This study strictly followed the inclusion and exclusion criteria (Supplementary Material S1) and enrolled a total of 15 patients who underwent low anal fistula resection. The patients were randomized into three groups (n = 5 per group): the experimental group (treated with Wugu Qilin Ointment-soaked gauze), the positive control group (treated with Kangfuxin Solution-soaked gauze), and the negative control group (treated with Vaseline-soaked gauze). All interventions commenced immediately after surgery, using a standardized dressing change protocol (twice daily with sterile dressing coverage and fixation), and continued until complete wound healing.

Wugu Qilin Ointment was prepared by the Preparation Center of the Affiliated Hospital of Shaanxi University of Chinese Medicine following the topical preparation standards outlined in the Pharmacopoeia of the People’s Republic of China (2020 edition). The ointment is composed of Chrysomya megacephala (30 g), Daemonorops draco (30 g), Coptis chinensis (30 g), and Arnebia euchroma (15 g), with a drug ratio of C. megacephala: Daemonorops draco: Coptis chinensis: Arnebia euchroma = 2:2:2:1 (w/w). All medicinal materials were verified using taxonomic databases (Supplementary Material S2). The preparation process was: C. megacephala, Coptis chinensis, and Arnebia euchroma were soaked in 400 mL of sesame oil for 3 days, then simmered over low heat until the herbs were charred and desiccated, which was followed by filtration. Daemonorops draco was then added and dissolved, and beeswax was gently heated and incorporated. After cooling, 40 pieces of 3 × 10 cm sterilized gauze were immersed in the ointment for 4 hours and stored at 4 °C for later use. Orthogonal fingerprint profiling was performed via liquid chromatography-mass spectrometry (LC-MS) in both positive and negative ion modes. The top ten characteristic peaks were selected based on scoring and used for total ion chromatogram (TIC) analysis (Supplementary Figures S1, S2). In positive ion mode, the detection of 7-hydroxy-3-phenyl-chromen-4-one (m/z 239.0703, retention time 381 s), a flavonoid component accounting for 42.7% of the peak area, suggests it as a major active constituent, consistent with the known bioactive compounds in Daemonorops draco and Coptis chinensis. The detection of Carnitine (m/z 162.1125) indicates the presence of amino acid metabolites. In negative ion mode, alpha-linolenic acid (m/z 277.2173) and linoleic acid (m/z 279.2330) together accounted for 68.3% of the total lipid content, confirming that the sesame oil matrix components are well preserved (Detailed data provided in Supplementary Material S3, 4; The ConPhyMP checklist can be found in Supplementary Material S4, 5).

Kangfuxin Solution Gauze: Sterile gauze strips (3 cm × 10 cm) were immersed in Kangfuxin Solution (manufactured by Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.; Approval No.: National Medicine Permit No. Z51021834).

Vaseline Gauze: Commercial Vaseline gauze (3 cm × 10 cm × 10 strips per pack) produced by Henan Yadu Industrial Co., Ltd. was used (Registration No.: National Food and Medical Device (Permit) 2014 No. 3641199).

The study protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Shaanxi University of Chinese Medicine (Approval No.: SZFYIEC-YJ-2023-[126]). Every participant gave informed consent in writing.

Statistical analyses were enabled by SPSS 26.0 and R 4.3.1. Measurement data are shown in mean ± standard deviation (SD) (x̄ ± s), and categorical ones were examined via the chi-square test. Regarding miRNA expression profiling, high-throughput sequencing data were analyzed with DESeq2 or edgeR, while microarray data were processed using the limma package. DEMs were identified based on |log₂FC| ≥ 1 and FDR <0.05, with results presented via volcano plots and hierarchical clustering. Inter-group comparisons were conducted using ANOVA or t-tests, with P < 0.05 denoting statistical significance. Every experiment was carried out three times independently. *P < 0.05, **P < 0.01, ***P < 0.001.

To unravel how miR-93-3p influences macrophage phenotypes, qPCR was employed to assess its relative expression in macrophages from every cohort. In contrast to the negative control cohort, the miR-93-3p mimic cohort exhibited elevated expression, whereas the inhibitor group displayed reduced levels (Figure 6A). These findings indicate that the transfection of miR-93-3p mimics and inhibitors successfully achieved effective modulation of miR-93-3p expression levels in macrophages. Specifically, the mimic group exhibited a marked upregulation of miR-93-3p expression, whereas the inhibitor group demonstrated a significant downregulation. These results confirm the stability and reliability of the experimental intervention model, thereby laying a sound foundation for subsequent investigations into the functional role of miR-93-3p in regulating macrophage polarization.

qPCR analysis revealed that the miR-93-3p mimic group exhibited reduced expression of M1 markers (CD86) and pro-inflammation cytokines (IL-1β, IL-6, TNF-α), alongside upregulation of M2 markers (Arg-1, CD206) and anti-inflammation cytokines (IL-10, TGF-β). However, the inhibitor group demonstrated the opposite pattern (Figures 6B,C). To further elucidate how miR-93-3p regulates MP, the expression of M1 marker CD86 and M2 markers Arg-1 and CD206 was assessed via FC (Figure 7). In contrast to the NC cohort, the miR-93-3p mimic group exhibited markedly fewer CD86⁺ cells (1.87% vs. 6.90%), while CD206⁺ and Arg-1⁺ cell proportions were significantly elevated (32.73%/32.76% vs. 6.52%/6.48%). The inhibitor group displayed the opposite trend. Meanwhile, the expression levels of key polarization-associated proteins were examined using WB (Figure 8). Quantitative analysis (Figure 8A) showed that relative to the NC group, the miR-93-3p mimic group exhibited marked downregulation of CD86 and upregulation of the Arg-1 and CD206, whereas the inhibitor cohort displayed the opposite trend. WB bands (Figure 8B) visually confirmed the downregulation of CD86 and upregulation of Arg-1 and CD206 in the mimic group, with the inverse pattern observed in the inhibitor group.

These findings confirm at the protein level that miR-93-3p promotes the MP from an M1 to an M2 phenotype through a bidirectional regulatory mechanism. Therefore, it was concluded that miR-93-3p facilitates the resolution of wound-associated inflammation. In the subsequent investigation, our study aimed to further elucidate its underlying regulatory mechanisms by examining its specific target gene interactions.

To gain a deeper understanding of the molecular mechanisms by which miR-93-3p promotes wound healing through the regulation of macrophage polarization, a systematic target gene prediction analysis was carried out for the DEMs. A total of 388 target genes were predicted for the upregulated miRNAs, while 287 target genes were identified for the downregulated miRNAs (Figure 9). These high-confidence target genes offer important clues for subsequent investigations.

Through a systematic cross-analysis of DEMs among the Wugu Qilin Ointment group, Kangfuxin Solution group, and Vaseline group, our study identified five miRNAs exhibiting the most significant differential expression, along with their corresponding target genes. Specifically, as a downstream effector of mTORC1, 4E-BP1 exerts its function by modulating the translational efficiency of pro-inflammatory and anti-inflammatory cytokines through its phosphorylation status (Byles et al., 2013). miR-6889-5p inhibits EIF4EBP2, which limits macrophage protein synthesis and proliferation (Sprenkle et al., 2023). miR-378d targets RAB10 and activates the NF-κB signaling pathway to facilitate pro-inflammation mediator secretion (Zhu et al., 2020). miR-451a modulates MIF, participating in the suppression of macrophage activity and immune regulation (Li et al., 2022). miR-32-3p targets TRAF6, facilitating the ubiquitin-mediated activation of the NF-κB pathway, which drives inflammatory responses and vascular injury (Lv et al., 2018). These target genes have been corroborated by multiple studies to play direct roles in regulating MP, activating key inflammatory signaling pathways, and remodeling the wound-healing microenvironment. The present analysis not only provides theoretical support for our experimental findings from the perspective of molecular interaction networks but also further validates the biological credibility and mechanistic relevance of our screening results through established literature.

To unveil the specific binding and mediating interaction between miR-93-3p and 3′UTR of the EIF4EBP1 gene, a rigorous functional validation was executed through a DLR system. Levene’s test for homogeneity of variance confirmed the assumption of equal variances across groups, thereby justifying subsequent analysis of variance (ANOVA). One-way ANOVA revealed a highly significant difference in luciferase activity ratios among the treatment groups. Further post hoc analysis using the least significant difference (LSD) method indicated that, under the wild-type 3′UTR context, luciferase activity in the miR-93-3p mimic cohort (Group B) was prominently lower than in the negative control cohort (Group A), with an inhibition rate of 63.5%. Conversely, under the mutant 3′UTR condition, the difference was insignificant across the miR-93-3p mimic cohort (Group D) and the control (Group C) (Figure 10). These results not only affirm the reliability of the experimental findings from a statistical standpoint but also substantiate, at a functional level, that miR-93-3p exerts its post-transcriptional regulatory effect by specifically binding to a target site within 3′UTR of EIF4EBP1. Mutation of this binding site completely abolished the regulatory effect. This provides direct molecular evidence supporting the therapeutic mechanism of the TCM-based “Euriching Pus for Tissue Growth” method in wound healing. The data further highlight the significance of the miR-93-3p/EIF4EBP1 axis in modulating MP and inflammatory responses, thereby offering a potential molecular target and theoretical foundation for the modernization of TCM.

To unveil the possible biological functions of DEMs, systematic enrichment analyses were carried out via GO and KEGG.

GO analysis examined gene function across three dimensions: biological process (BP), cellular component (CC), and molecular function (MF) (Figures 11A–F). Regarding BP, differentially expressed genes were notably enriched in pathways related to the regulation of inflammatory responses and MP. The CC analysis revealed that these genes are predominantly localized to the cell membrane and exosomes, while the MF analysis indicated enrichment in cytokine binding and protein kinase activity. This finding is highly consistent with the results presented in the earlier part of this study: miR-93-3p derived from wound exudate exosomes significantly upregulates the expression of M2 macrophage markers (Arg-1 and CD206) and anti-inflammatory cytokines (IL-10 and TGF-β) (P < 0.001), while concurrently suppressing the production of pro-inflammatory factors related to the M1 phenotype. This regulatory mechanism effectively promotes the phenotypic transition of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type and is critical in inhibiting excessive inflammatory responses.

Subsequent KEGG pathway analysis revealed marked enrichment of the target genes of DEMs in the Toll-like receptor, NF-κB, and wound healing pathways (Figures 11G,H). These findings, from a systems biology perspective, suggest that DEMs possibly contribute to wound repair by regulating key biological processes like inflammation and immune modulation. Furthermore, these signaling pathways are closely related to MP, thereby lending additional support to our hypothesis that differentially expressed exosomal miRNAs possibly inhibit inflammation and ameliorate wound healing through MP regulation.