Hepatic fibrosis (HF) is a pathological process characterized by excessive deposition and abnormal remodeling of extracellular matrix (ECM) after chronic liver injury. Repeated hepatocyte injury, inflammatory infiltration, cholestasis, lipotoxicity, or immune responses can activate hepatic stellate cells, causing them to transform into myofibroblast-like cells and secrete collagen, fibronectin, and proteoglycans. Continuous progression of fibrosis can lead to disruption of hepatic lobular architecture, pseudolobule formation, and cirrhosis, making it a key step in the progression of chronic liver disease toward end-stage liver disease.

Keywords: hepatic fibrosis; animal modeling; carbon tetrachloride; thioacetamide; bile duct ligation; NASH; hepatic stellate cells; TGF-β/Smad; extracellular matrix

1 Experimental Positioning of Hepatic Fibrosis Models

1.1 Core Objectives of Model Establishment

(1) Chronic hepatocyte injury and repair response

The core of hepatic fibrosis models is not a single episode of acute liver injury, but an imbalance in inflammatory repair after repeated injury. Chemical induction models such as carbon tetrachloride (CCl₄), thioacetamide (TAA), and dimethylnitrosamine can cause persistent hepatocyte necrosis, lipid peroxidation, and inflammatory responses, making them suitable for studying hepatic stellate cell activation and collagen deposition after toxic liver injury.

(2) Cholestasis and ductular reaction

The bile duct ligation model is used to simulate cholestatic liver injury. Bile acid accumulation can damage hepatocytes and bile duct epithelium, inducing ductular reactive proliferation, macrophage infiltration, and portal fibrosis. This model is suitable for studying cholestasis-related fibrosis, ductular reaction, and portal inflammation.

(3) Immune inflammation and metabolism-related fibrosis

Immune induction models are suitable for analyzing liver injury mediated by T cells, macrophages, and inflammatory factors. High-fat diet, choline-deficient diet, and fructose/alcohol combined interventions are more suitable for simulating fibrosis during the progression of metabolic dysfunction-associated steatohepatitis (MASH/NASH). Different models correspond to different etiologies, and model selection should not be based only on modeling speed.

1.2 Experimental Animals and Model Selection

Mice and rats are the most commonly used animals for hepatic fibrosis modeling. Mice are suitable for mechanistic studies, knockout models, or transgenic models. Rats are suitable for liver tissue sampling, serum biochemical testing, and long-term drug intervention. Model selection should consider research purpose, etiological matching, modeling duration, mortality, fibrosis location, histological features, and detection indicators.

Table 1 Selection Logic of Hepatic Fibrosis Animal Models

1.3 Common Inducing Factors and Modeling Positioning

Table 2 Common Inducing Factors and Modeling Positioning in Hepatic Fibrosis Animal Models

2 Chemically Induced Hepatic Fibrosis Models

2.1 Carbon Tetrachloride-Induced Model

(1) Model mechanism

After entering the liver, CCl₄ is mainly metabolized by the cytochrome P450 system to generate trichloromethyl radicals and related reactive intermediates, inducing lipid peroxidation, mitochondrial injury, and hepatocyte necrosis. Necrotic regions release damage-associated signals, activate Kupffer cells and inflammatory responses, and further promote the release of TGF-β1, PDGF, and inflammatory factors, causing quiescent hepatic stellate cells to transform into α-SMA-positive myofibroblast-like cells.

(2) Experimental interpretation

The CCl₄ model is relatively mature and suitable for studying toxic liver injury, oxidative stress, and HSC activation mechanisms. Its lesion severity is significantly affected by administration route, administration frequency, solvent, animal strain, and modeling duration. Evaluation should combine ALT, AST, liver tissue necrosis, inflammation score, Masson or Sirius Red staining, α-SMA, and Collagen I, rather than judging fibrosis severity only by serum transaminases.

2.2 Thioacetamide-Induced Model

(1) Model mechanism

After hepatic metabolism, TAA forms hepatotoxic intermediates that cause chronic hepatocyte injury, inflammatory infiltration, oxidative stress, and fibrotic deposition. Long-term TAA treatment can induce marked bridging fibrosis and, under some experimental conditions, gradually progress to cirrhosis-like changes.

(2) Experimental interpretation

The TAA model is suitable for studying chronic toxic liver injury, bridging fibrosis, and fibrotic progression. Compared with the CCl₄ model, the TAA model often shows more persistent inflammation and fibrosis accumulation, but animal tolerance and mortality are greatly affected by dose, administration cycle, and drinking water intake. If administered through drinking water, water intake and body weight changes should be recorded to avoid intra-group variation caused by intake differences.

2.3 Dimethylnitrosamine-Induced Model

Dimethylnitrosamine can induce hepatocyte injury, inflammatory responses, and fibrosis formation. The model progresses relatively rapidly but has strong toxicity and requires strict experimental safety, dose control, and animal condition monitoring. This model is not suitable as a routine preliminary screening model and is more often used in specific research designs requiring strong hepatotoxicity and rapid fibrotic responses.

Table 3 Comparison of Chemically Induced Hepatic Fibrosis Models

3 Cholestasis-, Immune-, and Metabolism-Related Models

3.1 Bile Duct Ligation Model

(1) Model mechanism

Bile duct ligation blocks bile drainage and causes bile acid accumulation in the liver, resulting in hepatocyte and bile duct epithelial injury. Ductular reactive proliferation, portal inflammation, macrophage infiltration, and HSC activation jointly promote portal fibrosis formation. This model is suitable for research on cholestatic liver disease, ductular reaction, and bile acid toxicity.

(2) Experimental interpretation

The BDL model progresses rapidly, but surgical operation greatly affects the results. Ligation site, bile duct injury extent, postoperative infection, bile leakage, and animal recovery status all affect model stability. Evaluation should combine ALP, total bile acids, bilirubin, CK19, portal inflammation, bile duct proliferation, and collagen deposition. It is not appropriate to summarize all lesions using only α-SMA or total collagen levels.

3.2 Immune Induction Models

(1) ConA-induced model

ConA can induce T cell-mediated acute immune liver injury, characterized by inflammatory factor release, hepatocyte necrosis, and immune cell infiltration. This model is more suitable for studying autoimmune hepatitis and inflammatory injury mechanisms. If it is used for fibrosis research, repeated stimulation or chronic modeling design is usually required; otherwise, it is more of an acute hepatitis model.

(2) Pig serum-induced model

Repeated pig serum injection can induce immune-related hepatic fibrosis and is mainly used to study chronic immune responses, inflammatory cytokines, and collagen deposition. This model is sensitive to immune response intensity and administration cycle. It is suitable for immune-mediated fibrosis research, but its etiology differs from toxic injury, cholestasis, or metabolic liver disease.

3.3 High-Fat Diet and NASH-Related Models

High-fat diet, MCD diet, and CDAHFD diet can be used to simulate steatohepatitis-related fibrosis. Lipotoxicity, insulin resistance, mitochondrial injury, Kupffer cell activation, and oxidative stress jointly promote HSC activation. The MCD diet can often rapidly induce steatosis and inflammation, but it does not fully match obesity and insulin resistance backgrounds. The high-fat diet is closer to metabolic conditions, but fibrosis formation is usually slower. The CDAHFD model is widely used in NASH fibrosis research and is suitable for observing the combined progression of steatosis, inflammation, and fibrosis.

3.4 Genetically Modified Models

Mdr2⁻/⁻ mice can spontaneously develop bile duct injury and portal fibrosis, making them suitable for research on bile duct lesions and cholestasis-related mechanisms. TGF-β1 overexpression models or pathway-related gene models can be used to directly analyze fibrotic signaling axes. The advantage of genetic models is clear mechanistic direction, while their limitations include high cost and single etiology. They cannot replace chemical, cholestatic, and metabolic models.

Table 4 Comparison of Cholestasis-, Immune-, and Metabolism-Related Hepatic Fibrosis Models

4 Mechanistic Evaluation of Hepatic Fibrosis Models

4.1 Hepatic Stellate Cell Activation

Hepatic stellate cells are the core effector cells in hepatic fibrosis formation. Quiescent HSCs mainly store vitamin A. After liver injury, stimulation by TGF-β1, PDGF, ROS, and inflammatory factors causes HSCs to transform into myofibroblast-like cells, characterized by increased α-SMA, Desmin, PDGFR-β, and Collagen I. The level of HSC activation directly affects ECM deposition and fibrotic progression.

4.2 TGF-β/Smad Pathway

After TGF-β1 binds to its receptors, it promotes Smad2/3 phosphorylation. Phosphorylated Smad2/3 forms a complex with Smad4 and enters the nucleus, inducing the expression of fibrosis-related genes such as COL1A1, COL3A1, α-SMA, and TIMP-1. Smad7 is a negative regulator of this pathway. The TGF-β/Smad axis is one of the most commonly studied signaling pathways in anti-fibrotic mechanism research. However, inhibiting this pathway alone may affect immune regulation, tissue repair, and tumor suppression-related functions.

4.3 Inflammation and NF-κB Pathway

In chronic liver injury, Kupffer cells, infiltrating monocytes, T cells, and neutrophils can release TNF-α, IL-1β, IL-6, and chemokines, activating NF-κB signaling and maintaining the inflammatory microenvironment. NF-κB not only participates in inflammatory amplification, but also affects HSC survival and anti-apoptotic status. Inflammatory indicators should be interpreted together with histological inflammation scores and fibrosis indicators.

4.4 Oxidative Stress and MAPK Pathway

CCl₄, alcohol, and lipotoxicity can all induce ROS accumulation, causing lipid peroxidation and mitochondrial dysfunction. Oxidative stress can activate ERK, JNK, and p38 MAPK pathways, promoting HSC proliferation, migration, and ECM synthesis. MDA, SOD, GSH, GSH-Px, and ROS detection are suitable for evaluating oxidative stress intensity, while p-ERK, p-JNK, and p-p38 can be used to analyze MAPK pathway activation status.

4.5 ECM Deposition and Imbalanced Matrix Degradation

The direct manifestation of hepatic fibrosis is excessive deposition of ECM components such as collagen, fibronectin, and proteoglycans. MMPs participate in matrix degradation, while TIMP-1/2 inhibit MMP activity and reduce ECM degradation. Collagen I, Collagen III, Fibronectin, hydroxyproline, MMP-2, MMP-9, and TIMP-1/2 can be jointly used to evaluate the balance between ECM synthesis and degradation.

5 Supplementary Applications of In Vitro and Ex Vivo Models

5.1 Hepatic Stellate Cell Models

LX-2 cells and primary hepatic stellate cells are commonly used for HSC activation mechanism studies and preliminary screening of anti-fibrotic drugs. TGF-β1, PDGF, lipopolysaccharide, oxidative stress stimulation, or conditioned medium can all induce HSC activation, characterized by increased α-SMA, Collagen I, TIMP-1, and Fibronectin. This model is suitable for mechanistic validation, but cannot fully simulate hepatic lobular structure, immune cell infiltration, or blood flow factors.

5.2 Hepatocyte Injury Models

HepG2, AML12, and primary hepatocytes can be used for research on CCl₄ metabolic toxicity, lipotoxicity, alcohol injury, and oxidative stress. Hepatocyte models are suitable for analyzing cell death, lipid deposition, mitochondrial injury, and inflammatory signaling, but they cannot independently represent hepatic fibrosis formation because the core effector cells of fibrosis are HSCs, and fibrosis requires interactions among hepatocytes, Kupffer cells, endothelial cells, and HSCs.

5.3 Co-Culture and Organoid Models

Hepatocyte-HSC co-culture, Kupffer cell-HSC co-culture, and liver organoid models can be used to analyze intercellular communication, inflammatory microenvironment, and fibrotic signal transmission. These models are closer to the tissue environment than single-cell models, but they still have difficulty fully simulating chronic injury, bile flow, blood flow shear stress, and systemic metabolic status.

Table 5 Application Positioning of In Vivo and In Vitro Models in Hepatic Fibrosis Research

6 Related Product and Material Selection

Table 6 Products Related to Liver Function Injury and Cholestasis Evaluation

Table 7 Products Related to Metabolism-Associated Hepatic Fibrosis Evaluation

7 Common Questions

7.1 How Should the CCl₄ Model and TAA Model Be Selected?

The CCl₄ model is more suitable for studying toxic liver injury, lipid peroxidation, and HSC activation mechanisms. Its modeling system is mature and suitable for preliminary screening of anti-fibrotic drugs. The TAA model is more suitable for studying chronic hepatotoxicity, bridging fibrosis, and cirrhosis-like progression, but it has higher requirements for controlling modeling duration and animal tolerance. If the research focus is oxidative stress and chemical toxic injury, CCl₄ can be prioritized. If the focus is chronic progression and bridging fibrosis, TAA can be considered.

7.2 Is the Bile Duct Ligation Model Suitable for All Hepatic Fibrosis Research?

No. The BDL model is more biased toward cholestasis and ductular reaction, and is suitable for research on bile acid toxicity, bile duct proliferation, portal inflammation, and cholestatic fibrosis. If the research focus is metabolism-associated steatohepatitis or toxic liver injury, BDL has insufficient etiological matching.

7.3 Why Do NASH-Related Fibrosis Models Usually Require a Long Modeling Period?

Metabolism-related fibrosis usually progresses through multiple stages, including steatosis, lipotoxicity, inflammatory response, HSC activation, and ECM deposition. The high-fat diet model is closer to obesity and metabolic syndrome backgrounds, but fibrosis formation is relatively slow. MCD and CDAHFD diets can accelerate inflammation and fibrosis, but nutritional status and body weight changes affect model interpretation.

7.4 Can Hepatic Fibrosis Models Be Evaluated Only by ALT and AST?

No. ALT and AST mainly reflect hepatocyte injury and do not directly represent fibrosis severity. Hepatic fibrosis evaluation should combine Masson or Sirius Red staining, hydroxyproline, α-SMA, Collagen I/III, and TGF-β/Smad-related indicators. If only transaminases are detected, acute injury can easily be misinterpreted as improvement or aggravation of fibrosis.

7.5 Does Increased α-SMA Equal Aggravated Fibrosis?

Increased α-SMA indicates activation of HSCs or myofibroblast-like cells, but it cannot independently represent final fibrosis severity. Aggravated fibrosis should also show increased collagen deposition, elevated hydroxyproline, expanded Masson or Sirius Red-positive area, and increased expression of ECM-related genes or proteins.

7.6 Why Is the TGF-β/Smad Pathway a Core Pathway in Hepatic Fibrosis Research?

The TGF-β/Smad pathway directly promotes the expression of fibrosis-related genes such as α-SMA, COL1A1, COL3A1, and TIMP-1, making it an important driving axis for HSC activation and ECM deposition. Most anti-fibrotic studies evaluate this pathway, but experimental interpretation should note that TGF-β also participates in immune regulation, tissue repair, and tumor-related processes.

7.7 Can In Vitro HSC Models Replace Animal Models?

They cannot fully replace animal models. LX-2 or primary HSC models are suitable for studying HSC activation, TGF-β/Smad signaling, and ECM synthesis mechanisms, but they cannot simulate liver tissue structure, inflammatory cell infiltration, cholestasis, blood flow changes, or systemic metabolic status. Key efficacy conclusions still require validation by animal models and histological results.

7.8 How Should Endpoints Be Set for Hepatic Fibrosis Efficacy Evaluation?

Efficacy evaluation should cover liver function, histology, collagen deposition, HSC activation, inflammation, and oxidative stress. Recommended combinations include ALT/AST, HE staining, Masson or Sirius Red staining, hydroxyproline, α-SMA, Collagen I, TGF-β1, and inflammatory factors. If NASH-related fibrosis is studied, metabolic evaluation indicators such as TG, TC, LDL-C, HDL-C, cholesteryl esters, and NAS score should also be included.

Hepatic fibrosis animal models should be selected according to toxic injury, cholestasis, immune inflammation, metabolic abnormalities, or specific pathway mechanisms. CCl₄ and TAA models are suitable for toxic injury and anti-fibrotic efficacy research, the BDL model is suitable for cholestatic fibrosis, dietary models are suitable for studying metabolism-associated steatohepatitis progression, and genetic models are used to analyze specific molecular pathways.

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