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

Sep 30, 2026

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Anal fistula is a frequently occurring anorectal disease, and Calvatia lilacina spore (CLS) has a long history in traditional Chinese medicine for wound treatment, yet its postoperative mechanism remains unclear. This study evaluates CLS efficacy and mechanism in promoting anal fistula wound healing by examining how CLS regulates interactions between macrophages and fibroblasts. Twenty patients undergoing anal surgery were analyzed using single-cell RNA sequencing with molecular and cellular assays, including ELISA and immunostaining, to link immune-inflammatory regulation with fibroblast transformation.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages17

Reading Time43 min

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Abstract

Background

Methods

Results

Conclusion

Introduction

Disease background and clinical burden

Therapeutic rationale: macrophage and fibroblast regulation

He etal. Chinese Medicine (2026) 21:14 [https://doi.org/10.1186/s13020-025-01293-w](https://doi.org/10.1186/s13020-025-01293-w) Chinese Medicine RESEARCH Open Access Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacinain promoting wound healing of anal fistula Tangtang He1,2†, Kewei Wang3*†, Ruiwen Mo1,2, Juntong Guo1,2, Bin Jiang3, Ruoyu Mu 1,2, Wen Min4, Lifeng Zhu5,6* and Jun Chen1,2,7* Abstract 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 ofWiskott-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. †Tangtang He and Kewei Wang have contributed equally to this work. *Correspondence: Kewei Wang kowi [1231@163.com](1231@163.com) Lifeng Zhu [zhulf2020@126.com](zhulf2020@126.com)[ ](zhulf2020@126.com)Jun Chen[chenjun75@163.com](chenjun75@163.com) Full list of author information is available at the end of the article © The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creativeco

FAQs

What problem does the study address about Calvatia lilacina spore (CLS)?

The study addresses the unclear mechanism of how CLS treats postoperative wounds in anal fistula, despite its traditional use for wound treatment.

How was the CLS effect studied in patients?

Twenty patients receiving anal surgery were enrolled, and granulation tissue samples with and without CLS treatment were compared using single-cell RNA sequencing, supported by ELISA, immunohistochemistry, western blot, immunofluorescence, flow cytometry, and cell co-culture.

What key signaling pathway links macrophages and fibroblasts in the results?

CLS enhanced macrophage–fibroblast intercellular communication by activating the JAK2/STAT3 signaling pathway, promoting migration ability of mouse skin fibroblasts.

Sep 30, 2026

Uploaded by Stanley