Fish infected with C. sinensis were purchased from Heng County, Guangxi, China. These fish were then digested with pepsin, centrifuged and resuspended in 100 μL PBS with 150 metacercariae for subsequent gavage.

BALB/c mice were selected and randomly divided into three groups, consisting of six mice per group. Each mouse in the infected group was intragastrically administered with 150 C. sinensis metacercariae. The colonic contents of the mice were collected at the 4th week and 16th week post-infection, respectively. Meanwhile, the colonic contents of normal control mice were collected for comparative analysis. The Cetyltrimethylammonium Bromide (CTAB) method was used to extract DNA from the colonic contents, and the V3-V4 region of 16S rRNA was amplified. Sequencing was performed using the NovaSeq 6000 platform. Post-sequencing data were filtered and denoised using QIIME 2 to obtain ASV (Amplicon Sequence Variant) feature sequences. Species annotation was conducted using the SILVA database with NT-16S. Subsequent analyses, including α-diversity, β-diversity, species differentiation, and Lefse analysis, were performed using R software (version 4.3.0).

Female BALB/c mice aged 6–8 weeks were randomly divided into three groups. Mice in the infection group were administered 100 μL of metacercariae suspension via gavage. Mice in the R.b group were subjected to a daily gavage of R. bromii (1 × 10⁹ colony forming units (CFUs)/200 μL/per mice). Normal Chow (NC) and C. sinensis group were given an equal volume of PBS solution by gavage every day.

For C. sinensis-infected mice treated with acetate, 200 mM sodium acetate was added to their daily drinking water post-infection (Qi et al., 2023). Mice in the NC group and the C. sinensis-only group were provided with double-distilled water as the drinking solution. After 8 weeks of feeding, the mice were sacrificed, and serum, tissue and fecal samples were collected for further analysis. All animal experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals in China, and approved by the ethical committee for animal research of Guilin Medical University (approval No. GLMC20230732).

The right lobe of the liver was fixed in 4% PFA for 48 h, followed by dehydration and embedding in paraffin. Sections were cut to 4 μm thick, and the liver was stained with hematoxylin-eosin (HE), Masson, and Sirius red trichrome, and the colon tissue was stained with HE and Alcian Blue Periodic Acid-Schiff (AB-PAS) stain. Images were captured using an Olympus BX53 microscope. Quantitative analysis of Masson trichrome staining and Sirius red staining was performed using ImageJ software.

The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hydroxyproline (HYP) were assessed using the ALT kit (cat no. C009-2-1, Jiancheng, China), AST kit (cat no. C010-2-1, Jiancheng, China), and HYP kit (cat no. A030-2-1, Jiancheng, China) according to the manufacturer’s instructions. Acetate mouse ELISA kit was used to detect acetate levels in the serum of mice.

Ruminococcus bromii ATCC51896 was anaerobically cultured in chopped meat carbohydrate medium at 37°C for 48 h. Once the OD600 of the culture reached 1, the culture broth was centrifuged at 5,000 g for 10 min at 4°C. Subsequently, the supernatant was filtered through a 0.22 μm filter to obtain the Ruminococcus bromii conditioned medium, henceforth referred to as R.b CM. The cells collected by centrifugation were washed three times with sterile anaerobic PBS and resuspended for oral administration in mice.

The human hepatic stellate cell line LX2 and the mouse hepatic stellate cell line JS1 were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO₂ until they reached 70–80% confluence. Subsequently, the medium was exchanged with serum-free medium to induce cellular starvation overnight. Afterwards, the control group was replenished with DMEM containing 10% FBS, while the experimental groups were incubated in DMEM supplemented with 50 μg/mL of ESPs and R.b CM, or sodium acetate (Sigma S2889) for 48 h. ESPs were extracted as described previously (Jin et al., 2014).

LX2 and JS1 cells were seeded in a 96-well plate at densities of 2 × 10³/100 μL and 3 × 10³/100 μL, respectively. Once the cells adhered, they were treated with R.b CM at different concentrations (10, 5, 2.5, and 1.25%) for 48 h. After this treatment period, the media was aspirated, and a solution composed of CCK-8 reagent and DMEM, in a 1:10 ratio, was added into each well. The plates were then incubated in a dark environment for 1 h. Finally, the optical density (OD) at 450 nm was measured for each group to calculate the cell proliferation rate.

Prepared cell slides and fixed the treated cells in 4% PFA. Permeabilized the cells with 0.5% Triton X-100 for 20 min and then blocked with 5% BSA at room temperature. Incubated overnight with α-SMA antibody (Abclonal) at a dilution of 1:200 at 4°C. Incubated with secondary antibody F488 (Proteintech) diluted in 5% BSA at a ratio of 1:500, and incubated in the dark at 37°C for 1 h. After washing with PBS, stained the nuclei with DAPI and observed the cells under a fluorescence microscope (Olympus).

Initially, pipetted 200 μL of conditioned medium or blank medium into a 1.5 mL centrifuge tube. Next, added 50 μL of 50% sulfuric acid solution and 200 μL of ether solution. Shake vigorously for 10 min, then ultrasonicated for 1 min. Centrifuged the mixture at 120,000 rpm for 20 min at 4°C. Carefully transferred the ether layer of the supernatant through anhydrous sodium sulfate before measurement. The concentration of short-chain fatty acids (SCFAs) was determined using gas chromatography–mass spectrometry (GC–MS). The raw data obtained from GC–MS was processed using ChromaTOF software (version 5.51, Leco Corp., United States) for peak integration, calibration, and quantification of each metabolite. iMAP software was used for subsequent PCA, and biomarker heatmap statistical analyses.

Total RNA was extracted from mouse liver tissue using TRIzol reagent (Takara, 9109). The quality and quantity of RNA were determined using a spectrophotometer. Complementary DNA was synthesized from 1,000 ng total RNA using the Complementary DNA Transformation Kit (cat no. MR0511; Monad). Real-time PCR was performed using SYBR Green qPCR Master Mix (cat no. MQ20301S; Monad). DNA was extracted from fecal samples using a kit (Solarbio), the collected DNA was processed according to the kit instructions for qRT-PCR, ormalized against 16 s rRNA. All the gene expression levels were calculated and quantified using the 2^−ΔΔCT method.

Trizol Reagent was used to extract total RNA of JS1 cell line for RNA sequencing (Illumina NovaSeq 6000 sequencing system), and Agilent 2100 bioanalyzer was used to detect the quality of RNA. After qualified quality control, the sequencing library was constructed. Library construction and RNA sequencing were performed by Novogene (Beijing, China). Data were expressed as Fragments Per Kilobase of transcript sequence per Millions base pairs (FPKM) and using DESeq2 (version 1.20.0). log2 fold change >1, p value < 0.05 and KEGG enrichment was performed using clusterProfile software (version 3.8.1).

Total protein was extracted using RIPA lysis buffer and quantified by the BCA method. The protein samples were then electrophoresed in an 8% or 10% SDS polyacrylamide gel and transferred to a 0.45 μm PVDF membrane. The membrane was blocked at room temperature for 2 h, followed by overnight incubation at 4°C with the primary antibody. After the primary antibody incubation, the strips were washed with TBST and incubated with a secondary antibody (1:5,000, cat no. RGAR001; Proteintech) at room temperature for 1 h. Subsequently, the strips were then developed using ECL reagent, and images were captured and analyzed for band density using Image Lab software (6.0.1). The primary antibodies employed in this study included α-SMA (cat no. A17910; Abclonal), CollagenI (cat no. 160043; Abcam), CollagenIII (cat no.184993; Abcam), Occludin (cat no. A24601; Abclonal), Claudin-1 (cat no. A21971; Abclonal), phospho-AKT (cat no. 80455-1; Proteintech) and AKT (cat no. 60203-2; Proteintech), phospho-PI3K (cat no. HA721672; Huabio) and PI3K (cat no. ET1608-70; Huabio), and GAPDH (cat no. AF7021; Affinity), all of which were diluted to a concentration of 1:1,000. GAPDH was used as the internal control.

Comparisons of numerical variables between the three groups were conducted using the Kruskal-Wallis test or Ordinary one-way ANOVA. The expression of total proteins was analyzed using two-way ANOVA or unpaired t test. All statistical analyses in this study were performed using GraphPad Prism 9.0 software or R software (version 4.3.0). Continuous variables were presented as the mean ± standard deviation (SD). When p < 0.05, the difference was considered statistically significant.

To delve into the intricacies of gut microbiota in mice infected with C. sinensis, we performed high-throughput sequencing of 16S rDNA derived from their colonic contents. The saturation of rarefaction curves for these samples underscored the adequacy of sequencing depth, ensuring the detection of a robust bacteria repertoire (Figure 1A). By leveraging Principal Coordinate Analysis (PCoA) to assess β-diversity, we quantitatively evaluated the similarity and dissimilarity among microbial communities, unveiling pronounced variations in microbial composition among the three experimental groups (Figure 1B). Our subsequent screening of the top 15 genera associated with infection, revealed statistically significant changes in nine of them. Notably, as the infection progressed, conditionally pathogenic bacteria such as Alistipes and Muribaculum exhibited an upsurge in their abundance. However, an increase in Lactobacillus and Odoribacter was exclusive to the gut microbiota infected for 4 weeks (Figure 1C). Remarkably, Ruminococcus displayed a distinctive temporal pattern, peaking in abundance at 4 weeks post infection before tapering off at 16 weeks. This pattern was reinforced by Lefse analysis, highlighting the unique enrichment of Ruminococcus solely at the 4-weeks mark (Figure 1D). Among the Ruminococcus genus, Ruminococcus bromii (R. bromii), a pivotal species, emerged as a subject of particular interest for further investigation. To validate the fluctuations in R. bromii abundance, we employed qRT-PCR to detect its expression in fecal samples of the three groups. The results concurred with our initial findings, confirming that R. bromii abundance indeed increased at 4 weeks and significantly decreased at 16 weeks (Figure 1E). Integrating these insights, we speculated that R. bromii may play a pivotal, potentially protective role during the fibrotic phase of C. sinensis infection.

To explore the effect of R. bromii supplementation on C. sinensis infection in mice, we established a BALB/c mouse model by gavage of 150 C. sinensis metacercariae per mouse, followed by daily gavage of either R. bromii (1 × 10⁹ CFU/mL) or PBS post infection (Figure 2A). Our results confirmed the successful colonization of R. bromii in mice for 8 weeks, as evidenced by qPCR analysis (Figure 2B). Notably, we observed a marked decline in the body weight of infected mice compared to the NC group, whereas R. bromii intervention slightly mitigated this weight loss (Figure 2E). Furthermore, serum hydroxyproline (HYP) levels surged in infected mice but underwent a significantly reduction in the R. bromii treated group, indicative of its potential to curb collagen synthesis associated with fibrosis. Moreover, R. bromii also significantly reduced serum levels of aminotransferase (ALT) and aspartate aminotransferase (AST) (Figure 2F). Histopathological examination of liver sections via HE staining revealed a stark contrast between the orderly hepatocyte arrangement and normal liver structure of the NC group and the disorganized, inflammatory-ridden state of the C. sinensis-infected group. Encouragingly, R. bromii treatment visibly reduced the lesion area (Figure 2C). To assess liver collagen deposition, we employed Masson’s trichrome and Sirius red staining, which unanimously demonstrated that R. bromii significantly reduced the area of collagen fiber deposition (Figure 2D). Additionally, the heightened expression of fibrosis markers such as α-SMA, Collagen I and Collagen III proteins in the C. sinensis infection group was markedly reduced upon R. bromii treatment (Figure 2G). This was also consistent with the expression of these genes at the transcriptional level (Supplementary Figure S1A). These results suggested that R. bromii has an inhibitory effect on C. sinensis induced liver fibrosis.

The analysis of pathological sections revealed profound alterations in the colon crypts of mice in C. sinensis infection group, characterized by their shallowness, the absence of goblet cells, and a conspicuous mucosal wall corrugation accompanied by extensive infiltration of inflammatory cells (Figure 3A). These findings underscored the capacity of C. sinensis infection to induce pathological changes in colon tissue, thereby compromising intestinal integrity. Notably, however, the deleterious effects were markedly mitigated intervention of R. bromii. Furthermore, employing the AB-PAS staining technique, we quantified goblet cells in the colon, revealing that R. bromii exhibited a remarkable ability to replenish the depleted goblet cells count (Figure 3B). Additionally, our analysis delved into the expression patterns of critical intestinal tight junction proteins, Occludin and Claudin-1. We observed that C. sinensis infection significantly downregulated the expression of these proteins, while R. bromii treatment counteracted this trend, upregulating their expression (Figure 3C). These findings underscored the potential therapeutic role of R. bromii in mitigating the adverse consequences of C. sinensis infection on intestinal architecture and function.

We initially used ESPs to activate HSCs in vitro, including both the human LX2 cell line and mouse JS1 cell line. Subsequently, we exposed these cells to varying concentrations of R. bromii conditioned medium (R.b CM). The ensuing analysis unveiled a pronounced, concentration-dependent suppression of HSCs proliferation by R.b CM. Specifically, when compared to ESPs-treated cells, the survival rates of LX2 exposed to 10 and 5% R.b CM diminished to 61 and 84% respectively, while similar reductions were observed in JS1 cells, with survival rates declining to 63 and 85% (Figures 4A,B). Furthermore, R.b CM emerged as a modulator of fibrosis-related protein expression, downregulating the levels of α-SMA, Collagen I and Collagen III (Figures 4C,D). This observation was further validated through immunofluorescence assays, which demonstrated a decrease in α-SMA fluorescence intensity within HSCs treated with 10% R.b CM (Figures 4E,F). Collectively, these results suggested that R.b CM possessed the capacity to suppress the proliferation and activation of HSCs, ultimately leading to a reduction in collagen deposition.

Considering that R. bromii is renowned for its production of short-chain fatty acids (SCFAs) (Baxter et al., 2019; Vital et al., 2018), we performed a targeted metabolic analysis focusing on SCFAs within R.b CM. Our analysis revealed significantly differences in the metabolic components of R.b CM compared to the blank medium control, with acetate standing out as the preeminent metabolite within R.b CM (Figures 5A,B). To validate the functional role of acetate, we treated LX2 and JS1 cells with sodium acetate, a stable form of acetate. Notably, acetate similarly reduced the expression of the fibrotic proteins α-SMA, Collagen I and Collagen III (Figures 5C,D). Overall, these findings underscored the pivotal role of acetate as a functional metabolic byproduct of R. bromii, effectively inhibiting the activation of HSCs induced by ESPs.

We raised the infected mice for 8 weeks by adding sodium acetate to their daily drinking water (Figure 6A). The results concurred with prior observations, acetate treatment reduced the liver to body weight ratio in mice (Figure 6B), and revealing a marked decrease in liver inflammation in the acetate treated group compared to the C. sinensis only group. Masson and Sirius red staining results showed that the collagen deposition around the intrahepatic bile ducts in the acetate group was significantly reduced (Figures 6C,D). Furthermore, the serum acetate levels in the acetate group were notably elevated (Figure 6E), while the serum biochemical markers indicative of fibrosis, namely HYP, ALT, and AST, exhibited a decrease (Figure 6F). Consistent with transcript levels (Supplementary Figure S1B), acetate reduced α-SMA, Collagen I, and Collagen III expression (Figure 6G). These compelling findings underscored the potential of acetate, a functional metabolite of R. bromii, to serve as a promising therapeutic agent against liver fibrosis.

Consistent with the previous results, acetate similarly reduced the intestinal inflammatory cell infiltration caused by C. sinensis infection, restored the intestinal structure, and increased the number of goblet cells (Figures 7A,B). Compared with C. sinensis-infected group, acetate up-regulated the expression of the intestinal tight junction proteins markers Occludin and Claudin-1, and decreased intestinal permeability (Figure 7C). The above results indicated that acetate produced by R. bromii restored intestinal barrier function.

To reveal the underlying mechanism, RNA sequencing was performed on HSC (JS1) with and without acetate treatment. Our analysis identified 308 differentially down-regulated genes and 925 up-regulated genes were found in the acetate-treated cells compared with the ESPs treatment group alone (Figure 8A). Further analysis revealed that the PI3K/AKT signaling pathway was significantly upregulated after acetate treatment (Figures 8B,C). Gene set enrichment analysis (GSEA) confirmed the upregulation of the PI3K/AKT signaling pathway in acetate-treated HSCs (Figure 8D). We then validated this pathway in two cell models and in mouse animal models. Consistent with the sequencing results, acetate significantly upregulated phosphorylated PI3K and phosphorylated AKT proteins in both HSCs (Figures 8E,F), and acetate promoted PI3K and AKT phosphorylation in animal tissues (Figure 8G). These findings suggested that the protective effect of acetate against C. sinensis induced liver fibrosis was mediated at least in through the activation of the PI3K/AKT signaling pathway.