ADP355+I/R组大鼠结扎冠状动脉建立I/R损伤模型,穿线结扎缺血30 min后,释放结扎线再灌注恢复血流4 h,sham组只穿线不结扎;称量大鼠体质量;ELISA法检测血清中胰岛素和APN含量;HE染色观察心肌组织病理学改变;Evan/TTC双染色测定心肌梗死面积;qRT-PCR法检测心肌组织中APN和AdipoR 1的mRNA表达水平;Western blot法检测心肌组织中p-AMPK/AMPK、p-mTOR/mTOR、LC3Ⅱ/Ⅰ和P62蛋白表达。结果与sham组相比,I/R组和ADP355+I/R组大鼠血清APN含量、心肌组织APN mRNA、AdipoR1 mRNA、P62蛋白表达均下降(P<0.05),而心肌组织损伤、心肌梗死面积、p-AMPK/AMPK、LC3Ⅱ/Ⅰ蛋白比值均上调(P<0.01),I/R组p-mTOR/mTOR上升(P<0.01);与I/R组相比,ADP355+I/R组大鼠血清胰岛素、心肌组织损伤、梗死面积、p-mTOR/mTOR均降低(P<0.05),AdipoR1的mRNA、p-AMPK/AMPK、LC3Ⅱ/Ⅰ蛋白比值、P62蛋白的表达水平均升高(P<0.05)。结论脂联素受体激动剂ADP355预处理对T2DM大鼠心肌I/R损伤有保护作用,其可能通过提高AdipoR 1表达,并激活AMPK途径促进自噬水平改善心肌损伤。

Objective To detect the expression levels of adiponectin(APN)and its receptor 1(AdipoR 1),and explore the protective effect and mechanism of adiponectin receptor agonist ADP355 against myocardial ischemia/reperfusion(I/R)injury in type 2 diabetes mellitus(T2DM)rats.Methods Sixty SD rats were fed with high fat and high sugar,and injected with streptozotocin(STZ)to establish T2DM models.T2DM model rats were randomly divided into 3 groups(n=20):sham diabetic group(sham group),diabetic myocardial I/R injury group(I/R group)and ADP355+I/R group.Rats were intraperitoneally injected with 2 ml ADP355(1 mg/kg)every week for 4 weeks in ADP355+I/R group,and the same volume of PBS in I/R group and sham group.After 4 weeks of continuous injection,I/R injury model was established by ligating the coronary arteries for 30 min ischemia and then reperfusion for 4 h in I/R group and ADP355+I/R group.Rats in sham group were only given ligatures but not ligation.The weight of rats was recorded.The contents of insulin and APN in serum were detected by ELISA.HE staining was used to detect the myocardial injury in rats.The size of myocardial infarction was determined by Evan/TTC double staining.The mRNA expression levels of APN and AdipoR 1 in myocardium were detected by qRT-PCR.The protein expression levels of p-AMPK/AMPK,p-mTOR/mTOR,LC3Ⅱ/Ⅰand P62 in myocardium were detected by Western blot.Results Compared with sham group,the content of APN in serum,the mRNA expression of APN,AdipoR1 and P62 protein in myocardial tissue were significantly decreased in I/R group and ADP355+I/R group(P<0.05),however,myocardial tissue injury,myocardial infarction area,p-AMPK/AMPK,LC3Ⅱ/Ⅰwere significantly up-regulated in I/R group and ADP355+I/R group(P<0.01),and p-mTOR/mTOR was significantly up-regulated in I/R group(P<0.01).Compared with I/R group,serum insulin,myocardial tissue injury,infarction area and p-mTOR/mTOR were significantly decreased in ADP355+I/R group(P<0.05),while AdipoR1 mRNA,p-AMPK/AMPK,LC3Ⅱ/Ⅰand P62 protein expression levels were significantly increased(P<0.05).Conclusion Pretreatment with adiponectin receptor agonist ADP355 has protective effect against myocardial I/R injury in T2DM rats by increasing AdipoR 1 expression and activating AMPK pathway thus promoting the autophagy.