Approach In Mechanism Which Oxidative Stress And DNA Methylation Participate In Homocysteine Induced Neural Tube Defects.

Background:Homocysteine (HCY), a sulfur amino acid, involves in one carbon metabolism. Neural tube defects (NTDs) are the most common and severe disease of birth defects in humans, which caused by disorder of neural tube genesis and differention because of genetic factors and enviromental factors in early developing embryo. Recent studies showed that increased HCY was closely related with the NTDs. However, the precise molecule mechanisms by which HCY induced NTDs are incompletely defined.Objective:Investigate the pathopoiesis mechanism of HCY-induced embryo malformations and raise prevention measure for the pathopoiesis mechanism and provide basement for the treat and prevention of NTDs.Methods:Investigate the effects of HCY on chick embryo neural tube development and the possibly molecule mechanism. Analyze the effect of HCY and co-treated with N-acetyl-cysteine (NAC) or choline (CHO) on chick embryo neural tube through microscope photographs, paraffin-embedded seotion and H-E staining. Measure the expression of Pax6 and Tuj1 through immunohistochemistry. Measure the effects of HCY on reactive oxygen species (ROS), Malondialdehyde (MDA) conten, Glutathion (GSSG), Superoxide Dismutase (SOD) activity and Glutathione peroxidasee (GPX) activity about oxidative stress. Measure the effects of HCY on DNA methylation by high performance liquid chromatography (HPLC), whole-mount immunofluorescence and western blotting; Measure the expression of miR-124 after HCY treatment by Taqman MicroRNA realtime PCR; Measure the expression of miR-124 target gene, SCP1, through immunohistochemistry; Knockdown and overpression of miR-124 through in ovo electroporation and analyse the effect on neural tube.Results:(1) The embryo viability was significantly decreased after HCY treatment. HCY induces neural precursor cell marker Pax6 upregulation and neuronal marker Tuj 1 protein downregulation. HCY could inhibit extraembryonic vascular development and induce hidden spinal bifida.(2) The above phenotype was partially rescued after co-treated with N-acetyl-cysteine (NAC) or choline (CHO) and these results indicated that HCY-induced spinal bifida was associated with oxidative stress and disturbed one carbon metabolism.(3) HCY could induce significant increase in ROS and MDA content and significant reduction in Mn SOD activity and GPX activity. When co-treated with NAC, these alterations was partially rescued. reduce in Mn SOD activity and GPX activity. When co-treated with NAC, these alterations was partially rescued.(4) HCY could cause global DNA hypomethylation, increase in S-adenosylhomocysteine (SAH), reduce in S-adenosylmethionine (SAM), SAM/SAH, expression of DNMT1 and DNMT3a; When co-treated with CHO, these alterations was partially rescued.(5) When co-treated with NAC, the global DNA hypomethylation and reduce in DNMT1 and DNMT3a were partically rescued, however, there was no significant alteration in SAM, SAH and SAM/SAH.(6) HCY could cause significant reduce in expression of miR-124. When co-treated with 5-Aza-2-Deoxycytidine, the expression of miR-124 was partically rescued. These results indicated the regulation of miR-124 was associated with hypermethylation in promoter region, immunohistochemistry results showed expression of SCP1 and Pax6 was significantly increased and expression of Tuj1 was significantly decreased.(7) Knockdown of miR-124 could cause spinfa bifida, upregulation of SCP1 and Pax6, and downregulation of Tuj1. These results indicate miR-124 may play important role in neural tube development.Conclusion:(1)This study found that HCY significantly reduced chick embryo viability, resulting in spina bifida malformation. The study first demonstrate HCY could influence DNA methylation through oxidative stress. And demonstrated NAC or CHO could protect HCY induced spinfa bifida.(2) By increasing reactive oxygen species, increased lipid peroxidation and reduce antioxidant capacity, HCY caused oxidative damage of chick embryo.HCY could cause hypomethylation through oxidative stress and disturbing DNA methylation along with hypermethylation in special area.(3) Downregulation of miR-124 may cause upregulation of its target gene SCP1 and increase of neural progenitor cell. These may cause abnormal thicking of neurepithelium and thus cause abnormal closure of neural tube. HCY may cause spinfa bifida through these mechanism.