Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regen...Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lnc RNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with mi R-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and mi R-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.展开更多
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 from H2O2-induced death.Conclusions At high concentrations,H2O2 induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.展开更多
基金supported by the National Natural Science Foundation of China,Nos.82271397(to MG),82001293(to MG),82171355(to RX),81971295(to RX)and 81671189(to RX)。
文摘Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lnc RNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with mi R-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and mi R-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.
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 from H2O2-induced death.Conclusions At high concentrations,H2O2 induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.
