氧化损伤对视网膜色素上皮细胞三联组氨酸核苷结合蛋白1(HINT1)表达的影响
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摘要
目的探讨过氧化氢(hydrogen peroxide,H_2O_2)诱导人视网膜色素上皮(retinal pigment epithelium,RPE)细胞氧化损伤对三联组氨酸核苷结合蛋白1(histidine triad nucleotide binding protein 1,HINT1)表达的影响。方法将人ARPE-19细胞系分别用0μmol·L~(-1)H_2O_2(对照组)、50μmol·L~(-1)H_2O_2、100μmol·L~(-1)H_2O_2、200μmol·L~(-1)H_2O_2、400μmol·L~(-1)H_2O_2、600μmol·L~(-1)H_2O_26个浓度梯度处理。采用Live/Dead荧光双染法和MTT法分别检测细胞活力和增殖能力;采用Western blot检测RPE细胞内BAX、BCL-2以及HINT1的表达水平。结果 H_2O_2处理6 h后,对照组及50μmol·L~(-1)、100μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)及600μmol·L~(-1)H_2O_2组RPE细胞死亡率分别为(0. 35±0. 11)%、(2. 03±0. 87)%、(3. 76±1. 41)%、(12. 17±1. 26)%、(17. 69±2. 24)%、(25. 22±0. 59)%,RPE细胞死亡率呈剂量依赖性升高(P <0. 05)。H_2O_2作用后对照组及50μmol·L~(-1)、100μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)、600μmol·L~(-1)H_2O_2组RPE细胞生存率分别为(40. 72±2. 71)%、(37. 75±0. 62)%、(35. 30±0. 68)%、(34. 10±0. 74)%、(33. 61±0. 71)%、(31. 05±1. 41)%,RPE细胞增殖明显减少(P <0. 05),具有剂量-效应关系。随着H_2O_2浓度的增加,BAX蛋白表达逐渐上升(P <0. 01),而抗凋亡BCL-2蛋白表达则逐渐下降(P <0. 01),BAX/BCL-2比值相比对照组分别升高1. 85倍(50μmol·L~(-1)H_2O_2组)、2. 35倍(100μmol·L~(-1)H_2O_2组)、2. 86倍(200μmol·L~(-1)H_2O_2组)、3. 97倍(400μmol·L~(-1)H_2O_2组)、8. 50倍(600μmol·L~(-1)H_2O_2组); HINT1在H_2O_2处理的RPE细胞中以剂量依赖性方式逐渐下降(P <0. 01),分别下降0. 78倍(50μmol·L~(-1)H_2O_2组)、0. 59倍(100μmol·L~(-1)H_2O_2组)、0. 45倍(200μmol·L~(-1)H_2O_2组)、0. 42倍(400μmol·L~(-1)H_2O_2组)、0. 38倍(600μmol·L~(-1)H_2O_2组)。结论 H_2O_2氧化损伤诱导RPE细胞活力显著减弱,细胞凋亡相关蛋白BAX表达升高;同时,细胞中HINT1表达下调,表明HINT1可能通过影响细胞凋亡参与年龄相关性黄斑变性的发生,为其药物作用的潜在靶点。
Objective To investigate the effects of hydrogen peroxide( H_2O_2) on the expression of histidine triad nucleotide-binding protein 1( HINT1) in oxidative damaged retinal pigment epithelial( RPE) cells in vitro.Methods ARPE-19 cell line was cultured conventionally and treated with six different concentrations of H_2O_2 including 0 μmol ·L~(-1) H_2O_2( control group),50 μmol·L~(-1) H_2O_2,100 μmol·L~(-1) H_2O_2,200 μmol·L~(-1) H_2O_2,400 μmol ·L~(-1) H_2O_2,600 μmol ·L~(-1) H_2O_2 of treatment groups.Live/Dead fluorescent staining and MTT were utilized to evaluate cell viability and proliferative capacity,respectively.The expression of HINT1,BAX and BCL-2 in RPE cell were examined by western blot.Results The death rates of RPE cells treated with 0 μmol·L~(-1),50 μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1) and 600 μmol·L~(-1) of H_2O_2 for 6 h were( 0.35 ± 0.11) %,( 2.03 ± 0.87) %,( 3.76 ±1.41) %,( 12.17 ± 1.26) %,( 17.69 ± 2.24) %,( 25.22 ± 0.59) %,respectively,which significantly increased in a dose-dependent manner( P< 0.05).The proliferation rates of RPE cell treated with 0 μmol·L~(-1),50μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1) and 600μmol·L~(-1) of H_2O_2 were( 40.72 ± 2.71) %,( 37.75 ± 0.62) %,( 35.30 ± 0.68) %,( 34.10 ± 0.74) %,( 33.61 ± 0.71) %,( 31.05 ± 1.41) %,respectively presenting with reduced proliferation ability in a dosedependent manner( P < 0.05).With the increase of H_2O_2 concentration,BAX protein level gradually increased( P < 0.01),while anti-apoptotic protein BCL-2 gradually decreased( P < 0.01); The relative ratio of BAX/BCL-2 enhanced 1.85-fold( 50 μmol·L~(-1) H_2O_2),2.35-fold( 100 μmol·L~(-1) H_2O_2),2.86-fold( 200 μmol ·L~(-1) H_2O_2),3.97-fold( 400μmol·L~(-1) H_2O_2),8.50-fold( 600 μmol·L~(-1) H_2O_2),respectively compared with control group; The level of HINT1 significantly decreased in H_2O_2 treated RPE cells in a dose dependent manner( P < 0.01),with decreased 0.78-fold( 50 μmol ·L~(-1) H_2O_2),0.59-fold( 100 μmol ·L~(-1) H_2O_2),0.45-fold( 200 μmol·L~(-1) H_2O_2),0.42-fold( 400 μmol·L~(-1) H_2O_2),0.38-fold( 600 μmol ·L~(-1) H_2O_2),respectively compared with control group.Conclusion Oxidative injured RPE cells induced by different concentrations of H_2O_2 show decreased cell viabilities and increased level of apoptosis-related proteins.Meanwhile,the expression of HINT1 in RPE cells is down-regulated after treatment of H_2O_2,indicating HINT1 may play an important role in the pathogenesis of age-related macular degeneration( AMD) by regulating apoptosis.This research provides potential target for AMD therapy and drug development.
Objective To investigate the effects of hydrogen peroxide( H_2O_2) on the expression of histidine triad nucleotide-binding protein 1( HINT1) in oxidative damaged retinal pigment epithelial( RPE) cells in vitro.Methods ARPE-19 cell line was cultured conventionally and treated with six different concentrations of H_2O_2 including 0 μmol ·L~(-1) H_2O_2( control group),50 μmol·L~(-1) H_2O_2,100 μmol·L~(-1) H_2O_2,200 μmol·L~(-1) H_2O_2,400 μmol ·L~(-1) H_2O_2,600 μmol ·L~(-1) H_2O_2 of treatment groups.Live/Dead fluorescent staining and MTT were utilized to evaluate cell viability and proliferative capacity,respectively.The expression of HINT1,BAX and BCL-2 in RPE cell were examined by western blot.Results The death rates of RPE cells treated with 0 μmol·L~(-1),50 μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1) and 600 μmol·L~(-1) of H_2O_2 for 6 h were( 0.35 ± 0.11) %,( 2.03 ± 0.87) %,( 3.76 ±1.41) %,( 12.17 ± 1.26) %,( 17.69 ± 2.24) %,( 25.22 ± 0.59) %,respectively,which significantly increased in a dose-dependent manner( P< 0.05).The proliferation rates of RPE cell treated with 0 μmol·L~(-1),50μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1) and 600μmol·L~(-1) of H_2O_2 were( 40.72 ± 2.71) %,( 37.75 ± 0.62) %,( 35.30 ± 0.68) %,( 34.10 ± 0.74) %,( 33.61 ± 0.71) %,( 31.05 ± 1.41) %,respectively presenting with reduced proliferation ability in a dosedependent manner( P < 0.05).With the increase of H_2O_2 concentration,BAX protein level gradually increased( P < 0.01),while anti-apoptotic protein BCL-2 gradually decreased( P < 0.01); The relative ratio of BAX/BCL-2 enhanced 1.85-fold( 50 μmol·L~(-1) H_2O_2),2.35-fold( 100 μmol·L~(-1) H_2O_2),2.86-fold( 200 μmol ·L~(-1) H_2O_2),3.97-fold( 400μmol·L~(-1) H_2O_2),8.50-fold( 600 μmol·L~(-1) H_2O_2),respectively compared with control group; The level of HINT1 significantly decreased in H_2O_2 treated RPE cells in a dose dependent manner( P < 0.01),with decreased 0.78-fold( 50 μmol ·L~(-1) H_2O_2),0.59-fold( 100 μmol ·L~(-1) H_2O_2),0.45-fold( 200 μmol·L~(-1) H_2O_2),0.42-fold( 400 μmol·L~(-1) H_2O_2),0.38-fold( 600 μmol ·L~(-1) H_2O_2),respectively compared with control group.Conclusion Oxidative injured RPE cells induced by different concentrations of H_2O_2 show decreased cell viabilities and increased level of apoptosis-related proteins.Meanwhile,the expression of HINT1 in RPE cells is down-regulated after treatment of H_2O_2,indicating HINT1 may play an important role in the pathogenesis of age-related macular degeneration( AMD) by regulating apoptosis.This research provides potential target for AMD therapy and drug development.
引文
[1]OWEN C G,JARRAR Z,WORMALD R,COOK DG,FLETCHER A E,RUDNICKA A R.The estimated prevalence and incidence of late stage age related macular degeneration in the UK[J].Br J Ophthalmol,2012,96(5):752-756.
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[4]KOPITZ J,HOLZ F G,KAEMMERER E,SCHUTT F.Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration[J].Biochimie,2004,86(11):825-831.
[5]SU T,SUZUI M,WANG L,LIN C S,XING W Q,WEINSTEIN I B.Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis[J].Proc Natl Acad Sci USA,2003,100(13):7824-7829.
[6]VARADARAJULU J,LEBAR M,KRISHNAMOORTHY G,HA-BELT S,LU J,BERNARD WEINSTEIN I.Increased anxiety-related behaviour in Hint1 knockout mice[J].Behav Brain Res,2011,220(2):305-311.
[7]BARBIER E,WANG J B.Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level[J].BMC Neurosci,2009,10(10):132.
[8]MARAZITA M C,DUGOUR A,MARQUIONI-RAMELLA M D,FIGUEROA J M,SUBURO A M.Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression in retinal pigment epithelial cells:Implications for Age-related Macular Degeneration[J].Redox Biol,2016,7(15):78-87.
[9]Age-Related Eye Disease Study 2 Research Group.Lutein+zeaxanthin and omega-3 fatty acids for age-related macular degeneration:The age-related eye disease study 2(areds2)randomized clinical trial[J].JAMA,2013,309(19):2005-2015.
[10]YU D Y,Cringle S J.Retinal degeneration and local oxygen metabolism[J].Exp Eye Res,2005,80(6):745-751.
[11]DROBEK S,OWIK M,KARCZEWICZ D,SAFRANOW K.The potential role of oxidative stress in the pathogenesis of the age-related macular degeneration(AMD)[J].Postepy Hig Med Dosw,2007,61(17):28-37.
[12]HANUS J,ANDERSON C,WANG S.RPE necroptosis in response to oxidative stress and in AMD[J].Ageing Res Rev,2015,24(Pt B):286-298.
[13]JARRETT S G,BOULTON M E.Consequences of oxidative stress in age-related macular degeneration[J].Mol Aspects Med,2012,33(4):399-417.
[14]RODIER F,CAMPISI J.Four faces of cellular senescence[J].JCell Biol,2011,192(4):547-556.
[15]CAI J,NELSON K C,WU M,STERNBERG P,JONES D P.Oxidative damage and protection of the RPE[J].Prog Retin Eye Res,2000,19(2):205-221.
[16]LI Z,DONG X,LIU H,CHEN X,SHI H,FAN Y,et al.Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phaseⅡenzymes through activation of PI3K/Akt[J].Mol Vis,2013,19(1):1656-1666.
[17]BARAK A,MORSE L S,GOLDKORN T.Ceramide:a potential mediator of apoptosis in human retinal pigment epithelial cells[J].Invest Ophthalmol Vis Sci,2001,42(12):247-254.
[18]KIM M H,CHUNG J,YANG J,CHUNG S M,KWAG N H,YOO JS.Hydrogen peroxide-induced cell death in a human retinal pigment epithelial cell line,ARPE-19[J].Korean J Ophthalmol,2003,17(6):19-28.
[19]AREND N,WERTHEIMER C,LAUBICHLER P,WOLF A,KA-MPIK A,KERNT M.Idebenone prevents oxidative stress,cell death and senescence of retinal pigment epithelium cells by stabilizing BAX/Bcl-2 ratio[J].Ophthalmologica,2015,34(2):146-147.
[20]MURTHY R K,RAVI K,BALAIYA S,BRAR V S,V CHALAMK.Lutein protects retinal pigment epithelium from cytotoxic oxidative stress[J].Cutan Ocul Toxicol,2014,33(2):132-137.
[21]OHNO-MATSUI K.Parallel findings in age-related macular degeneration and Alzheimer’s disease[J].Prog Retin Eye Res,2011,30(4):217-238.
[22]WEISKE J,HUBER O.The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription[J].J Cell Sci,2005,118(Pt 14):3117-3129.
[23]ABDEL-RASSOUL R,ALVES S,PANTESCO V,DE VOS J,MI-CHEL B,PERRET M,et al.Distinct transcriptome expression of the temporal cortex of the primate Microcebus murinus during brain aging versus Alzheimer’s disease-like pathology[J].PLoSOne,2010,5(5):3307-3314.
[24]ZHOU T,HU Y,CHEN Y,ZHOU KK,ZHANG B,GAO G,et al.The pathogenic role of the canonical Wnt pathway in age-related macular degeneration[J].Invest Ophthalmol Vis Sci,2010,51(13):4371-4379.
[25]WANG Y,XIAO Z,LIU X,BERK M.Venlafaxine modulates depression-induced behaviour and the expression of Bax mRNA and Bcl-xl mRNA in both hippocampus and myocardium[J].Hum Psychopharmacol,2011,26(2):95-101.
[2]LIM L S,MITCHELL P,SEDDON J M,HOLZ F G,WONG TY.Age-related macular degeneration[J].Lancet,2012,379(8927):1728-1738.
[3]WONG W L,SU X,LI X,CHEUNG C M G,KLEIN R,CHENG CY,et al.Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040:a systematic review and meta-analysis[J].Lancet Glob Heal,2014,2(2):e106-116.
[4]KOPITZ J,HOLZ F G,KAEMMERER E,SCHUTT F.Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration[J].Biochimie,2004,86(11):825-831.
[5]SU T,SUZUI M,WANG L,LIN C S,XING W Q,WEINSTEIN I B.Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis[J].Proc Natl Acad Sci USA,2003,100(13):7824-7829.
[6]VARADARAJULU J,LEBAR M,KRISHNAMOORTHY G,HA-BELT S,LU J,BERNARD WEINSTEIN I.Increased anxiety-related behaviour in Hint1 knockout mice[J].Behav Brain Res,2011,220(2):305-311.
[7]BARBIER E,WANG J B.Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level[J].BMC Neurosci,2009,10(10):132.
[8]MARAZITA M C,DUGOUR A,MARQUIONI-RAMELLA M D,FIGUEROA J M,SUBURO A M.Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression in retinal pigment epithelial cells:Implications for Age-related Macular Degeneration[J].Redox Biol,2016,7(15):78-87.
[9]Age-Related Eye Disease Study 2 Research Group.Lutein+zeaxanthin and omega-3 fatty acids for age-related macular degeneration:The age-related eye disease study 2(areds2)randomized clinical trial[J].JAMA,2013,309(19):2005-2015.
[10]YU D Y,Cringle S J.Retinal degeneration and local oxygen metabolism[J].Exp Eye Res,2005,80(6):745-751.
[11]DROBEK S,OWIK M,KARCZEWICZ D,SAFRANOW K.The potential role of oxidative stress in the pathogenesis of the age-related macular degeneration(AMD)[J].Postepy Hig Med Dosw,2007,61(17):28-37.
[12]HANUS J,ANDERSON C,WANG S.RPE necroptosis in response to oxidative stress and in AMD[J].Ageing Res Rev,2015,24(Pt B):286-298.
[13]JARRETT S G,BOULTON M E.Consequences of oxidative stress in age-related macular degeneration[J].Mol Aspects Med,2012,33(4):399-417.
[14]RODIER F,CAMPISI J.Four faces of cellular senescence[J].JCell Biol,2011,192(4):547-556.
[15]CAI J,NELSON K C,WU M,STERNBERG P,JONES D P.Oxidative damage and protection of the RPE[J].Prog Retin Eye Res,2000,19(2):205-221.
[16]LI Z,DONG X,LIU H,CHEN X,SHI H,FAN Y,et al.Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phaseⅡenzymes through activation of PI3K/Akt[J].Mol Vis,2013,19(1):1656-1666.
[17]BARAK A,MORSE L S,GOLDKORN T.Ceramide:a potential mediator of apoptosis in human retinal pigment epithelial cells[J].Invest Ophthalmol Vis Sci,2001,42(12):247-254.
[18]KIM M H,CHUNG J,YANG J,CHUNG S M,KWAG N H,YOO JS.Hydrogen peroxide-induced cell death in a human retinal pigment epithelial cell line,ARPE-19[J].Korean J Ophthalmol,2003,17(6):19-28.
[19]AREND N,WERTHEIMER C,LAUBICHLER P,WOLF A,KA-MPIK A,KERNT M.Idebenone prevents oxidative stress,cell death and senescence of retinal pigment epithelium cells by stabilizing BAX/Bcl-2 ratio[J].Ophthalmologica,2015,34(2):146-147.
[20]MURTHY R K,RAVI K,BALAIYA S,BRAR V S,V CHALAMK.Lutein protects retinal pigment epithelium from cytotoxic oxidative stress[J].Cutan Ocul Toxicol,2014,33(2):132-137.
[21]OHNO-MATSUI K.Parallel findings in age-related macular degeneration and Alzheimer’s disease[J].Prog Retin Eye Res,2011,30(4):217-238.
[22]WEISKE J,HUBER O.The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription[J].J Cell Sci,2005,118(Pt 14):3117-3129.
[23]ABDEL-RASSOUL R,ALVES S,PANTESCO V,DE VOS J,MI-CHEL B,PERRET M,et al.Distinct transcriptome expression of the temporal cortex of the primate Microcebus murinus during brain aging versus Alzheimer’s disease-like pathology[J].PLoSOne,2010,5(5):3307-3314.
[24]ZHOU T,HU Y,CHEN Y,ZHOU KK,ZHANG B,GAO G,et al.The pathogenic role of the canonical Wnt pathway in age-related macular degeneration[J].Invest Ophthalmol Vis Sci,2010,51(13):4371-4379.
[25]WANG Y,XIAO Z,LIU X,BERK M.Venlafaxine modulates depression-induced behaviour and the expression of Bax mRNA and Bcl-xl mRNA in both hippocampus and myocardium[J].Hum Psychopharmacol,2011,26(2):95-101.