Exploring the mechanism underlying negative pressure wound therapy in promoting the healing of diabetic foot ulcers via integrated multi-omics and bioinformatics approaches.

BACKGROUND: Diabetic foot ulcers (DFU) are one of the most serious complications of diabetes, closely associated with high amputation rates, mortality, and healthcare burdens, and the clinical treatment research is still limited. Negative Pressure Wound Therapy (NPWT) is a treatment modality for DFU, but its specific mechanisms promoting wound healing remain largely unknown. METHODS: The bulk RNA-seq and scRNA-seq data for DFU used in this study were obtained from the GEO database. First, this study used differential analysis and multiple machine learning algorithms to identify key genes activated by NPWT. Subsequently, this study used GSVA and AddModuleScore analysis to evaluate the mesenchymal-epithelial transition (MET) capability of keratinocytes (KCs), and used CytoTRACE and pseudotime trajectory analysis to construct MET differentiation trajectories. Finally, immunofluorescence (IF) was used to validate the expression of GRHL2 in DFU clinical samples. RESULTS: Differential expression analysis identified 975 dysregulated genes in DFU (530 upregulated, 445 downregulated). Through four machine learning algorithms, GRHL2 was consistently selected. GRHL2 was primarily expressed in skin at both RNA and protein levels. Single-cell analysis of 13 DFU samples confirmed GRHL2 is predominantly expressed in KCs. Specifically, NPWT activates GRHL2, thereby driving mesenchymal fibroblasts (MFs) into supraspinous_KCs. Additionally, molecular docking suggests that Parthenolide, MG-132, Mitoxantrone, and Irinotecan may have potential efficacy in treating DFU. Finally, IF results show that GRHL2 expression is consistent with transcriptomic analysis results. CONCLUSION: Overall, this study established a MET model of "MFs - supraspinous_KCs" in DFU repair. Additionally, the "NPWT - GRHL2 - MET - cell conversion" signaling axis proposed in this study supplements the molecular mechanism by which NPWT promotes wound healing through the regulation of cell fate determination, deepening the biological effects of physical stimulation from the macroscopic tissue repair phenotype to the microscopic cell conversion level, and further enriching the mechanism by which NPWT promotes wound healing.