Sanguisorba officinalis L. Ameliorates Hepatic Steatosis and Fibrosis by Modulating Oxidative Stress, Fatty Acid Oxidation, and Gut Microbiota in CDAHFD-Induced Mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases and encompasses non-alcoholic steatosis, steatohepatitis, and fibrosis. Sanguisorba officinalis L. (SO) roots have traditionally been used for their antioxidant properties and have beneficial effects on metabolic disorders, including diabetes and obesity. However, its effects on hepatic steatosis and fibrosis remain unclear. In this study, we explored the effects of a 95% ethanolic SO extract (SOEE) on NAFLD and fibrosis in vivo and in vitro. The SOEE was orally administered to C57BL/6J mice fed a choline-deficient, L-amino-acid-defined, high-fat diet for 10 weeks. The SOEE inhibited hepatic steatosis by modulating hepatic malondialdehyde levels and the expression of oxidative stress-associated genes, regulating fatty-acid-oxidation-related genes, and inhibiting the expression of genes that are responsible for fibrosis. The SOEE suppressed the deposition of extracellular matrix hydroxyproline and mRNA expression of fibrosis-associated genes. The SOEE decreased the expression of fibrosis-related genes in vitro by inhibiting SMAD2/3 phosphorylation. Furthermore, the SOEE restored the gut microbial diversity and modulated specific bacterial genera associated with NAFLD and fibrosis. This study suggests that SOEE might be the potential candidate for inhibiting hepatic steatosis and fibrosis by modulating oxidative stress, fatty acid oxidation, and gut microbiota composition.

Keywords: L-amino acid-defined; Sanguisorba officinalis L.; choline-deficient; fibrosis; gut microbiota; high-fat diet (CDAHFD); steatosis.

Figure 1

Effects of SOEE on NAFLD-related traits in CDAHFD-fed mice. (A) TG contents in liver tissue. (B) TC contents in liver tissue. (C) Representative image of H&E-stained liver section. Magnification is 200×. (D) Quantified surface area of lipid droplets in each group using ImageJ software. (E) Evaluated NAFLD activity score in H&E-stained liver sections using deep-learning-based CNN. Data are expressed as mean ± SEM (n = 10). One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05, and “ns” non-significant.

Figure 2

Effects of SOEE on oxidative-stress- and fatty-acid-oxidation-related markers in CDAHFD-fed mice. (A) Level of hepatic malondialdehyde (MDA), which is a biomarker of lipid peroxidation, measured using a colorimetric assay. (B,D) mRNA expression of oxidative-stress-related genes, namely, (B) catalase (Cat), (C) glutathione peroxidase I (Gpx1), and (D) superoxide dismutase 1 (Sod1), were quantified using qRT-PCR. mRNA expression levels of fatty-acid-oxidation-related genes were quantified using qRT-PCR. (E) Peroxisome proliferator-activated receptor alpha (Ppara), (F) carnitine palmitoyltransferase 1A (Cpt1a), and (G) lipoprotein lipase (Lpl) mRNA levels. Data are expressed as mean ± SEM (n = 10). One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05, and “ns” non-significant.

Figure 3

Effects of SOEE on fibrosis-related markers in the liver of CDAHFD-fed mice. (A) Representative image of Sirius-Red-stained liver section. Magnification was 200×. (B) Quantified Sirius-Red-stained area in liver tissue section (n = 40 images per group). (C) Hydroxyproline level in liver tissue (n = 10). (D–G) mRNA expression levels of fibrosis-related genes including (D) alpha-smooth muscle actin (Acta2), (E) collagen type I alpha 1 (Col1a1), (F) collagen type III alpha 1 (Col3a1), and (G) transforming growth factor beta 1 (Tgfb1) were quantified using qRT-PCR. Data are expressed as mean ± SEM (n = 10). One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05, and “ns” non-significant.

Figure 4

Effects of SOEE on mRNA expression levels of fibrosis-related genes and SMAD2/3 phosphorylation in LX-2 cells. To investigate the fibrosis inhibitory effect of SOEE, LX-2 cells were treated with TGF-β1 (TGFb1) at 5 ng/mL, and mRNA expression levels of (A) alpha-smooth muscle actin (ACTA2), (B) collagen type I alpha 1 (COL1A1), (C) TIMP metallopeptidase inhibitor 1 (TIMP1), and (D) collagen type III alpha 1 (COL3A1) were observed using qRT-PCR. (E) Protein levels of SMAD2/3 and p-SMAD2/3 were determined using Western blotting. Cells were pre-treated with SOEE for 24 h and then with TGF-β1 (5 ng/mL) and SOEE simultaneously for 30 min. (F) Protein expression levels were quantified using ImageJ software. Data are expressed as mean ± SEM. (n = 4). One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the TGF-β1 group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05.

Figure 5

Effect of SOEE on gut microbial diversity and composition in CDAHFD-fed mice. (A–C) Alpha diversity indices: (A) Shannon diversity, (B) Faith’s PD, and (C) observed ASV. One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. (D–F) Representative microbial genera, namely, (D) Butyricicoccus, (E) Acetivibrio ethanoligignens group, and (F) Lactobacillus show significantly differential abundance between the SOEE and CDAHFD groups. One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. (G) Heat map showing the correlations between the abundance of microbial genera and NAFLD/fibrosis-related markers. In taxonomic classification, the class and phylum level to which each genus belongs are denoted with different colors. p-values were adjusted using the Benjamini–Hochberg (BH) FDR procedure. p-value compared with the CDAHFD group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05, and “ns” non-significant.

Figure 5

Effect of SOEE on gut microbial diversity and composition in CDAHFD-fed mice. (A–C) Alpha diversity indices: (A) Shannon diversity, (B) Faith’s PD, and (C) observed ASV. One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. (D–F) Representative microbial genera, namely, (D) Butyricicoccus, (E) Acetivibrio ethanoligignens group, and (F) Lactobacillus show significantly differential abundance between the SOEE and CDAHFD groups. One-way ANOVA and Tukey’s post hoc multiple comparison test were performed for statistical analysis. p-value compared with the CDAHFD group. (G) Heat map showing the correlations between the abundance of microbial genera and NAFLD/fibrosis-related markers. In taxonomic classification, the class and phylum level to which each genus belongs are denoted with different colors. p-values were adjusted using the Benjamini–Hochberg (BH) FDR procedure. p-value compared with the CDAHFD group. “***” p < 0.001, “**” p < 0.01, “*” p < 0.05, and “ns” non-significant.