Chinese herbal decoction, Yi-Qi-Jian-Pi formula exerts anti-hepatic fibrosis effects in mouse models of CCl4-induced liver fibrosis

Abstract

Background: Yi-Qi-Jian-Pi Formula (YQJPF) is a herbal medicine that is used to treat patients with liver failure. However, scientific evidence supporting the treatment of hepatic fibrosis with YQJPF has not been forthcoming. The present study aimed to determine the mechanisms underlying the anti-fibrotic effects of YQJPF in mouse models of hepatic fibrosis.

Methods: Mice were randomly assigned to control, hepatic fibrosis model, silymarin (positive treated), and low-, medium- and high-dose YQJPF (7.5, 15, and 30 g/kg, respectively) groups. Liver function, inflammatory cytokines, and oxygen stress were analyzed using ELISA kits. Sections were histopathologically stained with hematoxylin-eosin, Masson trichrome, and Sirius red. Macrophage polarization was measured by flow cytometry and immunofluorescence. Potential targets of YQJPF against hepatic fibrosis were analyzed by network pharmacology of Chinese herbal compound and the effects of YQJPF on the transforming growth factor-beta (TGF-β)/Suppressor of Mothers against Decapentaplegic family member 3 (Smad3) signaling pathway were assessed using qRT-PCR and immunohistochemical staining. Finally, metagenomics and LC-MS/MS were used to detect the intestinal flora and metabolites of the mice, and an in-depth correlation analysis was performed by spearman correlation analysis. The data were compared by one-way ANOVA and least significant differences (LSDs) or ANOVA-Dunnett's T3 method used when no homogeneity was detected.

Results: We induced hepatic fibrosis using CCl₄ to establish mouse models and found that YQJPF dose-dependently increased body weight, improved liver function, and reversed hepatic fibrosis. Elevated levels of the pro-inflammatory factors IL-1β, IL-6, and TNF-α in the model mice were substantially decreased by YQJPF, particularly at the highest dose. Levels of serum malondialdehyde and superoxide dismutase (SOD) activity were elevated and reduced, respectively. The malondialdehyde concentration decreased and SOD activity increased in the high-dose group. M1 polarized macrophages (CD86) in the mouse models were significantly decreased and M2 polarization was mildly decreased without significance. However, high-dose YQJPF increased the numbers of M2 macrophages and inhibited TGF-β/Smad3 signaling. Metagenomic and non-targeted metabolomics detection results showed that YQJPF could regulate intestinal homeostasis, and Spearman correlation analysis showed that the abundance of Calditerrivibrio_nitroreducens was significantly negatively correlated with 18β-glycyrrhetinic acid. It is suggested that Calditerrivibrio_nitroreducens may reduce the anti-fibrosis effect of licorice and other Chinese herbs by digesting 18β-glycyrrhetinic acid.

Conclusions: YQJPF can reverse liver fibrosis by inhibiting inflammation, suppressing oxidative stress, regulating the immunological response initiated by macrophages, inhibiting TGF-β/Smad3 signaling and regulating intestinal flora homeostasis. Therefore, YQJPF may be included in clinical regimens to treat hepatic fibrosis.

Keywords: Hepatic fibrosis; Herbal medicine; Intestinal flora homeostasis; Network pharmacology.

Fig. 1

General status of mice. (A) Representative images of mice. (B) Changes in mouse body weight. Food intake (C), water intake (D), liver coefficients (E), representative gross appearance of liver (F), total liver to spleen ratio (G) and representative gross appearance of spleens (H) in all mice (*P < 0.05, **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. model).

Fig. 2

Hepatic fibrosis was reversed and liver function was improved by YQJPF. Serum levels of AST (A), ALT (B), ALB (C) and PTA (D). Representative images of liver tissues visualized by HE, Masson (E) and Sirius Red (F) staining (*P < 0.05, ^†P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. model). ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PTA, prothrombin activity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Inflammation was inhibited and oxidation activity was increased by YQJPF. Serum concentrations of IL-1β (A), IL-6 (B) and TNF-α (C) among groups. Activity of SOD (D) and MDA concentration (E) were measured using ELISA kits (*P < 0.05, **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. model). IL-1β, interleukin-1β; IL-6, interleukin-6; MDA, malondialdehyde; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.

Fig. 4

YQJPF enhances M2 macrophage polarization.Ratios (%) of CD86 (A) and CD206 (B) in macrophages. Representative flow cytometry plots of CD86 (C) and CD206 (D) (*P < 0.05, **P < 0.05 vs control; **P < 0.01 vs. model).

Fig. 5

Network pharmacology revealed the potential target of YQJPF in treating hepatic fibrosis (A) Venn diagram of the common targets of YQJPF and liver fibrosis; (B) PPI network of potential targets of YQJPF in the treatment of liver fibrosis; (C) KEGG pathway analysis of genes related to the anti-fibrosis treatment of YQJPF; (D) Enrichment analysis of GO-BP biological function in the treatment of liver fibrosis with YQJPF.

Fig. 6

Transforming growth factor beta/Smad3 signaling was inhibited by YQJPF.Relative levels of TGF-β mRNA (A) and Smad3 mRNA (B) in liver tissues. Representative images of immunohistochemical staining for TGF-β (C) and Smad3 (D) proteins.

Fig. 7

YQJPF regulates the structure and abundance of intestinal flora. (A) Differential metabolic pathways and pathway scores of Top10 between the model group and the control group; (B) Differential metabolic pathways and pathway scores of Top10 between YQJPF group and model group; On the left figure, the RichFactor on the X-axis represents the number of differential metabolites/all metabolites in the pathway. The dot size represents the number of differential metabolites in this Pathway. On the right, the Y-axis represents the name of the metabolic pathway, and the X-axis coordinates represent the differential abundance score (DAscore). DAscore is the global change of all metabolites in a metabolic pathway. A score of 1 indicates an up-regulation trend in the expression of all annotated differential metabolites in the pathway, and a score of negative 1 indicates a down-regulation trend in the expression of all annotated differential metabolites in the pathway. The length of the line segment represents the absolute value of DAscore, and the size of the dot at the end of the line segment represents the number of metabolites in the pathway. The larger the dot, the greater the number of metabolites. (C) Top5 ROC map of differential metabolites between model group and control group; (D) ROC chart of Top5 differential metabolites between YQJPF group and model group; The abscissa is 1-specificity and the ordinate is sensitivity. The area under the line is the AUC value. A larger AUC value indicates a more suitable metabolite as a biomarker.

Fig. 8

YQJPF regulates the composition of intestinal metabolites (A) The Top20 species and metabolites in the model group and the control group were highly correlated with each other; (B) The random forest analysis importance scores of different species and different metabolites in the model group and the control group are species or metabolites on the edge of the circle of the diagram and the connecting lines in the circle represent the correlation between species and metabolites, red is positive correlation, blue is negative correlation; (C) The correlation diagram of Top20 species and metabolites between YQJPF group and model group; (D) Random forest analysis importance scores of different species and metabolites in YQJPF group and model group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)