Abstract: BACKGROUND: Previous research has shown that Shixiang plaster is effective in promoting chronic refractory wound healing, but its precise mechanism in metabolomics is not fully understood. 
OBJECTIVE: To investigate the effects of Shixiang plaster on chronic refractory wounds using untargeted metabolomics and immunological methods. 
METHODS: Animal models of chronic refractory wounds (2 cm × 2 cm full-thickness skin defect + Staphylococcus aureus covered wound) were established in 36 male Sprague-Dawley rats. The day after modeling, model rats were divided into model (n=12), Beifuji (n=12), and Shixiang plaster (n=12) groups. The model group received only saline-moistened sterile dressings. Shixiang plaster or Beifuji (recombinant basic fibroblast growth factor as the main ingredient) was applied to the wounds in the latter two groups, respectively, each covered with sterile dressing and changed every 24 hours. Treatments continued for 14 days. Wound secretions were collected for detection of fibronectin levels using ELISA. Metabolic profiling of wound exudates was performed using liquid chromatography-mass spectrometry, and differential metabolites were screened and identified using principal component analysis and partial least squares-discriminant analysis. KEGG enrichment analysis of differential metabolites and Veen association analysis were performed.
RESULTS AND CONCLUSION: (1) With the prolongation of the administration time, the wound area was decreased in all the three groups, with the slowest wound recovery in the model group and the fastest in the Shixiang plaster group. The level of fibronectin in the Shixiang plaster group was higher than that in the model group at 7 and 14 days after administration (P < 0.05). (2) There were 118 unique differential metabolites screened between 7 days and 3 days after administration, 129 unique differential metabolites screened between 14 days and 3 days after administration, and 30 unique differential metabolites screened between 14 days and 7 days after administration. KEGG enrichment analysis showed that Shixiang plaster enhanced wound healing by modulating several metabolic pathways, especially the ABC transporter protein pathway, the cAMP signaling pathway, and the mTOR signaling pathway, which played crucial roles in cell proliferation, migration, immune response, and antioxidant processes. Veen correlation analysis identified common different metabolites, methoxsalen and 5'-methylthioadenosine, at 3, 7, and 14 days after administration. To conclude, Shixiang plaster may optimize ABC transporter proteins, cAMP signaling pathway and mTOR signaling pathway by modulating the expression of methoxsalen and 5'-methylthioadenosine, thus exerting anti-inflammatory, antioxidant and cell proliferation-promoting effects and accelerating wound healing.

Key words: Shixiang plaster, chronic refractory wounds, methoxsalen, 5'-methylthioadenosine, wound secretions, different metabolites, engineered tissue construction