Effect Of Sodium Arsenite On Differentiation Of Mouse Neural Stem Cells Induced By All-trans Retinoic Acid

Objective:To observe the effects of sodium arsenite（Na As O₂）on proliferation and differentiation of mouse neural stem cells（NE-4C）,and to explore the effects of Na As O₂on Hippo/YAP and Notch pathways related to NE-4C cell differentiation.Method:（1）Take NE-4C cells as test subjects,induce differentiation of NE-4C cells with all-trans retinoic acid（ATRA）,and determine the effects of different concentrations of ATRA（0,0.5,1.0,5.0μmol/L）on cell viability by CCK-8 method.The m RNA expression levels of differentiation-related specific genes Nestin,Sox2,NSE,Rbfox1,βⅢ-Tubulin,GFAP and MBP were detected by RT-PCR,and the optimal induction concentration of ATRA was screened to establish the differentiation model of neural stem cells.（2）On the basis of neural stem cell differentiation model,Na As O₂ intervention was added（the doses were 0,0.3,0.6,0.9,1.2μmol/L,respectively）to investigate the effect of Na As O₂ on neural stem cell differentiation.After 7 days of culture,CCK-8 method was used to detect the effect of Na As O₂ on cell viability.The differentiation status of NE-4C cells was determined by immunofluorescence staining.The expression rates of differentiation-related specific markers Nestin,NSE,βⅢ-Tubulin and GFAP were detected by flow cytometry.The expression levels of differentiation-related specific markers Sox2,βⅢ-Tubulin,GFAP and core proteins of Hippo/YAP signaling pathway were detected by Western Blot.The relative expression levels of Notch1 and PS1 genes were detected by RT-PCR.Results:（1）ATRA decreased the viability of NE-4C cells in concentration-dependent manner（P<0.05）,and the viability of NE-4C cells was the highest when ATRA was 0.5μmol/L.RT-PCR and flow cytometry showed that:Compared with the control group,the m RNA expression levels of neural stem cell specific genes Sox2 and Nestin and the expression rate of Nestin positive cells in ATRA induced group were decreased（P<0.05）.The m RNA expression levels of neuron specific genesβⅢ-Tubulin,NSE,Rbfox1,and the expression rates ofβⅢ-Tubulin and NSE positive cells were increased（P<0.05）,and the m RNA expression levels of differentiation markers and positive cells were the highest at 0.5μmol/L.The optimum concentration of NE-4C cells induced by ATRA was determined to be 0.5μmol/L,and the neural stem cell differentiation model was established successfully.（2）Na As O₂ decreased the activity of ARTA induced differentiation NE-4C cells（P<0.05）,and showed a dose-effect relationship with the increase of Na As O₂ concentration.The results of immunofluorescence and flow cytometry showed that the proportion of cells with positive differentiation related specific markers decreased with the increase of Na As O₂concentration（P<0.05）.Western Blot results showed that:Medium and high concentrations of Na As O₂（0.6,0.9,1.2μmol/L）could decrease the expression level of specific marker protein Sox2（P<0.05）.The expression levels ofβⅢ-Tubulin and GFAP were decreased in high concentration Na As O₂（0.9 and 1.2μmol/L）groups（P<0.05）.With the increase of Na As O₂ concentration,protein expression levels of LATS1,p-LATS1,SAV1,MOB1 and p-LATS1/LATS1 were gradually increased,while protein expression levels of YAP,p-YAP and p-YAP/YAP were gradually decreased（P<0.05）.High concentrations of Na As O₂（0.9 and 1.2μmol/L）could decrease p-YAP and p-YAP/YAP,while 1.2μmol/L could increase LATS1/p-LATS1 protein expression level（P<0.05）.Na As O₂ decreased the relative expression level of Notch1 gene（P<0.05）,but the expression level of PS1 gene was almost unaffected（P=1.78）.Conclusions:（1）ATRA（0.5μmol/L）-induced NE-4C differentiation model was established successfully.（2）Na As O₂decreased the activity of NE-4C cells induced by ATRA and inhibited the differentiation of NE-4C cells into neurons.The higher the concentration,the stronger the inhibition effect on proliferation and differentiation was.Na As O₂ can increase the core proteins LATS1,p-LATS1,SAV1 and MOB1 and decrease the expression level of YAP,p-YAP of Hippo/YAP signaling pathway and Notch1 of Notch signaling pathway.It is suggested that the inhibition of Na As O₂ neural stem cell differentiation may be related to Hippo/YAP and Notch1 pathway.