Abstract: BACKGROUND: Diabetic ulcers are prone to recurrent outbreaks and are difficult to heal. The occurrence and progression of these ulcers are associated with differential expression of miRNAs. Research on the impact of Zi-Zhu ointment on microRNA (miRNA) expression during the healing process may provide new insights into the treatment of chronic ulcers with traditional Chinese medicine. 
OBJECTIVE: To investigate the potential mechanism underlying the intervention of Zi-Zhu ointment in diabetic ulcers using high-throughput sequencing and bioinformatics analysis methods. 
METHODS: A total of six mice were used in each of the following groups: normal group, model group, and Zi-Zhu ointment group. In the latter two groups, high-fat feed combined with streptozotocin injection was performed to construct a diabetic model, and the skin was clipped on the back to form a chronic diabetic ulcer. The Zi-Zhu ointment group was given Zi-Zhu ointment at the dose of 0.032 g/cm², once a day, for 14 days. The modeling and wound healing indicators were assessed, and three mice from each group were randomly selected for sequencing. Differentially expressed miRNAs between the groups were analyzed using bioinformatics methods, and a preliminary regulatory network of the differentially expressed miRNAs, their target genes, and gene functions in diabetic ulcer was established. 
RESULTS AND CONCLUSION: (1) A total of 1 072 known miRNAs were screened in the model group tissues compared with the normal group, with 94 showing significant differences. Among these, the top three up-regulated miRNAs were miR-1298-5p, miR-29b-3p, and miR-151-5p, while the top three down-regulated miRNAs were miR-223-3p and miR-135a-5p. These miRNAs were primarily associated with cell proliferation, apoptosis, wound healing, angiogenesis, and anti-inflammation processes. (2) In diabetic mice before and after ulcer administration, a total of 1 056 known miRNAs were screened in ulcerated tissue, with 57 showing significant differences. The top three up-regulated miRNAs were miR-451a, miR-363-3p, and miR-122-5p, while the top three down-regulated miRNAs were miR-1964-3p, miR-5099, and miR-182-3p. These miRNAs were mainly associated with cell proliferation, migration, regulation of macrophages, and improvement of insulin resistance. (3) Gene ontology analysis revealed that the target genes were primarily implicated in cell development, neural development, protein binding, phosphatidylinositol 3-kinase activity, and serine/threonine protein kinase activity. (4) Furthermore, Kyoto Encyclopedia of Genes and Genomes functional analysis indicated that the differentially expressed target genes in the model group, compared with the normal group, were enriched in pathways such as mTOR signaling, PI3K-Akt signaling, AMPK signaling, the Wnt signaling pathway, and cancer-related pathways. Conversely, in the diabetic ulcer treated with Zi-Zhu ointment, enriched pathways included breast cancer and liver cancer-related pathways, as well as cAMP signaling, p53 signaling, Wnt signaling, and PI3K-Akt signaling pathways compared with the model group. The study preliminarily established a regulatory network involving differentially expressed miRNAs, their target genes, and gene functional pathways in diabetic ulcers. Zi-Zhu ointment can significantly promote the healing of diabetic ulcers. Diabetes induces differential expression of miRNAs, and treatment with Zi-Zhu Ointment also alters the miRNA profile. This lays a foundation for future research on the mechanisms of disease occurrence and development, as well as treatment strategies from the perspective of miRNAs.

Key words: Zi-Zhu ointment, diabetic ulcer, high-throughput sequencing, miRNA, bioinformatics analysis, engineered tissue construction