A phytotherapeutic agent demonstrates clinical efficacy in amelioration of murine colitis through gut microbiota modulation: mechanistic link to Lactobacillus gasseri-dependent inhibition of ferroptotic pathways

A phytotherapeutic agent demonstrates clinical efficacy in amelioration of murine colitis through gut microbiota modulation: mechanistic link to Lactobacillus gasseri-dependent inhibition of ferroptotic pathways | 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 A phytotherapeutic agent demonstrates clinical efficacy in amelioration of murine colitis through gut microbiota modulation: mechanistic link to Lactobacillus gasseri-dependent inhibition of ferroptotic pathways Cheng Cheng, Lei Zhu, Jingyi Hu, Wan Feng, Weiyang Li, Ryan Au, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7844488/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted 11 You are reading this latest preprint version Abstract Ulcerative colitis (UC), characterized by chronic intestinal inflammation and epithelial barrier dysfunction, remains a therapeutic challenge due to its complex etiology [1,2]. Among emerging mechanisms, ferroptosis—an iron-dependent form of regulated cell death driven by lipid peroxidation has recently been implicated in UC pathogenesis [3,4]. The gut microbiota plays a crucial role in maintaining intestinal homeostasis, and metabolism can result in pathological damage to the intestines [5, 6]. Metabolites act as important mediators of host-microbe interactions and are essential for the maintenance of the gut barrier [7, 8]. Targeting the microbiota-metabolic axis has emerged as a promising approach for managing UC. It has been demonstrated that QCHS significantly alleviated DSS-induced ferroptosis in the colon of UC mice, and that L. gasseri mediated this protective effect. For the first time, it was found that L. gasseri can act as a ferroptosis inhibitor, mitigating the progression of UC. In vitro, it was further demonstrated that L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells, with the mechanism involving activation of the GSH/GPX4 signaling pathway. This work provides compelling evidence for the regulatory role of QCHS on the microbiota-metabolome axis and ferroptosis in UC mice, and uncovering a novel function of L. gasseri as an inhibitor of ferroptosis, offering new insights into potential therapeutic strategies for UC. These findings suggest that through the microbiota modulators or ferroptosis inhibitors targeting Lactobacillus, QCHS may be promising candidates for the treatment of UC. Qing-Chang-Hua-Shi granule colitis Lactobacillus gasseri ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Ulcerative colitis (UC), characterized by chronic intestinal inflammation and epithelial barrier dysfunction, remains a therapeutic challenge due to its complex etiology involving dysregulated immune responses, microbiota dysbiosis, and aberrant cell death pathways [ 1 , 2 ]. Among emerging mechanisms, ferroptosis—an iron-dependent form of regulated cell death driven by lipid peroxidation—has recently been implicated in UC pathogenesis [ 3 , 4 ]. The gut microbiota plays a crucial role in maintaining intestinal homeostasis, and disruptions in its composition and metabolism can result in pathological damage to the intestines [ 5 , 6 ]. Metabolites act as important mediators of host-microbe interactions and are essential for the maintenance of the gut barrier [ 7 , 8 ]. Targeting the microbiota-metabolic axis has emerged as a promising approach for managing UC. For instance, bile acids derived from the gut microbiota can increase the proportion of regulatory T cells in the colon of mice, thereby reducing their susceptibility to colitis [ 9 ]. Short-chain fatty acids (SCFAs) produced by the microbiota can repair the intestinal epithelium by activating inflammasomes and influencing the secretion of interleukin (IL)-18 [ 10 ]. Additionally, studies have shown that fatty acids produced by gut microbes, especially unsaturated fatty acids, are closely associated with the inflammatory state of IBD [ 11 , 12 ]. Ferroptosis, a recently discovered form of regulated cell death (RCD), is characterized by iron dependence, lipid peroxidation, increased reactive oxygen species (ROS), and disruption of mitochondrial metabolism [ 13 , 14 ]. Glutathione peroxidase 4 (GPX4) is a key antioxidant enzyme that utilizes glutathione (GSH) as a substrate to catalyze the reduction of lipid peroxides to alcohol. The production of GSH relies on the cystine/glutamate antiporter system (System Xc−). Inhibition of GPX4/GSH or System Xc − activity is considered a central mechanism in the induction of ferroptosis [ 15 , 16 ]. Ferroptosis has been implicated as a potential pathogenic factor in IBD [ 17 ]. Studies have demonstrated ferroptosis-induced damage in both UC patients and colitic mice, and ferroptosis inhibitors are being explored as potential therapeutic agents for UC [ 18 – 20 ]. However, the relationship between the microbiota and ferroptosis in UC treatment remains largely unexplored. A phytotherapeutic agent, Qing-Chang-Hua-Shi granule (QCHS) is a traditional Chinese medicine (TCM) formula widely used in China for the treatment of UC. It is composed of Huanglian (Coptis chinensis Franch.), Huangqin (Scutellaria baicalensis Georgi), Baijiangcao (Patrinia scabiosaefolia Fisch.), Danggui (Angelica sinensis (Oliv.) Diels), Baishao (Paeonia lactiflora Pall.), Diyu (Sanguisorba officinalis L.), Zicao (Lithospermum erythrorhizon Siebold & Zucc.), Qiancao (Rubia cordifolia L.), Baizhi (Angelica dahurica (Fisch, ex Hoffm.) Benth. et Hook.f.), Muxiang (Aucklandia lappa Decne.), and Gancao (Glycyrrhiza uralensis Fisch.) in a ratio of 6:10:15:10:20:10:10:20:12:6:6. Our previous studies have shown that QCHS can regulate T cell differentiation and has superior anti-inflammatory effects compared to 5-aminosalicylic acid and sulfasalazine in experimental colitis models [ 21 , 22 ]. However, the effect of QCHS on the microbiota and metabolism requires further investigation. In this study, we evaluated the effects of QCHS on the gut microbiota and metabolism in UC mice. We demonstrated that QCHS acts as an intestinal microecological regulator by increasing the abundance of Lactobacillus gasseri (L. gasseri) and altering the profile of ferroptosis-related metabolites in feces. We also explored the correlation between L. gasseri and ferroptosis, and for the first time, we report that L. gasseri can ameliorate DSS-induced colitis by inhibiting ferroptosis. Our findings reveal a novel mechanism of action for QCHS and L. gasseri, suggesting that ferroptosis-targeted herbal medicine or probiotics could serve as a new treatment strategy for UC. Here, we hypothesize that QCHS ameliorates UC by reshaping the gut microbiome to inhibit ferroptosis. By integrating 16S rDNA sequencing, metabolomics, and functional assays, we reveal that QCHS enriches Lactobacillus gasseri, which drives GSH-dependent ferroptosis suppression. Our findings bridge the gap between TCM and modern mechanistic research, highlighting microbiota-ferroptosis axis as a novel target for UC therapy. 2. Materials and Methods 2.1. Reagents DSS (36000–50000 MW, CAS: 216011080) was obtained from MP Biomedicals. LPS (L2630) and FITC-Dextran (60842-46-8) were bought from Sigma-Aldrich. cDNA Synthesis Kit (R211-01), qRT-PCR SYBR Green Kit (Q221-01), and CCK-8 Cell Counting Kit (A311-01) were bought from Vazyme. Fetal Bovine Serum (04-001-1ACS), Trypsin EDTA solution (03-050-1A), and RPMI Medium 1640 (01-100-1ACS) were purchased from Biological Industries. Antibodies against FTH1(#4393) and β-Actin (#3700) were provided by CST. Antibodies against ZO-1 (sc-33725) was provided by Santa Cruz Biotechnology. Antibodies against Claudin-5 (ab131259), and Muc-2 (ab272692) were purchased from Abcam. Antibodies against 4-HNE (bs-6313R) were bought from Bioss. Antibodies against ACSL4 (66617-1), GPX4 (22401-1), and the secondary antibodies were bought from Proteintech. H&E (G1003), Alcian blue (G1027), and Prussian blue staining kit (G1029) were provided by Servicebio. SP Rabbit & Mouse HRP Kit (DAB, CW2069S) was purchased from CWBio. Iron Assay kit (TC1015) was provided by LEAGENE. Total Glutathione Assay Kit (S0052) and Lipid Peroxidation MDA Assay Kit (S0131S) were provided by Beyotime. All primers were provided by Generay Biotech (Shanghai) and the primer information is listed in the Supporting Information. 2.2. Preparation of Qing-Chang-Hua-Shi granule The 11 components of QCHS were provided by Jiangyin Tianjiang Pharmaceutical St (Jiangsu, China) in the form of granules, which were dissolved in double distilled water (ddH 2 O) and then diluted to three working concentrations before use. 2.3. Bacterial strain and culture conditions The L. gasseri (BNCC135322) strain was purchased from Bena Culture Collection (Henan, China) and confirmed by 16S rDNA sequencing in allwegene Tech, Ltd. (Beijing, China). L. gasseri were grown anaerobically in MRS broth at 37°C. The bacteria were centrifuged (4000×g, 10 min) and washed twice with sterile phosphate-buffered saline (PBS) solution, and finally resuspended in sterile PBS before use. 2.4. Detection of L. gasseri growth curve The fresh L. gasseri suspension was inoculated into 200 mL of MRS liquid medium at a 2% (v/v) inoculation volume. The OD600 value was measured every 3 hours (repeated 3 times with the average value taken), and the 24-hour growth curve of L. gasseri was plotted according to the incubation time and absorbance values. To determine QCHS’s effects on the growth of L. gasseri . the bacteria were cultured in MRS liquid medium with or without different concentrations of QCHS for 24 h. The OD600 value of the culture medium was measured and the survival rate of bacteria was calculated using the normal MRS culture group as a reference. 2.5. Animal experiments All mice (C57BL/6J, male, 20-22g) used were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd and were raised under SPF conditions at Nanjing University of Chinese Medicine (A standard 12h light/dark cycle, license number: SYXK(苏)2024-0049). The experiments were approved by the Animal Ethics Committee of NJUCM (Application Number: 202403A069) on 8th Mar, 2024. For experiments involving QCHS treatment, UC model was established using 3% DSS (dissolved in drinking water) for 7 days. Mice were then given different doses of QCHS (9.5, 19, 38 g/kg/day), or double distilled water (ddH 2 O) for another 7 days by gavage. For experiments involving L. gasseri intervention, mice were given 3% DSS with or without L. gasseri (5×10 8 CFU) for 7 days. The disease activity index (DAI) was determined using the body weight, fecal properties, and blood in the stool, as described in a previous publication [ 23 ]. The serum, colon tissue, and feces were collected for further analysis. 2.6. Measurement of FITC-Dextran 4 leakage Mice were fasted for 6 h before being gavaged with FITC-Dextran (40 mg/mL). After 4 h, serum was collected and the fluorescence value of serum samples were measured at 500 nm. A standard curve was drawn with FITC standards and the sample concentrations were calculated based on the fluorescence values. All samples were protected from light during the experiment. 2.7. Histological evaluation of colitis 0.5 cm of colon tissue was washed with PBS and fixed in paraformaldehyde for 48 h before being embedded in paraffin and cut into 4 µm thick sections for further analysis. H&E, Alcian blue, and Prussian blue staining were performed according to the staining kits’ protocol (Servicebio) after deparaffinization. Images were captured with a light microscope (Leica). The histological scores were performed as previously described based on crypt loss, lymphoid follicle formation, and inflammatory infiltration [ 23 ]. Histological scores were determined blindly based on the sum of the epithelium and infiltration scores. Epithelium score: 0 = normal; 1 = loss of goblet cells; 2 = loss of goblet cells in large areas; 3 = loss of crypts; 4 = loss of crypts in large areas. Infiltration score: 0 = normal; 1 = infiltration around crypt bases; 2 = infiltration reaching the muscularis mucosae; 3 = extensive infiltration reaching the muscularis mucosae; 4 = infiltration of the submucosa. 2.8. Immunostaining analysis After deparaffinization, antigen retrieval of colon tissue sections was performed using sodium citrate solution. Next, the sections were blocked with 2% BSA at 37°C for 1 h and then incubated with anti-Muc-2 (1:200), anti-Claudin-1 (1:200), and anti-4-HNE (1:100) at 4°C overnight. After washing with PBS for 3 times, the tissue sections were incubated with the corresponding secondary antibodies at 37°C for 1 h. For 4-HNE staining, sections were visualized according to the standard methods of the diaminobenzidine (DAB) solution kit. For Muc-2 and Claudin-1 staining, an anti-fade reagent was added after counterstaining the nucleus with DAPI. All images were analyzed using an inverted fluorescence microscope (Leica, DMi8). 2.9. Transmission electron microscopy (TEM) Colon tissues were segmented into 1 mm 3 sections on ice and fixed with 2.5% glutaraldehyde at 4°C overnight. Samples were then dehydrated in a gradient of ethanol concentrations and acetone, and then embedded in resin. 60–80 nm ultra-thin sections were cut from the resin block and transferred to 150 meshcopper grids coated with formvar film. The samples were stained with 2% uranyl acetate saturated alcohol solution followed by 2.6% lead citrate solution. Finally, the samples were observed using a transmission electron microscope (HITACHI HT7800, 120kV). 2.10. 16S rDNA sequence analysis The microbial sequencing analysis was performed by Allwegene Tech. Briefly, the genomic DNA was extracted and the integrity of DNA was inspected using 1% agarose gel electrophoresis. The V3-V4 regions of 16S rDNA gene were amplified and the PCR products were recovered using 1% agarose gel electrophoresis. Next, DNA were purified with Agencourt AMPure XP Nucleic Acid Purification Kit. DNA sequencing was performed based on the Illumina MiSeq platform (PE300). Chimeras and short sequences were removed from sequencing data to obtain high-quality sequences and operational taxonomic units (OTUs) were generated. Finally, OTUs with a similarity level of less than 97% were used for further bioinformatics analysis. 2.11. Quantification of Lactobacillus gasseri in stool The genomic DNA of feces were extracted using TIANamp Genomic DNA Kit (DP304, TIANGEN). PCR amplification of DNA was performed using L. gasseri- specific primers (F: 5’ -AATACTCCCGAAGCACGTCA-3’, R: 5’-TCATTGTGTTTGGCAATCGT-3’). PCR products were then checked with 1% agarose gel electrophoresis and recovered using the Magbead Gel Extraction Kit (CWBIO). Purified DNA was cloned using DH5α (CWBIO), and the plasmid was extracted as a standard for PCR quantification. The plasmid standard was diluted 10-fold from 10 1 -10 5 , and 2 µL of gradient was used as a template to establish a standard curve. The samples and standard DNA underwent Realtime PCR reaction, and then the DNA quantity was calculated according to the standard curve. All experiments were repeated three times. 2.12. Non-targeted metabolomic analysis 200 mg of fecal matter was mixed with pre-chilled 80% methanol, vortexed, incubated on ice for 5 min, then centrifuged at 15,000 x g at 4°C for 20 min. The supernatant was collected and then diluted with mass spectrometry-grade water so that the final concentration of methanol was 53%. Samples were then centrifuged at 15,000 x g at 4°C for 20 min. The supernatant was collected and injected into the UHPLC-MS/MS system (Thermo Fisher) with a Hypesil Gold column (C18, 100×2.1 mm, 1.9µm) for analysis. The mass spectrometer was operated in both positive and negative ionization mode with a mass range of 100 to 1500. The LC-MS/MS data were processed using Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking, and quantification for each metabolite. The peaks were matched with mzCloud ( https://www.mzcloud.org/),mzVaul t, and MassList databases to obtain accurate qualitative and relative quantitative results. The metabolites were annotated using the KEGG database and statistical analysis was performed using the statistical software R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6). Metabolomics analysis was completed by Novogene Co., Ltd. (Beijing, China). 2.13. Assessment for GSH, MDA, and Iron levels Protein samples were collected from colon tissues or NCM-460 cells and the concentrations were determined using the BCA Protein Assay Kit (Beyotime). The levels of Glutathione (GSH) were tested using a Total Glutathione Assay Kit (S0052, Beyotime). The contents of MDA were detected through the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime). An Iron Assay kit (TC1015, LEAGENE) was used to determine the iron levels of colon or NCM-460 cells. The final unit concentrations were calculated based on the sample protein concentrations. 2.14. Cell culture and model establishment Normal human colonic epithelial NCM-460 cells were cultured with RPMI 1640 medium (10% fetal bovine serum) under standard conditions. Inflammation was induced in NCM-460 cells using 1 µg/mL LPS. Briefly, cells (2×10 5 ) were treated with L. gasseri (10 5 -10 7 CFU/mL) for 12 h before LPS stimulation. RSL3 (3 µM) was utilized to induce ferroptosis in cells after L. gasseri treatment and the cells without any treatment were used as a negative control. Cell viability was detected using the standard procedures of the CCK8 assay (Vazyme). Total protein and RNA were collected for further analysis. 2.15. Cell viability assay NCM-460 cells (1×10 4 ) were plated into 96-well plates and incubated with QCHS-containing serum (10 5 -10 7 ) for 24h. Next, 10 µL CCK-8 was added into the plate and the absorbance of cells was measured at 450 nm after 2 h of incubation. 2.16. Statistical analysis All results were displayed as mean ± SEM. Statistical difference between multiple groups were analyzed by one-way ANOVA and P < 0.05 was considered significant. All statistical data were analyzed with GraphPad Prism 9.0. 3. Results 3.1. QCHS alleviated DSS-induced colitis in mice We first evaluated the therapeutic effect of QCHS using a 3% DSS-induced UC model. Mice in the QCHS-treated group showed significant weight recovery, lower DAI scores, and increased colon length compared to the DSS-only group (Fig. 1 A-D). The protective effects of QCHS were also shown to be dose-dependent. Histopathological images revealed that colonic mucosal disruption, inflammatory cell infiltration, and goblet cell loss were all restored in QCHS-treated mice. Moreover, the pathological scores of the QCHS-treated group were significantly lower than those of the DSS group (Fig. 1 E). As shown in Fig. 1 F-G, the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the inflammatory marker Lipocalin 2 (Lcn-2) were significantly increased in the colon of UC mice, and QCHS treatment reversed these abnormal inflammatory responses. QCHS treatment also reversed the DSS-induced abnormal secretion of P-Selectin and E-Selectin, adhesion molecules involved in the activation of cell proliferation, differentiation, and inflammation (Fig. 1 H-I). These results indicate that QCHS treatment significantly alleviated the DSS-induced colonic inflammatory response in mice. 3.2. QCHS improved intestinal barrier function in UC mice Disruption of intestinal barrier function is a key characteristic of UC. Therefore, we assessed the protective effect of QCHS on the intestinal barrier in UC mice. The concentration of FD-4 in the serum of QCHS-treated mice was significantly lower compared to the DSS group (Fig. 2 A), suggesting that QCHS can reduce intestinal permeability in UC mice. The protein levels of Mucin-2 (Muc-2) and tight junction proteins (ZO-1, claudin-5) in the colon of UC mice were significantly increased by QCHS treatment (Fig. 2 B). Alcian blue staining revealed a significant increase in colonic mucin production after QCHS treatment compared to the DSS group (Fig. 2 C). Furthermore, immunofluorescence staining showed that the expression of Muc-2 and Claudin-1 was reduced in colitic mice, but QCHS significantly restored their expression (Fig. 2 D). These results suggest that QCHS can improve intestinal barrier function in UC mice. 3.3. QCHS reprogrammed the gut microbiota and increased the relative abundance of L. gasseri The gut microbiome plays a crucial role in maintaining intestinal homeostasis. Therefore, we explored the effect of QCHS on the gut microbial composition in UC mice using 16S rDNA sequencing. The Venn diagram shown in Fig. 3 A illustrates the differences in the intestinal microbiota among the three groups of mice. The Chao1 index revealed that the alpha-diversity of the microbiome was affected by QCHS in mice with DSS-induced colitis (Fig. 3 B). Principal coordinates analysis (PCoA) showed differences in the gut microbial structure between the control group, the DSS group, and the QCHS group, indicating that QCHS treatment significantly altered the gut microbiota structure in UC mice (Fig. 3 C). In Fig. 3 D, the bar plot shows that Bacteroidota, Firmicutes, and Proteobacteria were the dominant phyla, and the ratio of Bacteroidota to Firmicutes was reversed after QCHS treatment. At the genus level (Fig. 3 E), the bacteria with the highest relative abundance were Lactobacillus, Bacteroides, Escherichia-Shigella, Romboutsia, and Turicibacter. The Wilcoxon test was used to compare the microbiota between the QCHS and DSS groups, showing significant differences. The results indicated that QCHS significantly decreased the relative abundance of Romboutsia and Turicibacter and dramatically increased the relative abundance of Lactobacillus (Fig. 3 F). More importantly, species-level analysis revealed that the relative abundance of Lactobacillus gasseri was significantly elevated by QCHS treatment (Fig. 3 G). LEfSe analysis (LDA score > 4) was performed to further explore which bacteria were significantly affected by QCHS. Notably, there were significant differences in the relative abundance of Lactobacillus at different taxonomic levels after QCHS treatment (Fig. 3 H). Quantitative PCR detection further confirmed that L. gasseri was significantly enriched by QCHS (Fig. 3 I). These results collectively indicate that QCHS altered the gut microbial composition and significantly increased the relative abundance of L. gasseri in colitic mice. 3.4. QCHS altered ferroptosis metabolism and increased Glutathione levels Metabolites are important mediators through which the gut microbiota exerts its effects. Therefore, we used UHPLC-MS/MS to examine the metabolomic changes in QCHS-treated UC mice. PCA analysis under both positive and negative ion modes demonstrated that the metabolic profiles of the fecal matter differed among the experimental groups (Fig. 4 A-B). The Venn diagram under the positive ion mode showed that 311 metabolites were significantly altered in the feces of mice after QCHS treatment (Fig. 4 C), while the negative ion mode revealed 178 differential metabolites between the QCHS and DSS groups (Fig. 4 D). The volcano plot shows the overall differences in fecal metabolites between the QCHS and DSS groups when both positive and negative ions were combined (Fig. 4 E). Cluster analysis was used to examine the expression levels of all differential metabolites, as shown in Fig. 4 F. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database is a powerful tool for analyzing in vivo metabolic networks. We performed KEGG pathway enrichment analysis to identify the major biochemical metabolic and signal transduction pathways involved in the differential metabolites. The top 20 enriched metabolic pathways after QCHS treatment are shown in Fig. 4 G, with significant pathways including Arachidonic acid metabolism (P = 0.0009), Biosynthesis of unsaturated fatty acids (P = 0.0010), Linoleic acid metabolism (P = 0.0335), and Ferroptosis (P = 0.0398). Interestingly, all these pathways are closely related to lipid metabolism and ferroptosis. Next, we analyzed the relative abundance of differential metabolites in these four metabolic pathways and found that QCHS significantly increased the level of Glutathione (GSH), which is a key regulator of ferroptosis (Fig. 4 H). This metabolomic analysis suggests that QCHS may influence GSH-mediated ferroptosis metabolism in colitic mice. 3.5. L. gasseri is significantly correlated with ferroptosis metabolism To explore the association between gut microbiota and metabolites affected by QCHS, we performed Pearson correlation analysis on the metabolites and differential bacteria at the genus level. The results showed that Lactobacillus was positively correlated with metabolites of the ferroptosis pathway (glutathione and mevalonic acid) and negatively correlated with metabolites in arachidonic acid metabolism (5-oxoicosatetraenoic acid, prostaglandin D2, and 16(R)-hydroxyeicosatetraenoic acid) (Fig. 5 A). Next, Spearman-based correlation analysis was performed between QCHS-mediated L. gasseri and four metabolites of the ferroptosis metabolic pathway. The results indicated that the relative abundance of L. gasseri was significantly positively correlated with GSH and significantly negatively correlated with mevalonic acid (MVA) (Fig. 5 B-E), suggesting that QCHS-mediated L. gasseri may potentially inhibit ferroptosis. 3.6. QCHS inhibited DSS-induced ferroptosis damage in UC mice The results of the metabolomic analysis show that QCHS significantly enriched ferroptosis metabolism in the feces. Based on these findings, we hypothesized that the therapeutic effect of QCHS in UC mice might be mediated through ferroptosis. To verify this hypothesis, we collected colon tissue and serum from UC mice for various assays. Electron microscopy analysis revealed shrunken mitochondria and reduced mitochondrial cristae in colonic epithelial cells after DSS induction, whereas QCHS significantly alleviated this ferroptosis-like injury in the mitochondria (Fig. 6 A).Lipid peroxidation plays a key role in the process of ferroptosis. 4-Hydroxynonenal (4-HNE), the end product of lipid peroxidation, is used as an indicator to detect lipid peroxidation. Immunohistochemical results showed that QCHS significantly reduced the abnormal increase of 4-HNE induced by DSS (Fig. 6 B). Additionally, lipid peroxidation, as indicated by MDA levels, was elevated after DSS induction but significantly decreased following QCHS treatment (Fig. 6 F). Ferroptosis is also dependent on the accumulation of iron ions. Both Prussian blue staining and iron assays demonstrated that QCHS reduced the iron overload caused by DSS (Fig. 6 C-D). Next, we assessed the mRNA levels of ferroptosis-related genes and found that their expression was upregulated following DSS induction, but remarkably decreased after QCHS treatment. Conversely, the levels of GSH and the catalytic enzyme GPX4 were significantly reduced in the colon of UC mice. Interestingly, QCHS promoted the expression of both GSH and GPX4 (Fig. 6 E and G). Moreover, after DSS induction, the protein levels of ACSL4 and FTH1, which are characteristic of ferroptosis, were significantly elevated, while GPX4 levels were reduced. QCHS treatment effectively reversed the levels of these ferroptosis-related proteins (Fig. 6 H). This data suggests that QCHS exerts a protective effect against DSS-induced ferroptosis in the colon. 3.7. QCHS promoted the proliferation of L. gasseri in vitro 16S rDNA sequencing results showed that QCHS increased the relative abundance of L. gasseri in the feces of UC mice, and the level of L. gasseri exhibited a significant positive correlation with GSH in the ferroptosis pathway. To further investigate the relationship between QCHS and L. gasseri, we cultured the L. gasseri strain with MRS medium in vitro (Fig. 6 A-B). Notably, the combination of QCHS (1000 µg/mL) and MRS medium significantly promoted the growth of L. gasseri (Fig. 6 C). 3.8. L. gasseri reduced DSS-induced intestinal injury in mice. Based on the above results, we speculated that the protective effect of QCHS on UC mice might be mediated by L. gasseri. To test this hypothesis, we administered L. gasseri (5×10⁸ CFU) to mice with or without DSS induction for 7 days to determine whether L. gasseri could influence the progression of UC in mice. L. gasseri-treated mice exhibited reduced body weight loss, lower DAI scores, restored colon length, and alleviated pathological damage compared to mice treated with DSS alone (Fig. 7 D-H). While DSS-treated mice showed higher serum FD-4 levels and reduced colonic mucin secretion, L. gasseri treatment significantly mitigated DSS-induced colonic barrier damage (Fig. 7 I-J). These findings suggest that L. gasseri can slow the progression of colitis and protect against intestinal injury in UC mice. 3.9. L. gasseri inhibited DSS-induced ferroptosis-like injury in mice. We also examined colonic ferroptosis impairment in L. gasseri -treated UC mice. Electron microscopy images showed that shrunken mitochondria and reduced mitochondrial crista in colonic epithelial cells could be significantly alleviated by treatment with L. gasseri ( Fig. 8 A ) . 4-HNE staining and Prussian blue staining of colon tissue showed that L. gasseri alleviated lipid peroxidation and excessive accumulation of iron ions caused by DSS, respectively ( Fig. 8 B-C ) . In addition, the L. gasseri treatment increased the levels of GSH, while effectively decreasing the levels of iron and MDA in DSS-induced colitis ( Fig. 8 D-F ) . Therefore, L. gasseri may attenuate intestinal injury in colitic mice by inhibiting ferroptosis. 3.10. L. gasseri inhibited LPS-induced inflammation in NCM-460 cells Next, we investigated the effect of L. gasseri on inflammation and ferroptosis through in vitro experiments. First, NCM-460 cells were pretreated with the indicated concentration of L. gasseri for 12 hours, followed by the addition of LPS (1 µg/mL) to induce cellular inflammation. As expected, L. gasseri significantly reduced the mRNA levels of IL-6, IL-1β, and TNF-α in LPS-induced NCM-460 cells (Fig. 9 A-C). 3.11. L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells Finally, RSL3, a known inhibitor of GPX4, was used to induce ferroptosis in NCM-460 cells. As shown in Fig. 9 D-F, RSL3 significantly increased the levels of iron and MDA, while decreasing GSH content in NCM-460 cells. However, pretreatment with L. gasseri effectively prevented iron overload and lipid peroxidation, and significantly increased GSH levels in the cells (Fig. 9 D-F). More importantly, the cell viability assay revealed that L. gasseri pretreatment significantly inhibited RSL3-induced cell death (Fig. 9 G). Additionally, analysis of ferroptosis-related molecules showed that L. gasseri increased the expression of GPX4 at both the mRNA and protein levels, while significantly inhibiting the expression of positive ferroptosis regulators such as FTH1 and ACSL4 (Fig. 9 H-I). These results suggest that L. gasseri may act as a potential ferroptosis inhibitor, offering protection against cellular injury. 4. Discussion The incidence of ulcerative colitis (UC) has been rising steadily in developing countries, placing a considerable burden on healthcare systems [ 24 , 25 ]. Given the challenges of controlling inflammation and promoting mucosal repair in UC treatment, there is a pressing need for the development of new therapeutic strategies. Traditional Chinese medicine (TCM) prescriptions, including QCHS, are increasingly being recognized as multi-targeted therapies. Key components identified in QCHS include berberine, baicalin, coumarin, ferulic acid, and paeoniflorin, among others. Detailed information about these chemical components has been provided in our previous study [ 22 ]. A randomized clinical trial has confirmed the efficacy and safety of QCHS in patients with moderately active UC [ 26 ]. However, the specific mechanisms through which QCHS exerts its effects remain largely unexplored. Dysbiosis in the gut microbiota is known to disrupt immune homeostasis, leading to abnormal immune responses and inflammatory cytokine release [ 27 , 28 ]. Inflammatory bowel disease (IBD) patients exhibit altered gut microbial diversity, such as an imbalance between Firmicutes and Bacteroidetes, an increase in Proteobacteria, and the depletion of Roseburia species [ 29 , 30 ]. The mucus layer, which interacts with both the microbiota and immune cells, plays a critical role in maintaining gut homeostasis. However, the expansion of pathogenic bacteria can break down this barrier, leading to “leaky gut” and increasing the risk of pathogens entering the lamina propria and bloodstream [ 31 – 33 ]. Dysbiosis may also disrupt the metabolome, further compromising the mucosal barrier and contributing to inflammation. For instance, the depletion of short-chain fatty acids (SCFAs) promotes the polarization of M1 macrophages, which drives intestinal inflammation [ 34 ]. Additionally, fecal metabolism of palmitoleic acid and tryptophan degradation have been linked to the production of TNF-α and interferon-gamma (IFN-γ), both of which are associated with inflammation [ 35 ]. Probiotics have been shown to restore microbial diversity, and their therapeutic potential in IBD has been demonstrated in both clinical and animal studies [ 36 , 37 ]. Lactobacillus species, in particular, are among the most widely used probiotics, and their depletion is associated with IBD progression [ 38 , 39 ]. Lactobacillus helps repair the intestinal epithelial barrier by upregulating tight junction proteins and reduces colonic inflammation in UC mice by inhibiting the TLR4-NF-κB-NLRP3 signaling pathway [ 40 , 41 ]. Notably, prebiotic therapy has been shown to promote probiotic growth, restoring gut function and alleviating IBD symptoms [ 42 , 43 ]. In this study, we demonstrated that QCHS treatment alleviated DSS-induced colitis by reducing colitis symptoms, inhibiting pro-inflammatory cytokine secretion, and repairing the colonic epithelial barrier. We utilized microbial sequencing and metabolomic analysis to explore the potential mechanisms of QCHS. Our results indicated that QCHS reshaped the gut microbiota, specifically increasing the relative abundance of Lactobacillus gasseri in the feces of UC mice. In vitro experiments confirmed that QCHS promoted the growth of L. gasseri strains. Furthermore, untargeted metabolomics revealed that QCHS altered the fecal metabolic profile of UC mice, with metabolites significantly enriched in ferroptosis and its related metabolic pathways. Notably, we are the first to report a correlation between Lactobacillus and ferroptosis, particularly the significant positive correlation between L. gasseri and glutathione (GSH) levels. The GPX4/GSH axis is a central regulator of ferroptosis, a form of regulated cell death (RCD) that is closely associated with lipid peroxidation. Disruptions in iron metabolism lead to the accumulation of intracellular free iron, which catalyzes the generation of reactive oxygen species (ROS) through the Fenton reaction. ROS then promote lipid peroxidation and trigger ferroptosis [ 44 ]. Additionally, polyunsaturated fatty acids (PUFAs), which are substrates for lipid peroxidation, influence ferroptosis susceptibility [ 45 ]. Our data showed that QCHS enriched pathways involved in arachidonic acid metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. Specifically, QCHS reduced the abundance of arachidonic acid, adrenic acid, linoleic acid, 16(R)-HETE, palmitic acid, prostaglandin F2α, prostaglandin D2, and other metabolites, which are implicated in both lipid peroxidation and inflammation. For example, arachidonic acid induces inflammation through its conversion to prostaglandins (PGs) via the cyclooxygenase (COX) pathway [ 46 ]. Emerging evidence suggests that ferroptosis plays a critical role in the pathogenesis of IBD, and inhibiting ferroptosis may offer a novel therapeutic approach for UC [ 47 , 48 ]. Our study demonstrated that QCHS significantly alleviated DSS-induced ferroptosis in the colon of UC mice, and that L. gasseri mediated this protective effect. For the first time, we found that L. gasseri can act as a ferroptosis inhibitor, mitigating the progression of UC. In vitro, we further demonstrated that L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells, with the mechanism involving activation of the GSH/GPX4 signaling pathway. Taken together, our study provides compelling evidence for the regulatory role of QCHS on the microbiota-metabolome axis and ferroptosis in UC mice (Fig. 10). We also uncover a novel function of L. gasseri as an inhibitor of ferroptosis, offering new insights into potential therapeutic strategies for UC. These findings suggest that through the microbiota modulators or ferroptosis inhibitors targeting Lactobacillus, QCHS may be promising candidates for the treatment of UC. Abbreviations UC, ulcerative colitis; QCHS, Qing-Chang-Hua-Shi granule; L. gasseri, Lactobacillus gasseri; IBD, inflammatory bowel disease; SCFAs, short-chain fatty acids; IL, interleukin; RCD, regulated cell death; ROS, reactive oxygen species; GPX4, Glutathione peroxidase 4; GSH, Glutathione; System Xc−, the cystine/glutamate antiporter system; TCM, traditional Chinese medicine; DSS, dextran sulfate sodium; DAI, Disease activity index; TNF-α, tumor necrosis factor-α; Lcn2, Lipocalin 2; FD-4, FITC-Dextran 4; Muc2, mucin2; 4-HNE, 4-Hydroxynonenal; MDA, malondialdehyde; ACSL4, acyl-CoA synthetase long-chain family member 4; FTH1, Ferritin heavy polypeptide 1; GSH, Glutathione; KEGG, Kyoto Encyclopedia of Genes and Genomes; MVA, Mevalonic acid; IFN-γ, interferon gamma; PUFAs, polyunsaturated fatty acids; PGs, prostaglandins. Declarations Author contributions L. G. and H. S. designed and funded the whole research. C. C., J. H., Z. L., W. L., R. A. Y. L., F. X., Y. W., and Y. C. collaborated to complete the experiments and data analysis. C. C., Z. L., R. A., Y. L.and L. G. edited the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. Declaration of interest The authors have declared no conflict of interest. Funding Declaration This work was supported by National Natural Science Foundation of China (82405296, 82274483, 82305158). Ethics approval and consent to participate The animal study protocol was approved by the Animal Care and Use Committee (ACUC) of [Nanjing University of Traditional Chinese Medicine], protocol number [ACU240404]. The study adhered to the guidelines set by the committee. Consent for publication This is to confirm that the work has not been and will not be submitted simultaneously to another journal, in whole or in part; it reports previously unpublished work. If accepted, neither the paper itself nor substantial parts of it, will be published in the same form, in any language, without the consent of the publishers. The submitted version of the manuscript has been approved by all the authors. Data Availability Statements The microbial and metabolomic raw data reported in this paper have been deposited in the China National Center for Bioinformation (https://ngdc.cncb.ac.cn) and the accession numbers are CRA009705 (animal microbiota) and OMIX002923 (animal metabolome). References J.D. Feuerstein, A.C. Moss, F.A. Farraye, Ulcerative Colitis, Mayo Clin Proc 94(7) (2019) 1357-1373. S. Danese, C. Fiocchi, Ulcerative colitis, N Engl J Med 365(18) 1713-25. G.P. Ramos, K.A. Papadakis, Mechanisms of Disease: Inflammatory Bowel Diseases, Mayo Clin Proc 94(1) (2019) 155-165. J.V. Patankar, C. Becker, Cell death in the gut epithelium and implications for chronic inflammation, Nat Rev Gastroenterol Hepatol 17(9) (2020) 543-556. J. Ni, G.D. Wu, L. Albenberg, V.T. Tomov, Gut microbiota and IBD: causation or correlation?, Nat Rev Gastroenterol Hepatol 14(10) (2017) 573-584. M. Lee, E.B. Chang, Inflammatory Bowel Diseases (IBD) and the Microbiome-Searching the Crime Scene for Clues, Gastroenterology 160(2) (2021) 524-537. A. Lavelle, H. Sokol, Gut microbiota-derived metabolites as key actors in inflammatory bowel disease, Nat Rev Gastroenterol Hepatol 17(4) (2020) 223-237. N. Gasaly, P. de Vos, M.A. Hermoso, Impact of Bacterial Metabolites on Gut Barrier Function and Host Immunity: A Focus on Bacterial Metabolism and Its Relevance for Intestinal Inflammation, Front Immunol 12 (2021) 658354. X. Song, X. Sun, S.F. Oh, M. Wu, Y. Zhang, W. Zheng, N. Geva-Zatorsky, R. Jupp, D. Mathis, C. Benoist, D.L. Kasper, Microbial bile acid metabolites modulate gut RORgamma(+) regulatory T cell homeostasis, Nature 577(7790) (2020) 410-415. L. Macia, J. Tan, A.T. Vieira, K. Leach, D. Stanley, S. Luong, M. Maruya, C. Ian McKenzie, A. Hijikata, C. Wong, Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome, Nature communications 6(1) (2015) 1-15. E.A. Scoville, M.M. Allaman, C.T. Brown, A.K. Motley, S.N. Horst, C.S. Williams, T. Koyama, Z. Zhao, D.W. Adams, D.B. Beaulieu, D.A. Schwartz, K.T. Wilson, L.A. Coburn, Alterations in Lipid, Amino Acid, and Energy Metabolism Distinguish Crohn's Disease from Ulcerative Colitis and Control Subjects by Serum Metabolomic Profiling, Metabolomics 14(1) (2018) 17. R. Marion-Letellier, G. Savoye, P.L. Beck, R. Panaccione, S. Ghosh, Polyunsaturated fatty acids in inflammatory bowel diseases: a reappraisal of effects and therapeutic approaches, Inflamm Bowel Dis 19(3) (2013) 650-61. J. Zheng, M. Conrad, The Metabolic Underpinnings of Ferroptosis, Cell Metab 32(6) (2020) 920-937. B.R. Stockwell, Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications, Cell 185(14) (2022) 2401-2421. X. Chen, J. Li, R. Kang, D.J. Klionsky, D. Tang, Ferroptosis: machinery and regulation, Autophagy 17(9) (2021) 2054-2081. W.S. Yang, R. SriRamaratnam, M.E. Welsch, K. Shimada, R. Skouta, V.S. Viswanathan, J.H. Cheah, P.A. Clemons, A.F. Shamji, C.B. Clish, L.M. Brown, A.W. Girotti, V.W. Cornish, S.L. Schreiber, B.R. Stockwell, Regulation of ferroptotic cancer cell death by GPX4, Cell 156(1-2) (2014) 317-331. S. Xu, Y. He, L. Lin, P. Chen, M. Chen, S. Zhang, The emerging role of ferroptosis in intestinal disease, Cell Death Dis 12(4) (2021) 289. M. Xu, J. Tao, Y. Yang, S. Tan, H. Liu, J. Jiang, F. Zheng, B. Wu, Ferroptosis involves in intestinal epithelial cell death in ulcerative colitis, Cell Death Dis 11(2) (2020) 86. Y. Chen, W. Yan, Y. Chen, J. Zhu, J. Wang, H. Jin, H. Wu, G. Zhang, S. Zhan, Q. Xi, T. Shi, W. Chen, SLC6A14 facilitates epithelial cell ferroptosis via the C/EBPβ-PAK6 axis in ulcerative colitis, Cell Mol Life Sci 79(11) (2022) 563. S. Dong, Y. Lu, G. Peng, J. Li, W. Li, M. Li, H. Wang, L. Liu, Q. Zhao, Furin inhibits epithelial cell injury and alleviates experimental colitis by activating the Nrf2-Gpx4 signaling pathway, Dig Liver Dis 53(10) (2021) 1276-1285. L. Zhu, L.M. Dai, H. Shen, P.Q. Gu, K. Zheng, Y.J. Liu, L. Zhang, J.F. Cheng, Qing Chang Hua Shi granule ameliorate inflammation in experimental rats and cell model of ulcerative colitis through MEK/ERK signaling pathway, Biomed Pharmacother 116 (2019) 108967. C. Cheng, J. Hu, Y. Li, Y. Ji, Z. Lian, R. Au, F. Xu, W. Li, H. Shen, L. Zhu, Qing-Chang-Hua-Shi granule ameliorates DSS-induced colitis by activating NLRP6 signaling and regulating Th17/Treg balance, Phytomedicine 107 (2022) 154452. C. Cheng, W. Zhang, C. Zhang, P. Ji, X. Wu, Z. Sha, X. Chen, Y. Wang, Y. Chen, H. Cheng, L. Shi, Hyperoside Ameliorates DSS-Induced Colitis through MKRN1-Mediated Regulation of PPARγ Signaling and Th17/Treg Balance, J Agric Food Chem 69(50) (2021) 15240-15251. S.C. Ng, H.Y. Shi, N. Hamidi, F.E. Underwood, W. Tang, E.I. Benchimol, R. Panaccione, S. Ghosh, J.C.Y. Wu, F.K.L. Chan, J.J.Y. Sung, G.G. Kaplan, Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies, Lancet 390(10114) (2017) 2769-2778. M.A. Kamm, Rapid changes in epidemiology of inflammatory bowel disease, Lancet 390(10114) (2017) 2741-2742. H. Shen, S. Zhang, W. Zhao, S. Ren, X. Ke, Q. Gu, Z. Tang, J. Xie, S. Chen, Y. Chen, J. Zou, L. Zhang, Z. Shen, K. Zheng, Y. Liu, P. Gu, J. Cheng, J. Hu, L. Zhu, Randomised clinical trial: Efficacy and safety of Qing-Chang-Hua-Shi granules in a multicenter, randomized, and double-blind clinical trial of patients with moderately active ulcerative colitis, Biomed Pharmacother 139 (2021) 111580. M. Alipour, D. Zaidi, R. Valcheva, J. Jovel, I. Martínez, C. Sergi, J. Walter, A.L. Mason, G.K. Wong, L.A. Dieleman, M.W. Carroll, H.Q. Huynh, E. Wine, Mucosal Barrier Depletion and Loss of Bacterial Diversity are Primary Abnormalities in Paediatric Ulcerative Colitis, J Crohns Colitis 10(4) (2016) 462-71. M. Alexander, Q.Y. Ang, R.R. Nayak, A.E. Bustion, M. Sandy, B. Zhang, V. Upadhyay, K.S. Pollard, S.V. Lynch, P.J. Turnbaugh, Human gut bacterial metabolism drives Th17 activation and colitis, Cell Host Microbe 30(1) (2022) 17-30.e9. S. Kumar, A. Kumar, Microbial pathogenesis in inflammatory bowel diseases, Microb Pathog 163 (2022) 105383. P. Qiu, T. Ishimoto, L. Fu, J. Zhang, Z. Zhang, Y. Liu, The Gut Microbiota in Inflammatory Bowel Disease, Front Cell Infect Microbiol 12 (2022) 733992. S. Yu, Y. Sun, X. Shao, Y. Zhou, Y. Yu, X. Kuai, C. Zhou, Leaky Gut in IBD: Intestinal Barrier-Gut Microbiota Interaction, J Microbiol Biotechnol 32(7) (2022) 825-834. L.C. Yu, Microbiota dysbiosis and barrier dysfunction in inflammatory bowel disease and colorectal cancers: exploring a common ground hypothesis, J Biomed Sci 25(1) (2018) 79. K. Parikh, A. Antanaviciute, D. Fawkner-Corbett, M. Jagielowicz, A. Aulicino, C. Lagerholm, S. Davis, J. Kinchen, H.H. Chen, N.K. Alham, N. Ashley, E. Johnson, P. Hublitz, L. Bao, J. Lukomska, R.S. Andev, E. Björklund, B.M. Kessler, R. Fischer, R. Goldin, H. Koohy, A. Simmons, Colonic epithelial cell diversity in health and inflammatory bowel disease, Nature 567(7746) (2019) 49-55. N.A. Scott, A. Andrusaite, P. Andersen, M. Lawson, C. Alcon-Giner, C. Leclaire, S. Caim, G. Le Gall, T. Shaw, J.P.R. Connolly, A.J. Roe, H. Wessel, A. Bravo-Blas, C.A. Thomson, V. Kästele, P. Wang, D.A. Peterson, A. Bancroft, X. Li, R. Grencis, A.M. Mowat, L.J. Hall, M.A. Travis, S.W.F. Milling, E.R. Mann, Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis, Sci Transl Med 10(464) (2018). M. Schirmer, S.P. Smeekens, H. Vlamakis, M. Jaeger, M. Oosting, E.A. Franzosa, R. Ter Horst, T. Jansen, L. Jacobs, M.J. Bonder, A. Kurilshikov, J. Fu, L.A.B. Joosten, A. Zhernakova, C. Huttenhower, C. Wijmenga, M.G. Netea, R.J. Xavier, Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity, Cell 167(4) (2016) 1125-1136.e8. Y. Zhu, Y. Xu, X. Wang, L. Rao, X. Yan, R. Gao, T. Shen, Y. Zhou, C. Kong, L. Zhou, Probiotic Cocktail Alleviates Intestinal Inflammation Through Improving Gut Microbiota and Metabolites in Colitis Mice, Front Cell Infect Microbiol 12 (2022) 886061. I. Bjarnason, G. Sission, B. Hayee, A randomised, double-blind, placebo-controlled trial of a multi-strain probiotic in patients with asymptomatic ulcerative colitis and Crohn's disease, Inflammopharmacology 27(3) (2019) 465-473. D.D. Heeney, M.G. Gareau, M.L. Marco, Intestinal Lactobacillus in health and disease, a driver or just along for the ride?, Curr Opin Biotechnol 49 (2018) 140-147. S. Selvamani, V. Mehta, H. Ali El Enshasy, S. Thevarajoo, H. El Adawi, I. Zeini, K. Pham, T. Varzakas, B. Abomoelak, Efficacy of Probiotics-Based Interventions as Therapy for Inflammatory Bowel Disease: A Recent Update, Saudi J Biol Sci 29(5) (2022) 3546-3567. Y. Chen, L. Zhang, G. Hong, C. Huang, W. Qian, T. Bai, J. Song, Y. Song, X. Hou, Probiotic mixtures with aerobic constituent promoted the recovery of multi-barriers in DSS-induced chronic colitis, Life Sci 240 (2020) 117089. L. Tong, X. Zhang, H. Hao, Q. Liu, Z. Zhou, X. Liang, T. Liu, P. Gong, L. Zhang, Z. Zhai, Y. Hao, H. Yi, Lactobacillus rhamnosus GG Derived Extracellular Vesicles Modulate Gut Microbiota and Attenuate Inflammatory in DSS-Induced Colitis Mice, Nutrients 13(10) (2021). X.F. Zhang, X.X. Guan, Y.J. Tang, J.F. Sun, X.K. Wang, W.D. Wang, J.M. Fan, Clinical effects and gut microbiota changes of using probiotics, prebiotics or synbiotics in inflammatory bowel disease: a systematic review and meta-analysis, Eur J Nutr 60(5) (2021) 2855-2875. P. Li, N. Xiao, L. Zeng, J. Xiao, J. Huang, Y. Xu, Y. Chen, Y. Ren, B. Du, Structural characteristics of a mannoglucan isolated from Chinese yam and its treatment effects against gut microbiota dysbiosis and DSS-induced colitis in mice, Carbohydr Polym 250 (2020) 116958. X. Chen, C. Yu, R. Kang, D. Tang, Iron Metabolism in Ferroptosis, Front Cell Dev Biol 8 (2020) 590226. J.Y. Lee, M. Nam, H.Y. Son, K. Hyun, S.Y. Jang, J.W. Kim, M.W. Kim, Y. Jung, E. Jang, S.J. Yoon, J. Kim, J. Kim, J. Seo, J.K. Min, K.J. Oh, B.S. Han, W.K. Kim, K.H. Bae, J. Song, J. Kim, Y.M. Huh, G.S. Hwang, E.W. Lee, S.C. Lee, Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer, Proc Natl Acad Sci U S A 117(51) (2020) 32433-32442. T. Wang, X. Fu, Q. Chen, J.K. Patra, D. Wang, Z. Wang, Z. Gai, Arachidonic Acid Metabolism and Kidney Inflammation, Int J Mol Sci 20(15) (2019). W. Gao, T. Zhang, H. Wu, Emerging Pathological Engagement of Ferroptosis in Gut Diseases, Oxid Med Cell Longev 2021 (2021) 4246255. J. Huang, J. Zhang, J. Ma, J. Ma, J. Liu, F. Wang, X. Tang, Inhibiting Ferroptosis: A Novel Approach for Ulcerative Colitis Therapeutics, Oxid Med Cell Longev 2022 (2022) 9678625. Additional Declarations No competing interests reported. Supplementary Files Graphicabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Revision requested 12 Nov, 2025 Reviews received at journal 12 Nov, 2025 Reviews received at journal 08 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviews received at journal 31 Oct, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviewers invited by journal 22 Oct, 2025 Editor assigned by journal 19 Oct, 2025 Submission checks completed at journal 18 Oct, 2025 First submitted to journal 13 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7844488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":535295499,"identity":"79770fec-9d56-45c5-a893-5198c21fedf8","order_by":0,"name":"Cheng Cheng","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Cheng","suffix":""},{"id":535295500,"identity":"0fcf86f7-b90a-4ddb-89f9-ec5f0e45c857","order_by":1,"name":"Lei Zhu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhu","suffix":""},{"id":535295501,"identity":"08d758b1-fd29-4a39-a38e-1b21af35b175","order_by":2,"name":"Jingyi Hu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Hu","suffix":""},{"id":535295502,"identity":"eb98417d-1827-4692-a6fe-809cf97e4901","order_by":3,"name":"Wan Feng","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"","lastName":"Feng","suffix":""},{"id":535295503,"identity":"33931473-5c0e-4652-b67c-9985afc8f0ff","order_by":4,"name":"Weiyang Li","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiyang","middleName":"","lastName":"Li","suffix":""},{"id":535295504,"identity":"45b73d4f-e4fb-41c0-b9f5-a195af79ecec","order_by":5,"name":"Ryan Au","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Au","suffix":""},{"id":535295505,"identity":"a67a3bc6-191c-4cf9-8dca-a55d967a7e89","order_by":6,"name":"Yanan Li","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Li","suffix":""},{"id":535295506,"identity":"2417578a-f850-4b16-94f2-1c8cc533e337","order_by":7,"name":"Feng Xu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""},{"id":535295507,"identity":"fde39cd7-05c7-4298-be61-4fc2c651ce9a","order_by":8,"name":"Yuguang Wu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuguang","middleName":"","lastName":"Wu","suffix":""},{"id":535295508,"identity":"ee2ff887-240d-40c5-b16e-eca599aa8bdf","order_by":9,"name":"yuan Cui","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"yuan","middleName":"","lastName":"Cui","suffix":""},{"id":535295509,"identity":"ddbb67a5-9d6f-44f9-9f73-68aa80cc0ce7","order_by":10,"name":"bin Li","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"bin","middleName":"","lastName":"Li","suffix":""},{"id":535295510,"identity":"9c27acb4-1d42-49da-9542-083ecabe9908","order_by":11,"name":"zhe Di","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"zhe","middleName":"","lastName":"Di","suffix":""},{"id":535295511,"identity":"3b900046-17e7-4b72-b671-d3213fdd27bd","order_by":12,"name":"yongchang Miao","email":"","orcid":"","institution":"second hospital of lianyungang","correspondingAuthor":false,"prefix":"","firstName":"yongchang","middleName":"","lastName":"Miao","suffix":""},{"id":535295512,"identity":"17682cec-2eb3-494b-ba2e-7b75c11c904f","order_by":13,"name":"yao Lin","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"yao","middleName":"","lastName":"Lin","suffix":""},{"id":535295513,"identity":"db61c107-022f-4ab6-a8c8-6a43fb534e38","order_by":14,"name":"lilin ge","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFAC5oYDDwwY5BgYDoC5jA2EtTA2HEgwYDAmTQtDAgNDYgOCSwDIuzc2HkgouJO+nfGM4acbDDayGw4wP3uAT4vhmYMghz3L3dlwxlg6hyHNeMMBNnMDvFpmJIK0HM7dcOCMGXMOw+HEDQd42CTwapn/EKwl3QCi5T9hLfIS4BA7nADVcoCwFgMeiMMMNxw4ViydY5BsPPMwmxl+W9oPH/7w4c9heYMbhzd+zqmwk+073vwMvy0HYCwJEAsUVMz41INsaYCx+BtwqxoFo2AUjIKRDQAVZVRR8iKi4AAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"lilin","middleName":"","lastName":"ge","suffix":""},{"id":535295514,"identity":"793559b6-a961-455d-8625-01f94413897c","order_by":15,"name":"hong shen","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"hong","middleName":"","lastName":"shen","suffix":""}],"badges":[],"createdAt":"2025-10-13 05:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7844488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7844488/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-025-01317-5","type":"published","date":"2026-01-26T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94963770,"identity":"92c2a783-4512-4529-b41c-f77f0b9f6328","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68200960,"visible":true,"origin":"","legend":"","description":"","filename":"Aphytotherapeuticagentdemonstratesclinicalefficacy.doc","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/5d3644d980b131c51420b6a8.doc"},{"id":94963741,"identity":"2545d355-e3d4-449d-9940-08f5f0608245","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14857,"visible":true,"origin":"","legend":"","description":"","filename":"b541683185874104ab3c7cf17c190594.json","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/1a3b8ae39617761f489226ad.json"},{"id":94988891,"identity":"597317fc-ff7e-459e-b714-716b9a7d542d","added_by":"auto","created_at":"2025-11-03 07:11:17","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154572,"visible":true,"origin":"","legend":"","description":"","filename":"b541683185874104ab3c7cf17c1905941enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/fea24232474665b82d8cdd22.xml"},{"id":94989373,"identity":"6868d424-0c20-426b-a571-aa5653d29a6e","added_by":"auto","created_at":"2025-11-03 07:12:42","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1981849,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/a7f0ae6f4959ad14b0a7a46e.png"},{"id":94963752,"identity":"99813294-fef3-4edb-8e23-e61d4d5c9965","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1745561,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/6e96cb135a7f053f9128eaf9.png"},{"id":94963751,"identity":"2842cced-bbb2-4adc-a219-b14c4f481b82","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9064038,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/02465609ae89c96c28fb4ed8.png"},{"id":94988298,"identity":"f3dd4269-d14c-4f81-b580-fbc5855d57d9","added_by":"auto","created_at":"2025-11-03 07:08:31","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13388446,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/20df4e8784ca6f3d2fa8d0db.png"},{"id":94963753,"identity":"027b9062-2d82-4a90-8b97-b4c58fd6e4fb","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2480457,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/03ed08e8a58317af84d669eb.png"},{"id":94988844,"identity":"d461c137-2efa-4c59-b1d2-98e30f3e3aef","added_by":"auto","created_at":"2025-11-03 07:11:06","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3678041,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/dedcccbe8c302ba7a256ba07.png"},{"id":94963760,"identity":"79352140-b58d-47fb-b1a1-0e037b3fa553","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3382074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/4bbc54a593b2a42585906137.png"},{"id":94963766,"identity":"15c76f90-d9c1-4828-94fd-88203a001ad3","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11602482,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/47dbb32a8b7b14bbf57796c7.png"},{"id":94963763,"identity":"87f3d872-1c85-40d0-975a-7ae2fe3deb78","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9631735,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/ce5a031e6ed717ebc91e8725.png"},{"id":94963765,"identity":"d8f157cf-8792-4a55-a6c5-418763068482","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10577348,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/347cc598d4e6151a1729a60a.png"},{"id":94963774,"identity":"0bc34494-97a6-405d-ae1a-3677b5d774da","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":300267,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/739555bc90c1a7afa2dd0c3a.png"},{"id":94963767,"identity":"6bfe1c8e-7a4d-484f-8156-c508351f6fd7","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":314547,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/ab8e54e438d8c7d1a793e854.png"},{"id":94988997,"identity":"038010e1-4986-4c7f-b1d5-edab94bd7d1a","added_by":"auto","created_at":"2025-11-03 07:11:39","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1264320,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/186a78f4e3c5bb49e14b5ba1.png"},{"id":94963762,"identity":"415912e6-14e7-463a-9f92-4a3c1f37b9bd","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1477548,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/17871fd36f6d21017d05076e.png"},{"id":94988239,"identity":"28051389-479e-4c2a-bbab-f4c8087e987e","added_by":"auto","created_at":"2025-11-03 07:07:52","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":484336,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/7a122e5ac33c93e75941d7bc.png"},{"id":94963769,"identity":"4480eb5f-8246-4e37-9afc-4426a4d4edba","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":620481,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/1111a319ca51939c5a6778d1.png"},{"id":94963772,"identity":"97f75068-0275-4626-9618-86e7e3f9b4bf","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":568294,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/263006b86a43aa92b03a04d0.png"},{"id":95000835,"identity":"e4de954b-f750-405c-86d5-76284dfc4c2d","added_by":"auto","created_at":"2025-11-03 09:00:28","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1667616,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/39feaca5e308c43fba892b20.png"},{"id":94988213,"identity":"b3ec699a-0d2b-4d3e-b1d7-05760e838fe7","added_by":"auto","created_at":"2025-11-03 07:07:31","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1350488,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/b8a0453dd378443f7b75b9d3.png"},{"id":94988960,"identity":"5205a184-8a24-43e8-b450-bec118981c87","added_by":"auto","created_at":"2025-11-03 07:11:30","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1662825,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/7cf1c4e02103fd8396bb37ed.png"},{"id":94988866,"identity":"22d6c607-5c18-4cea-93c7-c1395099be7e","added_by":"auto","created_at":"2025-11-03 07:11:11","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149233,"visible":true,"origin":"","legend":"","description":"","filename":"b541683185874104ab3c7cf17c1905941structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/454ea0a1a2141aa6ee80e6b1.xml"},{"id":94963764,"identity":"3780ff0b-aa64-4b2d-8e1f-0a8235e37e4e","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165450,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/262ed3e131e43fd1b54734f6.html"},{"id":94963745,"identity":"71d87095-b284-417a-8129-6f97ca6c29ac","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8613432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQCHS alleviated DSS-induced colitis in mice. (A) \u003c/strong\u003eBody weight change and \u003cstrong\u003e(B)\u003c/strong\u003e Disease activity index (DAI) evaluation. \u003cstrong\u003e(C-D)\u003c/strong\u003e Colon length. \u003cstrong\u003e(E)\u003c/strong\u003e H\u0026amp;E staining and pathological scoring of colon tissue, Scale bar: 200 μm. \u003cstrong\u003e(F) \u003c/strong\u003eThe mRNA levels of IL-6, TNF-α, and IL-1β were analyzed by real-time PCR. \u003cstrong\u003e(G-I)\u003c/strong\u003e The levels of inflammatory marker Lipocalin 2 (Lcn2) and cell adhesion molecules (P-selectin, E-selectin) were also detected by real-time PCR. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (one-way ANOVA).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/6179db623470c9472177d3a9.png"},{"id":94963749,"identity":"af13faa4-7d4c-496c-833f-72b27209afa1","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11780811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQCHS improved the integrity of the intestinal barrier in UC mice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eIntestinal permeability was measured by FITC-Dextran 4(FD-4) concentration in the serum. \u003cstrong\u003e(B)\u003c/strong\u003eThe protein levels of ZO-1, Muc-2, and Claudin-5 were examined by western blotting. \u003cstrong\u003e(C) \u003c/strong\u003eAlcian blue staining of colon tissue, scale bar: 200 μm. \u003cstrong\u003e(D) \u003c/strong\u003eImmunofluorescence images of Muc-2 and Claudin-1 in colon sections are shown. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001(one-way ANOVA).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/50e295d67f4d79e75527b212.png"},{"id":94988026,"identity":"958ed463-fbd4-40a1-aa47-f11d5a84b8bc","added_by":"auto","created_at":"2025-11-03 07:02:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2504182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQCHS reprogrammed the gut microbiota and increased the relative abundance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. gasseri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e The overlap of OTUs across groups were presented using a Venn diagram (n=8). \u003cstrong\u003e(B) \u003c/strong\u003eAlpha diversity indices (Chao1) and \u003cstrong\u003e(C)\u003c/strong\u003eBeta diversity index (principal co-ordinates analysis, PCoA) of gut microbial communities. \u003cstrong\u003e(D) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003erelative bacterial abundance at the phylum level and\u003cstrong\u003e (E)\u003c/strong\u003e genus level were shown. \u003cstrong\u003e(F) \u003c/strong\u003eWilcoxon test analyzed differential bacteria at the genus level of two groups. \u003cstrong\u003e(G)\u003c/strong\u003e The relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e at the species level. \u003cstrong\u003e(H) \u003c/strong\u003eLEfSe analysis identified the enriched bacteria in the gut microbiome of the two groups. (LDA score \u0026gt; 4 and a significance of \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05 determined by the Wilcoxon test.) \u003cstrong\u003e(I) \u003c/strong\u003e16s rDNA copies of \u003cem\u003eL. gasseri\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ein feces were measured by quantitative PCR. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001(one-way ANOVA).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/67fb1f5e746fb64b95b24bf3.png"},{"id":94963743,"identity":"8a719246-54aa-4dbd-aa23-daf54cad9e1c","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3641250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFerroptosis-related metabolic pathways were significantly enriched after QCHS treatment. (A-B) \u003c/strong\u003ePCA analysis of the metabolites in feces under positive and negative ion mode demonstrated distinct metabolite composition between different groups (n = 8). \u003cstrong\u003e(C-D) \u003c/strong\u003eVenn diagram analysis of differential metabolites in two ion modes. \u003cstrong\u003e(E) \u003c/strong\u003eVolcano plot showing metabolites with significant changes between QCHS and DSS groups, with each dot representing a metabolite. \u003cstrong\u003e(F) \u003c/strong\u003eClustering heat map of total differential metabolites in the feces of three groups. \u003cstrong\u003e(G)\u003c/strong\u003e KEGG Pathway enrichment analysis of differential metabolites (top 20, Metabolic pathways with significant differences are marked with red boxes). \u003cstrong\u003e(H)\u003c/strong\u003e Cluster heatmap of the metabolites in four metabolic pathways with significant differences.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/a4496cda4f6df50c0a1d8d48.png"},{"id":94963748,"identity":"5ab2f5c0-c96a-42f2-b061-414a4dae62a5","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3362558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. gasseri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand ferroptosis metabolism. (A) \u003c/strong\u003eCorrelation analysis of significantly different metabolites and significantly different bacteria in feces. \u003cstrong\u003e(B) \u003c/strong\u003eCorrelation analysis between metabolite levels in ferroptosis metabolic pathway and paired \u003cem\u003eL. gasseri\u003c/em\u003eabundance in feces. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (spearman analysis).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/a8895d85344748a5d025b259.png"},{"id":94963755,"identity":"cb0d463f-eede-43e9-8099-fa754dfb31ef","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11793355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQCHS attenuated DSS-induced ferroptosis in mice. (A)\u003c/strong\u003e Mitochondrial structure in colonic epithelial cells from UC mice were determined using transmission electron microscopy with yellow arrows indicating shrunken and disrupted mitochondria, Scale bar: 1.0-2.0μm. \u003cstrong\u003e(B)\u003c/strong\u003e Immunohistochemical staining was used to detect the expression of 4-HNE, Scale bar: 200 μm. \u003cstrong\u003e(C) \u003c/strong\u003eThe intracellular iron deposition was detected using Prussian blue staining, Scale bar: 100 μm. \u003cstrong\u003e(D)\u003c/strong\u003eIron assay kit was used to detect the levels of intracellular iron in the colon. \u003cstrong\u003e(E)\u003c/strong\u003e GSH levels of the colon tissue were measured by GSH assay. \u003cstrong\u003e(F)\u003c/strong\u003eMDA levels of the colon tissue were measured by MDA assay. \u003cstrong\u003e(G)\u003c/strong\u003e The mRNA levels of ferroptosis-related genes were analyzed by real-time PCR. (H) The levels of ferroptosis-related proteins (ACSL4, FTH1, and GPX4) were examined by immunoblotting. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001(one-way ANOVA).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/70ba7bd2b72fd5105a1f7197.png"},{"id":94963757,"identity":"45cc07bc-63eb-461f-907e-21661d67b21c","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9263181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQCHS-mediated L. gasseri alleviated DSS-induced intestinal injury in mice. (A) \u003c/strong\u003eGrowth curves of \u003cem\u003eL. gasseri\u003c/em\u003e at 37°C for 24 h in MRS broth. \u003cstrong\u003e(B)\u003c/strong\u003e Gram-stained \u003cem\u003eL. gasseri\u003c/em\u003e viewed under 63× magnification. \u003cstrong\u003e(C) \u003c/strong\u003eThe effect of QCHS on \u003cem\u003eL. gasseri\u003c/em\u003e survival was analyzed. DSS-induced colitis mouse model was used to evaluate the effect of \u003cem\u003eL. gasseri. \u003c/em\u003e\u003cstrong\u003e(D) \u003c/strong\u003eBody weight change, \u003cstrong\u003e(E)\u003c/strong\u003e Disease activity index (DAI) and \u003cstrong\u003e(F-G)\u003c/strong\u003e Colon length were evaluated. \u003cstrong\u003e(H)\u003c/strong\u003e H\u0026amp;E staining and pathological scoring of the colon, Scale bar: 200 μm. \u003cstrong\u003e(I) \u003c/strong\u003eIntestinal permeability was measured by FITC-Dextran 4 (FD-4) concentration in the serum. \u003cstrong\u003e(J) \u003c/strong\u003eAlcian blue staining of colon tissue, scale bar: 200 μm. All data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (one-way ANOVA).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/8137a749443084a43c36b80a.png"},{"id":94963759,"identity":"7ce57ba3-760b-4e85-882b-6db3c1c5a4f1","added_by":"auto","created_at":"2025-11-02 18:30:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11145718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eL. gasseri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduced DSS-induced ferroptosis in mice. (A)\u003c/strong\u003e Mitochondrial structure in colonic epithelial cells from UC mice were determined using transmission electron microscopy with yellow arrows indicating shrunken and disrupted mitochondria, Scale bar: 1.0-2.0μm. \u003cstrong\u003e(B)\u003c/strong\u003e Immunohistochemical staining was used to detect the expression of 4-HNE, Scale bar: 200 μm. \u003cstrong\u003e(C) \u003c/strong\u003eThe intracellular iron deposition was detected using Prussian blue staining, Scale bar: 100 μm. \u003cstrong\u003e(D)\u003c/strong\u003e Iron assay kit was used to detect the levels of intracellular iron in the colon. \u003cstrong\u003e(E)\u003c/strong\u003eGSH levels of the colon tissue were measured by GSH assay. \u003cstrong\u003e(F)\u003c/strong\u003e MDA levels of the colon tissue were measured by the MDA assay.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/422200d985ae68fd92456628.png"},{"id":94963750,"identity":"bc192f2f-5a2e-4fad-bda7-087d17902731","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1670002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eL. gasseri \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einhibited inflammation and RSL3-induced ferroptosis in NCM-460 cells.\u003c/strong\u003e \u003cstrong\u003e(A-C)\u003c/strong\u003e NCM-460 cells were pretreated with \u003cem\u003eL. gasseri\u003c/em\u003e for 12 h and then stimulated with or without LPS (500 ng/mL). The mRNA levels of IL-6, IL-1β, and TNF-α were detected by real-time PCR. \u003cstrong\u003e(D-F)\u003c/strong\u003e RSL3 was utilized to induce ferroptosis in NCM460 cells after \u003cem\u003eL. gasseri\u003c/em\u003e treatment and the level of intracellular iron, GSH, and MDA of NCM-460 cells were detected. \u003cstrong\u003e(G)\u003c/strong\u003eCell viability was analyzed by the CCK-8 assay. \u003cstrong\u003e(H)\u003c/strong\u003e Total RNA was extracted and the mRNA levels of ferroptosis-related genes were analyzed by real-time PCR. \u003cstrong\u003e(I)\u003c/strong\u003e The protein levels of ACSL4, FTH1, and GPX4 were examined by western blotting. Data are shown as mean ± SEM: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (one-way ANOVA).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/28f5e3dd35d685086c0d660c.png"},{"id":101690549,"identity":"52da65aa-1733-4d88-911c-686664a48b8d","added_by":"auto","created_at":"2026-02-02 16:05:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":67110548,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/f61fa8bd-60ae-4764-a58d-308ac965a6f0.pdf"},{"id":94963742,"identity":"5b2814bd-068a-42d5-baed-3b03b5606ac8","added_by":"auto","created_at":"2025-11-02 18:30:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":134095,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7844488/v1/764651df1337275c040e81c6.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"A phytotherapeutic agent demonstrates clinical efficacy in amelioration of murine colitis through gut microbiota modulation: mechanistic link to Lactobacillus gasseri-dependent inhibition of ferroptotic pathways","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eUlcerative colitis (UC), characterized by chronic intestinal inflammation and epithelial barrier dysfunction, remains a therapeutic challenge due to its complex etiology involving dysregulated immune responses, microbiota dysbiosis, and aberrant cell death pathways [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among emerging mechanisms, ferroptosis\u0026mdash;an iron-dependent form of regulated cell death driven by lipid peroxidation\u0026mdash;has recently been implicated in UC pathogenesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The gut microbiota plays a crucial role in maintaining intestinal homeostasis, and disruptions in its composition and metabolism can result in pathological damage to the intestines [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Metabolites act as important mediators of host-microbe interactions and are essential for the maintenance of the gut barrier [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Targeting the microbiota-metabolic axis has emerged as a promising approach for managing UC. For instance, bile acids derived from the gut microbiota can increase the proportion of regulatory T cells in the colon of mice, thereby reducing their susceptibility to colitis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Short-chain fatty acids (SCFAs) produced by the microbiota can repair the intestinal epithelium by activating inflammasomes and influencing the secretion of interleukin (IL)-18 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, studies have shown that fatty acids produced by gut microbes, especially unsaturated fatty acids, are closely associated with the inflammatory state of IBD [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFerroptosis, a recently discovered form of regulated cell death (RCD), is characterized by iron dependence, lipid peroxidation, increased reactive oxygen species (ROS), and disruption of mitochondrial metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Glutathione peroxidase 4 (GPX4) is a key antioxidant enzyme that utilizes glutathione (GSH) as a substrate to catalyze the reduction of lipid peroxides to alcohol. The production of GSH relies on the cystine/glutamate antiporter system (System Xc\u0026minus;). Inhibition of GPX4/GSH or System Xc\u0026thinsp;\u0026minus;\u0026thinsp;activity is considered a central mechanism in the induction of ferroptosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Ferroptosis has been implicated as a potential pathogenic factor in IBD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies have demonstrated ferroptosis-induced damage in both UC patients and colitic mice, and ferroptosis inhibitors are being explored as potential therapeutic agents for UC [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the relationship between the microbiota and ferroptosis in UC treatment remains largely unexplored.\u003c/p\u003e\u003cp\u003eA phytotherapeutic agent, Qing-Chang-Hua-Shi granule (QCHS) is a traditional Chinese medicine (TCM) formula widely used in China for the treatment of UC. It is composed of Huanglian (Coptis chinensis Franch.), Huangqin (Scutellaria baicalensis Georgi), Baijiangcao (Patrinia scabiosaefolia Fisch.), Danggui (Angelica sinensis (Oliv.) Diels), Baishao (Paeonia lactiflora Pall.), Diyu (Sanguisorba officinalis L.), Zicao (Lithospermum erythrorhizon Siebold \u0026amp; Zucc.), Qiancao (Rubia cordifolia L.), Baizhi (Angelica dahurica (Fisch, ex Hoffm.) Benth. et Hook.f.), Muxiang (Aucklandia lappa Decne.), and Gancao (Glycyrrhiza uralensis Fisch.) in a ratio of 6:10:15:10:20:10:10:20:12:6:6. Our previous studies have shown that QCHS can regulate T cell differentiation and has superior anti-inflammatory effects compared to 5-aminosalicylic acid and sulfasalazine in experimental colitis models [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the effect of QCHS on the microbiota and metabolism requires further investigation.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the effects of QCHS on the gut microbiota and metabolism in UC mice. We demonstrated that QCHS acts as an intestinal microecological regulator by increasing the abundance of Lactobacillus gasseri (L. gasseri) and altering the profile of ferroptosis-related metabolites in feces. We also explored the correlation between L. gasseri and ferroptosis, and for the first time, we report that L. gasseri can ameliorate DSS-induced colitis by inhibiting ferroptosis. Our findings reveal a novel mechanism of action for QCHS and L. gasseri, suggesting that ferroptosis-targeted herbal medicine or probiotics could serve as a new treatment strategy for UC.\u003c/p\u003e\u003cp\u003eHere, we hypothesize that QCHS ameliorates UC by reshaping the gut microbiome to inhibit ferroptosis. By integrating 16S rDNA sequencing, metabolomics, and functional assays, we reveal that QCHS enriches Lactobacillus gasseri, which drives GSH-dependent ferroptosis suppression. Our findings bridge the gap between TCM and modern mechanistic research, highlighting microbiota-ferroptosis axis as a novel target for UC therapy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Reagents\u003c/h2\u003e\u003cp\u003eDSS (36000\u0026ndash;50000 MW, CAS: 216011080) was obtained from MP Biomedicals. LPS (L2630) and FITC-Dextran (60842-46-8) were bought from Sigma-Aldrich. cDNA Synthesis Kit (R211-01), qRT-PCR SYBR Green Kit (Q221-01), and CCK-8 Cell Counting Kit (A311-01) were bought from Vazyme. Fetal Bovine Serum (04-001-1ACS), Trypsin EDTA solution (03-050-1A), and RPMI Medium 1640 (01-100-1ACS) were purchased from Biological Industries. Antibodies against FTH1(#4393) and β-Actin (#3700) were provided by CST. Antibodies against ZO-1 (sc-33725) was provided by Santa Cruz Biotechnology. Antibodies against Claudin-5 (ab131259), and Muc-2 (ab272692) were purchased from Abcam. Antibodies against 4-HNE (bs-6313R) were bought from Bioss. Antibodies against ACSL4 (66617-1), GPX4 (22401-1), and the secondary antibodies were bought from Proteintech. H\u0026amp;E (G1003), Alcian blue (G1027), and Prussian blue staining kit (G1029) were provided by Servicebio. SP Rabbit \u0026amp; Mouse HRP Kit (DAB, CW2069S) was purchased from CWBio. Iron Assay kit (TC1015) was provided by LEAGENE. Total Glutathione Assay Kit (S0052) and Lipid Peroxidation MDA Assay Kit (S0131S) were provided by Beyotime. All primers were provided by Generay Biotech (Shanghai) and the primer information is listed in the Supporting Information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of Qing-Chang-Hua-Shi granule\u003c/h2\u003e\u003cp\u003eThe 11 components of QCHS were provided by Jiangyin Tianjiang Pharmaceutical St (Jiangsu, China) in the form of granules, which were dissolved in double distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO) and then diluted to three working concentrations before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Bacterial strain and culture conditions\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eL. gasseri\u003c/em\u003e (BNCC135322) strain was purchased from Bena Culture Collection (Henan, China) and confirmed by 16S rDNA sequencing in allwegene Tech, Ltd. (Beijing, China). \u003cem\u003eL. gasseri\u003c/em\u003e were grown anaerobically in MRS broth at 37\u0026deg;C. The bacteria were centrifuged (4000\u0026times;g, 10 min) and washed twice with sterile phosphate-buffered saline (PBS) solution, and finally resuspended in sterile PBS before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Detection of L. gasseri growth curve\u003c/h2\u003e\u003cp\u003eThe fresh \u003cem\u003eL. gasseri\u003c/em\u003e suspension was inoculated into 200 mL of MRS liquid medium at a 2% (v/v) inoculation volume. The OD600 value was measured every 3 hours (repeated 3 times with the average value taken), and the 24-hour growth curve of \u003cem\u003eL. gasseri\u003c/em\u003e was plotted according to the incubation time and absorbance values.\u003c/p\u003e\u003cp\u003eTo determine QCHS\u0026rsquo;s effects on the growth of \u003cem\u003eL. gasseri\u003c/em\u003e. the bacteria were cultured in MRS liquid medium with or without different concentrations of QCHS for 24 h. The OD600 value of the culture medium was measured and the survival rate of bacteria was calculated using the normal MRS culture group as a reference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Animal experiments\u003c/h2\u003e\u003cp\u003eAll mice (C57BL/6J, male, 20-22g) used were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd and were raised under SPF conditions at Nanjing University of Chinese Medicine (A standard 12h light/dark cycle, license number: SYXK(苏)2024-0049). The experiments were approved by the Animal Ethics Committee of NJUCM (Application Number: 202403A069) on 8th Mar, 2024.\u003c/p\u003e\u003cp\u003eFor experiments involving QCHS treatment, UC model was established using 3% DSS (dissolved in drinking water) for 7 days. Mice were then given different doses of QCHS (9.5, 19, 38 g/kg/day), or double distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO) for another 7 days by gavage. For experiments involving \u003cem\u003eL. gasseri\u003c/em\u003e intervention, mice were given 3% DSS with or without \u003cem\u003eL. gasseri\u003c/em\u003e (5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU) for 7 days. The disease activity index (DAI) was determined using the body weight, fecal properties, and blood in the stool, as described in a previous publication [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The serum, colon tissue, and feces were collected for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Measurement of FITC-Dextran 4 leakage\u003c/h2\u003e\u003cp\u003eMice were fasted for 6 h before being gavaged with FITC-Dextran (40 mg/mL). After 4 h, serum was collected and the fluorescence value of serum samples were measured at 500 nm. A standard curve was drawn with FITC standards and the sample concentrations were calculated based on the fluorescence values. All samples were protected from light during the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Histological evaluation of colitis\u003c/h2\u003e\u003cp\u003e0.5 cm of colon tissue was washed with PBS and fixed in paraformaldehyde for 48 h before being embedded in paraffin and cut into 4 \u0026micro;m thick sections for further analysis. H\u0026amp;E, Alcian blue, and Prussian blue staining were performed according to the staining kits\u0026rsquo; protocol (Servicebio) after deparaffinization. Images were captured with a light microscope (Leica). The histological scores were performed as previously described based on crypt loss, lymphoid follicle formation, and inflammatory infiltration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHistological scores were determined blindly based on the sum of the epithelium and infiltration scores. Epithelium score: 0\u0026thinsp;=\u0026thinsp;normal; 1\u0026thinsp;=\u0026thinsp;loss of goblet cells; 2\u0026thinsp;=\u0026thinsp;loss of goblet cells in large areas; 3\u0026thinsp;=\u0026thinsp;loss of crypts; 4\u0026thinsp;=\u0026thinsp;loss of crypts in large areas. Infiltration score: 0\u0026thinsp;=\u0026thinsp;normal; 1\u0026thinsp;=\u0026thinsp;infiltration around crypt bases; 2\u0026thinsp;=\u0026thinsp;infiltration reaching the muscularis mucosae; 3\u0026thinsp;=\u0026thinsp;extensive infiltration reaching the muscularis mucosae; 4\u0026thinsp;=\u0026thinsp;infiltration of the submucosa.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Immunostaining analysis\u003c/h2\u003e\u003cp\u003eAfter deparaffinization, antigen retrieval of colon tissue sections was performed using sodium citrate solution. Next, the sections were blocked with 2% BSA at 37\u0026deg;C for 1 h and then incubated with anti-Muc-2 (1:200), anti-Claudin-1 (1:200), and anti-4-HNE (1:100) at 4\u0026deg;C overnight. After washing with PBS for 3 times, the tissue sections were incubated with the corresponding secondary antibodies at 37\u0026deg;C for 1 h. For 4-HNE staining, sections were visualized according to the standard methods of the diaminobenzidine (DAB) solution kit. For Muc-2 and Claudin-1 staining, an anti-fade reagent was added after counterstaining the nucleus with DAPI. All images were analyzed using an inverted fluorescence microscope (Leica, DMi8).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Transmission electron microscopy (TEM)\u003c/h2\u003e\u003cp\u003eColon tissues were segmented into 1 mm\u003csup\u003e3\u003c/sup\u003e sections on ice and fixed with 2.5% glutaraldehyde at 4\u0026deg;C overnight. Samples were then dehydrated in a gradient of ethanol concentrations and acetone, and then embedded in resin. 60\u0026ndash;80 nm ultra-thin sections were cut from the resin block and transferred to 150 meshcopper grids coated with formvar film. The samples were stained with 2% uranyl acetate saturated alcohol solution followed by 2.6% lead citrate solution. Finally, the samples were observed using a transmission electron microscope (HITACHI HT7800, 120kV).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. 16S rDNA sequence analysis\u003c/h2\u003e\u003cp\u003eThe microbial sequencing analysis was performed by Allwegene Tech. Briefly, the genomic DNA was extracted and the integrity of DNA was inspected using 1% agarose gel electrophoresis. The V3-V4 regions of 16S rDNA gene were amplified and the PCR products were recovered using 1% agarose gel electrophoresis. Next, DNA were purified with Agencourt AMPure XP Nucleic Acid Purification Kit. DNA sequencing was performed based on the Illumina MiSeq platform (PE300). Chimeras and short sequences were removed from sequencing data to obtain high-quality sequences and operational taxonomic units (OTUs) were generated. Finally, OTUs with a similarity level of less than 97% were used for further bioinformatics analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Quantification of Lactobacillus gasseri in stool\u003c/h2\u003e\u003cp\u003eThe genomic DNA of feces were extracted using TIANamp Genomic DNA Kit (DP304, TIANGEN). PCR amplification of DNA was performed using \u003cem\u003eL. gasseri-\u003c/em\u003especific primers (F: 5\u0026rsquo; -AATACTCCCGAAGCACGTCA-3\u0026rsquo;, R: 5\u0026rsquo;-TCATTGTGTTTGGCAATCGT-3\u0026rsquo;). PCR products were then checked with 1% agarose gel electrophoresis and recovered using the Magbead Gel Extraction Kit (CWBIO). Purified DNA was cloned using DH5α (CWBIO), and the plasmid was extracted as a standard for PCR quantification. The plasmid standard was diluted 10-fold from 10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e, and 2 \u0026micro;L of gradient was used as a template to establish a standard curve. The samples and standard DNA underwent Realtime PCR reaction, and then the DNA quantity was calculated according to the standard curve. All experiments were repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Non-targeted metabolomic analysis\u003c/h2\u003e\u003cp\u003e200 mg of fecal matter was mixed with pre-chilled 80% methanol, vortexed, incubated on ice for 5 min, then centrifuged at 15,000 x g at 4\u0026deg;C for 20 min. The supernatant was collected and then diluted with mass spectrometry-grade water so that the final concentration of methanol was 53%. Samples were then centrifuged at 15,000 x g at 4\u0026deg;C for 20 min. The supernatant was collected and injected into the UHPLC-MS/MS system (Thermo Fisher) with a Hypesil Gold column (C18, 100\u0026times;2.1 mm, 1.9\u0026micro;m) for analysis. The mass spectrometer was operated in both positive and negative ionization mode with a mass range of 100 to 1500. The LC-MS/MS data were processed using Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking, and quantification for each metabolite. The peaks were matched with mzCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mzcloud.org/),mzVaul\u003c/span\u003e\u003cspan address=\"https://www.mzcloud.org/),mzVaul\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003et, and MassList databases to obtain accurate qualitative and relative quantitative results. The metabolites were annotated using the KEGG database and statistical analysis was performed using the statistical software R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6). Metabolomics analysis was completed by Novogene Co., Ltd. (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Assessment for GSH, MDA, and Iron levels\u003c/h2\u003e\u003cp\u003eProtein samples were collected from colon tissues or NCM-460 cells and the concentrations were determined using the BCA Protein Assay Kit (Beyotime). The levels of Glutathione (GSH) were tested using a Total Glutathione Assay Kit (S0052, Beyotime). The contents of MDA were detected through the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime). An Iron Assay kit (TC1015, LEAGENE) was used to determine the iron levels of colon or NCM-460 cells. The final unit concentrations were calculated based on the sample protein concentrations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Cell culture and model establishment\u003c/h2\u003e\u003cp\u003eNormal human colonic epithelial NCM-460 cells were cultured with RPMI 1640 medium (10% fetal bovine serum) under standard conditions. Inflammation was induced in NCM-460 cells using 1 \u0026micro;g/mL LPS. Briefly, cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were treated with \u003cem\u003eL. gasseri\u003c/em\u003e (10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e CFU/mL) for 12 h before LPS stimulation. RSL3 (3 \u0026micro;M) was utilized to induce ferroptosis in cells after \u003cem\u003eL. gasseri\u003c/em\u003e treatment and the cells without any treatment were used as a negative control. Cell viability was detected using the standard procedures of the CCK8 assay (Vazyme). Total protein and RNA were collected for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Cell viability assay\u003c/h2\u003e\u003cp\u003eNCM-460 cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were plated into 96-well plates and incubated with QCHS-containing serum (10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e) for 24h. Next, 10 \u0026micro;L CCK-8 was added into the plate and the absorbance of cells was measured at 450 nm after 2 h of incubation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll results were displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical difference between multiple groups were analyzed by one-way ANOVA and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All statistical data were analyzed with GraphPad Prism 9.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1. QCHS alleviated DSS-induced colitis in mice\u003c/h2\u003e\u003cp\u003eWe first evaluated the therapeutic effect of QCHS using a 3% DSS-induced UC model. Mice in the QCHS-treated group showed significant weight recovery, lower DAI scores, and increased colon length compared to the DSS-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). The protective effects of QCHS were also shown to be dose-dependent. Histopathological images revealed that colonic mucosal disruption, inflammatory cell infiltration, and goblet cell loss were all restored in QCHS-treated mice. Moreover, the pathological scores of the QCHS-treated group were significantly lower than those of the DSS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G, the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the inflammatory marker Lipocalin 2 (Lcn-2) were significantly increased in the colon of UC mice, and QCHS treatment reversed these abnormal inflammatory responses. QCHS treatment also reversed the DSS-induced abnormal secretion of P-Selectin and E-Selectin, adhesion molecules involved in the activation of cell proliferation, differentiation, and inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). These results indicate that QCHS treatment significantly alleviated the DSS-induced colonic inflammatory response in mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2. QCHS improved intestinal barrier function in UC mice\u003c/h2\u003e\u003cp\u003eDisruption of intestinal barrier function is a key characteristic of UC. Therefore, we assessed the protective effect of QCHS on the intestinal barrier in UC mice. The concentration of FD-4 in the serum of QCHS-treated mice was significantly lower compared to the DSS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that QCHS can reduce intestinal permeability in UC mice. The protein levels of Mucin-2 (Muc-2) and tight junction proteins (ZO-1, claudin-5) in the colon of UC mice were significantly increased by QCHS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Alcian blue staining revealed a significant increase in colonic mucin production after QCHS treatment compared to the DSS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, immunofluorescence staining showed that the expression of Muc-2 and Claudin-1 was reduced in colitic mice, but QCHS significantly restored their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results suggest that QCHS can improve intestinal barrier function in UC mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3. QCHS reprogrammed the gut microbiota and increased the relative abundance of L. gasseri\u003c/h2\u003e\u003cp\u003eThe gut microbiome plays a crucial role in maintaining intestinal homeostasis. Therefore, we explored the effect of QCHS on the gut microbial composition in UC mice using 16S rDNA sequencing. The Venn diagram shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA illustrates the differences in the intestinal microbiota among the three groups of mice. The Chao1 index revealed that the alpha-diversity of the microbiome was affected by QCHS in mice with DSS-induced colitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Principal coordinates analysis (PCoA) showed differences in the gut microbial structure between the control group, the DSS group, and the QCHS group, indicating that QCHS treatment significantly altered the gut microbiota structure in UC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the bar plot shows that Bacteroidota, Firmicutes, and Proteobacteria were the dominant phyla, and the ratio of Bacteroidota to Firmicutes was reversed after QCHS treatment. At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), the bacteria with the highest relative abundance were Lactobacillus, Bacteroides, Escherichia-Shigella, Romboutsia, and Turicibacter. The Wilcoxon test was used to compare the microbiota between the QCHS and DSS groups, showing significant differences. The results indicated that QCHS significantly decreased the relative abundance of Romboutsia and Turicibacter and dramatically increased the relative abundance of Lactobacillus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). More importantly, species-level analysis revealed that the relative abundance of Lactobacillus gasseri was significantly elevated by QCHS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). LEfSe analysis (LDA score\u0026thinsp;\u0026gt;\u0026thinsp;4) was performed to further explore which bacteria were significantly affected by QCHS. Notably, there were significant differences in the relative abundance of Lactobacillus at different taxonomic levels after QCHS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Quantitative PCR detection further confirmed that L. gasseri was significantly enriched by QCHS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These results collectively indicate that QCHS altered the gut microbial composition and significantly increased the relative abundance of L. gasseri in colitic mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4. QCHS altered ferroptosis metabolism and increased Glutathione levels\u003c/h2\u003e\u003cp\u003eMetabolites are important mediators through which the gut microbiota exerts its effects. Therefore, we used UHPLC-MS/MS to examine the metabolomic changes in QCHS-treated UC mice. PCA analysis under both positive and negative ion modes demonstrated that the metabolic profiles of the fecal matter differed among the experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). The Venn diagram under the positive ion mode showed that 311 metabolites were significantly altered in the feces of mice after QCHS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), while the negative ion mode revealed 178 differential metabolites between the QCHS and DSS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The volcano plot shows the overall differences in fecal metabolites between the QCHS and DSS groups when both positive and negative ions were combined (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Cluster analysis was used to examine the expression levels of all differential metabolites, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF.\u003c/p\u003e\u003cp\u003eThe Kyoto Encyclopedia of Genes and Genomes (KEGG) database is a powerful tool for analyzing in vivo metabolic networks. We performed KEGG pathway enrichment analysis to identify the major biochemical metabolic and signal transduction pathways involved in the differential metabolites. The top 20 enriched metabolic pathways after QCHS treatment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, with significant pathways including Arachidonic acid metabolism (P\u0026thinsp;=\u0026thinsp;0.0009), Biosynthesis of unsaturated fatty acids (P\u0026thinsp;=\u0026thinsp;0.0010), Linoleic acid metabolism (P\u0026thinsp;=\u0026thinsp;0.0335), and Ferroptosis (P\u0026thinsp;=\u0026thinsp;0.0398). Interestingly, all these pathways are closely related to lipid metabolism and ferroptosis. Next, we analyzed the relative abundance of differential metabolites in these four metabolic pathways and found that QCHS significantly increased the level of Glutathione (GSH), which is a key regulator of ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). This metabolomic analysis suggests that QCHS may influence GSH-mediated ferroptosis metabolism in colitic mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.5. L. gasseri is significantly correlated with ferroptosis metabolism\u003c/h2\u003e\u003cp\u003eTo explore the association between gut microbiota and metabolites affected by QCHS, we performed Pearson correlation analysis on the metabolites and differential bacteria at the genus level. The results showed that Lactobacillus was positively correlated with metabolites of the ferroptosis pathway (glutathione and mevalonic acid) and negatively correlated with metabolites in arachidonic acid metabolism (5-oxoicosatetraenoic acid, prostaglandin D2, and 16(R)-hydroxyeicosatetraenoic acid) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, Spearman-based correlation analysis was performed between QCHS-mediated L. gasseri and four metabolites of the ferroptosis metabolic pathway. The results indicated that the relative abundance of L. gasseri was significantly positively correlated with GSH and significantly negatively correlated with mevalonic acid (MVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E), suggesting that QCHS-mediated L. gasseri may potentially inhibit ferroptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.6. QCHS inhibited DSS-induced ferroptosis damage in UC mice\u003c/h2\u003e\u003cp\u003eThe results of the metabolomic analysis show that QCHS significantly enriched ferroptosis metabolism in the feces. Based on these findings, we hypothesized that the therapeutic effect of QCHS in UC mice might be mediated through ferroptosis. To verify this hypothesis, we collected colon tissue and serum from UC mice for various assays. Electron microscopy analysis revealed shrunken mitochondria and reduced mitochondrial cristae in colonic epithelial cells after DSS induction, whereas QCHS significantly alleviated this ferroptosis-like injury in the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).Lipid peroxidation plays a key role in the process of ferroptosis. 4-Hydroxynonenal (4-HNE), the end product of lipid peroxidation, is used as an indicator to detect lipid peroxidation. Immunohistochemical results showed that QCHS significantly reduced the abnormal increase of 4-HNE induced by DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, lipid peroxidation, as indicated by MDA levels, was elevated after DSS induction but significantly decreased following QCHS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Ferroptosis is also dependent on the accumulation of iron ions. Both Prussian blue staining and iron assays demonstrated that QCHS reduced the iron overload caused by DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Next, we assessed the mRNA levels of ferroptosis-related genes and found that their expression was upregulated following DSS induction, but remarkably decreased after QCHS treatment. Conversely, the levels of GSH and the catalytic enzyme GPX4 were significantly reduced in the colon of UC mice. Interestingly, QCHS promoted the expression of both GSH and GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and G). Moreover, after DSS induction, the protein levels of ACSL4 and FTH1, which are characteristic of ferroptosis, were significantly elevated, while GPX4 levels were reduced. QCHS treatment effectively reversed the levels of these ferroptosis-related proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). This data suggests that QCHS exerts a protective effect against DSS-induced ferroptosis in the colon.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.7. QCHS promoted the proliferation of L. gasseri in vitro\u003c/h2\u003e\u003cp\u003e16S rDNA sequencing results showed that QCHS increased the relative abundance of L. gasseri in the feces of UC mice, and the level of L. gasseri exhibited a significant positive correlation with GSH in the ferroptosis pathway. To further investigate the relationship between QCHS and L. gasseri, we cultured the L. gasseri strain with MRS medium in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Notably, the combination of QCHS (1000 \u0026micro;g/mL) and MRS medium significantly promoted the growth of L. gasseri (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.8. L. gasseri reduced DSS-induced intestinal injury in mice.\u003c/h2\u003e\u003cp\u003eBased on the above results, we speculated that the protective effect of QCHS on UC mice might be mediated by L. gasseri. To test this hypothesis, we administered L. gasseri (5\u0026times;10⁸ CFU) to mice with or without DSS induction for 7 days to determine whether L. gasseri could influence the progression of UC in mice. L. gasseri-treated mice exhibited reduced body weight loss, lower DAI scores, restored colon length, and alleviated pathological damage compared to mice treated with DSS alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-H). While DSS-treated mice showed higher serum FD-4 levels and reduced colonic mucin secretion, L. gasseri treatment significantly mitigated DSS-induced colonic barrier damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI-J). These findings suggest that L. gasseri can slow the progression of colitis and protect against intestinal injury in UC mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.9. L. gasseri inhibited DSS-induced ferroptosis-like injury in mice.\u003c/h2\u003e\u003cp\u003eWe also examined colonic ferroptosis impairment in \u003cem\u003eL. gasseri\u003c/em\u003e-treated UC mice. Electron microscopy images showed that shrunken mitochondria and reduced mitochondrial crista in colonic epithelial cells could be significantly alleviated by treatment with \u003cem\u003eL. gasseri\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. 4-HNE staining and Prussian blue staining of colon tissue showed that \u003cem\u003eL. gasseri\u003c/em\u003e alleviated lipid peroxidation and excessive accumulation of iron ions caused by DSS, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. In addition, the \u003cem\u003eL. gasseri\u003c/em\u003e treatment increased the levels of GSH, while effectively decreasing the levels of iron and MDA in DSS-induced colitis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F\u003cb\u003e)\u003c/b\u003e. Therefore, \u003cem\u003eL. gasseri\u003c/em\u003e may attenuate intestinal injury in colitic mice by inhibiting ferroptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.10. L. gasseri inhibited LPS-induced inflammation in NCM-460 cells\u003c/h2\u003e\u003cp\u003eNext, we investigated the effect of L. gasseri on inflammation and ferroptosis through in vitro experiments. First, NCM-460 cells were pretreated with the indicated concentration of L. gasseri for 12 hours, followed by the addition of LPS (1 \u0026micro;g/mL) to induce cellular inflammation. As expected, L. gasseri significantly reduced the mRNA levels of IL-6, IL-1β, and TNF-α in LPS-induced NCM-460 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.11. L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells\u003c/h2\u003e\u003cp\u003eFinally, RSL3, a known inhibitor of GPX4, was used to induce ferroptosis in NCM-460 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-F, RSL3 significantly increased the levels of iron and MDA, while decreasing GSH content in NCM-460 cells. However, pretreatment with L. gasseri effectively prevented iron overload and lipid peroxidation, and significantly increased GSH levels in the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-F). More importantly, the cell viability assay revealed that L. gasseri pretreatment significantly inhibited RSL3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). Additionally, analysis of ferroptosis-related molecules showed that L. gasseri increased the expression of GPX4 at both the mRNA and protein levels, while significantly inhibiting the expression of positive ferroptosis regulators such as FTH1 and ACSL4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH-I). These results suggest that L. gasseri may act as a potential ferroptosis inhibitor, offering protection against cellular injury.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe incidence of ulcerative colitis (UC) has been rising steadily in developing countries, placing a considerable burden on healthcare systems [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Given the challenges of controlling inflammation and promoting mucosal repair in UC treatment, there is a pressing need for the development of new therapeutic strategies. Traditional Chinese medicine (TCM) prescriptions, including QCHS, are increasingly being recognized as multi-targeted therapies. Key components identified in QCHS include berberine, baicalin, coumarin, ferulic acid, and paeoniflorin, among others. Detailed information about these chemical components has been provided in our previous study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A randomized clinical trial has confirmed the efficacy and safety of QCHS in patients with moderately active UC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the specific mechanisms through which QCHS exerts its effects remain largely unexplored.\u003c/p\u003e\u003cp\u003eDysbiosis in the gut microbiota is known to disrupt immune homeostasis, leading to abnormal immune responses and inflammatory cytokine release [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Inflammatory bowel disease (IBD) patients exhibit altered gut microbial diversity, such as an imbalance between Firmicutes and Bacteroidetes, an increase in Proteobacteria, and the depletion of Roseburia species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The mucus layer, which interacts with both the microbiota and immune cells, plays a critical role in maintaining gut homeostasis. However, the expansion of pathogenic bacteria can break down this barrier, leading to \u0026ldquo;leaky gut\u0026rdquo; and increasing the risk of pathogens entering the lamina propria and bloodstream [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Dysbiosis may also disrupt the metabolome, further compromising the mucosal barrier and contributing to inflammation. For instance, the depletion of short-chain fatty acids (SCFAs) promotes the polarization of M1 macrophages, which drives intestinal inflammation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, fecal metabolism of palmitoleic acid and tryptophan degradation have been linked to the production of TNF-α and interferon-gamma (IFN-γ), both of which are associated with inflammation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProbiotics have been shown to restore microbial diversity, and their therapeutic potential in IBD has been demonstrated in both clinical and animal studies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Lactobacillus species, in particular, are among the most widely used probiotics, and their depletion is associated with IBD progression [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Lactobacillus helps repair the intestinal epithelial barrier by upregulating tight junction proteins and reduces colonic inflammation in UC mice by inhibiting the TLR4-NF-κB-NLRP3 signaling pathway [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Notably, prebiotic therapy has been shown to promote probiotic growth, restoring gut function and alleviating IBD symptoms [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated that QCHS treatment alleviated DSS-induced colitis by reducing colitis symptoms, inhibiting pro-inflammatory cytokine secretion, and repairing the colonic epithelial barrier. We utilized microbial sequencing and metabolomic analysis to explore the potential mechanisms of QCHS. Our results indicated that QCHS reshaped the gut microbiota, specifically increasing the relative abundance of Lactobacillus gasseri in the feces of UC mice. In vitro experiments confirmed that QCHS promoted the growth of L. gasseri strains. Furthermore, untargeted metabolomics revealed that QCHS altered the fecal metabolic profile of UC mice, with metabolites significantly enriched in ferroptosis and its related metabolic pathways. Notably, we are the first to report a correlation between Lactobacillus and ferroptosis, particularly the significant positive correlation between L. gasseri and glutathione (GSH) levels.\u003c/p\u003e\u003cp\u003eThe GPX4/GSH axis is a central regulator of ferroptosis, a form of regulated cell death (RCD) that is closely associated with lipid peroxidation. Disruptions in iron metabolism lead to the accumulation of intracellular free iron, which catalyzes the generation of reactive oxygen species (ROS) through the Fenton reaction. ROS then promote lipid peroxidation and trigger ferroptosis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, polyunsaturated fatty acids (PUFAs), which are substrates for lipid peroxidation, influence ferroptosis susceptibility [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our data showed that QCHS enriched pathways involved in arachidonic acid metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. Specifically, QCHS reduced the abundance of arachidonic acid, adrenic acid, linoleic acid, 16(R)-HETE, palmitic acid, prostaglandin F2α, prostaglandin D2, and other metabolites, which are implicated in both lipid peroxidation and inflammation. For example, arachidonic acid induces inflammation through its conversion to prostaglandins (PGs) via the cyclooxygenase (COX) pathway [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEmerging evidence suggests that ferroptosis plays a critical role in the pathogenesis of IBD, and inhibiting ferroptosis may offer a novel therapeutic approach for UC [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Our study demonstrated that QCHS significantly alleviated DSS-induced ferroptosis in the colon of UC mice, and that L. gasseri mediated this protective effect. For the first time, we found that L. gasseri can act as a ferroptosis inhibitor, mitigating the progression of UC. In vitro, we further demonstrated that L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells, with the mechanism involving activation of the GSH/GPX4 signaling pathway.\u003c/p\u003e\u003cp\u003eTaken together, our study provides compelling evidence for the regulatory role of QCHS on the microbiota-metabolome axis and ferroptosis in UC mice (Fig.\u0026nbsp;10). We also uncover a novel function of L. gasseri as an inhibitor of ferroptosis, offering new insights into potential therapeutic strategies for UC. These findings suggest that through the microbiota modulators or ferroptosis inhibitors targeting Lactobacillus, QCHS may be promising candidates for the treatment of UC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUC, ulcerative colitis; QCHS, Qing-Chang-Hua-Shi granule; L. gasseri, Lactobacillus gasseri; IBD, inflammatory bowel disease; SCFAs, short-chain fatty acids; IL, interleukin; RCD, regulated cell death; ROS, reactive oxygen species; GPX4, Glutathione peroxidase 4; GSH, Glutathione; System Xc−, the cystine/glutamate antiporter system; TCM, traditional Chinese medicine; DSS, dextran sulfate sodium; DAI, Disease activity index; TNF-α, tumor necrosis factor-α; Lcn2, Lipocalin 2; FD-4, FITC-Dextran 4; Muc2, mucin2; 4-HNE, 4-Hydroxynonenal; MDA, malondialdehyde; ACSL4, acyl-CoA synthetase long-chain family member 4; FTH1, Ferritin heavy polypeptide 1; GSH, Glutathione; KEGG, Kyoto Encyclopedia of Genes and Genomes; MVA, Mevalonic acid; IFN-γ, interferon gamma; PUFAs, polyunsaturated fatty acids; PGs, prostaglandins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. G. and H. S. designed and funded the whole research. C. C., J. H., Z. L., W. L., R. A. Y. L., F. X., Y. W., and Y. C. collaborated to complete the experiments and data analysis. C. C., Z. L., R. A., Y. L.and L. G. edited the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82405296, 82274483, 82305158).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the Animal Care and Use Committee (ACUC) of [Nanjing University of Traditional Chinese Medicine], protocol number [ACU240404]. The study adhered to the guidelines set by the committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is to confirm that the work has not been and will not be submitted simultaneously to another journal, in whole or in part; it reports previously unpublished work. If accepted, neither the paper itself nor substantial parts of it, will be published in the same form, in any language, without the consent of the publishers. The submitted version of the manuscript has been approved by all the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microbial and metabolomic raw data reported in this paper have been deposited in the China National Center for Bioinformation (https://ngdc.cncb.ac.cn) and the accession numbers are CRA009705 (animal microbiota) and OMIX002923 (animal metabolome).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ.D. Feuerstein, A.C. Moss, F.A. Farraye, Ulcerative Colitis, Mayo Clin Proc 94(7) (2019) 1357-1373.\u003c/li\u003e\n\u003cli\u003eS. Danese, C. Fiocchi, Ulcerative colitis, N Engl J Med 365(18) 1713-25.\u003c/li\u003e\n\u003cli\u003eG.P. Ramos, K.A. Papadakis, Mechanisms of Disease: Inflammatory Bowel Diseases, Mayo Clin Proc 94(1) (2019) 155-165.\u003c/li\u003e\n\u003cli\u003eJ.V. Patankar, C. Becker, Cell death in the gut epithelium and implications for chronic inflammation, Nat Rev Gastroenterol Hepatol 17(9) (2020) 543-556.\u003c/li\u003e\n\u003cli\u003eJ. Ni, G.D. Wu, L. Albenberg, V.T. Tomov, Gut microbiota and IBD: causation or correlation?, Nat Rev Gastroenterol Hepatol 14(10) (2017) 573-584.\u003c/li\u003e\n\u003cli\u003eM. Lee, E.B. Chang, Inflammatory Bowel Diseases (IBD) and the Microbiome-Searching the Crime Scene for Clues, Gastroenterology 160(2) (2021) 524-537.\u003c/li\u003e\n\u003cli\u003eA. Lavelle, H. Sokol, Gut microbiota-derived metabolites as key actors in inflammatory bowel disease, Nat Rev Gastroenterol Hepatol 17(4) (2020) 223-237.\u003c/li\u003e\n\u003cli\u003eN. Gasaly, P. de Vos, M.A. Hermoso, Impact of Bacterial Metabolites on Gut Barrier Function and Host Immunity: A Focus on Bacterial Metabolism and Its Relevance for Intestinal Inflammation, Front Immunol 12 (2021) 658354.\u003c/li\u003e\n\u003cli\u003eX. Song, X. Sun, S.F. Oh, M. Wu, Y. Zhang, W. Zheng, N. Geva-Zatorsky, R. Jupp, D. Mathis, C. Benoist, D.L. Kasper, Microbial bile acid metabolites modulate gut RORgamma(+) regulatory T cell homeostasis, Nature 577(7790) (2020) 410-415.\u003c/li\u003e\n\u003cli\u003eL. Macia, J. Tan, A.T. Vieira, K. Leach, D. Stanley, S. Luong, M. Maruya, C. Ian McKenzie, A. Hijikata, C. Wong, Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome, Nature communications 6(1) (2015) 1-15.\u003c/li\u003e\n\u003cli\u003eE.A. Scoville, M.M. Allaman, C.T. Brown, A.K. Motley, S.N. Horst, C.S. Williams, T. Koyama, Z. Zhao, D.W. Adams, D.B. Beaulieu, D.A. Schwartz, K.T. Wilson, L.A. Coburn, Alterations in Lipid, Amino Acid, and Energy Metabolism Distinguish Crohn\u0026apos;s Disease from Ulcerative Colitis and Control Subjects by Serum Metabolomic Profiling, Metabolomics 14(1) (2018) 17.\u003c/li\u003e\n\u003cli\u003eR. Marion-Letellier, G. Savoye, P.L. Beck, R. Panaccione, S. Ghosh, Polyunsaturated fatty acids in inflammatory bowel diseases: a reappraisal of effects and therapeutic approaches, Inflamm Bowel Dis 19(3) (2013) 650-61.\u003c/li\u003e\n\u003cli\u003eJ. Zheng, M. Conrad, The Metabolic Underpinnings of Ferroptosis, Cell Metab 32(6) (2020) 920-937.\u003c/li\u003e\n\u003cli\u003eB.R. Stockwell, Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications, Cell 185(14) (2022) 2401-2421.\u003c/li\u003e\n\u003cli\u003eX. Chen, J. Li, R. Kang, D.J. Klionsky, D. Tang, Ferroptosis: machinery and regulation, Autophagy 17(9) (2021) 2054-2081.\u003c/li\u003e\n\u003cli\u003eW.S. Yang, R. SriRamaratnam, M.E. Welsch, K. Shimada, R. Skouta, V.S. Viswanathan, J.H. Cheah, P.A. Clemons, A.F. Shamji, C.B. Clish, L.M. Brown, A.W. Girotti, V.W. Cornish, S.L. Schreiber, B.R. Stockwell, Regulation of ferroptotic cancer cell death by GPX4, Cell 156(1-2) (2014) 317-331.\u003c/li\u003e\n\u003cli\u003eS. Xu, Y. He, L. Lin, P. Chen, M. Chen, S. Zhang, The emerging role of ferroptosis in intestinal disease, Cell Death Dis 12(4) (2021) 289.\u003c/li\u003e\n\u003cli\u003eM. Xu, J. Tao, Y. Yang, S. Tan, H. Liu, J. Jiang, F. Zheng, B. Wu, Ferroptosis involves in intestinal epithelial cell death in ulcerative colitis, Cell Death Dis 11(2) (2020) 86.\u003c/li\u003e\n\u003cli\u003eY. Chen, W. Yan, Y. Chen, J. Zhu, J. Wang, H. Jin, H. Wu, G. Zhang, S. Zhan, Q. Xi, T. Shi, W. Chen, SLC6A14 facilitates epithelial cell ferroptosis via the C/EBP\u0026beta;-PAK6 axis in ulcerative colitis, Cell Mol Life Sci 79(11) (2022) 563.\u003c/li\u003e\n\u003cli\u003eS. Dong, Y. Lu, G. Peng, J. Li, W. Li, M. Li, H. Wang, L. Liu, Q. Zhao, Furin inhibits epithelial cell injury and alleviates experimental colitis by activating the Nrf2-Gpx4 signaling pathway, Dig Liver Dis 53(10) (2021) 1276-1285.\u003c/li\u003e\n\u003cli\u003eL. Zhu, L.M. Dai, H. Shen, P.Q. Gu, K. Zheng, Y.J. Liu, L. Zhang, J.F. Cheng, Qing Chang Hua Shi granule ameliorate inflammation in experimental rats and cell model of ulcerative colitis through MEK/ERK signaling pathway, Biomed Pharmacother 116 (2019) 108967.\u003c/li\u003e\n\u003cli\u003eC. Cheng, J. Hu, Y. Li, Y. Ji, Z. Lian, R. Au, F. Xu, W. Li, H. Shen, L. Zhu, Qing-Chang-Hua-Shi granule ameliorates DSS-induced colitis by activating NLRP6 signaling and regulating Th17/Treg balance, Phytomedicine 107 (2022) 154452.\u003c/li\u003e\n\u003cli\u003eC. Cheng, W. Zhang, C. Zhang, P. Ji, X. Wu, Z. Sha, X. Chen, Y. Wang, Y. Chen, H. Cheng, L. Shi, Hyperoside Ameliorates DSS-Induced Colitis through MKRN1-Mediated Regulation of PPAR\u0026gamma; Signaling and Th17/Treg Balance, J Agric Food Chem 69(50) (2021) 15240-15251.\u003c/li\u003e\n\u003cli\u003eS.C. Ng, H.Y. Shi, N. Hamidi, F.E. Underwood, W. Tang, E.I. Benchimol, R. Panaccione, S. Ghosh, J.C.Y. Wu, F.K.L. Chan, J.J.Y. Sung, G.G. Kaplan, Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies, Lancet 390(10114) (2017) 2769-2778.\u003c/li\u003e\n\u003cli\u003eM.A. Kamm, Rapid changes in epidemiology of inflammatory bowel disease, Lancet 390(10114) (2017) 2741-2742.\u003c/li\u003e\n\u003cli\u003eH. Shen, S. Zhang, W. Zhao, S. Ren, X. Ke, Q. Gu, Z. Tang, J. Xie, S. Chen, Y. Chen, J. Zou, L. Zhang, Z. Shen, K. Zheng, Y. Liu, P. Gu, J. Cheng, J. Hu, L. Zhu, Randomised clinical trial: Efficacy and safety of Qing-Chang-Hua-Shi granules in a multicenter, randomized, and double-blind clinical trial of patients with moderately active ulcerative colitis, Biomed Pharmacother 139 (2021) 111580.\u003c/li\u003e\n\u003cli\u003eM. Alipour, D. Zaidi, R. Valcheva, J. Jovel, I. Mart\u0026iacute;nez, C. Sergi, J. Walter, A.L. Mason, G.K. Wong, L.A. Dieleman, M.W. Carroll, H.Q. Huynh, E. Wine, Mucosal Barrier Depletion and Loss of Bacterial Diversity are Primary Abnormalities in Paediatric Ulcerative Colitis, J Crohns Colitis 10(4) (2016) 462-71.\u003c/li\u003e\n\u003cli\u003eM. Alexander, Q.Y. Ang, R.R. Nayak, A.E. Bustion, M. Sandy, B. Zhang, V. Upadhyay, K.S. Pollard, S.V. Lynch, P.J. Turnbaugh, Human gut bacterial metabolism drives Th17 activation and colitis, Cell Host Microbe 30(1) (2022) 17-30.e9.\u003c/li\u003e\n\u003cli\u003eS. Kumar, A. Kumar, Microbial pathogenesis in inflammatory bowel diseases, Microb Pathog 163 (2022) 105383.\u003c/li\u003e\n\u003cli\u003eP. Qiu, T. Ishimoto, L. Fu, J. Zhang, Z. Zhang, Y. Liu, The Gut Microbiota in Inflammatory Bowel Disease, Front Cell Infect Microbiol 12 (2022) 733992.\u003c/li\u003e\n\u003cli\u003eS. Yu, Y. Sun, X. Shao, Y. Zhou, Y. Yu, X. Kuai, C. Zhou, Leaky Gut in IBD: Intestinal Barrier-Gut Microbiota Interaction, J Microbiol Biotechnol 32(7) (2022) 825-834.\u003c/li\u003e\n\u003cli\u003eL.C. Yu, Microbiota dysbiosis and barrier dysfunction in inflammatory bowel disease and colorectal cancers: exploring a common ground hypothesis, J Biomed Sci 25(1) (2018) 79.\u003c/li\u003e\n\u003cli\u003eK. Parikh, A. Antanaviciute, D. Fawkner-Corbett, M. Jagielowicz, A. Aulicino, C. Lagerholm, S. Davis, J. Kinchen, H.H. Chen, N.K. Alham, N. Ashley, E. Johnson, P. Hublitz, L. Bao, J. Lukomska, R.S. Andev, E. Bj\u0026ouml;rklund, B.M. Kessler, R. Fischer, R. Goldin, H. Koohy, A. Simmons, Colonic epithelial cell diversity in health and inflammatory bowel disease, Nature 567(7746) (2019) 49-55.\u003c/li\u003e\n\u003cli\u003eN.A. Scott, A. Andrusaite, P. Andersen, M. Lawson, C. Alcon-Giner, C. Leclaire, S. Caim, G. Le Gall, T. Shaw, J.P.R. Connolly, A.J. Roe, H. Wessel, A. Bravo-Blas, C.A. Thomson, V. K\u0026auml;stele, P. Wang, D.A. Peterson, A. Bancroft, X. Li, R. Grencis, A.M. Mowat, L.J. Hall, M.A. Travis, S.W.F. Milling, E.R. Mann, Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis, Sci Transl Med 10(464) (2018).\u003c/li\u003e\n\u003cli\u003eM. Schirmer, S.P. Smeekens, H. Vlamakis, M. Jaeger, M. Oosting, E.A. Franzosa, R. Ter Horst, T. Jansen, L. Jacobs, M.J. Bonder, A. Kurilshikov, J. Fu, L.A.B. Joosten, A. Zhernakova, C. Huttenhower, C. Wijmenga, M.G. Netea, R.J. Xavier, Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity, Cell 167(4) (2016) 1125-1136.e8.\u003c/li\u003e\n\u003cli\u003eY. Zhu, Y. Xu, X. Wang, L. Rao, X. Yan, R. Gao, T. Shen, Y. Zhou, C. Kong, L. Zhou, Probiotic Cocktail Alleviates Intestinal Inflammation Through Improving Gut Microbiota and Metabolites in Colitis Mice, Front Cell Infect Microbiol 12 (2022) 886061.\u003c/li\u003e\n\u003cli\u003eI. Bjarnason, G. Sission, B. Hayee, A randomised, double-blind, placebo-controlled trial of a multi-strain probiotic in patients with asymptomatic ulcerative colitis and Crohn\u0026apos;s disease, Inflammopharmacology 27(3) (2019) 465-473.\u003c/li\u003e\n\u003cli\u003eD.D. Heeney, M.G. Gareau, M.L. Marco, Intestinal Lactobacillus in health and disease, a driver or just along for the ride?, Curr Opin Biotechnol 49 (2018) 140-147.\u003c/li\u003e\n\u003cli\u003eS. Selvamani, V. Mehta, H. Ali El Enshasy, S. Thevarajoo, H. El Adawi, I. Zeini, K. Pham, T. Varzakas, B. Abomoelak, Efficacy of Probiotics-Based Interventions as Therapy for Inflammatory Bowel Disease: A Recent Update, Saudi J Biol Sci 29(5) (2022) 3546-3567.\u003c/li\u003e\n\u003cli\u003eY. Chen, L. Zhang, G. Hong, C. Huang, W. Qian, T. Bai, J. Song, Y. Song, X. Hou, Probiotic mixtures with aerobic constituent promoted the recovery of multi-barriers in DSS-induced chronic colitis, Life Sci 240 (2020) 117089.\u003c/li\u003e\n\u003cli\u003eL. Tong, X. Zhang, H. Hao, Q. Liu, Z. Zhou, X. Liang, T. Liu, P. Gong, L. Zhang, Z. Zhai, Y. Hao, H. Yi, Lactobacillus rhamnosus GG Derived Extracellular Vesicles Modulate Gut Microbiota and Attenuate Inflammatory in DSS-Induced Colitis Mice, Nutrients 13(10) (2021).\u003c/li\u003e\n\u003cli\u003eX.F. Zhang, X.X. Guan, Y.J. Tang, J.F. Sun, X.K. Wang, W.D. Wang, J.M. Fan, Clinical effects and gut microbiota changes of using probiotics, prebiotics or synbiotics in inflammatory bowel disease: a systematic review and meta-analysis, Eur J Nutr 60(5) (2021) 2855-2875.\u003c/li\u003e\n\u003cli\u003eP. Li, N. Xiao, L. Zeng, J. Xiao, J. Huang, Y. Xu, Y. Chen, Y. Ren, B. Du, Structural characteristics of a mannoglucan isolated from Chinese yam and its treatment effects against gut microbiota dysbiosis and DSS-induced colitis in mice, Carbohydr Polym 250 (2020) 116958.\u003c/li\u003e\n\u003cli\u003eX. Chen, C. Yu, R. Kang, D. Tang, Iron Metabolism in Ferroptosis, Front Cell Dev Biol 8 (2020) 590226.\u003c/li\u003e\n\u003cli\u003eJ.Y. Lee, M. Nam, H.Y. Son, K. Hyun, S.Y. Jang, J.W. Kim, M.W. Kim, Y. Jung, E. Jang, S.J. Yoon, J. Kim, J. Kim, J. Seo, J.K. Min, K.J. Oh, B.S. Han, W.K. Kim, K.H. Bae, J. Song, J. Kim, Y.M. Huh, G.S. Hwang, E.W. Lee, S.C. Lee, Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer, Proc Natl Acad Sci U S A 117(51) (2020) 32433-32442.\u003c/li\u003e\n\u003cli\u003eT. Wang, X. Fu, Q. Chen, J.K. Patra, D. Wang, Z. Wang, Z. Gai, Arachidonic Acid Metabolism and Kidney Inflammation, Int J Mol Sci 20(15) (2019).\u003c/li\u003e\n\u003cli\u003eW. Gao, T. Zhang, H. Wu, Emerging Pathological Engagement of Ferroptosis in Gut Diseases, Oxid Med Cell Longev 2021 (2021) 4246255.\u003c/li\u003e\n\u003cli\u003eJ. Huang, J. Zhang, J. Ma, J. Ma, J. Liu, F. Wang, X. Tang, Inhibiting Ferroptosis: A Novel Approach for Ulcerative Colitis Therapeutics, Oxid Med Cell Longev 2022 (2022) 9678625.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Qing-Chang-Hua-Shi granule, colitis, Lactobacillus gasseri, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-7844488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7844488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUlcerative colitis (UC), characterized by chronic intestinal inflammation and epithelial barrier dysfunction, remains a therapeutic challenge due to its complex etiology [1,2]. Among emerging mechanisms, ferroptosis—an iron-dependent form of regulated cell death driven by lipid peroxidation has recently been implicated in UC pathogenesis [3,4]. The gut microbiota plays a crucial role in maintaining intestinal homeostasis, and metabolism can result in pathological damage to the intestines [5, 6]. Metabolites act as important mediators of host-microbe interactions and are essential for the maintenance of the gut barrier [7, 8]. Targeting the microbiota-metabolic axis has emerged as a promising approach for managing UC.\u003c/p\u003e\n\u003cp\u003eIt has been demonstrated that QCHS significantly alleviated DSS-induced ferroptosis in the colon of UC mice, and that L. gasseri mediated this protective effect. For the first time, it was found that L. gasseri can act as a ferroptosis inhibitor, mitigating the progression of UC. In vitro, it was further demonstrated that L. gasseri inhibited RSL3-induced ferroptosis in NCM-460 cells, with the mechanism involving activation of the GSH/GPX4 signaling pathway. This work provides compelling evidence for the regulatory role of QCHS on the microbiota-metabolome axis and ferroptosis in UC mice, and uncovering a novel function of L. gasseri as an inhibitor of ferroptosis, offering new insights into potential therapeutic strategies for UC. These findings suggest that through the microbiota modulators or ferroptosis inhibitors targeting Lactobacillus, QCHS may be promising candidates for the treatment of UC.\u003c/p\u003e","manuscriptTitle":"A phytotherapeutic agent demonstrates clinical efficacy in amelioration of murine colitis through gut microbiota modulation: mechanistic link to Lactobacillus gasseri-dependent inhibition of ferroptotic pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-02 18:29:56","doi":"10.21203/rs.3.rs-7844488/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-13T01:25:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T19:00:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-08T05:45:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110146564784749954574733261142965240399","date":"2025-11-05T06:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174327851515253573904058834127289052782","date":"2025-11-03T16:16:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T15:19:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229627809265707274310816079688360639794","date":"2025-10-22T05:29:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T04:19:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-19T04:01:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-18T07:20:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-10-13T04:58:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f8d6c8fe-c45c-4547-be86-dada923aa8cc","owner":[],"postedDate":"November 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:01:47+00:00","versionOfRecord":{"articleIdentity":"rs-7844488","link":"https://doi.org/10.1186/s13020-025-01317-5","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2026-01-26 15:58:24","publishedOnDateReadable":"January 26th, 2026"},"versionCreatedAt":"2025-11-02 18:29:56","video":"","vorDoi":"10.1186/s13020-025-01317-5","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01317-5","workflowStages":[]},"version":"v1","identity":"rs-7844488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7844488","identity":"rs-7844488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}