Construction method and application of metabolic disorder related fatty liver disease spleen-deficiency mouse model

Abstract

The invention discloses a construction method and application of a metabolic disorder related fatty liver disease spleen-deficiency mouse model, and relates to the technical field of biological model construction.The construction method is technically characterized in that a liquid medicine containing a large dose of radix rehmanniae recen and radix rehmanniae praeparata or a crude extract of the radix rehmanniae recen and the radix rehmanniae praeparata is added into a high-fat, high-sugar and high-cholesterol feed; an MASLD spleen-deficiency mouse model can be stably induced, and the increase of the weight of the mouse is not influenced; the mouse model is used for research on prevention and treatment of MASLD by traditional Chinese medicine.

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 metabolic disorder-related fatty liver disease spleen deficiency mouse model and application thereof. 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 models for spleen deficiency mainly include: overwork and dietary disorder, narcissine, reserpine, bitter cold purgation, and qi depletion and breaking qi. However, a common problem of these commonly used modeling methods is that they will cause mice to lose weight, which will affect the effect of the disease model when used for MASLD mouse 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 spleen deficiency syndrome and its application to solve the above 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 spleen deficiency mouse model of fatty liver disease associated with metabolic disorders and its application. By adding a medicinal solution containing raw rehmannia root and cooked rehmannia root or a crude extract of raw rehmannia root and cooked rehmannia root to a high-fat, high-sugar, and high-cholesterol feed, the weight of experimental mice in the MASLD spleen deficiency mouse model can be stably induced; the mouse model is used for research on the prevention and treatment of MASLD with traditional Chinese medicine.

[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 metabolic disorder-related fatty liver disease spleen deficiency mouse model, the method comprising the following steps:

[0009] S1. Create experimental groups: Create four experimental groups, feed the mice in each experimental group with the same amount of different feeds, and feed the mice in two experimental groups with high-fat, high-sugar, and high-cholesterol feed supplemented with Chinese medicine liquid and high-fat, high-sugar, and high-cholesterol customized medicated feed respectively;

[0010] S2. Analyze experimental data.

[0011] The invention discloses an application of a mouse model of metabolic disorder-related fatty liver disease and spleen deficiency syndrome, wherein the mouse model is used for research on the prevention and treatment of MASLD by traditional Chinese medicine.

[0012] The four experimental groups included a negative control group, a positive control group, a MASLD spleen deficiency observation group 1, and a MASLD spleen deficiency observation group 2;

[0013] The mice in the negative control group were fed with a normal control feed;

[0014] The mice in the positive control group were fed with a high-fat, high-sugar, and high-cholesterol diet;

[0015] The mice in the MASLD spleen deficiency observation group were fed with a high-fat, high-sugar, and high-cholesterol feed supplemented with Chinese medicine liquid;

[0016] The mice in the second MASLD spleen deficiency observation group were fed with a customized medicated feed containing high fat, high sugar and high cholesterol.

[0017] The control feed is a standard maintenance feed for experimental mice, consisting of 10% fat, 22% protein and 68% carbohydrates without fructose or cholesterol;

[0018] The high-fat, high-sugar, high-cholesterol feed is composed of 40% fat, 22% fructose and 2% cholesterol;

[0019] The Chinese medicine liquid is prepared by decocting and concentrating the Chinese medicine bag, which is composed of raw Rehmannia root and cooked Rehmannia root in a ratio of 1:1. Adults take 60g of each per day. The dosage for experimental mice is calculated based on the conversion rate of human and animal body surface areas, with reference to the "Methodology of Chinese Medicine Pharmacology Research";

[0020] The high-fat, high-sugar, and high-cholesterol customized medicated feed is composed of 40% fat, 22% fructose, 2% cholesterol, and 5.2% crude extract of traditional Chinese medicine, wherein the crude extract of traditional Chinese medicine is prepared by crudely extracting raw rehmannia root and cooked rehmannia root in a ratio of 30:1.

[0021] In this program,

[0022] Compared with the prior art, this solution has the following beneficial effects:

[0023] The invention can stably induce the weight of experimental mice in the MASLD spleen deficiency mouse model by adding a medicinal solution containing raw rehmannia root and cooked rehmannia root or a crude extract of raw rehmannia root and cooked rehmannia root to a high-fat, high-sugar and high-cholesterol feed, thereby improving the experimental accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a line graph of weight changes of mice in different experimental groups in the embodiment of the present invention;

[0025] Figure 2 is a bar graph of serum triglycerides (TG) and total cholesterol (TC) of mice in different experimental groups in the embodiment of the present invention;

[0026] 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;

[0027] Figure 4 Schematic diagram of liver tissue pathology of mice in different experimental groups in the embodiment of the present invention;

[0028] Figure 5 Schematic diagram of the anus of mice in different experimental groups in the embodiments of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] It should be noted that, in the absence of conflict, the embodiments and features of 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.

[0031] Example:

[0032] Create experimental groups;

[0033] Experimental Group 1: Male C57BL / 6 mice aged 6-8 weeks were given adequate 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 and gavaged with normal saline to serve as the negative control group (CON).

[0034] 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 after one week of adaptive feeding, and were gavaged with normal saline to serve as the MASLD positive control group (HFD).

[0035] Experimental Group 3: 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, Synergy Biological) for 12 weeks after one week of adaptive feeding. They were gavaged with Chinese herbal decoction concentrate (refer to "Methodology of Pharmacological Research of Traditional Chinese Medicine", standard conversion factor of experimental animals, mouse: human = 9.1, 60g each per day for adults, 120g for males, standard weight of adults 70kg, mouse dosage 120*9.1 / 70 = 15.6) as a group for observation of MASLD spleen deficiency (HFD-PX1).

[0036] Experimental group 4: Male C57BL / 6 mice aged 6-8 weeks were fed with a customized medicated feed with high fat, high sugar and high cholesterol (40% fat + 22% fructose + 2% cholesterol + 5.2% crude extract of traditional Chinese medicine, the ratio of raw Rehmannia root and cooked Rehmannia root crude extract was 30:1, calculated according to the previous dosage) for 12 weeks after adaptive feeding for one week. They were gavaged with normal saline as a group for observation of MASLD spleen deficiency (HFD-PX2). The general process of crude extraction was: raw material - cleaning - drying - spray drying - extraction - concentration - extract - crushing.

[0037] Results evaluation:

[0038] Result 1: The weight of mice is shown in Table 1. It can be seen that compared with the normal group in experimental group 1, the weight of mice in the normal group increased steadily, and the growth rate of experimental groups 2, 3, and 4 was significantly faster. The final weight increased significantly, and the difference was statistically significant compared with the normal group in experimental group 1 (P < 0.05). There was no significant difference among experimental groups 2, 3, and 4 (P > 0.05), which was consistent with the general situation of weight increase in MASLD model mice from the perspective of weight.

[0039] Table 1 Mouse weight changes

[0040]

[0041] 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, 3, and 4 were significantly increased, and the differences were statistically significant compared with the normal group in experimental group 1 (P < 0.05). There were no significant differences among experimental groups 2, 3, and 4 (P > 0.05), which is consistent with the abnormal lipid metabolism of MASLD model mice.

[0042] Table 2 Record of biochemical indexes of mice

[0043]

[0044] Note: **, compared with CON, P < 0.01; ***, compared with CON, P < 0.001.

[0045] Result 3: As shown in Table 2, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in experimental groups 2, 3, and 4 were significantly increased, and the differences were statistically significant compared with the normal group in experimental group 1 (P < 0.05). There were no significant differences among experimental groups 2, 3, and 4 (P > 0.05), which was consistent with the abnormal liver function of MASLD model mice.

[0046] 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, 3, and 4, there was obvious lipid droplet accumulation, fat vacuoles, ballooning, and inflammatory cell infiltration in the liver tissues of mice, which was consistent with the liver tissue pathology diagnosis of MASLD model mice.

[0047] 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 raw and cooked rehmannia in experimental groups 3 and 4 showed loose stools and dirty anus, which is consistent with the manifestation of spleen deficiency in MASLD.

[0048] in conclusion:

[0049] Neither the oral administration of a high-fat, high-sugar diet combined with large doses of raw and cooked rehmannia nor the incorporation of large doses of crude extracts of raw and cooked rehmannia into a high-fat, high-sugar diet affected the body weight of mice. Both diets could stably induce a mouse model of MASLD spleen deficiency.

[0050] 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 spleen deficiency syndrome, characterized by: The method comprises the following steps: S1. Create experimental groups: Create four experimental groups, feed the mice in each experimental group with the same amount of different feeds, and feed the mice in two experimental groups with high-fat, high-sugar, and high-cholesterol feed supplemented with Chinese medicine liquid and high-fat, high-sugar, and high-cholesterol customized medicated feed respectively; S2. Analyze experimental data.

2. The use of a metabolic disorder-related fatty liver disease spleen deficiency mouse model as claimed in claim 1, characterized in that: The mouse model is used for the study of various types of MASLD with spleen deficiency as the main symptom.

3. The method for constructing a metabolic disorder-related fatty liver disease spleen deficiency mouse model according to claim 1, characterized in that: The four experimental groups include a negative control group, a positive control group, a MASLD spleen deficiency observation group 1 and a MASLD spleen deficiency observation group 2; The mice in the negative control group were fed with a normal control feed; The mice in the positive control group were fed with a high-fat, high-sugar, and high-cholesterol diet; The mice in the MASLD spleen deficiency observation group were fed with a high-fat, high-sugar, and high-cholesterol feed supplemented with Chinese medicine liquid; The mice in the second MASLD spleen deficiency observation group were fed with a customized medicated feed containing high fat, high sugar and high cholesterol.

4. The method for constructing a metabolic disorder-related fatty liver disease spleen deficiency mouse model according to claim 3, characterized in that: The control feed is a standard maintenance feed for experimental mice; The high-fat, high-sugar, high-cholesterol feed is composed of 40% fat, 22% fructose and 2% cholesterol, and the fat is palm oil; The Chinese medicine liquid is prepared by decocting and concentrating the Chinese medicine bag, which is composed of raw Rehmannia root and cooked Rehmannia root in a ratio of 1:

1. Adults take 60g of each per day. The dosage for experimental mice is calculated based on the conversion rate of human and animal body surface areas, with reference to the "Methodology of Chinese Medicine Pharmacology Research"; The high-fat, high-sugar, and high-cholesterol customized medicated feed is composed of 40% fat, 22% fructose, 2% cholesterol, and 5.2% crude extract of traditional Chinese medicine, wherein the crude extract of traditional Chinese medicine is prepared by crudely extracting raw rehmannia root and cooked rehmannia root in a ratio of 30:1.

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