| ObjectiveThis study was conducted to investigate the effects of folic acid on NSCs differentiation in vitro, and to explore the underlying mechanism, so as to provide basic data for NSCs transplantation and directed differentiation.MethodsThe NSCs were isolated from the hippocampus of infant rats and were grown in serum-free medium in vitro. NSCs were stained using antibodies against SOX2/BrdU and divided into five groups:control group (folic acid-free, Comtrol), low folic acid group (8μg/mL folic acid, FA-L), high folic acid group(32μg/mL folic acid, FA-H), low folic acid and DNMT inhibitor zebularine group(8μg/mL folic acid and150nmol/mL zebularine, FAL-Z), and high folic acid and zebularine group(32μg/mL folic acid and150nmol/mL zebularine, FAH-Z). Cells were treated with zebularine on the first day of differentiation, exposed to zebularine for48h and then incubated without the drug for6d, before being harvested. The expressions of neuron-specific protein β-tubulin Ⅲ and astrocyte-specific protein GFAP in the neural cells were measured by immunofluorescent (IF) and wester blot (WB). Global DNA methylation levels were detected by the methyflashTM methylated DNA Quantification kit. DNMTs activity was measured by Active Motif’s DNA Methyltransferase Activity/Inhibition Assay kit. The protein expression levels of DNMTs were detected by Western blot. The intracellular concentrations of SAM and SAH were examined by HPLC method. Expressions of related protein in JAK/STAT signaling pathway: STAT1, STAT3were detected by Western blot. Methylation levels of GFAP gene promoter were determined by MeDIP-qPCR.ResultsAfter6d culture in serum-free medium, NSCs formed round neurospheres that remained suspended in the culture medium. Double immunfluorescence staining of cells showed colocalization of SOX2+and BrdU+. After1week of differentiation in vitro, the result of western blot and immunofluorescent showed that, compared with control, the expressions of β-tubulin Ⅲ increased in FA-L group and FA-H group, on the contrary, the expressions of GFAP decreased(P<0.05). The expressions of β-tubulin Ⅲ decreased in FAL-H group and FAH-Z group, on the contrary, the expressions of GFAP increased(P<0.05). Compared with FA-L group, the expressions of β-tubulin Ⅲ significantly decreased in FAL-Z, while the expression of GFAP significantly increased(.P<0.05). Compared with FA-H group, the expression of β-tubulin Ⅲ significantly decreased in FAL-Z group, while the expression of GFAP significantly increased(P<0.05). Global methylation results revealed a highly significant increase in FA-L group and FA-H group(P<0.05), and the methylation level of FA-H group was higher than that in FA-L group(P<0.05). The activities of DNMTs suggested that, compared to control group, DNMTs activities were significantly enhanced in FA-L group and FA-H group(P<0.05), while those were inhibited in FAL-Z group and FAH-Z group(P<0.05). Compared with FA-L group, those were significantly decreased in FAL-Z(P<0.05). And the same changes were observed between FA-H group and FAH-Z group(P<0.05). DNMTs expressions were examined by Western blot, and the results indicated that, compared with control, expressions of DNMT1and DNMT3a were significantly increased in FA-L group and FA-H group, the expression of DNMT3b were increased in FA-H group. HPLC results suggested that, compared with control group, the concentration of SAM increased, the concentration of SAH decreased, and SAM/SAH ratio increased in FA-L group and FA-H group(P<0.05). The expression of p-STAT3was significantly decreased in FA-H group. The expression of p-STATl had no difference among all groups. As we can concluded from the MeDIP-qPCR result that, compared with control, the GFAP promoter revealed a highly significant hypermethylation in FA-L group and FA-H group(P<0.05).ConclusionThese results confirmed that, folic acid could increase neural differentiation and decrease astrocytic differentiation in NSCs. |
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