The Effects Of Mecobalamin On Neural Stem Cells Under Hypoxia And Its Mechanisms

Objective:To investigate the effects of mecobalamin on neural stem cells(NSCs)that whether it could promote the survival,proliferation,differentiation to neurons and astrocytes and reduce the apoptosis or not under the hypoxic condition.Furthermore,to investigate the mechanisms that it may participate in.Above all,we may find a medicine or assistant medicine to deal with hypoxic-ischemic encephalopathy of newborns.Methods:Primary neural stem cells were used in this study.The hypoxic condition that included 90%N2,5%CO2 and 5%air was used to stimulate the NSCs.To investigate the effects of mecobalamin on NSCs under the hypoxic condition or not through MTT assay.EdU assay was used to estimate the rate of NSCs’ proliferation.Meanwhile,the stem cell gene-Sox2 was detected by Real-time Quantitative PCR.Western blotting of Sox2 to proved the results on the protein level.For apoptosis,immunofluorescence of cleaved-caspase3 was detected.NSCs have multidirectional differentiation potential.For central nervous system,they could keep balance of themselves,neurons and astrocytes.We used the neurons’ marker—Tuj1 and the astrocytes’ marker-GFAP,to confirm the rate of changes of such two cells.Real-time Quantitative PCR and western blotting were used to assume the changes of these markers like ngnl,NeuroD1,MAP2 and GFAP at RNA level and Tuj1 and GFAP at protein level.To investigate the mechanisms,we payed attention to the changes of epigenetic,especially to the methylation of H3 like H3K4me3 and H3K27me3 which are considered to have high relationship with NSCs’ fate definition.And mecobalamin participate in methionine cycle,which could give methyl to others.Western blotting was used to detect the level of H3K4me3 and H3K27me3.On the other hand,ERK pathway was related to the trimethylation of H3K4me3.SO,we assumed the phosphorylation of ERK by western blotting.The subjects were divided into four groups,included control group,mecobalamin treated group,hypoxic group and both mecobalamin treated and hypoxic group.Results:Mecobalamin had a great promotion of NSCs’ viability.MTT assay were used to detect the effects of different concentration of mecobalamin on NSCs.We found O.01μM and 0.1μM did not have statistical significance(P>0.05),however,1μM and 10μM had effects on the NSCs’ viability(P<0.05).Furthermore,we used the lowest concentration that had effect on NSCs,to investigate whether it could have protective effect on NSCs under hypoxic condition.We found that their viabilities were reduced under hypoxic condition and mecobalamin could rescue such conditions.Meanwhile,under hypoxic condition,the rate of NSCs’ proliferation was reduced,when mecobalamin was added,this situation was changed,it could significantly increase the NSCs’ proliferation.Through immunofluorescence of cleaved-caspase3,we found that cleaved-caspase3 positive cells were increased compared to control group under hypoxic condition.Further study of the ability of NSCs’ differentiation to neurons and astrocytes,we investigate the rate of neurons or astrocytes around all of the cells.What we found was mecobalamin could promote the differentiation to both neurons and astrocytes no matter on the hypoxic condition or not.We also proved the results at mRNA and protein levels.During fetal development,especially,the brain’s development,H3K4me3 and H3K27me3 could determine the fate of the brain’s cells basic.Through western blotting,we found the trimethylation of H3K4 was down regulated,the changes of trimethylation of H3K27 was not significant.When we used mecobalamin to treat them,we found the up regulation of H3K4me3,H3K27me3 still did not have significant changes.Western blotting proved the phosphorylation of ERK was reduced under the hypoxic condition,and mecobalamin could improve it after 24 hours added.Conclusions:Mecobalamin could protect the viability of NSCs which were under hypoxic condition,meanwhile it could promote the cells’ proliferation and the differentiation to neurons and astrocytes,inhibited the apoptosis under the hypoxic condition.It may have potential effect on the hypoxic-ischemic encephalopathy of newborn so that it may be an ideal medicine or assistant medicine to treat the disease.