天麻素通过蛋白激酶B/p38丝裂原活化蛋白激酶信号通路抑制H9c2心肌细胞凋亡
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摘要
目的采用血清剥夺/复灌的大鼠H9c2心肌细胞模拟心肌缺血/再灌注损伤(I/R),从细胞层面探讨天麻素(gastrodin)抗H9c2心肌细胞凋亡的作用及其机制。方法体外培养H9c2心肌细胞随机分为对照组、血清剥夺(0.5~6 h)处理组、血清剥夺/复灌(2/4 h)处理组、天麻素(10、20和40μmol/L)处理组、天麻素(20μmol/L)+渥曼青霉素(wortmannin)(1μmol/L)处理组、wortmannin (1μmol/L)处理组。采用免疫印迹法检测p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38MAPK(p-p38MAPK)、蛋白激酶B(Akt)、p-Akt以及凋亡相关蛋白cleaved Caspase-3、Bcl-2和Bax的表达变化;采用流式细胞术检测细胞凋亡;通过实时定量聚合酶链反应(Real-time PCR)检测Caspase-3、Bcl-2和Bax的mRNA表达变化;采用免疫荧光双标记法检测细胞内p-Akt蛋白水平表达的变化。结果血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理诱导细胞凋亡水平增加;加入不同浓度天麻素处理,与血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理组比较,凋亡相关蛋白cleaved Caspase-3表达降低,Bcl-2/Bax蛋白表达的比值上调(P<0.05),基因转录水平检测的Caspase-3和Bax表达量降低,Bcl-2表达量上调((P<0.05),流式细胞术凋亡检测结果也同样显示,天麻素能抑制血清剥夺/复灌(2/4 h)诱导的细胞凋亡(P<0.05);同时,血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理能抑制Akt/p38MAPK信号通路;加入不同浓度的天麻素(10、20和40μmol/L)处理后,p-Akt的表达水平增多,p-p38MAPK蛋白表达减少(P <0.05)。加入Akt的特异性抑制剂wortmannin(1μmol/L)处理后,天麻素对上述因子的调控作用被反转(P<0.05)。结论在H9c2心肌细胞中,天麻素通过激活Akt/p38MAPK信号通路,抑制血清剥夺/复灌诱导的细胞凋亡,从而发挥抗心肌I/R损伤的保护作用。
Objective To investigate the effect and precise molecular mechanisms for explaining how gastrodin suppresses the apoptosis in the serum deprivation/reperfusion-induced H9 c2 cardiomyocytes.Methods Rat H9 c2 cardiomyocytes were cultured and treated differently in vitro and randomly divided into following groups,including the control group,the serum deprivation-stimulated(0.5-6 hours) groups,the serum deprivation/reperfusion(2/4 hours)group,gastrodin(10,20 and 40 μmol/L) treatment groups,wortmannin(1 μmol/L) and gastrodin(20 μmol/L) cotreatment group,wortmannin(1 μmol/L) treatment group.The variation of protein expression levels of p-p38 mitogen actived protein kinase(MAPK),p-Akt and apoptosis related cleaved caspase-3,Bcl-2 and Bax were analyzed by Western blotting.H9 c2 cardiomyocytes were analyzed by flow cytometry.The level of RNA expression levels of apoptosis relatedCaspase-3,Bcl-2 and Bax were analyzed by Real-time PCR assay.The expression level of p-Akt was assayed by double immunofluorescence labeling.Results The results showed that serum deprivation(0.5-6 hours) and serum deprivation/reperfusion(2/4 hours) induced apoptosis increasing;Using Western blotting and Real-time PCR assay,we found that treatment with gastrodin significantly alleviated cleaved Caspase-3 activation and increased the ratio of Bcl-2/Bax in a concentration-dependent manner after serum deprivation and serum deprivation/reperfusion exposure.The same result has been showed by flow cytometer.Serum deprivation and serum deprivation/reperfusion inhibited the Akt/p38 MAPK signaling pathway;After treatment with gastrodin in a concentration-dependent manner induced the expression of p-Akt and decreased the expression of p-p38 MAPK.The regulation effects of gastrodin were reversed by the Akt specific inhibitor wortmannin.Conclusion In conclusion,the present study demonstrated that gastrodin exerted protective effects against serum deprivation/reperfusion-induced apoptosis in H9 c2 cardiomyocytes,at least partly via the Akt/p38 MAPK pathway.
Objective To investigate the effect and precise molecular mechanisms for explaining how gastrodin suppresses the apoptosis in the serum deprivation/reperfusion-induced H9 c2 cardiomyocytes.Methods Rat H9 c2 cardiomyocytes were cultured and treated differently in vitro and randomly divided into following groups,including the control group,the serum deprivation-stimulated(0.5-6 hours) groups,the serum deprivation/reperfusion(2/4 hours)group,gastrodin(10,20 and 40 μmol/L) treatment groups,wortmannin(1 μmol/L) and gastrodin(20 μmol/L) cotreatment group,wortmannin(1 μmol/L) treatment group.The variation of protein expression levels of p-p38 mitogen actived protein kinase(MAPK),p-Akt and apoptosis related cleaved caspase-3,Bcl-2 and Bax were analyzed by Western blotting.H9 c2 cardiomyocytes were analyzed by flow cytometry.The level of RNA expression levels of apoptosis relatedCaspase-3,Bcl-2 and Bax were analyzed by Real-time PCR assay.The expression level of p-Akt was assayed by double immunofluorescence labeling.Results The results showed that serum deprivation(0.5-6 hours) and serum deprivation/reperfusion(2/4 hours) induced apoptosis increasing;Using Western blotting and Real-time PCR assay,we found that treatment with gastrodin significantly alleviated cleaved Caspase-3 activation and increased the ratio of Bcl-2/Bax in a concentration-dependent manner after serum deprivation and serum deprivation/reperfusion exposure.The same result has been showed by flow cytometer.Serum deprivation and serum deprivation/reperfusion inhibited the Akt/p38 MAPK signaling pathway;After treatment with gastrodin in a concentration-dependent manner induced the expression of p-Akt and decreased the expression of p-p38 MAPK.The regulation effects of gastrodin were reversed by the Akt specific inhibitor wortmannin.Conclusion In conclusion,the present study demonstrated that gastrodin exerted protective effects against serum deprivation/reperfusion-induced apoptosis in H9 c2 cardiomyocytes,at least partly via the Akt/p38 MAPK pathway.
引文
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[7]Ran X,Diao JX,Sun XG,et al.Huangzhi oral liquid prevents arrhythmias by upregulating Caspase-3 and apoptosis network proteins in myocardial ischemia-reperfusion injury in rats[J].Evid Based Complement Alternat Med,2015,2015:518926.
[8]Jennings RB,Sommers HM,Smyth GA,et al.Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog[J].Arch Pathol,1960,70(1):68-79.
[9]Mozaffari MS,Liu JY,Abebe W,et al.Mechanisms of load dependency of myocardial ischemia reperfusion injury[J].Am JCardiovasc Dis,2013,3(4):180-196.
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[11]Zhou YP,Jia YH,Yang P,et al.Effect of dingxin recipe on cardiomyocyte apoptosis in rats with ischemia/reperfusion arrhythmia[J].Chinese Journal of Basic Medicine in Traditional Chinese Medicine,2007,13(7):515-516.(in Chinese)周玉平,贾钰华,杨萍,等.定心方对缺血再灌注心律失常大鼠心肌细胞凋亡的影响[J].中国中医基础医学杂志,2007,13(7):515-516.
[12]Zhang LP,Zhang J.Effect of tanshinoneⅡA on myocardial ischemia reperfusion injury in rats[J].Chinese Community Doctors(Medical Professional),2010,12(27):28.(in Chinese)张丽萍,张晶.丹参酮ⅡA对大鼠心肌缺血再灌注损伤的影响[J].中国社区医师(医学专业),2010,12(27):28.
[13]Wang LL,Huang Sh W,Dou LP,et al.Beneficial effects and the protential oxidative stress mechanism of tanshinoneⅡA on the noreflow phenomenon in rabbits after acute myocardial infarction and reperfusion[J].Chinese Archives of Traditional Chinese Medicine,2012,30(11):2513-2517.(in Chinese)王琳莉,黄抒伟,窦丽萍,等.丹参酮ⅡA磺酸钠对兔急性心梗再灌注后无复流的影响及与氧化应激的关系[J].中华中医药学刊,2012,30(11):2513-2517.
[14]Dai JN,Zong Y,Zhong LM,et al.Gastrodin inhibits expression of inducible NO synthase,cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-Stimulated microglia via MAPK pathways[J].PLoS One,2011,6(7):e21891.
[15]Li Y,Wen F,He H,et al.Protective of gastrodin pretreatment on rat isolated mycocardial/ischemia reperfusion injury[J].Acta Anatomica Sinica,2016,47(1):107-112.(in Chinese)李瑶,文飞,何花,等.天麻素预处理对大鼠离体心脏缺血/再灌注损伤的保护作用[J].解剖学报,2016,47(1):107-112.
[16]Mandlekar S,Kong AN.Mechanisms of tamoxifen-induced apoptosis[J].Apoptosis,2001,6(6):469-477.
[17]Yu G,Peng T,Feng Q,et al.Abrupt reoxygenation of microvascular endothelial cells after hypoxia activates ERK1/2 and JNK1,leading to NADPH oxidase-dependent oxidant production[J].Microcirculation,2007,14(2):125-136.
[18]Liu AL,Wang XW,Liu AH,et al.JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons[J].Exp Toxicol Pathol,2009,61(2):137-143.
[19]Shimada K,Nakamura M,Ishida E,et al.Roles of p38-and c-Jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiolinduced p53 induction and apoptosis[J].Carcinogenesis,2003,24(6):1067-1075.
[20]Li J,Yuan YP,Xu SC,et al.Arctiin protects against cardiac hypertrophy through inhibiting MAPKs and Akt signaling pathways[J].J Pharmacol Sci,2017,135(3):97-104.
[21]Matsui T,Li L,Monte FD,et al.Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and akt inhibits apoptosis of hypoxic cardiomyocytes in Vitro[J].Circulation,1999,100(23):2373-2379.
[22]Fujio Y,Nguyen T,Wencker D,et al.Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart[J].Circulation,2000,15,101(6):660-667.
[23]Shu J,Zhang W,Huang C,et al.Troxerutin protects against myocardial ischemia/reperfusion injury via PI3K/Akt pathway in rats[J].Cell Physiol Biochem,2017,44(5):1939-1948.
[24]Hang P,Zhao J,Su Z,et al.Choline inhibits ischemiareperfusion-induced cardiomyocyte autophagy in rat myocardium by activating akt/m TOR signaling.[J].Cell Physiol Biochem,2018,45(5):2136-2144.
[2]Braunwald E,Kloner RA.Myocardial reperfusion:a double-edged sword[J]?J Clin Invest,1985,76(5):1713-1719.
[3]Lopez AD,Murray CC.The global burden of disease,1990-2020[J].Nat Med,1998,4(11):1241-1243.
[4]Chen W,Guo LL,Wang J,et al.Research progress in preventing myocardial ischemia-reperfusion injury by traditional Chinese medicine[J].Liaoning Journal of Traditional Chinese Medicine,2012,39(6):1192-1195.(in Chinese)陈雯,郭丽丽,王阶.中药防治心肌缺血再灌注损伤研究进展[J].辽宁中医杂志,2012,39(6):1192-1195.
[5]Chinese Pharmacopoeia Commission.Pharmacopoeia of the People's Republic of China(PartⅠ)[S].Beijing:China Medical Science Press,2010:54-55.(in Chinese)中华人民共和国国家药典委员会.中华人民共和国药典(一部)[S].北京:中国医药科技出版社,2010:54-55.
[6]Chen J,Liu ShX,Hu GH,et al.Gastrodin injection in the treatment of vertigo:a multi-center single-blind ran-domized controlled trial[J].Chinese Journal of Evidence-Based Medicine,2004,4(12):864-870.(in Chinese)陈静,刘世喜,胡国华.天麻注射液治疗眩晕的多中心随机单盲对照试验[J].中国循证医学杂志,2004,4(12):864-870.
[7]Ran X,Diao JX,Sun XG,et al.Huangzhi oral liquid prevents arrhythmias by upregulating Caspase-3 and apoptosis network proteins in myocardial ischemia-reperfusion injury in rats[J].Evid Based Complement Alternat Med,2015,2015:518926.
[8]Jennings RB,Sommers HM,Smyth GA,et al.Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog[J].Arch Pathol,1960,70(1):68-79.
[9]Mozaffari MS,Liu JY,Abebe W,et al.Mechanisms of load dependency of myocardial ischemia reperfusion injury[J].Am JCardiovasc Dis,2013,3(4):180-196.
[10]Diwan A,Krenz M,Syed FM.et al.Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice[J].J Clin Invest,2007,117(10):2825-2833.
[11]Zhou YP,Jia YH,Yang P,et al.Effect of dingxin recipe on cardiomyocyte apoptosis in rats with ischemia/reperfusion arrhythmia[J].Chinese Journal of Basic Medicine in Traditional Chinese Medicine,2007,13(7):515-516.(in Chinese)周玉平,贾钰华,杨萍,等.定心方对缺血再灌注心律失常大鼠心肌细胞凋亡的影响[J].中国中医基础医学杂志,2007,13(7):515-516.
[12]Zhang LP,Zhang J.Effect of tanshinoneⅡA on myocardial ischemia reperfusion injury in rats[J].Chinese Community Doctors(Medical Professional),2010,12(27):28.(in Chinese)张丽萍,张晶.丹参酮ⅡA对大鼠心肌缺血再灌注损伤的影响[J].中国社区医师(医学专业),2010,12(27):28.
[13]Wang LL,Huang Sh W,Dou LP,et al.Beneficial effects and the protential oxidative stress mechanism of tanshinoneⅡA on the noreflow phenomenon in rabbits after acute myocardial infarction and reperfusion[J].Chinese Archives of Traditional Chinese Medicine,2012,30(11):2513-2517.(in Chinese)王琳莉,黄抒伟,窦丽萍,等.丹参酮ⅡA磺酸钠对兔急性心梗再灌注后无复流的影响及与氧化应激的关系[J].中华中医药学刊,2012,30(11):2513-2517.
[14]Dai JN,Zong Y,Zhong LM,et al.Gastrodin inhibits expression of inducible NO synthase,cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-Stimulated microglia via MAPK pathways[J].PLoS One,2011,6(7):e21891.
[15]Li Y,Wen F,He H,et al.Protective of gastrodin pretreatment on rat isolated mycocardial/ischemia reperfusion injury[J].Acta Anatomica Sinica,2016,47(1):107-112.(in Chinese)李瑶,文飞,何花,等.天麻素预处理对大鼠离体心脏缺血/再灌注损伤的保护作用[J].解剖学报,2016,47(1):107-112.
[16]Mandlekar S,Kong AN.Mechanisms of tamoxifen-induced apoptosis[J].Apoptosis,2001,6(6):469-477.
[17]Yu G,Peng T,Feng Q,et al.Abrupt reoxygenation of microvascular endothelial cells after hypoxia activates ERK1/2 and JNK1,leading to NADPH oxidase-dependent oxidant production[J].Microcirculation,2007,14(2):125-136.
[18]Liu AL,Wang XW,Liu AH,et al.JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons[J].Exp Toxicol Pathol,2009,61(2):137-143.
[19]Shimada K,Nakamura M,Ishida E,et al.Roles of p38-and c-Jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiolinduced p53 induction and apoptosis[J].Carcinogenesis,2003,24(6):1067-1075.
[20]Li J,Yuan YP,Xu SC,et al.Arctiin protects against cardiac hypertrophy through inhibiting MAPKs and Akt signaling pathways[J].J Pharmacol Sci,2017,135(3):97-104.
[21]Matsui T,Li L,Monte FD,et al.Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and akt inhibits apoptosis of hypoxic cardiomyocytes in Vitro[J].Circulation,1999,100(23):2373-2379.
[22]Fujio Y,Nguyen T,Wencker D,et al.Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart[J].Circulation,2000,15,101(6):660-667.
[23]Shu J,Zhang W,Huang C,et al.Troxerutin protects against myocardial ischemia/reperfusion injury via PI3K/Akt pathway in rats[J].Cell Physiol Biochem,2017,44(5):1939-1948.
[24]Hang P,Zhao J,Su Z,et al.Choline inhibits ischemiareperfusion-induced cardiomyocyte autophagy in rat myocardium by activating akt/m TOR signaling.[J].Cell Physiol Biochem,2018,45(5):2136-2144.
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