薯蓣皂素对缺氧诱导的H9c2大鼠心肌细胞内质网应激损伤的保护作用
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摘要
目的探究薯蓣皂素(Diosgenin, DG)对缺氧诱导的大鼠心肌细胞H9c2损伤及心肌缺血再灌注(Mycardial ischemia reperfusion, MI/R)模型大鼠的保护作用及作用机制。方法将细胞分为H9c2组、低氧诱导(Hypoxia)组、DG (10mg/L)、DG (20 mg/L)和DG (50 mg/L)组,缺氧诱导细胞损伤,并给予对应浓度的DG或溶媒处理,CCK8检测细胞增殖,流式检测细胞凋亡,Western blot检测C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CCAAT/enhancer-binding protein (C/EBP) homologous protein,CHOP)、Caspase-12、DNA损伤诱导蛋白(Growth arrest and DNA damage-inducible protein 34, GADD34)和免疫球蛋白重链结合蛋白(Immunoglobulin heavy-chain-binding protein, Bip)的表达,试剂盒检测上清液超氧化物歧化酶(Superoxide dismutase, SOD)和丙二醛(Malondialdehyde, MDA)浓度。复制大鼠MI/R模型,灌胃给予大鼠DG,检测大鼠心率和平均动脉压(Mean artery pressure, MAP)。检测大鼠血清肌酸激酶(creatine kinase, CK)、SOD和MDA浓度,HE染色检测组织损伤,Western blot检测内质网应激相关蛋白表达。结果与H9c2组比较,Hypoxia组细胞增殖速度显著降低,细胞凋亡率明显升高;与Hypoxia组比较,DG (10, 20, 50 mg/L)组细胞增殖速度明显升高,细胞凋亡率明显降低;同时,DG (10, 20, 50 mg/L)能显著减弱Hypoxia对CHOP、Caspase-12表达的诱导作用和对GADD34和BiP表达的抑制作用。此外,缺氧能显著升高上清液MDA浓度,降低SOD浓度;DG能明显减弱缺氧的作用。DG还能显著升高MI/R模型大鼠心肌功能,减轻心肌组织损伤,降低心肌组织CHOP和Caspase-12的表达,诱导GADD34和BiP的表达,降低血清MDA浓度,升高SOD浓度。结论 DG能通过抑制内质网应激减轻缺氧诱导的心肌细胞损伤及MI/R模型大鼠心肌组织损伤。
【Abstacr】 Objective To investigate the effects and mechanisms of diosgenin(DG) on hypoxiainduced injury of rat myocardial H9c2 cell. Methods Cells were divided into H9c2, DG, Hypoxia and Hypoxia + DG group. Cells were treated with hypoxia for inducing injury, and cells were treated with corresponding concentrations of DG or solvent. Cell proliferation was measured by CCK8 assay, apoptosis was determined by flow cytometry, the expressions of endoplasmic reticulum stress(ERS)-related proteins(CHOP, cleaved caspase-12, GADD34 and BiP) were determined by western blot. The concentrations of SOD and MDA were also measured. The MI/R rat model was produced, rats were treated with DG intragastrically,recorded heart rate(HR) and mean artery pressure(MAP). The concentrations of serum creatine kinase(CK),SOD and MDA were measured, the tissue injury was determined by HE staining, and the related proteins of ERS was measured by Western blot. Results Compared with H9c2 group, the proliferation rate was downregulated and apoptosis rate was increased in Hypoxia group; compared with the Hypoxia group, proliferation rate was up-regulated and apoptosis rate was decreased in Hypoxia + DG group. Meanwhile, the expression levels of CHOP and Caspase-12 in Hypoxia group were increased compared with the H9c2 group, but the expression levels of GADD34 and BiP were decreased. In addition, hypoxia decreased the concentration of MDA, but increased SOD significantly. DG alleviated the effects of hypoxia. Furthermore, DG enhanced cardiac function of model rats of MI/R, alleviated the injury of myocardial tissue, inhibited the expressions of CHOP and Caspase-12, induced the expressions of GADD34 and BiP, down-regulated the concentration of MDA, and increased SOD in serum of rats.Conclusion DG attenuates hypocia-induced myocardial cell injury and myocardial injury of model rats of MI/R through inhibiting ERS.
【Abstacr】 Objective To investigate the effects and mechanisms of diosgenin(DG) on hypoxiainduced injury of rat myocardial H9c2 cell. Methods Cells were divided into H9c2, DG, Hypoxia and Hypoxia + DG group. Cells were treated with hypoxia for inducing injury, and cells were treated with corresponding concentrations of DG or solvent. Cell proliferation was measured by CCK8 assay, apoptosis was determined by flow cytometry, the expressions of endoplasmic reticulum stress(ERS)-related proteins(CHOP, cleaved caspase-12, GADD34 and BiP) were determined by western blot. The concentrations of SOD and MDA were also measured. The MI/R rat model was produced, rats were treated with DG intragastrically,recorded heart rate(HR) and mean artery pressure(MAP). The concentrations of serum creatine kinase(CK),SOD and MDA were measured, the tissue injury was determined by HE staining, and the related proteins of ERS was measured by Western blot. Results Compared with H9c2 group, the proliferation rate was downregulated and apoptosis rate was increased in Hypoxia group; compared with the Hypoxia group, proliferation rate was up-regulated and apoptosis rate was decreased in Hypoxia + DG group. Meanwhile, the expression levels of CHOP and Caspase-12 in Hypoxia group were increased compared with the H9c2 group, but the expression levels of GADD34 and BiP were decreased. In addition, hypoxia decreased the concentration of MDA, but increased SOD significantly. DG alleviated the effects of hypoxia. Furthermore, DG enhanced cardiac function of model rats of MI/R, alleviated the injury of myocardial tissue, inhibited the expressions of CHOP and Caspase-12, induced the expressions of GADD34 and BiP, down-regulated the concentration of MDA, and increased SOD in serum of rats.Conclusion DG attenuates hypocia-induced myocardial cell injury and myocardial injury of model rats of MI/R through inhibiting ERS.
引文
[1]He J,Ma C,Liu W,et al.On-chip monitoring of skeletal myoblast transplantation for the treatment of hypoxia-induced myocardial injury[J].Analyst,2014,139(18):4482-4490.
[2]Hu J,Chu Z,Han J,et al.Phosphorylation-dependent mitochondrial translocation of MAP4 is an early step in hypoxia-induced apoptosis in cardiomyocytes[J].Cell Death Dis,2014,5(9):e1424.
[3]Terai K,Hiramoto Y,Masaki M,et al.AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress[J].Mol Cell Biol,2005,25(21):9554-9575.
[4]Wang J,Hu X,Jiang H.ER stress-induced apoptosis:a novel ther apeutic target in heart failure[J].Int J Cardiol,2014,177(2):564-565.
[5]Doroudgar S,Glembotski CC.New concepts of endoplasmic reticulum Ffunction in the heart:programmed to conserve[J].J Mol Cell Cardiol,2013,55(1):85-91.
[6]Minamino T,Komuro I M.Endoplasmic reticulum stress as a therapeutic target in cardiovascular disease[J].Circ Res,2010,107(9):1071-1082.
[7]Wang L,Ma T,Zheng Y,et al.Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes[J].Int J Clin Exp Pathol,2015,8(5):4830-4836.
[8]韩军,宣佳利,胡浩然,等.金丝桃苷预处理减轻大鼠心肌缺血再灌注损伤作用与PI3K/Akt信号通路的关系[J].中国中药杂志,2015,40(1):118-123.
[9]黄浪,徐策,许光宇.黄芩苷对冠心病大鼠缺血再灌注损伤的治疗作用及机制[J].山东医药,2018,58(37):40-44.
[10]Greco S,Gaetano C,Martelli F.Hypoxami R regulation and function in ischemic cardiovascular diseases[J].Antioxid Redox Signal,2014,21(8):1202-1219.
[11]Huang Z,Han Z,Ye B,et al.Berberine alleviates cardiac ischemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes[J].Eur J Pharmacol,2015,762:1-10.
[12]Prabhakar NR,Semenza GL.Adaptive and maladaptive cardiorespiratory responses to continuous and intermittent hypoxia mediated by hypoxia-inducible factors 1 and 2[J].Physiol Rev,2012,92(3):967-1003.
[13]Hao S,Xu R,Li D,et al.Attenuation of streptozotocin-induced lipid profile anomalies in the heart,brain,and mRNA expression of HMG-CoA reductase by diosgenin in rats[J].Cell Biochem Biophys,2015,72(3):741-749.
[14]Pi WX,Feng XP,Ye LH,et al.Combination of morroniside and diosgenin prevents high glucose-induced cardiomyocytes apoptosis[J].Molecules,2017,22(1):E163.
[15]Fonseca ACRG,Elisabete F,Oliveira CR,et al.Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40peptide in brain endothelial cells[J].Biochim Biophys Acta,2013,1832(12):2191-2203.
[16]Nakagawa T,Zhu H,Morishima N,et al.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloidbeta[J].Nature,2000,403(6765):98-103.
[17]Xu J,Hu H,Chen B,et al.Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes[J].Plos One,2015,10(8):e0136443.
[18]Tang JY,Jin P,He Q,et al.Naringenin ameliorates hypoxia/reoxygenation-induced endoplasmic reticulum stress-mediated apoptosis in H9c2 myocardial cells:involvement in ATF6,IRE1αand PERK signaling activation[J].Mol Cell Biochem,2016,424(1-2):111-122.
[19]Sano R,Reed J C.ER stress-induced cell death mechanisms[J].Biochim Biophys Acta,2013,1833(12):3460-3470.
[20]Miyazaki Y,Kaikita K,Endo M,et al.C/EBP homologous protein deficiency attenuates myocardial reperfusion injury by inhibiting myocardial apoptosis and inflammation[J].Arterioscler Thromb Vasc Biol,2011,31(5):1124-1132.
[21]Feng JF,Tang YN,Ji H,et al.Biotransformation of Dioscorea nipponica by Rat Intestinal Microflora and Cardioprotective Effects of Diosgenin[J].Oxid Med Cell Longev,2017,2017:4176518.
[22]Qin J,Kang Y,Xu Z,et al.Dioscin prevents the mitochondrial apoptosis and attenuates oxidative stress in cardiac H9c2 cells[J].Drug Res,2014,64(1):47-52.
[23]Sun L,Zang W J,Wang H,et al.Acetylcholine Promotes ROSDetoxification Against Hypoxia/reoxygenation-Induced Oxidative Stress Through FoxO3a/PGC-1αDependent Superoxide Dismutase[J].Cell Physiol Biochem,2014,34(5):1614-1625.
[2]Hu J,Chu Z,Han J,et al.Phosphorylation-dependent mitochondrial translocation of MAP4 is an early step in hypoxia-induced apoptosis in cardiomyocytes[J].Cell Death Dis,2014,5(9):e1424.
[3]Terai K,Hiramoto Y,Masaki M,et al.AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress[J].Mol Cell Biol,2005,25(21):9554-9575.
[4]Wang J,Hu X,Jiang H.ER stress-induced apoptosis:a novel ther apeutic target in heart failure[J].Int J Cardiol,2014,177(2):564-565.
[5]Doroudgar S,Glembotski CC.New concepts of endoplasmic reticulum Ffunction in the heart:programmed to conserve[J].J Mol Cell Cardiol,2013,55(1):85-91.
[6]Minamino T,Komuro I M.Endoplasmic reticulum stress as a therapeutic target in cardiovascular disease[J].Circ Res,2010,107(9):1071-1082.
[7]Wang L,Ma T,Zheng Y,et al.Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes[J].Int J Clin Exp Pathol,2015,8(5):4830-4836.
[8]韩军,宣佳利,胡浩然,等.金丝桃苷预处理减轻大鼠心肌缺血再灌注损伤作用与PI3K/Akt信号通路的关系[J].中国中药杂志,2015,40(1):118-123.
[9]黄浪,徐策,许光宇.黄芩苷对冠心病大鼠缺血再灌注损伤的治疗作用及机制[J].山东医药,2018,58(37):40-44.
[10]Greco S,Gaetano C,Martelli F.Hypoxami R regulation and function in ischemic cardiovascular diseases[J].Antioxid Redox Signal,2014,21(8):1202-1219.
[11]Huang Z,Han Z,Ye B,et al.Berberine alleviates cardiac ischemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes[J].Eur J Pharmacol,2015,762:1-10.
[12]Prabhakar NR,Semenza GL.Adaptive and maladaptive cardiorespiratory responses to continuous and intermittent hypoxia mediated by hypoxia-inducible factors 1 and 2[J].Physiol Rev,2012,92(3):967-1003.
[13]Hao S,Xu R,Li D,et al.Attenuation of streptozotocin-induced lipid profile anomalies in the heart,brain,and mRNA expression of HMG-CoA reductase by diosgenin in rats[J].Cell Biochem Biophys,2015,72(3):741-749.
[14]Pi WX,Feng XP,Ye LH,et al.Combination of morroniside and diosgenin prevents high glucose-induced cardiomyocytes apoptosis[J].Molecules,2017,22(1):E163.
[15]Fonseca ACRG,Elisabete F,Oliveira CR,et al.Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40peptide in brain endothelial cells[J].Biochim Biophys Acta,2013,1832(12):2191-2203.
[16]Nakagawa T,Zhu H,Morishima N,et al.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloidbeta[J].Nature,2000,403(6765):98-103.
[17]Xu J,Hu H,Chen B,et al.Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes[J].Plos One,2015,10(8):e0136443.
[18]Tang JY,Jin P,He Q,et al.Naringenin ameliorates hypoxia/reoxygenation-induced endoplasmic reticulum stress-mediated apoptosis in H9c2 myocardial cells:involvement in ATF6,IRE1αand PERK signaling activation[J].Mol Cell Biochem,2016,424(1-2):111-122.
[19]Sano R,Reed J C.ER stress-induced cell death mechanisms[J].Biochim Biophys Acta,2013,1833(12):3460-3470.
[20]Miyazaki Y,Kaikita K,Endo M,et al.C/EBP homologous protein deficiency attenuates myocardial reperfusion injury by inhibiting myocardial apoptosis and inflammation[J].Arterioscler Thromb Vasc Biol,2011,31(5):1124-1132.
[21]Feng JF,Tang YN,Ji H,et al.Biotransformation of Dioscorea nipponica by Rat Intestinal Microflora and Cardioprotective Effects of Diosgenin[J].Oxid Med Cell Longev,2017,2017:4176518.
[22]Qin J,Kang Y,Xu Z,et al.Dioscin prevents the mitochondrial apoptosis and attenuates oxidative stress in cardiac H9c2 cells[J].Drug Res,2014,64(1):47-52.
[23]Sun L,Zang W J,Wang H,et al.Acetylcholine Promotes ROSDetoxification Against Hypoxia/reoxygenation-Induced Oxidative Stress Through FoxO3a/PGC-1αDependent Superoxide Dismutase[J].Cell Physiol Biochem,2014,34(5):1614-1625.