No abstract

The classic carbon tetrachloride (CCl 4 )-induced liver injury model is widely used to study the pathogenesis of fibrosis and evaluate anti-fibrosis drugs. Here, we investigated the dynamic changes in the gut microbiota, bile acids (BAs) and the gut barrier over different fibrosis severities in a CCl 4 -based model. 16S rDNA sequencing demonstrated that the beneficial taxon Lactobacillus was always underrepresented, and pathogens including Escherichia_Shigella , Clostridium_sensu_stricto_1 , Colidextribacter , and Lachnospiraceae_UCG_010 were significantly overrepresented across liver fibrosis severities. Gut dysbiosis was more severe at the early stage of liver injury and advanced stage of fibrosis. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis revealed that with the progress of fibrosis, unconjugated BAs in faeces were significantly decreased and conjugated BAs in serum were significantly increased. The FXR-SHP signalling pathway in the liver and ileum was statistically repressed in the fibrosis groups. Determination of lipopolysaccharide (LPS) and fluorescein isothiocyanate (FITC)-dextran levels in plasma showed that the intestinal barrier remained relatively intact in the advanced fibrosis stage. The advances in knowledge of the gut-liver axis provided by this study yield new insights for application in research and drug evaluation.

ChiCTR-TRC-11001365.

Background Ganxianfang (GXF) formula as a traditional Chinese medicine (TCM) is used for liver fibrosis in clinical practice while its mechanism is unclear. The aim of this study is to explore the molecular mechanism of GXF against CCl4-induced liver fibrosis rats. Methods Detected the main compounds of GXF by UPLC-MS/MS. Evaluated the efficacy of GXF (1.58, 3.15, 4.73 g/kg/day) and Fuzheng Huayu (FZHY, positive control, 0.47 g/kg/day) through serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels and histopathological changes. Explored the underlying mechanisms by integrating our total liver RNA sequencing (RNA-seq) data with recent liver single-cell sequencing (scRNA-seq) studies. Verified potential pharmacodynamic substances of GXF by hepatic stellate cell (HSC)-T6 line. Results Main compounds were identified in GXF by UPLC-MS/MS, including baicalin, wogonoside and matrine etc. With GXF-high dose treatment, the elevation of ALT and AST induced by CCl4 were significantly reduced, and the protective effect of GXF-high dose treatment was better than FZHY. Liver histopathological changes were alleviated by GXF-high dose treatment, the ISHAK scoring showed the incidence of liver cirrhosis (F5/F6) decreased from 76.5 to 55.6%. The results of liver hydroxyproline content were consistent with the histopathological changes. RNA-seq analysis revealed the differential genes (DEGs) were mainly enriched in ECM-receptor interaction and chemokine signaling pathway. GXF effectively inhibited collagen deposition and significantly downregulated CCL2 to inhibit the recruitment of macrophages in liver tissue. Integrating scRNA-seq data revealed that GXF effectively inhibited the expansion of scar-associated Trem2+CD9+ macrophages subpopulation and PDGFRα+PDGFRβ+ scar-producing myofibroblasts in the damaged liver, and remodeled the fibrotic niche via regulation of ligand-receptor interactions including TGFβ/EGFR, PDGFB/PDGFRα, and TNFSF12/TNFRSF12a signaling. In vitro experiments demonstrated that baicalin, matrine and hesperidin in GXF inhibited the activation of hepatic stellate cells. Conclusions This study clarified the potential anti-fibrotic effects and molecular mechanism of GXF in CCl4-induced liver fibrosis rats, which deserves further promotion and application.

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