Potential Mechanism of HXSJ Decoction in the Treatment of Venous Leg Ulcer: Based on the Association between Venous Leg Ulcers and Ferroptosis

Abstract Background: Venous leg ulcer (VLU) represents one of the most severe clinical manifestations in the progression of chronic venous diseases (CVD), imposes substantial burdens on both patients and society. The etiology of VLU is associated with the impairment of vascular endothelial cells. Methods: In clinical, a total of 10 patients diagnosed with VLU were enrolled in this study, and 4 types of skin tissue samples were collected from each patient, including normal, hyperpigmentation, lipodermatosclerosis, and VLU areas. Subsequently, the iron content and GPX activity were quantified. In vitro, iron overload models of HUVECs were established by exogenous 100μM FAC or 100μM Hemin to simulate simple iron overload and hemoglobin exudation, respectively. And ferroptosis medel was induced by 10μM Erastin. Meanwhile, Huoxue Shengji Decoction (HXSJ Decoction) as an external Chinese herbal decoction used in VLU treatment, has been incorporated into our in vitro study. Followed by the lipid peroxidation damage was evaluated by the content of malondialdehyde, protein carbonylation, ferrous ion, DCFH-DA and BODIPY™ 581/591 C11 staining; mitochondrial function was determined through ATP content and mitochondrial membrane potential (MMP) of JC-1 staining; the activation of Nrf2/system Xc-/GPX4 axis was assessed through GPX activity, GSH content, qPCR and western blot. Results: The clinical results showed that, before progressing to VLU, iron deposition in the affected tissues of CVD gradually intensifies (P<0.05), and suddenly decreases in VLU stage (P<0.01). Meanwhile, in hyperpigmentation stage, the GPX activity increased significantly (P<0.05), with further deterioration of CVD, GPX activity was gradually suppressed (P<0.05). The in vitro results indicate that irrespective of iron overload or ferroptosis models, HXSJ Decoction effectively upregulated the expression of Nrf2, xCT, and GPX4 (P<0.05); inhibited the generation of malondialdehyde (P<0.01) and protein carbonylation (P<0.01), alleviated the accumulation of ferrous ions (P<0.05); restored MMP, promoted ATP production (P<0.05). Conclusions: Overall, this study suggested that iron accumulation-mediated inactivation of GPX4 is a significant contributing factor in VLU development through ferroptosis induction. Additionally, it revealed that the therapeutic mechanism of HXSJ Decoction potentially involves mitigating ferroptosis by activating the Nrf2/system Xc-/GPX4 pathway and alleviating the accumulation of ferrous ions.