Effects Of Folic Acid On Neural Stem Cells In Vitro Through MAPK Signaling Pathway After Hypoxia Damage

ObjectiveTo investigate the effects of folic acid on hypoxia neural stem cells and its related mechanisms in vitro.MethodsNSCs from Neonatal SD rats within 24 hours were cultured in vitro and divided into five groups which were control group, hypoxia model (Hypoxia) group, hypoxia +folate deficiency (Hypoxia+Folate-D) group, hypoxia+low dose folate (Hypoxia+Folate-L) group and hypoxia+high dose folate (Hypoxia+Folate-H) group. NSCs in all groups except those in the control group were duplicated into models of hypoxia. NSCs were collected after reoxygenation 3h,6h,12h,24h. NSCs morphology was observed under reverse microscope. Immunofluorescence staining method was used to identify the NSCs. NSCs proliferation activity was assayed using MTT, the expression level of ERK1/2 mRNA was measured by Fluorescence quantitative PCR and the total amount of pERK1/2 protein was detected by the method of Western blot.Added U0126, the inhibitor of mitogen-activated protein kinase (MAPK) pathway, NSCs were divided into six groups which were control group, hypoxia+ DMSO added (DMSO) group, hypoxia+U0126 added (U0126) group, hypoxia+ U0126+Folate deficiency (U0126+Folate-D) group, hypoxia+U0126+Folate low dose folate (U0126+Folate-L) group, hypoxia+U0126+Folate high dose folate (U0126+Folate-H) group. NSCs in all groups except those in the control group were duplicated into models of hypoxia. NSCs were collected after reoxygenation 3h,6h, 12h,24h. NSCs proliferation activity was assayed using MTT. Then NSCs were collected after reoxygenation 6h. The expression level of ERK1/2 mRNA was measured by Fluorescence Quantitative PCR and the total amount of pERK1/2 protein was detected by the method of Western blot.Results Under hypoxia conditions, NSCs could hardly form into neurospheres, more single cells under normoxia conditions. NSCs under microscope were like bright spots, had a clear boundary and strong reflected light. A large number of cells became suspended and irregular, some cells attaching to the wall, and a lot of cell fragments appeared after 24h. After that, most of suspended NSCs began to divide into neurospheres which consisted of the tens to thousands of cells. BrdU marked immunoflurescence showed NSCs was green.8h was confirmed to the hypoxia time by the method of MTT, and detected that reoxygenation 6h was at its proliferation peak, then the level of proliferation maintained at this level, and the addition of folic acid could promote the proliferation, the deficiency of folic acid could decrease the proliferation. The results of Fluorescence quantitative PCR and Western blot showed that 6h was at its expression peak, then the level of expression maintained at this level, and the addition of folic acid could promote the expression.ConclusionFolic acid could promote the proliferation of NSCs after hypoxia-reoxygenation, and proliferation peak occurred at 6h after reoxygenation. Reoxygenation after hypoxia could activite ERK1/2 phosphoryltion via ERK pathway. Folic acid supplementation could activate ERK1/2 and increased the expression levels of ERK1/2 mRNA and pERKl/2 protein. ERK signal pathway contributed to the process of ERK1/2 activity and promoted the survival of NSCs after reoxygenation.