Protective Effect Of Folic Acid On Embryonic And Neurodevelopmental Toxicity Induced By BPA

Bisphenol A(BPA)is widely used in the production of polycarbonate plastics and epoxy resins,and has strong neurodevelopmental toxicity to embryos.Notch signaling pathway is a key regulatory pathway in embryonic brain development,accurately regulating the development of nervous system.Folic Acid(FA)supplementation promote cell proliferation of embryonic neural stem cell by stimulating Notch signaling.At present,the protective mechanism of BPA induced neurotoxicity was unclear.Therefore,this search aims to study the protective effect of folic acid on BPA induced neurodevelopmental toxicity by regulating Notch signaling pathway.The specific research contents are as follows:1.Folic acid regulates Notch signaling pathway to alleviate BPA-induced neurodevelopmental toxicity in Xenopus laevis embryosIn order to study the teratogenic effects of BPA on embryos and neurodevelopmental disorders through Notch signaling pathway,a BPA exposure model was established with Xenopus laevis embryos,which were exposed to BPA(10 μM)for 24 h,48 h,72 h and 96 h with different concentrations of FA(2.5,5,10 μM).(1)At 96 h,embryos were counted for malformation rate and survival;(2)Statistical analysis of embryo development end indicators and basic movements;(3)After 96 h of embryo development,samples were collected,and the expression level of Notch1 and protein related genes Hes1 and Hey1 were detected by Western blot or qRT-PCR.The expression level of nerve cell markers Sox2,MAP2,β3-Tubulin mRNA was detected by qRT-PCR;(4)The embryonic motor neurons were labeled by whole-embryo staining of acetylated α-tubulin.Results:(1)Compared with BPA group,the survival rate of embryos was increased and malformation rate of embryos were decreased after FA supplementation;(2)Compared with BPA group,the embryonic of development toxicity and basal movement at end points were alleviated after added FA;(3)Under BPA exposure,FA significantly enhanced the Notch signaling pathway related proteins and mRNA expressions by Western blot or qRT-PCR;(4)Compared with BPA group,the arrangement of embryonic nerve segments regular and clear were promoted by added FA.These results suggested that FA alleviate the neurodevelopmental toxicity of BPA in embryos through Notch1-Sox2 axis.2.Effects of folic acid on stemness factors of P19 cells under BPA exposedIn order to further explore the regulatory effect of FA on BPA induced fetal neurodevelopmental toxicity through Notch signaling pathway,P19 teratoma cells were selected as the in vitro model.Con,100 μM BPA,5 μM FA and 100 μM BPA+5 μM FA groups were set up,and P19 cells were exposed for48 h.(1)Observe cell morphology and changes;The effects of FA and BPA on the activity of P19 cells were detected by CCK-8;(2)The protein and gene mRNA expression levels of Notch1,Jagged1,Sox2,Oct4 and Nanog were detected by Western blot and qRT-PCR;(3)The localization expression of Notch1 and Sox2 proteins were observed by immunofluorescence.Results:(1)After treated with BPA for 48 h,FA treatment the cell morphology restored,to a certain extent.The cell activity was significantly increased by CCK-8(P < 0.05);(2)The protein and mRNA expression levels of Notch1,Jagged1,Sox2,Oct4 and Nanog proteins and mRNA were significantly increased with FA was added at 100 μM BPA exposure;(3)Immunofluorescence analysis showed that FA enhanced the fluorescence intensity of Notch1 and Sox2 proteins in cells under BPA exposure(P < 0.01),the number of cells was increased.The results testified that folic acid alleviates the toxic effect of BPA on P19 cells,which is related to Notch signaling pathway,and restored the function of P19 cells.3.Effect of folic acid on the ability of BPA-exposed P19 cells and directionally differentiate neuronal cellsIn this study,P19 cells were selected as the research object,and retinoic acid(RA)was used to induce P19 cells to differentiate into nerve cells,and BPA exposure model was established in vitro.Con,30 μM BPA,1 μM FA,30 μM BPA+5 μM FA groups were divided into 4 groups during differentiation.(1)P19 cells were observed under the optical microscope to induce the formation of nerve balls at 4 days,and the surface area was calculated;(2)The mRNA and protein expression levels of neuralspheresassociated proteins Pax6,Nestin and MAP2,and neuron-like cell associated factors MAP2 and β3-Tubulin were detected by Western blot and qRT-PCR.The expression level of Notch related pathway genes Notch1,Jagged1,NICD,Hes1 and Hey1 mRNA were detected by qRT-PCR;(3)The expression and location of Pax6,Nestin,MAP2 and the localization of MAP2 and β3-Tubulin proteins were observed by immunofluorescence in neuron cells;(4)The length of neuron-like cell neurite was calculated.Results:(1)Under BPA exposure,the number of neurospheres were increased significantly by added FA(P < 0.05);(2)The protein expression level was significantly higher than that of BPA treatment group with FA treatment(P < 0.05);qRT-PCR results showed that FA significantly increased the mRNA expression levels of neurospheres marker proteins Pax6,Nestin and MAP2,neuronal related genes MAP2,β3-Tubulin and Neu-N,and Notch pathway related genes Notch1,Jagged1,NICD,Hes1 and Hey1;(3)Immunofluorescence analysis showed that the fluorescence intensity of BPA+FA group was significantly enhanced(Pax6: P < 0.01;Nestin,MAP2,β3-Tubulin: P < 0.05);(4)The length of neurite was significantly shortened under BPA exposure.After FA remission,the number of neurons and neurite length were significantly increased,and suggested that FA enhance the potential of neurite formation.These results indicated that FA therapy inhibited the differentiation of P19 cells into neural cells against BPA.In conclusion,FA alleviated BPA-induced embryonic neurotoxicity and the stemness function of P19 cells and the ability of directionally differentiate neuronal cells through activation of the Notch signaling pathway in the X.laevis embryos.This study will help to reveal the targets of BPA neurotoxicity in embryos and provide strong evidence for the screening of drugs against neurotoxic effects caused by environmental endocrine disruptors.