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[摘要]
目的 探究南葶苈子水提物(DS)对H2O2诱导的心肌H9c2细胞损伤的保护作用及潜在机制。方法 使用高效液相色谱与质谱联用方法分析鉴定DS中主要成分峰。采用H2O2处理制备H9c2细胞损伤模型,分为对照组、模型组、普罗布考组及DS 100、200、400 μg/mL组,MTT法检测H9c2细胞活力;流式细胞仪检测细胞凋亡率、细胞自噬水平、线粒体膜电位;试剂盒检测细胞氧化应激相关指标;Incell-Western法检测凋亡通路关键蛋白和自噬标志性蛋白表达水平。结果 共分析鉴定了DS中含量最高的7种成分。与模型组比较,DS可以提高H9c2细胞活力(P<0.05、0.01)与细胞存活率(P<0.01),改善线粒体膜电位水平(P<0.01),调节凋亡通路关键蛋白Caspase-3和Bax/Bcl-2蛋白表达(P<0.01),抑制心肌细胞凋亡;并能极显著降低细胞自噬水平(P<0.01),升高自噬标志性蛋白自噬微管相关蛋白轻链3B(LC3B)、p62表达水平(P<0.01),抑制心肌细胞自噬;降低心肌活性氧(ROS)水平(P<0.01),调节细胞内超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)活性及乳酸脱氢酶(LDH)、丙二醛(MDA)水平(P<0.01),改善心肌细胞氧化应激。结论 DS可以有效保护由H2O2引起的H9c2细胞损伤,其机制可能与改善细胞的氧化应激、抑制细胞凋亡和自噬有关,其物质基础可能与所含的黄酮苷类成分有关。
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[Abstract]
Objective To study the protective effect of aqueous extract from Descurainia Sophia (DS) on H2O2-induced H9c2 cardiomyocyte injury and to initially explore the potential mechanism.Methods The peaks of main components in DS were analyzed and identified by HPLC-MS. H9c2 cell injury model was established by H2O2. H9c2 cells were cultured in vitro and divided into control group, model group, probucol group, and DS at 100, 200, 400 μg/mL groups. In order to reveal the possible molecular mechanisms, the viability of H9c2 cells was measured by MTT assay; The apoptosis rate, autophagy rate, mitochondrial membrane potential, and reactive oxygen species (ROS) level were detected by flow cytometry; The relative indicators of cell oxidative stress were determined by biochemical kit; The expression levels of apoptosis-related protein and the autophagy-related protein were evaluated by Incell-western method.Results Seven components with the highest content were identified in DS through the results of mass spectrometry. Compared with the model group, DS can improve the cell viability (P < 0.05, 0.01) and survival rate of H9c2 cells (P < 0.01); At the same time, apoptosis was attenuated (P < 0.01), mitochondrial membrane potential was upregulated (P < 0.01), apoptosis related proteins Caspase-3, Bax/Bcl-2 were obviously downregulated (P < 0.01), autophagy phenomenon was attenuated (P < 0.01), autophagy related proteins LC3B and p62 were upregulated (P < 0.01). In addition, ROS level was decreased (P < 0.01), T-SOD and GSH-PX were upregulated and the levels of LDH and MDA were significantly decreased (P < 0.01).Conclusion This study suggests that DS can effectively protect H2O2-induced H9c2 cells injury, and the mechanism may be associated with improving oxidative stress in cells, inhibiting cell apoptosis and autophagy, which may be related to flavonoid glycosides.
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[基金项目]
国家重点基础研究发展计划(“973”计划)项目(2013CB531802);河南中医药大学2018年度博士科研基金(BSJJ2018-04)