Saccharomyces Boulardii Ameliorates Non-alcoholic Steatohepatitis in Mice Induced by a Methionine-Choline-Deficient Diet Through Gut-Liver Axis

Abstract

Non-alcoholic steatohepatitis (NASH) is affecting people worldwide. Changes in the intestinal microbiome are crucial to NASH. A previous study showed that eradicating intestinal fungi ameliorates NASH; however, the role of intestinal fungi in the development of NASH remains unclear. Saccharomyces boulardii (SB), a dietary supplement yeast, has been reported to restore the integrity of the intestine. Here, we tested the effect of SB in the treatment of NASH. For this study, we fed eight-week-old C57/BL6 male mice either a methionine-choline deficient (MCD) diet or a normal chow diet (NCD) for eight weeks. Half of the MCD diet-fed mice were gavaged with SB (5 mg/day) once daily. The remainder of the NCD-fed mice were gavaged with normal saline as a control. The MCD diet-fed mice on SB supplement showed better liver function, less hepatic steatosis, and decreased inflammation. Both hepatic inflammatory gene expression and fibrogenic gene expression were suppressed in mice with SB gavage. Intestinal damage caused by the MCD diet was tampered with, intestine inflammation decreased, and gut permeability improved in mice that had been given the SB supplement. Deep sequencing of the fecal microbiome showed a potentially increased beneficial gut microbiota and increased microbiota diversity in the SB-supplemented mice. The SB supplement maintains gut integrity, increases microbial diversity, and increases the number of potentially beneficial gut microbiota. Thus, the SB supplement attenuates gut leakage and exerts a protective effect against NASH. Our results provide new insight into the prevention of NASH.

Keywords: Saccharomyces boulardii; gut-liver axis; intestinal commensal fungi; methionine-choline deficiency diet; non-alcoholic steatohepatitis.

FIGURE 1

SB reduced MCD diet-caused steatohepatitis in mice. C57BL/6 male mice were fed an oral control diet (n = 10) or the MCD diet (n = 18) and given vehicle or SB. (A) Representative liver sections after H & E staining, scale bars: 100μm. (B) Plasma levels of alanine aminotransferase (ALT). (C) Hepatic triglyceride content. (D) NAFLD activity score (NAS). (E) Parameters of NAS: Steatosis, inflammation and ballooning. (F) Representative sections of Trichrome Masson staining, scale bar: 50μm, and fibrosis score based on the NAS scoring system. *p < 0.05.

FIGURE 2

Hepatic inflammatory responses caused by the MCD diet were suppressed by SB gavage. (A) Hepatic expressions of proinflammatory cytokines. (B) Representative liver sections of immunohistochemical staining of F4/80 and hepatic expression of F4/80. Scale bars: 50μm. *p < 0.05.

FIGURE 3

SB ameliorated MCD diet-caused hepatic fibrosis in mice. (A) Representative sections of Sirus Red staining (Scale bar: 100μm) and hepatic collagen 1a1 expression. (B) Representative sections of immunohistochemical staining ofα-SMA (Scale bar: 50μm) and hepatic expression ofα-SMA. (C) Hepatic fibrotic gene expressions, Tissue Inhibitor of Metalloproteinase 1 (Timp1) and Matrix Metallopeptidase 2 (Mmp2) *p < 0.05.

FIGURE 4

The MCD diet led to the shortening of the small intestine and intestinal villi, which were improved by the SB supplement. (A) Representative images of the small intestine of mice on the control diet (n = 10) or the MCD diet (n = 18), and also given vehicle or SB. The length of the small intestine improved after the SB supplement. (B) Representative sections of the distal small intestine after H & E staining. Both the intestine length of villi and the width of crypt improved after the SB supplement. Scale bars: 50μm. *p < 0.05.

FIGURE 5

SB ameliorated the MCD diet-caused intestinal inflammation. Intestinal expression of proinflammatory cytokines Il1b, Tnf-α, and CCL2 in the proximal, middle, distal small intestine (PSI, MSI, and DSI), and colon. *p < 0.05.

FIGURE 6

SB decreased the MCD diet-caused leaky gut with less Lipopolysaccharide translocation. (A) Fecal albumin measured by ELISA. (B) Plasma Lipopolysaccharide (LPS) level. (C) Intestinal expression of intestinal barrier gene, ZO-1, in the proximal, middle and distal small intestine. (D) Representative liver sections of ZO-1 immunohistochemical staining of the distal small intestine. Scale bars: 50μm. *p < 0.05.

FIGURE 7

SB affected the microbial configuration and restored intestinal microbiota diversity in the MCD diet-fed mice. (A) Topmost abundant taxa at the family level and (B) genus level. (C) The Shannon diversity index represents alpha diversity. (D) PCA represents beta diversity at the genus level. Each symbol represents one sample (n = 5–10 per group). (E) Discriminative biomarkers with an LDA score > 4.0.

FIGURE 8

The MCD diet altered the fungal composition in the intestine of mice. (A) Topmost abundant taxa at the family and (B) genus level, MCD diet feeding showed increased Pichia and Trichosporon. Alpha diversity is represented by Shannon (C) and Simpson’s (D) diversity index. (E) Beta diversity presented by PCA. Each symbol represents one sample (n = 4–6 per group).