CEBPD contributes to diabetic foot ulcer progression via transcriptional regulation of CXCL10: insights from in vitro and in vivo evidence.
Diabetic foot ulcer (DFU) is a severe complication of diabetes mellitus characterized by impaired wound healing and high amputation risk. Current treatments remain unsatisfactory, necessitating exploration of novel molecular mechanisms. The transcription factor CCAAT enhancer binding protein delta (CEBPD) regulates inflammatory responses and cellular stress pathways implicated in diabetic complications. However, its specific role and mechanism in DFU pathogenesis are poorly understood. Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (HG) to mimic diabetic conditions. C-X-C motif chemokine ligand 10 (CXCL10) and CEBPD mRNA expression were analyzed by quantitative real-time polymerase chain reaction. The protein expression of CXCL10, CEBPD, and glutathione peroxidase 4 (GPX4) was detected by western blotting assay. Cell viability was analyzed by a cell counting kit-8 assay. Cell proliferation was analyzed by a 5-ethynyl-2'-deoxyuridine assay. Cell apoptosis was detected by flow cytometry. Cell migration was analyzed by a wound-healing assay. Tube formation was analyzed by a tube formation assay. Fe2+ and malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were analyzed by colorimetric assays. Reactive oxygen species (ROS) levels were analyzed by fluorometric assay and flow cytometry. The chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter assay were used to analyze the association of CXCL10 and CEBPD. The effect of CEBPD knockdown on HG-induced cell injury was further analyzed using a DFU rat model. CXCL10 and CEBPD expression were significantly upregulated in DFU patients and HG-stimulated HUVECs. CXCL10 knockdown reversed HG-induced cellular damage, restoring proliferation, migration, and tube formation while suppressing apoptosis and altering ferroptosis-related indicators (evidenced by reduced Fe2+/MDA/ROS and elevated SOD/GPX4). Crucially, CEBPD transcriptionally activated CXCL10, confirmed by promoter binding (ChIP) and luciferase activity. Silencing CEBPD replicated the protective effects of CXCL10 knockdown, and CXCL10 overexpression attenuated CEBPD knockdown-induced effects in HG-treated HUVECs. In vivo, CEBPD knockdown accelerated diabetic wound closure in rats, enhancing collagen deposition and tissue regeneration. CEBPD exacerbated human umbilical vein endothelial cell dysfunction at least partly through the transcriptional regulation of CXCL10, and CEBPD knockdown accelerated diabetic wound healing in vivo, suggesting that targeting the CEBPD/CXCL10 interplay may serve as a potential therapeutic strategy for DFU.