依达拉奉保护H9c2心肌细胞对抗异丙肾上腺素诱导的氧化应激及内质网应激反应  

Myocardial protection of edaravone against isoprenaline-induced oxidative stress and endoplasmic reticulum stress in H9c2 cells

作　　者：黄涌[1] 阮经文[2] 杨春涛[3] 杨战利[3] 莫利球[4] 王秀玉[3] 董小变[5,6] 廖新学[5,6] 冯鉴强[3,4] 

机构地区：[1]中山大学附属第一医院东山院区内科,广东广州510080 [2]中山大学附属第一医院针灸科,广东广州510080 [3]中山大学中山医学院生理学教研室,广东广州510080 [4]中山大学附属第一医院黄埔院区麻醉科,广东广州510080 [5]中山大学附属第一医院心血管内科,广东广州510080 [6]中山大学附属第一医院高血压血管病科,广东广州510080

出　　处：《中国药理学通报》2011年第3期410-415,共6页Chinese Pharmacological Bulletin

基　　金：广东省科技计划资助项目(No2010B080701105;2009B08070101;2007B080701030)

摘　　要：目的探讨依达拉奉(EDA)能否保护H9c2心肌细胞对抗异丙肾上腺素(ISO)诱导的氧化应激和内质网应激(ERS)。方法用ISO处理H9c2心肌细胞,建立β1肾上腺素受体持续兴奋诱导心肌细胞毒性的体外模型。EDA在ISO处理心肌细胞前1 h加入培养基中作为预处理。CCK-8比色法检测细胞存活率;双氯荧光素(DCFH-DA)染色/荧光显微镜照相检测细胞内活性氧(ROS)的含量;罗丹明123(Rh123)染色/荧光显微镜照相检测线粒体膜电位(MMP);Western blot法检测葡萄糖调节蛋白78(GRP78)的表达。结果 ISO在20～100μmol.L-1浓度范围内处理H9c2心肌细胞48 h,呈剂量依赖性地降低细胞存活率;80μmol.L-1ISO处理H9c2心肌细胞可使细胞内ROS含量明显增多及MMP明显降低;80μmol.L-1 ISO处理H9c2心肌细胞0～24 h,可时间依赖性地上调内质网应激蛋白GRP78的表达,其中12 h达到高峰。分别用10、20和40μmol.L-1 EDA预处理1 h可以减弱80μmol.L-1 ISO处理H9c2心肌细胞48h引起的细胞存活率降低,500、1 000和2 000μmol.L-1氧自由基清除剂NAC分别预处理1 h也可减轻ISO诱导的心肌细胞毒性反应;40μmol.L-1的EDA预处理1 h可明显减轻ISO诱导的胞内ROS堆积及MMP降低,并明显抑制ISO引起的GRP78的表达上调。结论 EDA可保护H9c2心肌细胞对抗ISO诱导的损伤作用,其机制可能与抗氧化及抑制内质网应激反应有关。Aim To explore whether edaravone protected H9c2 myocardial cells（H9c2 cells） against oxi dative stress and endoplasmic reticulum stress（ERS） induced by isoprenaline（ISO）.Methods H9c2 cells were exposed to ISO to set up a myocardial toxicity model triggered by persistent excitation of β1 receptor.EDA was added before ISO-treatment.Cell viability was measured by using cell counter kit（CCK-8）.Intracellular reactive oxygen species（ROS） and mitochondrial membrane potential（MMP） were observed by 2′,7′-dichlorfluorescein-diacetate（DCFH-DA） staining and by rhodamine123（Rh123） staining,respectively,followed by photofluorography.The expression of GRP78 was evaluated by Western blot assay.Results Exposure of H9c2 cells to ISO at 20~100 μmol·L-1 for 48 h led to a concentration-dependent decrease in cell viability.When 80 μmol·L^-1 ISO was taken to further study,it was found that pretreatment with EDA at 10,20 and 40 μmol·L^-1 for 1 h could suppress the ISO-induced decrease in cell viability,and so did N-acetyl-L-cysteine（NAC）,a common ROS scavenger,at 500,1000 and 2000 μmol·L^-1.Treatment with 80 μmol·L^-1 ISO caused an intracellular ROS accumulation and MMP loss,and enhanced GRP78 expression time-dependently from 0 to 24 h in H9c2 cell,which was improved by pretreatment with 40 μmol·L^-1 EDA for 1 h.Conclusion EDA can protect H9c2 cells against myocardial injury induced by ISO,which may be associated with anti-oxidation and inhibition of ERS.

关 键 词：依达拉奉 异丙肾上腺素 内质网应激 氧化应激 线粒体膜电位 H9C2心肌细胞 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]