G蛋白偶联雌激素受体1对血管紧张素Ⅱ诱导心肌细胞肥大的影响及其机制  

Effect of G Protein Coupled Estrogen Receptor 1 on Angiotensin Ⅱ-induced Hypertrophy of Cultured Neonatal Rat Cardiomyocytes

作　　者：裴慧 王立启[2] 王磊[2] 王维[3] 毕秀萍[2] 才晓君[2] 苏国海[2] 赵卓[2] PEI Hui;WANG Li-qi;WANG Lei;WANG Wei;BI Xiu-ping;CAI Xiao-jun;SU Guo-hai;ZHAO Zhuo(Shandong University College of Clinical Medicine, Jinan （250012）, Shandong, China)

机构地区：[1]山东大学临床医学院,山东省济南市250012 [2]山东大学附属济南市中心医院心内科 [3]山东省胸科医院心脏中心

出　　处：《中国循环杂志》2018年第5期490-495,共6页Chinese Circulation Journal

基　　金：国家自然基金面上项目(81270715)

摘　　要：目的:观察激活G蛋白偶联雌激素受体1(GPER1)对血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大的影响,并探讨其可能的机制。方法:2~3日龄乳鼠心肌细胞体外原代培养。利用串联质谱标签(TMT)蛋白质谱技术检测蛋白表达差异,并作相关生物信息学分析,筛查其可能的调节机制。机制研究分6组,空白对照组、AngⅡ组、AngⅡ+GPER1激活剂(G1)组、AngⅡ+G1+GPER1抑制剂(G15)组、AngⅡ+G1+细胞外调节蛋白激酶(ERK)抑制剂(U0126)组和AngⅡ+G1+丝氨酸/苏氨酸激酶(AKT)抑制剂(MK2206)组,各组n=3。检测各组心肌细胞GPER1的表达,心房钠尿肽(ANP)和B型利钠肽(BNP)的m RNA水平,ERK、AKT蛋白的表达及其相互作用,以及对自噬相关蛋白胞浆型自噬标记轻链3(LC3II)和膜型自噬标记轻链3(LC3I)的调控。流式细胞技术检测GPER1对细胞凋亡的影响。结果:AngⅡ诱导心肌细胞肥大呈浓度依赖性,ANP和BNP m RNA水平梯度升高(P<0.05)。细胞免疫荧光染色显示,心肌细胞上存在GPER1蛋白表达。荧光定量逆转录聚合酶链式反应(q RT-PCR)结果显示,激活剂G1激活GPER1,并以浓度依赖方式抑制心肌细胞ANP和BNP m RNA水平(P<0.05);在AngⅡ+G1+G15组,心肌细胞ANP和BNP m RNA水平重新升高(P<0.05)。蛋白免疫印迹法(Western-blot)结果显示,与空白对照组或AngⅡ组相比,AngⅡ+G1组p-ERK、p-AKT蛋白表达增强(P<0.05);与AngⅡ+G1组相比,AngⅡ+G1+G15组p-ERK和p-AKT蛋白表达降低(P<0.05),AngⅡ+G1+MK2206组p-ERK、p-AKT蛋白表达水平和ANP、BNP m RNA水平降低(P<0.05);AngⅡ+G1+U0126组对G1诱导的p-AKT蛋白的表达无影响。流式细胞技术显示,AngⅡ+G1组与AngⅡ组心肌细胞凋亡水平无差异(P>0.05)。AngⅡ组较空白对照组LC3II/LC3I比值增大,其自噬水平显著升高(P<0.01)。而AngⅡ+G1组较AngⅡ组LC3II/LC3I比值降低,其心肌细胞自噬水平降低(P<0.01)。结论:GPER1的激活抑制心肌细胞肥大,其作用机制可能与AKT和ERK信号通路以及细Objectives： To observe the effect of activated G-protein coupled estrogen receptor 1（GPER1） on Angiotensin II（AngII）-induced hypertrophy of cultured neonatal rat cardiomyocytes and explore related mechanisms.Methods： Primary cardiomyocytes derived from 2-to 3-day-old neonatal rats were cultured in vitro. Tandem mass tags（TMT） protein mass spectrometry was used to examine protein expressions; relevant bioinformatics analysis was performed to screen the possible regulatory mechanisms. Cardiomyocytes were divided into 6 groups：（1）Blank control group,（2）AngII group,（3）AngII＋G1（GPER1 activator） group,（4）AngII＋G1＋G15（GPER1 inhibitor） group,（5）AngII＋G1＋U0126（extracellular ERK inhibitor） group and（6）AngII＋G1＋MK2206（AKT inhibitor） group（n=3 for each group）. Cardiomyocytes GPER1 expressions, m RNA levels of atrial natriuretic peptide（ANP） and B-type natriuretic peptide（BNP）, protein levelsof ERK, AKT with their interactions, autophagy-related proteins LC3II and LC3I were compared among different groups; impact of GPER1 on cardiomyocytes apoptosis was detected by flowcytometry. Results： AngII induced cardiomyocytes hypertrophy and upregulation of ANP and BNP m RNA levels in a dosedependent manner（P〈0.05）. GPER1 expression could be detected on cardiomyocytes by Immunofluorescence technique. q RT-PCR results showed that GPER1 was activated by the specific activator G1 and m RNA expressions of ANP and BNP were inhibited in a dose-dependent manner by the specific activator G1（P〈0.05）; m RNA levels of ANP and BNP were reelevated in AngII＋G1＋G15 group（P〈0.05）. Western blotting results showed that protein expression of p-ERK and p-AKT was significantly higher in AngII group and AngII＋G1 group than in blank control group（P〈0.05）, significantly reduced in AngII＋G1＋G15 group compared with AngII＋G1 group（P〈0.05）; decreased expressions of p-ERK, p-AKT and m RNA levels of ANP,BNP were also detected in AngII＋G

关 键 词：心肌细胞肥大 G蛋白偶联雌激素受体1 血管紧张素Ⅱ 

分 类 号：R54[医药卫生—心血管疾病]