| Bisphenol A（BPA）is one of environmental endocrine disruptor（Environmental Endocrine Disruptors,EEDs）and distributed typical and widely.BPA is used in many household products,electronic equipment and medical equipment,compelling involuntarily exposed.It had found that BPA can pass the placental barrier and the blood brain barrier,induced dangerous threat to embryonic development and brain tissue.According to previous experimental data and relative literature of BPA toxicity effects,BPA exposure can cause developmental toxicity in embryos and nervous system.But the molecule mechanism of toxicity has not been fully clarified.In order to further explore the toxicity mechanism of BPA,P19 cells were used as a test model in vitro.The following two aspects were studied.1 Molecular mechanism of BPA interference with the stemness of P19 cellsIn this study,P19 cells were used as cell model.P19 cells were exposed to different concentrations of BPA（0,0.01,0.1,1,10 and 100 μM）for 24 h.Morphological analysis of cells were observed under light microscope.CCK-8 assay was used to detect the effects of BPA on cell viability and proliferation.The expression levels of Oct4,Sox2,Nanog,Notch1 and Jagged1 protein and m RNA were detected by Western Blot and qRT-PCR.Immunofluorescence was used to observe the co-localization of Notch1 and Sox2 proteins and the protein localization of Jagged1,Oct4 and Nanog.After adding Notch1 inhibitor DAPT,Western Blot was used to detect the effects of BPA on the protein expression levels of Jagged1,Notch1,Oct4,Sox2 and Nanog protein expression.The results were as follows:（1）Compared with the control group,the morphology of P19 cells had little change in 0.01-10 μM BPA group.The morphology of P19 cells had aggregated and the density decreased in BPA 100 μM group.The cell viability of P19 cells BPA 0.01-10 μM group was increased.The cell viability of P19 cells was decreased in BPA 100 μM group.（2）Compared with the control group,the protein and gene expression of Oct4,Sox2 and Nanog was increased in μM BPA group（P < 0.05）.（3）The protein expression of Notch1 and Jagged1 was increased in BPA group（P < 0.05）.The gene expression of Notch1 and Jagged1 is disruptive.（4）The co-location fluorescence intensity of Notch1 and Sox2 proteins was increased in 10 μM BPA group.Jagged1,Oct4 and Nanog protein fluorescence intensity of P19 cells in all BPA treated groups was increased.（5）Compare with the BPA 100 μM,the expression levels of Notch1 and Nanog protein were decreased after DAPT treatment（P > 0.05）.The expression levels of Jagged1,Sox2 and Oct4 were significantly decreased（P > 0.01）.These results suggest that BPA can regulate the expression levels of Sox2,Oct4 and Nanog proteins through Notch1 signaling molecule and its ligand Jagged1.Thereby affect the stemness of P19 cells.2 BPA interferes with neural differentiation of P19 cellsP19 cells were differentiated into neuron-like cells by Retinoic acid to establish neural development model.BPA（0,0.1,1 and 10 μM）were used to treat the process of neural differentiation of P19 cells.The cell samples were collected on the 4th day（neurosphere）and 7th day（neuron-like cells）.When P19 cells were induced to neurospheres at the 2nd and 4th day,the morphology,number and size of neurospheres were observed under light microscope.When P19 cells were differentiated into neuron-like cells at the 7th day,the morphology were observed by light microscope.The protein and gene expression levels of Nestin,Map2 and Pax6 were detected by Western Blot and qRT-PCR in neurospheres.The protein and gene expression levels of Map2,Gfap,Tuj3 and NeuN were detected by Western Blot and qRT-PCR in neuron-like cells.Immunofluorescence was used to observe the localization of Nestin,Map2 and Pax6 in neurospheres.Immunofluorescence was used to observe the localization of Tuj3 and NeuN in neuron-like cells.The results were as follows:（1）Compared with the control group,neurospheres had irregular in 10μM BPA group on the 2ed day.The neurospheres showed dense inner and smooth surface in control group on the 4th day.The neurospheres showed rough surface in 1 and 10 μM BPA groups on the 4th day.The size of the neurospheres decreased in the BPA group on the 2ed day（P < 0.05）.The size of neruospheres decreased in BPA group on the 4th day（P < 0.01）.（2）Compared with the control group,the expression of Nestin protein was decreased in BPA groups（P < 0.05）.The fluorescence intensity of Nestin was decreased in BPA group.The protein and gene expression level of Pax6 was increased in the BPA group（P < 0.05）.The fluorescence intensity of Pax6 was increased in the BPA group.The protein expression expression level of Map2 decreased in BPA group（P < 0.05）.The fluorescence intensity of Map2 was decreased in the BPA group.（3）On the 7th day of differentiation,the neuron-like cells were abundant in the control group and 0.1 μM BPA group.Neuron-like cells had fewer numbers in 1 and 10 μM BPA groups.Compared with the control group,the protein and gene expression of Map2,Tuj3 and NeuN was decreased in BPA group（P < 0.05）.The fluorescence intensity of Tuj3 and NeuN proteins in BPA group was weaker than that in control group,and the number and length of neuron-like cell neurites were reduced.（4）Compared with the control group,the protein and gene expression of Gfap was increased in the BPA group,significantly increased in the 10 μM BPA group（P < 0.01）.The results showed that BPA exposure decreased the ability of P19 cells to differentiate into neuron-like cells and increased the number of astrocytes.Conclusions: BPA can activate Notch1 signaling molecules,and promote the protein expression levels of Oct4,Sox2 and Nanog.The expression levels of Oct4,Sox2 and Nanog proteins were inhibited by the addition of Notch1 inhibitor（DAPT）.These results suggest that BPA activates Notch1 to interfere with stemness of P19 cells.BPA reduced Nestin,Map2,Tuj3 and NeuN protein expression levels and increased the protein expression levels of Pax6 and Gfap.These results indicated that BPA inhibited the ability of P19 cells to differentiate into neurons,and interfered with the differentiation fate of P19 cell. |
