摘要
目的:探讨NADPH氧化酶4(NADPH oxidase 4,NOX4)在体外大鼠心肌细胞肥大中的作用及其信号机制。方法:分离SD乳大鼠原代心肌细胞,通过内皮素1(endothelin-1,ET-1;0.1μmol/L)诱导建立体外大鼠心肌细胞肥大模型,采用NOX4抑制剂GKT137831抑制肥大心肌细胞中NOX4表达。实验分为空白对照组、GKT137831组、ET-1组和GKT137831+ET-1组。采用RT-qPCR检测心肌细胞肥大标志物心房钠尿因子(atrial natriuretic factor,ANF)和β-肌球蛋白重链(β-myosin heavy chain,β-MHC)的mRNA表达水平。采用二氢乙啶(dihydroethidium,DHE)染色检测心肌细胞活性氧(reactive oxygen species,ROS)水平。采用Western blot法检测NOX催化亚基NOX4和调节亚基p47^(phox)的蛋白表达,以及蛋白激酶B(protein kinase B,PKB/Akt)、糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)和组蛋白脱乙酰酶4(histone deacetylase 4,HDAC4)的磷酸化水平。结果:ET-1可以诱导体外大鼠心肌细胞肥大,GKT137831显著抑制了ET-1诱导的心肌肥大,降低了心肌细胞表面积及肥大标志物ANF和β-MHC的mRNA表达水平(P<0.01)。GKT137831抑制了NOX中催化亚基NOX4和调节亚基p47^(phox)的蛋白表达(P<0.05或P<0.01),降低了ET-1诱导的心肌细胞中ROS的生成(P<0.05或P<0.01)。GKT137831抑制了ET-1诱导的心肌细胞中Akt、GSK-3β和HDAC4蛋白的磷酸化(P<0.05或P<0.01)。结论:抑制NOX4减轻了ET-1诱导的体外大鼠心肌细胞肥大,该作用可能与其抑制肥大信号通路Akt/GSK-3β的激活,进而抑制转录因子HDAC4蛋白的磷酸化有关。
AIM:To investigate the role of NADPH oxidase 4(NOX4)and its signaling mechanism in cardiac hypertrophy.METHODS:The primary cardiomyocytes of neonatal SD rats were isolated,and the cardiomyocyte hypertrophy model in vitro was induced by endothelin-1(ET-1).The expression of NOX4 in hypertrophic cardiomyocytes was inhibited by NOX4 inhibitor GKT137831.The cells were divided into control group,GKT137831 group,ET-1 group and GKT137831+ET-1 group.RT-qPCR was used to detect the mRNA expression levels of myocardial hypertrophy markers,atrial natriuretic factor(ANF)andβ-myosin heavy chain(β-MHC),in myocardial cells.Intracellular reactive reactive oxygen species(ROS)level was detected by dihydroethidium(DHE)staining.The protein levels of NOX4,p47^(phox),protein kinase B(PKB/Akt),glycogen synthase kinase-3β(GSK-3β)and histone deacetylase 4(HDAC4)were detected by Western blot.RESULTS:ET-1 induced rat cardiomyocyte hypertrophy in vitro.GKT137831 significantly alleviated cardiomyocyte hypertrophy induced by ET-1 and reduced cardiomyocyte surface area and ANF and β-MHC mRNA levels(P<0.01).GKT137831 decreased the production of ROS in cardiomyocytes induced by ET-1 and inhibited the protein expression of NOX catalytic subunit NOX4 and regulatory subunit p47^(phox)(P<0.05 or P<0.01).GKT137831 inhibited the phosphorylation of Akt,GSK-3β and HDAC4 in cardiomyocytes induced by ET-1(P<0.05 or P<0.01).CONCLUSION:Inhibition of NOX4 by GKT137831 suppresses ET-1-induced hypertrophy of rat cardiomyocytes in vitro,which may attribute to the inhibition of hypertrophic signaling pathway Akt/GSK-3βand transcription factor HDAC4 phosphorylation.
作者
王燕鸽
赵昕
张蕾
古心雨
雷婵豪
李瑞芳
WANG Yan-ge;ZHAO Xin;ZHANG Lei;GU Xin-yu;LEI Chan-hao;LI Rui-fang(Department of Pharmacy,School of basic Medicine,Henan University of Science and Technology,Luoyang 471023,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第11期1938-1944,共7页
Chinese Journal of Pathophysiology
基金
河南省自然科学基金资助项目(No.202300410150)。
