κ-阿片受体激动剂U50,488H抑制缺血/再灌注心肌线粒体分裂的作用及机制

κ-opioid receptor agonist U50, 488H inhibits mitochondrial fission induced by myocardial ischemia-reperfusion and its underlying mechanism

目的研究外源性κ-阿片受体激动剂U50,488H对缺血/再灌注心肌线粒体分裂的影响及机制。方法将SD大鼠随机分为6组:假手术(Sham)组,心肌缺血/再灌注(MI/R)组,MI/R+κ-阿片受体激动剂U50,488H(MI/R+U)组,MI/R+κ-阿片受体阻断剂nor-BNI+U50,488H(MI/R+N+U)组,MI/R+磷脂酰激醇3-激酶(pi 3-kinases,PI3K)抑制剂wortmannin+U50,488H(MI/R+W+U)组,MI/R+蛋白激酶B(protein kinase B,Akt)抑制剂MK2206+U50,488H(MI/R+M+U)组。血清肌酸激酶(creatine kinase,CK)试剂盒检测血清CK水平;三苯基氯化四氮唑(triphenyte-trazoliumchloride,TTC)-伊文氏蓝双染检测心肌梗死面积;Western blot检测细胞内磷酸化PI3K、磷酸化Akt(Ser 473)、磷酸化线粒体动力相关蛋白(dynamin-related protein,Drp)1(Ser 637)的表达;透射电镜观察线粒体形态。结果与Sham组相比,MI/R组血清CK水平明显升高(P<0.01),出现明显的心肌梗死(P<0.01),磷酸化PI3K、磷酸化Akt表达增加(P<0.05),磷酸化Drp1表达降低(P<0.01),线粒体分裂明显增加(P<0.01);与MI/R组相比,MI/R+U组血清CK水平明显降低(P<0.01),心肌梗死面积减小(P<0.01),磷酸化PI3K、磷酸化Akt和磷酸化Drp1的表达显著增加(P<0.01),线粒体分裂减少(P<0.01)。给予nor-BNI、wortmannin或MK2206后,U50,488H的上述作用均被阻断。结论缺血/再灌注可引起心肌损伤和线粒体分裂增加,κ-阿片受体激活后通过PI3K-Akt通路使Drp1磷酸化增加,抑制缺血/再灌注心肌的线粒体分裂,减轻心肌损伤。

AIM To investigate the effects of U50,488H , a selective kappa-opioid receptor agonist, on mitochondrial fission induced by myocardial ischemia/reperfusion ( MI/R) and its underlying mechanisms. METHODS Sprague Dawley rats were randomly assigned into six groups: Sham group;MI/R group;MI/R + U50,488H (MI/R + U) group;MI/R + nor-BNI ( a selective kappa-opioid receptor antagonist)+ U50,488 H (MI/R + N+U) group;MI/R + Wortmannin ( a PI3K inhibitor)+ U50,488H ( MI/R + W + U) group;and MI/R + MK2206 ( an Akt inhibitor)+ U50,488H (MI/R + M+U) group. Serum creatine kinase (CK) levels were determined and myocardial infarct size was analyzed by TTC/Evens blue double staining. Western blotting was used to examine the expression of phosphorylated PI3K, phosphory lated Akt ( Ser 473) and phosphorylated Drpl ( Ser 637 ) and a transmission electron microscope was used to evaluate mitochondrial morphology. RESULTS Compared with the sham group, MI/R group exhibited increased serum CK (P <0. 01), obvious infarct area and enhanced mitochondrial fission ( P <0. 01), which were accompanied by increased expressions of phosphorylated PI3K and phosphorylated Akt (P<0.05) and decreased expression of phosphorylated Drpl ( P < 0. 01 ). Compared with the MI/R, the MI/R + U group showed decreased serum CK ( P < 0. 01 ), reduced myocardial infarct size ( P < 0. 01 ), inhibited mitochondrial fission, and increased expression of phosphorylated PI3K, phosphorylated Akt and phosphorylated Drpl (P < 0. 01). Effects of U50,488H were blunted by nor-BNI, wortmannin, and MK2206. CONCLUSION MI/R causes myocardial injury and mitochondrial fission. Activation of K-opioid receptors reduces MI/R injury and inhibits mitochondrial fission by increasing the phosphorylation of Drpl at Ser 637 via PI3K/Akt signaling.