Differential expression of liver fibrosis-associated genes in Mice with CCl₄-induced liver fibrosis compared to healthy controls

To confirm the biological relevance of the selected genes, we performed a preliminary analysis using a publicly available dataset from a mouse model of CCl₄-induced liver fibrosis. A hierarchical clustering heat map (Fig. 1A) revealed distinct expression patterns between fibrotic and control liver samples. Notably, genes associated with fibrosis and inflammation including Stat3, Tlr4, Bmp6, Acta2, Il6, Dmt1, and Slc40a1 were upregulated in the liver fibrosis group, while Zo1 and Hamp were markedly downregulated compared to controls.

Fig. 1

Heatmap of selected liver fibrosis- and iron metabolism-related gene expression profiles (Stat3, Tlr4, Bmp6, α-SMA, Il6, Dmt1, Slc40a1, Zo1, and Hamp) comparing control and CCl₄-induced liver fibrosis groups. Red and blue colors indicate high and low expression levels, respectively (A). Principal component analysis (PCA) plot showing clear separation between control and liver fibrosis groups based on differential gene expression patterns (B)

Furthermore, PCA clearly separated the control and fibrotic groups based on their overall gene expression profiles (Fig. 1B), with the first PC1 accounting for 79.7% of the variance. These findings highlight the strong discriminatory power of the selected genes and validate their importance as targets for subsequent experimental analysis in our intervention study involving A. muciniphila, F. prausnitzii, and their CFS in mice with CCl₄-induced liver fibrosis.

Live and CFS forms of A. muciniphila and F. prausnitzii downregulated the expression of SLC40A1 and COL1A1 in TGFβ-stimulated LX-2 cells

To evaluation of the potential effect of A. muciniphila, F. prausnitzii and their CFS on the SLC40A1and COL1A1 genes, LX2 cells were stimulated with TGFβ, a potent fibrogenic factor known to activate quiescent HSCs. TGFβ-stimulated LX-2 cell were then treated with live forms of the studied bacteria at MOI of 10, 50, and 100, as well as with CFS at a concentration of 10% (v/v) overnight. As depicted in Fig. 2 (A-D) TGFβ stimulation significantly upregulated SLC40A1 and COL1A1 relative mRNA expression compared to unstimulated control cells. Treatment with A. muciniphila, F. prausnitzii, and their respective CFS significantly downregulated the TGFβ-induced mRNA expression of SLC40A1 and COL1A1 genes compared to the PBS. Notably, F. prausnitzii at MOI 50 and A. muciniphila at MOI 10 did not significantly alter SLC40A1 expression; however, all other bacterial treatments (live and CFS forms) led to a significant downregulation of SLC40A compared to the PBS (Fig. 2A and B). Similarly, with the exception of A. muciniphila at MOI 50 and F. prausnitzii at MOI 50, significant downregulation of COL1A1 gene was observed in all other treatment groups compared to the PBS control (Fig. 2C and D).

Fig. 2

Effect of live and CFS forms of Akkermansia muciniphila and Faecalibacterium prausnitzii on the expression of SLC40A1 and COL1A1 genes in TGF-β-activated LX-2 cells. (A, C) LX-2 cells were stimulated with TGF-β1 (1 ng/mL) to induce fibrogenic activation and treated with live bacteria (A. muciniphila or F. prausnitzii) at different multiplicities of infection (MOI 10, 50, and 100). (B, D) Parallel treatments were performed with 10% (v/v) cell-free supernatant (CFS) derived from the respective bacteria. Gene expression levels of SLC40A1 (A, B) and COL1A1 (C, D) were assessed by RT-qPCR and normalized to GAPDH as the internal control. Results are shown as fold change relative to control (unstimulated LX-2 cells). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Liver injury amelioration by live and CFS forms of A. muciniphila and F. prausnitzii in mice with CCl₄-induced liver fibrosis

To evaluate the protection effects of A. muciniphila, F. prausnitzii, and their CFS intervention on liver fibrosis induced by CCl₄, we performed histopathological analysis of liver tissues and assessed the expression of α-SMA, a key fibrogenic marker, using RT-qPCR and IHC techniques. Histopathological evaluation of liver sections stained with hematoxylin and eosin (H&E) and Masson’s trichrome staining showed that the liver tissue architecture was completely normal in the CNT and Olive mice groups (Fig. 3A and B). Necrosis of hepatocytes, high infiltration of inflammatory cells, extensive disruption of the hepatocyte plates, and an increase in interstitial connective tissue with collagen fibers and also elevated α-SMA were observed in the CCl₄ and PBS (Fig. 3A-E). Am, Fp, Am-CFS, and Fp-CFS groups exhibit less tissue damage and visibly reduced collagen staining compared to PBS group, with Fp and Fp-CFS showing more preserved liver architecture, suggesting better hepatoprotective effects (Fig. 3A and B). The relative mRNA levels of α-SMA (Acta2), a gene upregulated during liver fibrosis, were significantly lower in mice with liver fibrosis receiving live A. muciniphila, A. muciniphila CFS, F. prausnitzii, and F. prausnitzii CFS (Am, Am-CFS, Fp, and Fp-CFS groups) compared to the PBS group (Fig. 3C and D). As demonstrated in Fig. 3E, we also reported the reduced α-SMA in Fp, Fp-CFS and AM groups than in the PBS group, respectively. In mice with liver fibrosis treated with F. prausnitzii (Fp group) and F. prausnitzii CFS (Fp-CFS), low infiltration of inflammatory cells and α-SMA was identified in the structure of the liver tissue; the hepatic architecture was completely intact in most fields of the liver, and no histopathological changes were observed (Fig. 3A and E).

Fig. 3

Protective effects of live and CFS forms of Akkermansia muciniphila and Faecalibacterium prausnitzii on liver fibrosis in CCl₄-induced mice. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. (A) Hematoxylin and eosin (H&E) staining and (B) Masson’s trichrome staining of liver sections showing histopathological alterations and collagen deposition, respectively. (C, D) hepatic α-SMA (Acta2) mRNA expression in liver tissues were assessed by RT-qPCR and normalized to Rpl19 as the internal control. Results are shown as fold change relative to CNT group (E) Immunohistochemical (IHC) staining for α-SMA expression in liver tissues. Images were captured under light microscopy (scale bars = 50 μm). Yellow arrowheads indicate hepatocyte necrosis, yellow arrows indicate inflammatory infiltration, white arrowheads indicate collagen fibers, and black arrowheads indicate α-SMA-positive areas. Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on hepcidin and ferroportin expression in liver tissue of mice with CCl₄-induced liver fibrosis

Given the important role of the hepcidin-ferroportin axis in liver fibrosis, we evaluated the effects of A. muciniphila, F. prausnitzii, and their CFS on hepatic expression of hepcidin (Hamp) and ferroportin (Slc40a1) in mice liver tissue using RT-qPCR and IHC. RT-qPCR analysis revealed that hepatic mRNA expression of Hamp and Slc40a1 were significantly reduced in the CCl₄ group and the PBS group compared to the healthy control (CNT) group. We did not observe a significant difference in the relative mRNA expression of Hamp and Slc40a1 in the Olive group compared to the CNT group (Fig. 4A and D). Interestingly, with the exception of the non-significant change in hepatic Slc40a1 expression observed in mice with liver fibrosis treated with A. muciniphila CFS (Am-CFS group), intervention with both live and CFS forms of A. muciniphila and F. prausnitzii (Am, Am-CFS, Fp, and Fp-CFS groups) significantly increased hepatic Hamp and Slc40a1 expression compared to the PBS group (Fig. 4B and E). IHC staining supported the gene expression data CCl₄ and PBS groups show weaker staining for both Hamp and Fpn compared with CNT. Mild expression of Hamp in the histological structure of the liver was observed in the Am, and Am-CFS groups (Fig. 4C). The expression of Hamp in the hepatic architecture of the liver in CNT, Fp, and Fp-CFS groups was completely intact in most fields (Fig. 4C). Although a reduction in ferroportin expression was observed in the PBS, CCl₄, and Am-CFS groups, Fpn expression in the liver tissue of the CNT, Am, Fp, and Fp-CFS groups appeared normal in most histological fields (Fig. 4F).

Fig. 4

Effect of live and CFS forms of Akkermansia muciniphila and Faecalibacterium prausnitzii on hepatic Hamp and Fpn expression in CCl₄-induced liver fibrosis mice. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. (A, B, D and E) Relative mRNA expression of Hamp and ferroportin (Slc40a1) genes in liver tissue assessed by RT-qPCR and normalized to Rpl19 as the internal control. Results are shown as fold change relative to CNT group. (B, E) Comparison of Hamp and Fpn expression across intervention groups. (C, F) Immunohistochemically (IHC) staining for Hamp and Fpn in liver tissue (scale bar, 50 μm). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on serum iron and liver enzymes in mice with CCl₄-induced liver fibrosis

The serum levels of alanine aminotransferase (ALT), aspartate transaminase [37], lactate dehydrogenase (LDH), and iron were measured to assess the ameliorative effects of live A. muciniphila, F. prausnitzii, and their CFS on liver fibrosis. The serum levels of ALT, AST, LDH, and iron were significantly increased in the PBS group compared to the CNT group (Fig. 5A-D). Interestingly, the levels of these markers were markedly reduced in mice with liver fibrosis receiving F. prausnitzii and F. prausnitzii CFS (Fp and Fp-CFS groups) compared to the PBS group. Additionally, mice intervened with A. muciniphila CFS (Am-CFS group) showed significantly lower serum levels of AST, LDH, and iron compared to the PBS group, although ALT levels did not differ significantly. In the mice with liver fibrosis treated with live A. muciniphila (Am group), a noticeable reduction in ALT and LDH levels was observed, while AST and iron levels remained unchanged (Fig. 5A-D).

Fig. 5

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii and their CFS on serum biomarkers in mice with CCl₄-induced liver fibrosis. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. Serum levels of (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase [37], (C) lactate dehydrogenase (LDH), and (D) iron were measured. Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on the hepatic mRNA levels of Tlr4, Il6, Stat3, Dmt1, Bmp6, and Zo1 in liver tissue of mice with CCl₄-induced liver fibrosis

To investigate whether intervention with A. muciniphila, F. prausnitzii, and their CFS modulates the expression of genes involved in liver fibrosis and iron metabolism, we evaluated the hepatic mRNA expression of Tlr4, Il6, Stat3, Dmt1, Bmp6, and Zo1 in liver tissue of mice with liver fibrosis. The expression of Il6 and Tlr4 was significantly decreased in the PBS group compared to the CNT group, while Stat3 expression did not show a significant change (Figs. 6A-C).

Fig. 6

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii, and their CFS on hepatic mRNA expression of Stat3, Tlr4, Bmp6, Il6, Dmt1, and Zo1 genes in mice with CCl₄-induced liver fibrosis. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. Total RNA was extracted from liver tissues, and mRNA expression level of (A) Stat3, (B) Tlr4, (C) Il6, (D) Bmp6, (E) Dmt1 and (F) Zo1 were assessed by RT-qPCR and normalized to Rpl19 as the internal control. Results are shown as fold change relative to CNT group. Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Significant downregulation of Il6 and Tlr4 was observed in mice with liver fibrosis receiving A. muciniphila, A. muciniphila CFS, F. prausnitzii, and F. prausnitzii CFS (Am, Am-CFS, Fp, and Fp-CFS groups) compared to the PBS group (Fig. 6B and C). The hepatic mRNA expression of Stat3 was significantly increased in the Am group (mice receiving live A. muciniphila) and significantly decreased in the Am-CFS group compared to the PBS group. While Stat3 expression did not change significantly in the Fp group (mice receiving live F. prausnitzii), a significant upregulation was observed in the Fp-CFS group (mice receiving F. prausnitzii CFS) compared to the PBS group (Fig. 6A).

In addition, there was a significant reduction in Bmp6 and Dmt1 mRNA expression in the PBS group compared to the CNT group (Fig. 6D and E). This downregulation was also observed in the Am and Am-CFS groups compared to the PBS group. Although a significant reduction in Bmp6 expression was found in the Fp-CFS group, and a significant induction of Dmt1 was observed in the Fp group, no significant changes in hepatic Bmp6 expression were found in the Fp group, nor in Dmt1 expression in the Fp-CFS group (Fig. 6D and E).

Furthermore, a significant reduction in Zo1 transcript levels was observed in the liver tissue of PBS group compared to the CNT group. This reduction was significantly reversed in the Fp group (mice receiving F. prausnitzii) compared to the PBS group. However, no significant changes in hepatic Zo1 mRNA expression were detected following intervention with A. muciniphila, its CFS, or F. prausnitzii CFS (Am, Am-CFS, and Fp-CFS groups) compared to the PBS group (Fig. 6F).

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on mRNA levels of Zo1, Dmt1, and Slc40a1 in colonic tissue of mice with CCl₄-induced liver fibrosis

Given the critical role of genes involved in gut barrier integrity and intestinal iron absorption and transport, we investigated the effects of A. muciniphila, F. prausnitzii, and their CFS on the mRNA expression levels of Zo1, Dmt1, and Slc40a1 in the colonic tissue of mice with CCl₄-induced liver fibrosis.

A significant downregulation of Dmt1 gene expression was observed in the colonic tissue of the PBS group compared to the CNT group. While Dmt1 mRNA expression was significantly increased in mice with liver fibrosis receiving F. prausnitzii and its CFS (Fp and Fp-CFS groups) compared to the PBS group, no significant changes were detected in the Am and Am-CFS groups (mice treated with A. muciniphila and its CFS) (Fig. 7A). Although Slc40a1 expression did not differ significantly between the PBS and CNT groups, a significant downregulation of Slc40a1 was observed in the colonic tissue of the Am, Am-CFS, Fp, and Fp-CFS groups compared to the PBS group (Fig. 7B). Furthermore, compared to the PBS group, Zo1 mRNA expression was significantly upregulated in the colonic tissue of mice with liver fibrosis receiving F. prausnitzii and its CFS (Fp and Fp-CFS groups), while no significant effects were observed in the Am and Am-CFS groups (Fig. 7C).

Fig. 7

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii, and their CFS on the mRNA levels of Dmt1, Slc40a1, and Zo1 genes in the colon tissue of mice with CCl₄-induced liver fibrosis. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. Total RNA was extracted from colon tissues, and mRNA expression level of (A) Dmt1, (B) Slc40a1 and (C) Zo1 were assessed by RT-qPCR and normalized to Rpl19 as the internal control. Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on mRNA levels of Tlr4, Hamp, Dmt1, and Slc40a1 in brain tissue of mice with CCl₄-induced liver fibrosis

Given the importance of the gut–liver–brain axis in the context of liver fibrosis, we investigated the effects of live and CFS derived forms of A. muciniphila and F. prausnitzii on the expression of Tlr4, Hamp, Dmt1, and Slc40a1 transcripts in the brain tissue of mice with CCl₄-induced liver fibrosis.

Although Hamp expression was significantly downregulated in the PBS group compared to the CNT group, its mRNA level was significantly increased in the brain tissue of mice with liver fibrosis treated with F. prausnitzii CFS (Fp-CFS group) compared to the PBS group. In contrast, Hamp transcript levels remained unchanged in the brain tissue of mice receiving live A. muciniphila, A. muciniphila CFS, or live F. prausnitzii compared to the PBS group (Fig. 8A).

Fig. 8

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii, and their CFS on the mRNA levels of Hamp, Tlr4, Dmt1 and, Slc40a1 genes in the brain tissue of mice with liver fibrosis by RT-qPCR. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. Total RNA was extracted from brain tissues, and mRNA expression level of (A) Hamp, (B) Slc40a1, (C) Dmt1 and (D) Tlr4 were assessed by RT-qPCR and normalized to Rpl19 as the internal control. Results are shown as fold change relative to CNT group. Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS). Data represent the mean ± SEM. The significant results are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

In the brain tissue of mice receiving A. muciniphila CFS (Am-CFS), a significant reduction in Slc40a1 mRNA levels was observed compared to the PBS group, which showed significantly elevated Slc40a1 expression relative to the healthy control (CNT) group. No significant changes in Slc40a1 mRNA levels were observed in the other treatment groups (Fig. 8B). Although the PBS group showed significantly elevated Dmt1 mRNA levels in brain tissue compared to the CNT group, a significant downregulation of Dmt1 relative expression was observed in mice orally treated with F. prausnitzii CFS and A. muciniphila CFS (Fp-CFS and Am-CFS groups). In contrast, no significant changes in Dmt1 expression were observed in the brain tissue of mice with liver fibrosis receiving live A. muciniphila or F. prausnitzii compared to the PBS group (Fig. 8C). The transcription of the Tlr4 gene was significantly increased in the brain tissue of PBS-treated mice compared to the CNT group. A significant reduction in Tlr4 mRNA levels was observed in mice orally treated with live A. muciniphila and F. prausnitzii (Am and Fp groups) relative to the PBS group. In contrast, no significant changes in Tlr4 expression were detected in mice treated with the CFS forms of A. muciniphila and F. prausnitzii (Am-CFS and Fp-CFS groups) compared to the PBS group (Fig. 8D).

The impact of live and CFS forms of A. muciniphila and F. prausnitzii on the relative abundance of gut microbiota members in fecal samples of mice with CCl₄-induced liver fibrosis

To investigate whether A. muciniphila, F. prausnitzii, and their derived CFS can modulate the dysbiotic gut microbiota composition associated with CCl₄-induced liver fibrosis, we assessed the relative abundance of the target bacteria and the ratio of the two dominant bacterial phyla, Firmicutes and Bacteroidetes, in fecal samples. Quantification was performed using 16 S rRNA gene-targeted phylum- and species-specific primers via qPCR.

The relative abundance of A. muciniphila was significantly reduced in the PBS and CCl₄ groups compared to the CNT group. However, its abundance was significantly increased following administration of live A. muciniphila, A. muciniphila CFS, and F. prausnitzii CFS in the Am, Am-CFS, and Fp-CFS groups, respectively. A significant decrease in the relative abundance of F. prausnitzii was observed in the PBS and CCl₄ groups compared to the CNT group. Mice receiving A. muciniphila CFS (Am-CFS group) showed a significant increase in F. prausnitzii abundance compared to the PBS group. There was a significant elevation of E. coli in PBS mice group in comparison by CNT. A reduced abundance of E. coli was observed in mice with liver fibrosis receiving A. muciniphila CFS (Am-CFS group), F. prausnitzii CFS (Fp-CFS group) and live F. prausnitzii (Fp group) compared to the PBS group (Fig. 9). An elevated Firmicutes/Bacteroidetes ratio was observed in mice receiving F. prausnitzii (Fp group) and live and CFS forms of A. muciniphila (Am and Am-CFS groups), respectively. The highest and most significant increase in this ratio was found in the group receiving F. prausnitzii (Fp group) Fig. 9).

Fig. 9

Relative abundance of gut microbiota members assessed by qPCR in mice with CCl₄-induced liver fibrosis. Mice were pre-treated with A. muciniphila, F. prausnitzii, or their CFS starting 10 days before CCl₄ IP injection and continued until the end of experiment. Total DNA was extracted from fecal samples, and the relative abundance of A. muciniphila, F. prausnitzii, E. coli, Firmicutes, and Bacteroidetes was determined by qPCR using 16 S rRNA gene-specific primers. Data were normalized to the control group (CNT) and are presented as Z-score normalized values. A yellow-to-blue color gradient represents abundance levels (yellow indicates lower abundance; blue indicates higher abundance). Healthy mice (CNT), phosphate-buffered saline (PBS), A. muciniphila (Am), A. muciniphila CFS (Am-CFS), F. prausnitzii (Fp), and F. prausnitzii CFS (Fp-CFS)

Significant alterations in gut microbiota composition in liver fibrosis patients compared to healthy subjects identified via bioinformatics analysis

Using publicly available NGS data from the GMrepo database, we examined gut microbiota composition in patients with liver fibrosis compared to healthy controls. The analysis revealed that patients with liver fibrosis exhibited a higher relative abundance of several bacterial species, including Bacteroides thetaiotaomicron, Streptococcus salivarius, Haemophilus parainfluenzae, Veillonella parvula, Lactobacillus salivarius, Klebsiella pneumoniae, and Clostridium perfringens. In contrast, a significant reduction was observed in beneficial commensals such as Bacteroides eggerthii, Bacteroides massiliensis, Alistipes putredinis, Bacteroides plebeius, Bacteroides uniformis, Roseburia spp., and Faecalibacterium prausnitzii in liver fibrosis patients compared to healthy subjects.