OBJECTIVE To investigate the effects of Chuangling Ye (CLY) on diabetic foot ulcer (DFU) model mice based on the nuclear factor erythroid 2-related factor 2 (Nrf2)/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) pathway and explore its potential mechanism.

METHODS In animal experiments, streptozotocin (STZ)-induced diabetic mice with full-thickness skin defects were divided into control, model, positive control (recombinant human epidermal growth factor, rhEGF), and CLY low- and high-dose groups (n=10). After 14 days of intervention, wound healing rate was measured. Serum inflammatory factors (IL-6, TNF-α, IL-1β) were detected by ELISA, while oxidative stress markers (SOD, MDA, GSH) and tissue ferrous iron (Fe²⁺) levels were measured by colorimetric assays. Mitochondrial ultrastructure was observed via transmission electron microscopy (TEM). Nrf2, SLC7A11, and GPX4 expression in wound tissues were analyzed by qPCR and Western blot. In cell experiments, a high glucose (HG)-induced ferroptosis model in human umbilical vein endothelial cells (HUVECs) was established and divided into control, HG, CLY, and CLY combined with pathway inhibitors (ML385, Erastin, RSL3) groups.CCK-8 was used to detect cell viability, FerroOrange was used to detect Fe²⁺ content, DCFH-DA and C11-BODIPY 581/591 probes were used to detect intracellular ROS and lipid ROS content, and Western blot was used to detect the expression of proteins such as Nrf2/SLC7A11/GPX4.

RESULTS Animal experiments showed that compared with the model group, the wound healing rate in the low- and high-dose CLY groups was significantly improved (P < 0.01); the serum IL-6, IL-1β, and TNF-α levels were significantly decreased (P < 0.01); the MDA content was significantly reduced (P < 0.01), and the SOD activity and GSH content were significantly restored (P < 0.05). Colorimetric analysis showed that the low- and high-dose CLY significantly reduced the abnormally elevated Fe²⁺ levels in DFU wounds (P < 0.05, P < 0.001). Electron microscopy showed that the mitochondrial cristae structure was improved in the low- and high-dose groups. qPCR showed that high-dose CLY upregulated the expression levels of Rno-Nfe2l2 (Nrf2), Rno-Slc7a11, Rno-Gpx4, and Rno-Acsl4 mRNA, and inhibited the expression level of Rno-Acsl4 mRNA (P < 0.001). Western blot results showed that CLY upregulated the expression of Nrf2, SLC7A11, GPX4, and FTH1 in DFU wound tissues (P < 0.01), while downregulated the level of ACSL4 (P < 0.01). Cell experiments showed that treatment with CLY increased the survival rate of high glucose-stimulated HUVECs (P < 0.001). However, the protective effect of CLY was significantly inhibited after the addition of ML385, Erastin, or RSL3 (P < 0.01). Treatment with CLY significantly decreased the Fe²⁺ content (P < 0.001), which was reversed by ML385, Erastin, or RSL3. The levels of ROS and lipid ROS were also significantly reduced (P < 0.001), while the addition of ML385, Erastin, or RSL3 partially weakened the antioxidant effect of CLY. Western blot results showed that CLY significantly upregulated the expression of Nrf2、SLC7A11 and GPX4 (P < 0.001) and downregulated the expression of 4-HNE and COX2 (P < 0.01), and ML385, Erastin, or RSL3 could reverse this protective effect.

CONCLUSION CLY promotes DFU healing by activating the Nrf2/SLC7A11/GPX4 pathway to inhibit ferroptosis, mitigate oxidative stress, and suppress inflammation, providing a novel therapeutic target for DFU.