Hyperoside attenuates non-alcoholic fatty liver disease in rats via cholesterol metabolism and bile acid metabolism

Abstract

Introduction: Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic total cholesterol (TC) and total triglyceride (TG) accumulation. In our previous study, we found that rats treated with hyperoside became resistant to hepatic lipid accumulation.

Objectives: The present study aims to investigate the possible mechanisms responsible for the inhibitory effects of hyperoside on the lipid accumulation in the liver tissues of the NAFLD rats.

Methods: Label-free proteomics and metabolomics targeting at bile acid (BA) metabolism were applied to disclose the mechanisms for hyperoside reducing hepatic lipid accumulation among the NAFLD rats.

Results: In response to hyperoside treatment, several proteins related to the fatty acid degradation pathway, cholesterol metabolism pathway, and bile secretion pathway were altered, including ECI1, Acnat2, ApoE, and BSEP, etc. The expression of nuclear receptors (NRs), including farnesoid X receptor (FXR) and liver X receptor α (LXRα), were increased in hyperoside-treated rats' liver tissue, accompanied by decreased protein expression of catalyzing enzymes in the hepatic de novo lipogenesis and increased protein level of enzymes in the classical and alternative BA synthetic pathway. Liver conjugated BAs were less toxic and more hydrophilic than unconjugated BAs. The BA-targeted metabolomics suggest that hyperoside could decrease the levels of liver unconjugated BAs and increase the levels of liver conjugated BAs.

Conclusions: Taken together, the results suggest that hyperoside could improve the condition of NAFLD by regulating the cholesterol metabolism as well as BAs metabolism and excretion. These findings contribute to understanding the mechanisms by which hyperoside lowers the cholesterol and triglyceride in NAFLD rats.

Keywords: ACC, Acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; Apo, apolipoprotein; BAs, bile acids; BSH, bile salt hydrolase; Bile acid metabolism; CYP27A1, sterol 27-hydroxylase; CYP7A1, cholesterol 7α-hydroxylase; Cholesterol metabolism; FGF15/19, fibroblast growth factor 15/19; FXR, farnesoid X receptor; Hyperoside; LC-MS, the combination of high-performance liquid chromatography and mass spectrometry; LXRα, liver X receptor α; Label-free proteomics; NAFLD; NAFLD, non-alcoholic fatty liver disease; PMSF, phenylmethylsulfonyl fluoride; QC, quality control; SDS, sodium dodecyl sulfate; SHP, small heterodimer partner; SREBP1, sterol regulatory element-binding protein 1; SREBP2, sterol regulatory element-binding protein 2; SREBPs, sterol regulatory element binding proteins; TC, total cholesterol; TG, triglyceride; TGR5, Takeda G-protein-coupled receptor 5; Targeted metabolomics; VLDL, very low-density lipoprotein; WB, Western blot; pACC, phosphorylated ACC.

Graphical abstract

Fig. 1

The chemical structure of hyperoside.

Fig. 2

Proteomics analysis between model and hyperoside group. (a) Volcano map of differential protein. FC > 1.2 or < 0.83, and p < 0.05. (b) KEGG enrichment analysis differential protein between model and hyperoside group (p < 0.05). (c) GO enrichment analysis of differential protein between model and hyperoside group (p < 0.05).

Fig. 3

Hyperoside treatment increases the expression of proteins involved in bile acid biosynthesis, increases the expression of nuclear receptors, and decreases the expression of proteins involved in de novo lipogenesis. a. The protein expression of CYP7A1, CYP27A1, FXR, and LXRα, GAPDH was chosen as a loading control, and the density of the signals for CYP7A1, CYP27A1, FXR, and LXRα. b. The protein expression of ACC and pACC, GAPDH was chosen as a loading control, and the density of the signals for ACC and pACC. c. The protein expression of SREBP1 and SREBP2, GAPDH was chosen as a loading control, and the density of the signals for SREBP1 and SREBP2. n = 3, data are presented as mean ± SEM. ##, p < 0.01, #, p < 0.05, by unpaired Student t test compared with blank group, **, p < 0.01, *, p < 0.05, by unpaired Student t test compared with model group.

Fig. 4

The enhanced PCA and PLS-DA model for discrimination between blank, model and hyperoside groups. a. Score plot of the 3D PCA-DA model b. Score plot of the 3D PLS-DA model. c. Statistical validation of the PLS-DA model by permutation testing (n = 300) [Q² = −0.28, R² = 0.257]. d. Loading plot created from OPLS-DA modeling; red ball indicates BAs with VIP value greater than 1.

Fig. 5

The 15 BAs with significant contribution to the differentiation (a) The boxplot with points of 15 BAs with significant contribution to the differentiation (p < 0.05, Kruskal test); LCA, p = 0.0045, FDR = 0.0254; CDCA, p = 0.0051, FDR = 0.0254; 7-Keto-LCA, p = 0.0057, FDR = 0.0254; TωMCA, p = 0.0069, FDR = 0.0254; muroCA, p = 0.0083, FDR = 0.0254; THDCA, p = 0.0084, FDR = 0.0254; UDCA, p = 0.0091, FDR = 0.0254; TβMCA, p = 0.0103, FDR = 0.0254; ωMCA, p = 0.0104, FDR = 0.0254; NorCA, p = 0.0111, FDR = 0.0254; αMCA, p = 0.0130, FDR = 0.0272; DCA, p = 0.0176, FDR = 0.0337; CA, p = 0.0226, FDR = 0.0400; βMCA, p = 0.0256, FDR = 0.0420; ACA, p = 0.0419, FDR = 0.0642; FDR, false discovery rate (b) Heatmap with Z score of 15 BAs with significant contribution to the differentiation. The scale: blue colours indicate low Z values, whereas brown colours indicate high Z values.

Fig. 6

Hyperoside treatment increases concentration of total conjugated BAs(b) and TUDCA(e), decreases the concentration of total unconjugated BAs(a), DCA(c), and LCA(d) in rat liver. Data are presented as mean ± SEM. ##, p < 0.01, #, p < 0.05, by unpaired Student t test compared with blank group, **, p < 0.01, *, p < 0.05, by unpaired Student t test compared with model group.

Fig. 7

Hepatic pathology of liver sections stained with Hematoxylin & eosin staining,(H&E)from (a) Blank group, (b) Model group, (c) Hyperoside_1.5 mg/Kg group, and (d) Hyperoside_0.6 mg/Kg group, to assess liver damage, original magnification, 200×.

Fig. 8

The speculative mechanism of hyperoside for treatment of NAFLD in rats. Increased FXR expression is expected to increase the expression of UGT2, MDR3, BSEP, ECI1, ACADSB, and ApoB/E/AII/CI/CIII, and to decrease the expression of SREBP1. Enzymes BA synthesis is increased due to increased LXRα and conjugated BA level. OATs expression are also increased which would be expected to increase import of conjugated BA from intestine. ⊕, indicates up regulation; ⊖, indicates down regulation.