Mechanistic Study of Shixiang Plaster in DFUs Treatment: Network Pharmacology, Animal Experimentation, and LC-MS Analysis

Abstract DFUs are a common and debilitating complication of diabetes, often leading to poor wound healing and even amputations. Despite advances in treatment, healing remains slow due to factors like impaired angiogenesis and chronic inflammation. Shixiang plaster, a topical traditional Chinese medicine ointment has demonstrated promising effects in promoting the healing of DFUs. This study explored the underlying mechanisms of Shixiang plaster through a combination of network pharmacology, animal experiments, and LC-MS analysis. Network pharmacology identified 80 active ingredients and 90 potential therapeutic targets, with ESR1, RHOA, BCL2, CASP8, and MAPK8 emerging as key targets. Enrichment analyses highlighted pathways related to Apoptosis pathway, MAPK signaling pathway, C-type lectin receptor signaling pathway and so on. Molecular docking confirmed strong binding between active compounds and these targets. In vivo studies using the rat model revealed that Shixiang plaster significantly improved wound healing and reduced inflammation compared to controls. Immunohistochemistry showed elevated VEGF and CD34 expression, while WB analysis on day 14 indicated downregulation of MAPK8, p-p38, p38, p44/42, and p-p44/42. LC-MS analysis further identified daidzein, chrysin, and kaempferol as major bioactive compounds. These findings suggest that Shixiang plaster facilitates DFUs healing by modulating inflammatory pathways, promoting angiogenesis, and possibly exerting its effects through the downregulation of MAPK pathway-related proteins.