槲皮素保护H_2O_2诱导的H9C2心肌细胞氧化损伤的作用机制  

Protective Mechanism of Quercetin on Oxidative Injury in H9C2 Cardiomyocytes

作　　者：霍魁媛 常宏[1] 王勇[1] 李春[1] 唐炳华[1] 

机构地区：[1]北京中医药大学基础医学院,北京100029

出　　处：《中国实验方剂学杂志》2016年第15期122-127,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家"十二五"科技支撑计划项目(2012BAI29B07);国家自然科学基金项目(81202788;81473456;81470191;81302908;81530100);北京市自然科学基金项目(7142099);北京中医药大学优秀青年基金项目(2015-JYB-XYQ001)

摘　　要：目的:探讨在H_2O_2诱导的氧化应激损伤中槲皮素对H9C2心肌细胞存活率、活性氧分子(ROS)、还原型辅酶Ⅱ(NADPH)氧化酶、细胞凋亡等关键因子的影响,初步阐明槲皮素对心肌细胞的保护机制。方法:H_2O_2刺激H9C2心肌细胞建立氧化应激损伤模型,分为正常组、模型组、槲皮素组,采用噻唑蓝(MTT)法检测心肌细胞活力,活性氧检测试剂盒检测槲皮素处理后ROS变化,流式细胞术检测各组细胞凋亡率,实时荧光定量PCR(Real-time PCR)与蛋白免疫印迹法(Western blot)分别检测细胞氧化应激反应、细胞凋亡机制相关分子[NADPH氧化酶-4(NOX-4),NADPH氧化酶亚单位p22phox,B淋巴细胞瘤-2原瘤基因(Bcl-2),Bcl-2相关X基因(Bax),半胱胺酸蛋白酶-8(Caspase-8)]基因和蛋白表达水平。结果:10μmol·L-1槲皮素具有显著的抗氧化、抗凋亡作用。与模型组比较,槲皮素组细胞存活率明显提高(P<0.05),ROS水平与细胞凋亡率明显降低(P<0.05,P<0.01)。此外,槲皮素组中Bcl-2,NOX-4基因表达上调(P<0.05);p22phox,Bax,Caspase-8基因表达明显下调(P<0.05)。同时槲皮素能够抑制Bax,p22phox蛋白的表达(P<0.05),上调磷脂酰肌醇-3激酶(PI3K),Bcl-2蛋白的表达(P<0.05)。结论:10μmol·L-1槲皮素能有效减少H_2O_2介导的心肌细胞氧化损伤,进而抑制心肌细胞凋亡。该机制可能是通过调节NADPH氧化酶亚型NOX-4与p22phox,减少ROS生成,激活PI3K表达,升高Bcl-2实现。Objective： To preliminarily investigate the protective effects of quercetin on the survival rate of H_2O_2-induced oxidative injury in H9C2 cardiomyocytes,and such key factors as reactive oxygen species（ ROS）,nicotinamide adenosine denucleotide hydro-phosphoric acid（ NADPH） oxidase and apoptosis,in order to clarify the underlying mechanism. Method： A model of hydrogen peroxide H_2O_2-induced H9C2 cells oxidative injury was established in vitro,and divided into the normal group,the model group and the quercetin group. Cell viability was examined with an methylthiazolyldiphenyl-tetrazolium bromide（ MTT） assay to determine the available concentrations of H_2O_2 and quercetin. The effects of quercetin on ROS level and apoptosis rate were detected by2＇,7＇-Dichloroflurescin diacetate（ DCFH-DA） and flow-cytometry,respectively. We also used Real-time PCR and Western blot to examine the effect of quercetin on oxidative stress and apoptosis mechanism-related molecules（ NOX-4,p22 phox,Bcl-2,Bax and Caspase-8） genes and protein expression level. Result： Quercetin of 10μmol·L- 1has significant anti-oxidative and anti-apoptotic effect. Cell survival rate increased obviously in quercetin group compared with H_2O_2-model group（ P 〈0. 05）,ROS level and apoptosis rate significantly decreased（ P 〈0. 05,P 〈0. 01）. Moreover,quercetin had an up-regulation on PI3 K,Bcl-2 and NOX4（ P 〈0. 05） and downregulation of p22 phox,Bax and Caspase-8（ P 〈0. 05）. Meanwhile,quercetin can inhibit Bax,p22 phoxprotein expressions（ P 〈0. 05）,and up-regulate PI3 K,Bcl-2 protein expressions（ P 〈0. 05）. Conclusion： Quercetin protects H9C2 cells from H_2O_2-induced oxidative injury and inhibit myocardial cell apoptosis by regulating NOX-4p22 phox,reducing intracellular ROS level,activating PI3 K and increasing Bcl-2.

关 键 词：槲皮素 H2O2 氧化应激 凋亡 细胞存活率 活性氧分子 还原型辅酶Ⅱ氧化酶-4 

分 类 号：R285.5[医药卫生—中药学]