遵义医科大学学报2024，Vol.47Issue(4)：361-370,10.
20-HETE通过GPR75受体偶联Gαq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 Gαq signaling pathway
摘要
Abstract
Objective To investigate the role and mechanism of GPR75 receptor-coupled Gαq 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 IP3 and cAMP were quantified using ELISA.Apoptosis rate was evaluated by TUNEL assay.The intracellular Ca2+concentration,ROS content and mitochondrial membrane potential(△Ψm)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 de-creased 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 reduc-tion in mitochondrial membrane potential(△Ψm)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 IP3 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 IP3 production(P＜0.05).Knockdown of GPR75 expression and pretreatment with AAA or U73122 effectively inhibited the increase of Ca2+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 cardiomyocyte apoptosis(P＜0.05).Conclusion 20-HETE induces apoptosis of H9c2 cardiomyocytes through GPR75 receptor-coupled Gαq protein and its mediated PLC and EGFR/ERKl/2/AKT/GSK3β signaling pathways.关键词
20-羟二十烷四烯酸/G蛋白偶联受体75/Gαq蛋白/心肌细胞凋亡Key words
20-hydroxyicosaenoic acid/G-protein-coupled receptor 75/Gαq protein/cardiomyocyte apoptosis分类
医药卫生引用本文复制引用
刘恋恋,吉雨恬,刘娇莉,韩婧怡,李开远,张淳,韩勇..20-HETE通过GPR75受体偶联Gαq信号通路诱导H9c2心肌细胞凋亡的作用[J].遵义医科大学学报,2024,47(4):361-370,10.基金项目
国家自然科学基金资助项目(NO:32060201) （NO:32060201）
遵义医科大学研究生科研基金课题(NO:ZYK167) （NO:ZYK167）
遵义医科大学大学生创新创业训练计划项目(NO:S202310661002). （NO:S202310661002）
