Atherosclerosis animal model feed as well as preparation method and application thereof

Abstract

The invention relates to an atherosclerosis animal model feed. The atherosclerosis animal model feed comprises the following raw materials in parts by weight: 20-30 parts of casein, 0.2-0.4 part of sulfur-containing amino acid, 15-25 parts of corn starch, 5-10 parts of maltodextrin, 10-15 parts of fructose, 4-6 parts of cellulose, 15-30 parts of a high-fat substance, 0.2-0.3 part of choline tartrate, 1-2 parts of cholesterol, 3-6 parts of a mineral premix and 0.1-0.2 part of a vitamin premix, the raw material components can be directly mixed, or part of grease is pre-emulsified by adopting vitamin D3, and then the emulsified grease is mixed with other raw materials. According to the invention, the formula components of the feed are optimized, the lard oil and the anhydrous cream are used as high-fat components, and the fructose is used as a main carbon-water source; in addition, the step of adding high-fat substances is optimized, a process of pre-emulsifying part of grease is adopted, an animal model prepared from the feed is closer to the occurrence and development process of human atherosclerosis in the aspects of heredity, lipid metabolism and the like, and the animal model is more suitable for screening drugs for treating atherosclerosis.

Description

Technical Field

[0001] The invention relates to the technical field of animal model feed, and in particular to an atherosclerosis animal model feed and a preparation method and application thereof. Background Art

[0002] Cardiovascular diseases (CVDs) are an important cause of death worldwide. According to statistics from the World Health Organization, about 17 million people died from CVDs in 2019, accounting for 32% of the global death toll. Atherosclerosis (AS) is a chronic systemic inflammatory disease that occurs in blood vessels. It is an important pathological basis for a variety of CVDs such as coronary heart disease, angina pectoris, and ischemic heart disease. Its main feature is the formation of atherosclerotic fibrous plaques on the arterial wall. Risk factors for AS and its complications include persistently high levels of low-density lipoprotein cholesterol, hypertension, smoking, obesity, and diabetes. Although the pathological mechanism of AS is not yet fully understood, a large number of studies have shown that it is closely related to lipid metabolism disorders, inflammatory response, oxidative stress, endothelial cell dysfunction, and immune response disorders. It is reported that different types of cells, microenvironmental factors, and intracellular components are involved in the occurrence and development of AS. In the early stages of AS, risk factors invade arterial vessels, leading to endothelial dysfunction. Monocytes and macrophages pass through the damaged endothelium and aggregate and differentiate under the endothelium. Subsequently, lipid metabolism disorders aggravate the formation of macrophage-derived foam cells, inflammatory response, autophagy, and apoptosis, and ultimately lead to the formation of atherosclerotic plaques. Important progress has been made in clinical treatment methods for AS. Many advanced drugs are used to treat AS, such as lipid-lowering, antiplatelet, and anti-inflammatory drugs. In addition, organic nanoparticles and immune antibody therapy also have great potential. Despite the application of advanced treatment methods in clinical practice, the prevention and treatment of AS still face many challenges. The side effects of drugs gradually emerge, resulting in patients failing to achieve ideal treatment effects in clinical practice, and safer and more sustainable treatment methods are urgently needed.

[0003] Animal experimental atherosclerosis has become an important research tool. Although there have been many advances in the disease model, such as ApoE - / - Mouse, LDLr - / - Mice are widely used, but there is still a lack of clear evidence to prove that processes such as lipoprotein oxidation, inflammation, and immunity are important in the natural occurrence of human atherosclerosis. At the same time, the construction of the model in mice still has disadvantages such as less plaque formation, instability, and long modeling time. Therefore, it is still necessary to study a mouse model with more stable pathological manifestations, shorter modeling time, more stable plaques, and closer to the occurrence and development of human atherosclerosis in terms of genetics and lipid metabolism.

[0004] As an inducing component of the feed for atherosclerosis model, the price of purified feed for atherosclerosis model widely used has increased sharply due to the decline of global cocoa butter production, the increase of cocoa butter price and the weak global economy. Therefore, it is urgent to find a new oil and other feed raw materials that can replace cocoa butter to achieve the successful construction of diet-induced atherosclerosis model. Summary of the invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention provides an atherosclerosis animal model feed, which successfully establishes an atherosclerosis mouse model by optimizing its formula composition and preparation process, so as to solve the shortcomings of the animal model in the related art, such as small amount of plaque formation, instability, long modeling time and expensive formula raw material cost.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide an atherosclerosis animal model feed, which includes the following raw materials in parts by weight: 20 to 30 parts of casein, 0.2 to 0.4 parts of sulfur-containing amino acids, 15 to 20 parts of corn starch, 5 to 10 parts of maltodextrin, 10 to 15 parts of fructose, 4 to 5 parts of cellulose, 15 to 30 parts of high-fat substances, 0.2 to 0.3 parts of choline bitartrate, 1 to 2 parts of cholesterol, 3 to 6 parts of mineral premix, and 0.1 to 0.2 parts of vitamin premix.

[0008] Furthermore, the atherosclerosis animal model feed includes the following raw materials in parts by weight: 20 to 25 parts of casein, 0.3 to 0.4 parts of sulfur-containing amino acids, 20 to 25 parts of corn starch, 7 to 9 parts of maltodextrin, 12 to 14 parts of fructose, 5 to 6 parts of cellulose, 18 to 24 parts of high-fat substances, 0.2 to 0.25 parts of choline bitartrate, 1 to 1.5 parts of cholesterol, 4 to 5 parts of mineral premixes, and 0.1 to 0.15 parts of vitamin premixes.

[0009] Furthermore, the high-fat substance is selected from at least one of palm oil, tallow, lard, anhydrous butter, margarine, soybean oil, and corn oil; preferably, the high-fat substance is lard, anhydrous butter, or a combination of the two. When the high-fat substance consists of lard and anhydrous butter, the mass ratio of the lard to the anhydrous butter is 0.2 to 10:1, preferably the mass ratio of the two is 5 to 10:1; more preferably the mass ratio of the two is 6.2:1.

[0010] Furthermore, the sulfur-containing amino acid is at least one of methionine, cysteine ​​and cystine; preferably, the sulfur-containing amino acid is cystine.

[0011] Furthermore, the feed is composed of the following raw materials in parts by weight: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 20.06 parts of high-fat substances, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 4.75 parts of mineral premix, and 0.11 parts of vitamin premix; wherein the high-fat substance is at least one of lard and anhydrous butter; when the high-fat substance is composed of lard and anhydrous butter, the mass ratio of lard to anhydrous butter is 6.2:1.

[0012] Further, the mineral premix is ​​composed of 1.2-1.5 parts of calcium hydrogen phosphate, 0.5-0.7 parts of calcium carbonate, 1.5-2 parts of potassium citrate monohydrate, 0.2-0.3 parts of sodium chloride, and 0.4-0.6 parts of S10020 mineral premix; preferably, the mineral premix is ​​composed of 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, and 0.56 parts of S10020 mineral premix. The above-mentioned S10020 mineral premix is ​​purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.

[0013] Furthermore, the vitamin premix (V10001C vitamin premix) contains 8-12% tocopherol acetate, 2-4% niacin, 1-3% biotin, 1-2% vitamin B5, 0.5-1% vitamin D3, 0.08-0.12% vitamin B 12 , 0.8-1.5% vitamin A acetate, 0.5-1% vitamin B6, 0.5-1% riboflavin, 0.5-1% vitamin B1, 0.1-0.3% folic acid, 0.05-0.1% vitamin K and the remainder of corn starch; preferably, the vitamin premix contains 10% tocopherol acetate, 3% niacin, 2% biotin, 1.6% vitamin B5, 0.8% vitamin D3, 0.1% vitamin B 12 , 1% vitamin A acetate, 0.7% vitamin B6, 0.6% riboflavin, 0.6% vitamin B1, 0.2% folic acid, 0.08% vitamin K, and the vitamin premix also contains 79.32% corn starch.

[0014] The second aspect of the present invention is to provide a method for preparing an atherosclerosis animal model feed as described in any one of the first aspects, comprising the steps of: crushing raw materials with larger particles in advance; weighing a predetermined amount of raw materials, and mixing them in sequence according to the principle of large materials first and small materials later, and solid materials first and liquid materials later; after mixing for a predetermined time, wet granulating the mixed materials, drying and standing them to obtain the atherosclerosis animal model feed; wherein the raw materials with too small an addition amount are pre-mixed with the base material (casein, sucrose / fructose, maltodextrin) or corn starch and then mixed with other raw materials.

[0015] Furthermore, the crushed particle size reaches 30 mesh; the mixing time is 10 to 30 minutes; preferably, the raw materials are placed in a mixer and mixed for 15 minutes.

[0016] It is understandable that the above-mentioned operations such as pulverizing, wet granulation, drying, standing, and premixing are all carried out by conventional methods in the art.

[0017] Furthermore, in the preparation method, the high-fat substance is added by directly mixing it with other raw materials; or it can be added in the following manner: vitamin D3 is added to a part of the high-fat substance at 1200-1800 IU / kg (preferably 1500 IU / kg) for pre-emulsification, and after emulsification, it is added to the remaining part of the high-fat substance and stirred to mix, and all the high-fat substance after mixing is then mixed with other raw materials in the feed; wherein the emulsified part of the high-fat substance is 3-8% (preferably 5%) of the total mass of the high-fat substance.

[0018] Furthermore, the pre-emulsification step includes: adding vitamin D3 to distilled water and heating it to 80-85°C and stirring to mix; heating the high-fat substance to 80-85°C and weighing a portion of the high-fat substance; adding the stirred vitamin D3 to a portion of the high-fat substance, stirring to mix, and then pouring into an emulsifier and stirring at high speed to mix and emulsify.

[0019] It is understandable that the vitamin D3 used in the above-mentioned pre-emulsification step is an additionally added component and is not the vitamin D3 in the vitamin premix.

[0020] The third aspect of the present invention is to provide an application of the atherosclerosis animal model feed described in any one of the first aspects or the atherosclerosis animal model feed prepared by any one of the preparation methods described in the second aspect; wherein the feed is used to construct an atherosclerosis animal model.

[0021] The fourth aspect of the present invention is to provide a method for constructing an atherosclerosis animal model, which comprises the steps of: taking 6-8 week old mice, feeding them with atherosclerosis animal model feed for 8-12 weeks, and obtaining an atherosclerosis mouse model; wherein the mice are selected from C57BL / 6 mice, ApoE- / - Transgenic mice, LDLr - / - Transgenic mice; the atherosclerosis animal model feed is as described in any one of the first aspect or prepared by any one of the preparation methods described in the second aspect. Before feeding the feed, the mice generally need to be adaptively fed in a conventional manner for one week.

[0022] Furthermore, the mouse is ApoE - / - Transgenic mice.

[0023] Furthermore, after 8 to 12 weeks of feed feeding, the atherosclerosis mouse model is evaluated, and the evaluation indicators for its successful construction include: blood lipid level, aortic plaque area, aortic HE staining and aortic whole oil red O staining; preferably, the evaluation indicator is the aortic plaque area.

[0024] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0025] The present invention optimizes the components of the feed formula, which has the characteristics of high fat, high cholesterol, high fructose, etc. Lard and anhydrous butter are used as high-fat components, and the sources thereof are sufficient, which reduces the cost of the feed formula to a certain extent. Moreover, the feed formula removes some factors that induce the experimental animals to eat due to additional factors (flavors, etc.), and by replacing the added fructose substance as the main source of carbohydrates, the sweetness of the feed is increased, and the food intake of the animals is guaranteed to a certain extent, thereby facilitating the formation of aortic plaques in mice.

[0026] The present invention optimizes the step of adding high-fat substances in atherosclerosis model feed, and adopts a process of pre-emulsifying part of the oil, so as to better promote the absorption of high-fat components; the overall feed formula preparation process meets the requirements of animal welfare and guarantees the survival rate of experimental mice (such as gene knockout mice) to a certain extent; and the prepared feed simulates the eating habits of actual human diseased groups, ensures the modeling rate of animals, and enhances the uniformity of the model. The prepared animal model is closer to the occurrence and development process of human atherosclerosis in terms of genetics and lipid metabolism, and is more suitable for screening therapeutic drugs for atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 In one embodiment of the present invention, each group of ApoE - / - Oil red O staining results of the aorta of mice at week 8;

[0029] Figure 2 In one embodiment of the present invention, each group of ApoE - / - Oil red O staining results of the aorta of mice at week 10;

[0030] Figure 3 In one embodiment of the present invention, each group of ApoE - / - Oil red O staining results of aorta of mice at week 12;

[0031] Figure 4 In one embodiment of the present invention, each group of ApoE - / - HE staining results of mice at week 12. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0033] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0034] In certain embodiments of the present invention, a feed for an animal model of atherosclerosis is provided, which includes the following raw materials in parts by weight: 20-30 parts of casein, 0.2-0.4 parts of sulfur-containing amino acids, 15-25 parts of corn starch, 5-10 parts of maltodextrin, 10-15 parts of fructose, 4-6 parts of cellulose, 15-30 parts of high-fat substances, 0.2-0.3 parts of choline bitartrate, 1-2 parts of cholesterol, 3-6 parts of mineral premixes, and 0.1-0.2 parts of vitamin premixes; the above-mentioned mineral premixes and vitamin premixes can be conventional premixes or can be prepared separately; the above-mentioned high-fat substances are selected from at least one of palm oil, butter, lard, anhydrous butter, artificial butter, soybean oil, and corn oil; the above-mentioned sulfur-containing amino acids are at least one of methionine, cysteine, and cystine; and the above-mentioned raw material components are mixed to prepare feeding feed. The feeds prepared within the above-mentioned distribution ratio range can well meet the daily nutritional needs of experimental mice, and can successfully form animal models of atherosclerosis.

[0035] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0036] In the following examples, the determination indicators and methods used include:

[0037] (1) Histopathological examination: The aorta and aortic valve were immersed in a universal tissue fixative and sent to Nanjing Shuopu Biotechnology Co., Ltd. for routine paraffin embedding and staining. The aorta and aortic valve were stained with HE and Oil Red O.

[0038] (2) TC content analysis: After thawing the frozen serum, the T-CHO content in the mouse serum was determined according to the instructions of the total cholesterol (T-CHO) detection kit (Cat. No.: A111-1-1) of Nanjing Jiancheng Bioengineering Institute.

[0039] (3) Analysis of HDL-C and LDL-C contents: After thawing the frozen serum, the HDL-C and LDL-C contents in mouse serum were determined according to the instructions of the high-density lipoprotein cholesterol (HDL-C) detection kit (catalog number: A112-1-1) and the low-density lipoprotein cholesterol (LDL-C) detection kit (catalog number: A113-1-1) of Nanjing Jiancheng Bioengineering Institute.

[0040] (4) TG content analysis: After thawing the frozen serum, the TG content in the mouse serum was determined according to the steps in the instructions of the triglyceride (TG) detection kit (Cat. No.: A110-1-1) of Nanjing Jiancheng Bioengineering Institute.

[0041] (5) Detection of aortic plaque surface area: Use Image-Pro Plus 6.0 analysis software to select the same red area as the unified standard for judging plaques in all images, measure the plaque pixel area and the corresponding aortic pixel area in each image, and calculate the plaque area ratio = plaque pixel area / aortic pixel area*100%.

[0042] (6) Aortic sectioning and HE staining: 1) Dehydration: Remove the mouse aorta from the fixative and dehydrate it in a dehydrator with gradient ethanol in sequence. The program setting is: 75% ethanol for 4 hours → 85% ethanol for 2 hours → 90% ethanol for 2 hours → 95% ethanol for 1 hour → anhydrous ethanol for 30 minutes → anhydrous ethanol for 30 minutes → alcohol benzene for 10 minutes → xylene I for 10 minutes → xylene II for 10 minutes → paraffin I for 1 hour → paraffin II for 1 hour → paraffin III for 1 hour. 2) Embedding: ① Place the melted paraffin in the embedding frame, remove the tissue from the dehydration box before the paraffin solidifies, place it in the embedding frame according to the embedding requirements and attach the corresponding label; ② Place it in a -20℃ freezer to cool, remove it from the embedding frame after the wax block solidifies and trim it; ③ Place the embedded paraffin block in a 4℃ refrigerator to fix it, and determine the fixing time according to subsequent experiments. 3) Sectioning: ① After 10 hours of fixing, take out the tissue from the 4℃ refrigerator and fix it on the microtome. First, roughly adjust the thickness of the wax slice to 1 mm, determine the tissue section, and then fine-tune the section into 5μm paraffin sections; ② Use tweezers to clamp the cut sections into 45℃ constant temperature water and spread them. After the sections are spread flat, use a slide to remove the sections from the water, wait for them to dry naturally, and then put them in a 37℃ oven to dry. 4) Staining: After drying, take it out of the oven and immediately perform HE staining according to the following steps: xylene I dewaxing for 5 minutes → xylene II dewaxing for 5 minutes → xylene III dewaxing for 5 minutes → toluene IV dewaxing for 5 minutes → anhydrous ethanol dehydration for 5 minutes → 90% ethanol dehydration for 5 minutes → 80% ethanol dehydration for 5 minutes → 70% ethanol dehydration for 5 minutes → tap water washing 3 times (immersion washing, 1 minute each time) → hematoxylin staining for 5-10 minutes → tap water washing 3 times, 1 minute each time → 1% hydrochloric acid alcohol differentiation for 5-10 seconds → tap water washing 3 times, 1 minute each time → 37℃ constant temperature water bath box back to blue for 10 minutes → eosin staining for 2-3 minutes → tap water rapid washing → 80% ethanol dehydration for 10 seconds. 5) Sealing: ① Put the stained slices into a 37℃ drying machine for drying. After drying for 1 hour, take them out of the drying box and seal them with neutral gum. Be careful not to generate bubbles; ② Place them at room temperature and dry them naturally. 6) Photography: The aortic structure of each group of mice was observed under an optical microscope and photographed.

[0043] In the above-mentioned determination indicators and methods, Excel 2010 was used to organize the data, and SPSS23.0 statistical software was used for one-way ANOVA. Duncan's method was used for multiple comparisons when there was a significant difference. P ≤ 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0044] The technical solutions of the present invention are exemplified by the following embodiments.

[0045] Example 1 - Preparation of feed

[0046] This example shows some preferred feed compositions and preparations for atherosclerosis animal models. The feed formula is as follows:

[0047]

[0048] In the above feed formula, the mineral premix consists of 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, and 0.56 parts of S10020 mineral premix (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); the vitamin premix (V10001C vitamin premix) contains 10% tocopherol acetate, 3% niacin, 2% biotin, 1.6% vitamin B5, 0.8% vitamin D3, 0.1% vitamin B12, 1% vitamin A acetate, 0.7% vitamin B6, 0.6% riboflavin, 0.6% vitamin B1, 0.2% folic acid, 0.08% vitamin K, and 79.32% corn starch.

[0049] The preparation methods of the above feed formulas 1 to 5 are all as follows: weigh each raw material component, and the raw materials with larger particles need to be crushed to a 30-mesh sieve in advance; after weighing, add them to the mixer in sequence according to the principle of large materials first and small materials second, solid materials first and liquid materials second, among which the raw materials with too small addition amount need to be pre-mixed with the basic materials (casein, sucrose / fructose, maltodextrin) or corn starch before adding them to the mixer together with other raw materials; after mixing for 15 minutes, wet granulation can be carried out, and finally drying and standing.

[0050] Example 2 - Construction of an atherosclerosis animal model

[0051] In this embodiment, ApoE - / - Mice are used as experimental subjects to construct an atherosclerosis model, which specifically includes the following steps:

[0052] (1) Experimental groups;

[0053]

[0054] (2) Animals;

[0055] 48 ApoE - / - Mice (C57BL / 6J background), male, 6-8 weeks old. Before the experiment, all mice were fed adaptively for 1 week and lived under constant light (12 h light / dark cycle), temperature (22 ± 1 °C), and humidity (60 ± 10%) conditions, with free access to food and water.

[0056] After the adaptation feeding, the mice were weighed and randomly divided into cages (4 mice per cage). The mice were randomly divided into group A (conventional feed, control group), group B (induction feed 1), group C (induction feed 2) and group D (induction feed 3), with 12 mice in each group. The body weight and food intake were recorded at a fixed time every week, and the mental state, skin appearance, etc. were observed.

[0057] (3) Sample collection and measurement;

[0058] At weeks 8, 10, and 12, 4 ApoE - / - Mice, anesthetized mice that had been fasted and deprived of water for 12 hours in advance, and immediately removed the eyeballs to collect blood after anesthesia, let stand at room temperature for 30 minutes, centrifuged at 3000r / min, 4℃ for 10 minutes, collected serum, divided into 1.5mL centrifuge tubes, and stored at -80℃. After fasting and depriving of water for 24 hours in the 12th week, all remaining animals were separated and serum was collected according to the above method and stored at -80℃. Serum samples were used to detect total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels.

[0059] Four ApoE mice were randomly selected at week 8, week 10, and week 12. - / - Mice were killed and the full-length aorta was isolated. The whole aorta was stained with Oil Red O to observe the formation of aortic plaques in mice. All remaining animals were fasted and deprived of water for 24 hours at week 12, and the heart and the whole aorta were removed and divided into 2 sections. The heart and the root of the aortic arch connected to it were immediately embedded and frozen for histological analysis. HE-stained sections were used to analyze the length and wall thickness of the internal elastic layer of the proximal ascending aorta. Oil red-stained sections were used to analyze the fat content of the heart aortic valve.

[0060] (4) Experimental results;

[0061] 1) APOE - / - Effects of mouse body weight;

[0062] Table 1 - Effects of each group of feed on APOE - / - Effect of mouse body weight

[0063]

[0064] From the results in Table 1, it can be seen that there was no significant difference in the initial body weight of mice in each group (P>0.05). At the 8th and 12th weeks of the experiment, the body weight of mice in each experimental group was significantly higher than that in the control group (P<0.01). At the 10th week of the experiment, the body weight of mice in group C was significantly higher than that in the control group, group B, and group D (P<0.01).

[0065] 2) APOE - / - Effect of food intake on mice;

[0066] Table 2-Effects of each group of feed on APOE - / - Effect of food intake on mice

[0067]

[0068] From the results in Table 2, it can be seen that in the 8th and 10th weeks of the experiment, the food intake of mice in each experimental group was significantly higher than that of the control group (P < 0.01). In the 12th week of the experiment, the food intake of mice in group B was significantly higher than that of the control group (P < 0.05), but there was no significant difference in the food intake of mice in groups C and D compared with the control group (P > 0.05).

[0069] 3) APOE - / - Effects on blood lipids in mice;

[0070] Table 3 - Effects of each diet group on APOE at 8 weeks - / - Effects of blood lipids on mice

[0071]

[0072] Table 4 - Effects of each diet group on blood lipids in 10-week-old APOE- / - mice

[0073]

[0074] Table 5 - Effects of each diet group on blood lipids in 12-week-old APOE- / - mice

[0075]

[0076] From the results of Table 3, Table 4, and Table 5, it can be seen that the serum TG levels of mice in each test group were significantly increased compared with group A (control) at the 8th, 10th, and 12th weeks of the experiment (P<0.01), but at the 10th week, the serum TG level of mice in group C was not significantly higher than that of the control group (P>0.05). The test was carried out for the 8th, 10th, and 12th weeks. Compared with group A, the serum TC levels of mice in each test group were significantly increased (P<0..01). The test was carried out for the 8th week. Compared with group A, there was no significant difference in the serum HDL levels of mice in groups B and C (P>0.05), and the serum HDL level of mice in group D was significantly increased (P<0.05). At the 10th week of the experiment, the serum HDL levels of mice in groups B and D were significantly increased compared with group A (P<0.05). At the 12th week of the experiment, the serum HDL level of mice in group D was significantly higher than that in group A (P<0.05), while there was no significant difference between mice in groups B and C (P>0.05). The experiment was carried out until the 8th, 10th, and 12th weeks. Compared with group A, the serum LDL levels of mice in the remaining groups were extremely significantly increased (P<0.01).

[0077] 4) APOE - / - Effects on aortic plaque area in mice;

[0078] Table 6 - Effects of each diet group on APOE - / - Effects of aortic plaque size on mouse

[0079]

[0080] ApoE in each group - / - The results of Oil Red O staining of the aorta of mice at the 8th, 10th and 12th weeks were as follows: Figure 1 , Figure 2 , Figure 3 As shown. Figures 1 to 3 As shown in the results of Table 6, the proportion of aortic plaque area in mice in each experimental group increased significantly compared with group A at the 8th, 10th and 12th weeks of the experiment (P<0.01).

[0081] The experimental data in the above 1) to 4) sections showed that the food intake of mice in each test group was significantly increased compared with that in the control group. In the 8th, 10th and 12th weeks of the experiment, the TG, TC and LDL levels of mice in each test group were significantly increased compared with those in the control group. At the same time, the aortic plaque area of ​​each test group was significantly increased compared with that in the control group. Combining all indicators, it was found that the induced feed 1 (group B) was the optimal experimental formula for constructing an atherosclerotic disease model.

[0082] 5) APOE - / - Effect of HE staining of aorta at 12 weeks;

[0083] The results of HE staining of aorta of mice in each group are shown in Figure 4 As shown in the figure, the structures of each layer of vascular tissue of mice in group A are clear, the intima is thin, only a small number of endothelial cells are seen, the middle membrane is composed of multiple layers of smooth muscle and elastic fibers, and the cytoplasm of many smooth muscle cells is loose and lightly stained (black arrow), and the outer membrane is composed of loose connective tissue, with occasional lymphocyte infiltration (red arrow). In the vascular tissue of mice in group B, sheet-like vascular plaques can be seen, protruding into the lumen, and many foam cells can be seen on the surface of the plaque (black arrow), and many connective tissues proliferate in the deep layer, and a small number of pinhole-shaped cholesterol clefts can be seen (red arrow), local focal necrosis, nuclear dissolution (yellow arrow), small focal chondrocyte metaplasia (green arrow), uneven thickness of the middle membrane, irregular arrangement of elastic fibers and smooth muscle cells, and local elastic fiber rupture (blue arrow). In the vascular tissue of mice in group C, large-scale vascular plaque formation was observed, which bulged into the lumen, and the lumen was narrowed. A large number of foam cells were seen on the surface of the plaque (black arrow), and a large number of connective tissues proliferated in the deep layer, and a small number of pinhole-shaped cholesterol clefts (red arrows) were seen. There was local focal necrosis, nuclear dissolution (yellow arrows), small focal chondrocyte metaplasia (green arrows), uneven thickness of the middle membrane, and a large number of smooth muscle cells with loose and light stained cytoplasm (blue arrows). In the vascular tissue of mice in group D, the wall of the vessel was incomplete, and a large number of foam cells were seen under the endothelium (black arrows), bulging into the lumen to form plaques, the middle membrane was composed of multiple layers of smooth muscle and elastic fibers, and a large number of smooth muscle cells with loose and light stained cytoplasm (yellow arrows), the outer membrane was composed of loose connective tissue, and lymphocyte infiltration was occasionally seen (red arrows).

[0084] It can be seen from the above HE staining images that: at the 12th week of the experiment, vascular plaques formed in the aorta of APOE mice in each experimental group, bulging into the lumen, with a large amount of connective tissue hyperplasia in the deep layer, and a large number of foam cells were seen gathering, indicating that the atherosclerotic disease model in each experimental group was successfully constructed.

[0085] From the above experimental results, it can be seen that the purified feed for inducing atherosclerosis model with lard or anhydrous butter as the main fat source and fructose as the main carbohydrate source, combined with the deletion of the apolipoprotein gene in APOE mice, can successfully construct an atherosclerosis mouse model, among which the lard + fructose feed formula has a better effect.

[0086] Example 3 - Verification of the effect of feed formulation and preparation process optimization on the establishment of an atherosclerosis animal model

[0087] Based on the model building effect verification experiment in Example 2, this example further optimizes the feed formula and its preparation process, and verifies its modeling effect, specifically including the following steps:

[0088] (1) Optimization of feed formula;

[0089] Based on the fact that lard or anhydrous butter can be used as the main fat source to effectively construct an atherosclerosis mouse model, the high-fat substance in the formula was adjusted to a combination of lard and anhydrous butter, with a mass ratio of 6.2:1. The formula composition of the optimized feed was: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 17.27 parts of lard, 2.79 parts of anhydrous butter, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, 0.56 parts of S10020 mineral premix, and 0.11 parts of V10001C vitamin premix (the components are the same as in Example 1).

[0090] (2) Optimization of preparation process;

[0091] In actual application, it was found that the animal modeling effect was better after pre-emulsifying part of the lard with vitamin D3. Therefore, this embodiment optimized the step of adding oil: adding additional vitamin D3 at 1500 IU / kg to part of the high-fat substance (accounting for 5% of its total mass, if two high-fat substances are used, they need to be pre-mixed evenly) for pre-emulsification, adding it to the remaining total oil (accounting for 95% of its total mass) after emulsification, and stirring and mixing. The mixed oil is subsequently mixed with the raw materials according to the preparation method in Example 1; wherein, the specific steps of pre-emulsification are: adding vitamin D3 to distilled water, heating and stirring to 80-85°C, heating the oil in the feed to the same temperature of 80-85°C and taking 5% of the oil for standby use, adding the stirred and mixed vitamin D3 to the standby oil, stirring and mixing, and pouring into an emulsifier for high-speed stirring to mix and emulsify.

[0092] (3) Setting up the experimental and control groups;

[0093] Induction feed 1 + conventional preparation method (Group 1) The feed formula 1 in Example 1 is used: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 20.06 parts of lard, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, 0.56 parts of S10020 mineral premix, and 0.11 parts of V10001C vitamin premix; it is prepared by the preparation method described in Example 1.

[0094] Induction feed 1 + optimized preparation method (Group 2)The feed formula 1 in Example 1 is used: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 20.06 parts of lard, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, 0.56 parts of S10020 mineral premix, and 0.11 parts of V10001C vitamin premix; it is prepared by a preparation method of partial oil pre-emulsification (as described in the above-mentioned '(2) Optimization of the preparation process').

[0095] Optimized feed + optimized preparation method (Group 3) The feed formula described in the above '(1) Optimization of feed formula' was used: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 17.27 parts of lard, 2.79 parts of anhydrous butter, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, 0.56 parts of S10020 mineral premix, and 0.11 parts of V10001C vitamin premix; it was prepared by a preparation method of partial oil pre-emulsification (as described in the above '(2) Optimization of preparation process').

[0096] The above-mentioned feeds were all commissioned to Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. for production.

[0097] (4) Experimental grouping;

[0098] 36 ApoE- / - mice (C57BL / 6J background), male, 6-8 weeks old. Before the experiment, all mice were fed adaptively for 1 week and lived under constant light (12h light / dark cycle), temperature (22±1℃), and humidity (60±10%) conditions, with free access to food and water.

[0099] After the adaptation feeding, the mice were weighed and randomly divided into cages (4 mice per cage). The mice were randomly divided into the first group (induction diet 1 + conventional preparation method), the second group (induction diet 1 + optimized preparation method), and the third group (optimized diet + optimized preparation method), with 12 mice in each group. - / - The plaque area of ​​mouse aorta was counted.

[0100] (5) Experimental results;

[0101] Table 7 - Effects of each diet group on APOE - / - Effects of aortic plaque size on mouse

[0102]

[0103] From the results in Table 7 above, it can be seen that the granulation process using vitamin D3 pre-emulsification has a significant improvement effect on the construction of the atherosclerosis model; among them, the purified feed for inducing atherosclerosis model using a combination of lard and anhydrous butter as the main fat source, fructose as the main carbohydrate source and an optimized preparation process of partial oil pre-emulsification can better construct an atherosclerosis mouse model.

[0104] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. An atherosclerosis animal model feed, characterized in that: The feed comprises the following raw materials in parts by weight: 20-30 parts of casein, 0.2-0.4 parts of sulfur-containing amino acids, 15-25 parts of corn starch, 5-10 parts of maltodextrin, 10-15 parts of fructose, 4-6 parts of cellulose, 15-30 parts of high-fat substances, 0.2-0.3 parts of choline bitartrate, 1-2 parts of cholesterol, 3-6 parts of mineral premixes, and 0.1-0.2 parts of vitamin premixes.

2. The atherosclerosis animal model feed according to claim 1, characterized in that: The high-fat substance is selected from at least one of palm oil, butter, lard, anhydrous butter, margarine, soybean oil and corn oil.

3. The atherosclerosis animal model feed according to claim 1, characterized in that: The sulfur-containing amino acid is at least one of methionine, cysteine ​​and cystine.

4. The atherosclerosis animal model feed according to claim 1, characterized in that: The feed is composed of the following raw materials in parts by weight: 22.29 parts of casein, 0.33 parts of cystine, 23.63 parts of corn starch, 7.91 parts of maltodextrin, 13.87 parts of fructose, 5.57 parts of cellulose, 20.06 parts of high-fat substances, 0.22 parts of choline bitartrate, 1.25 parts of cholesterol, 4.75 parts of mineral premix, and 0.11 parts of vitamin premix; wherein the high-fat substance is at least one of lard and anhydrous butter.

5. The atherosclerosis animal model feed according to claim 4, characterized in that: The mineral premix is ​​composed of 1.45 parts of calcium hydrogen phosphate, 0.61 parts of calcium carbonate, 1.84 parts of potassium citrate monohydrate, 0.29 parts of sodium chloride, and 0.56 parts of S10020 mineral premix; The vitamin premix contains 10% tocopherol acetate, 3% niacin, 2% biotin, 1.6% vitamin B5, 0.8% vitamin D3, and 0.1% vitamin B 12 , 1% Vitamin A acetate, 0.7% Vitamin B6, 0.6% Riboflavin, 0.6% Vitamin B1, 0.2% Folic acid, 0.08% Vitamin K.

6. A method for preparing an atherosclerosis animal model feed according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: crushing raw materials with larger particles in advance; weighing a predetermined amount of raw materials, and mixing them in sequence according to the principle of large materials first and small materials second, solid materials first and liquid materials second; after mixing for a predetermined time, wet granulating the mixed materials, drying and standing them to obtain the atherosclerosis animal model feed; wherein the raw materials with too small an addition amount are premixed with the base material or corn starch and then mixed with other raw materials.

7. The preparation method according to claim 6, characterized in that: In the preparation method, the step of adding the high-fat substance is directly mixing with other raw materials; or, In the preparation method, the high-fat substance is added in the following manner: vitamin D3 is added to a portion of the high-fat substance at 1200-1800 IU / kg for pre-emulsification, and after the emulsification is completed, it is added to the remaining high-fat substance and stirred to mix, and all the high-fat substance after mixing is then mixed with other raw materials in the feed; wherein the emulsified portion of the high-fat substance is 3-8% of the total mass of the high-fat substance.

8. The preparation method according to claim 7, characterized in that: The pre-emulsification step includes: adding vitamin D3 to distilled water and heating it to 80-85° C. and stirring to mix; heating the high-fat substance to 80-85° C. and weighing a portion of the high-fat substance; adding the stirred vitamin D3 to a portion of the high-fat substance, stirring to mix, and then pouring into an emulsifier and stirring at high speed to mix and emulsify.

9. Use of the atherosclerosis animal model feed according to any one of claims 1 to 6 or the atherosclerosis animal model feed prepared by the preparation method according to any one of claims 6 to 8, characterized in that: The feed is used to construct an atherosclerosis animal model.

10. The use according to claim 9, characterized in that: The method for constructing an atherosclerosis animal model comprises the following steps: taking 6-8 week old mice, feeding them with atherosclerosis animal model feed for 8-12 weeks, and obtaining an atherosclerosis mouse model; wherein the mice are selected from C57BL / 6 mice, ApoE - / - Transgenic mice, LDLr - / - Transgenic mice.

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