Naringin Attenuates High Fat Diet Induced Non-alcoholic Fatty Liver Disease and Gut Bacterial Dysbiosis in Mice

Abstract

The incidence of non-alcoholic fatty liver disease (NAFLD) is rising annually, and emerging evidence suggests that the gut bacteria plays a causal role in NAFLD. Naringin, a natural flavanone enriched in citrus fruits, is reported to reduce hepatic lipid accumulation, but to date, no investigations have examined whether the benefits of naringin are associated with the gut bacteria. Thus, we investigated whether the antilipidemic effects of naringin are related to modulating the gut bacteria and metabolic functions. In this study, C57BL/6J mice were fed a high-fat diet (HFD) for 8 weeks, then fed an HFD with or without naringin administration for another 8 weeks. Naringin intervention reduced the body weight gain, liver lipid accumulation, and lipogenesis and attenuated plasma biochemical parameters in HFD-fed mice. Gut bacteria analysis showed that naringin altered the community compositional structure of the gut bacteria characterized by increased benefits and fewer harmful bacteria. Additionally, Spearman's correlation analysis showed that at the genus level, Allobaculum, Alloprevotella, Butyricicoccus, Lachnospiraceae_NK4A136_group, Parasutterella and uncultured_bacterium_f_Muribaculaceae were negatively correlated and Campylobacter, Coriobacteriaceae_UCG-002, Faecalibaculum and Fusobacterium were positively correlated with serum lipid levels. These results strongly suggest that naringin may be used as a potential agent to prevent gut dysbiosis and alleviate NAFLD.

Keywords: citrus fruits; gut dysbiosis; high-fat diet; lipogenesis; non-alcoholic fatty liver disease.

FIGURE 1

Effects of NAR on fat accumulation and body weight in HFD-fed mice. (A) Macroscopic view of livers from the different groups. Representative images of light microscopic (B) HE and (C) oil red O staining of liver tissues in the different groups, Bar = 50 μm. (D) Body weight, (E) liver weight, and (F) liver/body weight. Data are the mean ± SEM (n = 8). *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 2

Effects of NAR on serum lipid levels in HFD-fed mice. Serum levels of (A) TC, (B) TG, (C) HDL-C, and (D) LDL-C. Data are the mean ± SEM (n = 8). *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 3

Effects of NAR on serum ALT, AST, glucose, hsCRP and LPS levels, and liver F4/80 and MPO in HFD-fed mice. Serum levels of (A) ALT, (B) AST, (C) glucose, (D) hsCRP, and (E) LPS. Liver samples in four groups stained by (F) F4/80 and (G) MPO. Bar = 50 μm. The arrows in (F) indicate the F4/80-positive cells. The arrows in (G) indicate the MPO-positive cells. Data are the mean ± SEM (n = 8). *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 4

Effects of NAR on liver lipogenesis-related protein expression in HFD-fed mice. (A) Srebp1, Fas, Acc, and Scd1 expressions determined by western blot. (B) Quantitative analysis of Srebp1. (C) Quantitative analysis of Fas. (D) Quantitative analysis of Acc. (E) Quantitative analysis of Scd1. The blot image is representative of the four groups. The densitometry is an averaged result for the five animals, normalized to β-actin. Data are the mean ± SEM. *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 5

Effects of NAR on gut bacteria OTUs, diversity and similarity of species diversity. (A) OTU numbers of the gut bacteria from different parts of the gut in the four groups. (B) Alpha diversity of the gut bacteria assessed by the Shannon index for each group. PCoA analysis based on binary Jaccard distance algorithm of the gut bacteria in the (C) ileum, (D) cecum, and (E) colon. *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 6

Effects of NAR on gut bacteria composition. Relative abundances of Firmicutes/Bacteroidetes in the (A) ileum, (B) cecum, and (C) colon. *P < 0.05 vs. ND group. Heatmaps of the gut bacteria compositions (those with mean relative abundances ≥0.05%) and by comparison (ND vs. ND+NAR, ND vs. HFD, and HFD vs. HFD+NAR, q ≤ 0.05 after correcting for the p-value) in the (D) ileum, (E) cecum, and (F) colon at the genus level.

FIGURE 7

Effects of NAR on the relative abundances of several gut bacteria. Relative abundances of (A) Faecalibaculum and (B) Fusobacterium in the ileum, (C) Bacteroides, (D) Butyricicoccus, (E) Parasutterella, (F) uncultured_bacterium_f_Muribaculaceae, (G) Coriobacteriaceae_UCG-002, (H) Escherichia-Shigella, (I) Lachnospiraceae_XPB1014_group, (J) Faecalibaculum and (K) Fusobacterium in the cecum and (L) Allobaculum, (M) Alloprevotella, (N) Butyricicoccus, (O) Lachnospiraceae_NK4A136_group, (P) Parasutterella, (Q) uncultured_bacterium_f_Muribaculaceae, (R) Campylobacter, (S) Faecalibaculum and (T) Fusobacterium in the colon at the genus level, which in the HFD and HFD+NAR groups differed from ND group are presented. *P < 0.05 vs. ND group; #P < 0.05 vs. HFD group.

FIGURE 8

Correlation between the gut bacteria relative abundance and serum lipid level. Spearman rank correlations between altered gut bacteria and serum TG, TC, HDL-C, and LDL-C levels. Red indicates a positive correlation; blue indicates a negative correlation. Significant correlations are indicated by *P < 0.05 and marked with asterisks.

FIGURE 9

Proposed mechanisms for protective effects of NAR on HFD-fed mice. NAR reduces lipid synthesis and attenuates NAFLD parameters, possibly by modulating the gut bacteria composition via an unknown pathway.