High-Throughput Chemical Screen Identifies Epothilone B in Modulating Inflammatory Bowel Disease by Triggering Neutrophil Apoptosis

High-Throughput Chemical Screen Identifies Epothilone B in Modulating Inflammatory Bowel Disease by Triggering Neutrophil Apoptosis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 8 August 2025 V1 Latest version Share on High-Throughput Chemical Screen Identifies Epothilone B in Modulating Inflammatory Bowel Disease by Triggering Neutrophil Apoptosis Authors : Zhenting He , Ziling Deng , Huan Zhang , Yiming Xiang , Jiashi Guo , Yi Chen , Jingjing Qin , … Show All … , Ling Meng , Fengmin Xu , Xiaowen Wang , Siyuan Hou , Hao Yuan , and Chunguang Ren 0000-0003-3245-1159 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175464424.46609687/v1 Published Genes & Diseases Version of record Peer review timeline 168 views 122 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract characterized by complex interplay of genetic, environmental, and immunological factors. Neutrophils, as a key component of the innate immune system, play a critical role in IBD pathogenesis due to their dysregulated infiltration, impaired apoptosis, and resultant epithelial damage during the disease process.However, effective intervention strategies by modulating neutrophils to mitigate the severity of IBD have yet to be established. Methods High-throughput chemical screening was conducted utilizing neutrophil-specific transgenic zebrafish embryos to identify compounds capable of modulating neutrophil homeostasis. The identified compounds were subsequently evaluated in a dextran sodium sulfate (DSS)-induced zebrafish model of IBD by assessing intestinal damage, leukocyte infiltration, the expression of inflammatory cytokines, and alterations in intestinal bacterial microbiota. The effects of Epothilone B (Epo B) on neutrophil apoptosis were assessed via flow cytometry and whole-mount terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays, both in vivo and in vitro. Potential targets of Epo B in the context of IBD were identified through network pharmacology and molecular docking analyses, followed by validation at the protein level via Western blotting. Results Chemical screening identified four compounds, including Epo B, that influence neutrophil homeostasis. Epo B protects against IBD in zebrafish by promoting intestinal epithelial recovery, alleviating inflammatory responses, and rebalancing gut microbiota. Mechanistically, Epo B selectively stimulates neutrophil apoptosis by targeting and enhancing Caspase-3 cleavage, thereby inhibiting neutrophilic inflammation. Conclusion This study underscores the utility of zebrafish in drug screening for IBD and highlights Epo B as a promising candidate for repurposing in IBD treatment, offering a novel therapeutic strategy by targeting neutrophil apoptosis. High-Throughput Chemical Screen Identifies Epothilone B in Modulating Inflammatory Bowel Disease by Triggering Neutrophil Apoptosis Zhenting He 1, # , Ziling Deng 1, # , Huan Zhang 1, # , Yiming Xiang 1 , Jiashi Guo 1 , Yi Chen 2 , Jingjing Qin 1 , Ling Meng 1 , Fengmin Xu 3 , Xiaowen Wang 4 , Siyuan Hou 5 , Hao Yuan 2 , * , Chunguang Ren 1 , * 1 Laboratory of Developmental Biology, Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University & School of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400042, China 2 Shanghai Institute of Hematology, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. 3 Department of Clinical Laboratory, Nanchong Central Hospital (Nanchong Hospital of Beijing Anzhen Hospital, Capital Medical University), The Second Clinical Medical College of North Sichuan Medical College, Nanchong, Sichuan, 637000, China. 4 Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Chongqing, 400042, China. 5 Chongqing Depu Foreign Language School, Chongqing, 401320, China. # These authors contributed equally to this work. * Address correspondence to: Hao Yuan, E-mail: [email protected] & Chunguang Ren, E-mail: [email protected] . High-Throughput Chemical Screen Identifies Epothilone B in Modulating Inflammatory Bowel Disease by Triggering Neutrophil Apoptosis Background Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract characterized by complex interplay of genetic, environmental, and immunological factors. Neutrophils, as a key component of the innate immune system, play a critical role in IBD pathogenesis due to their dysregulated infiltration, impaired apoptosis, and resultant epithelial damage during the disease process.However, effective intervention strategies by modulating neutrophils to mitigate the severity of IBD have yet to be established. Methods High-throughput chemical screening was conducted utilizing neutrophil-specific transgenic zebrafish embryos to identify compounds capable of modulating neutrophil homeostasis. The identified compounds were subsequently evaluated in a dextran sodium sulfate (DSS)-induced zebrafish model of IBD by assessing intestinal damage, leukocyte infiltration, the expression of inflammatory cytokines, and alterations in intestinal bacterial microbiota. The effects of Epothilone B (Epo B) on neutrophil apoptosis were assessed via flow cytometry and whole-mount terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays, both in vivo and in vitro. Potential targets of Epo B in the context of IBD were identified through network pharmacology and molecular docking analyses, followed by validation at the protein level via Western blotting. Results Chemical screening identified four compounds, including Epo B, that influence neutrophil homeostasis. Epo B protects against IBD in zebrafish by promoting intestinal epithelial recovery, alleviating inflammatory responses, and rebalancing gut microbiota. Mechanistically, Epo B selectively stimulates neutrophil apoptosis by targeting and enhancing Caspase-3 cleavage, thereby inhibiting neutrophilic inflammation. Conclusion This study underscores the utility of zebrafish in drug screening for IBD and highlights Epo B as a promising candidate for repurposing in IBD treatment, offering a novel therapeutic strategy by targeting neutrophil apoptosis. Keywords: Epothilone B; High-throughput chemical screen; IBD; Neutrophil; Zebrafish; Introduction The etiology of inflammatory bowel disease (IBD), which encompasses Crohn’s disease (CD) and ulcerative colitis (UC), remains complex and involves genetic predispositions, alterations in microbiota, and immune dysregulation 1 . Although biological therapies, such as inhibitors of tumor necrosis factor and Janus kinase, are increasingly utilized in the treatment of patients with IBD, the disease continues to relapse and life-threatening, with rising incidence and prevalence worldwide. Novel therapeutic strategies are being explored to address these challenges. Neutrophils play a crucial role in the innate immune system of the gastrointestinal tract and are characterized by their infiltration of the intestinal mucosa during active IBD. Under homeostatic conditions, neutrophils typically undergo rapid apoptosis followed by phagocytosis by macrophages. However, in the context of inflammation, they undergo delayed apoptosis, allowing them to remain active in defending against pathogen invasion 2 . This prolonged survival, driven by excessive anti-apoptotic signals, results in the accumulation of neutrophils, which can lead to damage to the epithelial barrier and exacerbate inflammation through the release of reactive oxygen species (ROS) and cytotoxic granules, such as myeloperoxidase (MPO) and neutrophil extracellular traps (NETs) 2-4 . Notably, effective intervention strategies aimed at modulating neutrophil apoptosis to mitigate the severity of IBD have yet to be established. Given the pivotal role of neutrophils in the pathogenesis of IBD, large-scale drug screening for neutrophil modulators presents a promising avenue for developing of novel therapeutic approaches for this condition. The zebrafish genome exhibits a high degree of homology with the human genome, particularly with respect to its innate immune system. Approximately 84% of genes associated with human diseases have corresponding orthologs in zebrafish 5 . IBD-like conditions have been successfully established using chemicals such as dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS). These models closely emulate various characteristics of human IBD, particularly the similarities in compromised intestinal barrier function, elevated pro-inflammatory cytokine levels, inflammatory cell infiltration, oxidative stress, and histopathological features 6 . Additionally, zebrafish embryos and larvae are exceptionally useful in vivo model for high-throughput chemical screening of drugs for inflammatory diseases, because of their small size, optical transparency, rapid reproduction, and ease of exposure to drugs in fish culture media 78, . In this study, we conducted a high-throughput screening of 1,430 Food and Drug Administration (FDA)-approved drugs and identified several compounds that alter neutrophil homeostasis in zebrafish, resulting in either increased or decreased neutrophil counts. Subsequently, we evaluated the effects of these identified compounds in a DSS-induced zebrafish enterocolitis model and observed that Epothilone B (Epo B) provided protection against IBD by enhancing intestinal epithelial recovery, suppressing inflammatory responses, and restoring the gut microbiota in zebrafish. Furthermore, we demonstrated that Epo B selectively induced neutrophil apoptosis without affecting differentiation, leading to reduced neutrophilic inflammation and alleviation of gut injury in a zebrafish model of IBD. Our study suggests that Epo B could serve as a novel therapeutic agent for IBD by targeting neutrophil apoptosis. Materials and methods Zebrafish husbandry and transgenic lines The wild-type WT AB strain and transgenic zebrafish lines including Tg ( mpx : GFP ) i114 9 , Tg ( mpeg1 : Gal4 ) gl24 ; Tg ( UAS : Nfsb-mCherry ) i1 49 10 , Tg ( gata1 : DsRed ) 11 , Tg ( kdrl : mCherry ) is5 12 and Tg ( cd41 : GFP ) 13 were raised and maintained under standard conditions as previously described 14 . All animal studies were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University (Approval No. IACUG-CQMU-2024-0139). High-throughput screening and high-content image assay Zebrafish embryos collected at 30 hours post-fertilization (hpf) were treated with 2 mg/ml pronase (Roche) for 5 min to remove embryonic chorions. The embryos were subsequently placed into a Cell Carrier-96 well plate, with five embryos per well, containing 200 μl of E3 culture medium. A total of 1,430 chemicals from an FDA-approved drug library (L1300, Selleck Chemicals, Houston, TX, USA) were dissolved in dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) and added to wells of a 96-well plate using a JANUS liquid-handling workstation (PerkinElmer, Waltham, MA, USA). The plate was agitated for 1 min on a microplate shaker to ensure thorough mixing. The embryos were incubated in a culture chamber at 28.5 °C until 72 hpf. Prior to imaging, 2 μl of 0.4% Tricaine (A5040, Sigma-Aldrich, St. Louis, MO, USA) was added to each well for anesthetic purposes. The Opera Phenix high-content imaging system (PerkinElmer) was employed to scan and capture images, with one field of view per well. The resulting images were analyzed using Harmony 4.1 software to quantify the cell number and fluorescence intensity of green fluorescent protein (GFP)-positive neutrophils within each well. Fluorescence photography Following tricaine treatment, embryos and larvae were mounted on imaging plates using 4% methylcellulose. During imaging, embryos and larvae were maintained at 28.5°C. Fluorescence and bright-field images were captured using a Zeiss SteREO Discovery (V12) fluorescence stereoscope or a confocal laser scanning microscope (AX/AX R, Nikon, Tokyo, Japan). The imaging was performed in a blinded manner. Zebrafish model of DSS-induced enterocolitis The DSS-induced enterocolitis protocol was adapted and modified from a published method 15 . Briefly, to induce colitis, embryos at 3 days post-fertilization (dpf) were placed in freshly prepared 0.5% (w/v) DSS (molecular weight 36,000-50,000, 160110; MP Biomedical, Santa Ana, CA, USA) for 3 days. Dexamethasone (DEX) and Z-VAD-FMK (zVAD) were purchased from TargetMol (Boston, USA). All drugs were dissolved in DMSO and administered to the larvae for 24 h (5-6 dpf). The working concentrations of the hit compounds are listed in Table S1. Histological analysis Zebrafish larvae at 6 dpf were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4℃. Subsequently, the blocks of larvae were dehydrated through a standard ethanol series to 100%, embedded in paraffin, and sectioned at 5-μm intervals for staining with hematoxylin and eosin. Three intestinal segments—the intestinal bulb (IB), mid-intestine (MI), and posterior intestine (PI)—were histologically scored and synthetically graded per larva. The histological scoring method was adapted from previously described protocols, incorporating the following grading criteria: Impaired epithelial integrity (0–3), Deletion of villi (0–3), Expanded gut lumen (0–3), Degree of vacuolation (0–3), and Percentage of goblet cells (0–3) 1617, . At least five larvae were examined for each treatment condition. Resazurin reduction assay The assay was adapted and modified from a previously described method 15 . Briefly, larvae collected at 6 dpf were incubated in a solution of 1 mg/ml Alamar blue (R7017, Sigma-Aldrich) for 30 min. The larvae were anesthetized and individually imaged using a fluorescent microscope. The fluorescence intensity was quantified using Image J software version 1.43 (National Institutes of Health, Bethesda, MD, USA). Measurement of inflammatory cytokines Cytokine levels in zebrafish larvae were determined as previously described 18 . Interleukin (IL)-8 and IL-1β enzyme-linked immunosorbent assay kits were purchased from Jiangsu Meibiao Biological Technology Company (Jiangsu, China). Briefly, zebrafish larvae collected at 6 dpf were rinsed and homogenized with PBS, and then stored overnight at −20℃. Subsequently, the homogenates were centrifuged at 1000×g for 15 min at 4℃. The inflammatory cytokine levels in the supernatants were measured according to the manufacturer’s instructions. Flow cytometry Zebrafish larvae collected at 6 dpf were subjected to 30-min digestion with trypsin after being meticulously chopped up using a sterile scalpel blade. The resulting mixture was then washed with PBS and filtered through a 40 μm nylon mesh to obtain a cell suspension. Fluorescence-activated cell sorting was subsequently performed using the Guava® easyCyte system (Luminex Corporation, Austin, TX, USA). Data analysis was performed using the FlowJo software (TreeStar, Ashland, OR, USA). Whole-mount terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay Apoptotic neutrophils were detected using the TUNEL assay. Briefly, mpx : GFP larvae collected at 6 dpf were anesthetized and fixed in 4% PFA in PBS overnight at 4℃. After washing with 0.1% Tween-20 in PBS, the larvae were permeabilized with proteinase K (10 mg/ml) and refixed with 4% PFA. Apoptotic cells were detected using the One Step TUNEL Apoptosis Kit (Beyotime Biotechnology, Shanghai, China), following the manufacturer’s instructions. Fluorescence images were captured by a confocal laser scanning microscope. The number of apoptotic neutrophils was determined by counting TUNEL and GFP double-positive cells within the caudal hematopoietic tissue using ImageJ software. 16S rRNA sequencing of intestinal bacteria Zebrafish larvae collected at 6 dpf were subjected to microscopy-assisted dissection of the gastrointestinal tracts (GITs), followed by three washes with sterile PBS. The samples were immediately frozen in liquid nitrogen and subsequently stored at -80 °C. Total genomic DNA was extracted using a DNeasy PowerSoil Pro Kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer’s guidelines. DNA quality was verified by 1% agarose gel electrophoresis, and DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The V3-V4 regions of the 16S rRNA genes were amplified by PCR with primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified, quantified, and homogenized to generate a sequencing library. Quality control of the libraries was performed prior to sequencing using an Illumina NovaSeq 6000 PE250 system. The primary FastQ files were processed and quality-filtered using Trimmomatic, followed by sequence combination as described previously 19 . Operational taxonomic units (OTUs) were clustered using UPARSE (version 10.0) with a 97% similarity cut-off. Chimeric sequences were identified and deleted using UCHIME (version 8.1). Taxonomic analysis of each 16S rRNA gene sequence was performed by comparison with the Silva 16S rRNA (Release 128) database using the RDP Classifier algorithm (version 2.2), with a confidence threshold of 70%. Bioinformatics analysis was conducted using QIIME, whereas BugBase normalized the OTUs based on the predicted 16S copy number, subsequently predicting the microbial phenotype using the precomputed files. Statistical significance was defined as P < 0.05, with highly significant results defined as P < 0.01. A Venn diagram generated using Mothur was used to identify the core microbiome at the species level. Alpha diversity indices, including richness and Chao1 were calculated with 97% identity using Mothur (version 1.30). Beta diversity analysis was conducted using principal coordinate analysis (PCoA) based on BrayCurtis distances at the relative OTUs level, and unweighted UniFrac distances were used to compare different groups. STAMP v2.1.3 with Welsh’s t-test and one-way ANOVA were used to compare the relative abundances of the predominant bacteria across various groups. P ≤ 0.05 was considered statistically significant. Isolation of human neutrophils Whole blood was collected from healthy donors at The First Affiliated Hospital of Chongqing Medical University following the Declaration of Helsinki. Written, informed consent was obtained from each donor prior to the collection of human blood samples, which were de-identified before use. Neutrophils were isolated using a Ficoll-Hypaque gradient, sedimentation with dextran (3% w/v), and hypotonic lysis of erythrocytes, achieving a purity level of ≥ 90%, as assessed by flow cytometry using anti-human CD11b-APC (BioLegend, Cat# 980104, RRID: AB_2632619), CD66b-PE (BioLegend, Cat# 392903, RRID: AB_2750201), and CD16-APC antibodies (BioLegend, Cat# 980104, RRID: AB_2616904). This study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (Approval No. K2024-158-01). All donors provided written informed consent prior to specimen acquisition. Cell culture and treatment HL-60 human-derived myeloblastic leukemia cells (Cat# TCHu 23, RRID: CVCL_0002), THP-1 monocytic cells (Cat# SCSP-567, RRID: CVCL_0006), HEK embryonic kidney 293T cells (Cat# GNHu44, RRID: CVCL_KS57), NCM460 normal human colonic epithelial cell line (Cat# SNL-519, RRID: CVCL_0460), and RAW264.7 mouse-derived macrophages (Cat# SCSP-5036, RRID: CVCL_0493) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cell lines were cultured in RPMI-1640 or Dulbecco’s Modified Eagle’s medium (DMEM, Solarbio) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. The cells were regularly tested for mycoplasmas. All the cell lines were used within 10 passages. Neutrophils isolated from human peripheral blood were cultured in RPMI-1640 medium supplemented with 10% autologous serum. Cytotoxicity and apoptosis measurement For cytotoxicity measurement, cells were seeded in 96‑well plates at a density of 2 ×10 4 cells per well. Following treatment with Epo B (0-10 nM) for 6 h, the cells were washed with PBS. Subsequently, DMEM or RPMI-1640 medium, along with 10% Cell Counting Kit-8 (CCK-8) reagent (Solarbio), was added to each well according to the manufacturer’s instructions. The plates were incubated at 37 ˚C for 4 h, after which the absorbance was measured at 450 nm using a microplate reader (Tecan, Männedorf, Switzerland). To measure cell apoptosis, an Annexin V–FITC apoptosis kit was used according to the manufacturer’s instructions (BioLegend, London, UK). Cells (2 × 10 5 cells per well) were seeded in six-well plates and treated with Epo B (0, 2, or 5 nM) for 6 h. For human neutrophils, additional exposure groups for lipopolysaccharide (LPS; Sigma-Aldrich, USA) were established, in which LPS (100 ng/ml) was coincubated with the corresponding concentrations of Epo B for 2 h. After treatment, the cells were washed twice with cold PBS, centrifuged at 400×g for 5 min, and resuspended in 1× binding buffer. The cells were stained with 5 μl of Annexin V–FITC and 5 μl of the 7-aminoactinomycin D (7-AAD) reagent at 25℃ for 15 min before flow cytometry analysis. Differentiation assay of HL-60 cells HL-60 cells (3.5 × 10 5 ) were seeded in a T-25 flask and treated with 1.3% DMSO for 5 days to induce differentiation. The cells were also exposed to Epo B (0, 2 and 5 nM) for 24 h (day 4-5). The differentiation of HL-60 cells was assessed by measuring the expression level of CD11b using flow cytometry. Briefly, 1× 10 6 cells were washed twice with PBS, and stained with anti-CD11b-APC antibody for 30 min at 4 °C in the dark. Unstained cells and cells treated with APC Mouse IgG2b (BioLegend, London, UK) were used as controls. After staining, the cells were resuspended in PBS for flow cytometry analysis. The data were analyzed using the FlowJo software. Western blot Total proteins from the cell lines were extracted using RIPA lysis buffer containing the protease inhibitor phenylmethanesulfonyl fluoride and phosphatase inhibitors. Protein concentrations were quantified using a Bicinchoninic Acid (BCA) kit (Beyotime, Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% gel) was used to resolve proteins, which were transferred onto polyvinylidene fluoride (PVDF) membranes. An Enhanced Chemiluminescence Detection Kit (P0018FM, Beyotime) was used to visualize the protein strips. The primary antibodies used included: anti-PARP1 (ABclonal, Cat# A19596, RRID: AB_2862689), anti-Caspase-3 (ABclonal, Cat# A2156, RRID: AB_2862975), anti-BCL-2 (Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), anti-BAX (HUABIO, Cat# SZ3-07, RRID: AB_3069679), and anti-β-actin ( Proteintech, Cat# 20536-1-AP, RRID: AB_10700003). Network pharmacology analysis Drug targets for Epo B were sourced from the SwissTargetPrediction database, utilizing its the two-dimensional (2D) structure retrieved from PubChem (PubChem CID: 448013). Targets associated with IBD were identified by entering the keyword ”Inflammatory Bowel Disease” in the GeneCards database. Overlapping targets between Epo B and IBD were determined using bioinformatic analyses, and a corresponding Venn diagram was generated. The top 30 common targets were subsequently imported into the STRING database, and the protein category was specified as Homo sapiens . The minimum required interaction score was set to ”medium confidence (0.4),” and the option to ”hide disconnected nodes in the network” was selected to generate the protein-protein interaction (PPI) network visualizing by Cytoscape version 3.9.0. Functional characterization was performed via DAVID database, implementing comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment studies for the overlapping targets. Molecular docking verification Molecular docking analysis was performed using AutoDock Tools 1.5.6 software. The three-dimensional (3D) structure of Epo B was retrieved from PubChem. Crystal structures of the target proteins were obtained from the RCSB Protein Data Bank (PDB) database. These receptor structures were refined through the PyMOL software 2.5.0 (Schrödinger, LLC, New York, NY, USA) by removing the original ligands, solvent molecules, and extraneous organic molecules. Subsequent processing of both receptors and ligands was performed using AutoDock Tools, involving hydrogen addition, rotatable bond identification, and docking parameter configuration. Finally, PDBQT files were saved for modeling purposes, and docking scores were calculated to assess the Epo B-target interaction strength. The lowest energy conformation was identified as the most favorable binding model and visualized using PyMOL. Statistical analyses Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, Inc.). Data are presented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). All experiments were independently repeated at least three times. Comparisons between two groups were assessed using two sample t-test. Comparisons among multiple groups were assessed using one-way ANOVA followed by Tukey’s or Dunnett’s test post hoc test, or by two-way ANOVA analysis followed by Sidak’s multiple comparisons test. P ≤ 0.05 was considered statistically significant. Results An in vivo screening for chemicals modulating neutrophil homeostasis Dysregulation of normal developmental processes frequently influences the onset and progression of pathological changes. Many cellular and molecular mechanisms are shared between homeostasis and disease conditions, which is particularly evident in the extensive crosstalk between neutrophil homeostasis and inflammation 20 . For instance, granulocyte-colony stimulating factor, which is vital for neutrophil production, differentiation, and activation, significantly promotes inflammatory responses in rheumatoid arthritis 21 . Additionally, the inhibition of ErbB2 receptor tyrosine kinase, increases neutrophil apoptosis and thus reduces inflammation in murine models of acute lung injury 22 . These findings underscore the potential of identifying the modulators of neutrophilic inflammation by examining their roles in regulating neutrophil homeostasis. Therefore, in this study, a large-scale chemical screening was performed to identify neutrophil homeostasis modulators followed by validation of their effects on the pathogenesis of IBD. We developed a high-throughput chemical screening protocol using zebrafish neutrophil-specific transgenic line Tg ( mpx : GFP ) i114 . From an initial screening of 1,430 FDA-approved drugs (Fig. 1A) and subsequent manual validation, four chemicals, including Efavirenz (EFV), Irinotecan hydrochloride (IRI), Rosuvastatin calcium (RSVT), and Epo B were found to dose-dependently reduce neutrophil counts (Fig. 1B-F, Table S1). Notably, EFV exhibited biological toxicity at 10 μM, while demonstrating an inhibitory effect on neutrophil numbers at lower, safe dosages (Fig. 1B-C). Epo B and RSVT mitigate intestinal barrier damage induced by DSS To investigate the effects of the aforementioned four drugs on IBD, we established a zebrafish larval enterocolitis model by administering 0.5% DSS from 3 to 6 dpf (Fig. 2A). Dexamethasone (DEX), which ameliorates DSS-induced colonic damage and reduces acute inflammatory responses 23 , served as a positive control. Treated larvae were collected and processed for paraffin sectioning and hematoxylin and eosin staining. The DSS model group exhibited severe intestinal epithelial damage, as evidenced by a decreased depth of the epithelial layer, an increased number of goblet cells, and enlarged intestinal lumen compared with the control group. Notably, EFV, Epo B, and RSVT, similar to DEX, significantly restored the morphological epithelial injury caused by DSS treatment (Fig. 2B-C, Fig.S1). Subsequently, a resazurin reduction assay was performed to analyze intestinal cell proliferation. Significant fluorescent signals were observed in the gut of 6 dpf-control larvae, consistent with previous findings 15 . In comparison with the DMSO control, DSS exposure resulted in a marked reduction of proliferative cells in the intestine, which was partially and significantly restored by treatment with Epo B or RSVT (Fig. 2D-E). These findings suggest that Epo B and RSVT can effectively alleviate the pathological damage associated with DSS-induced enterocolitis and promote tissue repair. Epo B suppresses the inflammatory response in the zebrafish IBD model Given that the infiltration of inflammatory leukocytes is a hallmark of the acute phase of IBD, we further examined and quantified the neutrophils and macrophages labeled with mpx-GFP and mpeg1-mCherry transgenic lines, respectively, in the whole body and intestinal regions of larvae treated with DSS. IRI, Epo B, and RSVT significantly reduced the neutrophil levels in both whole larvae and intestinal tissue (Fig. 3A-C). In contrast, macrophage infiltration was minimally affected by these drugs (Fig. 3D-F). The impact of these drugs on IBD-associated inflammation was assessed by evaluating the expression levels of two pro-inflammatory cytokines, IL-8 and IL-1β. The results indicated that only Epo B treatment significantly attenuated the levels of both cytokines (Fig. 3G-H), similar to the effects observed in the DEX group. Collectively, the aforementioned pathogenic and cellular data suggest that Epo B may be the most effective drug for modulating IBD pathogenesis by suppressing neutrophil infiltration and the production of pro-inflammatory cytokines. Epo B specifically impairs neutrophil homeostasis The selective effect of Epo B in regulating neutrophil numbers, rather than macrophage numbers, in fish IBD models prompted us to investigate whether this effect arises from its intrinsic ability to modulate neutrophils under homeostatic conditions. We then examined the effect of Epo B on various hematopoietic lineages, including hematopoietic stem/progenitor cells (HSPCs), erythrocytes, thrombocytes, neutrophils, macrophages, and non-hematopoietic endothelial cells, using specific transgenic zebrafish reporter lines. Notably, larvae were treated from 5 to 6 dpf, a treatment condition consistent with that used in the DSS fish model. Treatment with Epo B resulted in a significant decrease in neutrophil numbers, as labeled with mpx-GFP (Fig. 4A), while it had no effect on the number of macrophages labeled with mpeg1-mCherry (Fig. 4B-D), erythrocytes labeled with gata1-DsRed (Fig. 4E-F), endothelial cells labeled with kdrl-mCherry (Fig. 4G-H), HSPCs labeled with cd41 : GFP low , or thrombocytes labeled with cd41 : GFP high (Fig. 4I-J). These data suggest that EpoB specifically inhibits neutrophil homeostasis in vivo. The effects of EpoB on neutrophil homeostasis were further investigated in vitro using various purified primary cells and cell lines. The CCK-8 assay quantified cell viability following a 6-h treatment with gradient concentrations of the drug. Primary human neutrophils were purified from the peripheral blood of healthy volunteers using the Ficoll density gradient centrifugation method. The resulting purity exceeded 97% when CD66b and CD16 were used as markers (Fig. S2). Purified human neutrophils exhibit greater sensitivity to Epo B treatment (IC 50 = 2.47 nM) than the closely related monocytic THP1 cells (Fig. S3A-C). Notably, Epo B had a minimal impact on the viability of RAW264.7 macrophages, NCM460 epithelial cells and 293T cells (Fig. S3D-F). These findings suggest that the modulation of neutrophil homeostasis by Epo B represents a specific biological regulatory mechanism, rather than a broad-spectrum or non-selective cytotoxic effect. Epo B stimulates neutrophil apoptosis To investigate the cellular and molecular mechanisms underlying Epo B treatment-induced neutropenia-like phenotypes, network pharmacology was utilized to predict the potential cellular processes and signaling pathways regulated by Epo B in IBD. Using the SwissTargetPrediction database, we predicted 289 potential targets of Epo B, and retrieved 8,525 IBD-related genes from the GeneCards database. Subsequently, 238 intersection targets were identified, as illustrated in the Venn diagram presented in Fig. S4A. GO and KEGG pathway enrichment analyses of these intersection targets indicated their involvement in various cellular processes including various biological functions and signaling pathways, such as the positive regulation of apoptotic processes, cell differentiation and the innate immune response, as well as the phosphoinositide 3-kinase-Akt and mitogen-activated protein kinase signaling pathways (Fig. S4B-C). We subsequently examined whether Epo B regulates neutrophil differentiation and cell death, with a particular focus on apoptosis. Initially, Epo B did not affect neutrophil differentiation in a DMSO-induced differentiation model of HL-60 cells, as evidenced by the expression of CD11b (Fig. S5). Next, we assessed the effect of Epo B on apoptosis in primary human neutrophils under homeostatic and LPS-stimulated conditions, using flow cytometric analysis with 7-AAD/Annexin V staining. Epo B treatment increased the apoptosis rate of human neutrophils in a dose-dependent manner (Fig. 5A-B). No such effect was observed in THP-1, RAW264.7, NCM460, or 293T cells (Fig. S6). These findings strongly indicate that Epo B has a preferential effect on neutrophil apoptosis. To further confirm the effect of Epo B on apoptosis, we analyzed the expression levels of apoptosis-related molecules. Epo B dose-dependently increased the expression of apoptotic markers, including cleaved poly (ADP-ribose) polymerase-1 (PARP1) and Bcl-2-associated X (BAX), while downregulating the expression of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2) (Fig. 5C-F). Finally, we investigated whether Epo B mediates neutrophil apoptosis in the zebrafish model of IBD using a whole-mount TUNEL assay. A greater number of mpx + neutrophils underwent apoptosis in Epo B-treated larvae than in the non-Epo B group (Fig. 6A-B). Notably, the co-treatment with pan-Caspase inhibitor zVAD, known for its anti-apoptotic properties, significantly restored the reduction in neutrophil abundance induced by Epo B (Fig. 6C-D). Collectively, these results indicate that Epo B preferentially promotes neutrophil apoptosis, rather than differentiation or maturation, under both homeostatic and inflammatory conditions. Importantly, these findings strongly suggest that the stimulatory roles of Epo B on neutrophil apoptosis in the context of inflammatory IBD conditions likely arise from its intrinsic ability to modulate neutrophils under homeostatic conditions. Epo B stimulates neutrophil apoptosis by targeting Caspase-3 To identify the direct target for Epo B in IBD, a PPI network was constructed with 238 intersection targets between Epo B and IBD using the network pharmacology analysis. The top 30 hub genes among the intersection targets were ranked using the degree method in Cytoscape (Fig. 7A; Table S2). Among these genes, CASP3, PPARG and PARP1 are directly associated with the apoptosis regulation. To elucidate the potential interactions between Epo B and these core targets, we conducted a molecular docking analysis with the top 10 hub genes from the PPI. Interestingly, the results revealed the lowest binding energy (-7.06 kcal/mol) between Epo B and the receptor protein Caspase-3 which involves the amino acid residues LEU33, HIS278 and SER36, through hydrogen bonds (Fig. 7B, Table S3). Consistently, the western blot results further confirmed that Epo B treatment promoted the cleavage of Caspase-3 (Fig. 7C-D). Given the cleavage of Caspase-3 is critical for its activation and function in promoting cell apoptosis, our results suggest that Epo B may directly target Caspase-3 to promote its cleavage and activation, thus triggering neutrophil apoptosis in IBD. Epo B restores gut microbiota defects in DSS-induced IBD The imbalance and dysfunction of gut microflora are recognized as key characteristics of DSS-induced colitis 24 . Following our demonstration of intestinal barrier repair and the immunomodulatory effects of Epo B, we further investigated whether Epo B could remodel the gut microbiota in IBD. We performed 16S rRNA sequencing to analyze the microbiota in the GIT of treated larvae. Exposure to DSS resulted in a significant reduction in the gut microbial diversity, as evidenced by a decrease in the number of OTUs, which was significantly restored by Epo B treatment (Fig. 8A). PCoA using unweighted UniFrac for β-diversity among each genotype revealed that Epo B effectively restored the disrupted gut microbiota induced by DSS to a composition that resembled a normal microbial community (Fig. 8B). Furthermore, Epo B treatment notably restored gut microbial richness and α-diversity, as indicated by α-diversity indices, including the Species Richness index and Chao1 index, which had been significantly reduced in the DSS-induced zebrafish inflammation model (Fig. 8C-D). At the phylum level, the bacterial community detected in all samples (≥ 90%) predominantly consisted of five phyla: Bacteroidota, Firmicutes, Proteobacteria, Actinobacteriota, and Verrucomicrobiota. DSS exposure led to a reduction in the relative abundance of Bacteroidota and an increase in Proteobacteria within the GITs (Fig. 8E). Remarkably, the relative abundances of these bacterial phyla were restored to levels comparable to those in the control group following Epo B treatment. At the order level, GITs in larvae treated with DSS exhibited decreased relative abundance of Bacteroidales and an increased relative abundance of Burkholderiales, both of which were restored by Epo B treatment (Fig. S7). These findings indicate that Epo B effectively mitigates DSS-induced dysbiosis of the gut microbiota in zebrafish colitis. Discussion In the current study, we performed a large-scale chemical screening of chemicals that modulate neutrophil numbers under homeostatic conditions. We further investigated the effects of four drugs identified in this screening using a DSS-induced zebrafish model of IBD. Notably, Epo B exhibited robust therapeutic effects in the zebrafish IBD model, leading to the alleviation of intestinal epithelial barrier disruption, inflammatory response, and rebalancing of the gut microbiota. Our findings also show that Epo B preferentially induces neutrophil apoptosis. Neutrophils are apoptosis-resistant and are involved in the release of ROS, MPO, and pro-inflammatory cytokines in IBD, which contribute to inflammation and intestinal damage 2-4 . Integrating these previous findings with our observations, we propose a model for the molecular mechanisms underlying Epo B-regulated IBD pathogenesis (Fig. S8). Specifically, Epo B enhances neutrophil apoptosis, possibly by targeting Caspase-3 and promoting its activation, resulting in a reduction in neutrophil abundance in the intestine and diminishing its role in exacerbating IBD pathogenesis. As a recurrent chronic condition, IBD therapy presents several challenges, including the need for long-term medication, significant risk of drug resistance, and high remission rates 125, . Consequently, exploring new therapeutic strategies for IBD becomes crucial. High-throughput chemical screening is an efficient and robust method for discovering new small-molecule drugs for various diseases 26 . However, only a limited number of studies have implemented chemical screening for IBD. Oehlers et al. conducted a high-content small-molecule screen using DSS-induced zebrafish models, to identify drugs affecting neutrophil numbers 27 . Nonetheless, their study did not address whether these identified compounds influenced intestinal tissue injury or the gut microbiota. In a separate study, Yu et al. used a TNBS-induced zebrafish IBD model to perform a small-scale screening of 74 formulations 28 . Their investigation quantified neutrophil numbers and ROS levels, and further explored the effects of these formulations on zebrafish intestinal epithelial barrier injury and inflammatory cytokine expression. However, neither of these studies examined the cellular and molecular mechanisms underlying the effects of the identified compounds. Here, we hypothesize that drugs capable of modulating neutrophil homeostasis could potentially be utilized in the treatment of IBD, given the critical role of neutrophils in its pathogenesis. To this end, we conducted a comprehensive high-content screening of 1,430 drugs was performed to identify modulators of neutrophil numbers under homeostatic conditions. Importantly, we validated the hit compounds in zebrafish IBD models by assessing epithelial tissue injury, inflammation, and gut microbiota. Additionally, we elucidated the cellular and molecular mechanisms by which Epo B regulates IBD severity. Drug repurposing has become an attractive research avenue because of the established safety profiles of existing medications, their low costs, and shortened development timelines 29 . In this study, we screened an FDA-approved drug library. We identified four drugs from the screening and confirmed their efficacy by dosage gradient testing. By investigating their effects on intestinal barrier injury in a DSS-induced zebrafish IBD model, we found that Epo B and RSVT exhibited significant protective effects against epithelial tissue damage. Additionally, these two drugs effectively suppressed neutrophil infiltration in the intestine, as well as the expression of inflammatory cytokines, including IL-8 and IL-1β. Notably, RSVT reportedly protects against DSS colitis by inhibiting mucosal inflammatory responses in mouse models 30 . These findings support the feasibility and effectiveness of our approach for identifying potential IBD modulators through chemical screening aimed at targeting neutrophil homeostasis. Given that Epo B has never been reported to affect IBD progression, we specifically focused on exploring the cellular and molecular mechanisms by which Epo B modulates DSS colitis. Epothilone is a natural 16-membered macrolide compound produced through the metabolism of the cellulose-degrading myxobacterium Sorangium cellulosum . Epo B and its derivatives are used as chemotherapeutic agents in various cancers because they interact with and stabilize tubulin, thereby inhibiting cancer cell division 31 . In the present study, Epo B significantly increased neutrophil apoptosis without affecting terminal differentiation. Given that mature neutrophils do not undergo cell division, the mechanism by which Epo B induces neutrophil death is unlikely to involve cell cycle arrest via tubulin targeting, which results in defective microtubule turnover. Instead, network pharmacology analysis supported our speculation that Epo B may target apoptosis-associated molecules, including CASP3 and PPARG. Furthermore, molecular docking results suggest that Epo B may directly interact with Caspase-3 to promote neutrophil apoptosis. Interestingly, Epo B preferentially stimulated neutrophil apoptosis compared to other hematopoietic lineages, as well as non-hematopoietic epithelial and endothelial cells, both in vivo in zebrafish larvae (Fig. 4) and in vitro in cell lines (Fig. S3 and S6). The mechanism underlying the selective pro-apoptotic effect of Epo B remains unknown and should be addressed in future studies. We speculate that this bias may arise from the intrinsic differences between neutrophils and other cell types, as mature and aged neutrophils are short-lived and exhibit a high rate of constitutive apoptosis. This predisposition to apoptosis in neutrophils is likely driven by the highly constitutive expression of several pro-apoptotic proteins of the Bcl-2 family, such as Bax and Bak 32 . These characteristics may render neutrophils more susceptible to Epo B stimulation. Epo B has also been reported to induce apoptosis in cancer cells 33 . However, the underlying macular mechanisms remain unknown. Our findings provide a mechanistic explanation for Epo B-mediated apoptosis. Notably, the clinical treatment of cancers with Epo B analogs often results in neutropenia 34-36 , which is considered a myelosuppressive effect common to many chemotherapy drugs. Our findings indicate a direct pro-apoptotic role for Epo B in neutrophils. However, based on our current data, we cannot rule out the possibility of general myelosuppressive mechanisms associated with Epo B, even though we did not observe suppression of the production of HSPCs, macrophages, erythrocytes, or platelets in the Epo B-treated zebrafish larvae. Although Epo B has primarily been used clinically for its antitumor activity, recent studies have demonstrated its anti-inflammatory and tissue repair effects. For instance, Epo B inhibits osteoclastogenesis via the signal transducer and activator of transcription 3 signaling pathway, thereby preventing LPS-induced inflammatory osteolysis 37 . Additionally, Epo B inhibits LPS-induced endothelial cell barrier dysfunction and reduces vascular leakage and pulmonary inflammation 38 . In the present study, Epo B treatment suppressed inflammation and injury associated with IBD by decreasing neutrophil abundance in the intestine through the stimulation of apoptosis. Notably, Epo B also induces apoptosis of mature osteoclasts 37 , which may contribute to the suppression of LPS-induced osteolysis. Together, these studies, along with our findings, indicate that Epo B plays a significant anti-inflammation role and may potentially be utilized in treating cancer patients suffering from inflammatory diseases such as IBD. Furthermore, apart from the induction of neutrophil apoptosis, whether Epo B directly affects neutrophil function to modulate IBD remains to be addressed in future studies. In the present study, the administration of Epo B effectively restored the intestinal bacterial microbiota in a zebrafish model of IBD. However, the precise mechanism underlying this phenomenon requires further investigation. Compelling evidence supports the existence of interactions between neutrophils and the gut microbiota within the context of inflammation 39 . During chronic inflammation, neutrophils promote the growth of pathogens, particularly facultative anaerobes, through the production of ROS, which is especially notable in the Enterobacteriaceae species from phylum Proteobacteria 4041, . Additionally, antimicrobial peptides from neutrophils play a role in regulating the interaction between gut microbiota and the host immune response such as LPS modification in Bacteroides 42 . Therefore, the effect of Epo B on regulating the balance of intestinal microbiota may stem from the direct action of neutrophils on bacteria or an indirect role in repairing the intestinal epithelial barrier. This study had several limitations. For instance, the therapeutic effects of Epo B on IBD were evaluated using a zebrafish model and have yet to be validated in higher model organisms, such as mouse models. Additionally, the reasons behind Epo B’s selective effect on neutrophil apoptosis remain unclear. Our network pharmacology, molecular docking and western blot analyses indicate that Epo B may directly target Caspase-3, an apoptosis-associated molecule to promote its cleavage and activation, thus enhancing neutrophil apoptosis and subsequent protection against IBD. However, the direct interaction between Epo B and Caspase-3 and its functional outcomes in IBD require further experimental validation. These limitations should be investigated in- depth in future studies. Despite these limitations, based on chemical screening and subsequent functional and mechanistic studies, we propose that Epo B is a promising therapeutic agent for the treatment of IBD, particularly in the context of co-occurring cancers. Conclusions This study identified the protective roles and mechanisms of action of Epo B in IBD through high-throughput screening in zebrafish, focusing on its effects on neutrophils. Epo B significantly alleviated the symptoms of DSS-induced enterocolitis by promoting neutrophil apoptosis. These findings provide novel insights and guidance for drug repurposing as well as the development of new therapeutic strategies for IBD. Funding This work was supported by National Natural Science Foundation of China (No. 32170766), Chongqing Municipal Science and Technology Bureau (No. CSTB2022NSCQ-MSX1682), the National Research Center for Translational Medicine at Shanghai (NO.NRCTM(SH)-2025-08) and China Postdoctoral Science Foundation (No.2024MD764043). CRediT authorship contribution statement Zhenting He: Conceptualization, Data curation, Methodology, Software, Visualization, Writing – original draft. Ziling Deng: Conceptualization, Data curation, Methodology, Visualization, Writing – review & editing. Huan Zhang: Conceptualization, Data curation, Visualization, Writing – review & editing. Yiming Xiang: Data curation, Writing – review & editing. Jiashi Guo: Data curation, Writing – review & editing. Yi Chen: Software, Validation. Jingjing Qin: Software, Validation. Ling Meng: Software, Validation. Fengmin Xu: Software, Validation. Xiaowen Wang: Software. Siyuan Hou: Validation. Hao Yuan: Conceptualization, Methodology, Writing – review & editing. Chunguang Ren: Conceptualization, Methodology, Writing – review & editing. Ethics declarations All animal studies were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University (Approval No. IACUG-CQMU-2024-0139). Collection of human peripheral blood sample was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (Approval No. K2024-158-01). All donors provided written informed consent prior to specimen acquisition. Consent for publication Not applicable. Conflict of interests The authors declare no competing interests. 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( A ) Schematic representation of experimental design for high-throughput chemical screening. ( B ) Representative images of Tg(mpx:GFP) zebrafish embryos at 3-dpf upon treatment of indicated drugs at 10 μM. Scale bar, 200 µm. ( C - F ) Quantification of total neutrophils in embryos exposed to the specified drugs at various concentrations. Each data point represents an individual embryo (n > 20). Error bars represent mean ± SD. **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DMSO group). hpf, hours post-fertilization; dpf, days post-fertilization. EFV, efavirenz; IRI, irinotecan hydrochloride; Epo B, epothilone B; RSVT, Rosuvastatin calcium; Conc, concentration. Figure 2. Epo B and RSVT mitigate intestinal barrier damage induced by DSS. ( A ) Schematic representation of experimental design for DSS-induced zebrafish enterocolitis model and drug treatment. ( B ) Representative images of transverse sections from the intestinal bulb (IB) of zebrafish larvae, stained with hematoxylin and eosin. Scale bar, 50 µm. Dashed blue ovals indicate intestinal lumens. ( C ) Quantification of histological scores for each treatment group outlined in (B) (n > 5). ( D - E ) Representative images (D) and quantitative analysis (E) of whole-mount resazurin staining of larvae subjected to the indicated drug treatments (n>30). Scale bar, 200 µm. Dashed white lines and white asterisks denote the intestinal area and otic vesicle, respectively. The concentration for each drug was listed in Table S1. Each data point represents an individual larva. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DSS+DMSO group). DEX, dexamethasone. Figure 3. Epo B suppresses the inflammatory response within the zebrafish IBD model. ( A - F ) Representative images (A, D) and quantitative analysis (B, E, whole body; C, F, intestine) of 6-dpf Tg(mpx:GFP) or Tg ( mpeg1 : Gal4 ); Tg ( UAS : Nfsb-mCherry ) zebrafish larvae subjected to DSS and/or the treatments of indicated drugs. Scale bar, 200 µm. ( G - H ) Quantitative analysis of IL-8 (G) and IL-1β (H) expression in 6-dpf wild-type zebrafish larvae subjected to the specified drug treatments. Each data point represents an individual larva (B, C, E, F) (n > 30) and a biological replicate (G-H) (n = 3), respectively. Error bars represent mean ± SD in (B, C, E, F) and SEM in (G-H). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DSS+DMSO group). Figure 4. Epo B impairs neutrophil homeostasis in zebrafish larvae. ( A - B ) Quantification of total fluorescent cells in 6-dpf Tg(mpx:GFP) (A) and Tg ( mpeg1 : Gal4 ); Tg ( UAS : Nfsb-mCherry ) (B), zebrafish larvae exposed to Epo B by manual counting of fluorescent cells. ( C - J ) Representative images (C, E, G, I) and quantitative analysis (D, F, H, J) of flow cytometry analysis of 6-dpf Tg(mpeg1:Gal4);Tg(UAS:Nfsb-mCherry) (C-D), Tg ( gata1 : DsRed ) (E-F), Tg ( kdrl : mCherry ) (G-H) and Tg ( cd41 : GFP ) (I-J) zebrafish larvae subjected to the indicated drug treatments. The c d41 : GFP low and cd41 : GFP high cells represent HSPCs and thrombocytes respectively. Each data point represents an individual larva (A, B) (n > 30) and a biological replicate (D, F, H, J) (n ≥ 4), respectively. Error bars represent mean ± SD in (A, B) and SEM in (D, F, H, J). **P < 0.01; ns, not significant (by unpaid t-test compared to DMSO group in A, B, D, F and H, by two-way ANOVA with Sidak’s multiple comparisons test compared to DMSO group in J). HSPCs, hematopoietic stem/progenitor cells. Figure 5. Epo B promotes neutrophil apoptosis in vitro . ( A - B ) Representative flow cytometric plots (A) and quantification (B) of Annexin V/7-AAD-stained primary human neutrophils treated with Epo B/LPS. ( C - F ) Representative immunoblots (C) and quantitative analysis of cleaved-PARP-1 (D), BCL-2 (E), and BAX (F) in primary human neutrophils treated with Epo B/LPS. Each data point represents an individual biological replicate (n ≥ 3). Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Tukey’s post hoc test). LPS, lipopolysaccharide. Figure 6. Epo B promotes neutrophil apoptosis in DSS-induced zebrafish IBD. ( A - B ) Representative images (A) and quantitative analysis (B) of Whole-mount TUNEL (Cy3) staining of 6-dpf Tg(mpx:GFP) zebrafish larvae subjected to the DSS/EpoB treatments. The caudal hematopoietic tissue (CHT) regions were presented, with the apoptosis rate defined as the percentages of TUNEL + GFP + neutrophil. Scale bar, 100 µm. ( C-D ) Quantitative analysis (C, whole body; D, intestine) of 6-dpf Tg(mpx:GFP) zebrafish larvae subjected to DSS/Epo B/zVAD (300 μM) treatments. The white arrowhead denotes the TUNEL and GFP double-positive cells. Each data point represents an individual fish larva (n ≥ 10). Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Tukey’s post hoc test). Figure 7. PPI network and molecular docking analysis of Epo B potential target. ( A ) PPI network of the top 30 hub target genes between Epo B and IBD. ( B ) Molecular docking patterns between Epo B and Caspase-3. ( C-D ) Representative immunoblots (C) and quantitative analysis of cleaved Caspase-3 (D) in primary human neutrophils treated with Epo B/LPS. Each data point represents an individual biological replicate (n ≥ 3). Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (by one-way ANOVA with Tukey’s post hoc test). Figure 8. Epo B rectifies gut microbiota defects in DSS-induced zebrafish. ( A ) Venn diagrams of bacterial OTUs in the GITs. ( B ) PCoA score plot based on unweighted UniFrac. ( C-D ) Richness index (C) and Chao1 index (D) of α-diversity. (E) Phylum-level relative abundance of the gut microbiota. Each data point represents an individual biological replicate (n = 5). *P < 0.05, ***P < 0.01, (by one-way ANOVA with Tukey’s post hoc test). OTUs, operational taxonomic units; GITs, gastrointestinal tracts. Supplementary Materials Figure S1. Epo B and RSVT mitigate DSS-induced intestinal pathological injury. ( A-B ) Representative images of transverse sections from the mid-intestine (MI) (A) and posterior intestine (PI) (B) of 6-dpf zebrafish larvae, stained with hematoxylin and eosin. Scale bar, 50 µm. The black arrowhead denotes goblet cells, and the red arrow indicates epithelial cells that have shed into the intestinal lumen. Figure S2. Purity validation of isolated human peripheral blood neutrophils. Flow cytometric Gating strategy for the examination of purity of human primary neutrophils identification of neutrophils by anti-CD11b, CD66b and CD16. Figure S3. Epo B preferentially suppresses neutrophil cell viability. (A - F ) Quantitative analysis of cell viability of Epo B-treated primary human neutrophils (A-B), THP-1 (C), RAW264.7 (D), NCM460 (E), and 293T cells (F) by CCK-8 assay. Each data point represents an individual biological replicate (n = 3). Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DMSO group). Figure S4. Network pharmacological analysis of Epo B in IBD. ( A ) Venn diagram of Epo B and IBD targets. ( B - C ) GO function (B) and KEGG pathway (C) enrichment analysis of the overlapping targets between Epo B and IBD. Figure S5. Minimal effect of Epo B on neutrophil differentiation. ( A-B ) Representative images (A) and quantitative analysis (B) from flow cytometry assessing the effects of Epo B on DMSO-induced neutrophil differentiation from HL-60 cells. The differentiation efficiency was quantified based on the ratio of CD11b+ cells. Each data point represents an individual biological replicate (n = 3). Error bars represent mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DMSO group). Figure S6. Minimal effects of Epo B on apoptosis in multiple cell lines. ( A - D ) Quantitative analysis of apoptosis, defined as the percentage of Annexin V-FITC-positive cells in THP-1 (A), RAW264.7 (B), NCM460 (C), and 293T cells (D). Each data point represents an individual biological replicate (n = 3). Error bars represent mean ± SD. *P < 0.05 and **P < 0.01; ns, not significant (by one-way ANOVA with Dunnett’s post hoc test compared to DMSO group). Figure S7. Diagram of a working model for Epo B-stimulated neutrophil apoptosis to mitigate IBD pathology. Table S1. Detailed information regarding the dosages and the associated morphological phenotypes of the identified hit compounds Table S2. Target degree values of Top 30 hub genes among the intersection targets between Epo B and IBD Table S3 . Affinities and amino acid sites of ligand-protein interaction in core targets detected by molecular docking Supplementary Material File (table s1.docx) Download 22.27 KB File (table s2.docx) Download 18.28 KB File (table s3.docx) Download 17.84 KB Information & Authors Information Version history V1 Version 1 08 August 2025 Peer review timeline Published Genes & Diseases Version of Record 1 Dec 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Zhenting He Chongqing Medical University View all articles by this author Ziling Deng Chongqing Medical University View all articles by this author Huan Zhang Chongqing Medical University View all articles by this author Yiming Xiang Chongqing Medical University View all articles by this author Jiashi Guo Chongqing Medical University View all articles by this author Yi Chen Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital View all articles by this author Jingjing Qin Chongqing Medical University View all articles by this author Ling Meng Chongqing Medical University View all articles by this author Fengmin Xu Nanchong Central Hospital Affiliated to North Sichuan Medical College View all articles by this author Xiaowen Wang The First Affiliated Hospital of Chongqing Medical University View all articles by this author Siyuan Hou Chongqing Depu Foreign Language School View all articles by this author Hao Yuan Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital View all articles by this author Chunguang Ren 0000-0003-3245-1159 [email protected] Chongqing Medical University View all articles by this author Metrics & Citations Metrics Article Usage 168 views 122 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zhenting He, Ziling Deng, Huan Zhang, et al. 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