Maternal Pregestational Diabetes Contributes to Neural Tube Defects in Mouse Fetuses Through H4K5ac-Mediated Regulation of Focal Adhesion Pathway

Objectives: To investigate the potential mechanisms of maternal pregestational diabetes-induced neural tube defects (NTDs) by integrating proteomic data and histone H4 lysine 5 acetylation (H4K5ac) ChIP-seq data from the mouse model. Methods: The diabetic mouse model was established by intraperitoneal injection of streptozotocin (STZ) into female friend leukemia virus B strain (FVB) mice, with subsequent blood glucose monitoring. Diabetic females were then mated with healthy males, and embryonic tissues were collected on embryonic day 9.5. Among the embryos obtained from diabetic pregnancies, six NTDs embryos and six control embryos were selected for protein expression profiling using tandem mass tag (TMT)-labeled liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as for assessment of H4K5ac modification by ChIP-seq. Multi-omics integration was performed to identify common differentially expressed genes, followed by functional enrichment analysis. Key genes were validated using RT-qPCR. Results: Proteomic analysis revealed that differentially expressed proteins were significantly enriched in focal adhesion pathway. Protein–protein interaction (PPI) network analysis indicated that these proteins (e.g., Integrin alpha 3 (Itga3), glycogen synthase kinase 3 beta (Gsk3b), mitogen-activated protein kinase 9 (Mapk9)) were associated with focal adhesion and cytoskeletal functions. Integrated multi-omics analysis identified 923 common differentially expressed genes, which were also significantly enriched in focal adhesion pathway. Within this pathway, the protein expression levels of Itga3, Gsk3b, and Mapk9 exhibited a consistent co-variation trend with H4K5ac enrichment. RT-qPCR results confirmed that Itga3 was significantly up-regulated, while Gsk3b was down-regulated in the NTDs group (p < 0.05). Conclusions: Maternal pregestational diabetes may contribute to NTDs by disrupting cytoskeletal reorganization, cell adhesion, and migration processes. This disruption is likely mediated through H4K5ac-regulated expression of key focal adhesion pathway genes such as Itga3 and Gsk3b.