| Hydrogen sulfide(H2S)is the third gaseous transmitter after nitric oxide and carbon monoxide.Accumulating evidence shows that H2S regulates a variety of pathophysiological processes and performs important biological functions in mammals.In the central nervous system,H2S acts as a neuromodulator,facilitating NMDA receptor-mediated long-term potentiation formation.However,it remains unclear whether H2S affects neurogenesis.Therefore,this studyis sought to examine whether H2S affects the proliferation and differentiation of neural stem cells in mouse SVZ and the mechanism involved.The findings would provide the experimental basis for neurobiological function of H2S in brain.Objective:To study the possible effects and underlying mechanism of H2S on neural stem cells proliferation and differentiation in mouse subventricular zone(SVZ).Methods:(1)Neural stem cells(NSCs)were isolated from SVZ of E13-14 C57BL mouse.For neurosphere counting experiment,the embryonic NSCs of the 2nd to 4th passage were used in this study.The NSCs specific markers nestin and Sox2 were used for identification and labeling of NSCs in immunoflluorescent study.(2)Immunofluorescent staining,western blot and RT-PCR were applied to identify the distribution and expression of H2S-producing enzymes including cystathionine beta synthase(CBS)and cystathionine gamma-lyase(CSE)in embryonic NSCs,adult SVZ area of C57BL mice and in the brain of embryonic E14.(3)Neurosphere counting and Brdu incorporation assay in combination with immunofluorescent study were applied to detect the self-renewal and proliferation of NSCs following treatment with H2S donors NaHS and GYY4137.The neuron specific marker Tuj-1 and the astrocyte specific marker GFAP were used in immunofluorescent study to determine the differentiation phenotypes of NSCs following treatment with H2S donors NaHS and GYY4137.MTT method was performed to detect the survival of NSCs.Caspase-3 immunofluorescent staining was used to assess the cell apoptosis of NSCs.Reverse transcription PCR was used to detect the mRNA level of glial derived neurotrophic factor of astrocytes.Western blotting was applied to evaluate the phosphorylation level of CREB.Results:(1)Immunofluorescent study showed that about 99%of the isolated embryonic NSCs were positively stained with NSCs specific markers nestin and Sox2.These NSCs were thus used for in vitro experimentation in this study.(2)Immunofluorescent staining revealed CBS expression and its co-localization with nestin in the adult SVZ of C57BL mice and in the brain slices of embryonic E14,as well as in vitro cultures of NSCs.This was confirmed by reverse PCR and western blotting study.Similarly,CSE was detected in the brain of embryonic E14 and in vitro cultures of embryonic NSCs,but not in the adult SVZ of C57BL mice.Real time PCR showed the mRNA level of CBS was about three-foldto that of CSE in isolated embryonic NSCs.(3)Neurosphere counting and Brdu incorporation assay showed that application with NaHS and H2S slow-releasing compound GYY4137 promoted the proliferation of NSCs,while’old’ GYY4137 did not produce this effect.Moreover,the in vivo Brdu incorporation assay displayed that intraperitoneal injection with NaHS tended to increase the NSCs proliferation in mouse SVZ in a dose-dependent manner.The in vitro differentiation study revealed that NaHS and GYY4137 promoted the NSCs differentiate into neurons.MTT assay and caspase-3 staining showed that NaHS(<500uM)has no significant effect on cell survival or apoptosis of NSCs.In addition,NaHS was observed to enhance the mRNA level of the glial derived neurotrophic factor in astrocytes concentration-dependently.Western blot showed that either NaHS or GYY4137 did not alter the phosphorylation of CREB in NSCs.Conclusion:Exogenous H2S can promote the proliferation of NSCs and facilitate the NSCs differentiate into neurons without affecting cell apoptosis.The underlying mechanisms may involve the increase of glial derived neurotrophic factor generation from astrocytes. |
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