U50,488H抑制缺氧/复氧心肌细胞线粒体分裂作用及机制

U50,488H inhibited mitochondrial division in hypoxia/reoxygenation cardiomyocytes and its mechanism

目的探讨外源性κ-阿片受体(κ-OR)激动剂U50,488H对缺氧/复氧(H/R) H9C2心肌细胞线粒体分裂的作用及机制。方法将H9C2心肌细胞随机分为常氧对照组(Control组)、H/R组、H/R+U50,488H组(H/R+U组)、H/R+κ-OR阻断剂(nor-BNI)+U50,488H组(H/R+N+U组)、H/R+磷脂酰肌醇3-激酶(PI3K)抑制剂wortmannin+U50,488H组(H/R+W+U组)、H/R+蛋白激酶B(Akt)抑制剂MK2206+U50,488H组(H/R+M+U组)。采用CCK8试剂盒检测细胞活力;流式细胞仪检测细胞凋亡率;激光共聚焦显微镜观察细胞线粒体形态;免疫印迹法检测细胞内PI3K、Akt、线粒体动力学分裂相关蛋白1(Drp1)的表达。结果与Control组比较,H/R组细胞活力降低,细胞凋亡率显著增加,线粒体分裂增加,磷酸化PI3K与Akt表达略增加,磷酸化Drp1 Ser637表达显著降低(P <0. 05);与H/R组比较,H/R+U组细胞活力增加,细胞凋亡率下降,线粒体分裂减少,磷酸化PI3K、Akt Ser473与Drp1 Ser637表达均显著增加(P <0. 05)。结论 H/R可引起心肌细胞凋亡及线粒体异常分裂,使用U50,488H激活κ-OR后,可通过PI3K-Akt通路促使Drp1 Ser637磷酸化,抑制H/R心肌细胞线粒体异常分裂,减少细胞凋亡。

Objective To investigate the effect and mechanism of exogenous κ-opioid receptor( κ-or) agonist U50,488 H on mitochondrial division in hypoxia/reoxygenation( H/R) H9C2 cardiomyocytes. Methods H9C2 cardiomyocytes were randomly divided into the normoxic Control group( Control group),H/R group,H/R + U50,488 H group( H/R + N + U group),H/R + κ-or blocker( nor-BNI) +U50,488 H group( H/R + W + U group),H/R + phosphatidylinositol 3-kinase( PI3K) inhibitor wortmannin + U50,488 H group( H/R +W + U group),H/R + protein kinase B( Akt) inhibitor MK2206 + U50,488 H group( H/R + M + U group). Cell activity was detected by CCK8 kit. The apoptosis rate was measured by flow cytometry. The morphology of mitochondria was observed by laser confocal microscopy. The expression of PI3K,Akt and mitochondrial kinetic division related protein 1( Drp1) in cells was detected by Western blot. Results Compared with the Control group,the H/R group showed decreased cell activity,significantly increased apoptosis rate,increased mitochondrial division,slightly increased phosphorylation of PI3K and Akt,and significantly reduced phosphorylation of Drp1 Ser637( P < 0. 05). Compared with the H/R group,the H/R + U group showed increased cell activity,reduced apoptosis rate,reduced mitochondrial division,and significantly increased phosphorylation of PI3K,Akt Ser473 and Drp1 Ser637( P < 0. 05).Conclusion H/R can cause apoptosis of cardiomyocytes and abnormal division of mitochondria. Via activating κ-or for U50,488 H,which can induce phosphorylation of Drp1 Ser637 through pi3k-akt pathway,inhibit abnormal division of mitochondria of H/R cardiomyocytes and reduce apoptosis.