Diet-induced mouse atherosclerosis modeling method

Abstract

The invention relates to the technical field of biology, in particular to a diet-induced mouse atherosclerosis modeling method. High-concentration food-grade sucralose is added into drinking water of an apolipoprotein E gene knockout (ApoE- / -) mouse, and high-fat diet is combined, so that the formation of atherosclerotic plaques is remarkably accelerated. According to the method, 8-week-old male ApoE <- / -> mice are adopted, and an experimental group drinks drinking water containing 2%-3% w / v sucralose after an adaptive period of one week and is fed with high-fat diet at the same time; the control group drinks purified water and is fed with high-fat diet. The intervention period is 6-8 weeks, and the plaque formation condition is evaluated by recording the change of the blood lipid level of the mouse and combining histological staining and molecular biological detection. Experimental results show that the modeling time can be effectively shortened, the plaque area and stability can be remarkably improved, and the method has good stability and repeatability and is suitable for animal model construction in atherosclerosis and intervention drug research.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for establishing a diet-induced atherosclerosis model in mice. Background Art

[0002] Atherosclerosis is the main pathological basis of cardiovascular diseases such as coronary heart disease and stroke. ApoE - / - Mice are used as a classical model for studying atherosclerosis. Usually, plaque formation is induced by a simple high-fat diet alone, but this method requires a long induction time, about 12 weeks to complete, and the time and material costs consumed are relatively high. In recent years, some studies have attempted to use different dietary interventions to accelerate the establishment of the model, but the effects and mechanisms of most interventions are still unclear.

[0003] Therefore, there is an urgent need to provide a modeling method with clear intervention effects and mechanisms, simple steps, effectiveness and good repeatability, so as to provide a more ideal animal model for atherosclerosis and related intervention research. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for establishing a diet-induced atherosclerosis model in mice.

[0005] The present invention provides a method for establishing a diet-induced atherosclerosis model in mice, which uses apolipoprotein E gene knockout (ApoE - / - ) mice as experimental animals, and accelerates the formation of atherosclerotic plaques by jointly administering a high-fat diet and adding sucralose of food grade to the drinking water; the concentration of sucralose in the drinking water is 2% - 3% w / v.

[0006] Furthermore,

[0007] The steps for establishing the model in ApoE - / - mice are as follows: male mice at 8 weeks old are grouped for experiments after a 1-week adaptation period. Among them, the control group is given a conventional high-fat diet and pure drinking water, and the experimental group is given drinking water containing sucralose under the same high-fat diet conditions.

[0008] The intervention period for establishing the model in ApoE - / - mice is 6 - 8 weeks.

[0009] The fat content in the high-fat diet is 15%, and the cholesterol content is 5%.

[0010] The present invention provides the application of the method for establishing a diet-induced atherosclerosis model in mice described above in the preparation of a model for studying atherosclerosis and vulnerable plaques.

[0011] The purpose of the present invention is to provide a method for establishing a diet-induced atherosclerosis model in mice, which only by- / - Adding food-grade sucralose to the drinking water of mice and combining it with a high-fat diet can increase the lipid metabolism stress of mice and accelerate the formation of atherosclerotic plaques, thus solving the problems of long time required and unstable results in the traditional simple high-fat diet-induced model.

[0012] To achieve the above object, specifically, the technical solution of the present invention is as follows:

[0013] 1. Select 8-week-old male ApoE - / - mice. After a 1-week adaptation period, they are randomly divided into an experimental group and a control group;

[0014] 2. The control group is fed with a high-fat diet (HFD) and drinks pure water at the same time;

[0015] 3. The experimental group drinks water added with 2% - 3% w / v food-grade sucralose under the same high-fat diet conditions;

[0016] 4. The intervention period is 6 - 8 weeks. During this period, indicators such as body weight, food intake, water intake, and blood lipids (such as total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density cholesterol) are regularly recorded;

[0017] 5. After the intervention, histological examinations (including HE staining, Oil Red O staining, and Masson staining) are performed on the aorta and liver of the mice, and image analysis software is used to quantitatively evaluate the plaque area, composition, and stability.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. Accelerate model establishment: By simply adding sucralose to the drinking water, which is a simple dietary factor, the formation time of atherosclerotic plaques can be significantly shortened on the basis of a high-fat diet, and the experimental period is shortened to 6 - 8 weeks;

[0020] 2. Stable model: By determining the concentration of sucralose and the intervention time, the consistency of the model establishment results among different batches of mice is ensured to be relatively high;

[0021] 3. Simple operation: Compared with the model establishment methods that require surgery or complex gene operations, this method has simple operation and is easy to popularize and apply;

[0022] 4. Research significance: The present invention discloses a brand-new diet-induced model establishment method, which further enriches the research means of regulating lipid metabolism and atherosclerotic process by dietary factors, and provides an ideal model for subsequent drug screening and mechanism of action research.

[0023] The present patented technology has a promoting effect on atherosclerosis by adding the artificial sweetener sucralose to the drinking water of mice and combining it with a high-fat diet condition, and there has been no report in the literature and public patents on using food-grade sucralose for ApoE - / - mouse modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Showing ApoE - / - Schematic diagram of the experimental process for intervening in ApoE mice;

[0025] Figure 2 Schematic diagram of the comparison of the levels of four blood lipids (total cholesterol, total triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein) in mice in three experimental groups and the blank control group after intervention;

[0026] Figure 3 Schematic diagram of the gross Oil Red O staining and quantitative comparison of the aorta of mice in each group after intervention;

[0027] Figure 4 Schematic diagram of the comparison of pathological staining of the aortic root of mice in each group after intervention (including Masson staining, Oil Red O staining, and HE staining);

[0028] Figure 5 Schematic diagram of the comparison of Oil Red O staining of the liver of mice in each group after intervention. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a method for establishing a diet-induced atherosclerosis mouse model. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0030] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with the embodiments:

[0031] Example 1

[0032] A method for establishing a diet-induced atherosclerosis mouse model, the process is as Figure 1 shown, specifically: Select 8-week-old male ApoE - / - mice. After a 1-week adaptation period, they are divided into an experimental group and a blank control group; in the experimental group, on the basis of a high-fat diet, they drink water containing sucralose, while the blank control group drinks pure water; the intervention period is 6-8 weeks; the detailed steps are as follows:

[0033] 1. Selection and Pre-treatment of Animal Models

[0034] Animal Selection: Male ApoE mice at 8 weeks of age were selected. - / -

[0035] Pre-treatment: All mice underwent a 1-week adaptation period to ensure environmental adaptation and basic health status.

[0036] Grouping: Randomly divided into an experimental group (n = 6) and a control group (n = 6).

[0037] 2. Feeding Regimen

[0038] High-Fat Diet: Both groups of mice were fed the prepared high-fat diet, with the fat content in the formula set at 15% and the cholesterol content at 5% to ensure plaque formation.

[0039] Drinking Water Treatment:

[0040] The control group drank pure water;

[0041] The experimental group drank drinking water supplemented with 2% w / v food-grade sucralose.

[0042] 3. Intervention Period and Monitoring

[0043] Period: The intervention period was 6 weeks.

[0044] Monitoring Indicators: The body weight, food intake, and water intake of the mice were recorded weekly; blood samples were collected before sacrifice to detect the levels of four lipid parameters (total cholesterol, total triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein).

[0045] 4. Endpoint Evaluation

[0046] Histological Detection: After the intervention, the mice were sacrificed, and aortic and liver tissues were taken for HE staining, Masson staining, and Oil Red O staining.

[0047] Quantitative Analysis: The gross Oil Red O area of the aorta was quantified using image analysis software, and a significance analysis was performed.

[0048] 5. Experimental Results

[0049] Results showed that the plaque area in the experimental group of mice was significantly larger than that in the control group within 6 weeks, and the lipid levels were significantly increased, demonstrating that the addition of sucralose to drinking water could significantly accelerate the formation of atherosclerosis under high-fat diet conditions.

[0050] Example 2

[0051] To verify the repeatability of the method and optimize the dose, the concentration of sucralose was further adjusted in different experimental batches, such as 2.5% w / v and 3% w / v, and the modeling effects among groups were compared.​

[0052] 1. Grouping and treatment

[0053] ApoE mice under the same conditions - / - The mice were randomly divided into three experimental groups and one control group, with n = 6 in each group.

[0054] The control group drank pure water;

[0055] The experimental groups drank drinking water containing 2%, 2.5%, and 3% w / v sucralose respectively, and other feeding conditions were the same.

[0056] 2. Data collection and analysis

[0057] Index monitoring: Record the weight changes, food intake and water intake of each group, and collect blood samples before sacrifice to detect the levels of four blood lipids (total cholesterol, total triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein).

[0058] Tissue detection: After the intervention, histological staining (Masson staining, Oil Red O staining, and HE staining) of the aorta gross, root, and liver was performed, and the area of Oil Red O staining of the aorta gross was quantified using image software.

[0059] 3. Results

[0060] Comparison of the levels of four blood lipids (total cholesterol, total triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein) in mice of the three experimental groups and the blank control group after intervention is as Figure 2 shown. The levels of total cholesterol, total triglyceride, and low-density lipoprotein cholesterol in the three experimental groups of mice were significantly higher than those in the blank control group (P < 0.05), while the level of high-density lipoprotein cholesterol was lower than that in the blank control group; this result indicates that sucralose intervention combined with a high-fat diet significantly exacerbated the blood lipid metabolism disorder in mice and promoted the process of atherosclerosis.

[0061] Comparison of Oil Red O staining and quantification of the aorta gross of mice in each group after intervention is as Figure 3 shown. Oil Red O staining showed that the aortic plaque area in the experimental group of mice was significantly larger than that in the control group, and the quantitative analysis results further confirmed that the plaque area in the experimental group increased significantly; this result indicates that the combined use of high-concentration sucralose and a high-fat diet can accelerate the formation of atherosclerotic plaques; and the effect of the 3% w / v group was the best, with the plaque area and inflammatory indexes significantly higher than those in the 2% and 2.5% groups, indicating that this method has good repeatability and dose-dependence, providing a reliable experimental model for subsequent mechanism research.

[0062] Comparison of pathological staining of the aortic root of mice in each group after intervention is as Figure 4Among them, Masson staining showed that the aortic wall of the experimental group mice was thickened and there was obvious infiltration of inflammatory cells; Oil Red O staining showed that the plaque area of the experimental group was significantly increased and there was abundant lipid deposition in the plaque; HE staining showed that the arterial wall structure of the experimental group mice was significantly damaged, the vascular endothelial cells were arranged disorderly, and the plaque was unstable; The results showed that sucralose intervention could significantly accelerate the formation of plaques and make the plaques unstable, with a high risk.

[0063] Comparison of Oil Red O staining of the livers of mice in each group after intervention is as Figure 5 shown. The lipid deposition in the livers of the experimental group mice was significantly higher than that of the control group, and the color of the plaque area was deeper, indicating that the lipid metabolism of the experimental group mice was abnormal and fatty liver occurred; The results further supported the interfering effect of the combined use of sucralose and high-fat diet on lipid metabolism and aggravated the lipid metabolism disorder of ApoE - / - mice.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A method for establishing a diet-induced atherosclerosis model in mice, characterized in that: Apolipoprotein E gene knockout (ApoE - / - ) mice were used as experimental animals, and the formation of atherosclerotic plaques was accelerated by combining a high-fat diet and adding food-grade sucralose to drinking water; the concentration of sucralose in the drinking water was 2% to 3% w / v.

2. The method according to claim 1, characterized in that ApoE - / - The mouse modeling procedure was to use 8-week-old male mice. After a 1-week adaptation period, the mice were divided into groups for testing. The control group was given a conventional high-fat diet and pure drinking water, while the experimental group was given drinking water containing sucralose under the same high-fat diet conditions.

3. The step according to claim 2, characterized in that: ApoE - / - The intervention period of mouse modeling is 6 to 8 weeks.

4. The step according to claim 2, characterized in that: The high-fat diet contains 15% fat and 5% cholesterol.

5. Use of the diet-induced mouse atherosclerosis modeling method according to claim 1 or 2 in preparing a model for studying atherosclerosis and vulnerable plaques.

Images