Protective Effects Of Folic Acid On Neural Stem Cells From Neonatal Rats Under Hypoxia In Vitro

ObjectiveTo investigate the protect effects of folic acid on hypoxia neural stem cells in vitro, and to explore the mechanisms of folic acid against hypoxia injury.MethodsThe NSCs taken from infant rats brain were cultured by serum-free medium in vitro and divided into five group which were Normal control group with folic acid4μg/ml in media, Hypoxia group with folic acid4μg/ml, Hypoxia+Folate-L group with folic acid8μg/ml, Hypoxia+Folate-H group with folic acid44μg/ml and Hypoxia+Folate-D group in which the concentration of folic acid is0.65μg/ml. Except the normal control group, the other groups were cultured in hypoxia condition for8hours on the third day of proliferation. The proliferation activity of NSCs after hypoxia damage was detected with MTT test. The homocysteine level in cell culture medium was determined by ELISA.The NSCs were collected after being cultured6days and then extracted the cell protein, examined the activity of SOD and GSH-Px, the content of MDA and ROS. The apotosis rate and the Mitochondrial transmembrane potential were detected by Flow cytometry. The expression of the related protein in Mitochondrial apoptosis pathway:activated Caspase-3、Bcl-2and Bax were detected by Western blot.ResultsThe morphological signs displayed that NSCs were damaged after hypoxia significantly, and folic acid could produce a protective effect. MTT result showed that in the different time after hypoxia, the proliferation acitivity of NSCs in Normol control group was much higher than Hypoxia model group and Hypoxia+Folate-D group(P<0.05), and Hypoxia+Folate-H group was significantly higher than Hypoxia+Folate-L group(P<0.05). The ELISA result displayed that the homocysteine level in Normol control group was lower than Hypoxia+Folate-L group and Hypoxia+Folate-H group(P<0.05). It also has the dose-response relationship. Compared with the Normol control group, the activity of SOD and GSH-Px decreased significantly in Hypoxia model group and Hypoxia+Folate-D group(.P<0.05), but the level of MDA and ROS increased(P<0.05); while the activity of SOD and GSH-Px increased in both Hypoxia+Folate-L group and Hypoxia+Folate-H group(P<0.05), but the level of MDA and ROS decreased(P<0.05). The Flow cytometry results illustrated that the apoptosis rate in Hypoxia model group was significantly higher than Normol control group and Hypoxia+Folate group(P<0.05), but lower than Hypoxia+Folate-D group(P<0.05), while the Mitochondrial transmembrane potential in Hypoxia model group was significantly lower than Normol control group and Hypoxia+Folate group(P<0.05), but much higher than Hypoxia+Folate-D group(.P<0.05); Western blot analyses illustrated that the protein expression of activated Caspase-3and Bax in Hypoxia model group were significantly higher than Normol control group and Hypoxia+Folate group(P<0.05), but lower than Hypoxia+Folate-D group(P<0.05), while the expression of Bcl-2in Hypoxia model group was significantly lower than Normol control group and Hypoxia+Folate group(P<0.05) but higher than Hypoxia+Folate-D group (P<0.05).ConclusionThe hypoxia model of NSCs in vitro was established successfully, folic acid supplement can promote the proliferation and reduce the apoptosis rate of damaged NSCs efficiently. Folic acid could reduce the Hcy level, improve the antioxidative capacity and inhibit the generation of ROS and MDA after hypoxia. Folic acid could improve the levels of Mitochondrial transmembrane potential and the Bcl-2protein expression, decrease the protein expression levels of activated Caspase-3and Bax, so that folic acid could produce a protective effect on the NSCs after hypoxia damage in vitro.