Feed, method for constructing non-alcoholic steatohepatitis animal model and application

Abstract

The invention discloses a feed, a method for constructing a non-alcoholic steatohepatitis animal model and application, and relates to the technical field of non-alcoholic steatohepatitis animal model construction. The feed cost is remarkably reduced, the modeling speed is increased, and the animal model constructed by adopting the formula is free of systemic emaciation and better conforms to the clinical manifestation of natural morbidity. The invention provides a reliable, economic and easy-to-popularize animal model basis for research and development of NASH related drugs.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology for non-alcoholic steatohepatitis (NAH). Specifically, it relates to a feed, a method for constructing an animal model of NHA, and its application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by hepatocellular steatosis and lipid accumulation in the absence of a history of excessive alcohol consumption. Its inflammatory subtype, non-alcoholic steatohepatitis (NASH), in addition to widespread inflammation and significant hepatocellular death, is often accompanied by liver fibrosis and can further develop into cirrhosis and hepatocellular carcinoma (HCC). It is estimated that more than 25% of the global population suffers from NAFLD and is at risk of progressing to NASH, placing a heavy burden on social healthcare and the economy. Research indicates that NASH is gradually replacing viral hepatitis as the leading cause of HCC. The pathogenesis of NASH involves complex interactions among multiple factors, including diet, gut microbiota, various metabolic organs (such as the pancreas, adipose tissue, and skeletal muscle), and the immune system. Diet-induced obesity is considered one of the key risk factors driving the development of NASH. Therefore, NASH animal models based on dietary intervention are widely considered to be the best means of simulating the pathological characteristics and dynamic evolution of human NASH.

[0003] In laboratory animals with conventional genetic backgrounds, the mainstream diet-induced NASH models currently fall into two main categories. The first category is the high-fat / high-carbohydrate Western diet (WD) model, such as the AMLN (Amylin LiverNASH) model and the GAN (Gubra Amylin NASH) model. These models typically consist of a high-fat diet (40 kcal fat, sourced from Primex trans fatty acids such as soybean oil and lard, or non-Primex trans fatty acids such as palm oil), high-sugar diet (22% fructose + 10% sucrose), 2% cholesterol, and a basal diet. They exhibit high similarity to human NASH at the histopathological and transcriptomic levels. The second category is the nutrient-deficient diet model, mainly including the choline-deficient high-fat diet (CDHFD), the choline-deficient L-amino acid-defined high-fat diet (NMCDH), and the methionine-choline-deficient diet (MCD) model. This type of model is based on a high-fat diet (45%–60% kcal fat, often using non-Primex trans fatty acids such as palm oil). It induces NASH by restricting the intake of choline and / or methionine or by adjusting the amino acid composition. It is characterized by significant liver histological changes and a short modeling period, and is widely used.

[0004] The advantages of Western dietary models include: easy control of animal experiments, high model success rate, low mortality rate, good reproducibility, simple modeling methods, progressive disease development and slow reversal after drug withdrawal, making them suitable for drug intervention development; at the same time, supplementing high fructose on a high-saturated fat and cholesterol diet can comprehensively simulate NASH characteristics and accelerate the progression of NAFLD to NASH. Its disadvantages include: long modeling period (usually more than 12 weeks to show typical NASH pathological manifestations); milder degree of liver fibrosis and inflammation, inconsistent steatosis, and often lack of evidence of perisinusoidal or central fibrosis; some models, such as high-fat, high-fructose diets, are difficult to induce significant obesity, limiting their application. The advantages of nutritional deficiency dietary models include: the MCD model can reproduce NASH histological changes highly similar to those in humans, such as severe steatosis, ballooning cell degeneration, and significantly elevated serum ALT; this model shows more pronounced responses to endoplasmic reticulum stress, oxidative stress, and autophagy, and can induce severe liver damage (including inflammation and fibrosis) within 8 weeks, making it suitable for rapid assessment of NASH mechanisms. Its disadvantages include: model animals typically exhibit weight loss, lack of obesity and insulin resistance, which does not match the metabolic characteristics of human NAFLD; the composition of MCD and NMCDH diets differs significantly from the daily human diet, making them more suitable for drug screening than mechanism discovery; long-term feeding of CDHFD only induces mild fibrosis, making it unsuitable for discovering fibrosis processes. Furthermore, these model diets are currently based on purified formulas, making them expensive (over 300 RMB / kg domestically and over 1000 RMB / kg internationally), and the processing technology presents significant barriers, further limiting their widespread application.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a feed, a method for constructing an animal model of non-alcoholic steatohepatitis (NASH), and its application to significantly reduce feed costs (by more than 50%), improve modeling speed, and provide a reliable, economical, and easily scalable animal model basis for the development of NASH-related drugs.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a feed for constructing an animal model of non-alcoholic steatohepatitis, comprising the following ingredients by weight percentage: 44-62% corn, 10-21.25% wheat, 10-15% wheat middlings, 1.2-1.4% lysine, 0.5-0.6% threonine, 7-8% glycine, 0.66-0.85% arginine, 0.3-0.35% histidine, 0.5-0.6% isoleucine, 0.8-0.95% leucine, 0.5-0.6% phenylalanine 0.15-0.2% tryptophan, 0.6-0.7% valine, 2-2.5% CaHPO42H2O, 1.5-1.8% limestone powder, 1-1.2% disodium hydrogen phosphate, 0.5-0.6% iodized salt, 2-2.5% cholesterol, 10-15% lard, 2-5% soybean oil, 0.1% trace element premix, and 0.1% compound vitamins.

[0008] Secondly, this invention provides the application of feed in constructing an animal model of non-alcoholic steatohepatitis.

[0009] Thirdly, the present invention provides a method for constructing an animal model of non-alcoholic steatohepatitis, comprising the following steps: The animals to be modeled were fed the above-mentioned feed to obtain an animal model of non-alcoholic steatohepatitis.

[0010] Fourthly, the present invention provides a method for constructing an animal model of non-alcoholic steatohepatitis (NAHH). The constructed NHAH animal model is used in screening drugs for the prevention or treatment of at least one of the following diseases: non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma.

[0011] The present invention has the following beneficial effects: This invention combines feed formulation technology and nutritional principles, integrates the advantages of Western dietary models and nutritional deficiency models, and utilizes natural feed ingredients to design a novel non-alcoholic steatohepatitis (NASH) model feed that is deficient in methionine and choline and high in fat content.

[0012] Compared to the methionine-choline deficiency diet (MCD) model, this invention can successfully establish a NASH animal model within 6 weeks. However, the body weight and feed intake of animals in the MCD group decreased significantly during the experiment, while the feed intake of animals in the experimental group using the formula of this invention maintained steady growth, and the animals did not experience systemic emaciation, which is more consistent with the clinical manifestations of natural disease. Compared to the high-fat / high-carbohydrate Western diet (WD), the time required to establish a NASH model with this invention is significantly shortened (only 6 weeks), while the WD model requires 20 weeks. In addition, the feed formula of this invention is significantly cheaper than foreign MCD and WD formulas, with a cost reduction of more than 50%.

[0013] The feed production process provided by this invention is simple and can be completed using conventional feed pelleting equipment, while foreign MCD and WD formulas cannot achieve this production process.

[0014] This invention ingeniously achieves a more severe pathological phenotype than purified feed by regulating key nutrients within the framework of a natural diet, while also possessing significant advantages such as low cost and simple process. Multi-omics data (transcriptomics, metabolomics) and pathological images show that the induced NASH model exhibits complete disintegration of the liver lobule structure, accompanied by diffuse ballooning degeneration, dense foam cell infiltration, and extensive necrosis and inflammation. Comprehensive transcriptomics and metabolomics analyses have elucidated the biological mechanisms by which this model induces NASH (such as retinol metabolism and enrichment of exogenous substance metabolic pathways) at the gene expression and metabolite levels. These multi-omics data not only validate the model's effectiveness but also provide valuable data resources and guidance for subsequent drug target discovery and mechanism research. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Statistical results of changes in body weight (A), liver weight (BC), and serum parameters (DG) in mice fed MCD, WD, and NMCDH diets; Figure 2 Graph showing changes in OGTT (AB), ITT (CD), and liver structure (EG) in mice fed MCD, WD, and NMCDH diets; Figure 3The transcriptome analysis results of liver tissues from mice fed different diets in Experiment Example 1 are shown in Figure A: Statistical results of gene upregulation and downregulation between different treatment groups; Figure B: Volcano plot. Figure 4 A graph showing the results of gene ontology (GO) enrichment analysis on differentially expressed genes; Figure 5 Figure showing the results of gene ontology (GO) enrichment analysis for differentially expressed genes in the WD and NMCDH groups; Figure 6 The KEGG pathway enrichment analysis revealed the different metabolic perturbations among the experimental groups. Figure 7 A comparison chart of RNA sequencing and qPCR results; Figure 8 The liver metabolomics profile was analyzed using non-targeted metabolomics techniques (A: PCA analysis diagram, B: comparative analysis results of differential metabolites (DMs)). Figure 9 The 20 metabolites that showed the most significant changes between NMCDH and the control group; Figure 10 The 20 metabolites that showed the most significant changes between the NMCDH and MCD groups; Figure 11 The graph shows the results of the most significantly enriched pathways between the MCD group and the control group, and between the NMCDH group and the control group. Figure 12 The diagram shows the results of comparing the most significantly enriched pathways between the MCD group and the NMCDH group. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0019] In a first aspect, the present invention provides a feed for constructing an animal model of non-alcoholic steatohepatitis, comprising the following ingredients by weight percentage: 44-62% corn, 10-21.25% wheat, 10-15% wheat middlings, 1.2-1.4% lysine, 0.5-0.6% threonine, 7-8% glycine, 0.66-0.85% arginine, 0.3-0.35% histidine, 0.5-0.6% isoleucine, 0.8-0.95% leucine, 0.5-0.6% phenylalanine 0.15-0.2% tryptophan, 0.6-0.7% valine, 2-2.5% CaHPO42H2O, 1.5-1.8% limestone powder, 1-1.2% disodium hydrogen phosphate, 0.5-0.6% iodized salt, 2-2.5% cholesterol, 10-15% lard, 2-5% soybean oil, 0.1% trace element premix, and 0.1% compound vitamins.

[0020] The feed provided by this invention exhibits significant comprehensive advantages in establishing a mouse NASH model, primarily due to its ability to efficiently and synchronously simulate the core pathological and metabolic characteristics of human NASH. Compared to the MCD diet, which leads to severe weight loss, and the WD diet, which induces liver damage more slowly, the non-alcoholic steatohepatitis animal model provided by this invention induces significant liver weight gain and an extremely high liver-to-body ratio without causing severe systemic emaciation, better simulating the clinical condition of human patients experiencing liver-specific damage in the context of obesity metabolism. This model can induce extremely severe liver pathological changes, including complete lobular disintegration, diffuse ballooning degeneration, dense foam cell infiltration, and extensive necrosis and inflammation, accompanied by a significant liver fibrosis phenotype, which is confirmed by its gene expression profile specifically enriched in collagen tissue. At the molecular level, NMCDH induces the most extensive transcriptome reprogramming and the most intense metabolic perturbations. Its specifically enriched pathways of retinol metabolism and exogenous substance metabolism provide rich insights into the mechanisms of NASH. Through comparison with clinical data, the formulation of this invention possesses more dimensions of data simulating clinical conditions.

[0021] The feed provided by this invention is suitable for drug development, long-term toxicity testing and therapeutic studies, large-scale drug screening, and in-depth exploration of NASH mechanisms based on multi-omics data in the late stage of NASH fibrosis. In summary, the NASH model constructed based on the feed provided by this invention can construct a severe NASH phenotype that simultaneously encompasses severe steatosis, significant inflammatory necrosis, active fibrotic processes, and metabolic disorders in a relatively short period of time, making it a more comprehensive, reliable, and translationally valuable preclinical research tool.

[0022] In a preferred embodiment of the present invention, the feed comprises the following raw materials by weight percentage: 48.59% corn, 10% wheat, 10% wheat middlings, 1.2% lysine, 0.5% threonine, 7% glycine, 0.66% arginine, 0.3% histidine, 0.5% isoleucine, 0.8% leucine, 0.5% phenylalanine, 0.15% tryptophan, 0.6% valine, 2% CaHPO4 2H2O, 1.5% limestone powder, 1% disodium hydrogen phosphate, 0.5% iodized salt, 2% cholesterol, 10% lard, 2% soybean oil, 0.1% trace element premix, and 0.1% compound vitamins.

[0023] The trace element premix includes manganese sulfate, copper sulfate, zinc sulfate, and calcium iodate added to corn starch. The mass ratio of manganese sulfate, copper sulfate, zinc sulfate, calcium iodate, and corn starch in the trace element premix is ​​0.185:0.03:0.03:0.013:0.742, respectively.

[0024] Each gram of multivitamin contains the following ingredients: 12250 IU of vitamin A, 1400 IU of vitamin D, 105 IU of vitamin E, 5.25 mg of vitamin K, 0.175 mg of biotin, 7 mg of folic acid, 78.75 mg of niacin, 29.75 mg of pantothenic acid, 17.5 mg of vitamin B2, 14 mg of vitamin B1, 10.5 mg of vitamin B6, and 0.035 mg of vitamin B12.

[0025] Preferably, the compound vitamins are selected from the vitamin premix of Zhonghua Multivitamin.

[0026] Secondly, this invention provides the application of feed in constructing an animal model of non-alcoholic steatohepatitis.

[0027] Thirdly, the present invention provides a method for constructing an animal model of non-alcoholic steatohepatitis, comprising the following steps: The animals to be modeled were fed the above-mentioned feed to obtain an animal model of non-alcoholic steatohepatitis.

[0028] In a preferred embodiment of the present invention, the animal model of non-alcoholic steatohepatitis has at least one of the following phenotypes: (1) The animal did not exhibit systemic emaciation; (2) The animal's liver weight increased; (3) The liver-to-body ratio of animals increases; (4) Elevated serum ALT and AST levels; (5) It lowered the blood sugar level in animals; (6) The animal's liver is significantly enlarged and pale in color; (7) Animal hepatocytes exhibit at least one of the following phenomena: complete lobular structure disintegration, diffuse ballooning degeneration, lipid droplets of varying sizes, dense foam cell infiltration, extensive necrosis with obvious inflammatory aggregation, obvious hepatocyte ballooning degeneration, and liver fibrosis. (8) Metabolism of retinol with specific enrichment and metabolism of exogenous substances with specific enrichment.

[0029] In a preferred embodiment of the present invention, the exogenous substance is selected from at least one of alanine, aspartic acid, glutamic acid, tryptophan and glutathione.

[0030] In a preferred embodiment of the present invention, the non-human mammal is selected from mice, rats, pigs, dogs, rabbits, guinea pigs, or monkeys.

[0031] In a preferred embodiment of the present invention, when the animal to be modeled is a mouse, mice aged 6-14 weeks are fed the above-mentioned feed for 6 weeks.

[0032] The feed provided by this invention can construct an animal model of severe NASH phenotype that simultaneously encompasses severe steatosis, significant inflammatory necrosis, active fibrosis, and metabolic disorders in a relatively short period of time.

[0033] In a preferred embodiment of the present invention, the animal is fed 5-8g of feed per day.

[0034] Fourthly, the present invention provides a method for constructing an animal model of non-alcoholic steatohepatitis (NAHH). The constructed NHAH animal model is used in screening drugs for the prevention or treatment of at least one of the following diseases: non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma.

[0035] Example 1 This embodiment provides a feed for constructing an animal model of non-alcoholic steatohepatitis, comprising the following ingredients by weight percentage: 48.59% corn, 10% wheat, 10% wheat middlings, 1.2% lysine, 0.5% threonine, 7% glycine, 0.66% arginine, 0.3% histidine, 0.5% isoleucine, 0.8% leucine, 0.5% phenylalanine, 0.15% tryptophan, 0.6% valine, 2% CaHPO42H2O, 1.5% limestone powder, 1% disodium hydrogen phosphate, 0.5% iodized salt, 2% cholesterol, 10% lard, 2% soybean oil, 0.1% trace element premix, and 0.1% compound vitamins.

[0036] The trace element premix was purchased from the Sichuan Academy of Animal Sciences, and the compound vitamins were purchased from the Sichuan Academy of Animal Sciences.

[0037] 1 kg of feed contains 0.1% trace element premix, which includes manganese sulfate, copper sulfate, zinc sulfate and calcium iodate added to corn starch. The composition table is as follows.

[0038]

[0039] Each gram of multivitamin contains the following ingredients: 12250 IU of vitamin A, 1400 IU of vitamin D, 105 IU of vitamin E, 5.25 mg of vitamin K, 0.175 mg of biotin, 7 mg of folic acid, 78.75 mg of niacin, 29.75 mg of pantothenic acid, 17.5 mg of vitamin B2, 14 mg of vitamin B1, 10.5 mg of vitamin B6, and 0.035 mg of vitamin B12.

[0040] The content of key ingredients in the above formula is shown in the table below:

[0041] The key components mentioned above are the actual measured content of the ingredients in the feed. The methionine and cystine content is the total content of these two amino acids actually measured in the feed; these two ingredients were not added separately to the raw materials.

[0042] Except for the nutritional requirements of methionine, cystine, and choline, the other nutritional requirements are designed according to the nutritional standards for maintenance feed of rats and mice (GB14923.4-2010).

[0043] According to the latest national standards, the crude fat requirement is 4%, and the feed design level provided by this invention is 14.67% (with 10% lard added to the formula); the methionine + cystine requirement is 0.53%, and the threonine design level in this formula is 0.29%; the choline requirement is 1250mg / kg, and the design level in this formula is 527mg / kg; in addition, 2% cholesterol is added to the formula.

[0044] Example 2 This embodiment provides a method for constructing an animal model of non-alcoholic steatohepatitis, which includes the following steps: Non-alcoholic steatohepatitis (NAH) animal models were obtained by feeding the animals to be modeled with the diet described in Example 1 above. This animal experiment was approved by the Animal Protection Advisory Committee of the Laboratory Animal Invention Institute of Sichuan Provincial People's Hospital and Sichuan Academy of Medical Sciences, affiliated with the University of Electronic Science and Technology of China, approval number AE2024010. Male wild-type (WT) C57BL / 6 mice were purchased from Chengdu Jicui Yaokang Biotechnology Co., Ltd.

[0045] Male wild-type mice were used in the experiment starting at 14 weeks of age, fed the diet described in Example 1 for 6 weeks. Mice were housed in an SPF-grade animal center, 5 mice per cage, with controlled temperature (21-25°C) and humidity (60-70%), and a 12-hour / 12-hour light / dark cycle. The experiment began after one week of acclimatization. All mice had free access to water and food. Body weight and feed intake were measured weekly.

[0046] Example 3 This embodiment provides a feed for constructing an animal model of non-alcoholic steatohepatitis, comprising the following ingredients by weight percentage: 46.09% corn, 12.5% ​​wheat, 10% wheat middlings, 1.2% lysine, 0.5% threonine, 7% glycine, 0.66% arginine, 0.3% histidine, 0.5% isoleucine, 0.8% leucine, 0.5% phenylalanine, 0.15% tryptophan, 0.6% valine, 2% CaHPO42H2O, 1.5% limestone powder, 1% disodium hydrogen phosphate, 0.5% iodized salt, 2% cholesterol, 10% lard, 2% soybean oil, 0.1% trace element premix, and 0.1% compound vitamins.

[0047] Comparative Example 1 MCD feed formulations from abroad.

[0048]

[0049] Comparative Example 2 The formula for WD (60% fat reduction) from overseas is as follows:

[0050] Experimental Example 1 This experiment compared mouse models of non-alcoholic steatohepatitis constructed with different diets, tested serum parameters, performed biochemical analyses of the liver and feces, conducted oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) analyses, calculated liver histology and non-alcoholic steatohepatitis activity score (NAS), and performed transcriptomic and non-targeted metabolomics analyses.

[0051] The specific experimental steps are as follows: 1. Animal experiments This animal experiment was approved by the Animal Protection Advisory Committee of the Institute of Laboratory Animal Invention, Sichuan Provincial People's Hospital Affiliated to the University of Electronic Science and Technology of China and Sichuan Academy of Medical Sciences, approval number AE2024010. To simulate human non-alcoholic steatohepatitis (NASH), mice in the MCD group and NMCDH (methionine-choline-deficient high-fat diet in a natural diet) group were started at 14 weeks of age, matching their final body weight to that of the 16-week-old control and WD (Western diet) groups to minimize the confounding effect of age on metabolic outcomes. Therefore, male wild-type (WT) C57BL / 6 mice (purchased from Chengdu Jicui Yaokang Biotechnology Co., Ltd.) were divided into the following four groups: Control group (n=30, starting at 4 weeks of age) fed a normal diet for 16 weeks; MCD group (n=30, starting at 14 weeks of age) fed a diet for 6 weeks; WD group (n=30, starting at 4 weeks of age) fed a diet for 16 weeks; and NMCDH group (n=30, starting at 14 weeks of age) fed a diet for 6 weeks.

[0052] Mice were housed in an SPF-grade animal center, five mice per cage, with controlled temperature (21-25℃) and humidity (60-70%), and a 12-hour / 12-hour light / dark cycle. Experiments began after one week of acclimatization. All mice had free access to water and food. Body weight and feed intake were measured weekly.

[0053] 2. Analysis and Measurement 2.1 Sample Collection and Tissue Preparation At the end of the experiment, mice were anesthetized and blood was collected via enucleation. Serum was then separated by centrifugation for serum parameter determination. Liver and fresh feces were rapidly removed, weighed, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis. Mouse liver tissue was also fixed with 4% paraformaldehyde and routinely embedded in paraffin. One animal was randomly selected from each of the six treatment groups (five mice per cage) for sample collection and downstream analysis, resulting in a final n=6 mice per group to ensure biological reproducibility.

[0054] 2.2 Biochemical Analysis Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) were measured using appropriate commercial kits (Nanjing Jiancheng Biotechnology Invention Institute, China) and detected by spectrophotometry using a Mindray BS-420 fully automated biochemical analyzer (Shenzhen, China).

[0055] 2.3 Analysis of oral glucose tolerance test (OGTT) and insulin tolerance test (ITT).

[0056] At 20 weeks of age, tail vein blood samples were measured using a portable blood glucose meter according to a pre-defined protocol (Huang et al., 2022), and oral glucose tolerance tests (OGTT) and intraperitoneal glucose tolerance tests (ITT) were performed over 120-minute time intervals (0, 15, 30, 60, 90, and 120 minutes). Mice were fasted overnight before the OGTT assessment, and then administered glucose solution (2 g / kg body weight) orally by gavage. Blood glucose was then measured at designated time points to quantify glucose load. For the ITT assessment, mice were fasted for 6 hours before receiving an intraperitoneal injection of insulin (0.75 U / kg body weight), followed by a series of blood glucose measurements to determine insulin load.

[0057] 2.4 Liver histology and calculation of nonalcoholic fatty liver disease activity score (NAS) Liver tissue samples were stained with hematoxylin and eosin (H&E) and observed at ×200 magnification. The NAS score was calculated using the method described in a previous invention (Shu and Hu, 2024). NAS is the sum of scores for steatosis (0: <5% liver tissue involvement; 1: 5%-33% liver tissue involvement; 2: 33%-66% liver tissue involvement; 3: >67% liver tissue involvement), lobular inflammation (0: no inflammatory foci / 20x field; 1: 1 inflammatory foci / 20x field; 2: 2-4 inflammatory foci / 20x field; 3: >4 inflammatory foci / 20x field), and hepatocellular ballooning degeneration (0: none; 1: few; 2: many). The total score ranged from 0 to 8. A NAS score >5 strongly suggests NASH.

[0058] 3. Experimental Results 3.1 Changes in body weight, liver weight, and serum parameters in mice fed MCD, WD, and NMCDH diets.

[0059] This invention analyzed wild-type mice previously fed MCD and NMCDH diets for 6 weeks, or control and Western diets (WD) for 16 weeks. Compared with the control group, mice fed MCD and NMCDH diets experienced sustained weight loss, ultimately resulting in a 27% and 3% decrease in body weight, respectively, while mice fed the WD diet showed a 28% increase in body weight (P<0.01). Figure 1 A in the middle.

[0060] The NMCDH group showed a continuous increase in food intake and a slight decrease in body weight, while the MCD group experienced a significant decrease in both food intake and body weight.

[0061] NMCDH group feed intake statistics:

[0062] The body weights of mice in the NMCDH group are shown in the table below:

[0063] The feed intake of the control group is shown in the table below:

[0064] The body weights of the control group are shown in the table below:

[0065] The feed intake of the MCD group is shown in the table below.

[0066] The weights of the MCD group are shown in the table below:

[0067] Furthermore, compared with the other two groups, the liver weight of the WD and NMCDH groups was significantly increased (P<0.001); Figure 1 (B) In the NMCDH group, the liver-to-body ratio (liver weight to body weight) was 1.5 times that of the control group and the MCD group (P<0.01); Figure 1 (C) Compared with the control group, serum ALT and AST levels were elevated in the MCD, WD, and NMCDH groups (P<0.01); Figure 1 (DE in the WD group). Compared with the control group, the serum total cholesterol (TC) level in the WD group increased significantly by 1.8 times (P<0.01). Figure 1 (F in the text). Furthermore, serum triglyceride (TG) levels in the WD diet group were significantly higher than in other groups (P<0.01). Figure 1 (G in the middle).

[0068] 3.2 Changes in OGTT, ITT and liver structure in mice fed MCD, WD and NMCDH diets.

[0069] Compared with the control group, the oral glucose tolerance test (OGTT) results showed that the WD diet significantly increased blood glucose levels in mice at five time points (15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes), while the MCD and NMCDH groups significantly decreased blood glucose levels in mice at three time points (60 minutes, 90 minutes, and 120 minutes). Figure 2 (A), while the WD diet showed the highest area under the curve (AUC) of OGTT (P<0.01); Figure 2 (B) In the insulin tolerance test (ITT), compared with the control group, the MCD and NMCDH diets significantly reduced blood glucose levels in mice at four time points (30 minutes, 60 minutes, 90 minutes, and 120 minutes). Figure 2 The WD diet lowered blood sugar levels at two time points (90 minutes and 120 minutes). Figure 2 (C). Therefore, the area under the ITT curve (AUC) was significantly lower in the MCD and NMCDH diet groups (P<0.01). Figure 2 (D). Figure 2 The livers in the WD and NMCDH groups were significantly enlarged and pale. Furthermore, liver histopathology showed group-specific changes (…). Figure 2 In the control group (MCD), the lobular structure remained intact with orderly hepatocyte arrangement. The MCD group showed disordered lobular structure, widespread hepatocyte ballooning, medium-sized lipid droplets (yellow arrows), foam cells (cyan arrows), scattered necrosis (blue arrows) with inflammation (black arrows). The WD group showed similar structural disruption, but compared to the MCD group, it showed heterogeneous lipid droplets (yellow arrows) and reduced necrosis / inflammation. The NMCDH samples showed the most severe pathological changes, including complete lobular disintegration, diffuse ballooning, lipid droplets of varying sizes (yellow arrows), dense foam cell infiltration (cyan arrows), and widespread necrosis (blue arrows) with significant inflammatory aggregation (black arrows). Furthermore, the NAFLD activity score (NAS) confirmed the presence of NASH; compared to the control group, the MCD, WD, and NMCDH groups had significantly higher scores (P<0.001), and more pronounced hepatocyte ballooning was observed in the NMCDH group. Figure 2 (G).

[0070] Experiment Example 2 Transcriptome analysis was performed on liver tissues from mice fed different diets in Experiment 1. The experimental methods are as follows: Transcriptome analysis was performed by Novogene Corporation (Beijing, China) according to standardized procedures. Total RNA was extracted from frozen liver tissue using TRIzol reagent (Ambion; Thermo Fisher Scientific, USA), and RNA integrity and concentration were verified by dual quantification using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). After purification and fragmentation, cDNA was synthesized using the SMARTer PCR cDNA Synthesis Kit (Clontech; TakaraBio, Japan). Before paired-end sequencing on an Illumina HiSeq 3000 platform, the constructed library (insertion fragment 370-420 bp) underwent quality control verification by capillary electrophoresis (Agilent 2100 Bioanalyzer) and quantitative real-time analysis (Qubit 3.0). Transcript quantification was performed by alignment with pre-defined genome annotations using StringTie. Differentially expressed genes (DEGs) were analyzed using DESeq2 (v1.38.3) with strict selection criteria (padj ≤ 0.05 and |log2(fold change)| ≥ 1.0). Benjamini-Hochberg multiple corrective tests were used in the differential expression analysis, followed by KEGG pathway enrichment analysis using clusterProfiler (v4.6.2).

[0071] like Figure 3 As shown, RNA sequencing analysis depicted the different gene expression profiles among the control group, MCD group, WD group, and NMCDH group. Comparative analysis showed that compared with the control group, 764, 606, and 854 genes were significantly downregulated in the MCD, WD, and NMCDH groups, respectively, while 1987, 593, and 976 genes were significantly upregulated, respectively. Figure 3 (A). The volcano plot further characterized differentially expressed genes (DEGs) between the experimental groups. Figure 3 (B)

[0072] Gene ontology (GO) enrichment analysis of differentially expressed genes showed that the experimental group had different functional characteristics in three ontology categories. Figures 4-5In biological processes (BP), compared to the control group, the MCD group was significantly enriched in leukocyte migration, while the WD and NMCDH groups were preferentially enriched in muscle system processes and exogenous substance metabolism, respectively. Cellular component (CC) analysis showed that the MCD group was enriched in extracellular matrix-related structures, the WD group in myofibril components, and the NMCDH group in collagen tissue. At the molecular function (MF) level, the MCD group showed enrichment in extracellular matrix structural components, while the WD and NMCDH groups mainly showed enrichment in actin-binding activity and monooxygenase activity, respectively. Compared to the MCD group, the WD group showed significant enrichment in the regulation of interleukin-6 production in BP analysis, while the NMCDH group showed significant enrichment in the positive regulation of cell activation. Cellular component (CC) analysis showed that the WD group was significantly enriched in extracellular matrix regulation-related items, while the NMCDH group was specifically enriched in external plasma membrane-related components. MF analysis showed that the WD and NMCDH groups were characterized by the regulation of monooxygenase activity. Compared with the WD group, the NMCDH group showed significant enrichment in all three GO categories: BP, which is related to steroid metabolism; CC, which involves MHC class II protein complexes; and MF, which is related to monooxygenase activity.

[0073] KEGG pathway enrichment analysis revealed different metabolic perturbations among the experimental groups. Figure 6 Compared to the control group, three characteristic pathways showed significant enrichment: the PI3K-Akt signaling pathway in the MCD group, the calcium signaling pathway in the WD group, and retinol metabolism in the NMCDH group. Comparative analysis between the MCD and WD groups identified significant enrichment of proteoglycans in the PI3K-Akt signaling pathway, while comparison between the MCD and NMCDH groups revealed cytokine-cytokine receptor interactions. Furthermore, comparison between the WD and NMCDH groups revealed steroid hormone biosynthesis. To validate these findings, six genes selected from differentially expressed genes (the ABC transporter pathway in the MCD group, linoleic acid metabolism in the WD group, and the protein digestion and absorption pathway in the NMCDH group) were confirmed by qPCR, and the results confirmed the sequencing results. Figure 7 ).

[0074] Experimental Example 3 Untargeted metabolomics analysis was performed on the liver tissues of mice fed different diets in Experiment 1.

[0075] Liver tissue was homogenized in liquid nitrogen and suspended in 80% methanol (v / v). After incubation on ice for 5 minutes, the sample was centrifuged at 15,000 × g for 20 minutes at 4°C. The resulting supernatant was adjusted to 53% methanol and centrifuged again under the same conditions. The final supernatant was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Chromatographic separations were performed on a Vanquish UHPLC system (Thermo Scientific) connected to an Orbitrap Q Exactive. TM HF mass spectrometer (ThermoScientific). Samples were loaded onto a Hypersil Gold column (C18) maintained at 40°C at a flow rate of 0.2 mL / min. A 12-minute linear gradient was used for positive ion mode analysis with mobile phases A (0.1% formic acid aqueous solution) and B (methanol). The gradient program was: 2% B (0–1.5 min), 2% B (1.5–3.0 min), 85% B (3.0–10.0 min), 100% B (10.0–10.1 min), holding 2% B (10.1–12.0 min), followed by reequilibration. Mass spectrometry parameters included: sheath gas flow rate 35 psi, spray voltage 3.5 kV, and capillary temperature 320°C. Raw data acquisition was performed in full scan mode (m / z 100–1500) at a resolution of 60,000. Multivariate analysis and modeling were performed on the mean-centered and scaled data using Robls software. Principal component analysis (PCA) was used for model construction. Partial least squares-discriminant analysis (PLS-DA) was used for metabolomics screening to select differentially expressed metabolites (DMs) that met the dual thresholds of variable importance projection (VIP) score > 1.0 and fold change (FC) > 1.5.

[0076] This invention employs non-targeted metabolomics technology to analyze the liver's metabolic profile and assesses the changes in liver metabolic profile induced by MCD, WD, and NMCDH diets. PCA plots show a clear separation between the control group, MCD group, and NMCDH group. Figure 8Comparative analysis of differentially expressed metabolites (DMs) revealed different metabolic profiles between the experimental groups. Compared with the control group, the MCD, WD, and NMCDH groups showed changes in 361, 294, and 468 differentially expressed metabolites, respectively. The MCD group showed 173 upregulated and 188 downregulated metabolites, while the WD group showed 123 upregulated and 171 downregulated metabolites. Notably, the NMCDH group showed the most significant metabolic perturbation, with 319 upregulated and 149 downregulated metabolites. Intergroup comparisons showed that compared with the WD group, the MCD group had 257 upregulated and 192 downregulated metabolites. Compared with the NMCDH group, the MCD group had 166 upregulated and 329 downregulated metabolites, while the WD group had 99 upregulated and 309 downregulated metabolites. Figure 8 (Middle B). The top 20 metabolites with the most significant changes among MCD, WD, NMCDH and control groups were identified. Figures 9-10Compared with the control group, the levels of guanidoacetic acid, PA (12:0 / 12:0(3-OH)), and glutathione were significantly increased in the MCD, WD, and NMCDH groups. Conversely, the levels of (5E)-8-hydroxytetradec-5-enoylcarnitine, (9Z,12Z)-3-hydroxyhexadecadienoylcarnitine, and 1-(9Z-eicosenoyl)-sn-glycero-3-phosphocholine were significantly decreased in these groups. Comparative analysis further revealed that, compared to the NMCDH group, 1-(9Z-eicosenoyl)-sn-glycerol-3-phosphocholine was the most significantly upregulated metabolite in both the MCD and WD groups, while glutathione and 2-methylbacteriohopanetetrol were the most significantly downregulated metabolites. Notably, compared to the WD group, the MCD group showed the most significant upregulation of guanidinoacetic acid and the most significant downregulation of 13,14-dihydro-15-oxo-20-carboxy-PGF2alpha. KEGG pathway enrichment analysis was used to identify metabolic pathways associated with intergroup differences. Compared to the control group, the most significantly enriched pathways in the MCD, WD, and NMCDH groups were the ABC transporter pathway, linoleic acid metabolism, and alanine, aspartate, and glutamate metabolisms, respectively. Similarly, comparative analysis showed a significant enrichment of necroptosis between the MCD and WD groups, a significant enrichment of sphingolipid metabolism between the MCD and NMCDH groups, and a significant enrichment of tryptophan metabolism between the WD and NMCDH groups. Figures 11-12 ).

[0077] In summary, the NMCDH diet demonstrates significant comprehensive advantages in establishing a mouse NASH model, primarily due to its ability to efficiently and synchronously mimic the core pathological and metabolic characteristics of human NASH. Compared to the MCD diet, which leads to severe weight loss, and the WD diet, which induces liver damage more slowly, the NMCDH model induces significant liver weight gain and a very high liver-to-body ratio without causing severe systemic emaciation, thus better mimicking the clinical condition of human patients experiencing liver-specific damage in the context of obesity metabolism. This model can induce extremely severe liver pathological changes, including complete lobular disintegration, diffuse ballooning degeneration, dense foam cell infiltration, and extensive necrosis and inflammation, accompanied by a significant liver fibrosis phenotype, which is confirmed by its gene expression profile specifically enriched in collagen tissue. At the molecular level, NMCDH induces the most extensive transcriptome reprogramming and the most severe metabolic perturbations. Its specifically enriched pathways of retinol metabolism and exogenous substance metabolism provide rich insights into the mechanisms of NASH. Through comparison with clinical data, the formulation of this invention possesses more dimensions of clinical simulation data (Table 1). This invention is more suitable for drug development in late-stage NASH fibrosis, long-term toxicity trials and therapeutic studies, large-scale drug screening, and in-depth exploration of NASH mechanisms based on multi-omics data. In summary, the NMCDH model can construct a severe NASH phenotype encompassing severe steatosis, significant inflammatory necrosis, active fibrotic processes, and metabolic disturbances in a relatively short time, making it a more comprehensive, reliable, and translationally valuable preclinical research tool.

[0078] Table 1. Pathological and metabolic characteristics of mouse models under different feed formulations.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A feed for constructing a non-alcoholic steatohepatitis animal model, characterized by, It comprises the following raw materials in percentage by weight: 44-62% of corn, 10-21.25% of wheat, 10-15% of secondary powder, 1.2-1.4% of lysine, 0.5-0.6% of threonine, 7-8% of glycine, 0.66-0.85% of arginine, 0.3-0.35% of histidine, 0.5-0.6% of isoleucine, 0.8-0.95% of leucine, 0.5-0.6% of phenylalanine, 0.15-0.2% of tryptophan, 0.6-0.7% of valine, 2-2.5% of CaHPO42H2O, 1.5-1.8% of stone powder, 1-1.2% of disodium hydrogen phosphate, 0.5-0.6% of iodized salt, 2-2.5% of cholesterol, 10-15% of lard, 2-5% of soybean oil, 0.1% of trace element premix and 0.1% of compound vitamin.

2. The feed according to claim 1, characterized in that, The feed comprises the following raw materials in percentage by weight: 48.59% of corn, 10% of wheat, 10% of secondary powder, 1.2% of lysine, 0.5% of threonine, 7% of glycine, 0.66% of arginine, 0.3% of histidine, 0.5% of isoleucine, 0.8% of leucine, 0.5% of phenylalanine, 0.15% of tryptophan, 0.6% of valine, 2% of CaHPO42H2O, 1.5% of stone powder, 1% of disodium hydrogen phosphate, 0.5% of iodized salt, 2% of cholesterol, 10% of lard, 2% of soybean oil, 0.1% of trace element premix and 0.1% of compound vitamin. Preferably, the trace element premix comprises manganese sulfate, copper sulfate, zinc sulfate and calcium iodate added in corn starch, and the mass ratio of manganese sulfate, copper sulfate, zinc sulfate, calcium iodate and corn starch in the trace element premix is 0.185:0.03:0.03:0.013:0.742 in turn. Each g of the compound vitamin comprises the following contents of raw materials: 12250IU of vitamin A, 1400IU of vitamin D, 105IU of vitamin E, 5.25mg of vitamin K, 0.175mg of biotin, 7mg of folic acid, 78.75mg of nicotinic acid, 29.75mg of pantothenic acid, 17.5mg of vitamin B2, 14mg of vitamin B1, 10.5mg of vitamin B6, 0.035mg of vitamin B12; Preferably, the compound vitamin is selected from the vitamin premix of Zhonghua Duowei.

3. The feed of any one of claims 1-2 for use in constructing an animal model of non-alcoholic steatohepatitis.

4. A method of constructing a non-alcoholic steatohepatitis animal model, characterized by, It comprises the following steps: feeding the animal to be modeled with the feed of any one of claims 1-2 to obtain an animal model of non-alcoholic steatohepatitis.

5. The method of constructing a non-alcoholic steatohepatitis animal model according to claim 4, wherein, The animal model of non-alcoholic steatohepatitis has at least one of the following phenotypes: (1) the animal has no systemic emaciation; (2) the liver weight of the animal is increased; (3) the liver-body ratio of the animal is increased; (4) the serum ALT and AST levels are increased; (5) the blood glucose level of the animal is decreased; (6) the liver of the animal is obviously enlarged and pale in color; (7) the animal liver cells exhibit at least one of the following phenomena: complete lobular structure collapse, diffuse ballooning, lipid droplets of various sizes, dense foam cell infiltration, extensive necrosis with obvious inflammatory aggregation, obvious hepatocyte ballooning and liver fibrosis; (8) specific enrichment of retinol metabolism, specific enrichment of exogenous substance metabolism.

6. The method of constructing a non-alcoholic steatohepatitis animal model according to claim 5, wherein, The exogenous substance is selected from at least one of alanine, aspartic acid, glutamic acid, tryptophan and glutathione.

7. The method of constructing a non-alcoholic steatohepatitis animal model according to claim 4, wherein, The non-human mammal is selected from mouse, rat, pig, dog, rabbit, guinea pig or monkey.

8. The method of constructing a non-alcoholic steatohepatitis animal model according to claim 7, wherein, When the animal to be modeled is a mouse, a 6-14 week old mouse is fed with the feed of any one of claims 1-2 for 6 weeks.

9. The method of claim 8, wherein the non-alcoholic steatohepatitis animal model is constructed by administering the compound of Formula 1 to the animal. The feed is fed at 5-8 g per mouse per day.

10. Use of the non-alcoholic steatohepatitis animal model constructed by the method of any one of claims 4-9 in screening drugs for preventing or treating at least one of the following diseases: non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, liver cirrhosis and hepatocellular carcinoma.

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