人参皂苷Rg1对心肌细胞的影响及其信号传导机制  

Effect of Ginsenoside-rg1 on Rat's Cardiomyocytes With its Mechanism of Signal Pathway in vitro

作　　者：刘燃[1] 宋蕊[1] 袁丽[1] 凌露[1] 杨萍[2] 郭家智[2] 张戈[1] 陆地[2] 孙林[1] 

机构地区：[1]昆明医科大学第二附属医院心内科三病区,云南省昆明市650101 [2]昆明医科大学生物医学工程中心

出　　处：《中国循环杂志》2015年第11期1096-1100,共5页Chinese Circulation Journal

基　　金：国家自然科学基金(81260061);云南省科技厅-昆明医科大学联合专项基金(2012FB046;2014FB047);云南省科技厅-昆明医科大学应用基础研究联合专项重点项目(2013FB101)

摘　　要：目的:从细胞和分子层面探讨人参皂苷Rg1(G-Rg1)对心肌细胞的影响及其信号传导机制。方法:体外培养大鼠H9c2心肌细胞,按实验要求随机分13组,每组5个平行孔,分别为空白对照组,单纯缺血缺氧2 h、6 h、12 h、24 h、48 h组,G-Rg1 5μmol/L、10μmol/L、50μmol/L组,缺氧诱导因子-1α(HIF-1α)的特异性抑制剂(YC-1)组,YC-1+G-Rg1组,蛋白激酶B(Akt)蛋白的磷酸化特异性抑制剂(Wortmannin)组,Wortmannin+G-Rg1组。检测G-Rg1、缺血缺氧以及YC-1对心肌细胞活力及心肌细胞损伤的影响。同时用逆转录聚合酶链反应(RT-PCR)检测心肌细胞内HIF-1α、葡萄糖载体蛋白-1(GLUT-1)和血红素氧合酶-1(HO-1)的信使核糖核酸mR NA表达水平变化。用蛋白质免疫印迹法检测HIF-1α、GLUT-1和HO-1、活化转录因子-6(ATF-6)、抑制CCAAT/增强子结合蛋白同源蛋白(CHOP)和Akt等细胞信号通路蛋白表达水平变化。结果:缺血缺氧时间与心肌细胞活力呈负相关(r=-0.8580,P<0.05),与乳酸脱氢酶溢出率呈正相关(r=0.9201,P<0.05)。G-Rg1 10μmol/L组较单纯缺血缺氧24 h组细胞活力明显回升(87.8%、62.6%,P<0.05),细胞培养上清乳酸脱氢酶(LDH)含量明显减少(25.0%、74.8%,P<0.05),且上调了HIF-1α、GLUT-1、HO-1的mR NA表达水平(P<0.05)。蛋白质免疫印迹法分析显示:YC-1+G-Rg1组较G-Rg1组,心肌细胞活力下降(68.0%,87.8%,P<0.05),细胞培养上清LDH含量增加(56.4%,25.0%,P<0.05),同时YC-1拮抗了G-Rg1对HIF-1α、GLUT-1、HO-1、ATF-6和CHOP的蛋白表达水平的调控(P<0.05);Wortmannin可拮抗G-Rg1对HIF-1α和CHOP的蛋白表达水平的调控(P<0.05),及对Akt两个磷酸化位点的激活作用(P<0.05)。结论:心肌细胞损伤程度与缺血缺氧时间有关;G-Rg1对心肌细胞具有保护作用,其机制激活HIF-1α及其下游因子的表达并抑制了内质网应激效应,可能与G-Rg1激活Akt有关。Objective： To investigate the effect of ginsenoside-rg1 （G-Rg1） on rat’s cardiomyocytes H9c2 with its mechanism of signal pathwayin vitro. Methods： H9c2 cells were cultured and treated in different conditions by following groups：①Blank control group,②Hypoxia alone group, the cells were treated for （2, 6, 12, 24, 48） hr respectively,③G-Rg1 group, the cells were treated by G-Rg1 at （5, 10, 50） μmol/L respectively,④YC-1 group, which is the speciifc inhibitor of hypoxia inducible factor-1α （HIF-1α）,⑤YC-1 ＋ G-Rg1 group,⑥Wortmannin group, which is the speciifc inhibitor for protein kinase B （Akt） phosphorylation and⑦Wortmannin ＋ G-Rg1 group. Each experiment was conducted with 5 replicates. The effects of G-Rg1, hypoxia and YC-1 on cell activity and injury were studied; intracellular mRNA expressions of HIF-1α, glucose＆amp;nbsp;transporter-1 （GLUT-1） and heme oxygenase-1 （HO-1） were examined by RT-PCR; protein expressions of HIF-1α, GLUT-1, HO-1, activating transcription factor-6 （ATF-6）, CCAAT/enhancer binding protein homologous protein （CHOP） and Akt with its signal pathway factors were measured by Western blot analysis. Results： The time of hypoxia was negatively related to cell activity （r=-0.8580,P〈0.05） and positively related to LDH overlfow rate （r=0.9201,P〈0.05）. G-Rg1 （10μmol/L） group showed increased cell activity than Hypoxia alone （24 hr） group （87.8% vs 62.6 %,P〈0.05）, while decreased LDH overlfow （25.0% vs 74.8%,P〈0.05）, and up-regulated mRNA expressions of HIF-1α, GLUT-1 and HO-1, P〈0.05. YC-1＋ G-Rg1 group had decreased cell activity than G-Rg1 group （68.0% vs 87.8%,P〈0.05）, while increased LDH overlfow （56.4% vs 25.0%,P〈0.05）. Meanwhile, YC-1 clashed the effect of G-Rg1 on protein expressions of HIF-1α, GLUT-1, HO-1, ATF-6 and CHOP,P〈0.05; wortmannin clashed the effect of G-Rg1 on protein expressions of HIF-1α, CHOP,P〈0.05 and suppressed the two phosphorylation sites for Akt ac

关 键 词：人参皂苷RG1 磷脂酰肌醇3激酶 缺氧诱导因子-1Α 内质网应激 凋亡 

分 类 号：R54[医药卫生—心血管疾病]