Objective Oxidative stress plays an important role in retinal pigmental epithelium （RPE） death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxyge...Objective Oxidative stress plays an important role in retinal pigmental epithelium （RPE） death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxygen species （ROS） including H2O2 can trigger apoptosis at lower concentrations and necrosis at higher concentrations,the exact molecular mechanism of RPE death is still unclear.The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS,especially at higher concentrations.Methods Cultured ARPE-19 cells were treated with H2O2 at different concentrations and cell viability was measured with the MTT assay.Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining.Furthermore,the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG.Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.Results H2O2 reduced the viability of ARPE-19 cells in a concentration-dependent manner,which was presented as a typical s-shaped curve.Cell death caused by high concentrations of H2O2 was confirmed to be programmed necrosis.Morphologically,dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane,positively detected with APOPercentage assay and PI staining.24-hour treatment with 500 ？mol/L H2O2 induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells.Moreover,antioxidant treatment using EGCG effectively protected cells from H2O2-induced injury,increasing cell viability from 14.17%±2.31% to 85.77%±4.58%.After H2O2 treatment,intracellular calcium levels were highly elevated with a maximum concentration of 1200nM.Significantly,the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway,rescuing the ARPE-19 cell fr展开更多
AIM: To observe the protective effect of astaxanthin(AST) against hydroquinone(HQ) mediated cell death in the apoptotic cascade and evaluate intracellular Ca2+ release, caspase-3, and-9 activation, reactive oxygen spe...AIM: To observe the protective effect of astaxanthin(AST) against hydroquinone(HQ) mediated cell death in the apoptotic cascade and evaluate intracellular Ca2+ release, caspase-3, and-9 activation, reactive oxygen species(ROS) production in ARPE-19 cells.METHODS: We cultured ARPE-19 cells in special mediums and performed MTT tests to determine protective effect of AST, before exposing the cells to HQ in an incubator. We analyzed intracellular Ca2+ release experiments, mitochondrial membrane depolarization, glutathione(GSH), glutathione peroxidase(GSH-Px) and ROS experiments, and apoptosis assay.RESULTS: ROS production ranges depend on the amount of cell death. We computed the correlation between ROS ranges and cell death by 20,70-dichlorofluorescein fluorescence, and Ca2+ levels by Fura-2-AM. HQ-induced cell death found out to rise ranges of caspase-3 and-9, and mitochondrial depolarization. These three steps were delayed by AST management.CONCLUSION: ARPE-19 cells are avoided from HQinduced ROS production and caspase-3 and-9 activation by AST. AST may limit the range of caspase synthesis, Ca2+ release and excess production of ROS with antiapoptotic effect. This study proposes a new therapeutic approach for the treatment of age-related macular degeneration.展开更多
AIM:To investigate the protective effect of heme oxygenase-1(HO-1)against H_(2)O_(2)-induced apoptosis in human ARPE-19 cells.METHODS:The lentiviral vector expressing HO-1 was prepared and transfected into apoptotic A...AIM:To investigate the protective effect of heme oxygenase-1(HO-1)against H_(2)O_(2)-induced apoptosis in human ARPE-19 cells.METHODS:The lentiviral vector expressing HO-1 was prepared and transfected into apoptotic ARPE-19 cells induced by H_(2)O_(2).Functional experiments including cell counting kit-8(CCK-8)assay,flow cytometry(FCM)and mitochondrial membrane potential assay were conducted.RESULTS:The ultrastructure of ARPE-19 cells was observed using transmission electron microscope(TEM).It was found that exogenous HO-1 significantly ameliorated H_(2)O_(2)-induced loss of cell viability,apoptosis and intracellular levels of reactive oxygen species(ROS)in ARPE-19 cells.The overexpression of HO-1 facilitated the transfer of nuclear factor erythroid-2-related factor 2(Nrf2)from cytoplasm to nucleus,which in turn upregualted expressions HO-1 and B-cell lymphoma-2(Bcl-2).Furthermore,HO-1 upregulation further inhibited H_(2)O_(2)-induced release of cysteinyl aspartate specific proteinase-3(caspase-3).CONCLUSION:Exogenous HO-1 protect ARPE-19 cells against H_(2)O_(2)-induced oxidative stress by regulating the expressions of Nrf2,HO-1,Bcl-2,and caspase-3.展开更多
基金supported by grants from the Natural Science Foundation of China (30801271)the Changchun Science and Technology Development Fund (08SF39)the International Joint Project from Science and Technology Bureau of Jilin Province (20090746)
文摘Objective Oxidative stress plays an important role in retinal pigmental epithelium （RPE） death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxygen species （ROS） including H2O2 can trigger apoptosis at lower concentrations and necrosis at higher concentrations,the exact molecular mechanism of RPE death is still unclear.The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS,especially at higher concentrations.Methods Cultured ARPE-19 cells were treated with H2O2 at different concentrations and cell viability was measured with the MTT assay.Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining.Furthermore,the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG.Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.Results H2O2 reduced the viability of ARPE-19 cells in a concentration-dependent manner,which was presented as a typical s-shaped curve.Cell death caused by high concentrations of H2O2 was confirmed to be programmed necrosis.Morphologically,dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane,positively detected with APOPercentage assay and PI staining.24-hour treatment with 500 ？mol/L H2O2 induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells.Moreover,antioxidant treatment using EGCG effectively protected cells from H2O2-induced injury,increasing cell viability from 14.17%±2.31% to 85.77%±4.58%.After H2O2 treatment,intracellular calcium levels were highly elevated with a maximum concentration of 1200nM.Significantly,the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway,rescuing the ARPE-19 cell fr
文摘AIM: To observe the protective effect of astaxanthin(AST) against hydroquinone(HQ) mediated cell death in the apoptotic cascade and evaluate intracellular Ca2+ release, caspase-3, and-9 activation, reactive oxygen species(ROS) production in ARPE-19 cells.METHODS: We cultured ARPE-19 cells in special mediums and performed MTT tests to determine protective effect of AST, before exposing the cells to HQ in an incubator. We analyzed intracellular Ca2+ release experiments, mitochondrial membrane depolarization, glutathione(GSH), glutathione peroxidase(GSH-Px) and ROS experiments, and apoptosis assay.RESULTS: ROS production ranges depend on the amount of cell death. We computed the correlation between ROS ranges and cell death by 20,70-dichlorofluorescein fluorescence, and Ca2+ levels by Fura-2-AM. HQ-induced cell death found out to rise ranges of caspase-3 and-9, and mitochondrial depolarization. These three steps were delayed by AST management.CONCLUSION: ARPE-19 cells are avoided from HQinduced ROS production and caspase-3 and-9 activation by AST. AST may limit the range of caspase synthesis, Ca2+ release and excess production of ROS with antiapoptotic effect. This study proposes a new therapeutic approach for the treatment of age-related macular degeneration.
基金Supported by Natural Science Foundation of Fujian Province(No.2017J01274)Health Science and Technology Program of Fujian Province(No.2015-ZQNJC-26)Quanzhou Science and Technology Plan Project(No.2016Z053)。
文摘AIM:To investigate the protective effect of heme oxygenase-1(HO-1)against H_(2)O_(2)-induced apoptosis in human ARPE-19 cells.METHODS:The lentiviral vector expressing HO-1 was prepared and transfected into apoptotic ARPE-19 cells induced by H_(2)O_(2).Functional experiments including cell counting kit-8(CCK-8)assay,flow cytometry(FCM)and mitochondrial membrane potential assay were conducted.RESULTS:The ultrastructure of ARPE-19 cells was observed using transmission electron microscope(TEM).It was found that exogenous HO-1 significantly ameliorated H_(2)O_(2)-induced loss of cell viability,apoptosis and intracellular levels of reactive oxygen species(ROS)in ARPE-19 cells.The overexpression of HO-1 facilitated the transfer of nuclear factor erythroid-2-related factor 2(Nrf2)from cytoplasm to nucleus,which in turn upregualted expressions HO-1 and B-cell lymphoma-2(Bcl-2).Furthermore,HO-1 upregulation further inhibited H_(2)O_(2)-induced release of cysteinyl aspartate specific proteinase-3(caspase-3).CONCLUSION:Exogenous HO-1 protect ARPE-19 cells against H_(2)O_(2)-induced oxidative stress by regulating the expressions of Nrf2,HO-1,Bcl-2,and caspase-3.
