Construction method and application of metabolic disorder related fatty liver disease damp-heat syndrome mouse model

Abstract

The invention discloses a construction method and application of a metabolic disorder related fatty liver disease damp-heat syndrome mouse model, and relates to the technical field of biological model construction.The construction method is technically characterized in that the damp-heat environment is simulated through a simple and convenient method of combining a silica gel heating plate with wet padding, and high-fat and high-sugar feed feeding is combined, so that the metabolic disorder related fatty liver disease damp-heat syndrome mouse model is obtained. The MASLD damp-heat syndrome mouse model is stably induced, and the equipment acquisition difficulty and the modeling cost in the research process are reduced; the mouse model is used for research on construction of MASLD of various symptom types mainly including damp-heat.

Description

Technical Field

[0001] The present invention relates to the technical field of biological model construction, and in particular to a method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome and its application. Background Art

[0002] In today's society, with the changes in lifestyle and dietary structure, the incidence of metabolic disorder-associated fatty liver disease (MASLD) has increased year by year and has become the primary cause of chronic liver disease in my country. In recent years, intervention strategies for MASLD are developing rapidly, but there is still a huge technical gap. The research and development of related therapeutic drugs has always been a research hotspot pursued by the international academic community and pharmaceutical companies.

[0003] Traditional Chinese medicine is a treasure of Chinese medicine. Due to its systemic and low toxicity and side effects, it is receiving more and more attention in the prevention and treatment of MASLD. However, in the research process of TCM prevention and treatment of MASLD, there is a long-standing problem, that is, when conducting preclinical efficacy and pharmacology studies on some clinically effective empirical prescriptions or classical prescriptions, there is a lack of animal models that can better meet the characteristics of TCM treatment of "symptom combination". From the perspective of TCM, MASLD belongs to the categories of "flank pain", "accumulation", "liver disorder", etc., and common clinical symptoms include spleen deficiency and liver depression, damp-heat accumulation, phlegm and blood stasis, etc.

[0004] At present, the mouse model of damp-heat syndrome is mainly given a high temperature and high humidity environment to simulate a damp-heat environment. Common conditions include: temperature 34℃~36℃, humidity 85%~95%, and daily intervention for 8-12 hours. This modeling method requires basic equipment, a small animal incubator. For many research groups with limited experimental platform conditions, it is difficult to obtain equipment, which is not convenient for conventional modeling.

[0005] To this end, the present invention aims to provide a method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome and its application to solve the above-mentioned problems. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems and provide a method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome and its application. By combining a silicone heating plate with a wet bedding material, a damp-heat environment can be simulated without using a small animal incubator. Combined with a high-fat and high-sugar feed, a MASLD damp-heat syndrome mouse model can be stably induced, reducing the difficulty of obtaining equipment and the cost of modeling during the research process. The mouse model is used to construct research on various types of MASLD with damp-heat as the main symptom.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome, the method comprising the following steps:

[0009] S1. Create experimental groups: Create three experimental groups, two of which are raised in a normal breeding environment, and the other is raised in a conventional SPF breeding cage. A silicone heating plate is attached to the top of the breeding cage, and the temperature is set to 36°C. The cage is irradiated for 12 hours a day, and the wet bedding is replaced every other day (humid heat simulation environment);

[0010] S2. Analyze experimental data.

[0011] The invention discloses an application of a mouse model of metabolic disorder-related fatty liver disease damp-heat syndrome, wherein the mouse model is used to construct a study on various types of MASLD with damp-heat as the main symptom.

[0012] The three experimental groups included a negative control group, a MASLD positive control group, and a MASLD damp-heat syndrome observation group;

[0013] The negative control group was fed with a normal control feed in a normal feeding environment for 12 weeks;

[0014] The negative control group was fed with a normal control feed for 12 weeks in a normal feeding environment, and the control feed was a standard maintenance feed for experimental mice;

[0015] The MASLD positive control group was fed with a high-fat, high-sugar, and high-cholesterol diet for 12 weeks in a normal feeding environment;

[0016] The MASLD damp-heat syndrome observation group was kept in a conventional SPF cage with a silicone heating plate attached to the top of the cage and the temperature set to 36°C. The mice were irradiated for 12 hours a day, the wet bedding was replaced every other day, and they were fed with a high-fat, high-sugar, and high-cholesterol feed for 12 weeks.

[0017] The high-fat, high-sugar and high-cholesterol feed consists of 40% fat, 22% fructose and 2% cholesterol, and the fat is palm oil.

[0018] Compared with the prior art, this solution has the following beneficial effects:

[0019] The present invention can simulate a hot and humid environment without using a small animal incubator by combining a silicone heating plate with a wet bedding material, and can stably induce a MASLD hot and humid syndrome mouse model by combining a high-fat and high-sugar feed, thereby reducing the difficulty of obtaining equipment and the cost of modeling during the research process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a line graph of weight changes of mice in different experimental groups in the embodiment of the present invention;

[0021] Figure 2is a bar graph of serum triglycerides (TG) and total cholesterol (TC) of mice in different experimental groups in the embodiment of the present invention;

[0022] Figure 3 is a bar graph of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice in different experimental groups in the embodiment of the present invention;

[0023] Figure 4 Schematic diagram of liver tissue pathology of mice in different experimental groups in the embodiment of the present invention;

[0024] Figure 5 Schematic diagram of the anus of mice in different experimental groups in the embodiments of the present invention. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0027] Example:

[0028] Create experimental groups;

[0029] Experimental Group 1: Male C57BL / 6 mice aged 6-8 weeks were given sufficient normal control feed and water, and allowed to grow freely without any intervention. After one week of adaptive feeding, they were fed with normal control feed for 12 weeks in a normal feeding environment with a temperature of 20℃-26℃, a humidity of 50%-60%, and a light of 12h / 12h, as a negative control group (CON). The control feed was a standard maintenance feed for experimental mice, consisting of 10% fat, 22% protein, and 68% carbohydrates without fructose or cholesterol.

[0030] Experimental Group 2: Male C57BL / 6 mice aged 6-8 weeks were fed with a high-fat, high-sugar, high-cholesterol diet (40% fat + 22% fructose + 2% cholesterol, XT310, Jiangsu Synergy Biological) for 12 weeks in a normal feeding environment after one week of adaptive feeding, serving as the MASLD positive control group (HFD).

[0031] Experimental Group 3: Male C57BL / 6 mice aged 6-8 weeks were fed with a high-fat, high-sugar, and high-cholesterol diet (40% fat + 22% fructose + 2% cholesterol, XT310, Jiangsu Synergy Biological) for 12 weeks in a simulated heat and humidity environment after one week of adaptive feeding as the MASLD heat and humidity observation group (HFD-SR).

[0032] Results evaluation:

[0033] Result 1: The weight of mice is shown in Table 1. It can be seen that compared with experimental group 1, the weight of normal group mice increased steadily, while the growth rates of experimental groups 2 and 3 were significantly faster, and the final weight increased significantly. The differences were statistically significant compared with experimental group 1 (P < 0.05). There was no significant difference between experimental groups 2 and 3 (P > 0.05), which was consistent with the general situation of weight increase in MASLD model mice from the perspective of weight.

[0034] Table 1 Mouse weight record

[0035]

[0036] Result 2: Blood lipids of mice As shown in Table 2, the levels of serum triglyceride (TG) and total cholesterol (TC) in experimental groups 2 and 3 were significantly increased, and the differences were statistically significant compared with the normal group in experimental group 1 (P < 0.05). There was no significant difference between experimental groups 2 and 3 (P > 0.05), which is consistent with the abnormal lipid metabolism of MASLD model mice.

[0037] Table 2 Record of biochemical indexes of mice

[0038]

[0039] Note: **, compared with CON, P < 0.01; ***, compared with CON, P < 0.001.

[0040] Result 3: As shown in Table 2, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in experimental groups 2 and 3 were significantly increased, and the differences were statistically significant compared with experimental group 1 (P < 0.05). There was no significant difference between experimental groups 2 and 3 (P > 0.05), which was consistent with the abnormal liver function of MASLD model mice.

[0041] Result 4: Mouse liver tissue pathology: In experimental group 1, the liver lobule structure of normal mice was clear, the hepatocytes were regularly arranged, there were fewer intracellular lipid droplets and no obvious oil red staining was observed. In experimental groups 2 and 3, there was obvious lipid droplet accumulation, fat vacuoles, ballooning and inflammatory cell infiltration in the liver tissue of mice, which was consistent with the liver tissue pathology diagnosis of MASLD model mice.

[0042] Result 5: The anus and stool morphology of the normal mice in experimental group 1 and the MASLD mice induced by high-fat diet alone in experimental group 2 were normal. The mice induced by high-fat diet combined with damp-heat simulated environment intervention in experimental group 3 all had sticky stools and red and swollen anus, which were consistent with the manifestations of MASLD damp-heat syndrome.

[0043] in conclusion:

[0044] The method of combining the silica gel heating plate with the wet bedding provided by the present invention can simulate a hot and humid environment without using a small animal incubator, and can stably induce a MASLD hot and humid mouse model in combination with a high-fat and high-sugar feed.

[0045] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome, characterized by: The method comprises the following steps: S1. Create experimental groups: Create three experimental groups, two of which are raised in a normal breeding environment, and the other is raised in a conventional SPF breeding cage. A silicone heating plate is attached to the top of the breeding cage, and the temperature is set to 36°C. The cage is irradiated for 12 hours a day, and the wet bedding is replaced every other day. S2. Analyze experimental data.

2. The use of a metabolic disorder-related fatty liver disease damp-heat syndrome mouse model as claimed in claim 1, characterized in that: The mouse model is used to construct a study on various types of MASLD characterized by dampness and heat.

3. The method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome according to claim 1, characterized in that: The three experimental groups included a negative control group, a MASLD positive control group, and a MASLD damp-heat syndrome observation group; The negative control group was fed with a normal control feed for 12 weeks in a normal feeding environment, and the control feed was a standard maintenance feed for experimental mice; The MASLD positive control group was fed with a high-fat, high-sugar, and high-cholesterol diet for 12 weeks in a normal feeding environment; The MASLD damp-heat syndrome observation group was kept in a conventional SPF cage with a silicone heating plate attached to the top of the cage and the temperature set to 36°C. The mice were irradiated for 12 hours a day, the wet bedding was replaced every other day, and they were fed with a high-fat, high-sugar, and high-cholesterol feed for 12 weeks.

4. The method for constructing a mouse model of metabolic disorder-related fatty liver disease and damp-heat syndrome according to claim 3, characterized in that: The high-fat, high-sugar and high-cholesterol feed consists of 40% fat, 22% fructose and 2% cholesterol, and the fat is palm oil.

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