Ling-Gui-Zhu-Gan decoction ameliorates nonalcoholic fatty liver disease via modulating the gut microbiota

Abstract

Numerous studies have supported that nonalcoholic fatty liver disease (NAFLD) is highly associated with gut microbiota dysbiosis. Ling-Gui-Zhu-Gan decoction (LG) has been clinically used to treat NAFLD, but the underlying mechanism remains unknown. This study investigated the therapeutic effect and mechanisms of LG in mice with NAFLD induced by a high-fat diet (HD). An HD-induced NAFLD mice model was established to evaluate the efficacy of LG followed by biochemical and histopathological analysis. Metagenomics, metabolomics, and transcriptomics were used to explore the structure and metabolism of the gut microbiota. LG significantly improved hepatic function and decreased lipid droplet accumulation in HD-induced NAFLD mice. LG reversed the structure of the gut microbiota that is damaged by HD and improved intestinal barrier function. Meanwhile, the LG group showed a lower total blood bile acids (BAs) concentration, a shifted BAs composition, and a higher fecal short-chain fatty acids (SCFAs) concentration. Furthermore, LG could regulate the hepatic expression of genes associated with the primary BAs biosynthesis pathway and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study suggested that LG could ameliorate NAFLD by altering the structure and metabolism of gut microbiota, while BAs and SCFAs are considered possible mediating substances.

Importance: Until now, there has still been no study on the gut microbiota and metabolomics of Ling-Gui-Zhu-Gan decoction (LG) in nonalcoholic fatty liver disease (NAFLD) mouse models. Our study is the first to report on the reshaping of the structure and metabolism of the gut microbiota by LG, as well as explore the potential mechanism underlying the improvement of NAFLD. Specifically, our study demonstrates the potential of gut microbial-derived short-chain fatty acids (SCFAs) and blood bile acids (BAs) as mediators of LG therapy for NAFLD in animal models. Based on the results of transcriptomics, we further verified that LG attenuates NAFLD by restoring the metabolic disorder of BAs via the up-regulation of Fgf15/FXR in the ileum and down-regulation of CYP7A1/FXR in the liver. LG also reduces lipogenesis in NAFLD mice by mediating the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which then contributes to reducing hepatic inflammation and improving intestinal barrier function to treat NAFLD.

Keywords: Ling-Gui-Zhu-Gan decoction; bile acid; gut microbiota; nonalcoholic fatty liver disease; short-chain fatty acids.

Fig 1

Effects of LG administration on NAFLD mice: (a) body weight gain rate with time (LG treatment started from the 16th week); (b) body fat ratio; (c) liver index; (d) hepatic TG level; (e) hepatic TC level; (f) hepatic free fatty acid level; (g) HE-stained liver section (magnification, ×100); (h) bar graph of the volume density of liver steatosis; (i) oil red O-stained liver section (magnification, ×200); (j) quantitative results of oil red O staining; (k) fasting blood glucose; (l) fasting serum insulin; (m) HOMA-IR; (n) serum ALT level; (o) serum AST level; (p) hepatic MDA level. # P < 0.05, ## P < 0.01 compared with the NC group. *P < 0.05, **PP < 0.01 compared with the HD group. Box plots display medians with interquartile ranges.

Fig 2

Effects of LG on the structure of gut microbiota. (a) Bacterial composition at the phylum level; (b) bacterial composition at the family level; (c) F/B ratio; (d) alpha-diversity analysis with the Simpson index; (e) principal coordinate analysis (PCoA) of three groups based on weighted UniFrac metrics, each spot representing one group with mean ± SD; (f) random forest analysis at the family level; (g) network analysis of highly abundant families with positive interactions in red and negative interactions in green. # P < 0.05, ## P < 0.01 compared with the NC group. *P < 0.05, **P < 0.01 compared with the HD group. Box plots display medians with interquartile ranges. (h) Spearman correlations between the gut microbial community at the family level and vital metabolic parameters related to NAFLD. + P < 0.05, *P < 0.01.

Fig 3

Effects of LG on the metabolites of gut microbiota. (a) Heatmap of different pathways between groups; (b) concentration of lipopolysaccharide-binding protein (LBP) in serum; (c) concentration of total BAs in serum; (d) relative abundance of different BAs in serum; (e) the ratio of primary/secondary BAs in serum; (f) high and low abundantly different components of BAs in serum; concentrations of acetic acid (g), propionic acid (h), and butyric acid (i) in feces. # P < 0.05, ## P < 0.01 compared with the NC group. *P < 0.05, **P < 0.01 compared with the HD group. Box plots display medians with interquartile ranges. (j) Spearman correlations between the gut microbial community at the family level and their major metabolites. + P < 0.05, *P < 0.01.

Fig 4

Potential molecular mechanism of LG in the treatment of NAFLD. (a) Enrichment analysis of KEGG metabolic pathway for the NC group in comparison with that of the HD group; (b) enrichment analysis of KEGG metabolic pathway for the LG group in comparison with that of the HD group; compared with the NC group, the relative expression of genes encoding for lipogenesis (c), fatty acid oxidation (d), lipolysis (e), inflammation (f), BAs biosynthesis (g) in the liver; (h) Western blotting analysis of PPARγ, CYP7A1 protein expression in the liver; cumulative densitometric analysis for PPARγ (i),CYP7A1 (j)in the liver of representative mice per each group. Band densities were normalized to the expression level of glycerol triphosphate dehydrogenase (GAPDH). Compared with the NC group, the relative expression of genes encoding for BAs biosynthesis (k), SCFAs receptor (l), intestinal permeability (m) in ileum; (n) Western blotting analysis of Fgf15 protein expression in the ileum; (o) cumulative densitometric analysis for Fgf15 in the ileum of representative mice per each group. Band densities were normalized to the expression level of GAPDH. (p) Immunohistochemical staining for Occludin protein expression in the terminal ileum (magnification, ×400). # P < 0.05, ## P < 0.01 compared with the NC group. *P < 0.05, **P < 0.01 compared with the HD group. Violin and Box plots display medians with interquartile ranges.

Fig 5

The proposed molecular mechanism by which LG ameliorates NAFLD. Left: The administration of HD increased the abundance of Peptococcaceae and decreased the expression of FXR in both the ileum and liver. Consequently, the activation of CYP7A1 in the liver was observed, leading to an accelerated biosynthesis of BAs. Additionally, HD caused a reduction in the abundance of S24-7 and inhibited the production of SCFAs. Furthermore, HD induced an increase in the expression of PPARγ in the liver, thereby promoting lipid synthesis. Right: The administration of LG resulted in a reduction in the abundance of Peptococcaceae and activation of FXR expression in both the ileum and liver. Consequently, the expression of CYP7A1 in the liver was suppressed, leading to the inhibition of BAs biosynthesis. Additionally, LG supplementation increased the abundance of S24-7 and facilitated the production of SCFAs. Furthermore, LG administration decreased the expression of PPARγ in the liver, thereby reducing lipid synthesis.