The Effect And Mechanism Of Folic Acid On The Proliferation, Differentiation And Neuronal Growth Of NSCs Treated With VPA

Valproic acid(VPA),a short-chain fatty acid,is widely used as a sedative to stabilize mood and treat epilepsy.It has been shown that offspring born to women who took VPA during pregnancy have higher rates of autism.In addition,studies have shown that VPA affects the proliferation and differentiation of neural stem cells(NSCs),the growth and morphological changes of neuronal protrusions.VPA may influence development of the nervous system through these mechanisms,leading to autistic behavior.Folic acid(FA),also known as vitamin B9,plays an important role in the early development of the nervous system as an important carbon transport unit in the body.Epidemiological studies have shown that folic acid supplementation in early pregnancy is neuroprotective.It has the ability to reduce the probability of autism in offspring,but the mechanism is still not clear.Objective:our previous studies have demonstrated that FA can significantly improve the autistic behavior of rodents obtained from VPA-induced autism models,but the corresponding cellular and molecular mechanisms are not clear.Therefore,this study will establish a culture system of primary neural stem cells and neurons to explore the effects of FA on proliferation and differentiation and neuron growth of VPA-treated NSCs,as well as the relevant cellular and molecular mechanisms,to provide certain theoretical and experimental basis for the prevention and treatment of autism.Methods:primary cultured NSCs and neurons were divided into four groups:blank control group(CT group),FA control group(FA group),VPA group,and FA preconditioning group(VPA+FA group).MTT,BrdU and other techniques were used to observe the proliferation of NSCs.The changes in differentiation trend of NSCs were observed by immunofluorescence staining with-tubulin and GFAP.Meanwhile,Western Blot was used to detect the expression levels of AKT,ERK1/2,Bax,Bcl-2,Caspase3 and other key signaling molecules in the signaling pathway related to cell proliferation and apoptosis.The growth of primary neurons was observed by MAP-2 immunofluorescence.Results:1.The primary NSCs and neuron culture system were established.Primary NSCs culture system:the results of Nestin immunofluorescence staining showed that 97%of the cultured primary cells were NSCs,and the cells showed good refractive property and spherical growth in the bright field.Immunofluorescence staining results of?-tubulin and GFAP showed that the cultured cells all contained immunostaining positive cells of ?-tubulin and GFAP,indicating that we had successfully cultured NSCs capable of proliferation and differentiation.Primary neuron culture system:the results of NeuN immunofluorescence staining showed that about 85%of the cultured primary cells were neurons,and the cell body was full,clear and prominent under the bright field.The results of MAP-2 immunofluorescence staining showed that the primary neurons were in good condition,and the protrusions were interwoven to form a network.2.FA pretreatment can reverse VPA-induced decrease in primary neural stem cell proliferation.The normal cultured NSCs were treated with 0.5-3mmol/L VPA,and cell viability was detected by MTT assay to screen the optimal VPA concentration.It was found that when the VPA concentration was 2mmoI/L,the cell survival rate was about 50%.The subsequent experiments was divided into four groups:control group,FA(90?mol/L)group,VPA(2mmol/L)group,FA+VPA(90 ?mol/L,2mmol/L)group.The results of MTT,BrdU and diameter of neurospheres measurements showed that,compared with the control group,the cell proliferation rate increased in the FA group(P<0.01,P<0.05,P<0.01).The proliferation rate of VPA group was significantly decreased(P<0.01,P<0.01,P<0.05).Compared with the VPA group,FA pretreatment(FA+VPA group)reversed the decrease of cell proliferation caused by VPA(P<0.05).Western blot analysis showed that.compared with the control group,the phosphorylation levels of ERK1/2 and AKT in FA group were significantly increased(P<0.05)while those in VPA group were significantly decreased(P<0.05).Compared with VPA group,the phosphorylation levels of ERK1/2 and AKT in FA+VPA group were increased(P<0.05).The results of apoptosis-related protein detection showed that,compared with the control group,the expression levels of Cleaved-Caspase3 and Bax/Bcl-2 protein in VPA group were significantly increased(P<0.05),while FA pretreatment partially reversed the increased expression levels of these proteins(P<0.05).3.FA pretreatment inhibited the differentiation of NSCs into neurons induced by VPA.The double-labeled immunofluorescence results of ?-tubulin and GFAP showed that,compared with the control group,the proportion of ?-tubulin positive cells in the VPA group increased significantly(P<0.05),while the proportion of ?-tubulin positive cells in the FA group decreased significantly(P<0.05).Compared with the VPA group,the proportion of ?-tubulin positive tubulin cells in the FA pretreatment group(FA+VPA group)was significantly reduced(P<0.05).4.FA inhibited VPA-induced the growth of primary mature neurons.Immunofluorescence results of MAP-2-DAPI showed that the growth of neurons in VPA group increased significantly compared with the control group(P<0.05).Compared with the VPA group,FA preconditioning(FA+VPA group)significantly reduced the growth of neurons(P<0.05).Conclusion:1.The culture system of primary NSCs and neurons was successfully established.2.Folic acid preconditioning can reverse the decreased proliferation of VPA-induced primary neural stem cells,while folic acid may regulate the proliferation of VPA-inhibited neural stem cells by regulating the expression of proliferation-related proteins and apoptosis.3.Folic acid can inhibit the differentiation of primary NSCs into neurons induced by VPA.4.Folic acid can inhibit the growth of primary neurons induced by VPA.