非酒精性脂肪肝 z-bo 2021-01-07 405

　　Nonalcoholic fatty liver disease (NAFLD, Nonalcoholic fatty liver disease) is a disease in which too much fat is stored in the liver. This kind of fat accumulation is not caused by heavy drinking. When heavy drinking causes liver fat accumulation, this condition is called alcoholic liver disease. There are two types of NAFLD: simple fatty liver and nonalcoholic steatohepatitis (NASH, nonalcoholic steatohepatitis). NASH is one of the most important causes of liver disease in the world. It has become the most common liver disease in developed countries, and its incidence in developing countries is also increasing year by year.

　　 is called "Year of NASH" in 2019. NASH drug candidates of 4 pharmaceutical companies have entered the phase III clinical development stage, and nearly a hundred drugs have entered the clinical trials (Clinical trials). Up to now, there are no approved effective NASH treatment drugs and programs. The FDA stated in a guidance document that successful treatment of NASH, improvement in fibrosis, or a combination of both are potential acceptable endpoints for approved NASH therapy. Nevertheless, which endpoint and which drug proved useful in the field first, there are still no accepted results.

　　 The main risk factors for NASH include obesity, type II diabetes, dyslipidemia and metabolic syndrome. In addition to liver steatosis, NASH also includes swelling, inflammation and fibrosis. In the middle and late stages of NASH, inflammation and liver cell damage can lead to liver fibrosis or scar formation, which can further develop into liver cirrhosis or liver cancer. Based on these characteristics, the development of animal models has also made great progress in recent years, especially in Liver fibrosis, which eventually develops to the stage of Hepatocellular carcinoma. Liver fibrosis refers to the accumulation of large amounts of scar (fibrosis) tissue in the liver due to repeated or long-term injury or inflammation. Although some animal studies have shown the potential of the liver to regenerate or heal itself, once the human liver is damaged, the liver usually does not heal. However, changes in drugs and lifestyle can help prevent the deterioration of fibrosis, and mouse models can be used for preclinical evaluation. The common reference index for liver fibrosis is the human liver fibrosis METAVIR scoring system, which divides fibrosis into 4 levels [1]. The fibrosis stages are from F0 to F4: F0: no fibrosis; F1: portal fibrosis without septal; F2: portal fibrosis with a small interval; F3: large interval without cirrhosis; F4: liver cirrhosis.

　　NASH mouse models can be divided into 4 categories: (1) diet induction model, (2) chemical substance induction model, (3) gene editing model, (4) the first two, or a comprehensive model combining the three methods The advantage of this type of model is that the pathological features of the disease model are the most typical and comprehensive. Since fibrosis improvement is a potential acceptable endpoint for approved NASH therapy, this article focuses on the fibrosis model.

　　1. Dietary induction model

　　Using high-fat, high-sugar, and/or high-cholesterol feed, or MCD (methionine choline deficient feed), can successfully induce obesity or the NASH model of nutritional deficiency. For example, for C57BL/6 mice, using Research Diets' D12492 feed (high-fat feed, 60% fat for energy) can induce obesity and basically no fibrosis. Adopt D09100310 feed (NASH feed, instead of FDA prohibits the use of feed AMLN DIET containing trans fatty acids, 40% fat for energy, mainly palm oil, 20% fructose and 2% cholesterol), 12 to 16 weeks to induce steatosis; 20 Inflammatory bodies are induced in ~26 weeks; fibrosis is induced in 26~34 weeks. The characteristic of the diet-induced NASH model is that it simulates the characteristics of obesity, type II diabetes, dyslipidemia and metabolic syndrome, but the liver fibrosis in mice is relatively weak, and HCC basically does not occur. In the diet induction model, adding a high proportion of fructose, glucose and cholesterol, the liver fibrosis of mice can reach the F2 standard (moderate moderate fibrosis), which is a preclinical mouse NASH model that is currently widely used .

　　2. Chemical substance induction model

　　Chemical-induced liver damage and liver fibrosis are very common, and have been used to construct mouse liver fibrosis models. The probability of such models developing to liver cirrhosis and liver cancer is also high [2]. Commonly used inducers are carbon tetrachloride (CCL4), thioacetamide (TAA) and streptozotocin (STZ). CCL4 and TAA are commonly used in adult mice, and STZ is used in newborn mice. The toxicity mechanism of CCl4 and TAA is not fully understood, but it involves the uptake and transformation of CCl4 and TAA by hepatocytes. The metabolites cause oxidative necrotic inflammation and the excessive activation and proliferation of collagen-secreting cells. Using neonatal mice with a specific background, combined with high-fat diet and STZ induction, a "STAM" mouse model can be established, which exhibits the characteristics of NASH at 8 weeks and fibrosis at 12 weeks, and finally nearly 100% of male rats show HCC[3].

　　3. Gene editing model

　　 Genes related to metabolism, or proteins that express damage to liver cells, researchers have developed many NASH gene editing models (see review [2]). Here are three types of NASH gene editing models, combined with a high-fat diet, NASH models can be obtained faster and in a higher proportion, with obvious fibrosis characteristics. The low-density lipoprotein receptor (Ldlr) gene family is composed of cell surface proteins involved in specific ligand receptor-mediated endocytosis. The serum cholesterol level of Ldlr KO mice is 2 to 4 times that of wild mice. Combined with high-fat (HFD-fed) feeding LDLR ko mice, it can induce metabolic syndrome characterized by obesity and hypercholesterolemia, hypertriglyceridemia and insulin resistance symptoms; similar to the human NASH model. model.

　　Mc4r (melanocortin 4 receptor) is a protein-coding gene. Mc4r homozygous knockout mice weighed up to 30 g at 6 to 8 weeks, showing obvious obesity. Combined with high-fat (HFD-fed) feeding Mc4r KO mice, it is easier to show liver steatosis, liver fibrosis and hepatocellular carcinoma, similar to the model characterized by the human NASH model [5].

　　Urokinase-type plasminogen activator (uPA) plays an important role in the degradation of extracellular matrix. uPA is toxic to liver cells and can cause liver damage. As early as 1992, Sandgren et al. made Alb-uPA transgenic mice to study liver cancer. However, due to the liver toxicity of uPA, Alb-uPA mice will show symptoms of hemolysis within 4 days of birth, and most of them will eventually die. In 2000, Weglarz et al. made improvements [6] and made MUP-uPA transgenic mice, using MUP enhancer/promoter to specifically express uPA in hepatocytes for studying hepatocyte transplantation. Similar to Alb-uPA, the expression of uPA will gradually decrease due to gene loss; the difference is that MUP-uPA mice will not begin to express uPA until 2-4 weeks, thereby reducing the mortality of newborn mice. A major study published in Nature in 2017 [7], MUP-uPA mice were backcrossed to the C57BL/6 background through 10 generations, fed a high-fat and high-sugar diet (WD-SW), and showed typical results for 3 months. NASH lesions, such as liver steatosis, steatohepatitis (including ballooning hepatocytes and Mallory-Denk bodies), after 6 to 11 months, 90% of mice develop HCC. From RNA-seq and histochemical data, it is proved that this model is the best model reported to simulate human NASH and HCC.

　　 Due to the wide range of causes of NASH, it is recognized that it is a heterogeneous disease, which also brings difficulties to the selection of models. Relieving fibrosis as an acceptable end point for potentially approved NASH therapy makes fibrosis indicators in animal models more important.