Targeting Grx1 by hyperoside regulates Ncf4 post-translational modification to enhance mitophagy and accelerate diabetic wound healing.

Diabetic wound healing impairment is a devastating diabetic complication with high amputation and mortality rates, and current therapies lack effective targeted agents. To define the pro-healing efficacy of hyperoside and elucidate its underlying molecular mechanism in diabetic wound repair. A type 2 diabetic mouse wound model was established via high-fat diet combined with low-dose streptozotocin. Mechanistic studies employed high glucose-stimulated bone marrow-derived macrophages, integrated DIA quantitative proteomics and single-cell RNA sequencing, molecular dynamics simulation, CETSA/DARTS binding assays, co-immunoprecipitation, and Grx1 gain/loss-of-function experiments. Hyperoside dose-dependently accelerated wound closure and ameliorated systemic metabolic disorders. Concurrently, hyperoside attenuated the intensity of inflammatory responses in the wound bed, upregulated the expression of collagens including Col1a1 and Col3a1, and promoted ordered collagen remodeling. It normalized redox homeostasis by elevating the GSH/GSSG and NADPH/NADP+ ratios, and suppressed the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. Integrated multi-omics analysis identified the Grx1/Ncf4/Bnip3l axis as the regulatory pathway. Mechanistically, in vitro studies demonstrated that hyperoside bound to Grx1 to enhance its protein stability, shifted the post-translational modification of Ncf4 from phosphorylation to glutathionylation to inhibit NOX2 activation, and augmented Bnip3l-mediated mitophagy to reverse macrophage dysfunction. Hyperoside exerted robust pro-healing effects via targeting the Grx1/Ncf4/Bnip3l axis, establishing it as a promising therapeutic candidate for refractory diabetic wounds.