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

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.

This study elucidated the molecular mechanisms of luteolin in diabetic wound healing through an integrated computational and experimental approach.

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.

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.

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.