自噬减少组蛋白介导的肾小管上皮细胞凋亡
|
摘要
目的:探讨自噬对组蛋白介导的人肾小管上皮细胞(HK-2)凋亡的效应。方法:以不同浓度组蛋白刺激HK-2,应用流式细胞仪和免疫印迹方法检测HK-2凋亡和自噬相关蛋白LC3Ⅱ、Beclin1的表达情况,初步了解组蛋白对HK-2凋亡和自噬的影响。以自噬抑制剂3-甲基腺嘌呤(3-MA)预处理后,观察组蛋白诱导的HK-2凋亡和caspase-3活化的情况,进一步明确自噬通路对凋亡的作用。结果:组蛋白25、50、100μg/mL组HK-2凋亡率和LC3Ⅱ、Beclin1表达水平均显著升高(P<0.05),并且组蛋白诱导HK-2凋亡具有浓度依赖效应。自噬抑制剂3-MA显著增强HK-2凋亡和caspase-3活化(P<0.05)。结论:自噬能够减轻组蛋白介导的HK-2凋亡,提示活化自噬通路在急性肾损伤中具有潜在的应用价值。
ObjectiveTo investigate the effect of autophagy on histone-mediated apoptosis of human proximal tubular endothelial cells(HK-2).MethodsTo investigate the effect of histones on the autophagy and apoptosis,HK-2 cells were treated with increasing concentrations of histones.The rate of apoptosis and the expressions of autophagy-related protien LC3Ⅱ and Beclin1 in HK-2 cells were detected by using flow cytometry and immunoblotting assay,respectively.To further confirm the effect of autophagy on apoptosis of HK-2 cells,cells were incubated with histones after one hour pretreatment with 10 mmol/L 3-MA,a pharmacological inhibitor.The rate of apoptosis and the activity of caspase-3 of HK-2 cells were detected separately by using flow cytometry and immunoblotting assay.ResultsHistones significantly enhanced apoptosis of HK-2 cells in a dose-dependent manner,with the increased expressions of LC3Ⅱ and Beclin1.Blockage of autophagy by 3-MA significantly increased the apoptosis of HK-2 cells and the activity of caspase3.ConclusionAutophagy in proximal tubules protects against apoptosis induced by histones,with potential value in acute kidney injury(AKI).
ObjectiveTo investigate the effect of autophagy on histone-mediated apoptosis of human proximal tubular endothelial cells(HK-2).MethodsTo investigate the effect of histones on the autophagy and apoptosis,HK-2 cells were treated with increasing concentrations of histones.The rate of apoptosis and the expressions of autophagy-related protien LC3Ⅱ and Beclin1 in HK-2 cells were detected by using flow cytometry and immunoblotting assay,respectively.To further confirm the effect of autophagy on apoptosis of HK-2 cells,cells were incubated with histones after one hour pretreatment with 10 mmol/L 3-MA,a pharmacological inhibitor.The rate of apoptosis and the activity of caspase-3 of HK-2 cells were detected separately by using flow cytometry and immunoblotting assay.ResultsHistones significantly enhanced apoptosis of HK-2 cells in a dose-dependent manner,with the increased expressions of LC3Ⅱ and Beclin1.Blockage of autophagy by 3-MA significantly increased the apoptosis of HK-2 cells and the activity of caspase3.ConclusionAutophagy in proximal tubules protects against apoptosis induced by histones,with potential value in acute kidney injury(AKI).
引文
[1]ALLAM R,SCHERBAUM C R,DARISIPUDI M N,et al.Histones from dying renal cells aggravate kidney injury viaTLR2 and TLR4[J].J Am Soc Nephrol,2012,23(8):1375-1388.
[2]李晨昊,张超,张越,等.腺苷及腺苷受体在急性肾损伤中的保护作用[J].实用医学杂志,2015,31(20):3451-3453.
[3]LIVINGSTON M J,DONG Z.Autophagy in acute kidney injury[J].Semin Nephrol,2014,34(1):17-26.
[4]LEVINE B,KLIONSKY D J.Development by self-digestion:molecular mechanisms and biological functions of autophagy[J].Dev Cell,2004,6(4):463-477.
[5]THORBURN A.Apoptosis and autophagy:regulatory connectionsbetween two supposedly different processes[J].Apoptosis,2008,13(1):1-9.
[6]JIANG M,LIU K,LUO J,et al.Autophagy is a renoprotectivemechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury[J].Am J Pathol,2010,176(3):1181-1192.
[7]SAFFARZADEH M,JUENEMANN C,QUEISSER M A,et al.Neutrophil extracellular traps directly induce epithelial andendothelial cell death:a predominant role of histones[J].PLo SOne,2012,7(2):e32366.
[8]HUANG H,EVANKOVICH J,YAN W,et al.Endogenoushistones function as alarmins in sterile inflammatory liver injurythrough Toll-like receptor 9 in mice[J].Hepatology,2011,54(3):999-1008.
[9]XU J,ZHANG X,PELAYO R,et al.Extracellular histones aremajor mediators of death in sepsis[J].Nat Med,2009,15(11):1318-1321.
[10]LEVINE B.Autophagy in cell death:an innocent convict[J].JClin Invest,2005,115(10):2679-2688.
[11]CHIEN C T,SHYUE S K,LAI M K.Bcl-x L augmentationpotentially reduces ischemia/reperfusion induced proximal anddistal tubular apoptosis and autophagy[J].Transplantation,2007,84(9):1183-1190.
[12]SUZUKI C,ISAKA Y,TAKABATAKE Y,et al.Participationof autophagy in renal ischemia/reperfusion injury[J].BiochemBiophys Res Commun,2008,368(1):100-106.
[13]CHEN Y,AZAD M B,GIBSON S B.Methods for detectingautophagy and determining autophagy-induced cell death Thisreview is one of a selection of papers published in a SpecialIssue on Oxidative Stress in Health and Disease.[J].Can JPhysiol Pharmacol,2010,88(3):285-295.
[14]LIANG C,FENG P,KU B,et al.Autophagic and tumoursuppressor activity of a novel Beclin1-binding protein UVRAG[J].Nat Cell Biol,2006,8(7):688-698.
[15]PERIYASAMY-THANDAVAN S,JIANG M,WEI Q,et al.Autophagy is cytoprotective during cisplatin injury of renalproximal tubular cells[J].Kidney Int,2008,74(5):631-640.
[16]KIMURA T,TAKABATAKE Y,TAKAHASHI A,et al.Autophagy protects the proximal tubule from degeneration andacute ischemic injury[J].J Am Soc Nephrol,2011,22(5):902-913.
[17]MEI S,LIVINGSTON M,HAO J,et al.Autophagy is activatedto protect against endotoxic acute kidney injury[J].Sci Rep,2016,6:22171.
[2]李晨昊,张超,张越,等.腺苷及腺苷受体在急性肾损伤中的保护作用[J].实用医学杂志,2015,31(20):3451-3453.
[3]LIVINGSTON M J,DONG Z.Autophagy in acute kidney injury[J].Semin Nephrol,2014,34(1):17-26.
[4]LEVINE B,KLIONSKY D J.Development by self-digestion:molecular mechanisms and biological functions of autophagy[J].Dev Cell,2004,6(4):463-477.
[5]THORBURN A.Apoptosis and autophagy:regulatory connectionsbetween two supposedly different processes[J].Apoptosis,2008,13(1):1-9.
[6]JIANG M,LIU K,LUO J,et al.Autophagy is a renoprotectivemechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury[J].Am J Pathol,2010,176(3):1181-1192.
[7]SAFFARZADEH M,JUENEMANN C,QUEISSER M A,et al.Neutrophil extracellular traps directly induce epithelial andendothelial cell death:a predominant role of histones[J].PLo SOne,2012,7(2):e32366.
[8]HUANG H,EVANKOVICH J,YAN W,et al.Endogenoushistones function as alarmins in sterile inflammatory liver injurythrough Toll-like receptor 9 in mice[J].Hepatology,2011,54(3):999-1008.
[9]XU J,ZHANG X,PELAYO R,et al.Extracellular histones aremajor mediators of death in sepsis[J].Nat Med,2009,15(11):1318-1321.
[10]LEVINE B.Autophagy in cell death:an innocent convict[J].JClin Invest,2005,115(10):2679-2688.
[11]CHIEN C T,SHYUE S K,LAI M K.Bcl-x L augmentationpotentially reduces ischemia/reperfusion induced proximal anddistal tubular apoptosis and autophagy[J].Transplantation,2007,84(9):1183-1190.
[12]SUZUKI C,ISAKA Y,TAKABATAKE Y,et al.Participationof autophagy in renal ischemia/reperfusion injury[J].BiochemBiophys Res Commun,2008,368(1):100-106.
[13]CHEN Y,AZAD M B,GIBSON S B.Methods for detectingautophagy and determining autophagy-induced cell death Thisreview is one of a selection of papers published in a SpecialIssue on Oxidative Stress in Health and Disease.[J].Can JPhysiol Pharmacol,2010,88(3):285-295.
[14]LIANG C,FENG P,KU B,et al.Autophagic and tumoursuppressor activity of a novel Beclin1-binding protein UVRAG[J].Nat Cell Biol,2006,8(7):688-698.
[15]PERIYASAMY-THANDAVAN S,JIANG M,WEI Q,et al.Autophagy is cytoprotective during cisplatin injury of renalproximal tubular cells[J].Kidney Int,2008,74(5):631-640.
[16]KIMURA T,TAKABATAKE Y,TAKAHASHI A,et al.Autophagy protects the proximal tubule from degeneration andacute ischemic injury[J].J Am Soc Nephrol,2011,22(5):902-913.
[17]MEI S,LIVINGSTON M,HAO J,et al.Autophagy is activatedto protect against endotoxic acute kidney injury[J].Sci Rep,2016,6:22171.