Mitigation of chemotherapy-induced gut dysbiosis and diarrhea by supplementation with heat-killed Bacteroides fragilis

Abstract

Background

The role of gut microbial dysbiosis in chemotherapy-induced diarrhea (CID) pathogenesis remains unclear in humans. This study investigates gut microbiota alterations in CID patients and evaluates the therapeutic potential of probiotic supplementation.

Methods

To establish a paired cohort for longitudinal comparison and minimize confounding factors in assessing CID-related microbiota changes, strict inclusion/exclusion criteria were applied to gastrointestinal cancer patients. Fecal samples from eligible participants underwent shotgun metagenomic sequencing to comprehensively profile the gut microbiome composition and function. To evaluate probiotic efficacy and mechanisms, we utilized 6–8-week-old male BALB/c and C57BL/6 mice in established 5-FU- or CPT-11-induced CID models. Probiotic efficacy was assessed using primary (diarrhea severity) and secondary endpoints (body weight change, intestinal permeability). Mechanistic studies were conducted in murine models, complemented by IEC-6 cells and intestinal organoid experiments to elucidate microbiota-host interactions.

Results

Analysis of paired fecal samples (pre- and post-chemotherapy) from 30 gastrointestinal cancer patients (n = 60) revealed chemotherapy-induced reduction of Bacteroides fragilis (B. f) via metagenomics sequencing, with baseline B. f relative abundance negatively correlating with CID severity (r =  − 0.93, p = 3.1e − 12). Building on these clinical observations, in 5-FU/CPT-11-induced CID murine models, oral gavage of heat-killed B. f (hk-B. f) outperformed live bacteria in diarrhea alleviation. Mechanistically, B. f-derived succinate exacerbated diarrhea, while its capsular polysaccharide (PSA) ameliorated mice diarrhea. This discovery explains the discrepant therapeutic effect between hk-B. f and live B. f. Fluorescence tracing confirmed hk-B. f transiently localized to the upper gastrointestinal tract without extraintestinal colonization. hk-B. f preserved epithelial integrity, mitochondrial function, and intestinal organoid development (higher budding count and larger organoid surface area). Moreover, hk-B. f upregulated the expression of BCL2 and downregulated the expression of BAX. Shifting the balance between BCL2 and BAX alleviates intestinal epithelial apoptosis. Caspase-3 inhibition or BCL2 silencing abrogated hk-B. f’s anti-apoptotic effects in IEC-6 cells.

Conclusions

Pathological process of CID can be partially explained by compositional alterations in the gut microbiota. Supplementation with hk-B. f reduces 5-FU-stimulated epithelial injury through mitochondrial apoptotic pathway in CID murine models. These preclinical findings suggest hk-B. f merits further investigation as a potential strategy for improving CID, pending clinical validation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-025-04233-5.

Background

Chemotherapeutic agents often cause gastrointestinal toxicity that may manifest as diarrhea, constipation, and mucositis, limiting patient tolerance and contributing to mortality [1, 2]. The risk for chemotherapy-induced diarrhea (CID) is significantly greater for chemotherapeutic regimens that include fluoropyrimidines, such as fluorouracil (5-FU) and irinotecan (CPT-11), with grade 3–4 diarrhea reported in up to 47% of patients [1, 2].

Several reviews and guidelines have been published on CID; however, most studies have focused on symptomatic management rather than mitigation of the underlying pathophysiological mechanisms. Chemotherapeutic agents used for cancer treatment primarily target rapidly dividing cells [1, 3], including cells of the gastrointestinal epithelium. These cells form a semipermeable barrier and are fundamentally important for maintaining the secretory, absorptive, and propulsive functions of the gastrointestinal tract [4–6]. The anti-cancer effects on rapidly dividing cells may shift the balance among functionally distinct cell types in the gastrointestinal epithelium and may cause changes in gastrointestinal physiology. The imbalance between absorption and secretion can lead to a large volume of fluid and electrolytes in the small bowel, which can overwhelm the absorptive capacity of the colon and result in CID [3, 6]. Recent study has shown changes in gut microbial composition (dysbiosis) in the pathogenesis of CID [7]. The relative abundances of Lactobacillus spp. and Bacteroides spp. reduced in patient fecal samples after chemotherapy [8, 9]. However, there has been no comprehensive assessment of dysbiosis in patients with CID. With the development of metagenomic sequencing and analysis, there is a growing demand for such in-depth analyses of alterations in gut microbes and their causal associations with symptoms.

In general, the pathogenesis of CID is poorly understood; however, based on the multidisciplinary literature review, adequate and appropriate use of loperamide is recommended for cancer patients suffering from gastrointestinal side effects [1, 2]. While loperamide remains a first-line therapeutic option for acute diarrhea management, its clinical application is constrained by limited high-quality evidence guiding optimal dosing strategies [1]. Current guidelines advise against exceeding 16 mg daily due to diminishing therapeutic returns and recommend transitioning to alternative interventions if symptoms persist beyond 48 h [1, 10]. This cautious approach is necessitated by loperamide’s dose-dependent opioid-like adverse effects, including central nervous system depression and respiratory depression [10, 11]. Other agents used for treating CID include octreotide and proteolytic enzymes; however, the underlying therapeutic mechanisms are not clearly understood [1, 2, 12, 13]. Alternatively, numerous probiotic organisms are known to modulate the severity of intestinal inflammation by altering the composition, metabolism, and functional properties of indigenous gut microbiota [14, 15], and many have been examined for the prevention and treatment of gastrointestinal conditions [1, 2]. These studies include randomized trials demonstrating that probiotic supplementation with Lactobacillus spp. or Bifidobacterium spp. can reduce the frequency of severe diarrhea and abdominal discomfort related to chemotherapy or radiotherapy without intolerable side effects [16, 17]. Nevertheless, the use of probiotics for preventing CID is also controversial because this strategy increases the risk of severe infections among immunocompromised patients [2]. Considering these uncertainties, innovative approaches that target other steps in the pathophysiology of CID are urgently required. Therefore, we conducted a detailed evaluation of CID-related dysbiosis in humans and mice and tested several probiotic treatment protocols in the mice models to identify potentially effective therapies. This study broadens the knowledge about gut microbes in patients with CID and provides new insights into therapeutic strategies.

Methods

Participant recruitment and sample collection

Patients hospitalized at oncology department in Nanfang hospital (the first affiliated hospital of Southern medical university, China) who met the inclusion and exclusion criteria were recruited in this research. The inclusion criteria were as follows: (1) age from 18 to 80 years; (2) pathologically diagnosed with gastrointestinal tumors; (3) undergoing chemotherapy (irinotecan, fluorouracil analog or the combination chemotherapy). The exclusion criteria were as follows: (1) received antibiotic drugs/endoscopy examination/radiation within 1 month; (2) received surgery within 3 months. For patients with cancer, pre-chemotherapy and post-chemotherapy samples were collected before the initiation or 12 h after the accomplishment of chemical regimen, respectively. All samples were storage at − 80℃. Human participants study protocol was approved by Ethics Committee of Southern Medical University Nanfang Hospital (NFEC-2020–269).

Animal models

Six to eight weeks male BALB/c and C57BL/6 mice were obtained from Experimental animal center of Southern Medical University. All mice were housed in SPF condition with no more than 5 mice per cage. A total of 10 mice per experimental group were included in this study, based on pilot data indicating an effect size (d = 1.5, α = 0.05, power = 0.8) with G Power 3.1-calculated minimum n = 9 per group and ethical compliance (reduction: G Power 3.1-calcul of group size). Animal experiments were carried out after 7 days of acclimation. For tumor bearing, BALB/c mice were inoculated orthotopically at the surface of the cecum with CT26 tumor cells (1 × 10⁶) in 0.04 mL of PBS for tumor development. The animals were assigned into different groups using an Excel-based randomization software performing stratified randomization based upon their body weights 11 days after inoculation for further research. For safety assessment of hk-B. f, C57BL/6 mice were orally administrated with 200 μL hk-B. f-AF647 or DIBO-AF647. For CID modeling, mice gently restrained using a tunnel device to minimize stress. BALB/c mice were intraperitoneally injected with 5-FU (50 mg/kg/day) or CPT-11 (60 mg/kg/day) for 5 days according to previous articles [18, 19]. Detailed modeling methods and grouping information are respectively shown in Fig. 2, Fig. 3, Fig. 8, Additional file 2: Fig. S4, and Additional file 2: Fig. S5. Probiotic efficacy was assessed in 5-FU- or CPT-11-induced CID murine models using primary (diarrhea severity) and secondary endpoints (body weight change, intestinal permeability). Treatment administration and outcome assessments were performed by separate investigators. Animal study protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University (K2021035).

Fig. 2.

The effect of probiotics on 5-FU induced CID model in mice. a Schematic diagram of the treatment schedule and timeline for CID model with 5-FU stimulation. b Body weight change in different experimental groups with 5-FU stimulation. p values were calculated with one-way ANOVA test. n = 10 mice for each experimental group. c The distribution of diarrhea grade of mice recorded at day 9 (before sacrifice) with 5-FU stimulation. Significant importance was calculated with Kruskal-Wallis H test among groups. Between group differences were accomplished with Mann-Whitney U test. The asterisk represents significant difference compared to 5-FU group. n = 10 for each group. b, c p < 0.05 indicates significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance. B. f, Bacteroides fragilis; hk-B. f, heat-killed Bacteroides fragilis; PB3, triple probiotics combination of living Bifidobacterium longum, Lactobacillus acidophilus and Streptococcus faecalis

Fig. 3.

The effect of different doses of hk-B. f on 5-FU stimulated CID model. a Schematic diagram of the treatment schedule and timeline for CID model. b Body weight change in different experimental groups (n = 10 for each group). c FITC-Dextran permeability in different experimental groups (n = 10 for each group). d The distribution of diarrhea grade of mice recorded from day 8 (end of modeling) to day 12 (end of experiment). Fecal consistency was classified based on the following visual grading scale: score = 0, normal; score = 1, soft feces or small black feces; score = 2, muddy feces; and score = 3, watery feces or mucous feces. Significant importance was calculated with Kruskal-Wallis H test among groups. Between groups differences were accomplished with Mann-Whitney U test. The asterisk represents significant difference compared to 5-FU group (n = 10 for each group). e–h Fecal wet weight (e, g) and fecal water content (f, h) at day 8 or day 12. Fecal water content was determined by the following equation: 1 − (dried solid content)/(fecal wet weight) × 100%. i Daily food intake recorded every 4 days for each experimental group. b, c, e–i Significant importance was calculated with one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance. hk-B. f, heat-killed Bacteroides fragilis; hk-B. f(L), low-dose of hk-B. f; hk-B. f(M), medium-dose of hk-B. f; hk-B. f(H), high-dose of hk-B. f; LPM, loperamide

Fig. 8.

The effect of B. f metabolites and cellular component TP2 on 5-FU stimulated CID model. a, i Schematic diagram of the experimental grouping and timeline for CID model. b, k Body weight change in different experimental groups (n = 10 for each group). The asterisk represents significant difference compared to 5-FU group. c, l The distribution of diarrhea grade of mice recorded at day 12 (end of experiment). Significant importance was calculated with Kruskal-Wallis H test among groups. Between groups differences were accomplished with Mann-Whitney U test. The asterisk represents significant difference compared to 5-FU group (n = 10 for each group). d, m Fecal water content at day 12. e, n FITC-Dextran permeability in different experimental groups (n = 10 for each group). f, o HE staining for ileal tissue in different experimental groups. Scale bars, 500 μm for × 4 images, 100 μm for × 20 images. g Volcano plot of non-targeted metabolomics analysis (B.f supernatant group vs blank culture medium group, n = 6 for each group). h Scatter plots of OPLSDA of non-targeted metabolomics analysis. i The heatmap and fold change (FC) value ranking of differently expressed metabolites in organic acid profile. B. f, Bacteroides fragilis; hk-B. f, heat-killed Bacteroides fragilis; TP2, capsular polysaccharides isolated and purified from Bacteroides fragilis strain ZY-312; PA, propionic acid; SA, succinic acid. *Indicates significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Bacteria strains

Bacteroides fragilis (B. f) strain ZY-312 and heat-killed B. f (hk-B. f) were obtained from ZYBio Co., Ltd. (Guangzhou, China). Bacteria were cultured in sterile tubes containing 10 ml of tryptone soy broth supplemented with 5% fetal bovine serum and incubated anaerobically at 37 °C. hk-B. f was the inactivation status of living B.f through thermal inactivation (80 °C for 30 min). Triple probiotics formulation (PB3), consisted of Bifidobacterium longum, Lactobacillus acidophilus, and Streptococcus faecalis, was obtained from Sinepharm Co., Ltd. (Shanghai, China).

Cell culture, cell transfection, and inhibitor administration

Rat small intestinal crypt epithelial cells IEC-6 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37℃. IEC-6 cells underwent siRNA-mediated FAS or BCL2 knockdown using Lipofectamine 3000 (Tsingke Biotech) per manufacturer protocols. Post-transfection (6 h), cells were maintained in DMEM/10% FBS for 48 h prior to 5-FU (5 μM, 24 h) and hk-B.f (1 × 10⁸ CFU/mL, 24 h) or PBS treatment (24 h). For BCL2, the siRNA target sequence was 5′–3′: AAGTTCGGTGGGGTCATGTGT. For FAS, the siRNA target sequence was 5′–3′: GCUAUUGUGGACGGAGGUATT. For caspase inhibition, cells were pre-treated with Z-VAD-FMK (50 μM, Selleck 187,389–52-2) for 6 h before 5-FU/hk-B. f exposure.

Mouse intestinal organoid culture

Intestinal organoids were isolated and cultured in vitro using IntestiCult™ Organoid Growth Medium (06005, STEMCELL) following the manufacturer’s instructions. Approximately 500 crypts were embedded in 50 μL Matrigel (356,231, Corning) per well of a 24-well plate and submerged in IntestiCult™ Organoid Growth Medium with included supplements. Organoids were then subcultured every 6–7 days at 37 ℃ in a 5% CO₂  incubator with a 1:4 splitting ratio.

Metagenomic sequencing

High-quality metagenomic sequencing was conducted on 60 paired fecal samples (pre-/post-chemotherapy, n = 30 patients) using standardized protocols. DNA extraction was performed with the MagPure Stool DNA KF Kit B (Magen, China): 200 mg fecal samples were transferred to sterile tubes containing grinding beads, homogenized with 1 mL Buffer ATL/PVP-10 using a grinding machine (Shanghai Jingxin Tech) at 65 °C for 20 min. The homogenate was centrifuged at 14000 × g (5 min, Eppendorf), and the supernatant was transferred to a new tube. After adding 0.6 mL Buffer PCI and vortexing (15 s), the mixture was centrifuged at 18213 × g (10 min). The supernatant was transferred to a deep-well plate containing 600 μL magnetic bead binding solution (20 μL Proteinase K + 5 μL RNase A). Sequential washes (700 μL Wash 1, 700 μL Wash 2, 700 μL Wash 3) and elution (100 μL Elution Buffer) were performed on the KingFisher Flex system (Thermo Fisher). Purified DNA was stored at − 80 °C.

Libraries were constructed from 200 ng DNA using the BGI Optimal DNA Library Prep Kit (BGI, China), involving fragmentation, single-stranded circularization, and rolling circle amplification to generate DNA nanoballs (DNBs). Sequencing was performed on the DNBSEQ-T7 platform (PE150, BGI) with DNB nanoarray loading. Raw data were processed through SOAPnuke v2.2.1 for quality trimming [20], followed by host read removal (SOAP2 alignment to hg38) [21]. High-quality reads were assembled de novo using MEGAHIT (k-mer 21–121) [22], retaining contigs ≥ 300 bp for MetaGeneMark gene prediction [23]. Non-redundant genes were filtered via CD-HIT (95% identity, 90% coverage) [24], with taxonomic annotation performed using Kraken2 (LCA algorithm) [25] against the NCBI NT sub-database k2_pluspf_16gb_20240112.

RNA sequencing

Total RNA from mice ilea was extracted using Trizol. In order to obtain mRNA, the extracted total RNA was purified with OligodT and DNA probes in order to eliminate the impact of nucleic acids as previously described [26]. The mRNA was then reverse transcribed into cDNA. PCR amplification system was established with a mixture of A-Tailing Mix, RNA Index Adapters, and the cDNA. An Agilent 2100 Bioanalyzer (Thermo Fisher Scientific) and the StepOnePlus Real-Time PCR System (ABI, Foster City, CA, USA) were adapted for assessing the quality of the constructed libraries. The high-throughput sequencing was accomplished with Illumina HiSeq4000 platform (Illumina San Diego, CA, USA) by the Beijing Genomics Institute (BGI, Shenzhen, China) on the basis of the high-quality libraries. The clean reads were obtained after filtering the low-quality or contaminated raw reads. The reads were then queried against the target genome sequences (GCF_000001405.37_GRCh38.p11) using Bowtie2. Finally, the gene expression levels were assessed with RSEM (BGI).

Diarrhea measurement

Diarrhea score and fecal water content were measured daily for evaluating diarrhea degree of mice during treatment. Diarrhea score/grading was determined based on previous scoring standard [27, 28]. Fecal consistency was classified based on the following visual grading scale: (1) score = 1, formed, stool maintains its shape, brown; (2) score = 2, semi-formed or soft, does not pour, yellow; and (3) score = 3, liquid, pours more easily. Fecal water content was determined by the equation: 1 − (dried solid content)/(total fecal content).

Preparation of fluorescently Labeled hk-B. f

B. f was cultured for 65 h at 37 °C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂) in an anaerobic glove box in basal peptone-yeast broth with GalNAz at a final concentration of 100 μM. Bacterium were spun down and washed three times in 1 × PBS supplemented with 1% bovine serum albumin (BSA) when the OD600 value ranged from 0.7 to 1.4. The bacteria pellet was resuspended in 10 ml PBS supplemented with 1% BSA and 20 mM DIBO-AF647 (100 μL per vial) and each vial of the bacterium suspension was kept separately. Incubate the resuspension in a shaker and protected from light for 5 h. Centrifuge the resuspension with 1 × PBS containing 3% BSA at 13000 rpm for 2 min, remove the supernatant, wash for five times. Resuspend the bacteria with 500 μL of 1 × PBS containing 1% BSA, inactivated by thermal inactivation (80 °C for 30 min), cooled and centrifuged at 13000 rpm for 2 min, remove the supernatant, add 300 μL of 30% glycerol broth solution, and store in a freezer set to maintain − 20 °C.

Optical imaging

Thirty-nine C57BL/6 mice were randomly divided into three groups: blank control group (3 mice), test group (18 mice), and positive control group (18 mice). The test group and positive control group were randomly divided into six groups (3 h, 6 h, 12 h, 24 h, 48 h, and 60 h) with three mice coded sequentially in each group. The mice in the test group were given fluorescently labeled hk-B. f by gavage, while the mice in the positive control group were given positive dye DIBO-AF647 by gavage, and the mice in the blank group were given PBS. Fluorescence intensity and distribution were measured at 3 h, 6 h, 12 h, 24 h, 48 h, and 60 h after gavage of the test interventions in mice. The fluorescence signals were detected by IVIS imaging system (PerkinElmer).

Intestinal permeability analysis

Intestinal permeability was evaluated with fluorescein isothiocyanate (FITC)-Dextran 4000 (78331, Sigma-Aldrich) as previously described [29]. Briefly, fasting mice were administered of FITC-Dextran 4000 (600 mg/kg) by oral gavage. Four hours after gavage, fluorescence intensity of FITC-Dextran 4000 in plasm was detected.

Antibodies

Primary antibodies against GAPDH (60004–1, Proteintech), Caspase3 (14220T, CST), Cleaved Caspase3 (9664T, CST), Occludin (91131S, CST), ZO-1 (402200, Invitrogen), Bax (60267–1, Proteintech), and BCL2 (ab32124, Abcam) were used in this article.

Non-targeted metabolomics analysis

Six blank culture medium and six B. f culture supernatant samples were collected. Samples were thawed on ice and vortexed. The extract containing the internal standard was added to the samples, and the supernatants were obtained by centrifugation (12000 r/min for 10 min at 4° C). Then sample supernatants were placed at − 20 °C for 30 min. Next, the supernatants were obtained by centrifugation (12000 r/min for 3 min at 4° C) for LC–MS analysis. All samples were collected by the LC–MS system according to machine instructions. UPLC column: Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm*100 mm). Then, column temperature, 40° C; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system containing water (0.1% formic acid) and acetonitrile (0.1% formic acid). The original data file acquired by LC–MS was converted into mzML format by ProteoWizard software. Peak extraction, peak alignment, and retention time correction were respectively performed by the XCMS program. The “SVR” method was used to correct the peak area and filter data. After that, metabolic identification information was obtained by searching the laboratory’s self-built database, integrated public database, AI database, and metDNA. The PCA scatter plot, volcano plot, and differential expression metabolites rank analysis were based on positive ion model data. Criteria for defining differential metabolites: VIP ≥ 1, P-value < 0.05 in Student’s t-test and fold change ≥ 2 or fold change ≤ 0.5 between two groups.

Extraction and purification of capsular polysaccharide TP2 from B. fragilis strain ZY-312

Zwitterionic polysaccharide (ZPS) with an average relative molecular weight of 70 kDa from ZY-312 was isolated and purified according to previous published article [30]. ZY312-derived capsular polysaccharide termed TP2. Polysaccharide TP2 was provided by Guangzhou Zhiyi Biotechnology Co., Ltd. (Guangzhou, China). The total sugar content of TP2 was 98%, the protein content was 1%, and the nucleic acid content was 0.5%. The repeating unit of TP2 consists of four monosaccharides: 2,4-dideoxy-4-amino-D-N-acetylfucose, D-N-acetylgalactosamine, D-galactopyranose, and D-galactofuranose with 4,6-pyruvate attached to the galactopyranose.

Statistical analysis

Normality was rigorously assessed using Shapiro–Wilk tests (n ≤ 50) and Kolmogorov–Smirnov tests with Q-Q plots (n > 50). Continuous variables were presented as mean ± standard error of mean (SEM) for normally distributed data and median (interquartile range, IQR) for non-normal distributions. Two-group comparisons used independent/paired t-tests (parametric) or Mann–Whitney U/Wilcoxon signed-rank tests (non-parametric), while multi-group analyses employed one-way ANOVA with Tukey’s post hoc testing (parametric) or Kruskal–Wallis with Dunn-Bonferroni correction (non-parametric). Correlation analysis was evaluated via Spearman’s ρ. All statistical analyses incorporated appropriate adjustments for multiple comparisons using either Bonferroni correction or false discovery rate (FDR) adjustment and were conducted in SPSS version 19.0 software. Graphs were generated by GraphPad Prism 9.0 and R version 4.2.2 software. p < 0.05 indicates a significant difference.

Results

Participant information

The self-controlled study design is schematically presented in Fig. 1a. From 30 CID patients, paired pre-/post-chemotherapy fecal samples were collected (n = 60 total), with microbiota composition compared through metagenomic sequencing (Fig. 1b). Clinico-demographic characteristics of this self-controlled cohort are detailed in Fig. 1c.

Fig. 1.

Bacterial diversity of the fecal microbiota associated with Pre- and Post-chemotherapy patients. a Participant selection and specimen collection process. b Study design and groups: 30 cancer patients with fecal samples both collected Pre- and Post-chemotherapy treatments (1). Samples were subjected to shotgun sequencing (2) and reads were mapped to reference databases (3) for sample-specific relative abundance calculation (4) and processed for nucleotide diversity and untargeted metabolomics (5). c Demographic information of the involving patients. d Fecal microbial alpha-diversity estimated by Chao1, Simpson, and Shannon index. p values were calculated by two-sided unpaired Wilcoxon test. e Fecal microbial beta-diversity calculated under Bray-Curtis distance. f Fecal microbial PCoA diagram calculated under Bray-Curtis distance. g Relative abundance of the bacteria at the genus level. h A Venn diagram displayed the overlaps between groups. i The relative abundance of microbial genera including Bacteroides spp., Bifidobacterium spp., Lactobacillus spp., and Akkermansia spp. between two groups. p values were calculated by two-sided paired Wilcoxon test. j The relative abundance of microbial species Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, and Akkermansia muciniphila. k Spearman’s correlation between the relative abundance of Bacteroides fragilis at baseline and patient’s diarrhea grade. d,e p values were calculated by two-sided unpaired Wilcoxon test. i, j p values were calculated by two-sided paired Wilcoxon test. Pre, pre-chemotherapy group; Post, post-chemotherapy group

Chemotherapy exposure alters gut microbiome composition

Metagenomic profiling of paired fecal samples revealed genus-level taxonomic differences (Pre vs. Post) as shown in Fig. 1g. While fecal α-diversity (Chao1, Simpson, Shannon indices) remained stable (Fig. 1d), β-diversity based on Bray–Curtis distances exhibited significant inter-group separation (Fig. 1e), visualized by PCoA diagram (Fig. 1f). A Venn diagram identified 328 core genera shared between groups, with 45 Pre-exclusive and 18 Post-exclusive taxa (Fig. 1h).

We then identified these differentially abundant taxa. At the genus level, Bacteroides spp. abundance was significantly reduced in Post group (Fig. 1i). Alternatively, neither Bifidobacterium spp. nor Lactobacillus spp. were significantly altered, despite their previously reported efficacy for improving CID. There was also no significant difference in the abundance of Akkermansia spp. At the species level, there was a significant decrease in the abundance of B. fragilis (B. f) in the Post group compared to the Pre group (Fig. 1j), whereas no substantial changes were observed in Bifidobacterium breve, Bifidobacterium longum, and Akkermansia muciniphila. The differential analysis with relative abundance is shown in Additional file 2: Fig. S1a, b. Correlation analysis (Fig. 1k) revealed that the abundance of B.f at baseline level had significantly negative correlation with patient’s diarrhea grade (R =  − 0.93, p < 0.001), which indicates that the relative abundance of B. f at baseline may have protective effects in patients. Overall, these data suggest that community-level dysbiosis may contribute to CID. Further, Bacteroides spp. and B. f were identified as key taxa contributing to compositional alterations after chemotherapy.

Functional analysis of fecal microbiota

To examine the functional and metabolic changes in fecal microbial communities associated with chemotherapy, all clean reads from metagenomic sequencing were aligned to the suggested database for KEGG and GO enrichment analyses (Additional file 2: Fig. S1c). Samples from Pre group displayed a few differentially enriched KEGG metabolic pathway terms compared with samples from Post group. Several pathway terms related to amino acid metabolism, fatty acid biosynthesis, and lipopolysaccharide biosynthesis were overrepresented in the Pre group. These results indicate that chemotherapy markedly alters the metabolic properties of gut microbiota.

Heat-killed B. f improved 5-FU-induced CID

Given the negative correlation between baseline B. f abundance and CID severity, we hypothesized that targeted B. f supplementation might mitigate CID. This mechanistic hypothesis was subsequently validated in preclinical murine models. Surprisingly, experiments comparing the efficacy of living B. f to heat-killed B. f (hk-B. f) and a combination probiotic (PB3) revealed that hk-B. f was most effective for restoring body weight loss and reducing diarrhea in 5-FU-induced mouse models of CID (Fig. 2a, b, c). Current clinical guidelines recommend LPM as first-line therapy for CID, yet explicitly contraindicate its use in bloody diarrhea—conditions recapitulated in our 5-FU-induced murine CID model (occult blood in 20% of mice in pre-experiment). This paradox prompted our comparative analysis of LPM and various doses of hk-B. f (Fig. 3a). Significantly decreased body weight and daily food intake (Fig. 3b, i) and increased intestinal permeability and fecal water content (Fig. 3c, f, h) were observed in mice stimulated with 5-FU, indicating that this model mice recapitulated the core features of CID. Different doses of hk-B. f and a standard dose of LPM maintained normal intestinal permeability (Fig. 3c); however, hk-B. f was more efficacious at reducing diarrhea grade. In both treatment groups, diarrhea grade fluctuated with time during treatment (Fig. 3d), and neither hk-B. f nor LPM improved diarrhea from day 8 to day 10. However, the high dose of hk-B. f, hk-B. f(H), significantly reduced the diarrhea score on days 11 and 12 (Fig. 3d). Moreover, hk-B. f(H) significantly decreased the fecal water content on day 12 (Fig. 3h) and increased feed consumption on average per cage (Fig. 3i), while LPM failed to improve diarrhea in the 5-FU-induced CID model.

We further performed 16S RNA sequencing of fecal samples from 5-FU-induced CID mice to explore whether different doses of hk-B.f alter mice intestinal microbiota composition. As shown in Additional file 2: Fig. S2a-c, low, medium, and high doses of hk-B. f did not alter α-diversity or β-diversity. Overall, as indicated in Additional file 2: Fig. S2d, e, there is a difference between the two batches of differential analysis. hk-B. f(H) significantly increased the relative abundance of Bacteroides spp. in batch B (Additional file 2: Fig. S2d, e), which provides additional evidence for the therapeutic effect of Bacteroides spp. on CID dysbiosis in mice.

High dose of hk-B. f improved ileal mitochondrial structure and function

Next, we analyzed the effects of hk-B. f supplementation on global gene expression and found that multiple pathways related to mitochondrial structure were enriched in the hk-B. f(H) sample group (Fig. 4a). Further analysis demonstrated that genes encoding mitochondrial respiratory complex proteins (Fig. 4b) were upregulated in the ileum of hk-B. f(H)-supplemented mice. Biochemical analysis of ileum tissue from 5-FU-induced CID model mice revealed a significant reduction in ATP concentration (Fig. 4d), whereas transmission electron microscopy (TEM) revealed a significant decline in mitochondrial number (Fig. 4c, e), and both these deficits were alleviated after hk-B. f(H) supplementation. The overall area occupied by mitochondria was also significantly larger in the 5-FU-induced CID model group than the control and hk-B. f(H) groups (Fig. 4c), suggesting that untreated CID is associated with mitochondrial swelling and dysfunction. Quantitative data analysis indicated that 5-FU-induced mitochondrial swelling which possibly disturbed proper function of mitochondria and can be ameliorated by hk-B. f(H) (Fig. 4f). Furthermore, we performed quantitative evaluation for mitochondrial ultrastructural changes with methods in previous published articles [32, 33]. Based on these criteria, we identified healthy and damaged mitochondria with the use of individual images. The results exhibited significantly lower levels of grey scale unit in hk-B. f(H) and control, which indicated healthier mitochondria (Fig. 4g). Overall, these results supported the prediction derived from gene expression data and illustrated that hk-B. f(H) protected ileal mitochondria which were damaged by 5-FU.

Fig. 4.

Ileal mitochondria destructions were ameliorated by the supplementation with high dose of hk-B. f. a Over-represented modules in GO enrichment shown in mice supplemented with high dose of hk-B. f (n = 3 for each group). b A heatmap showing the median expression of genes from the respiratory chain process in the ilea of mice. c Representative TEM images in ileal epithelial cells. Scale bars, 10 μm for the left image and 5 μm for the right image in each experimental group. d ATP concentration extracted from ileal tissue (n = 5 for each group). e–g Various metrics of mitochondria in the ileum of mice. n = 5 mice for each group in mitochondrial count. n = 100 mitochondria for each group in recording mitochondrial size and gray scale unit. Between-group significant importance for d–g were accomplished with Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance. hk-B. f, heat-killed Bacteroides fragilis; hk-B. f(H), high-dose of hk-B. f; LPM, loperamide. TEM, transmission electron microscopy

Heat-killed B. f protects against 5-FU-induced crypt damage and epithelial tight junction loss

We next explored the molecular mechanisms of mitochondrial protection by hk-B. f in the 5-FU-induced model of CID. Histological staining of ileal tissue from untreated model mice revealed substantial degeneration of mucosal integrity and crypt structure. Histological staining of ileal tissue from untreated model mice revealed substantial degeneration of mucosal integrity and crypt structure (Fig. 5b), and these injuries were partially mitigated by hk-B. f and LPM without interfering with the antitumor effects of 5-FU (Fig. 5a). Alcian blue staining revealed a decline in the number of mucus-filled cells in the 5-FU group (Fig. 5c). When treated with hk-B. f(H) or LPM, the number of mucus-filled cells significantly increased. Immunostaining also showed that the staining intensity of tight junction proteins ZO-1 and occludin were reduced and that the distributions of these proteins were aberrant and discontinuous in untreated model mice (Fig. 5d, e). hk-B. f(M), hk-B. f(H),and LPM restored ZO-1 localization to the apical regions, improved boundary continuity, and enhanced the staining intensities of ZO-1 and occludin. Consistent with immunostaining, western blot results confirmed that hk-B. f(H) and LPM reduced the decline in occludin expression (Fig. 5f, h), although there were no effects on ZO-1 expression compared to untreated model mice (Fig. 5f, g).

Fig. 5.

The effect of different interventions on intestinal histological structure in tumor-bearing CID model. a Representative tumor growth images for different experimental groups. Scale bars, 1 cm. Red arrows indicate tumor located on cecum. b HE staining for ileal tissue in different experimental groups. Scale bars, 500 μm for × 4 images, 200 μm for × 20 images. c Alcian blue staining for ileal tissue in different experimental groups. Scale bars, 200 μm. d, e Immunofluorescence section for Occludin (green) or ZO-1 (green) with DAPI (blue) staining of ileum. Scale bars, 200 μm. f–h Western blot and quantification for the expression level of tight junction proteins in ileal tissue. Significant differences for quantification were calculated with one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. a–f Images are representative of at least 5 mice per condition. f Western blot analysis was repeated for at least 3 times. hk-B. f, heat-killed Bacteroides fragilis; hk-B. f(L), low-dose of hk-B. f; hk-B. f(M), medium-dose of hk-B. f; hk-B. f(H), high-dose of hk-B. f; LPM, loperamide

Heat-killed B. f alleviates 5-FU-induced intestinal epithelial apoptosis by shifting the balance between anti-apoptotic BCL2 and proapoptotic BAX

We then examined the molecular mechanisms for mitochondrial protection by B. f. The number of TUNEL-positive (apoptotic) cells was significantly greater in tissues from the 5-FU model group than the control group, and most TUNEL-positive cells located in crypts (Fig. 6a). The epithelial cell apoptosis rate was reduced by hk-B. f and LPM (Fig. 6a). Western blot analysis indicated that these treatments reduced the expression of the apoptosis effector cleaved caspase-3 (Fig. 6d, g). We further investigated the upstream mechanism of caspase-3-mediated apoptosis and found that the expression of anti-apoptotic BCL2 significantly reduced in the 5-FU group compared to the control group, and that this downregulation was partially reversed by hk-B. f or LPM (Fig. 6b, d, e). Alternatively, expression of proapoptotic BAX was markedly enhanced by 5-FU exposure, and this effect was again reversed by hk-B. f and LPM (Fig. 6c, d, f). No significant changes were detected in the levels of inflammatory cytokines among groups (Fig. 6h). These data demonstrated that hk-B. f and LPM can ameliorate 5-FU-induced epithelial cell apoptosis by shifting the BCL2/BAX balance and thereby preventing activation of the mitochondrial apoptotic pathway.

Fig. 6.

The effect of different interventions on mitochondrial-apoptotic pathway. a Tunel-staining (red) with DAPI (blue) for ileal tissue in different experimental groups. Scale bars, 200 μm. White arrows indicated Tunel-positive cells. b, c Immunohistochemical sections for BCL2 or BAX staining of ileum. Scale bars, 200 μm. d–g Western blot and quantification for the expression level of mitochondrial-apoptotic proteins in ileal tissue. Significant differences for quantification were calculated with one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. h A heatmap displays the level of serous cytokines with ELISA assay. n = 10 mice for each group. Data were normalized with log10. a–d Images are representative of at least 5 mice per condition. d Western blot analysis was repeated for at least 3 times. hk-B. f, heat-killed Bacteroides fragilis; hk-B. f(L), low-dose of hk-B. f; hk-B. f(M), medium-dose of hk-B. f; hk-B. f(H), high-dose of hk-B. f; LPM, loperamide

Heat-killed B.f reversed 5-FU-stimulated apoptosis in in vitro epithelial barrier models

We further tested these effects in an in vitro barrier model established with the rat epithelial cell line IEC-6. Consistent with tissue immunoblotting results, 5-FU induced cell apoptosis as measured by the CCK-8 assay and barrier function loss as evidenced by the TEER assay, whereas hk-B. f treatment partially restored the proliferation rate and barrier integrity (Fig. 7a, b). In addition, Western blot analysis confirmed that 5-FU reduced BCL2, ZO-1, and occludin expression levels and that hk-B. f reversed these effects (Fig. 7c). Consistent with tissue studies, 5-FU treatment alone increased BAX and cleaved caspase-3 expression levels, and these effects were reversed by hk-B. f. Furthermore, flow cytometry analysis of Annexin V-FITC/PI-stained cells showed that hk-B. f co-treatment reduced the rate of apoptosis compared to 5-FU treatment alone (Fig. 7d). These results were further confirmed by scanning electron microscopy (SEM). As shown in Fig. 7e, after stimulation with 5-FU, IEC-6 cells exhibited swelling, nuclear fragmentation, and membrane rupture, and that all these signs were mitigated by hk-B. f.

Fig. 7.

hk-B. f improves 5-FU induced injury both in IEC-6 cell line and small intestinal organoids. a Proliferation curve detected by CCK-8 assay for IEC-6 cells. b TEER for IEC-6 cells. Black arrow indicates the time of 5-FU stimulation. c Western blot analysis for the expression of tight junction proteins and apoptotic proteins in IEC-6 cells. d Apoptosis rate of IEC-6 cells with different interventions detected by flow cytometry analysis. e Representative images of SEM observation for IEC-6 cells under different conditions. Scale bars, 50 μm. f Representative images of optic microscopy (left panel) and TEM (right panel) observations for small intestinal organoids with different interventions. Scale bars, 400 μm for optic microscope images, 2 μm for TEM images. g, h Statistical analysis for cross-section area and budding count of organoids. Between-group differences were calculated with Mann-Whitney U test. a–f Experiments were repeated for at least 3 times. a, b, d, g, h *indicates significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. hk-B. f, heat-killed Bacteroides fragilis; TEER, trans-epithelial elective resistance; SEM, scanning electron microscopy; TEM, transmission electron microscopy

Small intestinal organoids extracted from the small intestines of C57BL/6 mice grew and developed normally (Fig. 7f), while those stimulated by 5-FU were severely injured, with quantitatively decreased budding count and overall area (Fig. 7g, h). hk-B. f supplementation improved organoid growth and development as evidenced by significantly higher budding count and larger organoid surface area.

Silencing of BCL2 and caspase-3 interferes with the therapeutic effects of hk-B. f

To confirm that hk-B. f ameliorates 5-FU-induced gut epithelial damage by reducing mitochondrial apoptotic pathway activity, rescue experiments were conducted. As expected, hk-B. f failed to reduce 5-FU-induced apoptosis when the downstream effector cleaved caspase-3 was pharmacologically inhibited by Z-VAD-FMK or the upstream anti-apoptosis protein BCL2 was silenced by a targeted siRNA (Additional file 2: Fig. S3a–f). In contrast, siRNA-mediated knockdown of FAS, which acts on the upstream factor in the extrinsic apoptosis pathway, reduced baseline apoptosis rate following 5-FU treatment (Additional file 2: Fig. S3g, h, i) but did not interfere with the ameliorative effects of hk-B. f on cell proliferation and apoptosis. Western blot analyses (Additional file 2: Fig. S3j) also confirmed that hk-B. f restored the BCL2/BAX balance and prevented cleaved caspase-3 activation in 5-FU-treated cells.

B. f-derived succinic acid weakens the probiotic effect of capsule polysaccharides

Previous results showed that hk-B. f exerted stronger effects than the living state. Heat-inactivation partly preserves cellular components and denatures metabolites. Here, we supposed that B. f-derived metabolites played a key role in the discrepant therapeutic effect between hk-B. f and live B. f. Live B. f, hk-B. f, and B. f-derived culture supernatant were administered to CID model mice (Fig. 8a–f). Unexpectedly, we found that the supernatant aggravates weight loss (Fig. 8b) and pathological injury of mice ileum (Fig. 8f). To further probe the detrimental substances secreted by live B. f, we performed non-targeted metabolomics analysis on the culture supernatant from the blank medium and live B. f culture supernatants. Volcano plots and principal component analysis showed that these supernatants exhibited different metabolite profiles (Fig. 8g, h). We found that, succinic acid (SA) and propionic acid (PA) markedly enriched in live B. f supernatant and represented the two largest fold changes in organic acid profile (Fig. 8i). Collectively, these results indicated that SA or PA may participate in CID progression.

The most studied of the B. f capsular polysaccharide is polysaccharide A (PSA), which is considered as one of the sources of probiotic effects produced by B. f [34, 35]. We isolated and purified capsular PS from B. f strain ZY-312 and entitled TP2 [30]. TP2, PA, and SA were next orally administered to CID mice (Fig. 8j–o). Compared with CID group, body weight, diarrhea severity, intestinal permeability, and pathological injury of mice ileum were ameliorated by the administration of TP2 while aggravated by SA rather than PA. Collectively, this result indicates that B. f-secreted SA weakens the probiotic effect of TP2 on mice with CID, which may explains the discrepant therapeutic effect between hk-B. f and live B. f.

Heat-killed B. f improved CPT-11-induced CID

Experiments comparing the efficacy of living B. f to hk-B. f and PB3 revealed that hk-B. f was the most effective for restoring body weight loss and reducing diarrhea in CPT-11-induced mouse model of CID (Additional file 2: Fig. S4a-c). The CPT-11-induced CID model was also established to assess the relative efficacy of LPM and different doses of hk-B. f (Additional file 2: Fig. S5). Although neither treatment increased body weight, average feed consumption, or fecal wet weight (Additional file 2: Fig. S5b, S5d–h), both hk-B. f(M) and hk-B. f(H) were effective for improving diarrhea (Additional file 2: Fig. S5c), whereas LPM failed to improve diarrhea compared to the untreated model group. The fecal water content was significantly higher in the LPM group on day 12 (Additional file 2: Fig. S5c, g). To summarize, hk-B. f(H) was proved to be effective against CID induced by CPT-11, whereas LPM was ineffective.

Safety evaluation of hk-B. f

We investigated the safety of hk-B. f by monitoring the activity of fluorescently labeled specimens throughout the gastrointestinal tract post-dose. The fluorescence signals from Alexa Fluro 647 hk-B. f (hk-B. f-AF647) and DIBO-AF647 dye group were similar, reaching peak intensities at 3 h post-dose and gradually decreasing thereafter (Additional file 2: Fig. S6a, b), although hk-B. f-AF647 demonstrated stronger retention in vivo over time. However, at 60 h, DIBO-AF647 fluorescence increased abruptly, possibly because of coprophagia (Additional file 2: Fig. S6c, d). Quantitative analysis demonstrated that fluorescence intensities in extraintestinal organs (heart, liver, kidney, spleen, and lung) of hk-B. f-AF647 group showed no significant differences compared to DIBO-AF647 group. Therefore, we conclude that hk-B. f exhibits no evidence of ectopic colonization in extraintestinal tissues (Additional file 2: Fig. S6e). The distribution of hk-B. f-AF647 in the mouse GI tract is shown in Additional file 2: Fig. S7–S9. A strong fluorescence signal was observed in the stomach at 3 h post-dose, which decreased gradually until it was undetectable at 24 h post-dose (Additional file 2: Fig. S7b). hk-B. f-AF647 fluorescence signal began to appear in the ileum at 3 h post-dose and gradually increased, peaking at 6 h post-dose, and then gradually decreased until undetectable after 48 h post-dose. Thus, labeled hk-B. f stayed in the ileum for approximately 48 h (Additional file 2: Fig. S8b). The distribution of hk-B. f-AF647 in the mouse colon is shown in Additional file 2: Fig. S9b. hk-B. f-AF647 fluorescence signal was observed in the colon at 6 h post-dose. The signals were weak at other time points. To summarize, hk-B. f transiently stayed in the gastrointestinal tract (mainly in the upper digestive tract). In the meantime, hk-B. f did not colonize extra-gastrointestinal organs.

Discussion

This study provides the first comprehensive analysis of the microbial community structure in patients with cancer undergoing chemotherapy using metagenomic sequencing. A comparison of paired fecal samples before and after chemotherapy revealed significantly reduced β-diversity following chemotherapy, and Bacteroides spp., especially B. f, were identified as major contributors to this alteration.

Previous research has demonstrated reduced Lactobacillus spp. and Bacteroides spp. abundance in patients receiving chemotherapy compared with healthy controls [8, 9]. However, in our self-controlled pairwise study of the microbial composition before and after chemotherapy, we found no substantial differences in Bifidobacterium spp. or Lactobacillus spp. abundance, despite clinical trials reporting the efficacy of Bifidobacterium in preventing CID [16, 17]. We detected substantial differences in the abundance of Bacteroides spp. Based on these findings, we compared the therapeutic efficacy of bacterial supplementation against 5-FU- and CPT-11-induced CID in mice and found that heat-killed B. f was superior to living B. f and PB3. Moreover, hk-B. f was generally superior to LPM, which is the recommended first-line treatment for CID. In vivo fluorescence labeling experiment revealed that hk-B.f transiently stayed in gastrointestinal tract (mainly in the upper digestive tract) and did not colonize extra-gastrointestinal organs. Thus, hk-B. f supplementation may be a safe and effective strategy for CID warranting further clinical trials, particularly as the heat-killed formulation mitigates the potential risk of living bacteria in immunocompromised chemotherapy patients [2].

We present further evidence that hk-B. f improves CID by reducing 5-FU-induced activation of the mitochondrial apoptosis pathway in ileal epithelial cells. Apoptosis is a tightly controlled process that involves three major signaling pathways [36, 37]: mitochondrial, endoplasmic reticulum, and death receptor. The intrinsic mitochondrial pathway is regulated primarily by BCL2 proteins, which bind and inactivate proapoptotic pore-forming BAX proteins [38–40]. Activation of BAX at the mitochondrial surface leads to mitochondrial outer membrane permeabilization (MOMP) [41, 42], ensuring the release of apoptogenic proteins from the intermembrane space, and activation of the caspases, a family of cysteine proteases [37, 43–45]. Caspases are classified into two groups [43, 45]: initiator caspases such as caspase-9 and effector caspases such as caspase-3. Our TEM study of ileal tissue revealed that 5-FU damaged intestinal mitochondria and resulted in a relatively higher level of TUNEL-positive (apoptotic) cells, especially in the ileal crypt containing highly proliferative stem cells. This finding is consistent with a previous study showing that oxaliplatin-based chemotherapy-induced apoptosis mainly located at the crypts of human ilea [46]. We further demonstrated that hk-B. f could alleviate mitochondrial apoptosis by reversing the 5-FU-induced reduction in BCL2 expression and suppressing the 5-FU-induced upregulation of BAX expression. These results were further supported by in vitro experiments using the IEC-6 cell line and small intestinal organoids.

Many studies, including our previous work, have revealed that the living state of probiotics is essential for its beneficial effects, indicating that the secreted bioactive products play a key role in maintaining host homoeostasis [34, 47–52]. Heat-inactivation partly preserves cellular components and denatures metabolites [53]. Thus, it is an interesting finding that hk-B. f has better therapeutic effects than live B. f on CID model. It is worth mentioning that several inactivated probiotics, including Akkermansia muciniphila and Bifidobacterium bifidum, had better efficacy on diseases [54, 55]. How inactivation enhances the effects of live bacteria remains to be elucidated.

The underlying mechanism is complex and may involve several potential possibilities. One possibility is that a certain component in live B. f-derived metabolites is detrimental to CID, thus weakening the probiotic effect of bacterial structure. Therefore, we detected the main metabolite composition in live B. f culture supernatant. The content of SA and PA markedly changed. SA, one kind of organic acid secreted from live B.f has been detected to act as a danger factor driving the progression of CID. Previous studies also illustrated that succinate derived from mitochondrial metabolism is known to initiate and propagate danger signals from tissue injury [56, 57]. Unexpectedly, PA, one of the short-chain fatty acids (SCFAs), had no significant effect on 5-FU induced diarrhea despite it serves as a potent immunomodulator supplement in many other disease models [47, 58–60]. Further, we isolated and purified capsular PS from B. f strain ZY-312. B. f standard strain and its immunomodulatory capsular PSA have been extensively studied and shown to be equally effective in preventing colitis and experimental allergic encephalomyelitis in murine models [34, 61]. The immunomodulatory activities of PSA induce regulatory T cells secreting IL-10, a potent anti-inflammatory cytokine that restrains pathogenic inflammation in the gut [34, 62]. In this research, B. f strain ZY-312-derived capsular PSA, TP2, effectively maintain mice body weight, alleviate diarrhea, decrease fecal water content, and protect intestinal leakage. Collectively, these findings indicated that B.f-secreted succinic acid weakened the probiotic effect of capsule polysaccharides TP2 on CID which may explain the discrepant therapeutic effect between hk-B. f and live B. f.

This study has several limitations. First, while our metagenomic sequencing provides insights into taxonomic and functional shifts, we acknowledge that metagenomic binning was not performed. Second, animal experiments were conducted exclusively on male mice, so the relevance of these findings in female group requires future studies. Moreover, the mechanism by which hk-B. f/TP2 is recognized by immune cells and subsequently affects the mitochondrial apoptosis pathway in intestinal epithelial cells were not been carefully studied and elucidated. Future research is needed to address this issue.

Conclusions

Finally, our study demonstrates that the pathological process of CID can be at least partially explained by compositional alterations in gut microbiota, especially B. f. A hurdle to the use of live B. f is its high sensitivity to oxygen. Here, we show that non-replicative, hk-B. f had the satisfying effect on ameliorating diarrhea and intestinal apoptosis via BCL2-mediated mitochondrial-apoptotic pathway in preclinical CID models. These results pave the way for future human studies investigating hk-B. f as a therapeutic tool in the management of intolerable side effects from anti-tumor therapy.

Abbreviations

5-FU

5-Fluorouracil

B. f

Bacteroides fragilis

CID

Chemotherapy-induced diarrhea

CPT-11

Irinotecan

FITC-Dextran

Fluorescein isothiocyanate

hk-B. f

Heat-killed Bacteroides fragilis

hk-B. f(H)

High-dose of hk-B. f

hk-B. f(L)

Low-dose of hk-B. f

hk-B. f(M)

Medium-dose of hk-B. f

KEGG

Kyoto Encyclopedia of Genes and Genomes

LPM

Loperamide

MOMP

Mitochondrial outer membrane permeabilization

PA

Propionic acid

PB3

Triple probiotics combination of living Bifidobacterium longum, Lactobacillus acidophilus and Streptococcus faecalis

Post

Post-chemotherapy group

Pre

Pre-chemotherapy group

SA

Succinic acid

SCFAs

Short-chain fatty acids

SEM

Scanning electron microscopy

TEER

Trans-epithelial elective resistance

TEM

Transmission electron microscopy

TP2

Capsular polysaccharides isolated and purified from Bacteroides fragilis strain ZY-312

Z

Z-VAD-FMK

Authors’ contributions

XWY, WJL and FCZ conceptualized of the project. XWY, JHW and WJL managed the resources of clinical specimens. FW, YYL, PL and FCZ acquired research funding to the study. XWY and LJZ performed and analyzed the experiments.XWY and XLL wrote the manuscript with the input of co-authors. YL, YSL, CYH, BHS, HBL, RH, FYH, QZ, MYS, KL, FQZ, SL, YQL and WW provided the technical support. All authors read and approved the final manuscript. FCZ is the guarantor of this study.

Funding

This work was supported by Study on the Mechanism of FUT7 regulating CD15s + eTreg cells in the Pathogenesis of Ulcerative Colitis (Y20190159), Innovation Leading Team Project in Guangzhou (NO. 201809010014) and Key Technology Project in Guangzhou (NO.2024B03J1282).

Data availability

Metagenomic and RNA-Seq raw sequence data have been deposited in Sequence Read Archive with primary accession code PRJNA1010108. Non-targeted metabolomics raw sequence data have been deposited in MetaboLights with primary accession code MTBLS12404. 16S RNA raw sequence data have been deposited in Sequence Read Archive with primary accession code PRJNA1258387. These data are publicly available as of the date of publication. All data reported in this paper will be shared by the lead contact upon request.

Declarations

Ethics approval and consent to participate

All procedures involving pathological sample collection and usage were according to the guideline of the Ethics Committee of Nanfang Hospital of Southern Medical University (NFEC-2020–269). All participants gave their informed consent. All animal study protocols were approved by Institutional Animal Care and Use Committee at Southern Medical University (K2021035).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.