敲减G蛋白偶联受体75表达抑制20-HETE诱导的H9c2心肌细胞凋亡

目的:探讨G蛋白偶联受体75(GPR75)对20-羟二十烷四烯酸(20-HETE)诱导H9c2心肌细胞凋亡的影响及机制。方法:慢病毒转染敲减H9c2心肌细胞GPR75基因表达;TUNEL法检测细胞凋亡率;RT-qPCR法检测GPR75以及凋亡相关蛋白Bcl-2和Bax的mRNA表达;JC-1荧光探针检测线粒体膜电位水平;Western blot法检测GPR75、Bcl-2、Bax及细胞色素C(CytC)蛋白表达;发色底物法检测caspase-3的活性。结果:H9c2心肌细胞在mRNA及蛋白水平均表达GPR75,敲减GPR75抑制了20-HETE诱导的细胞凋亡(P<0.05);同时,敲减GPR75阻断了20-HETE诱导的Bax和CytC表达上调以及caspase-3活性增高作用(P<0.05),逆转了20-HETE诱导的Bcl-2表达下调和线粒体膜电位下降(P<0.05)。结论:20-HETE通过GPR75介导引起线粒体功能紊乱,诱导H9c2心肌细胞凋亡。

Knockdown of G-protein-coupled receptor 75 expression inhibits 20-HETE-induced apoptosis in H9c2 cardiomyocytes

AIM: To investigate the effects of G-protein-coupled receptor 75(GPR75) on 20-hydroxyeicosa-tetraenoic acid(20-HETE)-induced apoptosis in H9 c2 cardiomyocytes and to explore its underlying mechanisms. METHODS: Knockdown of GPR75 expression by lentiviral transfection in the H9 c2 cardiomyocytes was performed. TUNEL assay was used to analyze the apoptosis. The mRNA expression of GPR75 and apoptosis-related molecules Bcl-2 and Bax was measured by RT-qPCR. The fluorescent probe JC-1 was used to measure the mitochondrial membrane potential. Western blot was applied to evaluate the protein expression of GPR75, Bcl-2, Bax and cytochrome C(CytC). The activity of caspase-3 was determined by the method of chromogenic substrate assay. RESULTS: The expression of GPR75 was detected in the H9 c2 cardiomyocytes at mRNA and protein levels. Knockdown of GPR75 expression significantly inhibited the apoptosis of H9 c2 cardiomyocyte induced by 20-HETE(P<0.05). Meanwhile, GPR75 gene knockdown was able to inhibit 20-HETE-stimulated expression of Bax and CytC, and the activity of caspase-3(P<0.05). In contrast, GPR75 gene knockdown reversed the down-regulation of Bcl-2 at mRNA and protein levels, and the decrease in mitochondrial membrane potential induced by 20-HETE treatment(P<0.05). CONCLUSION: 20-HETE induces mitochondrial dysfunction by GPR75 signaling pathway and trigger apoptosis in the H9 c2 cardiomyocytes.