摘要
目的:探讨G蛋白偶联受体75(G-protein-coupled receptor 75,GPR75)介导的PLC/PKC信号通路在20-羟二十烷四烯酸(20-hydroxyeicosatetraenoic acid,20-HETE)诱导的乳鼠心肌细胞凋亡中的作用。方法:采用慢病毒转染敲减乳鼠心肌细胞GPR75基因表达;RT-q PCR和Western blot检测GPR75 m RNA及细胞色素C(cytochrome C,Cyt C)和caspase-3蛋白表达;ELISA法检测三磷酸肌醇(inositol triphosphate,IP_(3))含量以及蛋白激酶C(protein kinase C,PKC)和NADPH氧化酶活性;Fluo-3/AM探针法检测细胞内Ca^(2+)浓度;二氢乙啶(dihydroethidine,DHE)荧光探针检测细胞内活性氧簇(reactive oxygen species,ROS)的含量;TUNEL法检测细胞凋亡情况。结果:在培养的乳鼠心肌细胞中,GPR75在m RNA及蛋白水平均有表达,且20-HETE具有促进细胞内GPR75表达的作用(P<0.05);经20-HETE处理后,细胞内IP_(3)含量升高,而敲减GPR75或应用磷脂酶C(phospholipase C,PLC)抑制剂U73122可显著阻断20-HETE的如上效应,细胞内IP;含量降低(P<0.05);敲减GPR75表达、应用PLC抑制剂以及IP_(3)受体阻断剂2-氨基乙氧基二苯基硼酸酯(2-aminoethoxydiphenyl borate,2-APB)预处理,可抑制20-HETE诱导的细胞内Ca^(2+)浓度升高(P<0.05);敲减GPR75表达、应用PKC抑制剂GF109203X或NADPH氧化酶抑制剂apocynin处理后,20-HETE诱导的细胞内ROS生成被抑制(P<0.05);同时,敲减GPR75表达可降低20-HETE作用的细胞中PKC、NADPH氧化酶的活性(P<0.05);最后,敲减GPR75表达、应用U73122或GF109203X,均可减少20-HETE诱导的心肌细胞凋亡率及凋亡相关蛋白Cyt C和caspase-3的表达(P<0.05)。结论:GPR75介导的PLC/PKC信号通路通过引起心肌细胞钙超载、ROS生成增多,参与了20-HETE诱导的乳鼠心肌细胞凋亡进程。
AIM:To investigate the role of PLC/PKC signaling pathway mediated by G-protein-coupled receptor 75(GPR75)in 20-hydroxyeicosatetraenoic acid(20-HETE)-induced apoptosis in neonatal rat cardiomyocytes(NRC-Ms).METHODS:Knockdown of GPR75 expression was performed by lentiviral transfection in the NRCMs.The m RNA expression of GPR75 was detected by RT-q PCR,and the protein levels of GPR75,cytochrome C(Cyt C)and caspase-3were determined by Western blot.Enzyme-linked immunosorbent assay(ELISA)was performed to measure the content of inositol triphosphate(IP_(3))and the activity of protein kinase C(PKC)and NADPH oxidase.The Ca^(2+)-sensitive fluorescent probe Fluo-3/AM was applied to measure the intracellular concentration of Ca^(2+)([Ca^(2+)];).Dihydroethidium(DHE)staining was performed to determine the levels of reactive oxygen species(ROS).TUNEL assay was used to evaluate cardiomyocytes apoptosis.RESULTS:The GPR75 expression both at m RNA and protein levels was confirmed in the NRCMs,and it showed a significant increase after 20-HETE treatment(P<0.05).Treatment of NRCMs with 20-HETE induced a significant increase in IP;production,while knockdown of GPR75 gene expression or pre-treatment with phospholipase C(PLC)inhibitor U73122 significantly blocked the effects of 20-HETE mentioned above(P<0.05).Knockdown of GPR75 gene expression,pre-treatment with U73122 or 2-aminoethoxydiphenyl borate(2-APB;an inhibitor of IP_(3)receptor)significantly reduced 20-HETE-induced increase in[Ca^(2+)];(P<0.05).Moreover,knockdown of GPR75 gene expression or pre-treatment with PKC inhibitor GF109203X or NADPH oxidase inhibitor apocynin led to a decrease in ROS production induced by 20-HETE(P<0.05).Meanwhile,GPR75 gene knockdown was able to inhibit 20-HETE-stimulated activity of PKC and NADPH oxidase(P<0.05).More importantly,GPR75 gene knockdown,U73122 or GF109203X significantly reduced the percentage of apoptotic cells induced by 20-HETE,and down-regulated the expression of apoptosis-related proteins Cyt C and caspase-3(P<0.05).CONCLUSION:The
作者
刘娇莉
吉雨恬
毛凌
郭立荣
韩勇
LIU Jiao-li;JI Yu-tian;MAO Ling;GUO Li-rong;HAN Yong(Department of Physiology,Zunyi Medical University,Zunyi 563000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第1期65-73,共9页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.32060201)。
关键词
20-羟二十烷四烯酸
G蛋白偶联受体75
钙超载
活性氧簇
心肌细胞
细胞凋亡
20-Hydroxyeicosatetraenoic acid
G-protein-coupled receptor 75
Calcium overload
Reactive oxygen species
Cardiomyocytes
Apoptosis
