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, IHC staining, western blot, 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 level of TNF-α and increased the expression levels of VEGF, Collagen I in the granulation tissue. The single-cell sequencing revealed that the expression level of IL-6 and CXCL-8 was increased in the IL-6⁺ macrophages that promoted the expression of WASF3 in fibroblasts and further recruited ACTR2, ACTR3. Finally, CLS increased intercellular communication between macrophages and fibroblasts, enhancing MSF migration ability by activating JAK2/STAT3 signaling pathway. 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.