Method for rapidly constructing mouse atherosclerosis model by using sorafenib
By using sorafenib intervention combined with a high-fat diet and gavage administration, a mouse model of atherosclerosis was rapidly constructed, solving the problems of long modeling time and high cost in existing technologies, achieving significant atherosclerotic effects and shortening the modeling cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies suffer from long modeling times and high costs when constructing mouse atherosclerosis models, and existing methods are difficult to induce atherosclerosis models quickly and effectively.
Sorafenib was used as a drug intervention agent and administered by gavage in combination with a high-fat diet. The mice were divided into high-dose and low-dose groups and administered the drug every other day for 8 weeks to induce an ApoE-/- mouse atherosclerosis model, shortening the modeling period and significantly aggravating atherosclerotic lesions.
The modeling cycle was successfully shortened from 12 weeks to 8 weeks, significantly improving the success rate of modeling. The atherosclerotic effect was significant, with a significant increase in blood lipid levels and plaque area, providing a stable and simple experimental protocol.
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Figure CN121647217A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of basic medical research technology, specifically relating to a method for rapidly constructing a mouse atherosclerosis model using sorafenib. Background Technology
[0002] Despite the improvement of the national economy, the incidence and mortality rates of cardiovascular diseases in China continue to rise. Cardiovascular diseases mainly include hypertension, hyperlipidemia, myocardial infarction, and atherosclerosis. Atherosclerosis (AS) has a high incidence and mortality rate, and its overall incidence in the population has been increasing year by year in recent years, with a trend towards younger onset. It has become a major disease threatening human health. Therefore, in-depth research into the pathogenesis of AS and active exploration of new treatment options are of great significance and value.
[0003] Currently, the establishment of atherosclerosis models mainly involves the following methods: 1. High-fat diet induction: This can lead to anorexia in mice. Furthermore, high-fat feeding and other methods have drawbacks such as long plaque formation cycles and high experimental costs. 2. Gene knockout technology: Gene knockout technology is widely used in atherosclerosis research. The main gene knockout mouse for atherosclerosis is the ApoE mouse. Under normal dietary conditions, these mice can also form atherosclerotic plaques, but the degree of atherosclerosis is mild. Existing mouse atherosclerosis models require 12 weeks of high-fat feeding, which is time-consuming and resource-intensive. This invention uses sorafenib to aggravate atherosclerotic lesions, thus affecting the mouse atherosclerosis modeling process. This technology can accelerate the modeling speed and save animal feeding costs and experimental time. This invention proposes a modeling scheme with the characteristics of short modeling time and good stability. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method for constructing a mouse model of atherosclerosis using sorafenib. This method reduces experimental costs and modeling time, and has a high success rate. The resulting mouse models can be used more conveniently to study the pathogenesis of atherosclerosis.
[0005] This invention provides an application of sorafenib in constructing an atherosclerosis model.
[0006] Another embodiment of the present invention provides the application of sorafenib in constructing a mouse model of atherosclerosis. The mice preferentially receive ApoE. - / - Mice.
[0007] Another embodiment of the present invention provides the application of sorafenib in promoting the establishment of a mouse atherosclerosis model through direct high-fat diet feeding. In this application, the preferred dose of sorafenib is 7.5-15 mg / kg (more preferably 15 mg / kg), the preferred method of administration is gavage, and the preferred dosing cycle is every other day; the promotion mentioned in this application refers to shortening the modeling period from the original 12 weeks or longer to 8 weeks.
[0008] Another embodiment of the present invention provides a method for rapidly constructing a mouse atherosclerosis model using sorafenib, characterized by comprising the following steps:
[0009] (1) ApoE - / - Mice were randomly divided into three groups according to body weight: a high-dose sorafenib group (15 mg / kg), a low-dose sorafenib group (7.5 mg / kg), and a control group (physiological saline). The administration was by gavage, every other day for 8 weeks. All mice were fed a high-fat diet (feed formula: 83.5% basal diet + 15% lard + 1.5% cholesterol) for 8 weeks, with free access to water to induce atherosclerosis. Blood was collected from the tail vein every 4 weeks to detect blood lipids and various biochemical indicators.
[0010] (2) At different stages of atherosclerotic plaque formation, plasma TC, TG, and HDL-C in three groups of mice were measured using a kit;
[0011] (3) After the experiment, the animals were euthanized and the size of the atherosclerotic plaques, the composition of the diseased cells and the stability of the plaques were analyzed using histological and pathological methods, so as to observe the effect of sorafenib intervention on the occurrence and development of atherosclerosis.
[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention discloses for the first time that sorafenib can promote the direct construction of a mouse atherosclerosis model using a high-fat diet, shortening the original modeling cycle of 12 weeks or longer to 8 weeks, successfully solving the problem of long modeling time. (2) The modeling scheme of the present invention uses simple experimental techniques and is easy to operate. (3) The atherosclerosis mouse model constructed by the present invention has a very significant atherosclerotic effect (higher blood lipid levels of TC, TG and HDL-C); the aortic plaque area of atherosclerosis is larger and the vascular stenosis is more severe, and the formation of foam cells in the aortic valve is increased. Attached Figure Description
[0013] Figure 1 is a flowchart of the operation of inducing a mouse model with sorafenib;
[0014] Figure 2 shows the serum TC, TG, and HDL-C levels in mice after 8 weeks of induction with sorafenib.
[0015] Figure 3 shows the staining results of gross aortic lesions in mice after 8 weeks of sorafenib induction. The right figure shows that the percentage of aortic lesions (Aortic lesion %) in the low-dose intervention group (7.5 mg / kg) was significantly higher than that in the control group (Control), and the percentage of lesions in the high-dose intervention group (15 mg / kg) was further increased, indicating that this intervention measure is dose-dependent.
[0016] Figure 4 shows the Oil Red O staining results of aortic valves in mice after 8 weeks of sorafenib induction. The right figure shows that compared with the control group (Control), the lipid deposition in the aortic valves of the low-dose group (7.5 mg / kg) was significantly increased, and the number of foam cells filled with lipid droplets in the unit visual field was significantly increased. The lipid deposition in the high-dose group (15 mg / kg) was further aggravated on the basis of the low-dose aggravation, showing a dose-dependent "foaming" aggravation effect; the extremely high percentage of foaming is a direct manifestation of the drug aggravating the valve lipid deposition lesion at the cellular level.
[0017] * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 compared with the control group. Detailed implementation manners
[0018] The present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation manners.
[0019] The first part:
[0020] Describe the complete steps of the method of the present invention
[0021] Materials
[0022] 6-week-old male ApoE - / - mice, 20 - 22 g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2021 - 0006; sorafenib was purchased from: BAYER; TC and TG kits were purchased from Zhong Sheng Beikong Technology Co., Ltd., and the HDL-C kit was purchased from Nanjing Jiancheng Bioengineering Institute. All materials, reagents, etc. used can be obtained from commercial channels without special instructions.
[0023] (1) Purchase ApoE - / - mice, randomly divided into 3 groups according to body weight, the high-dose sorafenib group (15 mg / kg), the low-dose sorafenib group (7.5 mg / kg), and the control group (0 mg / kg). Blood was taken from the tail vein once every 4 weeks to detect blood lipids and various biochemical indexes.
[0024] (2) Feed all animals with a high-fat diet for 8 weeks to induce atherosclerosis.
[0025] (3) At different stages of atherosclerotic plaque formation, the plasma TC, TG, and HDL-C of three groups of mice were measured using a kit.
[0026] (4) After the experiment, the animals were euthanized and the size of the atherosclerotic plaques, the composition of the diseased cells and the stability of the plaques were analyzed using histological and pathological methods, so as to observe the effect of sorafenib intervention on the occurrence and development of atherosclerosis.
[0027] Part Two:
[0028] Example 1: Procedure for Sorafenib-Induced Mouse Model
[0029] Mice were housed in a clean environment at a temperature of 24 ± 1 ℃ and a relative humidity of 40% - 80%. Mice were divided into three groups: a control group (physiological saline), a low-dose sorafenib group (7.5 mg / kg), and a high-dose sorafenib group (15 mg / kg). Administration was by gavage every other day for 8 weeks. The procedure for inducing the sorafenib mouse model is as follows: Figure 1 As shown. All mice were fed a high-fat diet (feed formula: 83.5% basal diet + 15% lard + 1.5% cholesterol) and had free access to water.
[0030] Appendix Figure 1 The model was established for 8 weeks, during which the patient was fed a high-fat diet and received sorafenib by gavage every other day.
[0031] Example 2: Determination of TC, TG, and HDL-C levels in mouse serum
[0032] Experimental methods:
[0033] (1) After completing Example 1, the mice were fasted for 4 hours, an anticoagulant was added, and blood samples (200-300 μL) were collected from the tail using a capillary tube. The samples were centrifuged at 3000 r / min for 15 min to collect the plasma.
[0034] (2) Next, the contents of plasma TC, TG and HDL-C were measured using the kit and according to the instructions. Data were read using an ELISA reader at different wavelengths and analyzed using Excel.
[0035] (3) The experimental results are attached. Figure 2 .
[0036] Appendix Figure 2 The results showed that sorafenib significantly increased the levels of TC, TG, and HDL-C in mouse serum. Figure 2As shown in Figure A, compared with the control group, the total TC (TC) content in the sorafenib group was significantly increased, reaching 15 mg / kg in week 4 (p<0.05) and 15 mg / kg in week 8 (p<0.001), with the 15 mg / kg group showing the highest TC content; Figure 2 As shown in Figure B, compared with the control group, the TG level in the sorafenib 15 mg / kg group at week 8 was significantly increased (p<0.01), and the TG level was highest in the 15 mg / kg group; Figure 2 As shown in Figure C, compared with the control group, the HDL-C levels in the sorafenib administration groups at 7.5 mg / kg and 15 mg / kg were significantly increased in the fourth and eighth weeks, respectively (p<0.01), and the HDL-C level was highest when sorafenib was administered at 15 mg / kg.
[0037] Example 3: Gross staining experiment of mouse aortic lesions
[0038] Experimental methods:
[0039] 1. Place the separated aorta into a 6-well plate;
[0040] 2. Add 4% paraformaldehyde to a 6-well plate to fix the aorta for 15 minutes;
[0041] 3. Use a pipette to remove the paraformaldehyde, and wash with ddH2O 3 times, 1 min each time;
[0042] 4. Use a pipette to aspirate ddH2O, and soak the aorta in 60% isopropanol for 5 minutes to make the tissue isopropanolized so that Oil Red O can be stained.
[0043] 5. Use a pipette to aspirate 60% isopropanol and immerse the aorta in Oil Red O for 30 minutes;
[0044] 6. Use a pipette to remove Oil Red O, and rinse the aorta with 60% isopropanol to remove excess staining;
[0045] 7. Use a pipette to aspirate 60% isopropanol and rinse the aorta in ddH2O;
[0046] 8. Cut the aorta along its wall to expose its interior, take pictures, and calculate the plaque area.
[0047] 9. Experimental results are attached. Figure 3 .
[0048] Appendix Figure 3 Gross staining results of mouse aortic lesions showed that sorafenib intervention increased the gross area of atherosclerotic lesions in the mouse aorta; compared with the control group, the sorafenib 7.5 mg / kg (p<0.01) and 15 mg / kg groups significantly increased the gross area of atherosclerotic lesions in the mouse aorta (p<0.001).
[0049] One-way ANOVA was used to perform multiple comparisons and statistical significance analysis between different groups. GraphPadPrism 7 was used for statistical analysis.
[0050] Example 4: Mouse aortic valve Oil Red O staining experiment
[0051] Experimental methods:
[0052] 1. Remove the film from the -80℃ freezer and allow it to warm to room temperature for 30 minutes;
[0053] 2. Fix the slides in 4% paraformaldehyde for 15 minutes;
[0054] 3. Wash the slides three times with ddH2O, 1 min each time;
[0055] 4. Immerse the slide in 60% isopropanol for 5 minutes to isopropanize the tissue so that Oil Red O can be stained.
[0056] 5. Immerse the film in Oil Red O for 30 minutes;
[0057] 6. Soak the slides in 60% isopropanol to remove excess staining;
[0058] 7. Rinse the tablets with running purified water;
[0059] 8. Separate the tablets using glycerin gelatin sealing tablets and allow them to air dry;
[0060] 9. Observe using an inverted optical microscope at a magnification of 40x.
[0061] 10. Experimental results are attached. Figure 4 .
[0062] Appendix Figure 4 Oil Red O staining results showed that, compared with the control group, the sorafenib administration group had more severe intimal lipid accumulation at 7.5 mg / kg (p<0.001) and 15 mg / kg (p<0.001).
[0063] One-way ANOVA was used to perform multiple comparisons and statistical significance analysis between different groups. GraphPadPrism 7 was used for statistical analysis.
Claims
1. Application of sorafenib in constructing atherosclerosis models.
2. Application of sorafenib in constructing a mouse model of atherosclerosis.
3. Application of sorafenib in promoting the establishment of a mouse model of atherosclerosis by direct high-fat diet feeding.
4. The application according to claim 3, characterized in that... The preferred dosage of sorafenib is 7.5-15 mg / kg.
5. The application according to claim 4, characterized in that... The preferred dosage of sorafenib is 15 mg / kg.
6. The application according to any one of claims 3-5, characterized in that... Sorafenib is preferably administered by gavage.
7. The application according to any one of claims 3-6, characterized in that... The preferred dosing cycle for sorafenib is once every other day.
8. The application according to any one of claims 3-7, characterized in that... The aforementioned improvement refers to shortening the modeling cycle from the original 12 weeks or longer to 8 weeks.
9. The application according to any one of claims 2-8, characterized in that... The preferred mice are ApoE. - / - Mice.
10. The application according to any one of claims 1-9, characterized in that... Includes the following steps: (1) ApoE - / - Mice were randomly divided into three groups according to body weight: a high-dose sorafenib group (15 mg / kg), a low-dose sorafenib group (7.5 mg / kg), and a control group (physiological saline). The administration was by gavage, every other day for 8 weeks. All mice were fed a high-fat diet (feed formula: 83.5% basal diet + 15% lard + 1.5% cholesterol) for 8 weeks, with free access to water to induce atherosclerosis. Blood was collected from the tail vein every 4 weeks to detect blood lipids and various biochemical indicators. (2) At different stages of atherosclerotic plaque formation, plasma TC, TG, and HDL-C in three groups of mice were measured using a kit; (3) After the experiment, the animals were euthanized and the size of the atherosclerotic plaques, the composition of the diseased cells and the stability of the plaques were analyzed using histological and pathological methods, so as to observe the effect of sorafenib intervention on the occurrence and development of atherosclerosis.