Construction method and application of mouse non-alcoholic fatty liver model
By constructing a non-alcoholic fatty liver disease (NAFLD) model in mice through gavage administration of a high-nitrogen compound aqueous solution, this method solves the problems of long model cycles and high costs in existing technologies, and provides a stable and convenient research tool for NAFLD.
Patent Information
- Application Number
- CN202511054032.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-11-21
AI Technical Summary
The construction of existing animal models of non-alcoholic fatty liver disease is time-consuming and expensive, and the fatty liver characteristics of different animal models are inconsistent, which cannot fully reflect the pathological process of NAFLD.
A non-alcoholic fatty liver model was established by administering a high-nitrogen compound aqueous solution via oral gavage to mice using a gavage needle and syringe. BALB/c mice were used, and the dosage was 40-100 mg/kg body weight for 7 days, which simplified the model establishment process.
The constructed model is similar to clinical pathology, exhibiting a significant fatty liver phenotype. It does not require a high-fat diet, is low-cost, has good reproducibility, shortens the model establishment cycle, and saves research time and costs.
Smart Images
Figure CN120983404A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing a mouse non-alcoholic fatty liver model and its application. Background Technology
[0002] Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. It is characterized by disordered hepatic lipid homeostasis and macrovesicular steatosis in hepatocytes, occurring in the absence of a history of excessive alcohol consumption or other hepatotoxic factors. It is a common cause of cryptogenic cirrhosis, affecting approximately 1.27 billion people worldwide. With the global prevalence of obesity and related metabolic syndromes, the incidence of NAFLD is rising annually, becoming a major cause of chronic liver disease and hepatocellular carcinoma globally. Therefore, establishing a stable, reliable, convenient, and rapid animal model of nonalcoholic fatty liver disease is of great significance for studying the pathogenesis and treatment of NAFLD.
[0003] The mouse genome shares 93% genome sequence similarity with humans, and its advantages, including low cost of breeding, rapid reproduction, ease of modeling, minimal inter-individual variability, and convenient parallel experimental observation, make it the primary choice for constructing animal models. Currently known methods for constructing animal models of non-alcoholic fatty liver disease (NAFLD), whether using dietary intervention models or gene knockout models such as ApoE- / - mice and NR4A1- / - mice, all require induction with a high-fat diet after gene knockout to obtain the NAFLD animal model. This model construction cycle is long and cannot reflect the pathological process of NAFLD caused by non-dietary factors. Furthermore, animal models constructed using different animals have poor reliability, and the fatty liver characteristics exhibited by different animals are not entirely consistent, limiting their reference value. Therefore, there is an urgent need to establish a reliable model with a short breeding cycle that can comprehensively reflect the pathological characteristics of NAFLD, providing a flexible and convenient tool for studying the pathogenesis and treatment of NAFLD.
[0004] No identical technical solution to the present invention has been found in the prior art. Summary of the Invention
[0005] The present invention provides a method for constructing a mouse non-alcoholic fatty liver model and its application, aiming to overcome the problems of long modeling time and high cost in the existing non-alcoholic fatty liver animal models.
[0006] Therefore, the present invention provides a method for constructing a mouse non-alcoholic fatty liver disease model, comprising the following steps: S1. Select experimental mice; S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 4-10 mg / mL; S3. According to the dosage, administration time, and administration time, the prepared high-nitrogen compound aqueous solution was administered orally to obtain a non-alcoholic fatty liver animal model.
[0007] Preferably, the experimental mice are BALB / c mice.
[0008] Preferably, the BALB / c mice are male mice aged 6-8 weeks and weighing 20-25g.
[0009] Preferably, the BALB / c mice are housed in an SPF-grade laboratory animal room.
[0010] Preferably, the high-nitrogen compound is a modeling compound with the molecular formula C4H. 16 N 24 .
[0011] Preferably, the dosage is 1 ml / 100 g body weight, and the dose is 40-100 mg / kg body weight.
[0012] Preferably, the administration time is once a day for 7 days.
[0013] Preferably, the oral gavage administration includes the following steps: using a gavage needle and a syringe, administering the drug via oral gavage according to the dosage, inserting the tip of the gavage needle into the oral cavity of the experimental mouse, keeping it parallel to the body axis and slowly inserting it until a resistance is felt in the throat, after which it can smoothly enter the esophagus, and after the gavage needle reaches the stomach, slowly injecting the drug in the syringe to complete the administration.
[0014] Preferably, the process also includes liver function testing, anatomical observation, and pathological examination of the obtained non-alcoholic fatty liver animal model.
[0015] Preferably, the method for constructing the mouse non-alcoholic fatty liver model or the non-alcoholic fatty liver animal model obtained by the method for constructing the mouse non-alcoholic fatty liver model is used in screening or developing drugs for treating non-alcoholic fatty liver.
[0016] The beneficial effects of this invention are: The present invention provides a method for constructing a mouse non-alcoholic fatty liver disease (NAFLD) model and its application. The NAFLD animal model, obtained by oral administration of a prepared high-nitrogen compound aqueous solution, exhibits characteristics such as fatty liver and dyslipidemia, showing high similarity to clinical pathology and a significant fatty liver phenotype. The NAFLD animal model obtained through the method of this invention does not require a high-fat diet, is simple and easy to implement, low in cost, has good reproducibility, and is stable. It can effectively shorten the establishment cycle of NAFLD animal models, greatly saving time and costs in medical research on NAFLD, and providing a good animal model for studying the pathogenesis and treatment strategies of NAFLD. Attached Figure Description
[0017] The present invention will now be described in further detail with reference to the accompanying drawings.
[0018] Figure 1 This is a flowchart of the method for constructing a mouse model of non-alcoholic fatty liver disease; Figure 2 This is a gross anatomical diagram of the liver in Example 3; Figure 3 These are liver H&E staining images from the examples (control group, 40 mg / kg group, 80 mg / kg group, and 100 mg / kg group, respectively). Figure 4 The images shown are Oil Red O staining images of the liver in the examples (control group, 40 mg / kg group, 80 mg / kg group, and 100 mg / kg group, respectively). Detailed Implementation
[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0020] like Figure 1 As shown, a method for constructing a mouse model of non-alcoholic fatty liver disease includes the following steps: S1. Select experimental mice; S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 4-10 mg / mL; S3. According to the dosage, administration time, and administration time, the prepared high-nitrogen compound aqueous solution was administered orally to obtain a non-alcoholic fatty liver animal model.
[0021] Specifically, the non-alcoholic fatty liver disease (NAFLD) animal model obtained by oral administration of a prepared high-nitrogen compound aqueous solution exhibits characteristics such as fatty liver and dyslipidemia, showing high similarity to clinicopathological features and a significant fatty liver phenotype. The NAFLD animal model obtained through the method of this invention requires no high-fat diet, is simple and easy to implement, low in cost, highly reproducible, stable, and consistent, with a model establishment success rate of up to 100%. It effectively shortens the establishment cycle of NAFLD animal models, greatly saving time and costs in medical research on NAFLD, and providing a good animal model for studying the pathogenesis and treatment strategies of NAFLD.
[0022] Preferably, the experimental mice are BALB / c mice.
[0023] Specifically, BALB / c mice have high genotypic homozygosity, small inter-individual differences, and strong reproducibility of experimental results.
[0024] Preferably, the BALB / c mice are male mice aged 6-8 weeks and weighing 20-25g.
[0025] Specifically, mice aged 6-8 weeks are considered early adulthood, at which point their physiological functions are largely mature and stable, avoiding interference from the incomplete development of young mice or the functional decline of older mice. A body weight of 20-25g matches normal growth and development for this age group, indicating good nutritional status and no obvious developmental abnormalities, ensuring consistency among experimental individuals. Male BALB / c mice reduce the impact of physiological fluctuations caused by the female estrous cycle; the physiological state of males is relatively more stable, reducing experimental error.
[0026] Preferably, the BALB / c mice are housed in an SPF-grade laboratory animal room.
[0027] Specifically, mice raised in SPF-grade laboratory animal rooms can ensure both animal health and the accuracy and reproducibility of experimental data.
[0028] Preferably, the high-nitrogen compound is a modeling compound with the molecular formula C4H. 16 N 24 .
[0029] Specifically, this invention prioritizes high-nitrogen compounds as modeling compounds to establish an ideal animal model of non-alcoholic fatty liver for experimental research on the treatment of secondary liver injury.
[0030] Preferably, the dosage is 1 ml / 100 g body weight, and the dose is 40-100 mg / kg body weight.
[0031] Preferably, the administration time is once a day for 7 days.
[0032] Specifically, the dosage, administration time, and timing of this drug can better obtain the desired animal model of non-alcoholic fatty liver disease.
[0033] Preferably, the oral gavage administration includes the following steps: using a gavage needle and a syringe, administering the drug via oral gavage according to the dosage, inserting the tip of the gavage needle into the oral cavity of the experimental mouse, keeping it parallel to the body axis and slowly inserting it until a resistance is felt in the throat, after which it can smoothly enter the esophagus, and after the gavage needle reaches the stomach, slowly injecting the drug in the syringe to complete the administration.
[0034] Specifically, a 12-gauge gavage needle and a 1 mL syringe are used. The medication is administered orally at a dose of 1 mL / 100g body weight. The tip of the gavage needle is inserted into the oral cavity of the experimental mouse, parallel to the body axis, and slowly inserted until resistance is felt in the pharynx, at which point it smoothly enters the esophagus. Once the gavage needle reaches the stomach, the medication in the syringe is slowly injected. The procedure is simple.
[0035] Preferably, the process also includes liver function testing, anatomical observation, and pathological examination of the obtained non-alcoholic fatty liver animal model.
[0036] Specifically, pathological tissue examination is an important examination method that involves a series of processes, such as sampling, fixing, sectioning, and staining of diseased tissue, followed by observation of the morphological and structural changes of tissue cells under a microscope, thereby clarifying the diagnosis of the disease.
[0037] Preferably, the liver function tests include alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and total cholesterol.
[0038] Preferably, the method for constructing the mouse non-alcoholic fatty liver model or the non-alcoholic fatty liver animal model obtained by the method for constructing the mouse non-alcoholic fatty liver model is used in screening or developing drugs for treating non-alcoholic fatty liver.
[0039] Example 1: Based on the above, a method for constructing a mouse model of non-alcoholic fatty liver disease includes the following steps: S1. Select BALB / c mice; the BALB / c mice are male mice aged 6-8 weeks and weighing 20-25g, and the BALB / c mice are housed in an SPF-grade laboratory animal room. S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 4 mg / mL; S3. A non-alcoholic fatty liver animal model was obtained by administering a prepared high-nitrogen compound aqueous solution orally via gavage at a dosage of 1 ml / 100 g body weight and a dose of 40 mg / kg body weight. The oral gavage administration included the following steps: using a No. 12 gavage needle and a 1 mL syringe, the drug was administered orally at a dosage of 1 mL / 100 g body weight. The tip of the gavage needle was inserted into the oral cavity of the experimental mouse, kept parallel to the body axis, and slowly inserted until a resistance was felt in the throat, after which it could smoothly enter the esophagus. After the gavage needle reached the stomach, the drug in the syringe was slowly injected to complete the oral gavage administration.
[0040] Preferably, the process also includes liver function testing, anatomical observation, and pathological examination of the obtained non-alcoholic fatty liver animal model.
[0041] Preferably, the liver function tests include alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and total cholesterol.
[0042] Preferably, the method for constructing the mouse non-alcoholic fatty liver model or the non-alcoholic fatty liver animal model obtained by the method for constructing the mouse non-alcoholic fatty liver model is used in screening or developing drugs for treating non-alcoholic fatty liver.
[0043] Example 2: Based on the above, a method for constructing a mouse model of non-alcoholic fatty liver disease includes the following steps: S1. Select BALB / c mice; the BALB / c mice are male mice aged 6-8 weeks and weighing 20-25g, and the BALB / c mice are housed in an SPF-grade laboratory animal room. S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 8 mg / mL; S3. A non-alcoholic fatty liver animal model was obtained by administering a prepared high-nitrogen compound aqueous solution orally via gavage at a dosage of 1 ml / 100 g body weight and a dose of 80 mg / kg body weight for 7 days. The oral gavage administration included the following steps: using a No. 12 gavage needle and a 1 mL syringe, the drug was administered orally at a dosage of 1 mL / 100 g body weight. The tip of the gavage needle was inserted into the oral cavity of the experimental mouse, kept parallel to the body axis, and slowly inserted until a resistance was felt in the throat, after which it could smoothly enter the esophagus. After the gavage needle reached the stomach, the drug in the syringe was slowly injected to complete the oral gavage administration.
[0044] Preferably, the process also includes liver function testing, anatomical observation, and pathological examination of the obtained non-alcoholic fatty liver animal model.
[0045] Preferably, the liver function tests include alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and total cholesterol.
[0046] A method for constructing a mouse non-alcoholic fatty liver disease (NAFLD) model, or the application of an animal model of NAFLD obtained using the method for constructing the mouse NAFLD model in screening or developing drugs for treating NAFLD.
[0047] Example 3: Based on the above, a method for constructing a mouse model of non-alcoholic fatty liver disease includes the following steps: S1. Select BALB / c mice; the BALB / c mice are male mice aged 6-8 weeks and weighing 20-25g, and the BALB / c mice are housed in an SPF-grade laboratory animal room. S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 10 mg / mL; S3. A non-alcoholic fatty liver animal model was obtained by administering a prepared high-nitrogen compound aqueous solution orally via gavage at a dosage of 1 ml / 100 g body weight and a dose of 100 mg / kg body weight for 7 days. The oral gavage administration included the following steps: using a No. 12 gavage needle and a 1 mL syringe, the drug was administered orally at a dosage of 1 mL / 100 g body weight. The tip of the gavage needle was inserted into the oral cavity of the experimental mouse, parallel to the body axis, and slowly inserted until resistance was felt in the pharynx, at which point it could smoothly enter the esophagus. After the gavage needle reached the stomach, the drug in the syringe was slowly injected to complete the oral gavage administration.
[0048] Preferably, the process also includes liver function testing, anatomical observation, and pathological examination of the obtained non-alcoholic fatty liver animal model.
[0049] Preferably, the liver function tests include alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and total cholesterol.
[0050] A method for constructing a mouse non-alcoholic fatty liver disease (NAFLD) model, or the application of an animal model of NAFLD obtained using the method for constructing the mouse NAFLD model in screening or developing drugs for treating NAFLD.
[0051] Example 4: Based on the above embodiments, this embodiment evaluates the non-alcoholic fatty liver animal model obtained by the present invention and compares it with the model group and the control group.
[0052] Constructing an animal model of non-alcoholic fatty liver disease: 1) Experimental BALB / c mice; male; weight: 20-25g; 2) Husbandry conditions: SPF-grade laboratory animal room; 3) Instruments and materials: syringes, gavage needles, disinfectants, routine surgical instruments, routine medical consumables, etc.
[0053] 4) Drug preparation: Prepare aqueous solutions of high-nitrogen compounds with mass concentrations of 4, 8, and 10 mg / mL. Accurately weigh 40, 80, and 100 mg of the high-nitrogen compound and add drinking water to 10 mL, and dissolve thoroughly.
[0054] 5) Oral administration: Using a No. 12 gavage needle and a 1mL syringe, administer the drug orally at a dose of 1mL / 100g body weight. Insert the tip of the gavage needle into the mouse's mouth, keeping it parallel to the body axis, and slowly insert it. Once you feel resistance in the throat, it can smoothly enter the esophagus. After the gavage needle reaches the stomach, slowly inject the drug from the syringe.
[0055] 6) Dosage frequency and cycle: Administer once daily for 7 days; obtain blood and liver samples from the dosage group.
[0056] The control group consisted of blood and liver samples from 10 mice that did not receive the drug.
[0057] (a) Blood collection for liver function testing Liver function tests are shown in Table 1. Each group consisted of 10 mice. Data are expressed as mean ± standard deviation.
[0058] Table 1 Liver function tests
[0059] Note: *: P<0.05, P is the probability value.
[0060] As shown in Table 1, compared with the control group, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), and total cholesterol (TC) were all increased in recombinant mice at doses of 40, 80, and 100 mg / kg.
[0061] (II) Dissection: Gross observation and histopathological examination of the liver. Gross anatomical examination of mouse livers revealed that the livers of control group mice were generally reddish-brown, soft in texture, smooth in surface, and without obvious abnormalities; the livers of 40 and 80 mg / kg body weight mice were pale red in color, with slightly uneven texture; the livers of 100 mg / kg body weight mice were larger in volume, harder in texture, heavier, and had a distinctly yellow surface, with some areas showing mottled red and yellow patches, and blunted edges; see also... Figure 2 , Figure 2 Gross anatomical images of the livers of the control group and the 100 mg / kg body weight group.
[0062] The H&E staining results of the livers of mice in each dose group and the control group are as follows: Figure 3 As shown: Left upper control group: The liver lobule structure is clear, the hepatocytes are arranged regularly, the morphology of the hepatic cords and hepatic sinusoids is normal, the hepatocyte cytoplasm staining is uniform, the cell nuclei are normal in morphology and position, there are no obvious abnormalities such as degeneration and necrosis, and inflammatory cells are rare in the tissue; The low-dose group in the upper right corner (40mg / kg body weight): some hepatocytes showed slight changes, such as vacuoles in the cytoplasm of individual hepatocytes (indicated by arrows). The structure of the liver lobules was normal, but the arrangement of hepatocytes was slightly disordered. Compared with the control group, early signs of damage began to appear. Left lower medium dose group (80mg / kg body weight): Increased hepatocellular vacuolar degeneration ( Figure 3 (At the arrow) the size and distribution of vacuoles were uneven, the disorder of hepatocytes in the liver lobules was aggravated, a small number of inflammatory cells were infiltrated, and the hepatocyte damage was more obvious than in the 40mg / kg group; In the high-dose group (100 mg / kg body weight) in the lower right corner: hepatocyte vacuoles were widely present, hepatocyte structure was destroyed in some areas, vacuolar fusion or abnormal hepatocyte morphology was caused, liver lobule structure was unclear (damage was prominent at the arrow), and inflammatory cell infiltration was increased; suggesting that the pathological damage to liver tissue tends to worsen with increasing dose.
[0063] Oil Red O staining results of livers from mice in each dosage group and the control group are as follows: Figure 4 As shown: Left upper control group: In Oil Red O staining of liver tissue, there was almost no obvious red (lipid droplet staining was red) positive signal. In the liver lobule structure, the cytoplasm of hepatocytes was stained evenly, indicating that there was very little fat deposition in the liver and basically no abnormal lipid accumulation. The upper right low-dose group (40 mg / kg body weight): scattered, small amounts of red lipid droplets were visible, mainly distributed in the cytoplasm of hepatocytes. Compared with the control group, lipid deposition began to appear, but the area was small and the number was small, suggesting that mild lipid accumulation had already occurred in the liver under low-dose intervention. The lower left medium dose group (80mg / kg body weight): The red lipid droplet staining was significantly increased and more widely distributed. In the hepatocyte cytoplasm within the liver lobules, the number and area of lipid droplets were greater than those in the low dose group. In some areas, lipid droplets could be aggregated in small patches, indicating that the degree of liver fat deposition increased with increasing dose. The high-dose group in the lower right corner (100mg / kg body weight): red lipid droplets were widely and densely stained, almost diffusely distributed in the liver tissue. A large number of lipid droplets accumulated in the cytoplasm of hepatocytes. In some areas, the morphology of hepatocytes was changed due to excessive lipid droplets.
[0064] This embodiment provides a method for establishing and applying a mouse model of non-alcoholic fatty liver disease rapidly induced by high-nitrogen compounds.
[0065] The mouse non-alcoholic fatty liver disease (NAFLD) model constructed by this method has a high similarity to clinical pathology, does not require high-fat diet, is low-cost and highly efficient, and the fatty liver phenotype appears after 7 days of gavage administration. It can effectively shorten the establishment cycle of NAFLD animal models, solve the problems of long modeling time and high cost, and greatly save the time and cost of NAFLD medical research.
[0066] In the description of this invention, it should be understood that if terms such as "left," "upper," and "right" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, it does not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the accompanying drawings are for illustrative purposes only and should not be construed as limiting the invention.
[0067] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.
Claims
1. A method for constructing a mouse model of non-alcoholic fatty liver disease, characterized in that: Includes the following steps: S1. Select experimental mice; S2. Prepare an aqueous solution of a high-nitrogen compound with a mass concentration of 4-10 mg / mL; S3. According to the dosage, administration time, and administration time, the prepared high-nitrogen compound aqueous solution was administered orally to obtain a non-alcoholic fatty liver animal model.
2. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: The experimental mice were BALB / c mice.
3. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 2, characterized in that: The BALB / c mice were male mice aged 6-8 weeks and weighing 20-25g.
4. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 2, characterized in that: The BALB / c mice were housed in an SPF-grade laboratory animal room.
5. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: The high-nitrogen compound is a modeling compound with the molecular formula C4H. 16 N 24 .
6. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: The dosage is 1 ml / 100g body weight, and the dose is 40-100 mg / kg body weight.
7. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: The administration period is once daily for 7 days.
8. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: The oral gavage administration includes the following steps: using a gavage needle and a syringe, administer the drug via oral gavage according to the dosage. Insert the tip of the gavage needle into the oral cavity of the experimental mouse, keeping it parallel to the body axis and slowly inserting it. Once a resistance is felt when it reaches the throat, it can smoothly enter the esophagus. After the gavage needle reaches the stomach, slowly inject the drug from the syringe to complete the administration.
9. The method for constructing a mouse non-alcoholic fatty liver model as described in claim 1, characterized in that: It also includes liver function tests, anatomical observations, and pathological histological examinations of the obtained non-alcoholic fatty liver animal models.
10. The method for constructing a mouse non-alcoholic fatty liver model as described in any one of claims 1-9, or the use of the non-alcoholic fatty liver animal model obtained by the method for constructing the mouse non-alcoholic fatty liver model, in screening or developing drugs for treating non-alcoholic fatty liver.