Effect and Mechanism of Tauroursodeoxycholic Acid in Blue Fox Bile on Acute Alcohol-Associated Liver Injury in Mice

Summary

This study evaluated the hepatoprotective effects of blue fox bile on alcohol-induced liver injury in mice. Blue fox bile, containing TUDCA, UDCA, bilirubin, and TCDCA, reduced liver damage markers and acted via the AGE-RAGE pathway, AKT1, and PPARG, demonstrating anti-inflammatory and antioxidant properties.

Abstract

Animal bile, such as bear bile, have long been used in traditional medicine for its therapeutic benefits. Blue fox bile, similar to bear bile, is believed in certain traditional practices to possess hepatoprotective properties. This study examined the components and effects of blue fox bile on alcohol-associated liver injury in mice. Bile was collected from 30 blue foxes and processed into dried powder. The chemical composition of blue fox bile was analyzed using high-performance liquid chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Network pharmacology was employed to identify potential bioactive compounds, targets, and pathways associated with alcohol-associated liver injury. A mouse model of alcohol-associated liver injury was established using Kunming mice, which were administered blue fox bile powder at low and high doses. Serum alanine aminotransferase, aspartate aminotransferase, and total cholesterol levels were measured. Liver tissues were assessed by hematoxylin and eosin staining and malondialdehyde assay. Molecular docking was performed to predict the binding affinity between active compounds and core targets. Blue fox bile was found to contain tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid, bilirubin, and taurochenodeoxycholic acid. Histopathological analysis revealed no significant abnormalities or toxic effects in major organs following oral administration of blue fox bile powder. The core targets of blue fox bile included protein AKT1, PPARG, IGF1, MMP9, andCASP3. Blue fox bile treatment decreased serum ALT, AST, cholesterol, and MDA levels in mouse models of alcohol-related liver injury. Network pharmacology and molecular docking suggest that the hepatoprotective effects of blue fox bile may be related to the advanced glycation end product-receptor for advanced glycation end product signaling pathway. Overall, blue fox bile, with its TUDCA content similar to that of bear bile, targets proteins such as AKT1 and PPARG, demonstrating potential anti-inflammatory and antioxidant effects in the management of alcohol-associated liver injury.

Introduction

Alcohol-associated liver disease (ALD) encompasses a spectrum of liver injuries ranging from asymptomatic laboratory abnormalities to advanced liver disease with cirrhosis and, in its most severe form, life-threatening end-stage liver disease accompanied by multi-organ failure¹. The prevalence of ALD, including alcohol-associated fatty liver disease, represents a relatively mild and potentially reversible form of ALD¹. Alcohol-associated hepatitis is characterized by inflammation, with clinical presentations ranging from asymptomatic steatohepatitis to acute alcoholic hepatitis, which presents with acute jaundice and can....

Protocol

The study protocol was reviewed and approved by the Ethics Committee for Animal Experiments of Heilongjiang University of Chinese Medicine (approval #2023021101). All animal experiments were conducted in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) guidelines and relevant national and institutional guidelines for the care and use of laboratory animals. As this study involved animal experiments, informed consent was not applicable. All animal breeding and fur harvesting procedures strictly adhered to the Chinese Technical Regulations for the Management of Wild Animal Breeding. The study was approved by the Ethics Committee f....

Results

Identification of the active compounds in blue fox bile
Blue foxes and bears are both carnivores, sharing similar digestive physiology and metabolism²⁰. Therefore, their bile may have comparable compositions. Using the chromatographic conditions described above, each compound was well separated (Figure 2). In this experiment, the retention time of the TUDCA standard²¹ was used to identify TUDCA in the blue fox bile (

Discussion

According to the 2018 World Health Organization (WHO) Global Status Report on Alcohol and Health, the per-capita alcohol consumption in China increased from 4.1 L in 2005 to 7.2 L in 2016, representing a 76% rise. ALD caused by chronic alcohol consumption remains a major etiology of liver-related morbidity and mortality²². Bo et al. established a carbon tetrachloride (CCl₄)-induced liver injury model in mice and observed that blue fox bile significantly reduced AST, ALT, T-CHO, and MDA .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.03M Sodium dihydrogen phosphate	N/A	N/A	HPLC mobile phase A
0.1% Formic acid in water	Fisher Scientific, USA	N/A	Mobile phase for UPLC
0.22 μm membrane filter	N/A	N/A	Sample filtration
4% Paraformaldehyde	N/A	N/A	Tissue fixation
Acetonitrile, Chromatography Grade	Fisher Scientific, USA	N/A	Mobile phase for UPLC
ALT, AST, T-CHO kits	Shanghai Yuanye Biotechnology Co., Ltd., China	N/A	Serum biochemical analysis
Analytical balance	N/A	N/A	Animal weight measurement
Aurocholic acid	China Institute for Food and Drug Control	N/A	Internal standard for HPLC/MS
Bear bile powder	China Institute for Food and Drug Control, Beijing	N/A	Reference material
Bilirubin	Not specified	N/A	Identified active compound in bile
Blue fox	Suihua Fox Farm, Heilongjiang, China	N/A	Source of bile
Blue fox bile powder	In-house preparation	N/A	Sample for component analysis
C18 HPLC column	Agilent Technologies, USA	ZORBAX SB-C18 (4.6×150 mm, 5 μm)	HPLC separation
Cytoscape	https://cytoscape.org/	Version 3.9.1	Network visualization and topological analysis
DAVID	NIH	https://david.ncifcrf.gov/	GO/KEGG enrichment analysis
GeneCards	Weizmann Institute	https://www.genecards.org/	Disease-related gene retrieval
Hematoxylin and Eosin (H&E)	N/A	N/A	Histological staining
HPLC system	Agilent Technologies, USA	1200 Infinity	Composition analysis of bile
Kunming mice (SPF, 18–22g)	Liaoning Changsheng Biotechnology Co., Ltd.	SCXK (Liao) 2020-0010	Alcoholic liver injury model
Leucine-enkephalin	Sigma, USA	N/A	MS calibration standard (40 fmol/μL)
MDA detection kit	Shanghai Yuanye Biotechnology Co., Ltd., China	N/A	Liver oxidative stress measurement
Methanol, Chromatography Grade	Fisher Scientific, USA	N/A	Solvent for sample preparation
MOE	Chemical Computing Group	MOE 2022.02	Molecular docking analysis
OMIM	NIH	https://omim.org/	Disease-related gene retrieval
Optical microscope	N/A	N/A	Histological observation
PharmMapper	ECUST	http://www.lilab-ecust.cn/pharmmapper/	Prediction of active compound targets
Phosphoric acid	N/A	N/A	pH adjustment of mobile phase
PubChem	NCBI	https://pubchem.ncbi.nlm.nih.gov/	Compound structure retrieval
RCSB Protein Data Bank	https://www.pdbus.org/	N/A	Protein 3D structure source
Refrigerated centrifuge	N/A	N/A	Serum and tissue separation
Sodium tauroursodeoxycholate (TUDCA)	China Institute for Food and Drug Control	N/A	Internal standard for HPLC/MS
STRING	EMBL	http://version10.string-db.org/	Protein-protein interaction analysis
UniProt	EMBL-EBI	https://www.uniprot.org/	Target standardization
UPLC-Q-TOF-MS system	Waters, USA	UPLC HSS T3 (100×2.1 mm, 1.8 μm)	Component identification
Venny	CSIC	https://bioinfogp.cnb.csic.es/tools/venny/index.html	Target intersection visualization