葫芦茶苷保护活性氮诱导内皮细胞线粒体损伤的作用研究  

Study on the protective effect of Tadehaginoside on the damage of endothelial cell mitochondria induced by reactive nitrogen

作　　者：黄巧凡 吴昊霖 徐怡倩 樊好飞 方星悦 王爱萍 虞道锐 孟庆雯 刘启兵 HUANG Qiao-fan;WU Hao-lin;XU Yi-qian;FAN Hao-fei;FANG Xing-yue;WANG Ai-ping;YU Dao-rui;MENG Qing-wen;LIU Qi-bing(Department of Pharmacology,School of Basic Medicine and Life Sciences,Hainan Medical University,Haikou 571199,China;Key Laboratory of Brain Science Research and Transformation in Tropical Environment of Hainan Province,Haikou 571199 China)

机构地区：[1]海南医学院基础医学与生命科学学院药理学教研室,海南海口571199 [2]海南省热带脑科学研究与转化重点实验室,海南海口571199

出　　处：《海南医学院学报》2021年第24期1846-1851,共6页Journal of Hainan Medical University

基　　金：国家自然科学基金资助项目(81960663);2019年海南医学院大学生创新训练计划项目(X201911810036)。

摘　　要：目的:通过葫芦茶苷作用于活性氮致血管内皮细胞损伤模型,探讨其对血管内皮细胞损伤的保护作用。方法:采用MTT比色法检测葫芦茶苷在5~160μmol/L浓度范围对EA.hy 926内皮细胞存活率的影响;EA.hy 926内皮细胞预给药葫芦茶苷1 h后,给予0.5 mmol/L S-亚硝基谷胱甘肽(S-nitrosoglutathione,GSNO)损伤内皮细胞12 h,通过MTT比色法检测葫芦茶苷对EA.hy 926细胞存活率的影响;Real-time PCR法检测EA.hy 926细胞线粒体特异性因子细胞色素氧化酶1(COX-1)、NADH脱氢酶亚单位1(ND-1)和炎症因子白介素-1β(IL-1β)基因的表达效果,同时采用Western blot法检测Bcl-2关联X蛋白(Bax)和B细胞肿瘤因子2(Bcl-2)经时蛋白表达量的变化;通过线粒体膜电位荧光探针(JC-1)检测葫芦茶苷对EA.hy 926细胞线粒体膜电位的变化规律。结果:MTT法结果表明葫芦茶苷在5~160μmol/L范围内对EA.hy 926细胞无明显细胞毒性且最佳药物保护浓度为40μmol/L。Western Blot法显示GSNO经时损伤EA.hy 926细胞后Bax蛋白表达量呈时间依赖性增高,Bcl-2蛋白表达量与之相反。Real time-PCR结果显示,葫芦茶苷能明显上调COX-1基因(P<0.05),且能明显抑制活性氮GSNO诱导的ND-1(P<0.05)和IL-1β基因上调(P<0.01)。同时JC-1结果显示葫芦茶苷能明显保护线粒体膜电位免受GSNO损伤的作用。结论:活性氮GSNO损伤模型可能通过线粒体DNA损伤诱发Bax等促凋亡蛋白的增加,使抗凋亡因子Bcl-2表达降低。葫芦茶苷对活性氮诱导的内皮细胞线粒体损伤具有一定的保护作用,其机制与抑制ND-1和IL-1β基因表达,上调COX-1基因表达相关。Objective:To investigate the protective effect of Tadehaginoside on vascular endothelial cell injury induced by reactive nitrogen.Methods:MTT colorimetry was used to detect the effect of cucurbitacin at concentrations of 5-160μmol/L on the survival rate of EA.hy 926 endothelial cells.After EA.hy 926 endothelial cells were pre-administered with cucurbitacin for 1 h,0.5 mmol S-nitrosoglutathione(GSNO)was given to induce damages to the endothelial cells for 12 hours.Then effect of cucurbitacin on the survival rate of EA.hy 926 cells was detected by MTT colorimetry.Real time-PCR method was applied for detection of mitochondrial specific factors cytochrome oxidase 1(COX-1),NADH dehydrogenase subunit 1(ND-1)and inflammatory factor IL-1β(interleukin-1β)genes in EA.hy 926 cells.At the same time,Western blot was used to detect the changes in the protein expression of Bcl-2 associated X protein(Bax)and B cell tumor factor 2(Bcl-2)over time.The mitochondrial membrane potential kit(JC-1)was used to detect the change of Tadehaginoside on the mitochondrial membrane potential after GSNO induced damages in EA.hy 926 cell.Results:The results of the MTT method showed that Tadehaginoside had no obvious cytotoxicity on EA.hy 926 cells at concentrations of 5-160μmol/L,and the optimal protective concentration of the drug was 40μmol/L.Western Blot method showed that BAX protein expression increased in a time-dependent manner after GSNO induced damages in EA.hy 926 cells over time,while Bcl-2 protein expression showed the opposite trend.Real-time PCR results showed that Tadehaginoside can significantly up-regulate COX-1 gene(P<0.05),while significantly inhibit GSNO induced ND-1(P<0.05)and IL-1βgene up-regulation(P<0.01).At the same time,the results of JC-1 showed that Tadehaginoside could significantly protect the mitochondrial membrane potential from GSNO damage.Conclusion:The GSNO may induce the increase of Bax and other pro-apoptotic proteins through mitochondrial DNA damage and reduce the expression of anti-apoptotic factor Bc

关 键 词：活性氮 线粒体 葫芦茶苷 内皮细胞 

分 类 号：R285[医药卫生—中药学]