Identifying hepatoprotective mechanism and effective components of Yinchenzhufu decoction in chronic cholestatic liver injury using a comprehensive strategy based on metabolomics, molecular biology, pharmacokinetics, and cytology

Ethnopharmacological relevance: In Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice.Aim of the study: This study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI).Materials and methods: A chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydrox-ychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively.Results: YCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid meta-bolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-kappa B) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-kappa B1.Conclusion: YCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-kappa B signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.