Pathogenesis Mechanism On Myo-inositol Dysmetabolism In Neural Tube Defects

Neural tube defects(NTDs) are one of the most common and serious birth defects. It is also an important issue in China, for the high incidence and serious clinical consequences. Currently, the mechanism of this disease has not been elucidated, which is still one of the key problems of birth defects research. The effect of inositol and its metabolite on NTDs is one of etiologies remains undefined besides folic acid. Inositol belongs to the vitamin B group substance and it is an important nutrient which is necessary for cell growth and survival. Researches have confirmed that inositol is closely related with NTDs, inositol and its metabolite play an important role in embryonic development, especially during neural tube development, but the exact mechanism is still not clear.The present study utilized the established human NTD biological samples library, and interfered the key enzymes in inositol metabolism to induce inositol metabolic disordered NTD mouse model. Using gas chromatography mass spectrometry(GC-MS), immunohistochemistry, RT-PCR and Western Blotting techniques, we investigated the role and molecular mechanisms of inositol metabolism in NTDs. The results were as follows:1. The samples were randomly selected from the established human NTD samples library, including 40 pregnant woman serum samples and 9 embryo’s nervous tissue samples of NTD and control group, respectively. GC-MS technique was used for maternal inositol content detection and the Western Blotting method was used for the PI3K/AKT pathway activity. It was found that the inositol levels in NTD pregnant women was significantly lower than controls(1.8676±0.7270 mg/L & 2.5779±1.0951 mg/L, P<0.05); the phosphorylation level of AKT(p AKT-S473) was significantly higher in NTD fetuses(P<0.05), indicating the PI3K/AKT signaling pathway was activated extensively in NTDs fetuses.2. The inositol metabolic disordered NTD mouse model was successfully established in C57BL/6J mouse by intraperitoneal injection of Li2CO3 350 mg/kg on gestational day 7.5, which blocked the key enzyme(IMPase) in inositol metabolism during neural tube closure. The NTDs occurrence rate of the offspring was 26.7%, including the main phenotypes of encephalocele, anencephaly. The maternal inositol was at the lowest level within 4 h~8 h(P<0.01) and still lower than normal at 16h(P<0.05) after Li2CO3 injection. HE staining illustrated that neural tissue and cell morphological were changed in encephalocele mouse embryos and the lesion of neural tube in telencephalon was more serious compared with hindbrain.3. Based on the established NTD mouse model, PI3K/AKT signaling pathway activation status and related gene transcription levels were detected by immunohistochemistry, RT-PCR and Western Blotting methods to investigate the influence of inositol metabolic disorder on PI3K/AKT signaling pathway. It was found that p AKT-S473 antibody stained most of the cells located in neuroepithelial cells region, and the phosphorylation level of AKT(p AKT-S473) was significantly higher in NTD embryonic(P<0.05), indicating the PI3K/AKT signaling pathway was excessive activated in inositol metabolic disordered NTD mouse model. RT-PCR found that compared with the controls, Pik3 ca gene transcription level was significantly increased(P<0.05) and Pten gene transcription level was significantly decreased(P<0.05) in NTD mouse,while Inpp5 e and Akt1 gene transcription levels did not change significantly.The results of the present study indicated that maternal inositol metabolic disorder was closely related to NTDs. Inositol metabolic disordered NTD mouse model was established successfully and inositol deficiency might excessive activate PI3K/AKT signaling pathway in neuroepithelia of telencephalon possibly through the synergies of Pik3 ca and Pten genes, which affected the neural tissue structure and cell morphology and interfered in the normal neural tube closure process, leading NTDs occur. The results would contribute to further illustration of the mechanism on inositol deficiency in NTDs occurrence and early diagnosis and prevention of NTDs.