20-HETE通过GPR75受体偶联G_(αq)信号通路诱导H9c2心肌细胞凋亡的作用  

20-HETE induces apoptosis in H9c2 cardiomyocytes via GPR75 receptor coupled G_(αq) signaling pathway

作　　者：刘恋恋 吉雨恬 刘娇莉 韩婧怡 李开远 张淳 韩勇 Liu Lianlian;Ji Yutian;Liu Jiaoli;Han Jingyi;Li Kaiyuan;Zhang Chun;Han Yong(Department of Physiology,School of Basic Medical Sciences,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学基础医学院生理学教研室,贵州遵义563099

出　　处：《遵义医科大学学报》2024年第4期361-370,共10页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:32060201);遵义医科大学研究生科研基金课题(NO:ZYK167);遵义医科大学大学生创新创业训练计划项目(NO:S202310661002)。

摘　　要：目的 探讨GPR75受体偶联G_(αq)信号通路在20-HETE诱导的H9c2心肌细胞凋亡中的作用与机制。方法 H9c2心肌细胞采用慢病毒载体干扰GPR75表达,敲减效率通过RT-qPCR和Western blot法检测;ELISA法检测IP_(3)及cAMP含量;TUNEL法评估细胞凋亡率;分别使用Fluo-4/AM、DHE和JC-1荧光探针检测细胞内Ca^(2+)浓度、ROS含量以及线粒体膜电位(ΔΨm)水平;Western blot检测EGFR、ERK1/2、AKT、GSK3β、Bax、Bcl-2、Cyt C、Caspase-3蛋白表达。结果 慢病毒载体shGPR75转染后,H9c2心肌细胞GPR75受体mRNA和蛋白表达分别降低67.16%和63.99%(P<0.05)。敲减GPR75表达或应用AAA阻断其作用,显著抑制20-HETE诱导的H9c2心肌细胞凋亡(P<0.05),并可逆转ΔΨm下降及凋亡相关蛋白Bax、Cyt C、Caspase-3蛋白表达增高(P<0.05)。20-HETE处理后细胞内IP_(3)含量明显增加(P<0.05),但对cAMP生成无显著影响(P>0.05),而敲减GPR75表达、应用AAA或者PLC信号通路阻断剂U73122预处理后,可显著阻断细胞内IP_(3)生成(P<0.05)。敲减GPR75表达、应用AAA或者U73122预处理后,明显抑制20-HETE诱导的Ca^(2+)浓度升高和ROS生成效应(P<0.05)。20-HETE处理后,GPR75受体下游调控关键蛋白EGFR、ERK1/2、AKT、GSK3β磷酸化水平明显升高(P<0.05),敲减GPR75表达或应用AAA可有效阻断20-HETE上述效应(P<0.05)。最后,阻断PLC或PI3K信号通路,显著抑制20-HETE诱导的心肌细胞凋亡(P<0.05)。结论 20-HETE通过GPR75受体偶联的G_(αq)蛋白及其介导的PLC、EGFR/ERK1/2/AKT/GSK3β信号通路诱导H9c2心肌细胞凋亡。Objective To investigate the role and mechanism of GPR75 receptor-coupled G_(αq) signaling pathway in 20-HETE-induced apoptosis of H9c2 cardiomyocytes.Methods Lentiviral vector was employed to interfere GPR75 expression in H9c2 cardiomyocytes,and RT-qPCR and Western blot was utilized to assess the knockdown efficiency.The levels of IP_(3) and cAMP were quantified using ELISA.Apoptosis rate was evaluated by TUNEL assay.The intracellular Ca^(2+) concentration,ROS content and mitochondrial membrane potential(ΔΨm) were detected by Fluo-4/AM,DHE and JC-1 fluorescent probes,respectively.Protein expressions of EGFR,ERK1/2,AKT,GSK3β,Bax,Bcl-2,Cyt C,and Caspase-3 were analyzed via Western blot.Results After transfection of H9c2 cardiomyocytes with lentiviral vector shGPR75,the mRNA and protein expression of GPR75 receptor decreased by 67.16% and 63.99%,respectively(P<0.05).Knockdown of GPR75 expression or blockade of its effect by AAA significantly attenuated 20-HETE-induced apoptosis of H9c2 cardiomyocytes,reversed the reduction in mitochondrial membrane potential(ΔΨm) as well as the upregulation of apoptosis-related proteins Bax,Cyt C,and Caspase-3(P <0.05).After treatment with 20-HETE,the intracellular IP_(3) content was significantly increased(P <0.05),but there was no significant effect on cAMP generation(P >0.05).Knockdown of GPR75 expression,pretreatment with AAA,or inhibition of the PLC signaling pathway using U73122 effectively abrogated 20-HETE-induced IP_(3) production(P <0.05).Knockdown of GPR75 expression and pretreatment with AAA or U73122 effectively inhibited the increase of Ca^(2+) concentration and ROS generation induced by 20-HETE(P <0.05).After treatment with 20-HETE,the phosphorylation levels of EGFR,ERK1/2,AKT,and GSK3β exhibited a significant increase(P <0.05).Importantly,these effects induced by 20-HETE could be effectively attenuated either by knocking down GPR75 expression or by applying AAA(P <0.05).Finally,blocking PLC or PI3K signaling pathway significantly inhibited 20-HETE-induced car

关 键 词：20-羟二十烷四烯酸 G蛋白偶联受体75 G_(αq)蛋白 心肌细胞凋亡 

分 类 号：R363.2[医药卫生—病理学] R542.2[医药卫生—基础医学]