Dynamic alterations in the gut microbiota and metabolome during the development of methionine-choline-deficient diet-induced nonalcoholic steatohepatitis

Abstract

Aim: To investigate changes in gut microbiota and metabolism during nonalcoholic steatohepatitis (NASH) development in mice fed a methionine-choline-deficient (MCD) diet.

Methods: Twenty-four male C57BL/6J mice were equally divided into four groups and fed a methionine-choline-sufficient diet for 2 wk (Control 2w group, n = 6) or 4 wk (Control 4w group, n = 6) or the MCD diet for 2 wk (MCD 2w group, n = 6) or 4 wk (MCD 4w group, n = 6). Liver injury, fibrosis, and intestinal barrier function were evaluated after 2 and 4 wk of feeding. The fecal microbiome and metabolome were studied using 16s rRNA deep sequencing and gas chromatography-mass spectrometry.

Results: The mice fed the MCD diet presented with simple hepatic steatosis and slight intestinal barrier deterioration after 2 wk. After 4 wk of feeding with the MCD diet, however, the mice developed prominent NASH with liver fibrosis, and the intestinal barrier was more impaired. Compared with the control diet, the MCD diet induced gradual gut microbiota dysbiosis, as evidenced by a marked decrease in the abundance of Alistipes and the (Eubacterium) coprostanoligenes group (P < 0.001 and P < 0.05, respectively) and a significant increase in Ruminococcaceae UCG 014 abundance (P < 0.05) after 2 wk. At 4 wk, the MCD diet significantly reduced the promising probiotic Bifidobacterium levels and markedly promoted Bacteroides abundance (P < 0.05, and P < 0.01, respectively). The fecal metabolomic profile was also substantially altered by the MCD diet: At 2 wk, arachidic acid, hexadecane, palmitic acid, and tetracosane were selected as potential biomarkers that were significantly different in the corresponding control group, and at 4 wk, cholic acid, cholesterol, arachidic acid, tetracosane, and stearic acid were selected.

Conclusion: The MCD diet induced persistent alterations in the gut microbiota and metabolome.

Keywords: Gut microbiota; Metabolome; Methionine-choline deficient diet; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis.

Figure 1

Methionine-choline-deficient diet induced hepatic injury, fat accumulation, and macrophage infiltration. A-C: Representative liver histology assessed by HE staining (A), Oil red O staining (B), and macrophages (F4/80) staining (C). Scale bar: 250 μm. D: Representative staining intensities of Oil red O as designated by mean optical density. E: Percentage of F4/80 positive cells. The data are given as the means ± SEM. n = 6 per group. ^cP < 0.001 vs Control 2w group, ^eP < 0.01 and ^fP < 0.001 vs MCD 2w group, ^jP < 0.001 vs Control 4w group by post hoc ANOVA one-way statistical analysis. MCD: Methionine-choline-deficient.

Figure 2

Methionine-choline-deficient diet induced liver fibrosis after 4 wk of feeding. A-B: Representative liver histology assessed by Masson’s trichrome staining (A) and α-SMA staining (B). Scale bar: 250 μm. C: Percent of Masson’s trichrome-stained area as indicated by the fibrosis index (%). D: Representative staining intensities of α-SMA as designated by the mean optical density. The data are given as the means ± SEM. n = 6 per group. ^aP < 0.05 and ^bP < 0.01 vs Control 2w group, ^dP < 0.05 and ^eP < 0.01 vs MCD 2w group, ^hP < 0.05 and ⁱP < 0.01 vs Control 4w group by post hoc ANOVA one-way statistical analysis. MCD: Methionine-choline-deficient.

Figure 3

Methionine-choline-deficient diet resulted in gradual intestinal barrier impairment. Representative colon histology was assessed by ZO-1 immunofluorescence staining. Scale bar: 100 μm. The data are given as the means ± SEM. MCD: Methionine-choline-deficient.

Figure 4

Methionine-choline-deficient diet induced gut microbiota dysbiosis. A: PCoA plot of the microbiota based on unweighted UniFrac metric. Each symbol represents one sample (n = 6 per group). B-D: Top most abundant taxa at the phylum (B), family (C), and genus (D) levels. E: LEfSe cladogram represented taxon enriched in the Control 4w group (green) and in the MCD 4w group (red). Rings from the inside out represented taxonomic levels from phylum to genus. Sizes of the circles indicate the relative abundance levels of the taxa. F: Discriminative biomarkers with an LDA score > 4.8. MCD: Methionine-choline-deficient.

Figure 5

Methionine-choline-deficient diet altered the fecal metabolomic profile during nonalcoholic steatohepatitis progression. A: 4-state model of a PCA plot comparing between the Control 2w (cycle), MCD 2w (triangle), Control 4w (square), and MCD 4w groups (diamond). B: OPLS-DA score scatter plot comparing the Control 2w (cycle), MCD 2w (triangle), Control 4w (square), and MCD 4w groups (diamond). Each symbol represents one sample (n = 24 per group, with a quadruplicate technical replicates of 6 samples per group). C-D: S-plot constructed from the OPLS-DA model at 2 wk (C) and 4 wk (D). The selected metabolites (red triangles) are located in the upper far right and lower far left with considerable potential as biomarkers and marked discrimination between the groups. E-F: Euclidean distance hierarchical clustering analysis visualizing the different intensity levels of characteristic metabolites at 2 wk (E) and 4 wk (F). PCA: Principal component analysis; MCD: Methionine-choline-deficient; OPLS-DA: Orthogonal partial least-squares-discriminant analysis.