丹酚酸B调控NIX介导的线粒体自噬保护H9c2心肌细胞缺氧/复氧损伤  

Salvianolic acid B regulates mitochondrial autophagy mediated by NIX to protect H9c2 cardiomyocytes from hypoxia/reoxygenation injury

作　　者：辛高杰 付建华[1] 韩笑[1] 李磊[1] 郭浩[1] 孟红旭[1] 赵雨薇 贾飞凡 刘建勋[1] XIN Gao-jie;FU Jian-hua;HAN Xiao;LI Lei;GUO Hao;MENG Hong-xu;ZHAO Yu-wei;JIA Fei-fan;LIU Jian-xun(Institute of Basic Medicine,Xiyuan Hospital,Chinese Academy of Chinese Medical Sciences,Beijing 100091,China;School of Integrative Medicine,Binzhou Medical College,Yantai 264003,China)

机构地区：[1]中国中医科学院西苑医院基础医学研究所,北京100091 [2]滨州医学院中西医结合学院,山东烟台264003

出　　处：《中国中药杂志》2020年第12期2960-2965,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81774145);国家自然科学基金青年基金项目(81503292);国家自然科学基金重点项目(81530099);国家重点基础研究发展计划项目(2015CB554406);国家“重大新药创制”科技重大专项(2017ZX09301018)。

摘　　要：探讨丹酚酸B保护H9c2心肌细胞缺氧/复氧损伤的机制是否与调控NIX介导的线粒体自噬有关。体外培养H9c2心肌细胞,分为正常组、模型组、丹酚酸B组(50μmol·L-1),缺氧4 h复氧2 h建立缺氧/复氧损伤模型。正常组:高糖DMEM培养基培养;模型组:无糖无氧DMEM培养基培养,缺氧、复氧均不加任何药物;丹酚酸B组:缺氧同时加入用无糖DMEM培养基配制的丹酚酸B,其余过程同模型组。CCK-8法检测心肌细胞活力,微量酶标法检测心肌细胞乳酸脱氢酶(LDH)漏出量,化学荧光法检测细胞内活性氧(ROS)水平、线粒体膜电位(ΔΨm)变化,萤光素酶法检测细胞内三磷酸腺苷(ATP)水平,蛋白免疫印迹(Western blot)法检测自噬相关蛋白LC3-Ⅰ和LC3-Ⅱ、凋亡相关蛋白cleaved caspase-3及线粒体自噬受体蛋白NIX表达。与正常组相比,模型组H9c2心肌细胞活力、ATP水平降低(P<0.05),LDH漏出、ROS生成增多(P<0.01),ΔΨm降低(P<0.01),LC3-Ⅱ/LC3-Ⅰ比值、cleaved caspase-3、NIX蛋白表达均升高(P<0.05);与模型组相比,丹酚酸B可显著升高细胞活力、降低LDH漏出量和ROS水平(P<0.01),升高细胞内ATP含量(P<0.05)及ΔΨm(P<0.01),降低自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值(P<0.01),下调cleaved caspase-3和NIX蛋白表达(P<0.05)。丹酚酸B对H9c2心肌细胞缺氧/复氧损伤的保护作用与抑制线粒体自噬发生有关,其具体机制可能为抑制NIX介导的线粒体自噬激活,从而升高ΔΨm,降低ROS生成,降低cleaved caspase-3和LC3-Ⅱ蛋白表达,升高细胞活力。The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX.H9 c2 cardiomyocytes were cultured in vitro and divided into normal group,model group and salvianolic acid B group(50μmol·L-1).Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h.In normal group,high glucose DMEM medium was used for culture.Those in model group were cultured with DMEM medium without glucose and oxygen,and no drugs for hypoxia and reoxyge-nation.In salvianolic acid B group,salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia,and the other process was as same as the model group.The cell viability was evaluated by CCK-8 assay.The leakage of lactate dehydrogenase(LDH)was detected by microplate method.The levels of intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(ΔΨm)were measured by chemical fluorescence method.The level of intracellular adenosine triphosphate(ATP)was mea-sured by fluorescein enzyme method.The autophagy related proteins LC3-Ⅰ,LC3-Ⅱ,apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot.As compared with the normal group,the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05);LDH leakage and ROS production were increased(P<0.01);ΔΨm was decreased(P<0.01);LC3-Ⅱ/LC3-Ⅰratio,cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05)in the model group.As compared with the model group,the activity of cells andΔΨm were significantly increased(P<0.01);ATP level was increased(P<0.05);LDH leakage and ROS generation were decreased(P<0.01);LC3-Ⅱ/LC3-Ⅰratio was decreased(P<0.01);cleaved caspase-3 and NIX expression levels were decreased(P<0.05)in the salvianolic acid B group.The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of

关 键 词：丹酚酸B H9C2心肌细胞 线粒体自噬 NIX 自噬 

分 类 号：R285.5[医药卫生—中药学]