2.1 Alcoholic Liver Disease (ALD) Models

The impact of alcohol consumption on the onset and progression of alcoholic liver disease (ALD) is directly proportional to both the quantity consumed and the duration of exposure^[2]. The most commonly used animal models for studying chronic alcohol abuse are the Lieber-DeCarli liquid diet model, the intragastric ethanol infusion model, and the alcohol in drinking water model^[2]. The Lieber-DeCarli liquid diet method involves adding alcohol to a complete liquid diet for feeding. Researchers can adjust the nutritional elements according to their needs. The animals receive no food or drink other than the liquid diet containing ethanol. This modeling approach leads to early liver injury but does not progress to the most severe stages of ALD. The intragastric ethanol infusion model injects alcohol directly into the stomach. This method can sustain a high blood alcohol concentration. It also produces significant liver damage. However, this model is expensive. Additionally, it requires highly trained personnel to perform the procedure.

The alcohol in drinking water model most closely mimics the alcohol-dependent human drinking pattern. This model can be easily manipulated. It can also be investigated in conjunction with other cofactors (Such as iron, high-fat diet, vitamin supplements, LPS injections) ^[4].

Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by excess fat accumulation in the liver. This occurs in patients without a history of alcohol abuse. NAFLD is classified into two categories, simple steatosis and nonalcoholic steatohepatitis (NASH). At present, genetic models, dietary models and chemical models have been widely used in preclinical studies of NAFLD^[5][6].

In NASH, there is not only steatosis but also intra-lobular inflammation and hepatocellular ballooning, often accompanied by progressive fibrosis. Persistent NASH may evolve into cirrhosis and hepatocellular carcinoma. Dietary models are commonly used in NASH research. The C57BL/6 strain in mice is generally preferred for research. The Wistar and Sprague Dawley strains in rats are also commonly used. These strains have an intrinsic predisposition to develop obesity, type 2 diabetes (DM2), and non-alcoholic fatty liver disease (NAFLD). The time of onset of NAFLD varies based on several factors. The degree of NAFLD and accompanying metabolic features is also influenced by these factors. These factors include species, strain, sex, composition of gut microbiota, and the dietary intervention employed^[5].

The main causes of liver fibrosis include alcoholism, chronic hepatitis virus infection and non-alcoholic steatohepatitis, which can progress to cirrhosis and even hepatocellular carcinoma (HCC). The chemical injury liver fibrosis model is used to induce the formation of liver fibrosis. This is achieved by using chemical drugs that enter hepatocytes and produce toxic metabolites. These metabolites cause persistent liver injury. Currently, this model preparation mainly uses ethanol, carbon tetrachloride (CCl4), thioacetamide (TAA), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), or other liver toxins to induce liver fibrosis models^[8]. In most cases, intraperitoneal injection of these chemicals will induce liver fibrosis within a relatively short period. In contrast, oral administration or inhalation requires a longer development period.

CCl4 is the most widely used hepatotoxin in the study of liver fibrosis and cirrhosis in rodents. The damage it causes is similar to the important characteristics of human liver fibrosis, including inflammation, regeneration and fibrogenesis. It is often used to study acute liver injury, advanced liver fibrosis and fibrosis reversal. The CCl4 model can be applied to both rats and mice; however, mice are preferred due to their higher metabolic rate of CCl4 compared to rats. Additionally, the susceptibility of mice to CCl4-induced liver fibrosis is strain-dependent, with BALB/c mice showing more liver fibrosis upon CCl4 administration than C57BL/6 and DBA/2 mice^[9]. The CCl4-induced liver fibrosis model is highly reproducible and therefore an excellent candidate for drug screening.

DEN is a genotoxic carcinogen that is widely used to induce HCC in rodents. The time to HCC formation after a single dose of DEN is not only dose-dependent but also dependent on factors such as age, sex, and genetic background. Studies have shown that female mice are largely resistant to DEN-driven hepatocarcinogenesis^[18]. When mice are repeatedly exposed to DEN for an extended period, 100% of the male mice develop hepatic tumors. In contrast, only 30% of the female mice develop these tumors. For adult mice, a single administration of DEN may not be sufficient to induce hepatocellular carcinoma (HCC). In such cases, additional tumor promoters are required, including carbon tetrachloride (CCl4), phenobarbital, and high-fat diet feeding^[19]. In addition, the combined induction of DEN and CCl4 can improve the time-consuming disadvantage of traditional chemical induction and better simulate the development process of human liver cancer.

Of course, in addition to the common modeling methods of liver diseases at different development stages mentioned above, surgery, transgenic, immunization, diet and chemical methods can also be used to model liver diseases^[8][9]. For example, common bile duct ligation (BDL) can cause cholestatic injury and periportal biliary fibrosis; CCl4 can enhance the progression of NASH and fibrosis induced by HFD; and Mdr2-deficient mice can develop HCC at 4 to 6 months of age. Researchers should select models aligned with experimental objectives and consult literature for optimization.