[Mechanism of ethionine-induced neural tube defects via regulation of the Wnt/β-catenin pathway through the KDM5A/H3K4me3 axis].

Objective: To explore the molecular mechanisms by which ethionine induces neural tube defects (NTD) through regulating the lysine demethylase 5A (KDM5A)/trimethylated lysine 4 of histone H3 (H3K4me3) axis and modulating the Wnt/β-catenin signaling pathway. Methods: Specific pathogen-free adult male and female C57BL/6 mice (80 females and 35 males) were housed overnight in a 2∶1 female-to-male ratio. Vaginal plugs were examined the following morning to confirm pregnancy. Pregnant mice were randomly divided into two groups: the ethionine experimental group (ethionine group) and the control group. At embryonic day 7.5 (E7.5), the ethionine group received a single intraperitoneal injection of ethionine (500 mg/kg) to establish the NTD model, while the control group was injected with an equal volume of normal saline, with 21 mice in each group. At E10.5, pregnant mice were sacrificed and embryos were collected for observation and photography under a stereomicroscope. Brain tissues from embryos of both groups were harvested for transcriptome sequencing. The expression levels of KDM5A, H3K4me3, Wnt/β-catenin pathway components [β-catenin, transcription factor 4 (TCF4), axis inhibition protein 2 (Axin2), phosphorylated glycogen synthase kinase 3β (p-GSK3β)], and cell proliferation-related proteins [G1/S-specific cyclin-D1 (Cyclin D1), proliferating cell nuclear antigen (PCNA), Myc oncogene (c-Myc)] were assessed by Western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence. An additional KDM5A-specific inhibitor GSK-4J treatment group (GSK-4J group) and ethionine combined with GSK-4J treatment group (ethionine+GSK-4J group) were included to verify the regulatory role of the KDM5A/H3K4me3 axis. The pregnant mice in the GSK-4J group received intraperitoneal injection of GSK-4J (7 mg/kg) and the ethionine+GSK-4J group were injected with both 7 mg/kg GSK-4J and 500 mg/kg ethionine, with 12 mice in each group. Comparisons between the two groups were performed using independent samples t-test, one-way ANOVA was applied to evaluate the overall differences among multiple groups, and Tukey's honestly significant difference test was adopted for pairwise post-hoc comparisons. Results: The ethionine treated group exhibited 48.79% (121/248) incidence of incomplete brain structure and morphological malformations, while the malformation rate in the control group was 3.73% (10/268), with a statistically significant difference (P<0.001). Compared with the control group, the ethionine group showed elevated KDM5A mRNA and protein levels, accompanied by reduced H3K4me3 modification levels (all P<0.001); protein expression of β-catenin, TCF4, and Axin2 were decreased (P<0.01, P<0.05), whereas p-GSK3β was increased (P<0.01). Following GSK-4J intervention, the expression of β-catenin, TCF4, and Axin2 was partially restored (all P<0.01), and the percentage of Cyclin D1-positive cells, as well as the expression of PCNA and c-Myc were increased (all P<0.05). Conclusion: Ethionine participates in NTD development by upregulating KDM5A to decreased H3K4me3 levels, inhibiting Wnt/β-catenin signaling pathway, and reducing neuroepithelial cell proliferation. Thus, targeting the KDM5A/H3K4me3 axis may provide a new strategy for the prevention and treatment of NTD.