Construction method of animal model of progression-stage hepatic lesion

Abstract

The invention belongs to the field of animal model construction, and particularly relates to a construction method of a progression-stage hepatic lesion animal model. The construction method comprises the step of feeding model animals with high-fat and high-alcohol feed, wherein the high-fat and high-alcohol feed comprises the following components in percentage by mass: 60-75% of basal feed, 5-10% of cholesterol and 20-30% of fatty alcohol premix. According to the invention, model animals are fed with a novel high-fat and high-alcohol feed, so that the progression-stage liver diseases characterized by significant fat accumulation, infiltration of a large number of immune cells, steatohepatitis and hepatic fibrosis are successfully and stably induced in the animals.

Description

Technical Field

[0001] The present invention belongs to the field of animal model construction, and particularly relates to a method for constructing an animal model of advanced liver lesions. Background Art

[0002] Liver diseases pose a major threat to human health. There is no effective treatment for end-stage liver diseases and the fatality rate is high. Primary liver cancer is the seventh most common malignant tumor in the world, with a high number of deaths. In recent years, the incidence of metabolic liver diseases represented by alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), also known as metabolic associated fatty liver disease (MAFLD), has increased rapidly. Currently, the prevalence of fatty liver caused by ALD and NAFLD in China is relatively high. Therefore, it is of great significance to attach importance to the basic and translational research of liver diseases such as ALD and NAFLD. In this process, accurate and effective animal models are indispensable powerful tools.

[0003] Both ALD and NAFLD are metabolic syndromes caused by the long-term action of harmful factors on the liver. Such liver diseases include a long course of disease and similar evolution stages. Hepatic steatosis is a common early lesion in both ALD and NAFLD. With the chronic change of the microenvironment in the liver, the intrahepatic immune-inflammatory system is over-activated in some patients, resulting in alcoholic hepatitis (AH) or non-alcoholic steatohepatitis (NASH). Continuous inflammation can cause massive hepatocyte damage and induce liver fibrosis. If not effectively diagnosed and treated, ultimately a small number of patients will develop into end-stage liver diseases such as advanced liver cirrhosis or primary liver cancer, and the prognosis is poor at this time.

[0004] Metabolic liver diseases represented by ALD and NAFLD have a continuous and complex course of disease evolution, which poses high requirements for related animal models. An ideal animal model should be able to simulate all pathological stages of the occurrence and development of chronic liver diseases. Therefore, in-depth analysis of the advantages and limitations of existing methods will point the way for a new generation of animal diet models.

[0005] The Lieber-DeCarli (LD) diet, first introduced by Lieber et al. in 1963, is the most successful ALD animal model to date. Many important research results on ALD are based on the LD diet model. In view of the habit of rodents to dislike alcohol, this model uses a semi-liquid diet containing alcohol as the only available dietary source, inducing experimental animals to consume sufficient alcohol through eating. The NAFLD diet animal model simulates the high-fat, high-cholesterol diet of humans by increasing the fat energy supply ratio in the feed. Representative high-fat diet models include the simple high-fat diet model (High fat diet, HFD) and the Western diet model (Western diet, WD), and its stable modeling effect has been highly recognized by scientific researchers.

[0006] As the mainstream ALD model, the LD diet has the advantages of low cost, easy implementation, and stable effect. However, it is currently recognized that hepatic steatosis is the main pathological change mediated by the LD model. The inability of the LD diet alone to induce advanced liver lesions including fatty hepatitis and liver fibrosis is its main functional deficiency. Compared with the LD diet, the HFD and WD models only need to replace normal feed, with lower operation and maintenance costs and high safety. HFD and WD feeding can affect the systemic physiological condition of experimental animals, including significant increase in animal weight and hyperlipidemia. There are subtle differences in the effects of HFD and WD diets on the liver: it is generally believed that HFD mediates more severe intrahepatic fat accumulation, while the WD diet, although causing slightly milder hepatic steatosis, can be observed to have a significant inflammatory response. However, HFD or WD diet alone also cannot cause advanced liver disease changes such as liver fibrosis and liver tumors.

[0007] It can be seen that although the mainstream dietary model can cause liver damage efficiently and stably, it generally has the disadvantage of insufficient effectiveness: the use of any dietary model alone can only induce mild to moderate liver changes such as liver steatosis or mild hepatitis, which is particularly prominent in ALD research. Furthermore, there is currently no animal dietary model at home and abroad that can stably induce mid-to-late stage cirrhosis or liver tumors. Although some studies have reported that combining dietary models with other toxins can cause similar results, the introduction of other strong pathogenic factors has greatly increased the difficulty of interpreting the experimental results. Therefore, an animal dietary model that can stably mediate progressive liver disease has great scientific research value. Summary of the invention

[0008] In order to solve the above problems, the present invention has constructed a new type of high-fat and high-alcohol feed, and found that feeding it to model animals can stably induce progressive liver disease characterized by significant fat accumulation, massive immune cell infiltration, fatty hepatitis and liver fibrosis in the animals.

[0009] Specifically, the present invention provides a method for constructing an animal model of advanced liver lesions, characterized in that the construction method includes feeding a high-fat and high-alcohol diet to a model animal.

[0010] Wherein the high-fat and high-alcohol diet contains by mass percentage: 60-75% of basal diet, 5-10% of cholesterol, and 20-30% of fat-ethanol premix.

[0011] The fat-ethanol premix is a microsphere with a core-shell structure, the shell layer is glycidyl methacrylate-modified silk fibroin, and the core is an ethanol-in-oil emulsion.

[0012] Further, the model animal is a rodent.

[0013] Preferably, the rodent is a mouse or a rat.

[0014] Further, the ethanol-in-oil emulsion is composed of an aqueous ethanol solution, an edible oil, and an emulsifier.

[0015] Further, the mass ratio of the aqueous ethanol solution, the edible oil, and the emulsifier is 75-100:150-200:1.2-4.5.

[0016] Further, the volume percentage of ethanol in the aqueous ethanol solution is 40-65%.

[0017] Further, the edible oil includes edible vegetable oil or edible animal oil. Edible vegetable oil includes soybean oil, rapeseed oil, peanut oil, sesame oil, olive oil, corn oil, sunflower oil, and edible vegetable blended oil; edible animal oil includes lard, beef tallow, and fish oil.

[0018] Further, the emulsifier includes one or more of glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate.

[0019] Further, the emulsifier is compounded by glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate in a mass ratio of 5-7:1-2:1-2:3-8:0.5-1.5.

[0020] Further, the preparation method of the high-fat and high-alcohol diet includes the following steps:

[0021] (1) Thoroughly mix the edible oil and the emulsifier, add the aqueous ethanol to the mixed solution of the oil and the emulsifier, thoroughly mix to obtain a crude emulsion, and homogenize the crude emulsion to obtain a stable water-in-oil emulsion.

[0022] (2) Dissolve glycidyl methacrylate-modified silk fibroin in water, add a photoinitiator, and drop the resulting solution into the water-in-oil emulsion obtained in step (1) under stirring conditions, and stir well to mix evenly;

[0023] (3) Perform light treatment on the emulsion obtained in step (2) to promote crosslinking and curing, centrifuge to collect the microspheres, wash and dry them to obtain a fat-ethanol premix as the core-shell structure microspheres;

[0024] (4) Mix the required amounts of basal diet, cholesterol, and fat-ethanol premix evenly, and press them into pellets or blocks to obtain a construction method for constructing a liver disease animal model.

[0025] Further, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and the light irradiation is carried out with ultraviolet light of 365 - 405 nm.

[0026] As used herein, the basal diet is a common diet for daily feeding of non-human primates, which can ensure the energy required for the growth and activities of primates, such as non-human primate feed that can be purchased on the market. In a non-limiting example, the basal diet may contain the following components (1000 g): casein 140 g, L-cystine 1.8 g, corn starch 495.692 g, maltodextrin 125 g, sucrose 100 g, cellulose 50 g, soybean oil 40 g, tert-butylhydroquinone 0.008 g, mixed minerals 35 g, mixed vitamins 10 g, choline bitartrate 2.5 g.

[0027] The present invention also provides the use of the high-fat and high-alcohol diet as defined herein in constructing an animal model of advanced liver lesions.

[0028] Advantages of the Invention

[0029] By using the newly developed high-fat and high-alcohol diet, the present invention successfully constructs an animal model of advanced liver disease characterized by significant fat accumulation, massive infiltration of immune cells, steatohepatitis, and liver fibrosis. The novel high-fat and high-alcohol diet of the present invention has good stability, effectively reduces the smell of alcohol, is more in line with the olfactory preference of mice, and at the same time, the solid-phase encapsulation form can effectively ensure the solid form of the food, which is closer to the biting habit of mice. While maintaining the normal eating habits of mice, it increases the intake ratio of alcohol and fat, and simulates the formation process of food-induced advanced liver disease. Brief Description of the Drawings

[0030] Figure 1Showed that the new diet model for advanced liver disease in mice caused significant hepatic fat changes. A) Weight changes during the feeding of the new diet model. B) Comparison of body weights after feeding with the new diet model. C) Comparison of liver weights after feeding with the new diet model. D) Comparison of alanine aminotransferase after feeding with the new diet model. E) Comparison of aspartate aminotransferase after feeding with the new diet model. F) H&E staining of the liver after feeding with the new diet model. G) Oil red staining of the liver after feeding with the new diet model.

[0031] Figure 2 Showed that the new diet model for advanced liver disease in mice caused significant hepatic fat changes. A) Activation of neutrophils in the liver after feeding with the new diet model. B) Activation of macrophages in the liver after feeding with the new diet model. C) Sirius red staining showing hepatic fibrosis after feeding with the new diet model. D) Collagen showing hepatic fiber deposition after feeding with the new diet model. E) Detection of hepatic fibrosis marker levels by immunoprecipitation after feeding with the new diet model. F) Expression levels of marker genes related to inflammation and immune cells and G), H), I) protein levels. Detailed implementation manners

[0032] The present invention will be further illustrated below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0033] Example 1: Preparation of high-fat and high-alcohol feed

[0034] Preparation of glycidyl methacrylate-modified silk fibroin:

[0035] (1) Adjust the pH of an 8 wt% silk fibroin solution to about 8.5 with PBS buffer.

[0036] (2) Add an appropriate amount of glycidyl methacrylate (GMA) (the molar ratio of GMA to amino groups in SF is 5:1).

[0037] (3) React at room temperature or 37 °C for 12 hours.

[0038] (4) Use a dialysis bag with an MWCO of ~3500 Da to dialyze to remove unreacted GMA and by-products, and lyophilize to obtain glycidyl methacrylate-modified silk fibroin.

[0039] Preparation of fat-ethanol premix:

[0040] (1) Mix glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate in a mass ratio of 6:1.5:1.5:5:1 to obtain a compound emulsifier.

[0041] (2) Mix 40 parts of soybean oil, 20 parts of sunflower oil, 60 parts of liquid lard, and 60 parts of corn oil thoroughly with the compound emulsifier. Add 90 parts of aqueous ethanol with a volume percentage of 60% ethanol to the mixed solution of oil and emulsifier, and obtain a coarse emulsion through processing with a high-speed disperser. Homogenize the coarse emulsion using a dynamic high-pressure microfluidic homogenizer to obtain a stable water-in-oil emulsion. After testing, the water-in-oil emulsion remains stable without stratification for more than 30 days within the temperature range of -10 to 40 °C;

[0042] (3) Dissolve glycidyl methacrylate-modified silk fibroin (SilMA) in water to obtain a 20% (W / V) SilMA solution. Add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate as a photoinitiator with a final concentration of 0.25% (W / V). Drop the resulting solution into the water-in-oil emulsion obtained in step (2) under stirring conditions, and stir at 8000 rpm for 30 minutes;

[0043] (4) Treat the emulsion obtained in step (3) with 405 nm ultraviolet light for 10 min to promote crosslinking and curing. Centrifuge at 3000 rpm to collect the microspheres, wash them with distilled water, and then freeze-dry to obtain a fat-ethanol premix as core-shell structured microspheres.

[0044] Preparation of high-fat and high-alcohol diet:

[0045] Mix 60% of the basal diet, 10% of cholesterol, and 30% of the fat-ethanol premix thoroughly in a high-speed mixer, and press them into pellets to obtain a high-fat and high-alcohol diet for constructing a liver disease animal model.

[0046] The composition of the basal diet (1000 g): casein 140 g, L-cystine 1.8 g, corn starch 495.692 g, maltodextrin 125 g, sucrose 100 g, cellulose 50 g, soybean oil 40 g, tert-butylhydroquinone 0.008 g, mixed minerals 35 g, mixed vitamins 10 g, choline bitartrate 2.5 g.

[0047] Example 2: Construction of an advanced liver disease model

[0048] To induce advanced liver disease, 8-week-old male C57BL / 6J mice were divided into an experimental group and a control group, with 10 mice in each group. The experimental group was fed the new high-fat and high-alcohol diet, and the control group was fed the control diet. Both groups were given normal drinking water. Feed and water were freely available, and the mice were continuously fed for 12 weeks. After the feeding period, the mice were euthanized using a lethal dose of isoflurane. The body weights of the mice in the experimental group and the control group were measured weekly during the feeding period. After the feeding was completed, the mice were euthanized, and the effects of the diet on blood and liver changes were evaluated. The results showed that the new high-fat and high-alcohol diet caused a significant increase in the body weight of the mice ( Figure 1(A - B), in addition, the liver weight of mice fed with the feed also increased significantly. Figure 1 (C). The experimental diet caused an increase in the main transaminases, indicating significant liver damage. Figure 1 (D - E). H&E staining was used to evaluate the overall condition of the mouse liver. It was found that there was significant fat accumulation in the hepatocytes of the experimental group, and a large number of immune cells infiltrated between the hepatocytes. Figure 1 (F). Oil red staining confirmed obvious fat accumulation in the liver of the experimental group, indicating that the new mouse liver disease diet model can induce severe fatty liver. Figure 1 (G).

[0049] Liver inflammation is an important pathophysiological link in advanced liver diseases. The death of hepatocytes caused by various pathophysiological reasons releases inflammatory factors and recruits inflammatory cells from the periphery. The continuous inflammatory response in the liver activates more immune cells and initiates the process of liver fibrosis. After feeding with the new high - fat and high - alcohol diet, a large number of immune cells (detecting neutrophils and macrophages) in the mice invaded from the outside and were locally activated, indicating a strong inflammatory response in the liver. Figure 2 (A - B). In addition, the liver fibrosis of the mice was further detected. Sirius red staining and immunofluorescence staining showed a large amount of fibrous deposition in the liver, preliminarily indicating that the new liver disease diet model can induce obvious liver fibrosis. Figure 2 (C - D). Immunodeposition experiments were used to detect liver fibrosis markers, and it was found that their expression was significantly up - regulated in the livers of mice fed with the experimental feed, supporting the above view. Figure 2 (E).

[0050] Comparative Example 1:

[0051] The fat - ethanol premix was prepared according to Example 1, except that the addition amount of water - containing ethanol in step (2) was 70 parts.

[0052] The obtained fat - ethanol premix was used to prepare the high - fat and high - alcohol feed according to Example 1, and the advanced liver disease model was constructed according to Example 2. Since the addition amount of water - containing ethanol in the fat - ethanol premix was less, the effect of constructing the advanced liver disease model was poor.

[0053] Comparative Example 2:

[0054] The fat - ethanol premix was prepared according to Example 1, except that the addition amount of water - containing ethanol in step (2) was 110 parts.

[0055] The obtained fat - ethanol premix was used to prepare the high - fat and high - alcohol feed according to Example 1, and the advanced liver disease model was constructed according to Example 2. Since the addition amount of water - containing ethanol in the fat - ethanol premix was more, there was a leakage of alcohol smell in the high - fat and high - alcohol feed, resulting in less food intake of the mice during the construction of the advanced liver disease model, and the effect of constructing the model was poor.

[0056] It should be noted that the description and drawings of the present invention provide preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments do not serve as additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Moreover, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as falling within the scope described in the description of the present invention. Further, for those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for constructing an animal model of advanced liver lesions, characterized in that, The construction method includes feeding a high-fat and high-alcohol diet to a model animal, wherein the high-fat and high-alcohol diet contains, by mass percentage: 60-75% of a basal diet, 5-10% of cholesterol, and 20-30% of a fat-ethanol premix. The fat-ethanol premix is a microsphere with a core-shell structure, where the shell layer is glycidyl methacrylate-modified silk fibroin and the core is an ethanol-in-oil emulsion.

2. The construction method according to claim 1, wherein, The model animal is a rodent; Preferably, the rodent is a mouse or a rat.

3. The construction method according to claim 1, wherein The ethanol-in-oil emulsion is composed of an aqueous ethanol solution, an edible oil, and an emulsifier.

4. The construction method according to claim 3, characterized in that The mass ratio of the aqueous ethanol solution, the edible oil, and the emulsifier is 75-100:150-200:1.2-4.

5.

5. The construction method according to claim 3, characterized in that The volume percentage of ethanol in the aqueous ethanol solution is 40-65%.

6. The construction method according to claim 3, characterized in that The edible oil includes edible vegetable oil or edible animal oil. The edible vegetable oil includes soybean oil, rapeseed oil, peanut oil, sesame oil, olive oil, corn oil, sunflower oil, and edible vegetable blend oil; the edible animal oil includes lard, beef tallow, and fish oil.

7. The construction method according to claim 3, characterized in that The emulsifier includes one or more of glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate; Preferably, the emulsifier is compounded from glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate at a mass ratio of 5-7:1-2:1-2:3-8:0.5-1.

5.

8. The construction method according to any one of the preceding claims, characterized in that, The preparation method of the high-fat and high-alcohol diet includes the following steps: (1) Thoroughly mix the edible oil and the emulsifier, add the aqueous ethanol to the mixed solution of the oil and the emulsifier, and after thorough mixing, obtain a crude emulsion. Homogenize the crude emulsion to obtain a stable water-in-oil emulsion; (2) Dissolve glycidyl methacrylate-modified silk fibroin in water, add a photoinitiator, and drop the resulting solution into the water-in-oil emulsion obtained in step (1) under stirring conditions, and stir and mix thoroughly; (3) Perform a light treatment on the emulsion obtained in step (2) to promote crosslinking and curing, centrifuge to collect the microspheres, and after washing and drying, obtain the fat-ethanol premix as a core-shell structure microsphere; (4) Mix the required amounts of the basal diet, cholesterol, and fat-ethanol premix evenly, and press them into granules or blocks to obtain the construction method for constructing an animal model of liver disease.

9. The construction method according to claim 8, characterized in that, The photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and the light treatment is carried out using ultraviolet light with a wavelength of 365-405 nm.

10. Use of the high-fat and high-alcohol diet as defined in any one of claims 1-7 in constructing an animal model of advanced liver lesions.

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