Formononetin enhances angiogenesis in diabetic wounds by inhibiting ferroptosis through suppression of mtROS-mediated xCT/GPX4 upregulation

Background: Impaired angiogenesis critically contributes to delayed wound healing in diabetic patients. Mitochondrial damage in endothelial cells worsens this vascular dysfunction. Formononetin (FMN), a phytoestrogen from Astragalus roots, is traditionally used to enhance blood function and microcirculation; however, its mechanism remains unclear. This study investigates whether FMN protects human umbilical vein endothelial cells (HUVECs) from high glucose (HG)-induced Ferroptosis, and we also evaluated its potential to enhance vascular repair and accelerate diabetic wound healing.

Methods: We used Western Blotting to measure Ferroptosis markers including the cystine/gluta-mate antiporter system (xCT) and Glutathione Peroxidase 4 (GPX4) in both HUVECs and diabetic mouse wound tissue. To assess oxidative stress, we measured glutathione content (GSH), malondialdehyde production (MDA), and intracellular iron accumulation. Mitochondrial changes were evaluated through MitoSOX, TOMM20 protein expression, and JC-1 fluorescence. Wound tissue sections were processed for H&E staining to examine healing stages, and CD31 immunofluorescence was performed to visualize angiogenesis in the wound tissue.

Results: FMN effectively reduced Ferroptosis markers in HG-treated HUVECs, and Erastin treatment abolished this protective effect. The compound appeared to block Ferroptosis through two mechanisms: restoration of mitochondrial integrity and reactivation of the xCT/GPX4 antioxidant system. When we tested FMN in diabetic mice, wound closure rates improved substantially, the expression of xCT and GPX4 was increased, and CD31 expression in wound vessels increased, which matched what we observed in vitro.

Conclusion: Ferroptosis is critically involved in the pathogenesis of diabetic wounds. FMN exerts protective effects against HG-induced Ferroptosis in HUVECs via restoration of mitochondrial function and mitigation of mitochondrial Reactive Oxygen Species (mtROS) accumulation through xCT/GPX4 activation. This evidence suggests FMN could serve as a promising natural therapeutic agent for diabetic wound treatment.

Diabetic wound; Ferroptosis; Formononetin; Mitochondrial dysfunction.