JiangFuZhiXie pill ameliorates DSS-induced ulcerative colitis mice via modulating intestinal flora and inhibiting IL6/STAT3 signaling to reduce inflammatory response

JiangFuZhiXie pill ameliorates DSS-induced ulcerative colitis mice via modulating intestinal flora and inhibiting IL6/STAT3 signaling to reduce inflammatory response | 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 JiangFuZhiXie pill ameliorates DSS-induced ulcerative colitis mice via modulating intestinal flora and inhibiting IL6/STAT3 signaling to reduce inflammatory response Mengyuan Wang, Dengming Lu, Tingyu Zhang, Li Jin, Zhiming Ge, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9338335/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Current treatments for ulcerative colitis (UC) are unsatisfactory. JiangFuZhiXie pill (JFZX) is an in-hospital traditional Chinese medicine widely used in clinical practice for the treatment of UC, with excellent therapeutic effects. Purpose To explore the specific effects and underlying mechanisms of JFZX in the treatment of UC and further search for new therapeutic targets. Methods The UC mice model was established by free access to a 3% dextran sulfate sodium aqueous solution and intervention with JFZX via intragastric administration. Mouse serum was used to detect inflammatory factor levels. Mouse colon tissues were used for pathological, immunohistochemical, and immunofluorescence staining. Mouse colon tissues and intestinal contents were used for transcriptomic analysis and metagenomic testing, respectively. Gene expression was detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot. Results The intervention with JFZX significantly increased body weight and colonic tight junctions in UC mice, and decreased disease score, pathological score, and inflammatory level. Metagenomic analysis results showed that JFZX modulates the abundance of key bacterial populations and improves intestinal flora disorders in UC mice. The transcriptomic and RT-PCR analysis indicated that the key pathway regulated by JFZX in the treatment of UC is the IL6/STAT3 pathway. JFZX intervention significantly downregulated the expression of key targets, including Stat3, Il6, Lif, Il10, Il19, and Csf3. Conclusion JFZX has therapeutic effects in UC and effectively inhibits intestinal inflammation. Its mechanism of action involves rebalancing the intestinal flora and inhibiting the IL6/STAT3 signaling pathway, thus reducing intestinal inflammation damage. Ulcerative colitis Traditional Chinese medicine JiangFuZhiXie pill IL6/STAT3 signaling Intestinal flora Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Ulcerative colitis (UC) is characterized by symptoms including abdominal discomfort, diarrhea, hematochezia, and weight loss ( 1 ). In recent years, the incidence rate of UC has been steadily increasing, causing an increasingly heavy burden of disease ( 2 ). The precise etiological mechanisms underlying UC pathogenesis remain incompletely understood, while existing therapeutic approaches show restricted effectiveness and unfavorable long-term outcomes. Current clinical interventions, such as 5-ASA compounds, immunomodulatory agents, glucocorticoids, and biologic treatments, provide only symptomatic control rather than disease resolution, with some patients exhibiting suboptimal therapeutic responses ( 3 ). Thus, this clinical scenario underscores the pressing need for innovative treatment modalities in UC management. Traditional Chinese medicine (TCM) employs a distinctive therapeutic strategy that combines multiple herbal components to simultaneously target various pathological factors. This comprehensive approach offers particular benefits in managing ulcerative colitis (UC) cases ( 4 ). Based on TCM theory, most UC patients exhibit clinical manifestations consistent with yang deficiency and dampness accumulation ( 5 ). Consequently, herbal formulations with warming and dampness-eliminating properties are commonly prescribed, as they facilitate the removal of dampness and alleviate diarrhea by strengthening intestinal function ( 6 ). The present investigation focuses on JiangFuZhiXie pill (JFZX), a hospital-formulated TCM preparation widely used in clinical settings for the management of diarrhea and UC, demonstrating remarkable clinical efficacy ( 7 , 8 ). JFZX incorporates numerous bioactive constituents with potential UC-modulating effects, including Radix Ginseng, Dried Ginger‌, Largehead Atractylodes Rhizome, Liquorice Root, Prepared Common Monkshood Daughter Root, Malaytea Scurfpea Fruit, Nutmeg, Chinese Magnoliavine Fruit, Radix Saposhnikoviae, Dried Tangerine Peel, Rhizoma Coptis, Halloysit, Medicated Leaven, Hawthorn Fruit, and Chinese Thorowax Root. The herbal components of JFZX demonstrate significant therapeutic potential for UC through diverse mechanisms of action. Research indicates that Radix Ginseng alleviates UC symptoms by modulating gut microbiota composition ( 9 ); Dried Ginger's volatile oil facilitates intestinal mucosal repair by stimulating epithelial cell regeneration ( 10 ); Liquorice Root's primary active constituent provides mucosal protection through modulation of Nrf2 and NF-κB signaling pathways in UC models ( 11 ); Chinese Magnoliavine Fruit could protect against UC in mice via regulating gut microbiota and TLR4/NF-κB/NLRP3 pathway ( 12 ); Dried Tangerine Peel exhibits efficacy in restoring mucus barrier integrity, particularly in high-fat diet conditions ( 13 ); Coptisine from Rhizoma Coptis maintains colonic epithelial homeostasis in UC models through aryl hydrocarbon receptor activation, as evidenced in both in vivo and TNF-α-induced organoid studies ( 14 ); the polysaccharide derived from Crataegus pinnatifida, commonly known as hawthorn fruit, has demonstrated potential in mitigating UC through its ability to boost arginine production and modulate gut microbial composition ( 15 ). These findings provide additional evidence supporting the therapeutic efficacy of JFZX in the management of UC. Nevertheless, the precise mechanisms and therapeutic impacts of JFZX in UC treatment require further investigation. Dextran sulfate sodium (DSS; molecular weight range: 36–50 kDa) is widely employed to establish UC models due to its capacity to increase intestinal permeability, compromise the mucosal barrier, elevate pro-inflammatory cytokine levels, and induce gut microbiota imbalance ( 16 , 17 ). Based on a DSS-induced UC mouse model, this research aims to explore the effects of JFZX in the treatment of UC and to further elucidate its mechanism using multi-omics methodologies. 2. Methods 2.1 Chemical reagents Dextran sulfate sodium (DSS, molecular weight: 36–50 kDa) was acquired from MP Biomedicals (Santa Ana, US). JiangFuZhiXie pills (JFZX) were acquired from the Preparation Center of Yinchuan Traditional Chinese Medicine Hospital. 2.2 Preparation of JFZX The herbs of JFZX were wrapped in non-woven bags, and sufficient water was added to decoct them twice, each time for 2 hours, after which the liquid medicine was concentrated. 2.3 Chemical constituents of JFZX by HPLC-Q/TOF-MS analysis JFZX formulation incorporates sixteen distinct herbal components, with their specific quantitative ratios presented in Table 1 . Component characterization was performed utilizing a high-resolution Waters Synapt G2-Si Qtof mass spectrometer coupled with Unifi analytical software at Tsinghua University's Pharmaceutical Technology Research Center. The analytical methodology aligns with the protocol established in our prior investigation ( 18 ). Comprehensive data regarding the UHPLC‒MS/MS analytical outcomes are accessible in Supplementary Table S1 . Table 1 Constituents of JFZX Chinese name Latin name English name Proportion (g) Renshen Radix Panacis Ginseng‌ Radix Ginseng 3 Ganjiang Rhizoma Zingiberis Dried Ginger‌‌ 3 Baizhu Rhizoma Atractylodis Macrocephalae Largehead Atractylodes Rhizome 11 Gancao Glycyrrhizae Radix et Rhizoma‌ Liquorice Root‌‌ 3 Fuzi Aconiti Lateralis Radix Praeparata‌ ‌ Prepared Common Monkshood Daughter Root 3 Buguzhi Fructus Psoraleae Malaytea Scurfpea Fruit‌ 2 Roudoukou Myristica Fragrans Houtt Nutmeg 4 Wuweizi Fructus Schisandrae Chinensis Chinese Magnoliavine Fruit‌ 4 Fangfen Saposhnikovia Divaricata‌(Turcz.) Schischk Radix Saposhnikoviae 5 Chenpi Pericarpium Citri Reticulatae‌ Dried Tangerine Peel‌ 8 Huanglian Coptis Chinensis Franch Rhizoma Coptis 1 Chishizhi Halloysitum Rubrum Halloysit 4 Liushenqu Massa Medicata Fermentata‌ Medicated Leaven 11 Shanzha Fructus Crataegi Hawthorn Fruit 11 Chaihu Bupleuri Radix Chinese Thorowax Root 4 2.4 Experimental Animals Male C57BL/6J mice aged 4–6 weeks with body weights ranging between 18–22 grams were procured from Beijing Vital River Laboratory Animal Technology Company. The subjects were maintained in a sterile environment with controlled access and adhered to a 12-hour light/dark photoperiod. Environmental conditions were strictly regulated, with the ambient temperature kept at 22°C (± 3°C) and the humidity maintained at 50% (± 10%). Throughout the study period, the animals had ad libitum access to food and water. Following a seven-day adaptation phase, all experimental procedures were initiated. 2.5 Establishment of the ulcerative colitis model and grouping The experimental animals were divided into six distinct cohorts (6 mice per group) through a randomization process: the untreated control cohort, DSS-treated cohort, MSLZ-treated cohort (receiving Mesalazine at 0.5 g/kg from Ethypharm Company, China), JFZX-L cohort (administered 3.5 g/kg, matching the standard human therapeutic dose of JFZX), and JFZX-H cohort (given 7.5 g/kg, representing double the standard human dose of JFZX). Ulcerative colitis was experimentally induced by providing unrestricted access to a 3% DSS aqueous solution for 7 days. While the control and DSS groups received distilled water via oral gavage, the MSLZ, JFZX-L, and JFZX-H groups were given their respective medications using identical administration methods as illustrated in Fig. 2 A. Daily monitoring included comprehensive assessments of physical condition, body mass fluctuations, and fecal characteristics. Following a seven-day experimental period, all subjects were humanely euthanized, and biological specimens (including blood samples, fecal matter, and colonic tissue) were collected and cryopreserved at -80°C for subsequent investigations. 2.6 Disease activity index (DAI) analysis The DAI was determined according to the following rating sheet (Table 2 ): Table 2 The DAI rating sheet Score Body weight loss Stool morphology Fecal blood 0 no weight loss regular consistency normal 1 1–5% reduction soft but firm mild positive blood 2 6–10% reduction soft positive blood 3 11–18% reduction wet blood in the stool 4 reduction exceeding 18% watery diarrhea active bleeding 2.7 Chemiluminescence immunoassay detection Detected the IL6, IL-1β, and TNF-αin mice serum by Servicebio chemiluminescence immunoassay kits (GLM0004 Mouse IL-6 MPCLIA Kit, GLM0005 Mouse TNF-α MPCLIA Kit, GLM0010Mouse IL-1β MPCLIA Kit) according to the manufacturer’s instructions. 2.8 Histopathological staining Colon tissue from experimental mice was fixed in 4% paraformaldehyde (G1101, Servicebio, China) and rinsed under running water. Subsequently, the specimen should be dehydrated using graded ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd, China). The specimen should then be cleared using xylene (10023418, Sinopharm Chemical Reagent Co., Ltd, China). Following paraffin embedding and sectioning, hematoxylin and eosin (H&E) staining was performed using a Servicebio kit (G1004, Servicebio, China). Assessment of glycogen deposition in colon sections was performed using Periodic Acid-Schiff (PAS) staining with a specific kit (G1008, Servicebio, China). The slices are imaged and scanned by the slicing scanner. The positive results were then analyzed using ImageJ. And the HS score was determined according to the following rating sheet (Table 3 ): Table 3 The standard HS score Score Damaged area Mucodepletion of glands Tissue damage Inflammatory cell infiltration 0 \ None No mucosal damage Occasional inflammatory cells in the lamina propria 1 ≤ 25% Mild Discrete epithelial lesions Increased numbers of inflammatory cells in the lamina propria 2 ≤ 50% Moderate Surface mucosal erosion or focal ulceration Confluence inflammatory cells, extending into the submucosa 3 ≤ 75% Moderate Extensive mucosal damage and extension into deeper structures of the bowel wall Transmural extension of the infiltrate 4 ≤ 100% Severe \ \ 2.9 Immunohistochemistry (IHC) staining For immunohistochemical analysis, colon tissue sections underwent a series of preparatory steps, including deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inactivation, and blocking with normal goat serum. The primary antibody targeting CD68 (GB153109; Servicebio, China), a rabbit polyclonal antibody, was carefully applied and incubated in a humidified chamber at 4°C for approximately 24 hours. Following this incubation period, the sections were treated with both the reaction solution and horseradish peroxidase-conjugated anti-rabbit secondary antibody (GB23303; Servicebio, China), maintained at 37°C for half an hour. The chromogenic reaction was developed using diaminobenzidine (G1212; Servicebio, China), producing a distinct reddish-brown coloration. Cellular structures exhibiting cytoplasmic brown-yellow granular staining were identified as positive expression. Digital imaging of the sections was performed using a specialized scanning system, and subsequent quantitative analysis of positively stained regions and cells was conducted in ImageJ. 2.10 Immunofluorescence(IF)staining The tissue sections underwent identical procedures for both dewaxing and antigen retrieval. Initially, a self-fluorescence-quenching agent was applied, followed by a 5-minute incubation period. Subsequently, the specimens were washed under running water and incubated in serum for 30 minutes. Pre-formulated primary antibodies, specifically rabbit polyclonal antibodies against ZO-1 (GB15195; Servicebio, China) and Occludin (GB151401; Servicebio, China), were then added, and the samples were maintained at 4°C overnight. Tissue sections were then treated with species-matched secondary antibodies. This was followed by a 50-minute dark incubation at ambient temperature. Nuclear staining was subsequently performed using DAPI. Microscopic examination and data interpretation concluded the experimental process. 2.11 RNA sequencing Total RNA was extracted from the mice colon tissues using TRIzol® Reagent according to the manufacturer’s instructions (Invitrogen, US). RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA). The detailed information was described in our previous study ( 18 ). The data were analyzed using the free online platform of the Majorbio Cloud ( www.majorbio.com ). 2.12 Metagenomic sequencing Genomic DNA was isolated from 200 mg of murine colonic samples using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China) according to the manufacturer's standard protocol. The obtained DNA samples were then sheared into fragments averaging around 350 base pairs using mechanical disruption with the Covaris M220 (Gene Company Limited, China), preparing them for paired-end sequencing library preparation. Library preparation was performed using NEXTFLEX Rapid DNA-Seq reagents (Bioo Scientific, Austin, TX, USA). Sequencing was performed on the Illumina NovaSeq™ X Plus platform (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), using NovaSeq X Series 25B Reagent Kits according to the manufacturer's guidelines. Subsequent data processing was performed using the Majorbio Cloud online analysis platform ( www.majorbio.com ). 2.13 Quantitative Real-time PCR (qRT-PCR) The conversion of total RNA into complementary DNA was carried out utilizing a quantitative PCR reverse transcription master mix kit (TransGen, China). Quantitative reverse transcription polymerase chain reaction analysis was conducted on a real-time PCR instrument (Thermo Fisher, USA) employing SYBR Green-based real-time PCR master mix reagents (TransGen, China). Specific primer sequences employed for amplification are detailed in Supplementary Table S2 , with β-actin serving as the reference gene. All experimental procedures were conducted with three technical replicates and independently repeated across three separate experimental runs. 2.14 Western blot Protein samples were resolved using 10% SDS-PAGE gels (Epizyme Biomedical Technology, PG112, CN) followed by electroblotting onto PVDF membranes (Epizyme Biomedical Technology, WJ001, CN). Primary antibodies included STAT3-specific (Abclonal, A27918, 1:1000, CN) and GAPDH-detecting (MBL, M171-3, 1:5000, JPN) reagents. Detection was performed using HRP-linked secondary antibodies against mouse/rabbit IgG (H + L) (Proteintech, SA00001-1/1–2, 1:8000, US). Band intensity was quantified using ImageJ. 2.15 Statistical Analysis Graphical representation and statistical evaluation were performed with GraphPad Prism 8.0 (GraphPad Software, Inc., US). Results are presented as mean ± standard deviation. Two-group comparisons were performed using Student's t-test, while multiple-group analyses were performed using one-way ANOVA. Statistical significance was defined as P values below 0.05. 3. Results 3.1 Detection of active ingredients in JFZX The primary bioactive constituents of JFZX were characterized through high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS). Through comparative analysis with known standards, mass spectrometric techniques successfully detected 249 characteristic compounds, with comprehensive data presented in Table S1 . Figure 1 illustrates the typical chemical profile of JFZX. 3.2 Therapeutic Effects of JFZX on DSS-Induced Ulcerative Colitis in Mice The experimental induction of ulcerative colitis (UC) through DSS administration in murine subjects led to substantial body weight reduction and colonic tissue shrinkage, both recognized as key parameters for assessing pathological progression ( 19 ). Our investigation revealed that animals receiving DSS treatment began to show pronounced weight loss on the fifth day of observation. Importantly, therapeutic intervention with mesalazine (MSLZ) and JFZX at dosages of either 3.5 or 7.00 g/kg effectively attenuated this weight reduction phenomenon in UC-afflicted mice ( Fig. 2 B ) . Additionally, DSS exposure elevated disease activity index (DAI) measurements, while oral delivery of MSLZ and JFZX substantially lowered these scores in the experimental animals ( Fig. 2 C ) . Correspondingly, DSS-treated mice exhibited a considerable reduction in colonic length, a morphological alteration that was significantly ameliorated by MSLZ or JFZX treatment ( Fig. 2 D–E ) . These collective findings demonstrate the substantial therapeutic potential of JFZX in the management of UC symptoms. 3.3 JFZX relieved colon injury in UC mice Additional histopathological analyses were performed on murine colonic tissue using hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) staining. Examination of H&E-stained sections demonstrated substantial disruption of the villous architecture in colonic tissues from DSS-treated UC mice, reflected by elevated histopathological scores. However, administration of either MSLZ or JFZX substantially attenuated these pathological changes, leading to marked restoration of villous morphology and tissue organization ( Fig. 3 A &C) . PAS staining analysis showed that DSS exposure significantly decreased mucin production in the colonic mucosa, which was effectively counteracted by therapeutic intervention with MSLZ or JFZX ( Fig. 3 B &D) . These findings collectively demonstrate that JFZX exhibits potent protective properties against colonic tissue injury in the murine model. 3.4 JFZX increases the tight junctions in the UC mice colon Impairment of colonic tight junctions is a key pathological feature of DSS-induced ulcerative colitis ( 20 ). Our investigation evaluated the protein levels of two crucial tight junction components, ZO-1 and Occludin, in colonic tissue. Immunohistochemical analysis revealed substantial downregulation of both ZO-1 ( Fig. 4 A ) and Occludin ( Fig. 4 B ) expression in DSS-treated animals compared to healthy controls. Notably, administration of either MSLZ or JFZX effectively reversed this suppression, demonstrating their protective effects on intestinal barrier function. These findings suggest that JFZX treatment can mitigate tight junction disruption in experimental colitis models. 3.5 JFZX reduces the infiltration of inflammatory cells in the mouse colon and the inflammatory response Administration of DSS markedly elevated circulating levels of pro-inflammatory mediators such as TNFα, IL-6, and IL-1β, which were effectively attenuated by either MSLZ or JFZX administration ( Fig. 5 A-C ) . To assess immune cell recruitment, we examined CD68 immunoreactivity in colonic sections. Quantitative analysis demonstrated that both therapeutic interventions substantially reduced the number of inflammatory cells in colonic tissue ( Fig. 5 D ) . Collectively, these findings indicate that JFZX treatment produces notable anti-inflammatory effects in the UC mouse model. 3.6 JFZX rebalances intestinal flora in the UC mice model Since gut microbiota imbalance has been strongly linked to the development of UC ( 21 ), we performed metagenomic sequencing on colonic content specimens to investigate how JFZX influences microbial community composition in UC-afflicted mice. The Sobs index analysis demonstrated that DSS treatment markedly decreased microbial alpha diversity, while JFZX oral supplementation substantially reversed this reduction in UC model mice ( Fig. 6 A ) . Principal coordinates analysis and β-diversity assessments further revealed distinct microbial community patterns across experimental groups ( Fig. 6 B &C) . At the phylum taxonomic level, significant alterations in gut microbiota profiles were observed across treatment conditions, with JFZX administration effectively restoring microbial equilibrium. This restoration primarily involved elevating Bacteroidota and Actinomycetota populations and reducing the proportions of Bacillota, Verrucomicrobiota, and Uroviricota ( Fig. 6 D–G ) . To pinpoint specific bacterial variations among groups, we conducted a linear discriminant analysis effect size (LEfSe) analysis, focusing on bacterial taxa with LDA values exceeding 3. Following JFZX administration, the UC cohort showed marked increases in metabolic processes and secondary metabolite production pathways compared with the DSS control group ( Fig. 6 H ) . Collectively, these findings demonstrate that JFZX exerts therapeutic effects on UC by modulating gut microbial homeostasis. 3.7 The effect of JFZX in treating UC is associated with the JAK-STAT pathway To investigate how JFZX exerts therapeutic effects in UC, RNA sequencing of colonic tissue transcriptomes was performed. Gene expression quantification was achieved through the transcripts per million (TPM) method. The DESeq2 package was employed for identifying differentially expressed genes, with significance thresholds set at |log2(fold change)| ≥ 1 and adjusted p-value ≤ 0.05. Figures 7 A and 7 B illustrate the comparative analysis of gene expression patterns between the control and DSS groups, and between the DSS and JFZX-treated groups, respectively. Subsequent intersection analysis using Venn diagrams pinpointed 525 differentially expressed genes as potential therapeutic targets ( Fig. 7 C ) . Functional annotation of these 525 genes through KEGG pathway analysis and gene set enrichment analysis (GSEA) demonstrated that JFZX's mechanism predominantly involves modulation of the JAK-STAT signaling cascade ( Figs. 7 D &E) . The expression profile of relevant genes within this pathway is visually represented in Fig. 7 F, while Fig. 7 G specifically displays the GSEA enrichment pattern for the JAK-STAT pathway when comparing DSS and JFZX treatment groups. Collectively, these findings indicate that JFZX may alleviate ulcerative colitis by modulating the JAK-STAT signaling cascade. 3.8 JFZX inhibits the IL-6/STAT3 pathway in UC mice colon Following KEGG pathway analysis, 15 DEGs were identified within the JAK-STAT signaling cascade: Stat3, Il6, Lif, Ccnd1, Il6st, Omsr, Il4ra, Cntfr, Lepr, Il20rb, Il19, Il10, Csf3, Il15ra, and Pdgfra. Protein-protein interaction network analysis of these 15 genes revealed six central nodes: Stat3, Il6, Lif, Il10, Il19, and Csf3 ( Fig. 8 A ) . Quantitative PCR analysis was subsequently performed to measure mRNA levels of these key genes in murine colonic tissue. The transcriptional profiles of Il6, Stat3, Lif, Csf3, Il10, and Il19 showed marked elevation in DSS-treated animals compared to controls, with JFZX administration restoring expression to baseline levels ( Fig. 8 B ) , corroborating the RNA sequencing data. Western blot analysis demonstrated that JFZX treatment similarly reduced STAT3 protein abundance ( Fig. 8 C ) . These collective findings provide additional evidence that JFZX exerts therapeutic effects in ulcerative colitis by modulating the IL-6/STAT3 signaling axis. 4. Discussion According to statistical results, UC accounts for approximately 45% of the inflammatory bowel disease patient population, which has increasingly become a major disease burden worldwide ( 22 ). While the precise causes of UC remain unclear, researchers have identified potential links to genetic predisposition, immune dysfunction, and environmental influences ( 23 ). Current pharmaceutical treatments often produce adverse effects that compromise patients' well-being, highlighting the urgent need for alternative therapeutic strategies ( 24 ). JFZX, a TCM formulation with an extensive clinical history of use, is a promising candidate. Nevertheless, comprehensive studies examining JFZX's efficacy against UC and its underlying molecular mechanisms remain lacking. Our investigation employs animal models and multi-omics techniques to demonstrate for the first time that JFZX effectively mitigates DSS-induced UC in murine subjects by modulating gut microbiota composition and suppressing IL6/STAT3 pathway activation. The IL6/STAT3 signaling axis is central to STAT3 activation, influencing remodeling of the tumor microenvironment, angiogenesis, immune evasion, and therapy resistance ( 25 ). IL6 can promote the expression and activation of STAT3; furthermore, STAT3 can feed back to upregulate IL6 expression ( 25 ). It’s been confirmed that the IL6/STAT3 signal transduction pathway is crucial for regulating immune and inflammatory responses and plays an important role in the pathogenesis of UC ( 26 , 27 ). The progression of ulcerative colitis involves abnormal activation of multiple signaling pathways, and the IL6/STAT3 pathway is closely associated with repair of the intestinal mucosal barrier and maintenance of intestinal immune homeostasis, both of which play important roles in the pathogenesis of UC ( 28 ). Scientific investigations have revealed that metformin exerts protective effects against colitis by inhibiting STAT3 acetylation and decreasing acetyl-CoA levels ( 29 ). Similarly, Buzhongyiqi granules demonstrate therapeutic potential for the management of colitis by targeting the IL6/STAT3 signaling pathway ( 30 ). Research has demonstrated that both Jingfang granules effectively mitigate DSS-induced colitis by modulating the NF-κB/NLRP3/IL6/STAT3 signaling cascade ( 31 ). Shaoyao decoction has been shown to downregulate IL6, pSTAT3, and STAT3 levels in the colonic tissues of UC-afflicted mice ( 32 ). Additionally, Pulsatilla decoction exerts therapeutic effects on DSS-induced ulcerative colitis by regulating the IL6/STAT3 pathway ( 33 , 34 ). These findings collectively highlight the crucial involvement of the IL6/STAT3 signaling axis in UC pathogenesis and its potential as a therapeutic target. Our RNA sequencing analysis and subsequent gene expression verification identified the IL6/STAT3 pathway as the principal mechanism through which JFZX exerts its anti-UC effects. Treatment with JFZX led to a marked reduction in IL6 and STAT3 expression, as well as modulation of downstream gene activity. This regulatory influence could stem from either direct transcriptional control of IL6 and STAT3 by JFZX or an alternative indirect mechanism. Nevertheless, further in vitro investigations are necessary to elucidate the precise molecular interactions underlying these observations. Significantly, alterations in gut microbiota composition have been identified as a key factor in ulcerative colitis pathogenesis, with strong associations with activation of the IL6/STAT3 signaling cascade ( 35 , 36 ). The colonic microbial ecosystem is predominantly composed of the Bacteroidota and Bacillota phyla, with an increased Bacillota-to-Bacteroidota ratio implicated in UC development and progression ( 37 , 38 ). In our study, metagenomic sequencing results also suggested intestinal flora dysbiosis in DSS-induced UC, and treatment with JFZX increased the abundance of Bacteroidota and decreased that of Bacillota, indicating that the therapeutic effect of JFZX on UC is primarily associated with rebalancing the Bacillota/Bacteroidota ratio. Furthermore, the LEfSe analysis results showed that the JFZX treatment’s effect on the intestinal flora is correlated with the Metabolism and Biosynthesis of secondary metabolites pathway, indicating that microbial metabolites are closely related to JFZX and should be the focus of subsequent research. 5. Conclusion JFZX demonstrates significant efficacy in preventing DSS-induced ulcerative colitis by enhancing intestinal barrier integrity and reducing inflammatory responses in the colon. The therapeutic action of JFZX on UC involves downregulation of the IL6/STAT3 signaling cascade and restoration of gut microbiota equilibrium. These findings provide novel perspectives on JFZX's impact on UC pathogenesis, establishing a scientific basis for its potential as a UC treatment (Fig. 9 ). Abbreviations Ulcerative colitis (UC) Inflammatory bowel disease (IBD) Traditional Chinese Medicine (TCM) JiangFuZhiXie pill (JFZX) Dextran sulfate sodium (DSS) Mesalazine (MSLZ) Disease activity index (DAI) Hematoxylin and eosin (H&E) Periodic Acid Schiff (PAS) Immunohistochemistry (IHC) Differentially expressed genes (DEGs) Gene Ontology (GO) Kyoto Encyclopedia of Genes and Genomes (KEGG) Quantitative Real time PCR (qRT-PCR) Tight junctions (TJs) Declarations CRediT authorship contribution statement Mengyuan Wang and Dengming Lu: Writing-original draft, Writing-review & editing. Tingyu Zhang: Writing-review & editing, data analysis. Li Jin, Zhiming Ge, and Xianxian Fan: Visualization. Qian Zhang, Yuanyuan Yang, and Xinyue Ma: Conceptualization, Methodology. Zhao Ma, Lingyue Zhao, and Yanping Wang: Data curation. Xiaobin Zao: Writing-review & editing, Supervision, Funding acquisition. Yun Yang: Writing-review & editing, Supervision, Resources, Funding acquisition. Ethics approval and consent to participate The present animal experiment protocol has been approved by the Animal Welfare Ethics Review Committee of Beijing University of Chinese Medicine (No. BUCM-2025101502-4004). Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding The research was supported by the Ningxia Hui Autonomous Region Clinical Medical Research Center for Anorectal Diseases (Integrated Chinese and Western Medicine; grant no. 2022LCZX0013), the Science and Technology Basic Condition Construction Project of Ningxia Hui Autonomous Region (Grant no. 2025DPC05028), the National Natural Science Foundation of China (Grant no. 82560927), the Key Research and Development Program of Ningxia Hui Autonomous Region (Grant no. 2024BEG02024), the Natural Science Foundation for Ningxia Province(Grant no. 2024AAC05095), and the Science and Technology Program of Yinchuan City (Grant no. 2024SF025). Data availability Data supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements Figure 9 of this article was created on www.figdraw.com and has been authorized for use. References Wangchuk P, Yeshi K, Loukas A. 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Baitouweng decoction alleviates dextran sulfate sodium-induced ulcerative colitis by regulating intestinal microbiota and the IL-6/STAT3 signaling pathway. J Ethnopharmacol. 2021;265:113357. Zhong W, Wu K, Long Z, Zhou X, Zhong C, Wang S, Lai H, Guo Y, Lv D, Lu J, Mao X. Gut dysbiosis promotes prostate cancer progression and docetaxel resistance via activating NF-κB-IL6-STAT3 axis. Microbiome. 2022;10(1):94. Shan Y, Lee M, Chang EB. The Gut Microbiome and Inflammatory Bowel Diseases. Annu Rev Med. 2022;73:455–68. Yin Y, Yang T, Tian Z, Shi C, Yan C, Li H, Du Y, Li G. Progress in the investigation of the Firmicutes/Bacteroidetes ratio as a potential pathogenic factor in ulcerative colitis. J Med Microbiol. 2025;74(1). Peter R, Zhang F, Scott G, Hold GL, Gordon M, Iqbal TH, Hansen R. The Gut Microbiome at the Onset of Inflammatory Bowel Disease: A Systematic Review and Unified Bioinformatic Synthesis. Gastroenterology. 2026;170(3):539–56. CRediT. authorship contribution statement. Wang M, Lu D. Writing-original draft, Writing-review & editing. Tingyu Zhang: Writing-review & editing, data analysis. Li Jin, Zhiming Ge, and Xianxian Fan: Visualization. Qian Zhang, Yuanyuan Yang, and Xinyue Ma: Conceptualization, Methodology. Zhao Ma, Lingyue Zhao, and Yanping Wang: Data curation. Xiaobin Zao: Writing-review & editing, Supervision, Funding acquisition. Yun Yang: Writing-review & editing, Supervision, Resources, Funding acquisition. Additional Declarations No competing interests reported. Supplementary Files TableS2.RTPrimer.xlsx TableS1.TheinformationofcompoundsinJFZX.xlsx UncroppedWBimage.jpg Cite Share Download PDF Status: Posted Version 1 posted 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-9338335","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618544003,"identity":"df2eabb9-8999-4b8a-bd30-8f26c661159a","order_by":0,"name":"Mengyuan Wang","email":"","orcid":"","institution":"Yinchuan Traditional Chinese Medicine Hospital, Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengyuan","middleName":"","lastName":"Wang","suffix":""},{"id":618544004,"identity":"8aea1336-c239-4f7d-bb65-5681e10309a6","order_by":1,"name":"Dengming Lu","email":"","orcid":"","institution":"Yinchuan 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02:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9338335/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9338335/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106386241,"identity":"3fa8f1d4-222b-402c-9f57-f3c3c534ef80","added_by":"auto","created_at":"2026-04-08 06:20:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC/MS chromatographic analysis of JFZX under standard conditions. \u003c/strong\u003e(A) Detection in positive ionization mode. (B) Detection in negative ionization mode.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/959c2a0c077d45de9b3d293a.jpeg"},{"id":106404489,"identity":"c40cb9a4-f3a0-496a-b4d1-f36ef3e7c8f2","added_by":"auto","created_at":"2026-04-08 09:16:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of symptoms and detection of serum inflammatory factors in UC mice. \u003c/strong\u003e(A) Schematic diagram of the animal experimental process. (B) Changes in body weight of the mice in each group. (C) DAI score. (D) Typical images of the mouse colon. (E) Colon length in each group of mice.\u003cstrong\u003e \u003c/strong\u003ep \u0026lt; 0.01, compared to the Control; ***, p \u0026lt; 0.001, compared to the Control; #, p \u0026lt; 0.05, comprared to the DSS; ##, p \u0026lt; 0.01, comprared to the DSS; ###, p \u0026lt; 0.001, comprared to the DSS.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/1f98eb8b2b9ea5a157efd874.jpeg"},{"id":106403892,"identity":"34e32699-1028-4874-b0d2-b53502167f1d","added_by":"auto","created_at":"2026-04-08 09:15:10","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":586185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological examinations of the mouse colon. \u003c/strong\u003e(A) Representative images of H\u0026amp;E-stained mouse colon tissue samples. (B) Representative images of PAS-stained mouse colon tissue samples. (C) Histological score from H\u0026amp;E staining of the mouse colon tissues. (D) PAS-positive area of the mouse colon tissues.*, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/489db0262d024aeb9ebf35f4.jpeg"},{"id":106404271,"identity":"dda4f072-9cda-410b-b1de-1d5d0934fb41","added_by":"auto","created_at":"2026-04-08 09:15:45","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":435789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe IF analysis of ZO-1 and Occludin in the mouse colon.\u003c/strong\u003e (A) Representative IF images and positive area analysis of ZO1 in the colonic tissues of mice. (B) Representative IHC images and positive area analysis of Occludin in the colonic tissues of mice. **, p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/e8e87936a880d290c495cb63.jpeg"},{"id":106403673,"identity":"ffeea89b-5966-4135-a431-fbd5142dd843","added_by":"auto","created_at":"2026-04-08 09:14:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":246707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression detection of inflammatory factors in the mouse serum and IHC analysis of CD68 in the mouse colon. \u003c/strong\u003e(A-C) Expression of TNFα (A), IL-6 (B), and IL-1β (C) in mouse serum. (d) Representative IHC images of CD68 in the colonic tissues of mice and the positive cell number analysis of CD68. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/4801b3abfc2439224c443c7e.jpeg"},{"id":106403756,"identity":"d7d6642c-c02e-47ef-ad16-78d6eaab8168","added_by":"auto","created_at":"2026-04-08 09:14:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":398477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of JFZX on regulating intestinal flora in DSS-induced colitis mice.\u003c/strong\u003e(A) α-Diversity of the Sobs index. (B) β-Diversity of PCoA analysis. (C) Analysis of differences in β diversity. (D) The relative abundance of fecal bacteria at the phylum level. (E) Analysis of significantly different microbiota at the phylum level. (F) Relative abundance of Bacteroidota. (G) Relative abundance of Bacillota. (H) Significant bacterial communities were identified using LEfSe analysis (LDA \u0026gt; 3). *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/ed7083d76db7cc6575595c72.jpeg"},{"id":106403672,"identity":"09ed1c11-5f32-4cfc-9fbc-d9e7997eafd1","added_by":"auto","created_at":"2026-04-08 09:14:45","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":423603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome RNA-sequencing analysis in mice's colonic tissues. \u003c/strong\u003e(A\u0026amp;B) Volcano plots showing DEGs identified by RNA-sequencing in colonic tissues between groups. (C) Venn diagram of the DEGs. (D) Top 10 of KEGG pathways. (E) GSEA analysis between the DSS and JFZX groups. (F) Heatmap of gene expression in the JAK-STAT pathway. (G) GSEA enrichment plot of the JAK-STAT pathway between the DSS and JFZX groups.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/338e158632d03e9ed4c5218f.jpeg"},{"id":106404428,"identity":"f11d5642-8bf6-4088-8bb5-6ea485b4d451","added_by":"auto","created_at":"2026-04-08 09:15:59","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":280430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protein-protein interaction network and expression analysis of the key targets. \u003c/strong\u003e(A) The PPI network of the targets in the IL6/STAT3 pathway. (B) The mRNA levels of Il6, Stat3, Lif, Csf3, Il10, and Il19 in the colonic tissues of mice. (C) The protein expression of STAT3 in the colonic tissues of mice. *,p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/3d2076354ab9002d33611166.jpeg"},{"id":106404478,"identity":"606f0809-cf9c-446e-a625-8aa03ba0091d","added_by":"auto","created_at":"2026-04-08 09:16:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":316400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanism diagram of JFZX for the treatment of UC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/5479c7fa5a4d8429b39716d8.png"},{"id":109204547,"identity":"6ae44d26-e08f-4d03-a6db-2723da5e67e9","added_by":"auto","created_at":"2026-05-13 15:01:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3387151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/40e55de6-5165-4834-8346-2753a07fa90d.pdf"},{"id":106386230,"identity":"33925fba-d8e6-4b5e-89d5-cae619b31f07","added_by":"auto","created_at":"2026-04-08 06:20:25","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10464,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.RTPrimer.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/1c85c8a301d74b130b00a9bc.xlsx"},{"id":106403872,"identity":"3b24ba2d-6f02-4d7f-92b1-2c7fde17c303","added_by":"auto","created_at":"2026-04-08 09:15:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29092,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.TheinformationofcompoundsinJFZX.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/4ad882c41fa1020579fa7527.xlsx"},{"id":106404276,"identity":"89c15dc3-1708-4117-bdb3-dd6fd95063d6","added_by":"auto","created_at":"2026-04-08 09:15:45","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41591,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedWBimage.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9338335/v1/e8a165c0fb5aa8aa82c9aa35.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"JiangFuZhiXie pill ameliorates DSS-induced ulcerative colitis mice via modulating intestinal flora and inhibiting IL6/STAT3 signaling to reduce inflammatory response","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eUlcerative colitis (UC) is characterized by symptoms including abdominal discomfort, diarrhea, hematochezia, and weight loss (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In recent years, the incidence rate of UC has been steadily increasing, causing an increasingly heavy burden of disease (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The precise etiological mechanisms underlying UC pathogenesis remain incompletely understood, while existing therapeutic approaches show restricted effectiveness and unfavorable long-term outcomes. Current clinical interventions, such as 5-ASA compounds, immunomodulatory agents, glucocorticoids, and biologic treatments, provide only symptomatic control rather than disease resolution, with some patients exhibiting suboptimal therapeutic responses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Thus, this clinical scenario underscores the pressing need for innovative treatment modalities in UC management.\u003c/p\u003e \u003cp\u003eTraditional Chinese medicine (TCM) employs a distinctive therapeutic strategy that combines multiple herbal components to simultaneously target various pathological factors. This comprehensive approach offers particular benefits in managing ulcerative colitis (UC) cases (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Based on TCM theory, most UC patients exhibit clinical manifestations consistent with yang deficiency and dampness accumulation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Consequently, herbal formulations with warming and dampness-eliminating properties are commonly prescribed, as they facilitate the removal of dampness and alleviate diarrhea by strengthening intestinal function (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The present investigation focuses on JiangFuZhiXie pill (JFZX), a hospital-formulated TCM preparation widely used in clinical settings for the management of diarrhea and UC, demonstrating remarkable clinical efficacy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). JFZX incorporates numerous bioactive constituents with potential UC-modulating effects, including Radix Ginseng, Dried Ginger\u0026zwnj;, Largehead Atractylodes Rhizome, Liquorice Root, Prepared Common Monkshood Daughter Root, Malaytea Scurfpea Fruit, Nutmeg, Chinese Magnoliavine Fruit, Radix Saposhnikoviae, Dried Tangerine Peel, Rhizoma Coptis, Halloysit, Medicated Leaven, Hawthorn Fruit, and Chinese Thorowax Root.\u003c/p\u003e \u003cp\u003eThe herbal components of JFZX demonstrate significant therapeutic potential for UC through diverse mechanisms of action. Research indicates that Radix Ginseng alleviates UC symptoms by modulating gut microbiota composition (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e); Dried Ginger's volatile oil facilitates intestinal mucosal repair by stimulating epithelial cell regeneration (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e); Liquorice Root's primary active constituent provides mucosal protection through modulation of Nrf2 and NF-κB signaling pathways in UC models (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e); Chinese Magnoliavine Fruit could protect against UC in mice via regulating gut microbiota and TLR4/NF-κB/NLRP3 pathway (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e); Dried Tangerine Peel exhibits efficacy in restoring mucus barrier integrity, particularly in high-fat diet conditions (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e); Coptisine from Rhizoma Coptis maintains colonic epithelial homeostasis in UC models through aryl hydrocarbon receptor activation, as evidenced in both in vivo and TNF-α-induced organoid studies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e); the polysaccharide derived from Crataegus pinnatifida, commonly known as hawthorn fruit, has demonstrated potential in mitigating UC through its ability to boost arginine production and modulate gut microbial composition (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These findings provide additional evidence supporting the therapeutic efficacy of JFZX in the management of UC. Nevertheless, the precise mechanisms and therapeutic impacts of JFZX in UC treatment require further investigation.\u003c/p\u003e \u003cp\u003eDextran sulfate sodium (DSS; molecular weight range: 36\u0026ndash;50 kDa) is widely employed to establish UC models due to its capacity to increase intestinal permeability, compromise the mucosal barrier, elevate pro-inflammatory cytokine levels, and induce gut microbiota imbalance (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Based on a DSS-induced UC mouse model, this research aims to explore the effects of JFZX in the treatment of UC and to further elucidate its mechanism using multi-omics methodologies.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical reagents\u003c/h2\u003e \u003cp\u003eDextran sulfate sodium (DSS, molecular weight: 36\u0026ndash;50 kDa) was acquired from MP Biomedicals (Santa Ana, US). JiangFuZhiXie pills (JFZX) were acquired from the Preparation Center of Yinchuan Traditional Chinese Medicine Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of JFZX\u003c/h2\u003e \u003cp\u003eThe herbs of JFZX were wrapped in non-woven bags, and sufficient water was added to decoct them twice, each time for 2 hours, after which the liquid medicine was concentrated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Chemical constituents of JFZX by HPLC-Q/TOF-MS analysis\u003c/h2\u003e \u003cp\u003eJFZX formulation incorporates sixteen distinct herbal components, with their specific quantitative ratios presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Component characterization was performed utilizing a high-resolution Waters Synapt G2-Si Qtof mass spectrometer coupled with Unifi analytical software at Tsinghua University's Pharmaceutical Technology Research Center. The analytical methodology aligns with the protocol established in our prior investigation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Comprehensive data regarding the UHPLC‒MS/MS analytical outcomes are accessible in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConstituents of JFZX\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChinese name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLatin name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnglish name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProportion (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenshen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadix Panacis Ginseng\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRadix Ginseng\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGanjiang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRhizoma Zingiberis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDried Ginger\u0026zwnj;\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaizhu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRhizoma Atractylodis Macrocephalae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLargehead Atractylodes Rhizome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGancao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycyrrhizae Radix et Rhizoma\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLiquorice Root\u0026zwnj;\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFuzi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAconiti Lateralis Radix Praeparata\u0026zwnj; \u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrepared Common Monkshood Daughter Root\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBuguzhi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFructus Psoraleae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMalaytea Scurfpea Fruit\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoudoukou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMyristica Fragrans Houtt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNutmeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWuweizi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFructus Schisandrae Chinensis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChinese Magnoliavine Fruit\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFangfen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaposhnikovia Divaricata\u0026zwnj;(Turcz.) Schischk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRadix Saposhnikoviae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChenpi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePericarpium Citri Reticulatae\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDried Tangerine Peel\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuanglian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCoptis Chinensis Franch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRhizoma Coptis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChishizhi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHalloysitum Rubrum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHalloysit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiushenqu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMassa Medicata Fermentata\u0026zwnj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedicated Leaven\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShanzha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFructus Crataegi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHawthorn Fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChaihu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBupleuri Radix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChinese Thorowax Root\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental Animals\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice aged 4\u0026ndash;6 weeks with body weights ranging between 18\u0026ndash;22 grams were procured from Beijing Vital River Laboratory Animal Technology Company. The subjects were maintained in a sterile environment with controlled access and adhered to a 12-hour light/dark photoperiod. Environmental conditions were strictly regulated, with the ambient temperature kept at 22\u0026deg;C (\u0026plusmn;\u0026thinsp;3\u0026deg;C) and the humidity maintained at 50% (\u0026plusmn;\u0026thinsp;10%). Throughout the study period, the animals had ad libitum access to food and water. Following a seven-day adaptation phase, all experimental procedures were initiated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Establishment of the ulcerative colitis model and grouping\u003c/h2\u003e \u003cp\u003eThe experimental animals were divided into six distinct cohorts (6 mice per group) through a randomization process: the untreated control cohort, DSS-treated cohort, MSLZ-treated cohort (receiving Mesalazine at 0.5 g/kg from Ethypharm Company, China), JFZX-L cohort (administered 3.5 g/kg, matching the standard human therapeutic dose of JFZX), and JFZX-H cohort (given 7.5 g/kg, representing double the standard human dose of JFZX). Ulcerative colitis was experimentally induced by providing unrestricted access to a 3% DSS aqueous solution for 7 days. While the control and DSS groups received distilled water via oral gavage, the MSLZ, JFZX-L, and JFZX-H groups were given their respective medications using identical administration methods as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Daily monitoring included comprehensive assessments of physical condition, body mass fluctuations, and fecal characteristics. Following a seven-day experimental period, all subjects were humanely euthanized, and biological specimens (including blood samples, fecal matter, and colonic tissue) were collected and cryopreserved at -80\u0026deg;C for subsequent investigations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Disease activity index (DAI) analysis\u003c/h2\u003e \u003cp\u003eThe DAI was determined according to the following rating sheet (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe DAI rating sheet\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBody weight loss\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStool morphology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFecal blood\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eno weight loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eregular consistency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;5% reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esoft but firm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emild positive blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u0026ndash;10% reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u0026ndash;18% reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eblood in the stool\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereduction exceeding 18%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewatery diarrhea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eactive bleeding\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Chemiluminescence immunoassay detection\u003c/h2\u003e \u003cp\u003e Detected the IL6, IL-1β, and TNF-αin mice serum by Servicebio chemiluminescence immunoassay kits (GLM0004 Mouse IL-6 MPCLIA Kit, GLM0005 Mouse TNF-α MPCLIA Kit, GLM0010Mouse IL-1β MPCLIA Kit) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Histopathological staining\u003c/h2\u003e \u003cp\u003eColon tissue from experimental mice was fixed in 4% paraformaldehyde (G1101, Servicebio, China) and rinsed under running water. Subsequently, the specimen should be dehydrated using graded ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd, China). The specimen should then be cleared using xylene (10023418, Sinopharm Chemical Reagent Co., Ltd, China). Following paraffin embedding and sectioning, hematoxylin and eosin (H\u0026amp;E) staining was performed using a Servicebio kit (G1004, Servicebio, China). Assessment of glycogen deposition in colon sections was performed using Periodic Acid-Schiff (PAS) staining with a specific kit (G1008, Servicebio, China). The slices are imaged and scanned by the slicing scanner. The positive results were then analyzed using ImageJ. And the HS score was determined according to the following rating sheet (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe standard HS score\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDamaged area\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eMucodepletion of glands\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTissue damage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eInflammatory cell infiltration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\\\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNo mucosal damage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOccasional inflammatory cells in the lamina propria\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eDiscrete epithelial lesions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncreased numbers of inflammatory cells in the lamina propria\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSurface mucosal erosion or focal ulceration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eConfluence inflammatory cells, extending into the submucosa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eExtensive mucosal damage and extension into deeper structures of the bowel wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTransmural extension of the infiltrate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\\\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\\\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunohistochemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eFor immunohistochemical analysis, colon tissue sections underwent a series of preparatory steps, including deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inactivation, and blocking with normal goat serum. The primary antibody targeting CD68 (GB153109; Servicebio, China), a rabbit polyclonal antibody, was carefully applied and incubated in a humidified chamber at 4\u0026deg;C for approximately 24 hours. Following this incubation period, the sections were treated with both the reaction solution and horseradish peroxidase-conjugated anti-rabbit secondary antibody (GB23303; Servicebio, China), maintained at 37\u0026deg;C for half an hour. The chromogenic reaction was developed using diaminobenzidine (G1212; Servicebio, China), producing a distinct reddish-brown coloration. Cellular structures exhibiting cytoplasmic brown-yellow granular staining were identified as positive expression. Digital imaging of the sections was performed using a specialized scanning system, and subsequent quantitative analysis of positively stained regions and cells was conducted in ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Immunofluorescence(IF)staining\u003c/h2\u003e \u003cp\u003eThe tissue sections underwent identical procedures for both dewaxing and antigen retrieval. Initially, a self-fluorescence-quenching agent was applied, followed by a 5-minute incubation period. Subsequently, the specimens were washed under running water and incubated in serum for 30 minutes. Pre-formulated primary antibodies, specifically rabbit polyclonal antibodies against ZO-1 (GB15195; Servicebio, China) and Occludin (GB151401; Servicebio, China), were then added, and the samples were maintained at 4\u0026deg;C overnight. Tissue sections were then treated with species-matched secondary antibodies. This was followed by a 50-minute dark incubation at ambient temperature. Nuclear staining was subsequently performed using DAPI. Microscopic examination and data interpretation concluded the experimental process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 RNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the mice colon tissues using TRIzol\u0026reg; Reagent according to the manufacturer\u0026rsquo;s instructions (Invitrogen, US). RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer\u0026rsquo;s instructions (Illumina, San Diego, CA). The detailed information was described in our previous study (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The data were analyzed using the free online platform of the Majorbio Cloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.majorbio.com\u003c/span\u003e\u003cspan address=\"http://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Metagenomic sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA was isolated from 200 mg of murine colonic samples using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China) according to the manufacturer's standard protocol. The obtained DNA samples were then sheared into fragments averaging around 350 base pairs using mechanical disruption with the Covaris M220 (Gene Company Limited, China), preparing them for paired-end sequencing library preparation. Library preparation was performed using NEXTFLEX Rapid DNA-Seq reagents (Bioo Scientific, Austin, TX, USA). Sequencing was performed on the Illumina NovaSeq\u0026trade; X Plus platform (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), using NovaSeq X Series 25B Reagent Kits according to the manufacturer's guidelines. Subsequent data processing was performed using the Majorbio Cloud online analysis platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.majorbio.com\u003c/span\u003e\u003cspan address=\"http://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Quantitative Real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe conversion of total RNA into complementary DNA was carried out utilizing a quantitative PCR reverse transcription master mix kit (TransGen, China). Quantitative reverse transcription polymerase chain reaction analysis was conducted on a real-time PCR instrument (Thermo Fisher, USA) employing SYBR Green-based real-time PCR master mix reagents (TransGen, China). Specific primer sequences employed for amplification are detailed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e, with β-actin serving as the reference gene. All experimental procedures were conducted with three technical replicates and independently repeated across three separate experimental runs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Western blot\u003c/h2\u003e \u003cp\u003eProtein samples were resolved using 10% SDS-PAGE gels (Epizyme Biomedical Technology, PG112, CN) followed by electroblotting onto PVDF membranes (Epizyme Biomedical Technology, WJ001, CN). Primary antibodies included STAT3-specific (Abclonal, A27918, 1:1000, CN) and GAPDH-detecting (MBL, M171-3, 1:5000, JPN) reagents. Detection was performed using HRP-linked secondary antibodies against mouse/rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Proteintech, SA00001-1/1\u0026ndash;2, 1:8000, US). Band intensity was quantified using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical Analysis\u003c/h2\u003e \u003cp\u003eGraphical representation and statistical evaluation were performed with GraphPad Prism 8.0 (GraphPad Software, Inc., US). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Two-group comparisons were performed using Student's t-test, while multiple-group analyses were performed using one-way ANOVA. Statistical significance was defined as P values below 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Detection of active ingredients in JFZX\u003c/h2\u003e \u003cp\u003eThe primary bioactive constituents of JFZX were characterized through high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS). Through comparative analysis with known standards, mass spectrometric techniques successfully detected 249 characteristic compounds, with comprehensive data presented in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the typical chemical profile of JFZX.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Therapeutic Effects of JFZX on DSS-Induced Ulcerative Colitis in Mice\u003c/h2\u003e \u003cp\u003eThe experimental induction of ulcerative colitis (UC) through DSS administration in murine subjects led to substantial body weight reduction and colonic tissue shrinkage, both recognized as key parameters for assessing pathological progression (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Our investigation revealed that animals receiving DSS treatment began to show pronounced weight loss on the fifth day of observation. Importantly, therapeutic intervention with mesalazine (MSLZ) and JFZX at dosages of either 3.5 or 7.00 g/kg effectively attenuated this weight reduction phenomenon in UC-afflicted mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Additionally, DSS exposure elevated disease activity index (DAI) measurements, while oral delivery of MSLZ and JFZX substantially lowered these scores in the experimental animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Correspondingly, DSS-treated mice exhibited a considerable reduction in colonic length, a morphological alteration that was significantly ameliorated by MSLZ or JFZX treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E\u003cb\u003e)\u003c/b\u003e. These collective findings demonstrate the substantial therapeutic potential of JFZX in the management of UC symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 JFZX relieved colon injury in UC mice\u003c/h2\u003e \u003cp\u003eAdditional histopathological analyses were performed on murine colonic tissue using hematoxylin-eosin (H\u0026amp;E) and periodic acid-Schiff (PAS) staining. Examination of H\u0026amp;E-stained sections demonstrated substantial disruption of the villous architecture in colonic tissues from DSS-treated UC mice, reflected by elevated histopathological scores. However, administration of either MSLZ or JFZX substantially attenuated these pathological changes, leading to marked restoration of villous morphology and tissue organization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e\u0026amp;C)\u003c/b\u003e. PAS staining analysis showed that DSS exposure significantly decreased mucin production in the colonic mucosa, which was effectively counteracted by therapeutic intervention with MSLZ or JFZX \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e\u0026amp;D)\u003c/b\u003e. These findings collectively demonstrate that JFZX exhibits potent protective properties against colonic tissue injury in the murine model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 JFZX increases the tight junctions in the UC mice colon\u003c/h2\u003e \u003cp\u003eImpairment of colonic tight junctions is a key pathological feature of DSS-induced ulcerative colitis (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Our investigation evaluated the protein levels of two crucial tight junction components, ZO-1 and Occludin, in colonic tissue. Immunohistochemical analysis revealed substantial downregulation of both ZO-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and Occludin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e expression in DSS-treated animals compared to healthy controls. Notably, administration of either MSLZ or JFZX effectively reversed this suppression, demonstrating their protective effects on intestinal barrier function. These findings suggest that JFZX treatment can mitigate tight junction disruption in experimental colitis models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.5 JFZX reduces the infiltration of inflammatory cells in the mouse colon and the inflammatory response\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAdministration of DSS markedly elevated circulating levels of pro-inflammatory mediators such as TNFα, IL-6, and IL-1β, which were effectively attenuated by either MSLZ or JFZX administration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e. To assess immune cell recruitment, we examined CD68 immunoreactivity in colonic sections. Quantitative analysis demonstrated that both therapeutic interventions substantially reduced the number of inflammatory cells in colonic tissue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Collectively, these findings indicate that JFZX treatment produces notable anti-inflammatory effects in the UC mouse model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 JFZX rebalances intestinal flora in the UC mice model\u003c/h2\u003e \u003cp\u003eSince gut microbiota imbalance has been strongly linked to the development of UC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), we performed metagenomic sequencing on colonic content specimens to investigate how JFZX influences microbial community composition in UC-afflicted mice. The Sobs index analysis demonstrated that DSS treatment markedly decreased microbial alpha diversity, while JFZX oral supplementation substantially reversed this reduction in UC model mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Principal coordinates analysis and β-diversity assessments further revealed distinct microbial community patterns across experimental groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e\u0026amp;C)\u003c/b\u003e. At the phylum taxonomic level, significant alterations in gut microbiota profiles were observed across treatment conditions, with JFZX administration effectively restoring microbial equilibrium. This restoration primarily involved elevating Bacteroidota and Actinomycetota populations and reducing the proportions of Bacillota, Verrucomicrobiota, and Uroviricota \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;G\u003cb\u003e)\u003c/b\u003e. To pinpoint specific bacterial variations among groups, we conducted a linear discriminant analysis effect size (LEfSe) analysis, focusing on bacterial taxa with LDA values exceeding 3. Following JFZX administration, the UC cohort showed marked increases in metabolic processes and secondary metabolite production pathways compared with the DSS control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. Collectively, these findings demonstrate that JFZX exerts therapeutic effects on UC by modulating gut microbial homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 The effect of JFZX in treating UC is associated with the JAK-STAT pathway\u003c/h2\u003e \u003cp\u003eTo investigate how JFZX exerts therapeutic effects in UC, RNA sequencing of colonic tissue transcriptomes was performed. Gene expression quantification was achieved through the transcripts per million (TPM) method. The DESeq2 package was employed for identifying differentially expressed genes, with significance thresholds set at |log2(fold change)| \u0026ge; 1 and adjusted p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB illustrate the comparative analysis of gene expression patterns between the control and DSS groups, and between the DSS and JFZX-treated groups, respectively. Subsequent intersection analysis using Venn diagrams pinpointed 525 differentially expressed genes as potential therapeutic targets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Functional annotation of these 525 genes through KEGG pathway analysis and gene set enrichment analysis (GSEA) demonstrated that JFZX's mechanism predominantly involves modulation of the JAK-STAT signaling cascade \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u003cb\u003e\u0026amp;E)\u003c/b\u003e. The expression profile of relevant genes within this pathway is visually represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, while Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG specifically displays the GSEA enrichment pattern for the JAK-STAT pathway when comparing DSS and JFZX treatment groups. Collectively, these findings indicate that JFZX may alleviate ulcerative colitis by modulating the JAK-STAT signaling cascade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8 JFZX inhibits the IL-6/STAT3 pathway in UC mice colon\u003c/h2\u003e \u003cp\u003eFollowing KEGG pathway analysis, 15 DEGs were identified within the JAK-STAT signaling cascade: Stat3, Il6, Lif, Ccnd1, Il6st, Omsr, Il4ra, Cntfr, Lepr, Il20rb, Il19, Il10, Csf3, Il15ra, and Pdgfra. Protein-protein interaction network analysis of these 15 genes revealed six central nodes: Stat3, Il6, Lif, Il10, Il19, and Csf3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Quantitative PCR analysis was subsequently performed to measure mRNA levels of these key genes in murine colonic tissue. The transcriptional profiles of Il6, Stat3, Lif, Csf3, Il10, and Il19 showed marked elevation in DSS-treated animals compared to controls, with JFZX administration restoring expression to baseline levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, corroborating the RNA sequencing data. Western blot analysis demonstrated that JFZX treatment similarly reduced STAT3 protein abundance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. These collective findings provide additional evidence that JFZX exerts therapeutic effects in ulcerative colitis by modulating the IL-6/STAT3 signaling axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAccording to statistical results, UC accounts for approximately 45% of the inflammatory bowel disease patient population, which has increasingly become a major disease burden worldwide (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). While the precise causes of UC remain unclear, researchers have identified potential links to genetic predisposition, immune dysfunction, and environmental influences (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Current pharmaceutical treatments often produce adverse effects that compromise patients' well-being, highlighting the urgent need for alternative therapeutic strategies (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). JFZX, a TCM formulation with an extensive clinical history of use, is a promising candidate. Nevertheless, comprehensive studies examining JFZX's efficacy against UC and its underlying molecular mechanisms remain lacking. Our investigation employs animal models and multi-omics techniques to demonstrate for the first time that JFZX effectively mitigates DSS-induced UC in murine subjects by modulating gut microbiota composition and suppressing IL6/STAT3 pathway activation.\u003c/p\u003e \u003cp\u003eThe IL6/STAT3 signaling axis is central to STAT3 activation, influencing remodeling of the tumor microenvironment, angiogenesis, immune evasion, and therapy resistance (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). IL6 can promote the expression and activation of STAT3; furthermore, STAT3 can feed back to upregulate IL6 expression (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). It\u0026rsquo;s been confirmed that the IL6/STAT3 signal transduction pathway is crucial for regulating immune and inflammatory responses and plays an important role in the pathogenesis of UC (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The progression of ulcerative colitis involves abnormal activation of multiple signaling pathways, and the IL6/STAT3 pathway is closely associated with repair of the intestinal mucosal barrier and maintenance of intestinal immune homeostasis, both of which play important roles in the pathogenesis of UC (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Scientific investigations have revealed that metformin exerts protective effects against colitis by inhibiting STAT3 acetylation and decreasing acetyl-CoA levels (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Similarly, Buzhongyiqi granules demonstrate therapeutic potential for the management of colitis by targeting the IL6/STAT3 signaling pathway (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Research has demonstrated that both Jingfang granules effectively mitigate DSS-induced colitis by modulating the NF-κB/NLRP3/IL6/STAT3 signaling cascade (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Shaoyao decoction has been shown to downregulate IL6, pSTAT3, and STAT3 levels in the colonic tissues of UC-afflicted mice (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Additionally, Pulsatilla decoction exerts therapeutic effects on DSS-induced ulcerative colitis by regulating the IL6/STAT3 pathway (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). These findings collectively highlight the crucial involvement of the IL6/STAT3 signaling axis in UC pathogenesis and its potential as a therapeutic target.\u003c/p\u003e \u003cp\u003eOur RNA sequencing analysis and subsequent gene expression verification identified the IL6/STAT3 pathway as the principal mechanism through which JFZX exerts its anti-UC effects. Treatment with JFZX led to a marked reduction in IL6 and STAT3 expression, as well as modulation of downstream gene activity. This regulatory influence could stem from either direct transcriptional control of IL6 and STAT3 by JFZX or an alternative indirect mechanism. Nevertheless, further in vitro investigations are necessary to elucidate the precise molecular interactions underlying these observations.\u003c/p\u003e \u003cp\u003eSignificantly, alterations in gut microbiota composition have been identified as a key factor in ulcerative colitis pathogenesis, with strong associations with activation of the IL6/STAT3 signaling cascade (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The colonic microbial ecosystem is predominantly composed of the Bacteroidota and Bacillota phyla, with an increased Bacillota-to-Bacteroidota ratio implicated in UC development and progression (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In our study, metagenomic sequencing results also suggested intestinal flora dysbiosis in DSS-induced UC, and treatment with JFZX increased the abundance of Bacteroidota and decreased that of Bacillota, indicating that the therapeutic effect of JFZX on UC is primarily associated with rebalancing the Bacillota/Bacteroidota ratio. Furthermore, the LEfSe analysis results showed that the JFZX treatment\u0026rsquo;s effect on the intestinal flora is correlated with the Metabolism and Biosynthesis of secondary metabolites pathway, indicating that microbial metabolites are closely related to JFZX and should be the focus of subsequent research.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eJFZX demonstrates significant efficacy in preventing DSS-induced ulcerative colitis by enhancing intestinal barrier integrity and reducing inflammatory responses in the colon. The therapeutic action of JFZX on UC involves downregulation of the IL6/STAT3 signaling cascade and restoration of gut microbiota equilibrium. These findings provide novel perspectives on JFZX's impact on UC pathogenesis, establishing a scientific basis for its potential as a UC treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUlcerative colitis (UC)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eInflammatory bowel disease (IBD)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTraditional Chinese Medicine (TCM)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJiangFuZhiXie pill (JFZX)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDextran sulfate sodium (DSS)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMesalazine (MSLZ)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDisease activity index (DAI)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHematoxylin and eosin (H\u0026amp;E)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePeriodic Acid\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSchiff (PAS)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eImmunohistochemistry (IHC)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDifferentially expressed genes (DEGs)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGene Ontology (GO)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKyoto Encyclopedia of Genes and Genomes (KEGG)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQuantitative Real\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etime PCR (qRT-PCR)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTight junctions (TJs)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMengyuan Wang and Dengming Lu:\u0026nbsp;\u003c/strong\u003eWriting-original draft, Writing-review \u0026amp; editing. \u003cstrong\u003eTingyu Zhang:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, data analysis. \u003cstrong\u003eLi Jin, Zhiming Ge, and Xianxian Fan:\u0026nbsp;\u003c/strong\u003eVisualization. \u003cstrong\u003eQian Zhang, Yuanyuan Yang, and Xinyue Ma:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology. \u003cstrong\u003eZhao Ma, Lingyue Zhao, and Yanping Wang:\u0026nbsp;\u003c/strong\u003eData curation. \u003cstrong\u003eXiaobin Zao:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Supervision, Funding acquisition. \u003cstrong\u003eYun Yang:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Supervision, Resources, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present animal experiment protocol has been approved by the Animal Welfare Ethics Review Committee of Beijing University of Chinese Medicine (No. BUCM-2025101502-4004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the Ningxia Hui Autonomous Region Clinical Medical Research Center for Anorectal Diseases (Integrated Chinese and Western Medicine; grant no. 2022LCZX0013), the Science and Technology Basic Condition Construction Project of Ningxia Hui Autonomous Region (Grant no. 2025DPC05028), the National Natural Science Foundation of China (Grant no. 82560927), the Key Research and Development Program of Ningxia Hui Autonomous Region (Grant no. 2024BEG02024), the Natural Science Foundation for Ningxia Province(Grant no. 2024AAC05095), and the Science and Technology Program of Yinchuan City (Grant no. 2024SF025). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 9 of this article was created on www.figdraw.com and has been authorized for use.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWangchuk P, Yeshi K, Loukas A. 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The Gut Microbiome and Inflammatory Bowel Diseases. Annu Rev Med. 2022;73:455\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin Y, Yang T, Tian Z, Shi C, Yan C, Li H, Du Y, Li G. Progress in the investigation of the Firmicutes/Bacteroidetes ratio as a potential pathogenic factor in ulcerative colitis. J Med Microbiol. 2025;74(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeter R, Zhang F, Scott G, Hold GL, Gordon M, Iqbal TH, Hansen R. The Gut Microbiome at the Onset of Inflammatory Bowel Disease: A Systematic Review and Unified Bioinformatic Synthesis. Gastroenterology. 2026;170(3):539\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCRediT. authorship contribution statement.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Lu D. Writing-original draft, Writing-review \u0026amp; editing. Tingyu Zhang: Writing-review \u0026amp; editing, data analysis. Li Jin, Zhiming Ge, and Xianxian Fan: Visualization. Qian Zhang, Yuanyuan Yang, and Xinyue Ma: Conceptualization, Methodology. Zhao Ma, Lingyue Zhao, and Yanping Wang: Data curation. Xiaobin Zao: Writing-review \u0026amp; editing, Supervision, Funding acquisition. Yun Yang: Writing-review \u0026amp; editing, Supervision, Resources, Funding acquisition.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ulcerative colitis, Traditional Chinese medicine, JiangFuZhiXie pill, IL6/STAT3 signaling, Intestinal flora","lastPublishedDoi":"10.21203/rs.3.rs-9338335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9338335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCurrent treatments for ulcerative colitis (UC) are unsatisfactory. JiangFuZhiXie pill (JFZX) is an in-hospital traditional Chinese medicine widely used in clinical practice for the treatment of UC, with excellent therapeutic effects.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo explore the specific effects and underlying mechanisms of JFZX in the treatment of UC and further search for new therapeutic targets.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe UC mice model was established by free access to a 3% dextran sulfate sodium aqueous solution and intervention with JFZX via intragastric administration. Mouse serum was used to detect inflammatory factor levels. Mouse colon tissues were used for pathological, immunohistochemical, and immunofluorescence staining. Mouse colon tissues and intestinal contents were used for transcriptomic analysis and metagenomic testing, respectively. Gene expression was detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe intervention with JFZX significantly increased body weight and colonic tight junctions in UC mice, and decreased disease score, pathological score, and inflammatory level. Metagenomic analysis results showed that JFZX modulates the abundance of key bacterial populations and improves intestinal flora disorders in UC mice. The transcriptomic and RT-PCR analysis indicated that the key pathway regulated by JFZX in the treatment of UC is the IL6/STAT3 pathway. JFZX intervention significantly downregulated the expression of key targets, including Stat3, Il6, Lif, Il10, Il19, and Csf3.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eJFZX has therapeutic effects in UC and effectively inhibits intestinal inflammation. Its mechanism of action involves rebalancing the intestinal flora and inhibiting the IL6/STAT3 signaling pathway, thus reducing intestinal inflammation damage.\u003c/p\u003e","manuscriptTitle":"JiangFuZhiXie pill ameliorates DSS-induced ulcerative colitis mice via modulating intestinal flora and inhibiting IL6/STAT3 signaling to reduce inflammatory response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 06:20:20","doi":"10.21203/rs.3.rs-9338335/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd4c97b3-6ce6-40b5-9f11-04e8180ffde8","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-12T10:57:06+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T11:26:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 06:20:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9338335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9338335","identity":"rs-9338335","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}