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

Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula Tangtang He, Kewei Wang, Ruiwen Mo, Juntong Guo, Bin Jiang, Ruoyu mu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6830339/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted 8 You are reading this latest preprint version 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, 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. postoperative wound Traditional Chinese medicine Single-cell transcriptomics cell interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Anal fistula is one of the most common and frequently occurring diseases in the anorectal department. The pathogenesis is still unclear, and it has very few well-identified influence factors [1]. Nowadays, the hypotheses such as immune abnormalities [2], an obstruction of anal crypt [3], and bacterial translocation have been proposed [4,5].There are also studies indicating a significant correlation between the anal fistula and diet, and daily routine [6]. In clinical settings, patients often experience symptoms such as anal pus discharge, fistula defecation, and pain [7]. The commonly used treatment for anal fistula is surgical removal of the fistula, resulting in the formation of a wound. However, due to the special location of the affected area, the wound is chronic, and there is a strong risk of infection [8], as well as a high recurrence rate after anal fistula surgery [9]. It will be a heavy burden on the patient individual and the whole medical system. Consequently, promoting wound healing plays an essential role in the treatment of anal fistula [10]. The regulation of macrophage and fibroblast functions is the core strategy for wound healing. Macrophages in the early stage engulf pathogens and necrotic tissues, regulate inflammatory responses, and secrete related factors that not only promote angiogenesis, but also activate fibroblast proliferation [11]. During the proliferation phase, they migrate extensively to the wound, synthesize extracellular matrix such as collagen, and differentiate into myofibroblasts to mediate wound contraction, ultimately promoting tissue remodeling and barrier function recovery together [12]. Targeting the signaling pathways of both (such as TGF-β/Smad, Wnt/β-catenin) or intercellular interactions can optimize the repair process, reduce complications, and provide a key entry point for the treatment of wounds [13]. Puffballs are a class of mushrooms that are found worldwide. Some species of puffball are edible when they are fresh, and other species, such as Calvatia lilacina, have been used as traditional medicine worldwide. Calvatia lilacina spores (CLS) used as traditional Chinese medicine (TCM) have been proven to be used for the treatment of hemostasis, throat pain and cough [14]. Historical literature used its powder to treat various wounds, and it is mostly used as wound dressings, because dry, mature spores have a positive effect on wound healing[15,16] And it had been confirmed that puffballs promoted the healing of diabetes ulcers by balancing oxidative stress, accelerating angiogenesis and regulating wound microbiota[17,18]. In addition, the CLS extracts had also been shown to inhibit cancer cell proliferation [19].The CLS has spiky and hollow structure which gives it strong adsorption ability [20]. Therefore, CLS has great potential in the treatment of anal fistula wounds due to its diverse pharmacological effects and unique structures. In the current study sought to evaluate the molecular mechanism of CLS on anal fistula wound. In addition, the study has constructed single-cell transcription maps of granulation tissue from control and treatment groups. Single-cell RNA sequencing analysis is enabling the identification of cell-type specific changes of gene expression and high-throughput quantification with individual cells between two groups [21]. Notably, a novel approach to unraveling the the intercellular heterogeneity of anal fistula granulation tissue and analysis of cell-cell communication using CellChat [22], exploring the key mechanisms of promoting wound healing based on the complex components and special structure of traditional Chinese medicine CLS.. Method And Materials Reagents and antibodies TNF-α, COL1A1, VEGF Elisa Kit (EK182, EK183, EK1C01, Multi Sciences, China); WASF3 Antibody (67620-1-lg, Proteintech, China); α-SMA (19245s, CST, USA); CD14 Antibody (ab183322, abcam, Britain ); CD11b Antibody (ab133357, abcam, Britain); IL-6 flow Antibody (562050, BD Pharmingen, USA); JAK2 (WL02188, Wanleibio, China); p-JAK2 (WL02997, Wanleibio, China); STAT3 (WL03207, Wanleibio, China); p-STAT3 (WL06214, Wanleibio, China); Preparation of CLS, composition analysis and characterization Calvatia lilacina (Mont.et Berk.) Lloyd was purchased from (blind for peer review) and authenticated by (blind for peer review) based on the Pharmacopoeia of the People’s Republic of China (Part I, 2020 version). After removing the shell and impurities, the obtained spore powder was sieved through a filter screen and then subjected to radiation sterilization for subsequent experiments. Due to its complex composition, we determined the composition of CLS via UV, GC, HPLC and UPLC-MS. In addition, we conducted the CLS morphology related indices and structural characterization (fig.1, Supplementary Table 3). Sample collection The study enrolled a total of twenty patients who received anal surgery in Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, and randomly divided into the control group and the treatment group. The control group, patients on the second day after surgery were routinely disinfected with 0.5% iodine cotton balls on the wound, and sterilized Vaseline oil gauze was applied to the wound, which was covered and fixed with sterile dry gauze outside. On the basis of the control group, the treatment group evenly applied 0.04 g/cm 2 of CLS onto sterilized vaseline oil gauze and covered the wound. The dressing was changed twice a day, and collected granulation tissue at different time. The protocol was approved by the Ethics Committee of the hospital (KY2023302). All enrolled participants provided written informed consent to participate in the study under the principles of Declaration Helsinki. In addition, written informed consents were obtained from all the participants before study procedures began, and participants were free to withdraw from the study at any stage. Enzyme-Linked Immunosorbent Assays According to the instructions of the ELISA kit, added 100 μL of detection antibody to the standard and sample wells, except for the blank well, incubated at 37 °C for 1 hour. Then, added 250-300 μL of washing solution to each well for 1 minute, discarded the liquid. Subsequently, each well added color reagent and incubated in the dark for 15 minutes. Afterwards, added 50 µL of termination solution to the plate. Finally, the OD value was determined by the enzyme marker at 450 nm. Immunohistochemistry staining of granulation tissues Obtaining granulation tissue slides consistent with the above method for subsequent detection of CD14 and CD11b. After dewaxed and dehydrated the tissue slides, performed antigen repair at high temperatures, they were blocked with serum, then incubated overnight at 4 ℃ with primary antibody. Incubating secondary antibodies corresponding the species. Stained the cell nucleus with hematoxylin, slides dehydrated, sealed and then examined with a microscope. The expression of specific proteins be analyzed by detecting the mean gray value of immunohistochemistry photographs. Tissue dissociation and preparation for single-cell RNA sequencing The fresh tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron) on ice after the surgery within 30 mins. The specimens were washed with Hanks Balanced Salt Solution (HBSS) for three times, minced into small pieces, and then digested with 3 mL sCelLiveTM Tissue Dissociation Solution (Singleron) by Singleron PythoN™ Tissue Dissociation System at 37 °C for 15 min. The cell suspension was collected and filtered through a 40-micron sterile strainer. Afterwards, the GEXSCOPE® red blood cell lysis buffer (RCLB, Singleron) was added, and the mixture [Cell: RCLB=1:2 (volume ratio)] was incubated at room temperature for 5-8 min to remove red blood cells. The mixture was then centrifuged at 300 × g 4 ℃ for 5 mins to remove supernatant and suspended softly with PBS. RT & Amplification & Library Construction Single-cell suspensions (2×10 5 cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) (Ref). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads. Primary analysis of raw read data Raw reads were processed to generate gene expression profiles using CeleScope V3.0.1 (Singleron Biotechnologies) with default parameters. Briefly, Barcodes and UMIs were extracted from R1 reads and corrected. Adapter sequences and poly A tails were trimmed from R2 reads and the trimmed R2 reads were aligned against the GRCh38 (hg38) transcriptome using STAR (v2.6.1b). Uniquely mapped reads were then assigned to genes with FeatureCounts(v2.0.1). Successfully Assigned Reads with the same cell barcode, UMI and gene were grouped together to generate the gene expression matrix for further analysis. Quality control, dimension-reduction and clustering Scanpy v1.8.1 was used for quality control, dimensionality reduction and clustering under Python 3.7. For each sample dataset, we filtered expression matrix by the following criteria: 1) cells with gene count less than 200 or with top 2% gene count were excluded; 2) cells with top 2% UMI count were excluded; 3) cells with mitochondrial content > 10% were excluded; 4) genes expressed in less than 5 cells were excluded. After filtering, 81068 cells were retained for the downstream analyses, with on average 1004 genes and 2183 UMIs per cell. The raw count matrix was normalized by total counts per cell and logarithmically transformed into normalized data matrix. Top 2000 variable genes were selected by setting flavor = ‘seurat’. Cells were separated into 22 clusters by using Louvain algorithm and setting resolution parameter at 1.2. Cell clusters were visualized by using Uniform Manifold Approximation and Projection (UMAP). Batch effect between samples was removed by Harmony v1.0. Cell-cell Interaction Analysis CellChat (version 0.0.2) was used to analyze the intercellular communication networks from scRNA-seq data. A CellChat object was created using the R package process. Cell information was added into the meta slot of the object. The ligand-receptor interaction database was set, and the matching receptor inference calculation was performed. Pseudotime Trajectory Analysis: monocle2 Cell differentiation trajectory of Fibroblasts subtypes was reconstructed with the Monocle2 v 2.10.0 (ref). For constructing the trajectory, top 2000 highly variable genes were selected by Seurat(v3.1.2) FindVairableFeatures() and dimension-reduction was performed by DDRTree(). The trajectory was visualized by plot_cell_trajectory() function in Monocle2. UCell Gene Set Scoring Gene set scoring was performed using the R package UCell v 1.1.0. UCell scores are based on the Mann-Whitney U statistic by ranking query genes’ in order of their expression levels in individual cells. Because UCell is a rank-based scoring method, it is suitable to be used in large datasets containing multiple samples and batches. Culture and intervention of THP-1 Cells were incubated in a 5% CO 2 cell culture incubator at 37 °C. Cell selection during logarithmic growth phase, added PMA (500nM) and induced for 24 hours in a 12 well plate with a concentration of 10 6 cells/ml. Then the control group was given single culture, while the CLS group was incubated with particles at different dosages 0.04, 0.08 particles/μm 2 , the SiO 2 group was 0.08 particles/μm 2 for 24 h. Then cells and culture supernatant were collected for subsequent experiments. Culture of MSF with conditioned medium and transewell migration assay The supernatant of the four groups of macrophages were mixed with culture medium, to form conditioned medium. After culturing 24 hours, proteins were collected for subsequent detection. Meanwhile 500 ul of the conditioned medium took to the lower chamber, 100ul of MSF suspension added to the transwell chamber at a cell concentration of 2×10 5 cells/ml for 24 hours, then fixed with methanol for 15 minutes, and stained the cells with 1% crystal violet for 15 minutes. After washing with PBS, wiped off the cells in the small chamber with a cotton swab, and finally took photos and performed quantitative analysis. Flow Cytometry Adherent cells were incubated with trypsin EDTA at 37 °C for 1 minutes and gently dislodged by pipetting for cell harvest, washed the cells twice with PBS buffer. Then 1 μl of the BD Horizo Fixable Viability Stain 660 Stock Solution was added for 10 mintunites. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block. The cells were fixed, permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody at 4 °C for 30 minutes. Finally, the cells were centrifuged, the supernatant was discarded, washed three times with PBS, and resuspended in 250 μl PBS for fluorescence intensity detection. Immunofluorescence staining of granulation tissues The granulation tissue slides for antigen repair, then blocked them with BSA. Incubating overnight with WASF3 primary antibody (proteintech, 67620-1-Ig, 1:500), followed by secondary antibody incubation and DAPI staining. Then added anti-fluorescence quencher and sealed the slide for fluorescence microscopic examination. The gray value of each pixel represents the fluorescence intensity of the point, and the mean gray value of a certain fluorescence channel on the immunofluorescence photograph can be quantified using ImageJ. Western Blot The conditioned medium formed by macrophage culture was added to MSF and cultured for 24 hours. Cell proteins were extracted with RIPA lysis buffer and separated by SDS polyacrylamide gel electrophoresis. After electrophoresis, the protein were transferred onto the PVDF membrane, blocked it with a blocking solution for 1 hour, and then washed it with TBST. Add the first antibody and incubated overnight at 4 ℃. The next day, the membrane was washed with TBST, added the second antibody corresponding to the first antibody, incubated the mixture at room temperature for 1 hour, then the protein was exposed to color development by enhanced chemiluminescence (ECL) reagent, and used ImageJ to calculate the grayscale value. Statistical analysis The statistical analysis was made by SPSS 22.0 software. Descriptive statistics were reported as mean ± SD. A P-value of less than 0.05 was considered significant. Results Component analysis and structural characterization In this study, the results showed that total protein (38.74 ± 6.01 mg/g), total steroids (15.15 ± 0.45 mg/g), total polysaccharides (11.03 ± 0.54 mg/g) and total polyphenols (8.53 ± 0.32 mg/g) in CLS (Table 1, supplementary results S1-4). For fatty acid analysis, GC-2010 plus was used. The saturated fatty acid content was analyzed as 0.2007% (C16:0 methyl palm 0.1270%, C18:0 stearic acid methyl ester 0.0616%, C24:0 methyl tetracosanoate 0.0121%). Monounsaturated fatty acids and polyunsaturated fatty acids were analyzed as 0.3050% and 0.0815% (C18:1n-9c methy1-9-oleic acid methyl ester, C18:2n-6c linoleic acid methyl ester) (Supplementary Table 1, S7). Cysteine (0.1465%) and valine (0.0803%) were determined as major components of amino acids in the CLS (Supplementary Table 2). UPLC-MS and HPLC detected the representative monomeric components ergosterol (0.142%) and ergosterone (0.023%) in CLS (Supplementary results S5, S6). The spore powder has a uniform particle size and is spiky (Fig.1). While in the study observed the micromeritics characteristics of CLS (Supplementary Table 3). Table 1 Summary of the contents of total steroids, total polyphenols, total polysaccharides, and protein in the CLS (mean ± SD, n = 3) contents total protein（contain amino acids, mg/g） total steroids（mg/g） total polysaccharides（mg/g） total polyphenols（mg/g） Calvatia lilacina 26.32 ± 0.71 15.15 ± 0.45 11.03 ± 0.54 8.53 ± 0.32 CLS promoted wound healing in anal fistula by reducing TNF-α-driven inflammation and promoting Collagen I Synthesis, angiogenesis To verify the role of CLS in collagen generation, angiogenesis, and inflammation, we detected the expression levels of TNF-α, VEGF, and Collagen I in granulation tissue by ELISA. As shown in the results (Fig 2A-C), CLS significantly inhibited the levels of inflammatory cytokine TNF-α with the most significant effect observed on day 14. Meanwhile, CLS also had the effect of promoting collagen generation related cytokine Collagen I levels, and had a slight effect on the expression of vascular factor VEGF. We further performed immunohistochemical staining on granulation tissue on days 7 and 14 to observe the status of inflammation related cells (Fig 2D). Consistent with the ELISA results, there was a significant decrease in the positive expression rates of CD14 and CD11b on day 14. As shown by histological results, positive high expression was observed on days 7 and 14, indicating that immune and inflammation related cells are the main functional groups in the early stage of anal fistula wound healing (Fig 2E, F). (A-C) ELISA detection of TNF-α, VEGF, and Collagen I expression levels in granulation tissue; (D) Immunohistochemical staining of CD14 and CD11b; (E,F) Immunohistochemical quantitative analysis. * P < 0.05, ** P < 0.01, *** P < 0.001,**** P 0.05) vs.the control group; n = 10. Single-cell atlas of anal fistula granulation tissue revealed macrophages were the main cell population of MPs To obtain a better understanding of the effect of CLS on the makeup of cell types and transcriptional patterns in granulation tissue, we created single-cell atlases for the granulation tissue. GEXSCOPE® was used to convert single-cell suspensions of the scRNA-seq samples to barcoded libraries. A total of 81068 viable cells were obtained after quality control for subsequent analyses. To identify each cell type, we used the Seurat v2.3.4 packages to perform clustering on the sequencing data, then annotated each cell type based on the expression levels of canonical cell-type-specific markers. In general, we identified 22 cell clusters in granulation tissue that could be classified into eight major cell types: ECs (identified by PECAM1, VMF, CDH5), Fibroblasts (identified by COL1A1, DCN, COL1A2), MuralCells (identified by ACTA2, CALD1, MCAM), BCells (CD79A, MS4A1, CD19), TCells (CD3D, CD3E, CD3G), Neutrophils (FCGR3B, S100A9, S100A8), MPs (CSF1R, CD14, CD68), pDCs (IL3RA, CLEC4C, LILRA4).(Fig.3 A-C). In summary, we developed a tissue characterization of the cellular diversity of the granulation tissue and established a comprehensive framework. Single-cell results showed a significant reduction in the number of macrophages and monocytes in the treatment group, indicating that CLS has a certain anti-inflammatory effect in the early stage (Fig. 3D-F). Therefore, we focused on macrophages as the main cell population for further dimensionality reduction analysis. CLS induced an IL-6 + macrophage subset with coordinated expression of IL-6 and CXCL-8 during wound healing A total of 13023 viable macrophages were obtained to recluster on the sequencing data, and we identified 7 cell clusters in macrophages. The results showed that there was a certain increase in the number of IL-6 + macrophages, meanwhile IL-6 + macrophages regulate inflammation function significantly stronger than other subgroups (Fig.4 A-C). Research report suggested that the coexistence of IL-6 and CXCL-8 in macrophages could activate the WASF3 [23]. Therefore, we further compared the expression of IL-6 and CXCL-8 in the macrophage, the resulted the IL-6 and CXCL-8 had co-high expression only in IL-6 + macrophages (Fig.4 D). This also suggested that IL-6 + macrophages may be a special cell population after CLS intervention. The results further showed that the expression of IL-6 and CXCL-8 was significantly increasing in the treatment group (Fig.4 E,F). CLS enhanced macrophages and fibroblasts communication through IL-6R and CXCL-8R1 receptors CLS adhered to macrophages and spike SiO 2 also had the effect of adhering to cells. The flow cytometry results showed that CLS increased the number of IL-6 + macrophages, and the SiO 2 group also had a certain effect (Fig.5 A,B). This result reminded us that the spike structure of CLS could stimulate immune activity that consistent with previous reports [24]. The elisa results showed that CLS significantly increased the expression levels of IL-6 and CXCL-8 (Fig.5 C,D). In addition, studies had shown that ergosterol can activate the signal expression of AP-1 protein and AP-1 was required for CXCL-8 activation[25]. Meanwhile, we found CLS enhanced the strength of cell interaction between macrophages and fibroblasts (Fig.5 E, F). The gene sequence of mouse CXCL-1 had high homology with human CXCL-8 at the amino acid level. The elisa results showed that CLS significantly increased the expression levels of IL-6 and CXCL-1 (supplementary results S8). IL-6 and CXCL8 activated downstream signaling pathways by binding to receptors, the results showed that there were multiple receptors for IL-6 + macrophages in fibroblasts, among which IL-6R_IL-6ST and CXCL-8R1 were the main functional receptors. CLS significantly enhanced the inter population communication intensity of IL-6 and CXCL-8 signaling pathways (Fig.5 G,H). CLS promoted macrophages to co-upregulate IL-6 and CXCL-8, thereby accelerating downstream WASF3-mediated wound closure During the process of wound healing, fibroblasts are the main cell group to regulate wound closure [26]. we subset fibroblasts for reintegrating and subclustering. Altogether, 15679 cells contributed to 7 clusters (Fig.6 A,B). We found that the expression of ACTR2, ACTR3 and WASF3 in the treatment group was significantly higher than the control group (Fig.6 C, supplementary results S9 D,E). Our further immunofluorescence results showed that after CLS intervention, the expression levels of α-SMA and WASF3 increased in granulation tissue, consistent with single-cell detection data (Fig.6 D,F). The gene expression pattern across pseudotime showed that the expression curves of ACTR2 and ACTR3 were relatively smoother in the treatment group than in the control group. Meanwhile, the overall expression level of ACTR2 in the treatment group was higher than that in the control group (Fig.6 G,H). ACTR2, ACTR3 are components of the Arp2/3 complex, which is involved in regulating various cellular processes. WASF3 plays a critical role in regulating actin cytoskeleton dynamics, and previous study showed that cell migration through increased the expression of WASF3 to recruit the Arp2/3 complex, increasing the formation of dendritic protrusions and thus driving cell migration [23]. Therefore, we reasonably speculated that CLS enhanced the expression of IL-6 and CXCL-8 in the IL-6 + macrophage, thereby increasing the expression level of WASF3 in fibroblasts and accelerating wound closure. The conditioned medium derived from macrophage cultures stimulated MSF migration by activating the JAK2/STAT3 signaling pathway The transwell experiment results showed that the conditioned medium for macrophage culture promoted the migration of MSF (Fig.7. A-B). The JAK2/STAT3 signaling pathway regulates multiple cell functions, such as proliferation, differentiation, migration and immune function. Several studies have confirmed that activated JAK2/STAT3 signaling pathway can promote wound healing [27]. Therefore, we next investigated whether the migration effect of CLS involved the JAK2/STAT3 pathway. To this point, our data indicated that CLS activated the JAK2/STAT3 pathway. As a classic prosurvival signaling axis, the JAK2/STAT3 pathway possesses the capacity to promote cell migration. In addition, the western blot results showed that CLS promoted the phosphorylation of JAK2/STAT3 (Fig.7 C-D), thereby promoting the expression of downstream proteins WASF3 to accelerate cell migration. Discussion Due to the complexity of the pathogenesis of anal fistula, there is a huge challenge in promoting wound healing after surgical treatment. In clinical, there are no specific drugs for anal fistula wounds and most of them are only given conventional drugs after debridement to promote healing [28]. Fumigation, rubbing and external washing are common treatment method, which greatly increases the pain of dressing changes for patients. In addition, due to the special location of the anal fistula wound which the treatment effect is often poor [29]. Puffballs can promote the healing of diabetes ulcers through multiple components and targets, which is confirmed by our previous research [17]. In this study, we demonstrated that the CLS offered a safe and well-tolerated treatment option for anal fistula wounds. Spore powder is amazing substance in nature that plays a fundamental role in the plants' reproduction. Spore powder is very uniform in size and shape. According to their particular structure, spore powder have essential characteristics such as acid and alkyl resistance, mucosal adhesion, and great stability [30]. Based on this, spore powder is increasingly being used for disease treatment. Spore powder have the usability in the targeting of the immune system and causing allergies, stimulating inflammatory responses in the immune system based on the related mechanisms. Ganoderma lucidum spore powder (GLSP) could regulat CD4 + CD8 + T cell ratio and macrophage function, especially the expression of PD-1 in TIME [31]. Inonotus hispidus spore powder (IHS) influenced T cells in ApcMin/ + mice by regulating the IL-5, -6, and -10 levels, thus suppressing tumor development [32]. Spore-mimetic metal–organic frameworks (MOFs) had greater cell attachment and faster and more efficient phagocytosis in cells, which resulted in greater expressions of pro-inflammatory cytokines [33]. Excessive inflammation and impaired fibroblast function are important reasons why wounds are difficult to healing [34]. Inflammatory and immune related cells play an important role as the main functional cell groups in the early stage of wound healing. And CLS increased the expression level of IL-6 and CXCL-8 in the IL-6 + macrophage. Previous research had shown that ERK3-mediated CXCL-8 secretion is critical for the chemotaxis of inflammatory cells to the epithelium, locally used recombinant protein accelerated wound healing[35,36]. And furthermore, the study demonstrated that IL-6 signaling was required for chemotaxis and promoted generation of a new subset of tissue repair macrophage[37,38]. Importantly, IL-6 and CXCL-8 promoted the migration of MCF10A cells and HER2-positive breast cancer cells [39]. These suggested IL-6 and CXCL-8 could promote migration in the neighboring cell and influence the migratory behavior of the entire wound microenvironment. Palmitate in fatty acids had the ability to promote IL-6 via coordinated acetylation of H3K9/H3K18, p300, and RNA Polymerase II [40].Steroids are crucial components of anti-inflammatory effects by neutralizing pro-inflammatory cytokine production in human monocytes, and are also found in CLS constituents such as ergosterol and ergosterone [41]. CLS simultaneously regulated the function of IL-6 + macrophages that enhanced mutual communication with fibroblasts to promote wound contraction. Wound contraction is a process that occurs during the healing of an open wound. Modified fibroblasts were first observed in the granulation tissue of healing wounds which led to the suggestion that these cells have a role in the production of the contractile force that is involved in this process. The myofibroblast had a role in the synthesis of ECM and in force generation, which resulted in ECM reorganization and wound contraction [42]. Related research showed that polysaccharides effectively promoted the formation of granulation tissue, collagen deposition [43]. Polyphenols inhibited collagenase inhibitory activities, which play a critical role in preventing collagen breakdown [44]. A recent study observed that saliva promoted fibroblast migration by increasing the secretion of IL-6 and CXCL-8 [45]. Consistent with this, our research firstly found that CLS promoted signal exchange between fibroblasts and macrophages, which increased the expression level of IL-6 and CXCL-8 in the IL-6 + macrophage, then promoted the expression of WASF3 in fibroblasts via JAK2/STAT3 pathway, thereby promoting wound contraction and accelerating wound healing. Our metabolic analysis showed that CLS significantly enhances lysine degradation, glycosaminoglycan biosynthesis-keratin sulfate, D-glutamine and D-glutamate metabolism, and selenocompound metabolism had a significant impact (supplementary results S10 A-C). Lysine degradation was an important metabolic process and research had confirmed that lysine was able to synthesize glutamate [46]. More recent work had indicated that fibroblasts were the primary producers of collagen and synthesize proline from glutamate rather than arginine. Glutamate is known to be the core of almost all metabolic pathways required at different stages of inflammatory wounds. We have already demonstrated in our previous research that Glutamate promoted the proliferation of HaCaT cells, migration of MSFs and macrophage polarization [18]. The fibroblasts synthesize collagen and GAGs which were components of the ECM. Myofibroblasts produced keratansulfate which assisted in collagen polymerization [47]. This study had several limitations. The treatment group had the effect of promoting angiogenesis and collagen regeneration on the 14th day. Previous research showed that CXCL-8 stimulated angiogenesis in the skin [36]. The causal relationship and specific mechanism still need to be clarified. Conclusion In this paper, we have applied single-cell RNA sequencing to deeply explore that CLS reduced the number of monocytes to decrease the inflammatory state of the wound, while increased the communication between IL6 + macrophages and fibroblasts, thus accelerated the transition from the inflammatory phase to the proliferative phase of the wound. These novel results may also guide future research on TCM in anal fistula wound. Meanwhile, these findings provided key information and guidance for further investigation on the clinical applications of CLS. Abbreviations ACTR2 Actin-Related Protein 2 ACTR3 Actin-Related Protein 3 GAGs Glycosaminoglycans CLS Calvatia lilacina spore ECs Endothelial cells GC Gas Chromatography HE Hematoxylin and eosin HPLC High-performance liquid chromatography IF Immunofluorescence IHC Immunohistochemistry MPs Mononuclear phagocyte system Q-TOF-MS/MS Quadrupole Time-of-Flight Mass Spectrometry SEM Scanning Electron Microscope TCM Traditional Chinese Medicine TNF-α Tumor Necrosis Factor-alpha U-MAP Uniform manifold approximation and projection UPLC-MS Ultra Performance Liquid Chromatography-Mass Spectrometry UV Ultraviolet VEGF Vascular endothelial growth factor WASF3 Wiskott-Aldrich syndrome protein family member 3 Declarations Author contributions Tangtang He wrote the paper draft. Jun Chen, Lifeng Zhu corrected the draft. Kewei Wang, Bin Jiang, Ruoyu Mu, Wen Min supervised the experimentators. Tangtang He, Ruiwen Mo, Juntong Guo performed the experiments. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. Funding This work was financially supported by the National Natural Science Foundation of China (No. 82474200; No. 82173994), Leading Program of Traditional Chinese Medicine First-class Discipline (ZYXYL2024-014), Jiangsu Province Traditional Chinese Medicine Development Project (MS2022006), Natural Science Research Project of the Jiangsu Higher Education Institutions of China (No. 22KJA360002), Priority Academic Program Development of Jiangsu Higher Education Institutions, Nanjing Traditional Chinese Medicine Youth Talent Training Program (ZYQ20042), the Graduate Student Scientific Research Innovation Projects in Jiangsu Province (KYCX24_2284). Data availability The datasets analyzed during this study are available from the corresponding author on reasonable request. Human ethics and consent to participate This study was performed following the standard of International Coordinating Committee on Global Partnerships and the revised edition of the Declaration of Helsinki. The present study was approved by the Ethics Committee of Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine (Ethic code: KY2023302).. It was registered in ITMCTR (ITMCTR20250001078). Every participant endorsed informed consent voluntarily. Consent for publication We, the undersigned authors of the manuscript titled “[Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula]”submitted to “Chinese medicine”, unconditionally consent to its publication in this journal, understanding it will be the publisher’s property and made public per journal policies. We confirm all content is original, not previously published except as disclosed, and we’ve obtained permissions for non‑own copyrighted materials. When the manuscript involves human/animal subjects or sensitive info, we’ve secured ethical approvals and protected all rights and privacy as per laws. We recognize the publisher’s right to make editorial changes for style, format, and ethics compliance and will fully cooperate, providing any needed additional info or clarifications. Competing Interest There are no potential conflicts of interest relevant to this article. References Assaraf J, Lambrescak E, Lefèvre JH, De Parades V, Bourguignon J, Etienney I, et al. Increased Long-term Risk of Anal Fistula After Proctologic Surgery: A Case-Control Study. Ann Coloproctol. 2021;37:90–3. Fitzpatrick DP, Kealey C, Brady D, Goodman M, Gately N. Treatments for the amelioration of persistent factors in complex anal fistula. Biotechnol Lett. 2022;44:23–31. Hamadani A, Haigh PI, Liu I-LA, Abbas MA. Who is at risk for developing chronic anal fistula or recurrent anal sepsis after initial perianal abscess? Dis Colon Rectum. 2009;52:217–21. Jaiswal P, Sharma S, Pratap A, Ansari M, Shukla VK, Basu S, et al. Significant presence of biofilm-producing gut-derived bacteria in anal fistula of chronic duration. Int Wound J. 2021;18:519–24. Sugrue J, Nordenstam J, Abcarian H, Bartholomew A, Schwartz JL, Mellgren A, et al. Pathogenesis and persistence of cryptoglandular anal fistula: a systematic review. Tech Coloproctol. 2017;21:425–32. Devaraj B, Khabassi S, Cosman BC. Recent smoking is a risk factor for anal abscess and fistula. Dis Colon Rectum. 2011;54:681–5. Owen HA, Buchanan GN, Schizas A, Cohen R, Williams AB. Quality of life with anal fistula. Ann R Coll Surg Engl. 2016;98:334–8. Kang J, Liu Y, Peng S, Tang X, Liu L, Xie Z, et al. Efficacy and safety of traditional Chinese medicine external washing in the treatment of postoperative wound of diabetes complicated with anal fistula: Study protocol of a randomized, double-blind, placebo-controlled, multi-center clinical trial. Front Pharmacol. 2022;13:938270. Zubaidi AM. Anal fistula. Past and present. Saudi Med J. 2014;35:937–44. Limura E, Giordano P. Modern management of anal fistula. World J Gastroenterol. 2015;21:12–20. Sharifiaghdam M, Shaabani E, Faridi-Majidi R, De Smedt SC, Braeckmans K, Fraire JC. Macrophages as a therapeutic target to promote diabetic wound healing. Mol Ther. 2022;30:2891–908. Younesi FS, Miller AE, Barker TH, Rossi FMV, Hinz B. Fibroblast and myofibroblast activation in normal tissue repair and fibrosis. Nat Rev Mol Cell Biol. 2024;25:617–38. Li S, Ding X, Zhang H, Ding Y, Tan Q. IL-25 improves diabetic wound healing through stimulating M2 macrophage polarization and fibroblast activation. Int Immunopharmacol. 2022;106:108605. Tsay J-G, Chung K-T, Yeh C-H, Chen W-L, Chen C-H, Lin MH-C, et al. Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells. J Agric Food Chem. 2009;57:1579–88. Cicha-Jeleń M, Muszynska B, Kala K, Sulkowska-Ziaja K. Medicinal Potential of the Giant Puffball Mushroom Calvatia gigantea (Agaricomycetes): A Review. Int J Med Mushrooms. 2024;26:13–25. Lee D, Lee S, Jang YS, Ryoo R, Kim JK, Kang KS, et al. N,N-Dimethyl-anthranilic Acid from Calvatia nipponica Mushroom Fruiting Bodies Induces Apoptotic Effects on MDA-MB-231 Human Breast Cancer Cells. Nutrients. 2023;15:3091. He T, Sun P, Liu B, Wan S, Fang P, Chen J, et al. Puffball spores improve wound healing in a diabetic rat model. Front Endocrinol (Lausanne). 2022;13:942549. Ding X, Yang C, Li Y, He T, Xu Y, Cheng X, et al. Reshaped commensal wound microbiome via topical application of Calvatia gigantea extract contributes to faster diabetic wound healing. Burns Trauma. 2024;12:tkae037. Zeng Q, Singh R, Ye Y, Cheng S, Fan C, Zeng Q. Calvatia Lilacina Extracts Exert Anti-Breast-Cancer Bioactivity through the Apoptosis Induction Dependent on Mitochondrial Reactive Oxygen Species and Caspase Activation. Nutr Cancer. 2022;74:1058–70. Wang H, Lu Z, Xu Y, Kong L, Shi J, Liu Y, et al. Genome-wide characterization of SPL family in Medicago truncatula reveals the novel roles of miR156/SPL module in spiky pod development. BMC Genomics. 2019;20:552. Wu C, Tang H, Cui X, Li N, Fei J, Ge H, et al. A single-cell profile reveals the transcriptional regulation responded for Abelmoschus manihot (L.) treatment in diabetic kidney disease. Phytomedicine. 2024;130:155642. Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan C-H, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021;12:1088. Jayatilaka H, Tyle P, Chen JJ, Kwak M, Ju J, Kim HJ, et al. Synergistic IL-6 and IL-8 paracrine signalling pathway infers a strategy to inhibit tumour cell migration. Nat Commun. 2017;8:15584. Chen H-J, Hang T, Yang C, Liu D, Su C, Xiao S, et al. Functionalized Spiky Particles for Intracellular Biomolecular Delivery. ACS Cent Sci. 2019;5:960–9. Xu Y, Lin L, Zheng H, Xu S, Hong X, Cai T, et al. Protective effect of Amauroderma rugosum ethanol extract and its primary bioactive compound, ergosterol, against acute gastric ulcers based on LXR-mediated gastric mucus secretions. Phytomedicine. 2024;123:155236. Su J, Wei Q, Ma K, Wang Y, Hu W, Meng H, et al. P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/ CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts. Burns Trauma. 2023;11:tkad010. Cao W, Meng X, Cao F, Wang J, Yang M. Exosomes derived from platelet-rich plasma promote diabetic wound healing via the JAK2/STAT3 pathway. iScience. 2023;26:108236. Simpson JA, Banerjea A, Scholefield JH. Management of anal fistula. BMJ. 2012;345:e6705. Pingping M, Wenzhe F, Peng S, Wenxiu Z, Yu Z. Clinical research progress in Traditional Chinese Medicine in treating wound healing after anal fistula surgery. J Tradit Chin Med. 2023;43:1047–54. Ourani-Pourdashti S, Azadi A. Pollens in therapeutic/diagnostic systems and immune system targeting. J Control Release. 2021;340:308–17. Wu T-T, Chen Y-Y, Yuan Z-C, Yang G-W, Zhang G-L. Synergy of de-walled Ganoderma Lucidum spore powder (GLSP) on targeted therapy in advanced non-squamous non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutant: protocol for a randomized, double-blind, placebo-controlled study. BMC Complement Med Ther. 2024;24:125. Yang H, Li S, Qu Y, Li L, Li Y, Wang D. Anti-Colorectal Cancer Effects of Inonotus hispidus (Bull.: Fr.) P. Karst. Spore Powder through Regulation of Gut Microbiota-Mediated JAK/STAT Signaling. Nutrients. 2022;14:3299. Chen P-M, Pan W-Y, Luo P-K, Phung HN, Liu Y-M, Chiang M-C, et al. Pollen-Mimetic Metal-Organic Frameworks with Tunable Spike-Like Nanostructures That Promote Cell Interactions to Improve Antigen-Specific Humoral Immunity. ACS Nano. 2021;15:7596–607. Wu B, Pan W, Luo S, Luo X, Zhao Y, Xiu Q, et al. Turmeric-Derived Nanoparticles Functionalized Aerogel Regulates Multicellular Networks to Promote Diabetic Wound Healing. Adv Sci (Weinh). 2024;11:e2307630. Bogucka K, Pompaiah M, Marini F, Binder H, Harms G, Kaulich M, et al. ERK3/MAPK6 controls IL-8 production and chemotaxis. Elife. 2020;9:e52511. Cambier S, Gouwy M, Proost P. The chemokines CXCL8 and CXCL12: molecular and functional properties, role in disease and efforts towards pharmacological intervention. Cell Mol Immunol. 2023;20:217–51. Matsumura F, Polz R, Singh S, Matsumura A, Scheller J, Yamashiro S. Investigation of Fascin1, a Marker of Mature Dendritic Cells, Reveals a New Role for IL-6 Signaling in CCR7-Mediated Chemotaxis. J Immunol. 2021;207:938–49. Li Z, Xiao J, Xu X, Li W, Zhong R, Qi L, et al. M-CSF, IL-6, and TGF-β promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery. Sci Adv. 2021;7:eabb6260. Kim D, Cooper JA, Helfman DM. Loss of myosin light chain kinase induces the cellular senescence associated secretory phenotype to promote breast epithelial cell migration. Sci Rep. 2024;14:25786. Kochumon S, Jacob T, Koshy M, Al-Rashed F, Sindhu S, Al-Ozairi E, et al. Palmitate Potentiates Lipopolysaccharide-Induced IL-6 Production via Coordinated Acetylation of H3K9/H3K18, p300, and RNA Polymerase II. J Immunol. 2022;209:731–41. Tada H, Kawahara K, Osawa H, Song L-T, Numazaki K, Kawai J, et al. Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes. Biochem Biophys Res Commun. 2022;636:1–9. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3:349–63. Li S, Xu W, Zhu W, Wang J, Shi J, Tang J, et al. Superior hemostatic and wound-healing properties of tetrastigma polysaccharide. Materials & Design. 2024;241:112967. Panichakul T, Ponnikorn S, Tupchiangmai W, Haritakun W, Srisanga K. Skin Anti-Aging Potential of Ipomoea pes-caprae Ethanolic Extracts on Promoting Cell Proliferation and Collagen Production in Human Fibroblasts (CCD-986sk Cells). Pharmaceuticals (Basel). 2022;15:969. Rodrigues Neves C, Buskermolen J, Roffel S, Waaijman T, Thon M, Veerman E, et al. Human saliva stimulates skin and oral wound healing in vitro. J Tissue Eng Regen Med. 2019;13:1079–92. Guo Y, Wu J, Wang M, Wang X, Jian Y, Yang C, et al. The Metabolite Saccharopine Impairs Neuronal Development by Inhibiting the Neurotrophic Function of Glucose-6-Phosphate Isomerase. J Neurosci. 2022;42:2631–46. Kosir MA, Quinn CC, Wang W, Tromp G. Matrix glycosaminoglycans in the growth phase of fibroblasts: more of the story in wound healing. J Surg Res. 2000;92:45–52. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Supplementaryresults.docx Cite Share Download PDF Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Revision requested 08 Oct, 2025 Reviews received at journal 27 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 18 Jun, 2025 Submission checks completed at journal 18 Jun, 2025 First submitted to journal 05 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6830339","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482095064,"identity":"dcacd233-f8fd-42ba-ab41-5d4a89622e80","order_by":0,"name":"Tangtang He","email":"","orcid":"","institution":"Jiangsu Provincial Engineering Research Center of Traditional Chinese Medicine External Medication Development and Application","correspondingAuthor":false,"prefix":"","firstName":"Tangtang","middleName":"","lastName":"He","suffix":""},{"id":482095065,"identity":"8979b11b-010c-419a-ae0d-120aeb97f17b","order_by":1,"name":"Kewei Wang","email":"","orcid":"","institution":"Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kewei","middleName":"","lastName":"Wang","suffix":""},{"id":482095066,"identity":"41bf8fcd-5510-4e24-942f-fdcc531d3438","order_by":2,"name":"Ruiwen Mo","email":"","orcid":"","institution":"Jiangsu Provincial Engineering Research Center of Traditional Chinese Medicine External Medication Development and Application","correspondingAuthor":false,"prefix":"","firstName":"Ruiwen","middleName":"","lastName":"Mo","suffix":""},{"id":482095067,"identity":"ddfec813-7e68-48db-950c-4810c3745523","order_by":3,"name":"Juntong Guo","email":"","orcid":"","institution":"Jiangsu Provincial Engineering Research Center of Traditional Chinese Medicine External Medication Development and Application","correspondingAuthor":false,"prefix":"","firstName":"Juntong","middleName":"","lastName":"Guo","suffix":""},{"id":482095068,"identity":"7720df6d-ff01-4909-b5e0-d5eed296da05","order_by":4,"name":"Bin Jiang","email":"","orcid":"","institution":"Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Jiang","suffix":""},{"id":482095069,"identity":"3f02bb4e-4e78-4c9b-b060-0472ddbce7bf","order_by":5,"name":"Ruoyu mu","email":"","orcid":"","institution":"Jiangsu Provincial Engineering Research Center of Traditional Chinese Medicine External Medication Development and Application","correspondingAuthor":false,"prefix":"","firstName":"Ruoyu","middleName":"","lastName":"mu","suffix":""},{"id":482095070,"identity":"2f7a7c6c-3c1d-40b7-bda5-544988bb5c86","order_by":6,"name":"Wen min","email":"","orcid":"","institution":"Department of Bone Injury of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"min","suffix":""},{"id":482095073,"identity":"a9f88c67-39b8-4982-b40a-24d100cdfc6f","order_by":7,"name":"Lifeng zhu","email":"","orcid":"","institution":"Key Laboratory of Drug Target and Drug for Degenerative Disease","correspondingAuthor":false,"prefix":"","firstName":"Lifeng","middleName":"","lastName":"zhu","suffix":""},{"id":482095077,"identity":"9e722a1b-a296-42cf-bb01-3986a994c7dd","order_by":8,"name":"Jun chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACCQiu39/e2PjwAylaGDfwHG42liBaCwNIi0R6mwAPMTr4Zzcfe2DZdofZXPJhG1C/nZxuAyFL7hxLN5Bse8ZmOTux7UEBQ7Kx2QECWgwkcswkJNsO8zDcTmw3kGA4kLiNsJb8byAtEgw3D7ZJ8BCnJYcNpMXA4AYjkVokbqSZSUicO5wg2ZMIDGQDIvzCPyP5mbRE2eEEfvbjDx9+qLCTI6gFBJgRMWhAhHIQYCQunYyCUTAKRsGIBQByq0BV33bkygAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangsu Provincial Engineering Research Center of Traditional Chinese Medicine External Medication Development and Application","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"chen","suffix":""}],"badges":[],"createdAt":"2025-06-05 14:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6830339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6830339/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-025-01293-w","type":"published","date":"2026-01-07T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86316718,"identity":"db067afd-bdef-4b30-b288-02ed6986f96c","added_by":"auto","created_at":"2025-07-09 09:03:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44590862,"visible":true,"origin":"","legend":"\u003cp\u003eCalvatia lilacina Structural characterization.(A) Calvatia lilacina spore powder; (B-C) SEM, particle size analyze observation of Calvatia lilacina spore powder.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/5ca6149e08b0c24b5abc2418.png"},{"id":86316719,"identity":"02568524-9aea-4f37-aaa2-4ac212258d36","added_by":"auto","created_at":"2025-07-09 09:03:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59559289,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of CLS on inflammation, vascular, and collagen at different time points.\u003c/p\u003e\n\u003cp\u003e(A-C) ELISA detection of TNF-α, VEGF, and Collagen I expression levels in granulation tissue; (D) Immunohistochemical staining of CD14 and CD11b; (E,F) Immunohistochemical quantitative analysis. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001,****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, and ns: not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) vs.the control group; n = 10.\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/6ba712fa2e3a1b1137d28e1d.png"},{"id":86316722,"identity":"cb60714f-e4da-4737-8de4-0fbda0969900","added_by":"auto","created_at":"2025-07-09 09:03:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28715090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eSingle-cell RNA-seq analysis of granulation tissue samples reveals the cellular landscape of anal fistula wound healing. (A) Overview of the scRNA-seq samples from granulation tissue; (B,C) Uniform Manifold Approximation and Projection (UMAP) plot for dimension reduction of all cells colored by their cell type/identity and example marker genes are highlighted, dark represents high expression; light represents low expression; (D) UMAP plot of different MPs colored by their cell type in two groups; (E,F) Bar charts of the proportion of different MPs clusters in two groups.\u003c/u\u003e\u003c/p\u003e","description":"","filename":"fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/3b3e105a4212fc5a67f52568.png"},{"id":86316717,"identity":"abca4a3e-4867-488a-b9d3-102a18969542","added_by":"auto","created_at":"2025-07-09 09:03:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20379223,"visible":true,"origin":"","legend":"\u003cp\u003eClustering and functional annotation of macrophage. (A) UMAP plot of different macrophages colored by their cell type; (B) Bar charts of the proportion of different macrophages clusters in two groups; (C) Boxplots of inflammatory by macrophages subtypes; (D) The expression of IL-6, CXCL-8 in macrophages subtypes; (E,F) Violin plot of IL-6, CXCL-8 distribution in IL-6\u003csup\u003e+\u003c/sup\u003e macrophages across 2 groups.\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/560f8292657c31e6c3c19ae2.png"},{"id":86316720,"identity":"d288e0f3-6870-42fe-8c58-fd3ab4acd70b","added_by":"auto","created_at":"2025-07-09 09:03:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":19471788,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry and SEM detection of adhesion and effect of CLS on macrophages. (A) Flow cytometry was used to determine the ratio of IL-6 positive cells in macrophage after extraction. (B) Quantitative analysis the rate of IL-6 positive macrophage. (C-D) Elisa detection of IL-6 and CXCL-8 in cell supernatant. (E-F) Intensity of intercellular communication between control group and treatment group. (G) Analysis of IL-6\u003csup\u003e+\u003c/sup\u003emacrophage and fibroblast receptor-ligand binding. (H) Analysis of cell population interaction intensity of IL-6, CXCL-8 signaling pathway. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ns: not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) vs.the control group; n = 3.\u003c/p\u003e","description":"","filename":"fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/8e24636fdd3cac5284adfe57.png"},{"id":86316723,"identity":"c203cc60-bcc4-4095-a4a2-d123112cacd0","added_by":"auto","created_at":"2025-07-09 09:03:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":56028951,"visible":true,"origin":"","legend":"\u003cp\u003eClustering annotation of fibroblast and changes of the key regulatory factors of wound closure. (A) UMAP plot of different fibroblast colored by their cell type; (B) Bar charts of the proportion of different fibroblast clusters in two groups; (C) Violin plot of WASF3 distribution across the control and treatment groups; (D) Immunofluorescence staining of WASF3; (E) Immunofluorescence quantitative analysis. (G,H) Cluster-defined fibroblast related gene expression of different subclusters on pseudo timeline; *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ns: not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) vs.the control group; n = 10.\u003c/p\u003e","description":"","filename":"fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/8118be2d26c4ce0e6fb192ec.png"},{"id":86316721,"identity":"5452dc2f-b262-44ac-b4b3-57142534a4f3","added_by":"auto","created_at":"2025-07-09 09:03:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":45446229,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of cell migration ability and related signaling pathways protein expression. (A) Schematic diagram and results of the coculture and grouping of MSF with the conditioned medium for macrophage culture using Transwell constructs. (B) Quantitative analysis of cell migration quantity. (C-E) Protein levels of JAK2, p-JAK2, STAT3 and p-STAT3 protein levels in MSF. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001,****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, and ns: not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) vs.the control group; n = 3.\u003c/p\u003e","description":"","filename":"fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/4e63ac9b0d5f504c1e90a59b.png"},{"id":86316715,"identity":"e3604309-fac5-45e0-b143-f591c2266232","added_by":"auto","created_at":"2025-07-09 09:03:22","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5329206,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/e45c17e2d3a455de0f1b5039.png"},{"id":86316724,"identity":"8a1ef8ba-6b80-45d9-90b7-d9f25346bb46","added_by":"auto","created_at":"2025-07-09 09:03:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":214889976,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryresults.docx","url":"https://assets-eu.researchsquare.com/files/rs-6830339/v1/0f0272a2645ba3585a46e0d5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnal fistula is one of the most common and frequently occurring diseases in the anorectal department.\u0026nbsp;The pathogenesis is still unclear, and it has very few well-identified influence factors [1]. Nowadays, the hypotheses such as immune abnormalities [2], an obstruction of anal crypt [3], and bacterial translocation have been proposed [4,5].There are also studies indicating a significant correlation between the anal fistula and diet, and daily routine [6]. In clinical settings, patients often experience symptoms such as anal pus discharge, fistula defecation, and pain [7]. The commonly used treatment for anal fistula is surgical removal of the fistula, resulting in the formation of a wound. However, due to the special location of the affected area, the wound is chronic, and there is a strong risk of infection [8], as well as a high recurrence rate after anal fistula surgery [9].\u0026nbsp;It will be a heavy burden on the patient individual and the whole medical system.\u0026nbsp;Consequently,\u0026nbsp;promoting wound healing plays an essential role in the treatment of anal fistula\u0026nbsp;[10].\u003c/p\u003e\n\u003cp\u003eThe regulation of macrophage and fibroblast functions is the core strategy for wound healing. Macrophages in the early stage engulf pathogens and necrotic tissues, regulate inflammatory responses, and secrete related factors that not only promote angiogenesis, but also activate fibroblast proliferation [11]. During the proliferation phase, they migrate extensively to the wound, synthesize extracellular matrix such as collagen, and differentiate into myofibroblasts to mediate wound contraction, ultimately promoting tissue remodeling and barrier function recovery together [12]. Targeting the signaling pathways of both (such as TGF-β/Smad, Wnt/β-catenin) or intercellular interactions can optimize the repair process, reduce complications, and provide a key entry point for the treatment of wounds [13].\u003c/p\u003e\n\u003cp\u003ePuffballs are a class of mushrooms that are found worldwide. Some species of puffball are edible when they are fresh, and other species, such as \u003cem\u003eCalvatia lilacina,\u003c/em\u003e have been used as traditional medicine worldwide. Calvatia lilacina spores (CLS) used as traditional Chinese medicine (TCM) have been proven to be used for the treatment of hemostasis, throat pain and cough [14]. Historical literature used its powder to treat various wounds, and it is mostly used as wound dressings, because dry, mature spores have a positive effect on wound healing[15,16] And it had been confirmed that puffballs promoted the healing of diabetes ulcers by balancing oxidative stress, accelerating angiogenesis and regulating wound microbiota[17,18].\u0026nbsp;In addition, the CLS extracts had also been shown to inhibit cancer cell proliferation\u0026nbsp;[19].The CLS has spiky and hollow structure which gives it strong adsorption ability\u0026nbsp;[20].\u0026nbsp;Therefore, CLS has great potential in the treatment of anal fistula wounds due to its diverse pharmacological effects and unique structures.\u003c/p\u003e\n\u003cp\u003eIn the current study sought to evaluate the molecular mechanism of CLS on anal fistula wound. In addition, the study has constructed single-cell transcription maps of granulation tissue from control and treatment groups. Single-cell RNA sequencing analysis is enabling the identification of cell-type specific changes of gene expression and high-throughput quantification with individual cells between two groups [21]. Notably, a novel approach to unraveling the the intercellular heterogeneity of anal fistula granulation tissue and analysis of cell-cell communication using CellChat [22], exploring the key mechanisms of promoting wound healing based on the complex components and special structure of traditional Chinese medicine CLS..\u003c/p\u003e"},{"header":"Method And Materials","content":"\u003cp\u003e\u003cstrong\u003eReagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTNF-α, COL1A1, VEGF Elisa Kit (EK182, EK183, EK1C01, Multi Sciences, China); WASF3 Antibody (67620-1-lg, Proteintech, China); α-SMA (19245s, CST, USA); CD14 Antibody (ab183322, abcam, Britain ); CD11b Antibody (ab133357, abcam, Britain); IL-6 flow Antibody (562050, BD Pharmingen, USA); JAK2 (WL02188, Wanleibio, China); p-JAK2 (WL02997, Wanleibio, China); STAT3 (WL03207, Wanleibio, China); p-STAT3 (WL06214, Wanleibio, China);\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of CLS, composition analysis and characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCalvatia lilacina (Mont.et Berk.) Lloyd was purchased from (blind for peer review) and authenticated by (blind for peer review) based on the Pharmacopoeia of the People’s Republic of China (Part I, 2020 version). After removing the shell and impurities, the obtained spore powder was sieved through a filter screen and then subjected to radiation sterilization for subsequent experiments. Due to its complex composition, we determined the composition of CLS via UV, GC, HPLC and UPLC-MS. In addition, we conducted the CLS\u0026nbsp;morphology related indices and structural characterization (fig.1, Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study enrolled a total of twenty patients who received anal surgery in Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, and randomly divided into the control group and the treatment group. The control group, patients on the second day after surgery were routinely disinfected with 0.5% iodine cotton balls on the wound, and sterilized Vaseline oil gauze was applied to the wound, which was covered and fixed with sterile dry gauze outside. On the basis of the control group, the treatment group evenly applied 0.04 g/cm\u003csup\u003e2\u003c/sup\u003e of CLS onto sterilized vaseline oil gauze and covered the wound. The dressing was changed twice a day, and collected granulation tissue at different time.\u0026nbsp;The protocol was approved by the Ethics Committee of the hospital (KY2023302).\u0026nbsp;All enrolled participants provided written informed consent to participate in the study under the principles of Declaration Helsinki. In addition, written informed consents were obtained from all the participants before study procedures began, and participants were free to withdraw from the study at any stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the instructions of the ELISA kit, added 100 μL of detection antibody to the standard and sample wells, except for the blank well, incubated at 37 °C for 1 hour. Then, added 250-300 μL of washing solution to each well for 1 minute, discarded the liquid. Subsequently, each well added color reagent and incubated in the dark for 15 minutes. Afterwards, added 50 µL of termination solution to the plate. Finally, the OD value was determined by the enzyme marker at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry staining of granulation tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eObtaining granulation tissue slides consistent with the above method for subsequent detection of CD14 and CD11b. After dewaxed and dehydrated the tissue slides, performed antigen repair at high temperatures, they were blocked with serum, then incubated overnight at 4 ℃ with primary antibody. Incubating secondary antibodies corresponding the species. Stained the cell nucleus with hematoxylin, slides dehydrated, sealed and then examined with a microscope. The expression of specific proteins be analyzed by detecting the mean gray value of immunohistochemistry photographs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue dissociation and preparation for single-cell RNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fresh tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron) on ice after the surgery within 30 mins. The specimens were washed with Hanks Balanced Salt Solution (HBSS) for three times, minced into small pieces, and then digested with 3 mL sCelLiveTM Tissue Dissociation Solution (Singleron) by Singleron PythoN™ Tissue Dissociation System at 37 °C for 15 min. The cell suspension was collected and filtered through a 40-micron sterile strainer. Afterwards, the GEXSCOPE® red blood cell lysis buffer (RCLB, Singleron) was added, and the mixture [Cell: RCLB=1:2 (volume ratio)] was incubated at room temperature for 5-8 min to remove red blood cells. The mixture was then centrifuged at 300 × g 4 ℃ for 5 mins to remove supernatant and suspended softly with PBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT \u0026amp; Amplification \u0026amp; Library Construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions (2×10\u003csup\u003e5\u003c/sup\u003e cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) (Ref). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary analysis of raw read data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw reads were processed to generate gene expression profiles using CeleScope V3.0.1 \u0026nbsp;(Singleron Biotechnologies) with default parameters. Briefly, Barcodes and UMIs were extracted from R1 reads and corrected. Adapter sequences and poly A tails were trimmed from R2 reads and the trimmed R2 reads were aligned against the GRCh38 (hg38) \u0026nbsp; transcriptome using STAR (v2.6.1b). Uniquely mapped reads were then assigned to genes with FeatureCounts(v2.0.1). Successfully Assigned Reads with the same cell barcode, UMI and gene were grouped together to generate the gene expression matrix for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuality control, dimension-reduction and clustering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScanpy v1.8.1 was used for quality control, dimensionality reduction and clustering under Python 3.7. For each sample dataset, we filtered expression matrix by the following criteria: 1) cells with gene count less than 200 or with top 2% gene count were excluded; 2) cells with top 2% UMI count were excluded; 3) cells with mitochondrial content \u0026gt; 10% were excluded; 4) genes expressed in less than 5 cells were excluded. After filtering, 81068 cells were retained for the downstream analyses, with on average 1004 genes and 2183 UMIs per cell. The raw count matrix was normalized by total counts per cell and logarithmically transformed into normalized data matrix. Top 2000 variable genes were selected by setting flavor = ‘seurat’. Cells were separated into 22 clusters by using Louvain algorithm and setting resolution parameter at 1.2. Cell clusters were visualized by using Uniform Manifold Approximation and Projection (UMAP). Batch effect between samples was removed by Harmony v1.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell-cell Interaction Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellChat (version 0.0.2) was used to analyze the intercellular communication networks from scRNA-seq data. A CellChat object was created using the R package process. Cell information was added into the meta slot of the object. The ligand-receptor interaction database was set, and the matching receptor inference calculation was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePseudotime Trajectory Analysis: monocle2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell differentiation trajectory of Fibroblasts subtypes was reconstructed with the Monocle2 v 2.10.0 (ref). For constructing the trajectory, top 2000 highly variable genes were selected by Seurat(v3.1.2) FindVairableFeatures() and dimension-reduction was performed by DDRTree(). The trajectory was visualized by plot_cell_trajectory()\u0026nbsp;function in Monocle2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUCell Gene Set Scoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene set scoring was performed using the R package UCell v 1.1.0. UCell scores are based on the Mann-Whitney U statistic by ranking query genes’ in order of their expression levels in individual cells. Because UCell is a rank-based scoring method, it is suitable to be used in large datasets containing multiple samples and batches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture and intervention of THP-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e cell culture incubator at 37 °C. Cell selection during logarithmic growth phase, added PMA (500nM) and induced for 24 hours in a 12 well plate with a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/ml. Then the control group was given single culture, while the CLS group was incubated with particles at different dosages 0.04, 0.08 particles/μm\u003csup\u003e2\u003c/sup\u003e, the SiO\u003csub\u003e2\u003c/sub\u003e group was 0.08 particles/μm\u003csup\u003e2\u003c/sup\u003e for 24 h. Then cells and culture supernatant were collected for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture of MSF with conditioned medium and transewell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatant of the four groups of macrophages were mixed with culture medium, to form conditioned medium. After culturing 24 hours, proteins were collected for subsequent detection. Meanwhile 500 ul of the conditioned medium took to the lower chamber, 100ul of MSF suspension added to the transwell chamber at a cell concentration of 2×10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells/ml for 24 hours, then fixed with methanol for 15 minutes, and stained the cells with 1% crystal violet for 15 minutes. After washing with PBS, wiped off the cells in the small chamber with a cotton swab, and finally took photos and performed quantitative analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdherent cells were incubated with trypsin EDTA at 37 °C for 1 minutes and gently dislodged by pipetting for cell harvest, washed the cells twice with PBS buffer. Then 1 μl of the BD Horizo Fixable Viability Stain 660 Stock Solution was added for 10 mintunites. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block. The cells were fixed, permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody at 4 °C for 30 minutes. Finally, the cells were centrifuged, the supernatant was discarded, washed three times with PBS, and resuspended in 250 μl PBS for fluorescence intensity detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining of granulation tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe granulation tissue slides for antigen repair, then blocked them with BSA. Incubating overnight with WASF3 primary antibody (proteintech, 67620-1-Ig, 1:500), followed by secondary antibody incubation and DAPI staining. Then added anti-fluorescence quencher and sealed the slide for fluorescence microscopic examination. The gray value of each pixel represents the fluorescence intensity of the point, and the mean gray value of a certain fluorescence channel on the immunofluorescence photograph can be quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conditioned medium formed by macrophage culture was added to MSF and cultured for 24 hours. Cell proteins were extracted with RIPA lysis buffer and separated by SDS polyacrylamide gel electrophoresis. After electrophoresis, the protein were transferred onto the PVDF membrane, blocked it with a blocking solution for 1 hour, and then washed it with TBST. Add the first antibody and incubated overnight at 4 ℃. The next day, the membrane was washed with TBST, added the second antibody corresponding to the first antibody, incubated the mixture at room temperature for 1 hour, then the protein was exposed to color development by enhanced chemiluminescence (ECL) reagent, and used ImageJ to calculate the grayscale value.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analysis was made by SPSS 22.0 software. Descriptive statistics were reported as mean ± SD. A P-value of less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eComponent analysis and structural characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the results showed that total protein (38.74 \u0026plusmn; 6.01 mg/g), total steroids (15.15 \u0026plusmn; 0.45 mg/g), total polysaccharides (11.03 \u0026plusmn; 0.54 mg/g) and total polyphenols (8.53 \u0026plusmn; 0.32 mg/g) in CLS (Table 1, supplementary results S1-4). For fatty acid analysis, GC-2010 plus was used. The saturated fatty acid content was analyzed as 0.2007% (C16:0 methyl palm 0.1270%, C18:0 stearic acid methyl ester\u0026nbsp;0.0616%, C24:0 methyl tetracosanoate\u0026nbsp;0.0121%). Monounsaturated fatty acids and polyunsaturated fatty acids were analyzed as\u0026nbsp;0.3050% and 0.0815% (C18:1n-9c\u0026nbsp;methy1-9-oleic acid methyl ester, C18:2n-6c\u0026nbsp;linoleic acid methyl ester) (Supplementary Table 1, S7). Cysteine (0.1465%) and valine (0.0803%) were determined as major components of amino acids in the CLS (Supplementary Table 2).\u0026nbsp;UPLC-MS and HPLC detected the representative monomeric components ergosterol (0.142%) and ergosterone (0.023%) in CLS (Supplementary results S5, S6). The spore powder has a uniform particle size and is spiky (Fig.1). While in the study observed the micromeritics characteristics of CLS (Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003eTable 1 Summary of the contents of total steroids, total polyphenols, total polysaccharides, and protein in the CLS (mean \u0026plusmn; SD, n = 3)\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003econtents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etotal protein（contain amino acids, mg/g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etotal steroids（mg/g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etotal polysaccharides（mg/g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etotal polyphenols（mg/g）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCalvatia lilacina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.32 \u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.15 \u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.03 \u0026plusmn; 0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.53 \u0026plusmn; 0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLS promoted wound healing in anal fistula by reducing TNF-\u0026alpha;-driven inflammation and promoting Collagen I Synthesis, angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the role of CLS in collagen generation, angiogenesis, and inflammation, we detected the expression levels of TNF-\u0026alpha;, VEGF, and Collagen I in granulation tissue by ELISA. As shown in the results (Fig 2A-C), CLS significantly inhibited the levels of inflammatory cytokine TNF-\u0026alpha; with the most significant effect observed on day 14. Meanwhile, CLS also had the effect of promoting collagen generation related cytokine Collagen I levels, and had a slight effect on the expression of vascular factor VEGF. We further performed immunohistochemical staining on granulation tissue on days 7 and 14 to observe the status of inflammation related cells (Fig 2D). Consistent with the ELISA results, there was a significant decrease in the positive expression rates of CD14 and CD11b on day 14. As shown by histological results, positive high expression was observed on days 7 and 14, indicating that immune and inflammation related cells are the main functional groups in the early stage of anal fistula wound healing (Fig 2E, F).\u003c/p\u003e\n\u003cp\u003e(A-C) ELISA detection of TNF-\u0026alpha;, VEGF, and Collagen I expression levels in granulation tissue; (D) Immunohistochemical staining of CD14 and CD11b; (E,F) Immunohistochemical quantitative analysis. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001,****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, and ns: not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) vs.the control group; n = 10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell atlas of anal fistula granulation tissue revealed macrophages were the main cell population of MPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain a better understanding of the effect of CLS on the makeup of cell types and transcriptional patterns in granulation tissue, we created single-cell atlases for the granulation tissue.\u0026nbsp;GEXSCOPE\u0026reg; was used to convert single-cell suspensions of the scRNA-seq samples to barcoded libraries. A total of 81068 viable cells were obtained after quality control for subsequent analyses.\u0026nbsp;To identify each cell type, we used the Seurat v2.3.4 packages to perform clustering on the sequencing data, then annotated each cell type based on the expression levels of canonical cell-type-specific markers. In general, we identified 22 cell clusters in granulation tissue that could be classified into eight major cell types: ECs (identified by PECAM1, VMF, CDH5), Fibroblasts (identified by COL1A1, DCN, COL1A2), MuralCells (identified by ACTA2, CALD1, MCAM), BCells (CD79A, MS4A1, CD19), TCells (CD3D, CD3E, CD3G), Neutrophils (FCGR3B, S100A9, S100A8), MPs (CSF1R, CD14, CD68), pDCs (IL3RA, CLEC4C, LILRA4).(Fig.3 A-C). In summary, we developed a tissue characterization of the cellular diversity of the granulation tissue and established a comprehensive framework.\u003c/p\u003e\n\u003cp\u003eSingle-cell results showed a significant reduction in the number of macrophages and monocytes in the treatment group, indicating that CLS has a certain anti-inflammatory effect in the early stage (Fig. 3D-F). Therefore, we focused on macrophages as the main cell population for further dimensionality reduction analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLS induced an IL-6\u003csup\u003e+\u003c/sup\u003e macrophage subset with coordinated expression of IL-6 and CXCL-8 during wound healing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 13023 viable macrophages were obtained to recluster on the sequencing data, and we identified 7 cell clusters in macrophages.\u0026nbsp;The results showed that there was a certain increase in the number of IL-6\u003csup\u003e+\u003c/sup\u003e macrophages, meanwhile IL-6\u003csup\u003e+\u003c/sup\u003e macrophages regulate inflammation function significantly stronger than other subgroups (Fig.4 A-C). Research report suggested that the coexistence of IL-6 and CXCL-8 in macrophages could activate the WASF3 [23]. Therefore, we further compared the expression of IL-6 and CXCL-8 in the macrophage, the resulted the IL-6 and CXCL-8 had co-high expression only in IL-6\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.4 D). This also suggested that IL-6\u003csup\u003e+\u003c/sup\u003e macrophages may be a special cell population after CLS intervention. The results further showed that the expression of IL-6 and CXCL-8 was significantly increasing in the treatment group (Fig.4 E,F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLS enhanced macrophages and fibroblasts communication through IL-6R and CXCL-8R1 receptors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCLS adhered to macrophages and spike SiO\u003csub\u003e2\u003c/sub\u003e also had the effect of adhering to cells. The flow cytometry results showed that CLS increased the number of IL-6\u003csup\u003e+\u003c/sup\u003e macrophages, and the SiO\u003csub\u003e2\u003c/sub\u003e group also had a certain effect (Fig.5 A,B). This result reminded us that the spike structure of CLS could stimulate immune activity that consistent with previous reports [24]. The elisa results showed that CLS significantly increased the expression levels of IL-6 and CXCL-8 (Fig.5 C,D). In addition, studies had shown that ergosterol can activate the signal expression of AP-1 protein and AP-1 was required for CXCL-8 activation[25]. Meanwhile, we found CLS enhanced the strength of cell interaction between macrophages and fibroblasts (Fig.5 E, F). The gene sequence of mouse CXCL-1 had high homology with human CXCL-8 at the amino acid level. The elisa results showed that CLS significantly increased the expression levels of IL-6 and CXCL-1 (supplementary results S8). IL-6 and CXCL8 activated downstream signaling pathways by binding to receptors, the results showed that there were multiple receptors for IL-6\u003csup\u003e+\u003c/sup\u003emacrophages in fibroblasts, among which IL-6R_IL-6ST and CXCL-8R1 were the main functional receptors. CLS significantly enhanced the inter population communication intensity of IL-6 and CXCL-8 signaling pathways (Fig.5 G,H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLS promoted macrophages to co-upregulate IL-6 and CXCL-8, thereby accelerating downstream WASF3-mediated wound closure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the process of wound healing, fibroblasts are the main cell group to regulate wound closure [26]. we subset fibroblasts for reintegrating and subclustering. Altogether, 15679 cells contributed to 7 clusters (Fig.6 A,B). We found that the expression of ACTR2, ACTR3 and WASF3 in the treatment group was significantly higher than the control group (Fig.6 C, supplementary results S9 D,E). Our further immunofluorescence results showed that after CLS intervention, the expression levels of \u0026alpha;-SMA and WASF3 increased in granulation tissue, consistent with single-cell detection data (Fig.6 D,F). The gene expression pattern across pseudotime showed that the expression curves of ACTR2 and ACTR3 were relatively smoother in the treatment group than in the control group. Meanwhile, the overall expression level of ACTR2 in the treatment group was higher than that in the control group (Fig.6 G,H). ACTR2, ACTR3 are components of the Arp2/3 complex, which is involved in regulating various cellular processes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWASF3 plays a critical role in regulating actin cytoskeleton dynamics, and previous study showed that cell migration through increased the expression of WASF3 to recruit the Arp2/3 complex, increasing the formation of dendritic protrusions and thus driving cell migration [23]. Therefore, we reasonably speculated that CLS enhanced the expression of IL-6 and CXCL-8 in the IL-6\u003csup\u003e+\u003c/sup\u003e macrophage, thereby increasing the expression level of WASF3 in fibroblasts and accelerating wound closure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe conditioned medium derived from macrophage cultures stimulated MSF migration by activating the JAK2/STAT3 signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transwell experiment results showed that the conditioned medium for macrophage culture promoted the migration of MSF (Fig.7. A-B). The JAK2/STAT3 signaling pathway regulates multiple cell functions, such as proliferation, differentiation, migration and immune function. Several studies have confirmed that activated JAK2/STAT3 signaling pathway can promote wound healing [27]. Therefore, we next investigated whether the migration effect of CLS involved the JAK2/STAT3 pathway. To this point, our data indicated that CLS activated the JAK2/STAT3 pathway. As a classic prosurvival signaling axis, the JAK2/STAT3 pathway possesses the capacity to promote cell migration. In addition, the western blot results showed that CLS promoted the phosphorylation of JAK2/STAT3 (Fig.7 C-D), thereby promoting the expression of downstream proteins WASF3 to accelerate cell migration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the complexity of the pathogenesis of anal fistula, there is a huge challenge in promoting wound healing after surgical treatment.\u0026nbsp;In clinical, there are no specific drugs for anal fistula wounds and most of them are only given conventional drugs after debridement to promote healing [28]. Fumigation, rubbing and external washing are common treatment method, which greatly increases the pain of dressing changes for patients. In addition, due to the special location of the anal fistula wound which the treatment effect is often poor [29].\u0026nbsp;Puffballs can promote the healing of diabetes ulcers through multiple components and targets, which is confirmed by our previous research\u0026nbsp;[17]. In this study, we demonstrated that the CLS offered a safe and well-tolerated treatment option for anal fistula wounds.\u003c/p\u003e\n\u003cp\u003eSpore powder is amazing substance in nature that plays a fundamental role in the plants' reproduction. Spore powder is very uniform in size and shape. According to their particular structure, spore powder have essential characteristics such as acid and alkyl resistance, mucosal adhesion, and great stability [30]. Based on this, spore powder is increasingly being used for disease treatment. Spore powder have the usability in the targeting of the immune system and causing allergies, stimulating inflammatory responses in the immune system based on the related mechanisms. Ganoderma lucidum spore powder (GLSP) could regulat CD4\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell ratio and macrophage function, especially the expression of PD-1 in TIME\u0026nbsp;[31]. Inonotus hispidus spore powder (IHS) influenced T cells in ApcMin/\u003csup\u003e+\u003c/sup\u003e mice by regulating the IL-5, -6, and -10 levels, thus suppressing tumor development\u0026nbsp;[32]. Spore-mimetic metal–organic frameworks (MOFs) had greater cell attachment and faster and more efficient phagocytosis in cells, which resulted in greater expressions of pro-inflammatory cytokines\u0026nbsp;[33].\u003c/p\u003e\n\u003cp\u003eExcessive inflammation and impaired fibroblast function are important reasons why wounds are difficult to healing [34]. Inflammatory and immune related cells play an important role as the main functional cell groups in the early stage of wound healing. And CLS increased the expression level of IL-6 and CXCL-8 in the IL-6\u003csup\u003e+\u003c/sup\u003e macrophage. Previous research had shown that ERK3-mediated CXCL-8 secretion is critical for the chemotaxis of inflammatory cells to the epithelium, locally used recombinant protein accelerated wound healing[35,36]. And furthermore, the study demonstrated that IL-6 signaling was required for chemotaxis and promoted generation of a new subset of tissue repair macrophage[37,38]. Importantly, IL-6 and CXCL-8 promoted the migration of MCF10A cells and HER2-positive breast cancer cells\u0026nbsp;[39]. These suggested IL-6 and CXCL-8 could promote migration in the neighboring cell and influence the migratory behavior of the entire wound microenvironment. Palmitate in fatty acids had the ability to promote IL-6 via coordinated acetylation of H3K9/H3K18, p300, and RNA Polymerase II\u0026nbsp;[40].Steroids are crucial components of anti-inflammatory effects by neutralizing pro-inflammatory cytokine production in human monocytes, and are also found in CLS constituents such as ergosterol and ergosterone\u0026nbsp;[41].\u003c/p\u003e\n\u003cp\u003eCLS simultaneously regulated the function of IL-6\u003csup\u003e+\u003c/sup\u003e macrophages that enhanced mutual communication with fibroblasts to promote wound contraction. Wound contraction is a process that occurs during the healing of an open wound. Modified fibroblasts were first observed in the granulation tissue of healing wounds which led to the suggestion that these cells have a role in the production of the contractile force that is involved in this process. The myofibroblast had a role in the synthesis of ECM and in force generation, which resulted in ECM reorganization and wound contraction [42].\u0026nbsp;Related research showed that polysaccharides\u0026nbsp;effectively promoted the formation of granulation tissue, collagen deposition\u0026nbsp;[43]. Polyphenols inhibited collagenase inhibitory activities, which play a critical role in preventing collagen breakdown\u0026nbsp;[44]. A recent study observed that saliva promoted fibroblast migration by increasing the secretion of IL-6 and CXCL-8\u0026nbsp;[45]. Consistent with this, our research firstly found that CLS promoted signal exchange between fibroblasts and macrophages, which increased the expression level of IL-6 and CXCL-8 in the IL-6\u003csup\u003e+\u003c/sup\u003e macrophage, then promoted the expression of WASF3 in fibroblasts via JAK2/STAT3 pathway, thereby promoting wound contraction and accelerating wound healing.\u003c/p\u003e\n\u003cp\u003eOur metabolic analysis showed that CLS significantly enhances lysine degradation, glycosaminoglycan biosynthesis-keratin sulfate, D-glutamine and D-glutamate metabolism, and selenocompound metabolism had a significant impact (supplementary results S10 A-C). Lysine degradation was an important metabolic process and research had confirmed that lysine was able to synthesize glutamate [46]. More recent work had indicated that fibroblasts were the primary producers of collagen and synthesize proline from glutamate rather than arginine. Glutamate is known to be the core of almost all metabolic pathways required at different stages of inflammatory wounds.\u0026nbsp;We have already demonstrated in our previous research that\u0026nbsp;Glutamate promoted the proliferation of HaCaT cells, migration of MSFs and macrophage polarization\u0026nbsp;[18]. The fibroblasts synthesize collagen and GAGs which were components of the ECM. Myofibroblasts produced keratansulfate which assisted in collagen polymerization\u0026nbsp;[47].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study had several limitations. The treatment group had the effect of promoting angiogenesis and collagen regeneration on the 14th day. Previous research showed that CXCL-8 stimulated angiogenesis in the skin [36]. The causal relationship and specific mechanism still need to be clarified.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this paper, we have applied single-cell RNA sequencing to deeply explore that CLS \u0026nbsp;reduced the number of monocytes to decrease the inflammatory state of the wound, while increased the communication between IL6\u003csup\u003e+\u003c/sup\u003emacrophages and fibroblasts, thus accelerated the transition from the inflammatory phase to the proliferative phase of the wound. These novel results may also guide future research on TCM in anal fistula wound. Meanwhile, these findings provided key information and guidance for further investigation on the clinical applications of CLS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACTR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eActin-Related Protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACTR3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eActin-Related Protein 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGAGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlycosaminoglycans\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCalvatia lilacina spore\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eECs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEndothelial cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGas Chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHematoxylin and eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHPLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHigh-performance liquid chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eImmunohistochemistry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMononuclear phagocyte system\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQ-TOF-MS/MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQuadrupole Time-of-Flight Mass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eScanning Electron Microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTraditional Chinese Medicine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNF-α\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTumor Necrosis Factor-alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eU-MAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUniform manifold approximation and projection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUPLC-MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUltra Performance Liquid Chromatography-Mass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUltraviolet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWASF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWiskott-Aldrich syndrome protein family member 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTangtang He wrote the paper draft. Jun Chen, Lifeng Zhu corrected the draft. Kewei Wang, Bin Jiang, Ruoyu Mu, Wen Min supervised the experimentators. Tangtang He, Ruiwen Mo, Juntong Guo performed the experiments. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No. 82474200; No. 82173994), Leading Program of Traditional Chinese Medicine First-class Discipline (ZYXYL2024-014), Jiangsu Province Traditional Chinese Medicine Development Project (MS2022006), Natural Science Research Project of the Jiangsu Higher Education Institutions of China (No. 22KJA360002), Priority Academic Program Development of Jiangsu Higher Education Institutions, Nanjing Traditional Chinese Medicine Youth Talent Training Program (ZYQ20042), the Graduate Student Scientific Research Innovation Projects in Jiangsu Province (KYCX24_2284).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman ethics and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed following the standard of International Coordinating Committee on Global Partnerships and the revised edition of the Declaration of Helsinki. The present study was approved by the Ethics Committee of Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine (Ethic code: KY2023302).. It was registered in ITMCTR (ITMCTR20250001078). Every participant endorsed informed consent voluntarily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe, the undersigned authors of the manuscript titled \u0026ldquo;[Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula]\u0026rdquo;submitted to \u0026ldquo;Chinese medicine\u0026rdquo;, unconditionally consent to its publication in this journal, understanding it will be the publisher\u0026rsquo;s property and made public per journal policies. We confirm all content is original, not previously published except as disclosed, and we\u0026rsquo;ve obtained permissions for non‑own copyrighted materials. When the manuscript involves human/animal subjects or sensitive info, we\u0026rsquo;ve secured ethical approvals and protected all rights and privacy as per laws. We recognize the publisher\u0026rsquo;s right to make editorial changes for style, format, and ethics compliance and will fully cooperate, providing any needed additional info or clarifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no potential conflicts of interest relevant to this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAssaraf J, Lambrescak E, Lef\u0026egrave;vre JH, De Parades V, Bourguignon J, Etienney I, et al. Increased Long-term Risk of Anal Fistula After Proctologic Surgery: A Case-Control Study. Ann Coloproctol. 2021;37:90\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eFitzpatrick DP, Kealey C, Brady D, Goodman M, Gately N. Treatments for the amelioration of persistent factors in complex anal fistula. Biotechnol Lett. 2022;44:23\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eHamadani A, Haigh PI, Liu I-LA, Abbas MA. Who is at risk for developing chronic anal fistula or recurrent anal sepsis after initial perianal abscess? Dis Colon Rectum. 2009;52:217\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eJaiswal P, Sharma S, Pratap A, Ansari M, Shukla VK, Basu S, et al. Significant presence of biofilm-producing gut-derived bacteria in anal fistula of chronic duration. Int Wound J. 2021;18:519\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eSugrue J, Nordenstam J, Abcarian H, Bartholomew A, Schwartz JL, Mellgren A, et al. Pathogenesis and persistence of cryptoglandular anal fistula: a systematic review. Tech Coloproctol. 2017;21:425\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eDevaraj B, Khabassi S, Cosman BC. Recent smoking is a risk factor for anal abscess and fistula. Dis Colon Rectum. 2011;54:681\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eOwen HA, Buchanan GN, Schizas A, Cohen R, Williams AB. Quality of life with anal fistula. Ann R Coll Surg Engl. 2016;98:334\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eKang J, Liu Y, Peng S, Tang X, Liu L, Xie Z, et al. Efficacy and safety of traditional Chinese medicine external washing in the treatment of postoperative wound of diabetes complicated with anal fistula: Study protocol of a randomized, double-blind, placebo-controlled, multi-center clinical trial. Front Pharmacol. 2022;13:938270.\u003c/li\u003e\n\u003cli\u003eZubaidi AM. Anal fistula. Past and present. Saudi Med J. 2014;35:937\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eLimura E, Giordano P. Modern management of anal fistula. World J Gastroenterol. 2015;21:12\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eSharifiaghdam M, Shaabani E, Faridi-Majidi R, De Smedt SC, Braeckmans K, Fraire JC. Macrophages as a therapeutic target to promote diabetic wound healing. Mol Ther. 2022;30:2891\u0026ndash;908.\u003c/li\u003e\n\u003cli\u003eYounesi FS, Miller AE, Barker TH, Rossi FMV, Hinz B. Fibroblast and myofibroblast activation in normal tissue repair and fibrosis. Nat Rev Mol Cell Biol. 2024;25:617\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eLi S, Ding X, Zhang H, Ding Y, Tan Q. IL-25 improves diabetic wound healing through stimulating M2 macrophage polarization and fibroblast activation. Int Immunopharmacol. 2022;106:108605.\u003c/li\u003e\n\u003cli\u003eTsay J-G, Chung K-T, Yeh C-H, Chen W-L, Chen C-H, Lin MH-C, et al. Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells. J Agric Food Chem. 2009;57:1579\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eCicha-Jeleń M, Muszynska B, Kala K, Sulkowska-Ziaja K. Medicinal Potential of the Giant Puffball Mushroom Calvatia gigantea (Agaricomycetes): A Review. Int J Med Mushrooms. 2024;26:13\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eLee D, Lee S, Jang YS, Ryoo R, Kim JK, Kang KS, et al. N,N-Dimethyl-anthranilic Acid from Calvatia nipponica Mushroom Fruiting Bodies Induces Apoptotic Effects on MDA-MB-231 Human Breast Cancer Cells. Nutrients. 2023;15:3091.\u003c/li\u003e\n\u003cli\u003eHe T, Sun P, Liu B, Wan S, Fang P, Chen J, et al. Puffball spores improve wound healing in a diabetic rat model. Front Endocrinol (Lausanne). 2022;13:942549.\u003c/li\u003e\n\u003cli\u003eDing X, Yang C, Li Y, He T, Xu Y, Cheng X, et al. Reshaped commensal wound microbiome via topical application of Calvatia gigantea extract contributes to faster diabetic wound healing. Burns Trauma. 2024;12:tkae037.\u003c/li\u003e\n\u003cli\u003eZeng Q, Singh R, Ye Y, Cheng S, Fan C, Zeng Q. Calvatia Lilacina Extracts Exert Anti-Breast-Cancer Bioactivity through the Apoptosis Induction Dependent on Mitochondrial Reactive Oxygen Species and Caspase Activation. Nutr Cancer. 2022;74:1058\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eWang H, Lu Z, Xu Y, Kong L, Shi J, Liu Y, et al. Genome-wide characterization of SPL family in Medicago truncatula reveals the novel roles of miR156/SPL module in spiky pod development. BMC Genomics. 2019;20:552.\u003c/li\u003e\n\u003cli\u003eWu C, Tang H, Cui X, Li N, Fei J, Ge H, et al. A single-cell profile reveals the transcriptional regulation responded for Abelmoschus manihot (L.) treatment in diabetic kidney disease. Phytomedicine. 2024;130:155642.\u003c/li\u003e\n\u003cli\u003eJin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan C-H, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021;12:1088.\u003c/li\u003e\n\u003cli\u003eJayatilaka H, Tyle P, Chen JJ, Kwak M, Ju J, Kim HJ, et al. Synergistic IL-6 and IL-8 paracrine signalling pathway infers a strategy to inhibit tumour cell migration. Nat Commun. 2017;8:15584.\u003c/li\u003e\n\u003cli\u003eChen H-J, Hang T, Yang C, Liu D, Su C, Xiao S, et al. Functionalized Spiky Particles for Intracellular Biomolecular Delivery. ACS Cent Sci. 2019;5:960\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eXu Y, Lin L, Zheng H, Xu S, Hong X, Cai T, et al. Protective effect of Amauroderma rugosum ethanol extract and its primary bioactive compound, ergosterol, against acute gastric ulcers based on LXR-mediated gastric mucus secretions. Phytomedicine. 2024;123:155236.\u003c/li\u003e\n\u003cli\u003eSu J, Wei Q, Ma K, Wang Y, Hu W, Meng H, et al. P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/ CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts. Burns Trauma. 2023;11:tkad010.\u003c/li\u003e\n\u003cli\u003eCao W, Meng X, Cao F, Wang J, Yang M. Exosomes derived from platelet-rich plasma promote diabetic wound healing via the JAK2/STAT3 pathway. iScience. 2023;26:108236.\u003c/li\u003e\n\u003cli\u003eSimpson JA, Banerjea A, Scholefield JH. Management of anal fistula. BMJ. 2012;345:e6705.\u003c/li\u003e\n\u003cli\u003ePingping M, Wenzhe F, Peng S, Wenxiu Z, Yu Z. Clinical research progress in Traditional Chinese Medicine in treating wound healing after anal fistula surgery. J Tradit Chin Med. 2023;43:1047\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eOurani-Pourdashti S, Azadi A. Pollens in therapeutic/diagnostic systems and immune system targeting. J Control Release. 2021;340:308\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eWu T-T, Chen Y-Y, Yuan Z-C, Yang G-W, Zhang G-L. Synergy of de-walled Ganoderma Lucidum spore powder (GLSP) on targeted therapy in advanced non-squamous non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutant: protocol for a randomized, double-blind, placebo-controlled study. BMC Complement Med Ther. 2024;24:125.\u003c/li\u003e\n\u003cli\u003eYang H, Li S, Qu Y, Li L, Li Y, Wang D. Anti-Colorectal Cancer Effects of Inonotus hispidus (Bull.: Fr.) P. Karst. Spore Powder through Regulation of Gut Microbiota-Mediated JAK/STAT Signaling. Nutrients. 2022;14:3299.\u003c/li\u003e\n\u003cli\u003eChen P-M, Pan W-Y, Luo P-K, Phung HN, Liu Y-M, Chiang M-C, et al. Pollen-Mimetic Metal-Organic Frameworks with Tunable Spike-Like Nanostructures That Promote Cell Interactions to Improve Antigen-Specific Humoral Immunity. ACS Nano. 2021;15:7596\u0026ndash;607.\u003c/li\u003e\n\u003cli\u003eWu B, Pan W, Luo S, Luo X, Zhao Y, Xiu Q, et al. Turmeric-Derived Nanoparticles Functionalized Aerogel Regulates Multicellular Networks to Promote Diabetic Wound Healing. Adv Sci (Weinh). 2024;11:e2307630.\u003c/li\u003e\n\u003cli\u003eBogucka K, Pompaiah M, Marini F, Binder H, Harms G, Kaulich M, et al. ERK3/MAPK6 controls IL-8 production and chemotaxis. Elife. 2020;9:e52511.\u003c/li\u003e\n\u003cli\u003eCambier S, Gouwy M, Proost P. The chemokines CXCL8 and CXCL12: molecular and functional properties, role in disease and efforts towards pharmacological intervention. Cell Mol Immunol. 2023;20:217\u0026ndash;51.\u003c/li\u003e\n\u003cli\u003eMatsumura F, Polz R, Singh S, Matsumura A, Scheller J, Yamashiro S. Investigation of Fascin1, a Marker of Mature Dendritic Cells, Reveals a New Role for IL-6 Signaling in CCR7-Mediated Chemotaxis. J Immunol. 2021;207:938\u0026ndash;49.\u003c/li\u003e\n\u003cli\u003eLi Z, Xiao J, Xu X, Li W, Zhong R, Qi L, et al. M-CSF, IL-6, and TGF-\u0026beta; promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery. Sci Adv. 2021;7:eabb6260.\u003c/li\u003e\n\u003cli\u003eKim D, Cooper JA, Helfman DM. Loss of myosin light chain kinase induces the cellular senescence associated secretory phenotype to promote breast epithelial cell migration. Sci Rep. 2024;14:25786.\u003c/li\u003e\n\u003cli\u003eKochumon S, Jacob T, Koshy M, Al-Rashed F, Sindhu S, Al-Ozairi E, et al. Palmitate Potentiates Lipopolysaccharide-Induced IL-6 Production via Coordinated Acetylation of H3K9/H3K18, p300, and RNA Polymerase II. J Immunol. 2022;209:731\u0026ndash;41.\u003c/li\u003e\n\u003cli\u003eTada H, Kawahara K, Osawa H, Song L-T, Numazaki K, Kawai J, et al. Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes. Biochem Biophys Res Commun. 2022;636:1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eTomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3:349\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eLi S, Xu W, Zhu W, Wang J, Shi J, Tang J, et al. Superior hemostatic and wound-healing properties of tetrastigma polysaccharide. Materials \u0026amp; Design. 2024;241:112967.\u003c/li\u003e\n\u003cli\u003ePanichakul T, Ponnikorn S, Tupchiangmai W, Haritakun W, Srisanga K. Skin Anti-Aging Potential of Ipomoea pes-caprae Ethanolic Extracts on Promoting Cell Proliferation and Collagen Production in Human Fibroblasts (CCD-986sk Cells). Pharmaceuticals (Basel). 2022;15:969.\u003c/li\u003e\n\u003cli\u003eRodrigues Neves C, Buskermolen J, Roffel S, Waaijman T, Thon M, Veerman E, et al. Human saliva stimulates skin and oral wound healing in vitro. J Tissue Eng Regen Med. 2019;13:1079\u0026ndash;92.\u003c/li\u003e\n\u003cli\u003eGuo Y, Wu J, Wang M, Wang X, Jian Y, Yang C, et al. The Metabolite Saccharopine Impairs Neuronal Development by Inhibiting the Neurotrophic Function of Glucose-6-Phosphate Isomerase. J Neurosci. 2022;42:2631\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eKosir MA, Quinn CC, Wang W, Tromp G. Matrix glycosaminoglycans in the growth phase of fibroblasts: more of the story in wound healing. J Surg Res. 2000;92:45\u0026ndash;52.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"postoperative wound, Traditional Chinese medicine, Single-cell transcriptomics, cell interaction","lastPublishedDoi":"10.21203/rs.3.rs-6830339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6830339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAnal 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTwenty 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe 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\u003csup\u003e+\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur 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.\u003c/p\u003e","manuscriptTitle":"Single-cell RNA sequencing reveals the therapeutic mechanism of Calvatia lilacina in promoting wound healing of anal fistula","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 09:03:17","doi":"10.21203/rs.3.rs-6830339/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-09T01:38:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T00:59:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175825589119476763257296777553312269870","date":"2025-07-17T03:00:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157639221855009902630929247774463029025","date":"2025-07-08T01:43:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-08T00:29:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T06:34:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-18T06:33:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-06-05T14:48:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9def4348-841e-4982-abd8-43fa000c3d46","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:07:40+00:00","versionOfRecord":{"articleIdentity":"rs-6830339","link":"https://doi.org/10.1186/s13020-025-01293-w","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2026-01-07 15:57:56","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-07-09 09:03:17","video":"","vorDoi":"10.1186/s13020-025-01293-w","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01293-w","workflowStages":[]},"version":"v1","identity":"rs-6830339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6830339","identity":"rs-6830339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}