Gavage agent for constructing metabolism-related fatty liver disease animal model as well as preparation method and application of gavage agent

Abstract

The invention relates to the technical field of animal model construction, and particularly provides a gavage agent for constructing a metabolism-related fatty liver disease animal model as well as a preparation method and application of the gavage agent. The gavage agent comprises cholesterol, sodium cholate, Tween 80 and corn oil according to a specific ratio, a quantitative gavage administration mode is adopted, and the technical problems that an existing MAFLD animal model is long in modeling period, high in cost and large in individual difference, and a single pathogenic factor cannot be accurately researched are solved. The gavage agent is used for building a rat model and can efficiently and stably induce an MAFLD animal model with typical liver fatty degeneration and lipid disorder characteristics within 8 weeks, the modeling period is shortened by more than 4 weeks compared with that of a traditional high-fat feed method, the individual difference of the model is remarkably reduced through accurate dosage control, and the method is suitable for large-scale popularization and application. And meanwhile, the single-day modeling cost is controlled at a relatively low level.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically providing a gavage agent for constructing an animal model of metabolic-related fatty liver disease, its preparation method, and its application. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common chronic liver disease with a spectrum ranging from simple hepatic steatosis to steatohepatitis, and even liver fibrosis, cirrhosis, and hepatocellular carcinoma. In-depth research into the pathogenesis of MAFLD and the development of effective treatment strategies heavily rely on animal models that can accurately mimic the pathological characteristics of the human disease.

[0003] Currently, the most mainstream modeling method is high-fat diet feeding. This method simulates the high-fat diet in humans by feeding animals a diet with extremely high fat content. On the one hand, high-fat diets are usually composed of a mixture of various fat sources, cholesterol, and other components, making it difficult to identify which specific component plays a dominant role in the development of the disease. This is not conducive to precise exploration of the mechanism of a single pathogenic factor (such as cholesterol). On the other hand, since it relies on animals' free access to food, differences in food intake between individuals are inevitable, leading to varying degrees of disease severity within the model group and large individual differences, which affects the stability and reproducibility of experimental results. Furthermore, this modeling method usually requires 12 to 16 weeks or even longer to induce stable hepatic steatosis and other phenotypes, resulting in a long experimental cycle and high costs.

[0004] There are also genetically engineered animal models, such as transgenic mice with specific gene knockouts or overexpressions. While these models can provide tools for studying the function of specific genes, their preparation process is technically complex, time-consuming, and extremely expensive. Furthermore, their genetic background often does not match the polygenic inheritance characteristics of human diseases, limiting their widespread application in large-scale drug screening and general mechanism studies.

[0005] In addition, some studies have attempted to use methods such as drug induction or surgery, but these methods often cannot well simulate the natural occurrence and development of MAFLD as a metabolic disease in the body, and the pathological characteristics do not match human disease well. Summary of the Invention

[0006] The purpose of this invention is to address the problems of long cycle, high cost, large individual differences, and inability to accurately study single pathogenic factors in existing MAFLD animal model modeling techniques, and to provide a gavage agent for constructing an animal model of metabolic-related fatty liver disease, its preparation method, and its application.

[0007] The first aspect of this invention provides an oral gavage for constructing an animal model of metabolic-related fatty liver disease, wherein each 100 ml of the oral gavage contains: 5g-15g cholesterol crystals, 2g-6g sodium cholate powder, 18mL-25mL Tween 80, and the remainder corn oil.

[0008] This invention provides a gavage agent for constructing an animal model of metabolic-associated fatty liver disease (MAFLD). The agent comprises a specific ratio of cholesterol, sodium cholate, Tween 80, and corn oil, and is administered via quantitative gavage. This addresses the technical problems of existing MAFLD animal models, such as long modeling cycles, high costs, significant individual variability, and the inability to accurately study single pathogenic factors. When used in rat model construction, this gavage agent can efficiently and stably induce MAFLD animal models with typical hepatic steatosis and dyslipidemia characteristics within 8 weeks. It not only shortens the modeling cycle by more than 4 weeks compared to traditional high-fat diet methods but also significantly reduces individual variability through precise dosage control, while keeping the daily modeling cost low.

[0009] Furthermore, each 100 ml of the oral gavage contains: 8g-12g cholesterol crystals, 4g-6g sodium cholate powder, 20mL-25mL Tween 80, and the remainder corn oil.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned gavage agent for constructing an animal model of metabolic-related fatty liver disease, comprising the following steps: mixing cholesterol crystals, sodium cholate powder, Tween 80 and corn oil, heating and stirring at 50-70°C.

[0011] The third aspect of this invention provides a method for constructing an animal model of metabolic-related fatty liver disease, wherein the experimental animals are gavaged with the above-mentioned gavage agent at a dose of 4-6 mL / kg body weight for a gavage cycle of 6-10 weeks.

[0012] Furthermore, the experimental animal was a rat.

[0013] The fourth aspect of this invention provides the use of the above-described gavage agent in constructing an animal model of metabolic-associated fatty liver disease.

[0014] The fifth aspect of the present invention provides the use of the above-described gavage preparation in the preparation of a pharmaceutical composition for screening or evaluating remedies for the prevention and / or treatment of metabolic-related fatty liver disease.

[0015] The sixth aspect of the present invention provides the use of the above-described gavage preparation in the preparation of reagents for studying the mechanism of action of cholesterol metabolism disorders in the development and progression of metabolism-related fatty liver disease.

[0016] The seventh aspect of the present invention provides a method for inducing hepatocyte steatosis in vivo for non-diagnostic and non-therapeutic purposes, by administering the above-mentioned gavage agent to experimental animals.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a gavage preparation composed of cholesterol, sodium cholate, Tween 80, and corn oil in a specific ratio, administered via quantitative gavage. This addresses the technical problems of existing MAFLD animal models, such as long modeling cycles, high costs, significant individual variability, and the inability to accurately study single pathogenic factors. When used to construct a rat model, this gavage preparation can efficiently and stably induce an MAFLD animal model with typical hepatic steatosis and dyslipidemia characteristics within 8 weeks. This not only shortens the modeling cycle by more than 4 weeks compared to traditional high-fat diet methods but also significantly reduces individual variability through precise dosage control, while keeping the daily modeling cost low. Furthermore, this model enables precise control and study of cholesterol as a single pathogenic factor, providing a reliable and efficient experimental platform for elucidating the specific role of cholesterol in the pathogenesis of MAFLD and screening targeted therapeutic drugs. Attached Figure Description

[0018] Figure 1 This is a line graph showing the changes in body weight of rats in the model group and control group in the MAFLD model establishment method of the present invention. Con represents the control group, Mod represents the model group, the vertical axis represents body weight, and the horizontal axis represents the number of experimental days.

[0019] Figure 2 In the MAFLD model establishment method of the present invention, the visual examination representative photos and liver coefficients of rat livers in the model group and control group are used, where Con is the control group and Mod is the model group.

[0020] Figure 3 This is a bar chart showing the results of blood lipids in the model group and control group rats in the MAFLD model establishment method of the present invention: total cholesterol (T-CHO), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C), where Con represents the control group, Mod represents the model group, the vertical axis represents the concentration, and the horizontal axis represents the item.

[0021] Figure 4 This is a bar chart showing the results of liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group and control group rats in the MAFLD model establishment method of the present invention. Con represents the control group, Mod represents the model group, the vertical axis represents enzyme activity, and the horizontal axis represents the item.

[0022] Figure 5 The image shows H&E staining of liver tissue from rats in the model group and control group in the MAFLD model establishment method of this invention. Con represents the control group and Mod represents the model group. The magnification is 200 times.

[0023] Figure 6 This is an Oil Red O staining image of rat liver tissue from the model group and control group in the MAFLD model establishment method of the present invention, where Con represents the control group and Mod represents the model group, and the magnification is 100 times.

[0024] Figure 7 This is a statistical result of the proportion of fat staining caused by Oil Red O dye in the liver tissue of rats in the model group and the control group in the MAFLD model establishment method of the present invention. Con represents the control group and Mod represents the model group. The magnification is 100 times.

[0025] Figure 8 Line graphs showing the changes in body weight of mice in the control group and the high-fat diet group during the establishment of the MAFLD model using a traditional high-fat diet. Control represents the control group, and HFD represents the high-fat diet group. The vertical axis represents body weight, and the horizontal axis represents the number of experimental weeks.

[0026] Figure 9 To establish the MAFLD model using a conventional high-fat diet, line graphs were plotted showing the changes in food intake between the control group and the high-fat diet group. The Control group represents the control group, and the HFD group represents the high-fat diet group. The vertical axis represents the daily food intake per mouse, and the horizontal axis represents the number of experimental weeks.

[0027] Figure 10 To establish a MAFLD model using a conventional high-fat diet, visual examination images of the livers and liver coefficients of mice in the control group and the high-fat diet group were collected. The control group was the control group, and the high-fat diet group was the HFD group.

[0028] Figure 11 Bar chart showing the results of blood lipid T-CHO, LDL-C, and HDL-C in mice in the control group and the high-fat diet group during the establishment of the MAFLD model using a traditional high-fat diet. The control group represents the control group, and the high-fat diet group represents the HFD group.

[0029] Figure 12 In the process of establishing a MAFLD model using a traditional high-fat diet, the H&E chromosomes of the livers of mice in the control group and the high-fat diet group were measured. The control group was the control group, and the high-fat diet group was the high-fat diet group. The magnification was 100 times. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0031] The first embodiment of this invention provides an oral gavage for constructing an animal model of metabolic-related fatty liver disease, wherein each 100 ml of the oral gavage contains: 5g-15g cholesterol crystals, 2g-6g sodium cholate powder, 18mL-25mL Tween 80, and the remainder corn oil.

[0032] This invention addresses the technical challenges of existing MAFLD animal models, such as long modeling cycles, high costs, significant individual variability, and the inability to precisely study single pathogenic factors, by providing a gavage preparation composed of cholesterol, sodium cholate, Tween 80, and corn oil in specific ratios and administering the medication via quantitative gavage. When used to construct a rat model, this gavage preparation efficiently and stably induces an MAFLD animal model with typical hepatic steatosis and dyslipidemia characteristics within 8 weeks. This not only shortens the modeling cycle by more than 4 weeks compared to traditional high-fat diet methods but also significantly reduces individual variability through precise dosage control, while keeping daily modeling costs low. Furthermore, this model enables precise control and study of cholesterol as a single pathogenic factor, providing a reliable and efficient experimental platform for elucidating the specific role of cholesterol in the pathogenesis of MAFLD and screening targeted therapeutic drugs.

[0033] In some embodiments, each 100 ml of the oral gavage contains: 8g-12g cholesterol crystals, 4g-6g sodium cholate powder, 20mL-25mL Tween 80, and the remainder corn oil.

[0034] Studies have found that the proportions of each ingredient are a key factor affecting the quality of animal model construction. Controlling the proportions within a reasonable range helps induce high-quality MAFLD animal models. For example, in some embodiments, each 100 ml of the gavage solution contains: 5 g cholesterol crystals, 6 g sodium cholate powder, 25 mL Tween 80, and the remainder corn oil. In other embodiments, each 100 ml of the gavage solution contains: 15 g cholesterol crystals, 2 g sodium cholate powder, 18 mL Tween 80, and the remainder corn oil. Both methods successfully constructed MAFLD animal models, and the gavage solution formed a homogeneous and stable solvent.

[0035] The second aspect of this embodiment provides a method for preparing the above-mentioned gavage agent for constructing an animal model of metabolic-related fatty liver disease, comprising the following steps: mixing cholesterol crystals, sodium cholate powder, Tween 80 and corn oil, heating and stirring at 50-70°C.

[0036] This preparation method effectively solves the key technical problems of easy precipitation, layering, and poor homogeneity of high-cholesterol gavage by heating and stirring a specific ratio of cholesterol, sodium cholate, Tween 80, and corn oil at 50-70℃. Heating promotes lipid dissolution and forms a stable microemulsion through emulsification, ultimately achieving uniform and stable physical properties of the gavage and a significant improvement in bioavailability.

[0037] The third embodiment of this invention provides a method for constructing an animal model of metabolic-related fatty liver disease. The experimental animals are administered the above-mentioned gavage agent by gavage at a dose of 4-6 mL / kg body weight for a gavage cycle of 6-10 weeks.

[0038] By administering a specially formulated high-cholesterol emulsion via quantitative gavage, a MAFLD model exhibiting typical hepatic steatosis, dyslipidemia, and low-grade inflammation can be efficiently induced within 6-10 weeks, shortening the modeling cycle by approximately one-third compared to traditional high-fat diets. Precise control of cholesterol intake (4-6 mL / kg body weight) effectively addresses individual variability issues caused by free-feeding, significantly improving model consistency and reproducibility.

[0039] In some embodiments, the experimental animal is a rat.

[0040] The fourth aspect of this embodiment provides the use of the above-described gavage agent in constructing an animal model of metabolic-associated fatty liver disease.

[0041] By regularly administering the aforementioned gavage to experimental animals, the synergistic effect of cholesterol and sodium cholate, along with the high bioavailability of Tween 80, can actively and reliably reproduce the core pathological features of human MAFLD in vivo, including hepatocellular steatosis, dyslipidemia, and low-grade inflammation.

[0042] The fifth aspect of this embodiment provides the use of the above-described gavage agent in the preparation of pharmaceutical compositions for screening or evaluating remedies for the prevention and / or treatment of metabolic-related fatty liver disease.

[0043] The sixth aspect of this embodiment provides the use of the above-described gavage agent in the preparation of reagents for studying the mechanism of action of cholesterol metabolism disorders in the development and progression of metabolism-related fatty liver disease.

[0044] The seventh aspect of this embodiment provides a method for inducing hepatocyte steatosis in vivo for non-diagnostic and non-therapeutic purposes, by administering the above-mentioned gavage to experimental animals.

[0045] To better understand the above technical solution, the following more detailed implementation methods are provided for further explanation: Example 1 Example 1 provides a method for constructing a rat MAFLD model by gavage with a 10% cholesterol solution.

[0046] Specifically: (1) Twenty-four 21-day-old SPF-grade male SD rats (weighing 100-120 g) were purchased from Shandong Pengyue Experimental Animal Co., Ltd. Specific feeding conditions were as follows: temperature: 20-25℃, humidity: 50%-65%, 12h light / 12h dark diurnal cycle, with ample fresh feed and drinking water provided daily. After one week of acclimatization, the rats were randomly divided into two groups: a model group (n=12) and a control group (n=12).

[0047] (2) Prepare the 10% cholesterol solution for gavage in the model group and the solution for gavage in the control group according to the formula. 10% cholesterol solution: Weigh 5g cholesterol crystals and 2g sodium cholate powder, add 33mL corn oil and 10mL Tween 80, heat the mixture in a 60℃ water bath until fully dissolved, and prepare the 10% cholesterol gavage solution. Control group gavage solution: Measure 33mL corn oil and 10mL Tween 80, add 7mL distilled water, heat the mixture in a 60℃ water bath until fully dissolved, and prepare the control group gavage solution.

[0048] (3) After the experiment began, the rats’ weight was recorded daily. The model group was given 5 mL / kg body weight of 10% cholesterol solution by gavage daily, while the control group was given 5 mL / kg body weight of gavage solution by gavage daily for 8 consecutive weeks.

[0049] (4) At the end of the 8th week after gavage, the rats in each group were fasted for 12 hours and their fasting weight was measured. The rats were then anesthetized with a 20% urethane solution. The formula for the 20% urethane solution is as follows: Weigh 10g of urethane crystals, add 50mL of physiological saline, stir to dissolve completely, and administer anesthesia via intraperitoneal injection at a dose of 5mL / kg.

[0050] (5) After anesthesia, blood was collected from the heart and placed in an EDTAK2 anticoagulant tube. The blood was centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant plasma was collected for subsequent biochemical index detection. Simultaneously, rat livers were removed, and the livers of each group were photographed for observation. The entire liver was placed in PBS to rinse off the blood on the surface, and the surface moisture was wiped dry with filter paper before weighing and recording the weight. This weight was used to calculate the liver coefficient. Liver coefficient = liver weight / fasting rat weight * 100%.

[0051] (6) Take a soybean-sized tissue sample from the central part of the right lobe of the liver of each group of rats, fix it in 4% paraformaldehyde, embed it in paraffin and section it for H&E staining. The specific method is as follows: Dewax the tissue sections with xylene (twice, 5 minutes each), then hydrate them stepwise with a gradient of ethanol (100%, 95%, 80%, 70%) to distilled water (2 minutes each), immerse the sections in hematoxylin staining solution for 5 minutes to stain the cell nuclei (blue), differentiate them with 1% hydrochloric acid ethanol for a few seconds, immediately place them in warm water or dilute ammonia water to return to blue, and then rinse with distilled water. Immerse the sections in eosin staining solution for 3 minutes to make the cytoplasm red, then dehydrate them with a gradient of ethanol (70%, 80%, 95%, 100%) (1-2 minutes each), clear them with xylene (twice, 5 minutes each), mount them with neutral resin, and finally observe and photograph them under an optical microscope.

[0052] (7) Take a soybean-sized tissue sample from the central part of the right lobe of the liver of each group of rats, embed it in OCT embedding medium and perform serial frozen sectioning for Oil Red O staining. The specific method is as follows: immerse the section in pre-filtered Oil Red O staining solution for 15 minutes, rapidly differentiate it 2-3 times with 60% isopropanol (until the background is colorless), rinse with distilled water, stain the cell nuclei with hematoxylin staining solution for 3 minutes, wash with water, differentiate with hydrochloric acid ethanol, then blue again, wash with water, mount with glycerol gelatin, and observe and photograph under an optical microscope.

[0053] (8) The levels of T-CHO, LDL-C, and HDL-C in rat plasma were detected using a kit, and the activities of liver function indicators AST and ALT in rat plasma were detected using a kit.

[0054] Male SD rats (100-120 g) aged 21 days were selected and randomly divided into a model group (n=12) and a control group (n=12) after one week of acclimatization. The rats were housed under standard international conditions (temperature: 20-25℃, humidity: 50%-65%, 12h light / 12h dark diurnal cycle, continuous feeding, fresh feed provided daily, and ample water supply). Rats' weight was recorded daily. The model group received 5 mL / kg body weight of 10% cholesterol solution via gavage daily, while the control group received 5 mL / kg body weight of control solution via gavage daily for 8 consecutive weeks. At the end of the 8th week, the rats were fasted for 12 hours, then sacrificed, and blood and liver tissue were collected for relevant indicator testing.

[0055] The formula for the 10% cholesterol gavage solution is as follows: Weigh 5g of cholesterol crystals and 2g of sodium cholate powder, add 33mL of corn oil and 10mL of Tween 80, and heat the above mixture in a 60℃ water bath to fully dissolve it, thus preparing a 10% cholesterol gavage solution.

[0056] The cholesterol concentration selection and calculation are as follows: Total solution mass = Corn oil (33 mL × 0.92 g / mL ≈ 30.36 g) + Tween 80 (10 mL × 1.08 g / mL ≈ 10.8 g) + Cholesterol (5.0 g) + Sodium cholate (2.0 g) ≈ 48.16 g. Cholesterol mass percentage concentration = 5.0 g / 48.16 g × 100% ≈ 10.4% (i.e., "10% cholesterol solution"). A dosage of 5 mL / kg ensures a cholesterol intake of 0.52 g / kg / day (10% solution × 5 mL / kg), which is approximately 5-10 times the average daily cholesterol intake for humans (simulating extreme metabolic disorders).

[0057] The components of the described gavage solution function as follows: corn oil, acting as a lipid-soluble carrier, increases the solubility of cholesterol crystals, mimics the intestinal lipid absorption environment, and promotes cholesterol deposition in the liver; Tween 80 reduces surface tension, forming cholesterol microemulsions, improving solubility and stability (preventing cholesterol precipitation); sodium cholate activates intestinal FXR receptors, inhibits bile acid synthesis feedback, and simultaneously promotes the expression of cholesterol cotransporter protein (NPC1L1), significantly improving cholesterol absorption efficiency. The gavage solution is heated to 60°C to promote complete dissolution of cholesterol crystals, ensuring uniform gavage dosage and inactivating potential microbial contamination. This prepared gavage solution exhibits high stability, improves cholesterol solubility and bioavailability, and costs only 1 / 3 of that of commercial high-fat diet models (single gavage cost <0.5 yuan / animal / day).

[0058] The control group gavage solution was formulated as follows: 33 mL of corn oil, 10 mL of Tween 80, and 7 mL of distilled water were added to maintain the same volume as the solution administered to the model group. The mixture was heated in a 60°C water bath to ensure complete dissolution, thus preparing the control group gavage solution. This process eliminates the interference of the solvent itself on lipid metabolism, ensuring that the pathological changes in the experimental group originate from the synergistic effect of cholesterol and sodium cholate.

[0059] The gavage time setting mentioned above is based on the verification of preliminary experimental results. After 8 weeks of gavage, the liver coefficient of the model group rats can be induced to increase significantly, and significant hepatic steatosis can be observed in the liver of the model group rats. The high-fat diet model shortens the time by 4 weeks.

[0060] The relevant index detection includes: (1) weighing the rats before sacrifice on an empty stomach, collecting the livers of each group of rats, washing the surface blood in PBS, wiping the surface moisture with filter paper, weighing them, and calculating the liver coefficient. Liver coefficient = liver weight / rat fasting body weight * 100%; (2) histopathological examination of liver tissue, taking a soybean-sized tissue from the central part of the right lobe of the liver of each group of rats, fixing it in 4% paraformaldehyde, embedding it in paraffin and sectioning it, using H&E staining, mounting it and observing the changes in liver tissue structure under an optical microscope. Similarly, a soybean-sized tissue was taken from the central part of the right lobe of the liver of each group of rats, embedded in OCT embedding medium, and then continuously frozen sectioned and stained with Oil Red O. After mounting, the tissue was observed under an optical microscope and the proportion of fat stained with Oil Red O was counted. (3) Plasma lipid level detection: Plasma of each group of rats was taken and the levels of total cholesterol (T-CHO), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) were detected. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma were also detected.

[0061] This invention established a rat MAFLD model induced by high cholesterol intake. The body weight of the rats in this model was not different from that of the normal control group. Figure 1 However, in MAFLD model rats, the liver tissue was visibly enlarged, with increased fat and palpable lumps. Figure 2 Compared with the control group, MAFLD model rats showed significantly increased plasma total cholesterol (T-CHO) and low-density lipoprotein (LDL-C) levels, while significantly decreased high-density lipoprotein (HDL-C) levels. Figure 3 This indicates lipid metabolism disorder; simultaneously, plasma AST levels in MAFLD model rats were significantly elevated, while ALT levels showed no significant difference compared to the control group. Figure 4 This suggests that liver function in the model group rats may be impaired. Finally, liver tissue staining and microscopic examination confirmed that the liver tissue structure of the MAFLD model rats was disordered, with fat accumulation and hepatic steatosis, while the liver tissue structure of the control group rats was normal and no hepatic steatosis was observed. Figures 5-7 Based on the latest MAFLD diagnostic criteria, the above results demonstrate that the method described in this patented paper successfully constructed a rat model of MAFLD. This model has a short rearing period, lower cost than traditional high-fat diet models, higher efficiency, and smaller individual variability. Simultaneously, it simulates the unhealthy dietary habits of high cholesterol intake in the general population, facilitating research on the role of cholesterol as a single factor in the progression of MAFLD.

[0062] The method for establishing MAFLD in this invention uses quantitative high cholesterol intake as the core inducing factor to isolate the complex effects of high-fat diet components on the liver, which facilitates in-depth research on the key role of cholesterol in the pathogenesis and progression of MAFLD.

[0063] The MAFLD modeling method of this invention can produce obvious and stable fatty liver symptoms in just eight weeks. The cholesterol intake of each rat can be quantified by gavage. The modeling effect is relatively stable, with small individual differences, short feeding cycle, and high efficiency, which can effectively improve the establishment cycle of MAFLD model.

[0064] The MAFLD model of this invention is characterized by high blood lipids, lipid metabolism disorders, and liver inflammation, which are consistent with the clinicopathological features of human MAFLD.

[0065] The MAFLD model of this invention has a low cost and improves cholesterol utilization by gavage, with a cost of about 0.5 yuan per animal per day, which facilitates large-scale mechanism research and drug screening.

[0066] The MAFLD model of this invention has minimal systemic toxicity to animals during its establishment process, a 100% survival rate, and does not affect the function of other organs in the animals.

[0067] Example 2 Laboratory animals and grouping (1) Thirty healthy 21-day-old SPF-grade male BALB / c mice (weighing 15-20g) were selected and purchased from Shandong Pengyue Experimental Animal Co., Ltd. The specific feeding conditions were as follows: temperature: 20-25℃, humidity: 50%-65%, 12h light / 12h dark day-night cycle, and sufficient drinking water was provided daily. After one week of acclimatization feeding, the mice were randomly divided into two groups according to their weight: a traditional high-fat diet feeding group and a control group, with 10 mice in each group.

[0068] (2) After the experiment began, the weight of the mice was recorded daily. The mice in the traditional high-fat diet group were fed the traditional high-fat diet, while the control group was fed the normal diet and allowed to eat freely. The amount of food consumed by each group of mice was recorded daily and the intervention lasted for 20 weeks.

[0069] (3) The formula of traditional high-fat feed is: 10% lard, 5% sucrose, 1.5% cholesterol, 1.25% bile salts, 10% egg yolk powder, and 73.25% basic feed.

[0070] (4) At the end of week 20, after fasting for 12 hours, the fasting weight of mice in each group was measured. Mice were anesthetized with isoflurane, and blood was collected from the orbital cavity after anesthesia. The blood was placed in an EDTAK2 anticoagulant tube and centrifuged at 3000 rpm for 10 min at 4°C. The supernatant plasma was collected for subsequent biochemical index detection. At the same time, the livers of mice in each group were removed and photographed. The whole liver was placed in PBS to rinse the blood off the surface of the liver. After drying the surface of the liver with filter paper, it was weighed and the weight was recorded. This weight was used to calculate the liver coefficient. Liver coefficient = liver weight / fasting weight of mouse * 100%.

[0071] (5) Take a soybean-sized tissue sample from the central part of the right lobe of the liver of each group of mice, fix it in 4% paraformaldehyde, embed it in paraffin, and section it for H&E staining. The specific method is as follows: Dewax the tissue sections with xylene (twice, 5 minutes each), then hydrate them stepwise with a gradient of ethanol (100%, 95%, 80%, 70%) to distilled water (2 minutes each), immerse the sections in hematoxylin staining solution for 5 minutes to stain the cell nuclei (blue), differentiate them with 1% hydrochloric acid ethanol for a few seconds, immediately place them in warm water or dilute ammonia water to return to blue, and then rinse with distilled water. Immerse the sections in eosin staining solution for 3 minutes to make the cytoplasm red, then dehydrate them with a gradient of ethanol (70%, 80%, 95%, 100%) (1-2 minutes each), clear them with xylene (twice, 5 minutes each), mount them with neutral resin, and finally observe and photograph them under an optical microscope.

[0072] (6) The levels of T-CHO, LDL-C and HDL-C in mouse plasma were detected using a kit.

[0073] Test data like Figures 8-12 As shown, the experimental results indicate that during the model establishment period, the food intake of mice in the high-fat diet group was significantly lower than that in the control group, suggesting that traditional high-fat diet induction models have the problem of difficulty in precisely controlling intake, and that there are large differences in food intake among individuals. In contrast, the rat model established by gavage with 10% cholesterol solution in this invention can achieve precise dosage control.

[0074] After 20 weeks of high-fat diet feeding, the macroscopic liver manifestations of the mice did not exhibit the typical pathological features comparable to those of the 10% cholesterol gavage rat model of this invention. Plasma biochemical analysis showed that although the high-fat diet-fed mice had significantly elevated levels of total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) was also abnormally elevated. This phenomenon is significantly contradictory to the typical lipid profile of human MAFLD (decreased HDL-C), indicating that this model differs from the pathophysiological process of human MAFLD.

[0075] Histopathological observation further confirmed that H&E staining of the livers of mice fed a high-fat diet showed a much lower degree of lipid droplet accumulation in hepatocytes compared to the cholesterol-gavaged rat model of this invention. Notably, even after 20 weeks of continuous high-fat diet feeding, the high-fat diet model failed to produce the significant lipid droplet accumulation observed in the model of this invention.

[0076] Comprehensive comparative analysis shows that, compared with the traditional high-fat diet induction model, the 10% cholesterol solution gavage method used in this invention has the following significant advantages: (1) the dosage is precisely controllable, avoiding experimental errors caused by differences in food intake; (2) it is more consistent with the typical lipid metabolism characteristics of human MAFLD; (3) the degree of lipid accumulation in the liver is higher and more stable; and (4) the pathological phenotype appears in a shorter time. These advantages make the model of this invention closer to the clinical characteristics of MAFLD, providing a more reliable research tool for the development of related drugs.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gavage agent for constructing an animal model of metabolic-related fatty liver disease, characterized in that, Each 100 ml of the above-mentioned gavage contains: 5g-15g cholesterol crystals, 2g-6g sodium cholate powder, 18mL-25mL Tween 80, and the remainder corn oil.

2. The gavage agent according to claim 1, characterized in that, Each 100 ml of the above-mentioned gavage contains: 8g-12g cholesterol crystals, 4g-6g sodium cholate powder, 20mL-25mL Tween 80, and the remainder corn oil.

3. The method for preparing the gavage agent for constructing an animal model of metabolic-related fatty liver disease according to claim 1, characterized in that, The process includes the following steps: mixing cholesterol crystals, sodium cholate powder, Tween 80, and corn oil, heating and stirring at 50-70°C.

4. A method for constructing an animal model of metabolism-related fatty liver disease, characterized in that, Experimental animals were administered the gavage solution according to claim 1 or 2 by gavage at a dose of 4-6 mL / kg body weight for 6-10 weeks.

5. The method according to claim 4, characterized in that, The experimental animal was a rat.

6. Use of the gavage agent as described in claim 1 or 2 in the construction of an animal model of metabolic-associated fatty liver disease.

7. Use of the gavage preparation of claim 1 or 2 in the preparation of a pharmaceutical composition for screening or evaluating remedies for the prevention and / or treatment of metabolic-related fatty liver disease.

8. Use of the gavage preparation of claim 1 or 2 in the preparation of reagents for studying the mechanism of action of cholesterol metabolism disorders in the development and progression of metabolism-related fatty liver disease.

9. A method for inducing hepatocyte steatosis in vivo for non-diagnostic and non-therapeutic purposes, characterized in that, Administer the gavage agent as described in claim 1 or 2 to the experimental animals.

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