Mechanisms On Abnormal Embryonic Neural Tube Development In Inositol Imbalance Mice

Neural tube defects is a common malformation caused by the neural tube did not closed or closed incompletely in the early period of embryonic development,clinical consequences are serious.The mechanism of NTDs is not clear.Inositol,also known as cyclohexyl hexanol,belongs to the vitamin B family of nutrients,plays an important role in the process of nerve tube closed and embryonic nerve development,inositol is closely related with NTDs.In this study,we used the inhibitor to intervene the important target enzyme of the inositol metabolism process,based on the established NTD mice model of inositol metabolic disorders.GC-MS,HE staining,immunohistochemistry,protein chip,PI3K enzyme activity detection,bioinformatics and western blotting methods were used to further study the mechanism on inositol metabolism disorder in the process of mice NTDs.C57BL/6J mice were selected to establish animal models of inositol metabolic disorders by intraperitoneal injection of 350 mg/kg dose of Li₂CO₃,and the results of the occurrence of NTDs had good reproducibility.The incidence of encephalocele was the highest in the NTDs embryos.The result of different levels of pathological changes of embryonic neural tube showed that the proliferation of neuroepithelial cells was abnormal.The PI3K enzyme activity in NTDs,normal and control group of mouse on E13.5 days showed an increasing trend.The results showed that PI3K enzyme activity was increased in the brain,placenta,heart,liver and spleen of pregnant mice at 8h after injected Li₂CO_3。The neural stem cells of mice were cultured for vitro experiments.With the increase of the inositol concentration,the activity of PI3K enzyme decreased gradually,and the activity of the enzyme was the lowest when the inositol concentration was 5 mM（P<0.01）,then gradually increased,but still lower than the normal level.We used the protein chip to detect the protein phosphorylation levels of samples among NTDs,normal and control group of mouse on E13.5 days,and the PI3K/Akt pathway was activated abnormally.In this study,PI3K/Akt protein chip was used to detect the phosphorylation of PI3K/Akt pathway protein in embryonic nerve tissue of the three groups.After inositol intervention,the phosphorylation level of related protein showed an increasing trend in the normal and NTD group,such as Akt、Bcl-2、GSK-3β、P53、p70S6K、mtor protein.The activity of PI3K/Akt signal pathway in embryonic nerve tissue of three groups of mice was further detected.The expression of pAkt-Ser473 and Akt protein in embryonic nerve tissue of NTD group was significantly higher than that of normal and control group.While the expression of GSK3βprotein was inhibited,and the expression level of pGSK3β-Ser9 protein in NTD and normal group was higher than the control groups（P<0.05）,P53,Caspase-3 and PTEN protein expression showed a decreasing trend in control,normal and NTD group（P<0.05）.Compared with the immunohistochemical results of pAKT-Ser473 protein,the positive/negative cells ratio of forebrain and hindbrain were significantly higher in the control group than that of normal and NTD mice,and hindbrain was particularly evident（P<0.05）.The positive/negative cells ratio of pGSK3β-Ser9 protein showed an increasing trend（P<0.01）,while GSK3βprotein showed a decreasing trend in the embryonic forebrain and hindbrain of control,normal and NTD group,the result was consistent with protein chip and western blotting,PI3K/Akt signaling pathway was abnormal activated.The results showed that maternal inositol metabolism was closely related to NTDs,PI3K/Akt signaling pathway was abnormal activated,its phosphorylation regulated the activity of downstream proteins,caused changes in cell cycle and apoptosis to affect the closure of the neural tube.In this study,the NTD mice model of inositol metabolic disorders was further established and improved.These results might make a contribution for the prevention and treatment of NTDs from PI3K/Akt signaling pathway.