Application of high-fat, high-glucose and high-cholesterol feed in construction of metabolism-related steatohepatitis rat model with insulin resistance

Abstract

The invention relates to a high-fat, high-sugar and high-cholesterol feed which is prepared from the following raw materials: maintenance basal feed, lard, fructose, maltodextrin, whole milk powder, casein, multivitamins, microcrystalline cellulose, calcium hydrophosphate, cholesterol, sodium cholate and mountain flour. A metabolism-related steatohepatitis rat model with insulin resistance constructed on the basis of the diet formula can simulate liver fatty degeneration, inflammatory infiltration and fibrosis induced by bad diet, and has obvious insulin resistance characteristics; the method accords with human dietary habits, reflects pathogenesis, pathogenesis, pathology and disease development and characteristics of human metabolism-related steatohepatitis, and is low in price, high in survival rate, mild in process, simple and stable in model establishment and short in time consumption.

Description

Technical Field

[0001] The invention relates to a high-fat, high-sugar and high-cholesterol feed and application thereof in constructing a metabolic-related fatty hepatitis rat model accompanied by insulin resistance. Background Art

[0002] With the improvement of people's living standards, changes in living habits and dietary structure, the incidence of metabolic-related fatty liver disease has increased year by year, and it is showing a trend of younger age. It will progress to metabolic-related fatty liver disease, leading to progressive fibrosis, sometimes leading to cirrhosis, and then leading to liver decompensation and hepatocellular carcinoma (HCC). The causes of metabolic-related fatty liver disease are complex, and insulin resistance, hyperlipidemia, obesity, diabetes, etc. induced by high-sugar, high-fat, and high-cholesterol diets are considered to be the main high-risk factors. Among them, insulin resistance, as one of its important pathogenesis, may aggravate the appearance of clinical symptoms. In addition, it will also lead to metabolic disorders in the body, promote the occurrence and development of cardiovascular and endocrine diseases such as atherosclerosis, coronary heart disease, and diabetes, and increase the risk of extrahepatic diseases such as chronic kidney disease and osteoporosis. The occurrence of these complications is closely related to the severity and duration of insulin resistance. At the same time, liver lesions increase the sensitivity of the body and increase the risk of clinical medication, which seriously affects human medical health.

[0003] Common treatment methods generally include controlling diet and losing weight. Indirect control drugs such as insulin sensitizers, vitamin E, lipid-lowering and glucose-lowering drugs, and probiotics can slow down the progression of liver disease to a small extent. However, for most patients, it is difficult to maintain a long-term regular life and lose weight, and most indirect drugs usually target a single pathway and their safety is difficult to guarantee. In recent years, the development of safe and effective anti-metabolism-related fatty liver drugs has gradually become a hot topic. In order to ensure that the developed drugs are suitable for clinical use, it is necessary to explore models of pathogenesis and symptoms that are close to clinical practice.

[0004] Given that clinical sample collection is subject to ethical restrictions, research on metabolic-related fatty liver disease has progressed slowly. Preclinical animal models are an important means to study the pathogenesis of metabolic-related fatty liver disease and screen effective prevention and treatment measures. The use of a high-fat diet to induce an animal model of metabolic-related fatty liver disease is currently the most commonly used method, which has the advantages of being easy to operate and consistent with human pathogenesis and symptoms; but it also has defects such as long modeling time and mild inflammation and fibrosis; while the methionine-choline deficiency diet has a short modeling time and good results, but is expensive, and the diet does not conform to the human dietary structure, and has certain differences from the pathogenesis and symptoms of human metabolic-related fatty liver disease. High starch and high sugar induce mild inflammatory reactions and no insulin resistance. This model can only be used for drug screening, not for pathogenesis research. Although the modeling time is short when high-concentration fat emulsion is gavaged or drugs such as carbon tetrachloride are intraperitoneally injected to cause acute liver lesions to induce metabolic-related fatty liver disease, the success rate of modeling is low, the animal mortality rate is high, and the pathogenesis, pathological process and histological morphological changes are all quite different from human metabolic-related fatty liver disease. High-fat diet plus tetracycline induction does not conform to human dietary habits. The pathogenesis, course of disease evolution and final tissue morphological changes are quite different from those of humans. Animals are prone to poisoning and death, and no fibrosis occurs. High-fat diet combined with streptozotocin induces lobular inflammation and fibrosis and causes severe liver damage, but it is suitable for the study of diabetes combined with metabolic-related fatty liver hepatitis. Using fa / fa rats, ob / ob mice, BHE rats, leptin-resistant mice, PTEN knockout mouse models and other strains with certain genetic characteristics can form fatty liver disease related to sugar and fat metabolism, but the cycle is long or requires acquired cultivation, the price is extremely expensive, and the practicality is limited.

[0005] The current common metabolic-related fatty liver hepatitis models still have the following problems: long modeling cycle, high cost, poor effect, not in line with human dietary habits, high mortality rate, pathogenesis, course of disease evolution and final tissue morphological changes are quite different from those of humans, and most of them have no or only less obvious pathological features of insulin resistance and fibrosis, which are quite different from clinical practice. Summary of the invention

[0006] In view of the above problems existing in the prior art, the present invention provides a high-fat, high-sugar, and high-cholesterol feed. A rat model of metabolic-related fatty liver hepatitis with insulin resistance is constructed based on the dietary formula, which can simulate the liver fatty degeneration, inflammatory infiltration and fibrosis induced by a poor diet, has obvious insulin resistance characteristics, conforms to human dietary habits, reflects the pathogenesis, etiology, pathology, disease development and characteristics of human metabolic-related fatty liver hepatitis, has a low price, a high survival rate, a mild process, and the model establishment is simple, stable and time-saving.

[0007] The first aspect of the present invention provides a feed for inducing a metabolic-related fatty hepatitis rat model, which is made of the following raw materials: calculated by the percentage content of each raw material, 27%-31% maintenance basic feed, 23%-27% lard, 8%-12% fructose, 1%-1.5% maltodextrin, 10%-12% whole milk powder, 9%-11% casein, 1%-3% complex vitamins, 3%-5% microcrystalline cellulose, 3%-5% calcium hydrogen phosphate, 1%-1.5% cholesterol, 0.3%-0.7% sodium cholate, and 0.5%-1% stone powder.

[0008] The raw materials are all commercially available products.

[0009] The second aspect of the present invention provides the use of the feed described in the first aspect in constructing a metabolic-related fatty liver disease rat model, and the model construction method is as follows: Wistar rats are fed with the feed in a free-feeding manner for 16 weeks.

[0010] The third aspect of the present invention provides a method for constructing a metabolic-related fatty liver disease rat model, comprising the following steps: feeding Wistar rats with the feed described in the first aspect in a free-feeding manner for 16 weeks.

[0011] The fourth aspect of the present invention provides the use of the metabolism-related fatty liver disease rat model obtained by the construction method described in the third aspect in screening therapeutic drugs for metabolism-related fatty liver disease.

[0012] The metabolism-related fatty liver disease rat model in the first, second, third and fourth aspects of the present invention is a metabolism-related fatty liver disease rat model with fibrosis.

[0013] Preferably, the rat model of non-alcoholic fatty liver disease with fibrosis also has the characteristics of insulin resistance.

[0014] Preferably, the rats are healthy male Wistar rats, 6-8 weeks old, weighing 180-220 grams.

[0015] Beneficial Effects

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) While shortening the modeling cycle, there is obvious fatty degeneration, inflammatory infiltration and fibrosis, with obvious insulin resistance characteristics, and the effect is good.

[0018] (2) An appropriate proportion of sodium cholate is added to the formula to promote the absorption of cholesterol and fat, and it is in an optimal ratio with cholesterol.

[0019] (3) An appropriate proportion of fructose is added to the formula. This proportion is relatively high and can appropriately improve the palatability of experimental animals. Animals are not prone to anorexia after long-term consumption.

[0020] (4) Maltodextrin can promote the accumulation of fat in the liver, and whole milk powder provides high fat and high energy, which accelerates the development of metabolic-related fatty liver disease.

[0021] (5) High-fat, high-sugar, and high-cholesterol feed is in line with human eating habits.

[0022] (6) It is consistent with the pathogenesis of human non-alcoholic fatty liver disease and can reflect the etiology, pathology, symptoms, disease development and characteristics of human metabolic-related fatty liver disease.

[0023] (7) Low price and 100% survival rate.

[0024] (8) The modeling method is simple and stable, and the process is gentle.

[0025] (9) The constructed animal model can be used to study the role of diet in the pathogenesis of insulin resistance-related metabolic fatty liver disease and screen for preventive and therapeutic measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the appearance of rat liver.

[0027] Figure 2 The results of HE staining and picrosirius red staining of rat liver sections (×200).

[0028] Figure 3 This is a data chart of oral glucose tolerance and intraperitoneal insulin tolerance. DETAILED DESCRIPTION

[0029] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0030] Example 1 Construction of a rat model of nonalcoholic fatty liver disease with fibrosis

[0031] Healthy male Wistar rats aged 6-8 weeks and weighing 180-220 grams were selected and randomly divided into a model group and a blank group, with 8 rats in each group.

[0032] Model group: After one week of adaptive feeding, the mice were allowed to eat high-fat, high-sugar, and high-cholesterol feed ad libitum for 16 consecutive weeks.

[0033] Blank group: The mice were provided with basic feed and allowed to eat freely for 16 consecutive weeks.

[0034] After 16 weeks of continuous feeding, no rats in the model group and the blank group died.

[0035] The high-fat, high-sugar, and high-cholesterol feed is made of the following raw materials: calculated by the percentage of each raw material, 29.05% maintenance basic feed, 25% lard, 10% fructose, 1.3% maltodextrin, 10.8% whole milk powder, 10% casein, 2% multivitamins (Beijing B. Braun Agriculture and Animal Husbandry Technology Co., Ltd., item number: 0321), 5% microcrystalline cellulose, 4.3% calcium hydrogen phosphate, 1.25% cholesterol, 0.5% sodium cholate, and 0.8% stone powder.

[0036] The basic feed (Beijing SPF Biotechnology Co., Ltd., item number: SPF-F02-001) was composed of 29% first-grade flour, 4.1% high-gluten flour, 40.84% ​​puffed corn / corn, 2% fish meal, 16.06% soybean meal, 2% soybean oil, and 6% premix.

[0037] Example 2 Evaluation of the results of modeling of nonalcoholic fatty liver disease with fibrosis in rats

[0038] 1. Specific operations

[0039] At week 6, two rats in the model group were randomly killed, and the livers were removed, collected and stored in liquid nitrogen or fat-specific fixative and 4% paraformaldehyde for histological analysis.

[0040] At the 16th week, the body weight and insulin resistance indexes of the rats in the blank group and the remaining rats in the model group were measured, and then the rats were killed, the liver was removed, the liver morphology was observed and the liver weight was measured, and the liver was collected and stored in liquid nitrogen or fat-specific fixative and 4% paraformaldehyde for histological analysis (HE staining, picrosirius red staining).

[0041] 2. Evaluation results

[0042] 2.1 Body weight, liver weight, and liver-to-body weight ratio

[0043] Table 1 Body weight, liver weight, and liver-to-body weight ratio of rats

[0044]

[0045]

[0046] Note: Compared with the blank group, # P<0.05, ## P<0.01, ### P<0.001.

[0047] Compared with the blank group, the body weight, liver weight and liver-to-body weight ratio of rats in the model group were significantly increased (P<0.05).

[0048] 2.2 Liver morphology

[0049] like Figure 1 As shown, compared with the blank group, the liver volume of the rats in the model group was significantly increased, the capsule was tense, the edges were rounded and blunt, the color was creamy yellow, and it felt greasy and heavy.

[0050] 2.3 Histopathology

[0051] like Figure 2 As shown, at the 16th week, the results of HE staining and picrosirius red staining showed that the liver tissue and cell structure of the rats in the blank group were normal and complete, with clear cell nuclei, abundant cytoplasm, and normal-sized sinusoidal waves. There was no degeneration, ballooning, or fibrosis.

[0052] At week 6, HE staining and picrosirius red staining results showed that the model group rats had lipid accumulation in the liver. At week 16, the liver tissue structure of the model group was abnormal, the central vein in the field of vision was significantly enlarged, and cell edema was visible around it. In addition, the inflammatory infiltration area was clearly visible, and the number was large, and fibrosis appeared.

[0053] 2.4 Insulin resistance indicators

[0054] like Figure 3 As shown in Table 2, compared with the blank group, the oral glucose tolerance, intraperitoneal insulin tolerance, and serum insulin level of the rats in the model group were significantly increased (p<0.001), the insulin resistance index was significantly increased (p<0.001), and the insulin sensitivity index was significantly decreased (p<0.001).

[0055] Table 2 Insulin resistance indexes in rats

[0056]

[0057] Note: Compared with the blank group, # P<0.05, ## P<0.01, ### P<0.001.

[0058] 3. Conclusion

[0059] According to the method provided by the present invention, rats were modeled, and no animal died, with a success rate of 100%. It can be seen that the method for establishing the animal model of the present invention is safe and reliable, with a high success rate, shortening the modeling cycle, and having obvious fatty degeneration, inflammatory infiltration and fibrosis, and having obvious insulin resistance characteristics, with good effects; it is consistent with the pathogenesis of human metabolic-related fatty liver disease, and can reflect the etiology, pathology, symptoms, disease development and characteristics of human metabolic-related fatty liver disease, and has good application prospects, and provides an important strategy for exploring models of pathogenesis and symptoms close to clinical practice and treatment measures.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A feed for inducing a rat model of metabolic-related fatty hepatitis with insulin resistance, characterized in that: The feed is made of the following raw materials: according to the percentage content of each raw material, 27%-31% maintenance basic feed, 23%-27% lard, 8%-12% fructose, 1%-1.5% maltodextrin, 10%-12% whole milk powder, 9% to 11% casein, 1% to 3% multivitamins, 3% to 5% microcrystalline cellulose, 3% to 5% calcium hydrogen phosphate, 1%~1.5% cholesterol, 0.3%~0.7% sodium cholate, 0.5%~1% stone powder.

2. The feed according to claim 1, characterized in that The feed is made of the following raw materials: calculated by the percentage of each raw material, 29.05% maintenance basic feed, 25% lard, 10% fructose, 1.3% maltodextrin, 10.8% whole milk powder, 10% casein, 2% complex vitamins, 5% microcrystalline cellulose, 4.3% calcium hydrogen phosphate, 1.25% cholesterol, 0.5% sodium cholate, and 0.8% stone powder.

3. Use of the feed according to claim 1 or 2 in constructing a rat model of metabolic-related fatty hepatitis with insulin resistance, characterized in that: The model was constructed as follows: Wistar rats were fed with the feed in a free-feeding manner for 16 weeks.

4. A method for constructing a rat model of metabolic-related fatty liver disease with insulin resistance, comprising the following steps: The feed according to claim 1 or 2 is used to feed Wistar rats ad libitum for 16 weeks to obtain the obtained product.

5. Use of the metabolic-related fatty liver disease rat model obtained by the construction method of claim 4 in screening therapeutic drugs for metabolic-related fatty liver disease.

6. The feed according to claim 1 or 2, or the use according to claim 3 or 5, or the method according to claim 4, characterized in that: The metabolic-related fatty liver disease rat model with insulin resistance is also accompanied by fibrosis characteristics.

7. The feed, use or method according to any one of claims 1 to 6, characterized in that The rats are healthy male Wistar rats, 6-8 weeks old, weighing 180-220 grams.

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