Study On Inhibition Of GARFT Mice To Establish NTDs Animal Model And Its Mechanism

Background:A large number of studies have shown that folic acid deficiency is closely related to the occurrence of NTDs,adequate folic acid supplementation during pregnancy can effectively prevent aboub 50-70% of NTDs,therefore,using folic acid as the starting point,has important significance for preveting NTDs and study of specific pathogenesis of NTDs.Notably,there has been no report on the successful establishment of an pure diet control animal model of NTDs induced by folic acid deficiency so far.According to our previous hypothesis,folic acid deficiency is a cause of NTDs and other development disorders that indirectly through the dysbolism of folic acid in different metabolic pathway,we block the corresponding folic acid metabolic pathway,successfully prepared dihydrofolate reductase,ribonucleotide reductase,thymidylate synthase,methyltransferase related dysbolism pathways mice animal models of NTDs,and confirmed that the above key enzymes involved dysbolism pathways can lead to NTDs independently.Providing a model for further exploration of NTDs and birth defects caused by folic acid deficiency,through the comparison of different models,we can further confirm the metabolic mechanism of NTDs and birth defects caused by different metabolic pathways of folic acid.This paper related to glycinamide ribonucleotide formyltransferase(GARFT)is one of the key enzymes involved in metabolic pathways of folate acid realize its biological effects,inhibits its activity lead to related metabolic disorders,whether it can induce NTDs and its preliminary molecular mechanism are the scientific problems to be solved in this paper.Objectives:1.Intervention of pregnant mice by Glycinamide ribonucleotide formyl transferase(GARFT)specific inhibitor Lometrexol(DDATHF),induce the abnormal metabolism of purine in mice of NTDs.2.Investigate the activity of GARFT and levels of purine in NTDs embryo tissues induced by DDATHF.3.Cell proliferation and apoptosis were investigated in DDATHF induced NTDs by interfering the de novo purine synthesis.4.Investigate the proliferation and apoptosis in GAR-FGAR metabolic disorder embryonic stem cells induced by DDATHF.Methods:Pregnant mice were randomly divided into two groups,the experimental groups were treated with DDATHF by intraperitoneal injection on gestation day7.5(E7.5),establishment of animal model of neural tube defects,the control group was intraperitoneally injected with saline according to the weight.Pregnant mice were cervical dislocation executed on gestation day11.5(E11.5),all embryos were carefully dissected and examined under a dissect microscope,to observe the external characteristics of NTDs embrys,after fixation,sectioning and staining,teras situation and neural tube morphological changes were observed under light microscope;the activity of GARFT and the content of ATP,GTP,d ATP and d GTP in different time points(0h,6h,24 h,48h,96h)were measured by GARFT enzymatic reation and high performance liquid chromatortaphy(HPLC);Immunohistochemistry,western-blot and RT-q PCR were performed to analyze proliferation and apoptosis in murine NTDs induced by DDATHF.CCK-8,flow cytometry and comet assay were performed to analyze proliferation and apoptosis in NTDs murine embryonic stem cells induced by DDATHF.Results:1.DDATHF has been successfully induced mouse NTDs,the optimal dose is45mg/kg,the incidence of NTDs is about 31.7%,lack of closure in the hindbrain was primarily observed in embryos with NTDs,the other malformations were craniofacial abnormalities and growth retardation,HE staining showed that the afterbrain bubble of fetal rats with NTDs was not closed.2.GARFT activity was significantly decreased after 6 hour of DDATHF injection on gestational day 7.5 and remain continuously low even after 96 hour of the injection.3.HPLC results show that the levels of ATP,GTP,d ATP and d GTP in NTDs embryonic brain tissue gradually decreased after DDATHF treatment and remain continuously low even after 96 hour of the injection.Among them,changes of ATP and GTP were more pronounced than d GTP and d ATP.4.We performed PH3 staining and Casp-3 to assess the proliferation and apoptosis of neuroepithelial cells by the use of immunohistochemical assay,which showed that PH3 staining in NTD embryos was less obvious than that in control embryos,casp-3 in NTD embryos was more obvious than that in the control embryos(P<0.05).5.The protein levels of PH3 and casp-3 in embryonic brain tissue were analyzed by western blot,and the results showed that the expression of PH3 was significantly reduced than that in the control embryos,the expression of cleaved Caspase-3 protein was significantly increased in embryonic brain tissue of NTDs(P<0.5).6.The proliferation-related genes(Pcna,Foxg1 and Ptch1)and apoptosis-related genes(Bax,Casp8 and Casp9)were determined to investigate the proliferation and apoptosis in NTDs by RT-q PCR.Compared to control group,results showed significantly decreased expression of proliferation-related genes and increased expression of apoptosis-related genes(P<0.5).7.CCK-8,Brdu flow cytometry showed that DDATHF inhibited proliferation of m ESC(P<0.5).8.DDATHF induced apoptosis of m ESC was detected by Annexin V/PI,mitochondrial membrane potential and comet assay(P<0.5).Conclusions:1.NTDs mice model was successfully induced by DDATHF.2.The study indicates that the purine dysmetabolism was associated with the development of NTDs,which is an important evidence for the mechanism between folate deficiency and NTDs.3.DDATHF induced mice NTDs could be by the influence on the apoptosis and proliferation of neuroepithelial cells.Disturb the balance of the proliferation and apoptosis during the development of neural tube,finally resulted in NTDs,that may be one of the pathological mechanism of NTDs.4.DDATHF induced GAR-FGAR metabolic disorder in D3 mouse embryonic stem cell proliferation inhibition,apoptosis increased.