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.
出处
《中国药理学通报》
CAS
CSCD
北大核心
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)
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
edaravone
isoprenaline
endoplasmic reticulum stress
oxidative stress
mitochondrial membrane potential
H9c2 myocardial cells
