Integrated bioinformatics and experimental validation reveal Luteolin targets the PI3K/AKT/NRF2 Axis in diabetic wound healing.

BACKGROUND: Diabetic wound healing represents a critical global health challenge, with India harbouring over 77 million diabetic adults and diabetic foot ulcer prevalence ranging from 4.5 to 15%. The complex pathophysiology involves chronic inflammation, impaired angiogenesis, and excessive oxidative stress that fundamentally disrupts normal healing cascades. Luteolin, a naturally occurring flavonoid with documented anti-inflammatory and antioxidant properties, has emerged as a promising therapeutic candidate. OBJECTIVE: This study elucidated the molecular mechanisms of luteolin in diabetic wound healing through an integrated computational and experimental approach. METHODS: Transcriptomic analysis was conducted on three diabetic wound datasets (GSE80178, GSE134431, GSE199939) using differential gene expression analysis. Swiss Target Prediction identified luteolin targets, followed by the construction of a protein-protein interaction network and molecular docking with 200 ns molecular dynamics simulations. Experimental validation involved HS27 human dermal fibroblasts with MTT viability assays, flow cytometry-based ROS measurement, apoptosis analysis, LC-MS/MS metabolomics profiling, scratch wound healing assays, Western blotting, and immunofluorescence. RESULTS: Analysis identified 520 common differentially expressed genes among three datasets and PIK3R1 as a candidate therapeutic target through bioinformatics, machine learning and target prediction. Molecular docking revealed strong binding affinities: PIK3R1 (-9.62 kcal/mol), mTOR (-9.10 kcal/mol), and AKT1 (-8.42 kcal/mol). Treatment with luteolin (25 μg/mL) significantly enhanced cell viability to 130%, markedly reduced intracellular ROS levels (p < 0.001) and promoted wound closure by nearly 40%. Western blot analysis further confirmed the molecular basis of these effects, demonstrating pronounced upregulation of p-PI3K, AKT, NRF2 and Bcl-2 along with concomitant downregulation of GSK-3β and Bax. Metabolomic profiling identified luteolin-responsive metabolites with significant alterations in specific metabolic pathways, including L-leucine and sphingomyelin-related metabolites, were of particular interest given their established association with PI3K/AKT signalling. CONCLUSION: Our study provides a hypothesis linking luteolin to PIK3R1 in diabetic wound-associated dermal fibroblasts, supported by computational, cellular, and metabolomic evidence, and suggests the potential of luteolin as a therapeutic approach for diabetic wound healing.