Study On Neural Tube Defects Induced By FIDAS-5 And Its Mechanism

BackgroundNeural tube defects(NTDs)are a series of congenital central nervous system defects that occur during early embryonic development due to the failure of neural tube closure caused by various biological and physical factors.Among the many uncertain factors,it is believed that timely and appropriate folic acid supplementation may reduce the incidence of NTDs.However,folic acid supplementation alone does not completely prevent NTDs,and therefore,the direction of exploring folic acid metabolism rather than disorders of folic acid occurrence is proposed.When key enzymes in the folate metabolism pathway are inhibited,NTDs can be induced to occur independently,as has been demonstrated in the pre-subject period.Fluorinated N,N-dialkylaminostilbenes(FIDAS-5),an inhibitor of methionine adenosyltransferase(MAT),causes methionine in the folate cycle(FIDAS-5 can interfere with the production of S-adenosylhomocysteine(SAM),the body’s methyl donor,and theoretically can further affect a range of biochemical reactions not limited to those involving nucleic acids and proteins.A range of biochemical reactions are involved.It is unknown whether inhibition of MAT causes disruption of methionine metabolism,whether it interferes with neural tube development,and if so,what the molecular mechanisms of abnormal neural tube closure are.Therefore,this study aims to establish an animal model of NTDs by using FIDAS-5,an inhibitor of MAT,to interfere with the methionine cycle and to explore the mechanism of its occurrence,so as to lay the foundation for exploring the occurrence of neural tube malformations from different perspectives.Objectives1.To prepare an animal model of NTDs using FIDAS-5,a MAT inhibitor.2.To observe the effect of FIDAS-5 on the proliferation and apoptosis of mouse embryonic neuroepithelial cells.3.To explore the role of Wnt/PCP-JNK pathway in the development of FIDAS-5-induced NTDs.Methods1 、 Construction of an animal model of NTDs using FIDAS-5 and detection of related metabolite levelsC57 mice were randomly grouped and caged together.At 7.5 days of gestation(E7.5),the control group was injected intraperitoneally with saline containing 1% DMSO,and the experimental group was injected with different doses of FIDAS-5 solution,and the embryos were detached and observed under a body microscope at 11.5 days of gestation.The total number of embryos and the number of malformed embryos were recorded,and the top-rump length and weight of embryos were measured.The brain tissue of mouse embryos was sectioned and stained with HE to observe the development of neural tube.2.To detect the changes of neuroepithelial cell proliferation and apoptosis in embryonic brain tissue sections after FIDAS-5 injectionImmunohistochemistry was used to detect the proliferation of neuroepithelial cells in embryonic tissue sections;TUNEL was used to detect the apoptosis level of neuroepithelial cells;protein immunoblotting was used to detect the expression of PCNA and Cleaved Caspase-3 in mouse embryonic brain tissues.3.To detect the effect of Wnt/PCP-JNK pathway in the induction of NTDs by FIDAS-5Immunohistochemistry and immunofluorescence were applied to detect the contents of DVL1 and RhoA in mouse embryonic brain tissues,respectively;Western Blot immunoblotting was used to detect the expression of pathway-related proteins DVL1,RhoA,JNK and p-JNK in mouse embryonic brain tissues.CCK8 was used to detect the proliferation of HT-22 cells;AO/EB staining and Annexin V-FITC/PI double-staining were used to detect the apoptosis level;Western Blot was used to detect the changes in the expression of proliferation and apoptosis-related proteins PCNA and Cleaved Caspase-3 in HT-22 cells.Results1.A FIDAS-5-induced mouse NTDs model was established,and the highest incidence of NTDs(34.8%)was observed at a dose of 20 mg/kg,which was used as the optimal modeling dose.The mice with NTDs were mainly characterized by hindbrain malformations,but other malformations such as microphthalmia and developmental delay were also observed.The levels of SAM and SAH and the SAM/SAH ratio in the brain tissue of NTDs embryos were lower than those of the control group as determined by ELISA.2.The results of section staining of embryonic brain tissues showed that the neural tube of NTDs embryos failed to close,the wall thickness of the lumen was uneven,the lumen was irregular,the parietal plate was only wrapped by amnion and mesenchyme,and the arrangement of neuroepithelial cells was disordered;the brain tissues of control embryos were well developed,the neural tube closed completely,the lumen was symmetrical,and the neuroepithelial cells were closely arranged.Immunohistochemical results showed that the expression of proliferating cell nuclear antigen(PCNA)in brain tissue sections of NTDs embryos was significantly less than that of the control group(P<0.05).The results of TUNEL assay for apoptosis showed a significant increase of apoptotic cells in embryonic brain tissues of NTDs group(P<0.01).western blot assay for apoptosis-related protein Cleaved Caspase-3 in embryonic brain tissues showed a higher level of apoptosis in embryonic brain tissues of NTDs(P<0.01).4.Immunohistochemistry and immunofluorescence methods were used to detect DVL1 and RhoA in the Wnt/PCP-JNK pathway,and the number of positive cells for both was found to be significantly increased in the experimental group(P<0.01);Western Blot method was used to quantify the expression levels of key proteins of the signaling pathway,and the results showed that the expression levels of DVL1,RhoA,JNK and p-JNK expression levels were significantly increased in the NTDs group.5.CCK8 results showed that the survival rate of HT-22 cells was halved at a FIDAS-5 concentration of 4 umol/L.AO-EB staining assay and flow cytometry were used to detect the apoptosis level,and both results indicated that the HT-22 apoptosis level was higher in the experimental group.The results of protein immunoblotting showed that the expression of PCNA was reduced(P<0.05)and the expression of Cleaved Caspase-3 was increased(P<0.01)in the HT-22 cells of the experimental group.Conclusion1.A mouse model of NTDs was successfully established by applying the MAT inhibitor FIDAS-5.2.FIDAS-5 can cause the imbalance of cell proliferation and apoptosis.3.The Wnt/PCP-JNK pathway mediated the induction of NTDs by FIDAS-5.