Integrated metabolomic and transcriptomic analysis reveals P4HA3-associated 4-Hydroxyproline reduction in a mouse model of LiCl-induced cleft palate.

OBJECTIVE: Lithium chloride (LiCl) induces cleft palate by disrupting palatal shelf elevation, but the underlying metabolic mechanisms remain unclear. This study aims to investigate these mechanisms using an integrated multi-omics approach. DESIGN: A LiCl-induced cleft palate mouse model was established by intraperitoneal injection of LiCl (0.4 mg/g/day) from embryonic day (E) 10.5 to E13.5. Palatal shelves were collected at E16.5 for targeted metabolomics using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and transcriptome sequencing. Integrated pathway analysis was performed to identify key pathways. Immunofluorescence, reverse transcription quantitative PCR (RT-qPCR), and Hydroxyproline assay validated findings in vivo and in human embryonic palatal mesenchymal (HEPM) cells. Further temporal analysis was conducted at E13.5, E14.0, and E14.5 to identify the key stage. RESULTS: In LiCl-induced cleft palate mice, multi-omics identified 24 differentially abundant metabolites and 595 differentially expressed genes, with arginine and proline metabolism as the most enriched pathway. Within this pathway, 4-Hydroxyproline was downregulated, proline was upregulated, accompanied by downregulation of P4HA3, a key enzyme for 4-Hydroxyproline synthesis. Immunofluorescence revealed increased β-catenin and decreased P4HA3 in cleft palate tissues, with partial colocalization. Temporal analysis showed these changes became significant at E14.0, coinciding with disrupted palatal elevation. In vitro, LiCl simultaneously upregulated β-catenin and downregulated P4HA3 in HEPM cells. CONCLUSION: LiCl-induced cleft palate in mice involves β-catenin-associated downregulation of P4HA3, which contributes to a reduction in 4-Hydroxyproline and impaired palatal elevation. These findings reveal a novel metabolic mechanism in cleft palate pathogenesis.