Phocaeicola dorei ameliorates progression of steatotic liver disease by regulating bile acid, lipid, inflammation and proliferation

Abstract

Gut microbiota and their metabolites are known to influence the pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). In this study, we investigated the potential beneficial effects of Phocaeicola dorei in modulating MASLD progression, beginning with clinical observations and followed by mechanistic validation in animal models. Human data (49 healthy controls and 129 MASLD patients) were collected to investigate gut microbial biomarkers. The relative abundance of P. dorei was found to significantly vary with MASLD severity in human. Western diet-induced MASLD mice supplemented with P. dorei (12 weeks, 10⁹ CFU/g twice/week) or 100 μl of P. dorei cell-free supernatant (CFS, 5 times/week) were utilized. STAM^TM mice (10 weeks, 10⁸ CFU/g four times/week) and RAW 264.7 cells were used for the validation. MASLD severity was determined based on liver/body weight, pathology, and biochemistry markers. Cecum feces were collected for 16S rRNA gene sequencing and metabolite profiles. In the animal model, P. dorei oral administration and its CFS alleviated lipid accumulation by increasing β-oxidation gene expression and inhibited inflammatory response from fatty liver to hepatitis progression. In the STAM^TM model, P. dorei decreased nuclear atypia and cell proliferation. Additionally, P. dorei CFS inhibited TNF-α and CXCL10 in activated macrophages, and this result was consistent with the results of animal models. P. dorei and its metabolites ameliorate MASLD progression by modulating bile acid, lipid accumulation, inflammation, and proliferation. P. dorei could be a promising candidate for novel microbiota-based therapeutic strategies against MASLD.

Keywords: Metabolic dysfunction-associated steatotic liver disease; Phocaeicola dorei; gut; metabolites; microbiota.

Graphical abstract

Figure 1.

Taxonomic profile of gut microbiota according to the progression of MASLD. (A) Study design. (B) Box plots represent alpha diversity based on the Shannon index in different clinical conditions. Beta diversity was generated using relative OUT abundance data according to the UniFrac. (C) The pie chart shows relative abundance at the phylum level within each group. (D) Genus-level difference among groups. (E) RAs of P. dorei according to disease progression. All data are expressed as the means with SEM.

Figure 2.

Oral administration of P. dorei and probiotics improves the Western diet induced liver damage. (A) Scheme of the animal experiment. (B) Body weight and liver weight. (C) Representative images of liver tissues and H&E staining (D) NAFLD activity scores on liver tissues (P. dorei, p = 0.0111; L. lactis, p = 0.0023; UDCA, p = 0.0050). Serum endotoxin biochemistry analysis. (E) Serum liver enzyme analysis (F) Gene expression of hepatic proinflammatory cytokines and lipid metabolism in mice liver. Data are expressed as the means with SEM. * p < 0.05, ** p < 0.01.

Figure 3.

Gut microbiota and liver RNA transcription are changed by P. dorei administration. Stool samples and liver tissues were collected from mice to perform 16S rRNA and RNA-Seq analysis. (A) Phylogenetic diversity based on species richness in cecal samples (n = 3–5 per group). (B) Phylum-level microbial composition in cecal samples from NC, WD, P. dorei. L. lactis, and UDCA. Abundance and relative abundance (%) of the two major phyla. (C) Beta diversity was plotted as a PCoA plot showing the similarity of bacterial community structure based on Bray Curtis (D) Heatmap analysis for significantly different species. (E) Comparative analysis of the estimated functional profiles based on KEGG orthology in different experimental group. (F) Expression similarity between samples by principal component analysis. (G) Venn diagram showing specific or common differentially expressed genes between Western/normal and P. dorei+Western/Western (n = 3 per group). (H) Heatmap showing genes commonly up- or down-expressed (n = 3 per group). (I) Bar chart of genes involved in GO biological processes up- and down-regulated by P. dorei administration Gene set enrichment analysis for KEGG items (n = 3 per group). (j) Gene set enrichment analysis for KEGG items. Data are expressed as the means with SEM. *p < 0.05, **p < 0.01.

Figure 4.

Differential metabolic signatures in mouse cecum across diet and administration groups. (A) Chemical classification of identified metabolites in mouse caecum provided by HMDB (http://www.hmdb.ca). The 176 compounds (96.7%) are categorized into nine superclasses. (B) The score scatter plot of 182 cecal metabolites by partial least squares-discriminant analysis (PLS-DA). (C) Dendrogram of hierarchical cluster analysis of cecum metabolomics data from Western diet group (n = 5), administration group (P. dorei administration group, n = 5; UDCA administration group, n = 5), and normal chow diet (n = 5) group. Each sample on the y-axis reflects one cecum sample. The x-axis shows the similarity levels expressed as Pearson distances. Horizontal and vertical lines depict differences and clustering of samples in the distances, respectively. (D) The metabolites show specific abundance patterns in other groups compared to the Western diet group. (E) The bar plots show the fold-change of group-specific metabolites in the log₂ scale compared to the Western diet (p < 0.05). (F) The network is constructed based on chemical structural similarity (Tanimoto score) and KEGG reaction pair (substrate-product relation), which results in distinctive metabolic modules indicated by the box. Red and blue colors present significantly higher or lower abundance in P. dorei, and UDCA groups, respectively, compared to the Western diet (Student’s t-test; p < 0.05: red, blue; p < 0.1: pink, sky-blue). The node sizes are determined by the ratios. Volcano plot for identification of metabolites with significant differences in the P. dorei, and UDCA, respectively, compared to the Western diet group. The X-axis presents the fold change in the log₂ scale, and the Y-axis indicates the statistical significance (value of P) in the log₁₀ scale based on the student’s t-test. Red and blue colors present significantly higher or lower abundance in other groups, respectively, compared to the Western diet (p < 0.05; 0.05< p < 0.1).

Figure 5.

Evaluating group-specific metabolites across diet and administration. (A) An appropriate number of K-means clusters was determined based on simple structure index (SSI) in the median values of metabolic features on the normal chow diet, P. dorei administration, and Western diet group. (B) The abundance patterns of metabolic features were grouped into five clusters. (C) The abundance and correlation patterns of individual metabolites were visualized on a heatmap (normal chow diet: 0, P. dorei administration: 1, and the Western diet: 2).

Figure 6.

P. dorei inhibits nuclear atypia in the STAM mouse model. (A) Scheme of the animal experiment. (B) Representative H&E stained liver sections. Atypia grade and staging grade. (C) IHC with Ki-67 (D) mRNA expression of Ki-67. (E) Serum endotoxin level. (F) Proliferation markers (cdc20 and Ccnb2) analysis (G) Expression of chemokines and cytokines (h) in-vitro analysis for antififlammatory effect of P. dorei. All data are expressed as the means with SEM. *p < 0.05, **p < 0.01.

Figure 7.

P. dorei CFS reduces LPS-induced inflammation in Raw 264.7 cells. (A) Metabolomic comparison between RCM control and cell-free supernatant of P. dorei. (B-D) expression of proinflammatory cytokines, chemokines genes, and p-p38. (E) The significant features were identified in the comparison of the Western diet and P. dorei diet within the cecum and media. (F) Relative abundance of metabolites (e.g., deoxycholic acid, hypoxanthine) in cecum samples. Statistical significance in linear regression is determined based on p < 0.05.

Figure 8.

P. dorei CFS attenuates hepatic injury in the Western diet-induced MASH mouse model. (A) Scheme of the animal experiment. (B) Representative images on H&E-stained liver tissues, and comparison of NAS. (C) Change of weight. (D) Liver enzyme analysis. (E) Expression of proinflammatory cytokines and chemokines genes. (F) Expression of lipid metabolism markers. All data are expressed as the means with SEM. *p < 0.05, **p < 0.01.