Abstract: BACKGROUND: Angiogenesis is one of the critical issues in chronic ulcer healing. Previous studies have indicated that Zizhu ointment can increase the expression of vascular endothelial growth factor in wounds, promote wound healing, and stimulate angiogenesis in high-glucose and high-lipid cell models.
OBJECTIVE: To investigate the effects of Zizhu ointment on angiogenesis in diabetic ulcer model mice and elucidate its underlying mechanisms in promoting wound healing. 
METHODS: Twenty-four mice were randomly divided into four groups: normal control, model control, saline-treated, and Zizhu ointment groups. The diabetic mouse model was established in the latter three groups through a high-fat diet combined with streptozotocin injections. After blood glucose stabilization, full-thickness dorsal skin was removed to simulate diabetic ulcers, with regular monitoring of body mass and blood glucose levels. In the Zizhu ointment group, wound dressings were changed daily. Wound conditions were documented photographically on days 3, 7, 11, and 14 post-injury, followed by skin sample collection on day 14. Histological analyses were performed using hematoxylin-eosin and Masson’s staining to evaluate wound healing. Angiogenesis was assessed by immunofluorescence staining for vascular endothelial growth factor A and CD34. Quantitative real-time PCR and western blot assay were used to analyze mRNA and protein expression of angiogenesis-related factors, including vascular endothelial growth factor A, angiopoietin-2, sprouty-related EVH1 domain-containing protein 1, and phosphoinositide 3-kinase regulatory subunit 2. 
RESULTS AND CONCLUSION: (1) The diabetic ulcer mouse model was established successfully. Wound healing was significantly delayed in the model group. In contrast, the Zizhu ointment treatment group showed markedly accelerated healing compared with the model group at days 3, 7, 11, and 14 (P < 0.01), with particularly higher healing rates than those in the saline-treated group at days 7, 11, and 14 (P < 0.01). (2) Histopathological examination through hematoxylin-eosin and Masson’s staining revealed that compared with the control group, the model group exhibited enhanced inflammatory infiltration, reduced dermal appendages, and diminished neovascularization, while the Zizhu ointment group demonstrated improved tissue regeneration characterized by increased appendage formation and enhanced vascular cluster development. (3) Immunofluorescence staining for CD34 and vascular endothelial growth factor A revealed a reduction in positive cells within ulcerated tissue in the model group, which increased following treatment with Zizhu ointment. The mean microvascular density in the Zizhu ointment group was significantly higher than that in both the model group and the saline-treated group (P < 0.05). (4) Compared with the control group, the model group displayed downregulated vascular endothelial growth factor A and angiopoietin-2 expression (P < 0.01) along with upregulated sprouty-related EVH1 domain-containing protein 1 and phosphoinositide 3-kinase regulatory subunit 2 levels (P < 0.001). Compared with the model and saline-treated groups, Zizhu ointment significantly increased vascular endothelial growth factor A and angiopoietin-2 expression (P < 0.05) while reducing the expression of sprouty-related EVH1 domain-containing protein 1 and phosphoinositide 3-kinase regulatory subunit 2 (P < 0.05 or P < 0.01). To conclude, streptozotocin-induced diabetic foot ulcer mice exhibit delayed wound healing accompanied by impaired neovascularization. Treatment with Zizhu ointment significantly accelerates ulcer healing, promotes angiogenesis, and modulates the expression of angiogenesis-related genes and proteins. Our preliminary results indicate that topical application of Zizhu ointment facilitates the healing of diabetic ulcers in mice, potentially through its regulatory effects on wound angiogenesis.

Key words: diabetic ulcer, Zizhu ointment, angiogenesis, mouse model, ulcer healing