The combination of berberine and evodiamine ameliorates high-fat diet-induced non-alcoholic fatty liver disease associated with modulation of gut microbiota in rats

Abstract

Nonalcoholic fatty liver disease (NAFLD) is considered to be a manifestation of hepatic metabolic syndrome. Some studies on the pathogenesis of NAFLD by targeting gut microbiota have attracted wide attention. Previous studies have demonstrated the positive effects of berberine and evodiamine on metabolic diseases and gut microbiota dysbiosis. However, it is not known whether the combination of berberine and evodiamine (BE) can prevent the development of high-fat diet (HFD)-induced NAFLD. Therefore, we aimed to explore the protective effects of BE on the development of HFD-induced NAFLD from the perspective of the gut microbiota. Gut microbiota profiles were established by high throughput sequencing of the bacterial 16S ribosomal RNA gene. The effects of BE on liver and intestinal tissue, intestinal barrier integrity, and hepatic inflammation were also investigated. The results showed that the abundance and diversity of gut microbiota were enriched by BE treatment, with an increase in beneficial bacteria, such as Lactobacillus, Ruminococcus, and Prevotella, and a decrease in pathogenic bacteria such as Fusobacterium and Lachnospira. In addition, BE effectively improved liver fat accumulation and tissue damage, inhibited the apoptosis of intestinal epithelial cells, increased the contents of intestinal tight junction proteins, and decreased the expression of pro-inflammatory factors. Consequently, BE treatment could be an effective and alternative strategy for alleviating NAFLD by modulating gut microbiota and safeguarding the intestinal barrier.

Figure 1. Experimental design. SCS: sodium carboxymethyl cellulose; BE: berberine and evodiamine; FF: fenofibrate.

Figure 2. Effect of berberine and evodiamine (BE) at high, medium, and low doses (BEH, BEM, BEL) or fenofibrate (FF) on lipid accumulation and pathological lesions of the liver of animals fed a high-fat diet (HFD). A, Changes in body weights, (B) epididymal fat weights, and (C) liver/body weight ratio. Serum levels of (D) triglyceride (TC), (E) total cholesterol (TG), (F) alanine aminotransferase (ALT), (G) aspartate aminotransferase (AST), (H) alkaline phosphatase (ALP), and (I) γ-glutamyl transferase (γ-GT). J, Representative macroscopic images of livers; K, Oil-Red-O staining; and L, hematoxylin and eosin (HE) staining images of the liver (magnification: 200×, scale bar: 100 µm). The black arrows represent circular vacuoles; the blue arrows represent inflammatory cell infiltration. Data are reported as means±SD. ***P<0.001 (one-way ANOVA).

Figure 3. Effect of berberine and evodiamine (BE) at high, medium, and low doses (BEH, BEM, BEL) or fenofibrate (FF) on the diversity of gut microbiota of animals fed a high-fat diet (HFD). Alpha diversity analysis of Chao1 (A) and Shannon indexes (B) on operational taxonomic units (OTU). C, Beta diversity analysis, principal component analysis (PCA) plot and (D) principal co-ordinates analysis (PCoA) based on Bray-Curtis distances on OTU level. Data are reported as means±SD. **P<0.01, ***P<0.001 (one-way ANOVA).

Figure 4. Effect of berberine and evodiamine (BE) at high, medium, and low doses (BEH, BEM, BEL) or fenofibrate (FF) on the structure of gut microbiota animals fed a high-fat diet (HFD). Bacterial taxa of relative abundance at (A) phylum level and (B) genus level. C, Heatmap of abundance at genus level. D, Linear discriminant analysis (LDA) effect size (LEfSe) score.

Figure 5. Effect of berberine and evodiamine (BE) at high, medium, and low doses (BEH, BEM, BEL) or fenofibrate (FF) on intestinal integrity of animals fed a high-fat diet (HFD). A, Hematoxylin and eosin (HE) staining of ileum tissues (magnification: 200×, scale bar: 100 µm). The blue arrows indicate lymphocyte infiltration, and the yellow arrows indicate submucosa edema. B, mRNA expression of intestine fatty acid binding protein (I-FABP) by qRT-PCR. C, Immunofluorescence images of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) (green fluorescence) as an indicator of apoptosis of ileum epithelial cells (magnification: 10×, scale bar: 100 µm). D, mRNA expression of occludin and (E) zonula occludens-1 (ZO-1) by qRT-PCR. Data are reported as means±SD. **P<0.01; ***P<0.001 (one-way ANOVA).

Figure 6. Berberine and evodiamine (BE) at high, medium, and low doses (BEH, BEM, BEL) or fenofibrate (FF) treatment alters inflammatory cytokine expressions and inhibits oxidative stress of animals fed a high-fat diet (HFD). A, Lipopolysaccharide (LPS), (B) tumor necrosis factor (TNF-α), (C) interleukin (IL) 1β, (D) IL-6, (E) IL-10, and (F) IL-4 cytokines levels, (G) malondialdehyde (MDA) content, and (H) superoxide dismutase (SOD) activity were evaluated by enzyme-linked immunosorbent assay (ELISA). Data are reported as means±SD. *P<0.05, **P<0.01; ***P<0.001 (one-way ANOVA).