Regulation mechanism of Rosa roxburghii Tratt. (Cili) fruit vinegar on non-alcoholic fatty liver disease

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by the excessive accumulation of lipids as a pathological feature. Previous studies have demonstrated that Rosa roxburghii Tratt. fruit vinegar (RFV) played an important role in intervening in obesity and related complications by regulating the intestinal microbiota in high-fat diet mice.

Methods: This study investigated the mechanisms by which RFV improves NAFLD from multiple perspectives. Potential targets were predicted by network pharmacology and molecular docking analyses. Intestinal microbial communities were detected and analyzed using 16S rRNA gene sequencing technology. Liver metabolites were detected and analyzed using ultra high performance liquid chromatography quadrupole-exactive high field-X mass spectrometer (UHPLC-Q-Exactive HF-X) and Progenesis QI software. Hepatic protein expression levels were detected and quantified using Western blotting analysis and gray-value analysis, respectively.

Results: The results indicated that, RFV could improve the diversity of intestinal microbiota in NAFLD mice, reduce the ratio of Firmicutes to Bacteroidetes (F/B), and reverse the relative abundance of differential bacteria genera related to lipid accumulation and energy metabolism. The intestinal microbiota was correlated with the levels of lipid metabolism and oxidative stress in the serum and liver of mice with NAFLD. The primary bacteria genera involved were Allobaculum, Faecalibaculum, Dubosiella, Blautia, and unclassified_f_Lachnospiraceae. A total of 441 liver metabolites were identified in NAFLD mice and participating in 21 metabolic pathways. Glycerophospholipid metabolism may be an important pathway regulating NAFLD by RFV. Phosphatidylcholines (PC) and lysophosphatidylcholinergic (LPC) metabolites were significantly regulated by RFV and had significant correlation with differential microbiota. RFV may improve NAFLD by regulating lipid synthesis in the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway. Western blotting analysis showed that, RFV could activate the AMPK phosphorylation, and reduce the expression of fatty acid synthase (FASN) and sterol regulatory element-binding protein 1 (SREBP-1c), resulting in the inhibition of fatty acids de novo synthesis and lipid accumulation.

Conclusion: As a functional food, RFV has been proven to be effective in improving NAFLD. The underlying mechanisms involve the modulation of the intestinal microbiota and metabolites balance, and regulation on lipid disorders through AMPK signaling pathway.

Keywords: AMPK signal pathway; Rosa roxburghii Tratt.; fruit vinegar; intestinal microbiota; metabolomics; non-alcoholic fatty liver disease.

Figure 1

Results of network pharmacology and molecular docking. Venn diagram of potential targets of RFV on NAFLD (A); PPI network diagram of key targets (B); GO function enrichment analysis of potential targets (C); KEGG pathway enrichment of potential targets (D); Compound-key target-pathway visualization network (E); The molecular docking diagram of AMPK and ellagic acid, gallic acid, quercetin. AMPK-ellagic acid (−9.1 kcal/mol), gallic acid (−5.9 kcal/mol), and quercetin (−8.7 kcal/mol) (F); The molecular docking diagram of FASN and ellagic acid, gallic acid, quercetin. FASN-ellagic acid (−8.2 kcal/mol), gallic acid (−6.6 kcal/mol), and quercetin (−8.4 kcal/mol) (G).

Figure 2

Effect of RFV on intestinal microbiota in NAFLD mice. Venn diagram of OTUs (A); NMDS analysis of β diversity (B); Chao1 indices of Alpha diversity (C); Simpson indices of Alpha diversity (D). The Control, NAFLD, RRJ, RFV-L, and RFV-H were normol diet group, high-fat diet group, Rosa roxburghii juice, low dosage Rosa roxburghii fruit vinegar group, and high dosage Rosa roxburghii fruit group, respectively. ^#p<0.05, compared with Control group, ^*p<0.05, compared with NAFLD group (n=6).

Figure 3

Species composition and bacteria with significant differences in relative abundanceat at the phylum and genus level. Histogram of relative abundance at phylum level (A); Histogram of relative abundance at genus level (B). Relative abundance of Firmicutes and Bacteroidetes (C); Ratio of F to B (F/B) (D); The relative abundance at genus level of Allobaculum (E); Blautia (F); Coriobacteriaceae_UCG-002 (G); Dubosiella (H); Faecalibaculum (I); Unclassified_f_Lachnospiraceae (J). ^#p<0.05, compared with Control group, ^*p<0.05, compared with NAFLD group.

Figure 4

Effect of RFV on liver metabolites in NAFLD mice. PLS-DA score chart (A); PLS-DA displacement test map (B); Pie chart of differential metabolite classification (C); The trend of metabolites change in Control vs. NAFLD (D); The trend of metabolites change in RFV-H vs. NAFLD (E) (n=6).

Figure 5

Analysis of enriched pathways of differential metabolites. Topology analysis diagram (A,B); Glycerophospholipid metabolism pathway diagram (C); Histogramgraph of the relative abundance of metabolites in each group (D). ^#p<0.05, compared with Control group, ^*p<0.05, compared with NAFLD group. C00350: phosphatidylethanolamine (PE), C00416: phosphatidic acid (PA; 18:0/18:2 (9Z and 12Z)), C04308: PE-NMe2 (14:0/14:0), C01233: Sn-glycoro-3-Phosphatethanolamine, C0015: phosphatidylcholine (PC), C00344: glycerophosphate (PGs), C04230: lysophosphatidylcholine (LysoPC), C02737: Phosphatidylserine (PS; 16:1(9Z)/(24:0)), C00670: Glycerophosphocholine and Glycerylphosphorylcholine.

Figure 6

The correlation among intestinal microbiota, liver metabolites, and NAFLD-related indexes based on heatmap correlation analysis. The correlation between Bacteria at each taxonomic level and mouse lipid metabolism and oxidative stress indicators (A,B); The correlation between differential metabolites and differential microflora (C); p ≤ 0.05 is marked as *; p ≤ 0.01 is marked as **; p ≤ 0.001 is marked as ***. Blue box: dominant intestinal microbiota; Red box: significant metabolites.

Figure 7

The protein expression and relative abundance of in liver. Protein bands diagram (A); the gray values of P-AMPK/AMPK (B); SREBBP-1c/β -actin (C); FASN/β -actin (D). #p<0.05, compared with Control group, *p<0.05, compared with NAFLD group (n=3).