Potential mechanism underlying HXSJ decoction in the treatment of venous leg ulcers: based on the association between venous leg ulcers and ferroptosis

Background: Venous leg ulcer (VLU) is among the most severe clinical manifestations of chronic venous disease (CVD) and imposes substantial burdens on both patients and society. VLU pathogenesis is closely associated with the impairment of vascular endothelial cells. In this study, tissue samples from the patients' s skins around wound at different stages of CVD were collected during operation and used to elucidate the involvement of Ferroptosis in the pathogenesis of VLU. Also the potential mechanism through which Huoxue Shengji (HXSJ) decoction alleviates Ferroptosis in human umbilical vein endothelial cells (HUVECs) was investigated.

Methods: During surgical procedure such as great saphenous vein high ligation and stripping surgery, matched skin tissues from the normal, hyperpigmentation (HPT), lipodermatosclerosis (LDS), and VLU regions were collected from 10 patients with VLU, and the levels of iron and Glutathione Peroxidase 4 (GPX4) were quantified to evaluate Ferroptosis. In vitro, HUVECs were used to iron overload induction using exogenous 100 µM ferric ammonium citrate (FAC) or 100 µM hemin, or Ferroptosis induction with 10 µM erastin, and treated with 10 µg/ml HXSJ decoction for 24 h. Subsequently, lipid peroxidation (LPO) damage, mitochondrial function, and key genes involved in Ferroptosis were assessed.

Results: Iron deposition in the affected skin of patients with CVD gradually increased before progression to VLU and significantly decreased during the VLU stage. Moreover, GPX4 expression increased significantly in the HPT stage but was gradually suppressed with further deterioration of CVD. The in vitro results indicated that in both the iron overload and Ferroptosis models, HXSJ decoction effectively upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), solute carrier family 7 member 11 (SLC7A11, xCT), and GPX4, which was accompanied by the inhibition of malondialdehyde and protein carbonylation production, the alleviation of ferrous ion accumulation, and the restoration of mitochondrial function.

Conclusions: This study demonstrated that iron accumulation-mediated inactivation of GPX4 serves as a crucial mechanism in VLU formation through Ferroptosis induction. Notably, the therapeutic mechanism through which HXSJ decoction alleviates Ferroptosis involves Nrf2/xCT/GPX4 pathway activation and a reduction in ferrous ion accumulation. These findings may provide novel insights into VLU pathogenesis and give possible reason for developing traditional Chinese medicine therapies targeting Ferroptosis and VLUs.

Chinese herbal compound; Ferroptosis; HUVECs; Iron overload; Venous leg ulcer.