南葶苈子抑制氧化应激与自噬通路抗H_2O_2诱导的H9c2细胞损伤作用研究
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摘要
目的探究南葶苈子水提物(DS)对H_2O_2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H_2O_2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、400μg/m L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H_2O_2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。
Objective To study the protective effect of aqueous extract from Descurainia Sophia(DS) on H_2O_2-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 H_2O_2. 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 LC3 B 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 H_2O_2-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.
Objective To study the protective effect of aqueous extract from Descurainia Sophia(DS) on H_2O_2-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 H_2O_2. 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 LC3 B 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 H_2O_2-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.
引文
[1]国家心血管疾病中心.中国心血管病报告2016[M].北京:中国百科全书出版社,2016
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[7]孙利,谷春华,高学东,等.芪苈强心胶囊对不同病因所致慢性心力衰竭患者心功能的影响[J].河北中医,2013,35(6):904-905.
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[10]王志军,曹佳佳,刁增利,等.芪苈强心胶囊改善心肌梗死后心力衰竭患者的临床症状[J].中成药,2015,37(1):44-48.
[11]颜蕾,薛文海,尹立俊.益心汤对慢性心功能不全患者心功能影响的临床研究[J].实用中医内科杂志,2012,26(12):1-2.
[12]沈淑静,冼绍祥,黄衍寿,等.益气温阳活血利水中药对心力衰竭兔的血流动力学影响和配伍研究[J].中国实验方剂学杂志,2013,19(5):195-199.
[13]郭娟,陈长勋.葶苈子对压力负荷性大鼠心室重构及神经内分泌因子和心肌I、III型胶原的影响[J].中药材,2007,30(8):963-967.
[14]王晴川,刘广芬,李常春.播娘蒿种子(华东葶苈子)的强心作用、生物活性、吸收、蓄积及毒性反应[J].福建医学院学报,1964(Z1):27-33.
[15]周喜丹,唐力英,周国洪,等.南北葶苈子的最新研究进展[J].中国中药杂志,2014,39(24):4699-4708.
[16]张国顺,白义萍,郑晓珂,等.葶苈子抗心衰有效组分筛选及其作用机制分析[J].中国实验方剂学杂志,2017,23(4):118-125.
[17]姜艳,尹学艳,李晋,等.高效液相色谱法同时测定南葶苈子中6种成分含量[J].天津中医药,2016,33(12):750-755.
[18]孙凯.南葶苈子的化学成分及其生物活性研究[D].沈阳:沈阳药科大学,2005.
[19]王爱芹,王秀坤,赵海誉,等.南葶苈子化学成分与质量研究[J].中国药物与临床,2005,5(1):5-6.
[20]Zhou X,Tang L,Wu H,et al.Chemometric analyses for the characterization of raw and processed seeds of Descurainia sophia(L.)based on HPLC fingerprints[J].J Pharm Biomed Anal,2015,doi:10.1016/j.jpba.2015.03.010.
[21]Meng Z H,Qiao L,Whang Z Q,et al.Analysis of the constituents in Semen Descurainiae by UPLC/Q-TOF-MS/MS[J].J Chin Pharm Sci,2015,24(5):303-309.
[22]Kukhta V K,Marozkina N V,Sokolchik I G,et al.Molecular mechanisms of apoptosis[J].Ukr Biokhim Zh2003,75(6):5-9.
[23]Fan Y J,Zong W X.The cellular decision between apoptosis and autophagy[J].Chin J Cancer,2013,32(3):121-129.
[24]Corbalan J J,Vatner D E,Vatner S F.Myocardial apoptosis in heart disease:Does the emperor have clothes?[J].Basic Res Cardiol,2016,doi:10.1007/s00395-016-0549-2.
[25]鹿欣伦,胡成云,张雨梅.心力衰竭机制的研究进展[J].山西医药杂志,2011,40(8):766-767.
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[28]Ren S Y,Xu X.Role of autophagy in metabolic syndrome-associated heart disease[J].Biochim Biophys Acta,2015,1852(2):225-231.
[29]Matsui Y,Kyoi S,Takagi H,et al.Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion[J].Autophagy,2008,4(4):409-415.
[30]Xu X,Hua Y,Nair S,et al.Macrophage migration inhibitory factor deletion exacerbates pressure overloadinduced cardiac hypertrophy through mitigating autophagy[J].Hypertension,2014,63(3):490-499.
[31]Song Y,Li W,Peng X,et al.Inhibition of autophagy results in a reversal of taxol resistance in nasopharyngeal carcinoma by enhancing taxol-induced caspase-dependent apoptosis[J].Am J Transl Res,2017,9(4):1934-1942.
[32]Dutta D,Xu J,Dirain M L,et al.Calorie restriction combined with resveratrol induces autophagy and protects26-month-old rat hearts from doxorubicin-induced toxicity[J].Free Rad Biol Med,2014,doi:10.1016/j.freeradbiomed.2014.06.011.
[33]Xie Z L,Lau K,Eby B,et al.Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice[J].Diabetes,2011,60(6):1770-1778.
[34]Liu S,Pereira N A,Teo J J,et al.Mitochondrially targeted Bcl-2 and Bcl-X(L)chimeras elicit different apoptotic responses[J].Mol Cells,2007,24(3):378-387.
[35]Saita S,Shirane M,Nakayama K I,et al.Selective escape of proteins from the mitochondria during mitophagy[J].Nat Commun,2013,doi:10.1038/ncomms2400.
[36]Hua Y,Zhang Y,Ceylan-Isik A F,et al.Chronic Akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction:Role of autophagy[J].Basic Res Cardiol,2011,106(6):1173-1191.
[37]Xu X,Hua Y,Nair S,et al.Akt2 knockout preserves cardiac function in high-fat diet-induced obesity by rescuing cardiac autophagosome maturation[J].J Mol Cell Biol,2013,5(1):61-63.
[38]Xu X,Bucala R,Ren J.Macrophage migration inhibitory factor deficiency augments doxorubicin-induced cardiomyopathy[J].J Am Heart Assoc,2013,2(6):e000439.
[39]Hariharan N,Maejima Y,Nakae J,et al.Deacetylation of FoxO by sirt1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes[J].Circ Res,2010,107(12):1470-1482.
[40]Tsutsui H,Kinugawa S,Matsushima S.Oxidative stress and heart failure[J].Am J Physiol Heart Circ Physiol,2011,301(6):H2181-H2190.
[41]Martindale J J,Metzger J M.Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization[J].J Mol Cell Cardiol,2014,doi:10.1016/j.yjmcc.2013.12.008.
[42]Bhatt D L,Pashkow F J.Introduction.Oxidative stress and heart disease[J].Am J Cardiol,2008,doi:10.1016/j.amjcard.2008.02.001.
[43]Sokolova N,Pan S,Provazza S,et al.ADP protects cardiac mitochondria under severe oxidative stress[J].PLoS One,2013,8(12):e83214.
[44]Mokwatsi G G,Schutte A E,Kruger R.A biomarker of tissue damage,lactate dehydrogenase,is associated with fibulin-1 and oxidative stress in blacks:The SAfrEICstudy[J].Biomarker,2016,21(1):48-55.
[45]Filomeni G,de Zio D,Cecconi F.Oxidative stress and autophagy:The clash between damage and metabolic needs[J].Cell Death Differ,2015,22(3):377-388.
[46]Cai Q,Wei J,Zhao W,et al.Toxicity of evodiae fructus on rat liver mitochondria:The role of oxidative stress and mitochondrial permeability transition[J].Molecules,2014,19(12):21168-21182.
[2]陈伟伟,高润霖,刘力生,等.《中国心血管病报告2016》概要[J].中国循环杂志,2017,32(6):521-530.
[3]张文,张艳,礼海.葶苈子在慢性心衰治疗中的应用[J].世界中西医结合杂志,2010,5(4):349.
[4]潘九英,金芝贵,吴飞华.葶苈子及其复方治疗心血管系统疾病的研究进展[J].上海中医药杂志,2008,42(12):83-85.
[5]石衍梅,李洁,张庆蕊,等.基于中医传承辅助平台的治疗慢性心力衰竭方剂组方规律分析[J].中国实验方剂学杂志,2016:22(2):191-194.
[6]冯志毅,王小兰,郑晓珂.葶苈子的本草考证[J].世界科学技术-中医药现代化,2014,16(9):1938-1941.
[7]孙利,谷春华,高学东,等.芪苈强心胶囊对不同病因所致慢性心力衰竭患者心功能的影响[J].河北中医,2013,35(6):904-905.
[8]王萧.十枣汤合葶苈子方治疗心力衰竭的临床与实验研究[D].广州:广州中医药大学,2000.
[9]李克忠.生脉强心汤治疗慢性心衰40例[J].长春中医药大学学报,2009,25(1):88.
[10]王志军,曹佳佳,刁增利,等.芪苈强心胶囊改善心肌梗死后心力衰竭患者的临床症状[J].中成药,2015,37(1):44-48.
[11]颜蕾,薛文海,尹立俊.益心汤对慢性心功能不全患者心功能影响的临床研究[J].实用中医内科杂志,2012,26(12):1-2.
[12]沈淑静,冼绍祥,黄衍寿,等.益气温阳活血利水中药对心力衰竭兔的血流动力学影响和配伍研究[J].中国实验方剂学杂志,2013,19(5):195-199.
[13]郭娟,陈长勋.葶苈子对压力负荷性大鼠心室重构及神经内分泌因子和心肌I、III型胶原的影响[J].中药材,2007,30(8):963-967.
[14]王晴川,刘广芬,李常春.播娘蒿种子(华东葶苈子)的强心作用、生物活性、吸收、蓄积及毒性反应[J].福建医学院学报,1964(Z1):27-33.
[15]周喜丹,唐力英,周国洪,等.南北葶苈子的最新研究进展[J].中国中药杂志,2014,39(24):4699-4708.
[16]张国顺,白义萍,郑晓珂,等.葶苈子抗心衰有效组分筛选及其作用机制分析[J].中国实验方剂学杂志,2017,23(4):118-125.
[17]姜艳,尹学艳,李晋,等.高效液相色谱法同时测定南葶苈子中6种成分含量[J].天津中医药,2016,33(12):750-755.
[18]孙凯.南葶苈子的化学成分及其生物活性研究[D].沈阳:沈阳药科大学,2005.
[19]王爱芹,王秀坤,赵海誉,等.南葶苈子化学成分与质量研究[J].中国药物与临床,2005,5(1):5-6.
[20]Zhou X,Tang L,Wu H,et al.Chemometric analyses for the characterization of raw and processed seeds of Descurainia sophia(L.)based on HPLC fingerprints[J].J Pharm Biomed Anal,2015,doi:10.1016/j.jpba.2015.03.010.
[21]Meng Z H,Qiao L,Whang Z Q,et al.Analysis of the constituents in Semen Descurainiae by UPLC/Q-TOF-MS/MS[J].J Chin Pharm Sci,2015,24(5):303-309.
[22]Kukhta V K,Marozkina N V,Sokolchik I G,et al.Molecular mechanisms of apoptosis[J].Ukr Biokhim Zh2003,75(6):5-9.
[23]Fan Y J,Zong W X.The cellular decision between apoptosis and autophagy[J].Chin J Cancer,2013,32(3):121-129.
[24]Corbalan J J,Vatner D E,Vatner S F.Myocardial apoptosis in heart disease:Does the emperor have clothes?[J].Basic Res Cardiol,2016,doi:10.1007/s00395-016-0549-2.
[25]鹿欣伦,胡成云,张雨梅.心力衰竭机制的研究进展[J].山西医药杂志,2011,40(8):766-767.
[26]Green D R,Levine B.To be or not to be?How selective autophagy and cell death govern cell fate[J].Cell,2014,157(1):65-75.
[27]Shintani T,Klionsky D J.Autophagy in health and disease:A double-edged sword[J].Science,2004,306(5698):990-995.
[28]Ren S Y,Xu X.Role of autophagy in metabolic syndrome-associated heart disease[J].Biochim Biophys Acta,2015,1852(2):225-231.
[29]Matsui Y,Kyoi S,Takagi H,et al.Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion[J].Autophagy,2008,4(4):409-415.
[30]Xu X,Hua Y,Nair S,et al.Macrophage migration inhibitory factor deletion exacerbates pressure overloadinduced cardiac hypertrophy through mitigating autophagy[J].Hypertension,2014,63(3):490-499.
[31]Song Y,Li W,Peng X,et al.Inhibition of autophagy results in a reversal of taxol resistance in nasopharyngeal carcinoma by enhancing taxol-induced caspase-dependent apoptosis[J].Am J Transl Res,2017,9(4):1934-1942.
[32]Dutta D,Xu J,Dirain M L,et al.Calorie restriction combined with resveratrol induces autophagy and protects26-month-old rat hearts from doxorubicin-induced toxicity[J].Free Rad Biol Med,2014,doi:10.1016/j.freeradbiomed.2014.06.011.
[33]Xie Z L,Lau K,Eby B,et al.Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice[J].Diabetes,2011,60(6):1770-1778.
[34]Liu S,Pereira N A,Teo J J,et al.Mitochondrially targeted Bcl-2 and Bcl-X(L)chimeras elicit different apoptotic responses[J].Mol Cells,2007,24(3):378-387.
[35]Saita S,Shirane M,Nakayama K I,et al.Selective escape of proteins from the mitochondria during mitophagy[J].Nat Commun,2013,doi:10.1038/ncomms2400.
[36]Hua Y,Zhang Y,Ceylan-Isik A F,et al.Chronic Akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction:Role of autophagy[J].Basic Res Cardiol,2011,106(6):1173-1191.
[37]Xu X,Hua Y,Nair S,et al.Akt2 knockout preserves cardiac function in high-fat diet-induced obesity by rescuing cardiac autophagosome maturation[J].J Mol Cell Biol,2013,5(1):61-63.
[38]Xu X,Bucala R,Ren J.Macrophage migration inhibitory factor deficiency augments doxorubicin-induced cardiomyopathy[J].J Am Heart Assoc,2013,2(6):e000439.
[39]Hariharan N,Maejima Y,Nakae J,et al.Deacetylation of FoxO by sirt1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes[J].Circ Res,2010,107(12):1470-1482.
[40]Tsutsui H,Kinugawa S,Matsushima S.Oxidative stress and heart failure[J].Am J Physiol Heart Circ Physiol,2011,301(6):H2181-H2190.
[41]Martindale J J,Metzger J M.Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization[J].J Mol Cell Cardiol,2014,doi:10.1016/j.yjmcc.2013.12.008.
[42]Bhatt D L,Pashkow F J.Introduction.Oxidative stress and heart disease[J].Am J Cardiol,2008,doi:10.1016/j.amjcard.2008.02.001.
[43]Sokolova N,Pan S,Provazza S,et al.ADP protects cardiac mitochondria under severe oxidative stress[J].PLoS One,2013,8(12):e83214.
[44]Mokwatsi G G,Schutte A E,Kruger R.A biomarker of tissue damage,lactate dehydrogenase,is associated with fibulin-1 and oxidative stress in blacks:The SAfrEICstudy[J].Biomarker,2016,21(1):48-55.
[45]Filomeni G,de Zio D,Cecconi F.Oxidative stress and autophagy:The clash between damage and metabolic needs[J].Cell Death Differ,2015,22(3):377-388.
[46]Cai Q,Wei J,Zhao W,et al.Toxicity of evodiae fructus on rat liver mitochondria:The role of oxidative stress and mitochondrial permeability transition[J].Molecules,2014,19(12):21168-21182.