中国中药杂志

2020, v.45(12) 2960-2965

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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

辛高杰;付建华;韩笑;李磊;郭浩;孟红旭;赵雨薇;贾飞凡;刘建勋;
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;School of Integrative Medicine, Binzhou Medical College;

摘要(Abstract):

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(50 μmol·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-Ⅰ ratio, 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 and ΔΨm 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-Ⅰ ratio 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 H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.

关键词(KeyWords): 丹酚酸B;H9c2心肌细胞;线粒体自噬;NIX;自噬
salvianolic acid B;H9c2 cardiomyocytes;mitochondrial autophagy;NIX;autophagy

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 辛高杰;付建华;韩笑;李磊;郭浩;孟红旭;赵雨薇;贾飞凡;刘建勋;
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;School of Integrative Medicine, Binzhou Medical College;

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