Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula.

BACKGROUND: Anal fistula is one of the most common and frequently occurring diseases in the anorectal department. Calvatia lilacina spore (CLS) has been applied for wound treatment with a long history as a traditional Chinese medicine (TCM). However, the mechanism of CLS to treat postoperative wound of anal fistula remains unclear. The present study aims to investigate the efficacy and mechanism of CLS in promoting anal fistula wound healing from the perspective of regulating the interaction between macrophages and fibroblasts. METHODS: Twenty patients who received anal surgery were recruited in Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. We presented a single-cell atlas of granulation tissue, comparing samples with and without CLS treatment, utilizing single-cell RNA sequencing. The pharmacological effects and mechanism of CLS on anal fistula wound were assessed using elisa, Immunohistochemistry (IHC) staining, western blot, Immunofluorescence (IF) staining, flow cytometry assays and cell co-culture. RESULTS: The CLS had a uniform particle size and contained components mainly including proteins, steroids, polysaccharides and polyphenols. CLS reduced the expression levels of Tumor Necrosis Factor-alpha (TNF-α) and increased the expression levels of Vascular Endothelial Growth Factor (VEGF) and Collagen Type I Alpha 1 (COL1A1) in the granulation tissue. The single-cell sequencing revealed that the expression level of interleukin 6 (IL-6) and C-X-C Motif Chemokine Ligand 8 (CXCL-8) was increased in the IL-6(+) macrophages that promoted the expression of Wiskott-Aldrich syndrome protein family member 3 (WASF3) in fibroblasts and further recruited Actin-Related Protein 2 (ACTR2), Actin-Related Protein 3 (ACTR3). Finally, CLS enhanced intercellular communication between macrophages and fibroblasts by activating the Janus Kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, thereby promoting mouse skin fibroblasts (MSF) migration ability. CONCLUSION: Our study objectively demonstrated the pharmacological effects of CLS in promoting the wound healing of anal fistula and investigated its mechanisms in terms of regulating the immune inflammatory process of macrophages increases signal communication with fibroblasts while promoting fibroblast transformation.