Phlegm-blood stasis type myocardial infarction or cerebral infarction animal model and construction method and application thereof

Abstract

The invention discloses a construction method of an intermingled phlegm and blood stasis type myocardial infarction or cerebral infarction animal model, which comprises the following steps: selecting a healthy mouse, inducing an intermingled phlegm and blood stasis state by adopting a method of combining high-fat feeding with epinephrine hydrochloride subcutaneous injection, and feeding for 28 days; 1 mg / kg of epinephrine hydrochloride is subcutaneously injected every day on the 21th day, and the injection is continued for 7 days; establishing a myocardial infarction model by adopting a left coronary artery anterior descending branch ligation method, and establishing a cerebral infarction model by adopting a middle cerebral artery suture method; the invention further discloses an animal model of phlegm and blood stasis type myocardial infarction or cerebral infarction and application of the animal model to preparation of drugs for preventing or treating phlegm and blood stasis type myocardial infarction or cerebral infarction. According to the method, the phlegm-blood stasis type myocardial infarction or cerebral infarction animal model is successfully induced, the standard and objective judgment of the model state is realized by matching with an evaluation scale, the modeling method is simple, the repeatability is high, the evaluation system is scientific and reasonable, and the method has good research and application prospects.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis, its construction method, and its application. Background Technology

[0002] Myocardial infarction and cerebral infarction are major cardiovascular and cerebrovascular diseases threatening human health today, with their incidence and mortality rates increasing year by year, seriously endangering public health. The common pathological basis of both is focal ischemic necrosis caused by the formation of atherosclerotic plaques and thrombotic obstruction. However, due to the different sites of onset, they are often studied as two independent diseases in modern medical research. Existing models mostly focus on the replication of local ischemic foci in the myocardium or brain tissue, which can simulate physical damage relatively well, but it is difficult to reflect the systemic pathological changes in the body, especially the internal environment imbalance characteristics related to the traditional Chinese medicine pathogenesis of "phlegm" and "blood stasis".

[0003] In the theoretical system of Traditional Chinese Medicine (TCM), although myocardial infarction and cerebral infarction have different locations, they both fall under the category of "phlegm-blood stasis syndrome." Phlegm is a yin evil, mainly causing stickiness and obstruction of the collaterals; blood stasis is a yang evil, mainly causing obstruction of blood flow. Phlegm can lead to blood stasis, and blood stasis can also generate phlegm; the two are mutually causal and mutually reinforcing, leading to qi stagnation and meridian blockage. Clinically, the main manifestations of phlegm-blood stasis syndrome include obesity, chest tightness and shortness of breath, thick and greasy tongue coating, dark purple tongue, and choppy pulse, commonly seen in patients with arteriosclerosis and cardiovascular and cerebrovascular diseases. Phlegm-blood stasis syndrome is a systemic pathological understanding of complex metabolic and circulatory disorders in TCM, and its essence is highly correlated with the process described in modern medicine as "hyperlipidemia—vascular endothelial damage—thrombosis."

[0004] Existing animal models mostly focus on a single pathological link: (1) High-fat diet model: mainly used to simulate the endogenous state of phlegm and dampness, but it is difficult to induce significant changes in blood rheology and microcirculation; (2) Adrenaline or norepinephrine injection model: can cause vasoconstriction, erythrocyte aggregation and blood hypercoagulability, but has a weak interference with lipid metabolism; (3) Thrombotic myocardial infarction or cerebral infarction model: focuses more on local ischemic injury, while ignoring the formation of the systemic phlegm and blood stasis pathological environment. None of these models can reproduce the dual pathogenesis of phlegm and dampness accumulation and blood stasis obstruction in the same animal at the same time, which makes it difficult to fully reflect the essence of the phlegm and blood stasis syndrome.

[0005] Furthermore, traditional model studies often focus on single disease types, lacking a systematic experimental foundation for research on "different diseases with the same symptoms." Although some scholars have attempted to construct phlegm-blood stasis models by combining a high-fat diet with adrenaline injection, these efforts have mostly remained at the rat level and have failed to integrate myocardial infarction and cerebral infarction models, thus failing to reveal the molecular basis for their coexistence in different diseases. Mice, as model animals, have significant advantages in terms of stable genetic background and mature molecular tools, but to date, there is still a lack of standardized phlegm-blood stasis models suitable for mice, especially for their application in combined research on "different diseases with the same symptoms" in myocardial infarction and cerebral infarction.

[0006] Therefore, there is an urgent need for a composite model that uses mice as experimental subjects, can stably replicate the dual pathogenesis of phlegm-dampness and blood stasis, and can jointly model myocardial infarction and cerebral infarction. This model can not only realistically reflect the pathological characteristics of the "phlegm-blood stasis syndrome" in traditional Chinese medicine, but also provide a quantifiable and verifiable experimental tool for modern medical research, thereby providing experimental evidence for the integrated prevention and treatment of cardiovascular and cerebrovascular diseases using traditional Chinese and Western medicine and the theory of "different diseases with the same syndrome". Summary of the Invention

[0007] This invention designs and develops an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis. The purpose of this invention is to solve the problem of standardized phlegm and blood stasis model in mice, especially the model of "different diseases with the same symptoms" in myocardial infarction and cerebral infarction.

[0008] This invention designs and develops a method for constructing an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis.

[0009] This invention also designed and developed an animal model of myocardial infarction or cerebral infarction with phlegm and blood stasis in order to prepare drugs for the prevention or treatment of myocardial infarction or cerebral infarction with phlegm and blood stasis in combination.

[0010] The technical solution provided by this invention is as follows:

[0011] A method for constructing an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis includes the following steps:

[0012] Healthy mice were selected and induced to have a state of phlegm and blood stasis by high-fat diet combined with subcutaneous injection of epinephrine hydrochloride for 28 days.

[0013] On day 21, administer 1 mg / kg of epinephrine hydrochloride subcutaneously daily for 7 consecutive days.

[0014] A myocardial infarction model was established by ligation of the left anterior descending coronary artery, and a cerebral infarction model was established by suture occlusion of the middle cerebral artery.

[0015] Preferably, it also includes: daily subcutaneous injection of 1 mg / kg of epinephrine hydrochloride into the back for 7 consecutive days on day 21 to induce a state of phlegm and blood stasis.

[0016] Preferably, the concentration of the epinephrine hydrochloride is 0.54 mg / mL.

[0017] Preferably, the mice are SPF-grade male C57BL / 6J mice, weighing 19-20g, kept in a normal temperature and humidity environment, and the experiment begins after 7 days of acclimatization feeding.

[0018] Preferably, the mice are randomly divided into a normal group, a phlegm-blood stasis group, a myocardial infarction group, a cerebral infarction group, a phlegm-blood stasis myocardial infarction group, and a phlegm-blood stasis cerebral infarction group.

[0019] Preferably, the ligation position for the left anterior descending coronary artery ligation method is 2-3 mm away from the root of the left atrial appendage.

[0020] Preferably, the middle cerebral artery suture method is used, with the suture marking point reaching the bifurcation point, the occlusion time being 60 minutes, and the sample being taken 24 hours after reperfusion.

[0021] An animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis is constructed using the method described above.

[0022] An animal model of phlegm-blood stasis type myocardial infarction or cerebral infarction is used in the preparation of drugs for preventing phlegm-blood stasis type myocardial infarction or cerebral infarction.

[0023] An animal model of phlegm-blood stasis type myocardial infarction or cerebral infarction is used in the preparation of drugs for treating phlegm-blood stasis type myocardial infarction or cerebral infarction.

[0024] The beneficial effects of this invention are as follows: This invention provides a method for constructing an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis. By using a combination of high-fat feeding, adrenaline injection, and myocardial infarction or cerebral infarction modeling, the animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis is successfully induced. The accompanying evaluation scale realizes the standardization and objective judgment of the model status. The modeling method is simple, highly reproducible, and the evaluation system is scientific and reasonable, with good research and application prospects. Attached Figure Description

[0025] Figure 1 This is a rating chart of the phlegm-blood stasis intermingling animal model described in this invention;

[0026] Figure 2a The graph shows the total cholesterol (TC) detection results for each experimental group described in this invention.

[0027] Figure 2b The graph shows the triglyceride (TG) detection results for each experimental group described in this invention.

[0028] Figure 2cThe graph shows the detection results of high-density lipoprotein cholesterol (HDL-C) in each experimental group described in this invention.

[0029] Figure 2d The graph shows the detection results of low-density lipoprotein cholesterol (LDL-C) in each experimental group described in this invention.

[0030] Figure 3 This is a graph showing the detection results of platelet-activating factor TXB levels in each experimental group described in this invention.

[0031] Figure 4 HE staining image of normal mouse brain tissue as described in this invention;

[0032] Figure 5 HE staining image of brain tissue from the mouse model of cerebral infarction caused by phlegm and blood stasis as described in this invention;

[0033] Figure 6 HE staining image of normal mouse myocardial tissue as described in this invention;

[0034] Figure 7 HE staining image of myocardial tissue in a mouse model of myocardial infarction caused by phlegm and blood stasis as described in this invention. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0036] This invention provides a method for constructing an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis, comprising the following steps:

[0037] Step 1: Animal selection: SPF-grade male C57BL / 6J mice, weighing 19-20g, were selected and kept in a normal temperature and humidity environment. After 7 days of acclimatization feeding, the experiment was started and the mice were randomly divided into normal group, phlegm-blood stasis group, myocardial infarction group, cerebral infarction group, phlegm-blood stasis myocardial infarction group, and phlegm-blood stasis cerebral infarction group.

[0038] Step Two: The Modeling Stage of Phlegm and Blood Stasis Intermingling

[0039] High-fat diet feeding: Feed continuously for 28 days to induce the internal generation of phlegm and dampness;

[0040] Blood stasis induction: Starting from day 21, subcutaneous injections of epinephrine hydrochloride (concentration 0.54 mg / mL, dose 1 mg / kg) were administered into the back daily for 7 consecutive days to induce vasoconstriction, microcirculatory disturbance and hypercoagulable state.

[0041] Step two induces mice to develop a pathological state characterized by both "phlegm and dampness accumulation" and "blood stasis obstructing the collaterals," manifested as dull fur, lethargy, purplish tongue, and obesity.

[0042] Step 3: Modeling stage of myocardial infarction / cerebral infarction

[0043] After the phlegm and blood stasis condition was established, surgical modeling was performed on the 28th day:

[0044] Myocardial infarction model: Ligation of the left anterior descending coronary artery under anesthesia caused focal ischemic necrosis of the myocardium;

[0045] Cerebral infarction model: The middle cerebral artery (MCAO) was blocked using the middle cerebral artery suture occlusion method, causing focal ischemia in the cerebral cortex;

[0046] After the model was replicated, it was fed normally for 24 hours, and its behavioral and tongue appearance changes were observed. Heart and brain tissue were also taken for testing.

[0047] In another embodiment, the left anterior descending coronary artery is ligated 2-3 mm from the root of the left atrial appendage.

[0048] In another embodiment, the middle cerebral artery suture method is used, with the suture marking point reaching the bifurcation point, the occlusion time being 60 minutes, and the sample being taken 24 hours after reperfusion.

[0049] In another embodiment, after the model is established, a comprehensive assessment is made based on the mouse's general condition, blood lipid levels, platelet activating factor, and histopathological changes. Preferably, blood lipid levels are assessed by detecting total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Platelet activating factor is assessed by detecting TXB. Histopathological changes are observed by HE staining to detect focal necrosis, vascular lesions, and cell morphological changes in myocardial and brain tissues.

[0050] In another embodiment, as shown in Table 1, in order to facilitate the evaluation of the phlegm-blood stasis intermingling mouse model, the present invention also provides an evaluation scale for the phlegm-blood stasis intermingling animal model. This scale can be used for quantitative assessment of the model status and verification of its correlation with biochemical indicators. Each item is scored from 0 to 2 points, where 0 points is normal, 1 point is mildly abnormal, and 2 points is significantly abnormal. The total score is 20 points. When the total score is ≥10 points, it is determined to be a phlegm-blood stasis intermingling state.

[0051] Table 1 Evaluation scale for animal models of phlegm-blood stasis syndrome

[0052]

[0053]

[0054] The present application will be further described in detail below with reference to specific embodiments.

[0055] Example

[0056] I. Materials and Methods

[0057] 1. Experimental Animals

[0058] The animals selected for this experiment were 5-week-old SPF-grade male C57BL / 6J mice, weighing 19 - 20 g. All mice were provided by Beijing Huafukang Biotechnology Co., Ltd. (Animal License No.: SCXK Beijing 2024 - 0003), and were housed in the Experimental Center of Tianjin University of Traditional Chinese Medicine. The housing conditions were 5 mice per cage, room temperature (23 ± 3°C), and humidity (40 ± 5%); the Experimental Animal Ethics License No. (TCM - LAEC2025217H2066); they were randomly divided into a normal group, a phlegm and stasis syndrome group, a myocardial infarction group, a cerebral infarction group, a phlegm and stasis syndrome with myocardial infarction group, and a phlegm and stasis syndrome with cerebral infarction group, with 6 mice in each group. After 1 week of adaptive feeding, they were reserved for use.

[0059] 2. Main Reagents

[0060] High-fat diet (Koa协力 (Tianjin) Co., Ltd., Custom Feed 12109C, which contains: 200 g casein, 3 g cystine, 212 g corn starch, 124.41 g sucrose, 71 g maltodextrin, 50 g cellulose, 155 g cocoa butter, 25 g soybean oil, 16.5 g potassium citrate, 13 g calcium hydrogen phosphate, 5.5 g calcium carbonate, 5 g multi-mineral S10020, 2.59 g sodium chloride, 2 g choline tartrate, 1 g multi-vitamin V10001C, 11.25 g cholesterol, 4.5 g sodium cholate); adrenaline hydrochloride (Shanghai Aladdin Biochemical Technology Co., Ltd., L303968 - 1 g); 4% paraformaldehyde fixative (Hefei Biosharp); HE stain (Beijing Solarbio); neutral resin (China National Pharmaceutical Corporation); sodium citrate anticoagulant (Beijing Regen Biotechnology Co., Ltd., R10127 - 100 ml); P-selectin test kit, total cholesterol (CHO) kit, triglyceride (TG) kit, low-density lipoprotein cholesterol (LDL-C) kit, and high-density lipoprotein cholesterol (HDL-C) kit, all provided by Nanjing Jiancheng Technology Co., Ltd.

[0061] 3. Main Instruments

[0062] Gas anesthetic machine (Beijing Zhongshi Technology Co., Ltd.); MCAO model wire embolism (Shenzhen Rewod Life Science and Technology Co., Ltd.); tissue dehydrator (Leica, Germany); paraffin embedding machine (Leica, Germany); paraffin slicer (Leica, Germany); anti-drop coverslips (Jiangsu Shitai Experimental Equipment Co., Ltd.); baking machine (Leica, Germany); incubator (Hunan Xiangyi Experimental Instrument Development Co., Ltd.); pathological section digital scanner (Ningbo Jiangfeng Bioinformatics Co., Ltd.).

[0063] 4. Experimental Methods

[0064] Mice were induced to develop a state of phlegm and blood stasis by a high-fat diet combined with subcutaneous injection of epinephrine hydrochloride, and then focal ischemia modeled in myocardial or cerebral tissue was established.

[0065] (1) Starting from day 0 of the experiment, the body was fed a high-fat diet for 28 consecutive days to cause lipid metabolism disorder and elevated blood lipids, thereby forming the pathological basis of "phlegm and dampness accumulation".

[0066] Starting from day 21, mice were subcutaneously injected with epinephrine hydrochloride solution on their backs daily at a dose of 1 mg / kg and a solution concentration of 0.54 mg / mL, using physiological saline as a solvent, once daily for 7 consecutive days. Epinephrine injection caused strong vasoconstriction, microcirculatory disturbances, and a hypercoagulable state, thereby inducing the pathological changes of "blood stasis obstructing the collaterals." The combined effects of a high-fat diet and epinephrine stimulation resulted in mice exhibiting symptoms of phlegm and blood stasis, such as dull fur, lethargy, obesity, and a purplish-dark tongue, suggesting an imbalance in the internal environment where phlegm and blood stasis coexist.

[0067] (2) After the phlegm-blood stasis state was established, surgical modeling was initiated on day 28:

[0068] The myocardial infarction model was constructed using the left anterior descending coronary artery ligation method: mice were anesthetized by isoflurane gas inhalation, fixed in a supine position, and the left anterior descending coronary artery was exposed by making an incision in the 3rd to 4th intercostal space on the left chest. The left anterior descending coronary artery was ligated with 6-0 sutures about 2 mm from the apex of the heart. The model was considered successful when the apex of the heart turned white and the ST segment was elevated after ligation.

[0069] The cerebral infarction model was constructed using the middle cerebral artery suture occlusion method: Mice were anesthetized with isoflurane gas inhalation and fixed in a supine position on the operating table. A longitudinal incision of approximately 1 cm was made along the right side of the midline of the neck. Subsequently, the muscles, nerves, and fascia were bluntly dissected using ophthalmic forceps until the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were clearly separated. After carefully lifting the CCA, a ligation suture was used to ligate its proximal end, and the blood flow to the ECA was clamped with a vascular clamp. At the bifurcation point between the arterial artery (A) and the intracranial artery (ICA), a surgical suture is left for subsequent fixation of the suture plug. Then, at the CCA position 3mm from the bifurcation, a small incision is made using a syringe needle, and the suture plug is inserted. Immediately after the suture plug enters the ICA, the arterial clamp is released, and the suture plug is pushed forward until the suture plug marker reaches the bifurcation, at which point the insertion depth is appropriate. The suture plug is then bound to the CCA with pre-prepared surgical suture to prevent displacement or bleeding at the insertion site. After cleaning the surgical wound, the neck incision is sutured with surgical suture. After 60 minutes of ischemia, the mouse is anesthetized and the suture plug is removed to achieve reperfusion of the ischemic brain tissue.

[0070] To ensure the stability and reliability of the experiment and reduce the variability in cerebral infarction results, all mouse surgeries were performed by the same operator; as a control, the normal group mice also had their blood vessels and vagus nerves separated, but the blood vessels were not ligated or embolized.

[0071] (3) After the model was prepared, samples were collected 24 hours post-surgery: Mice were euthanized by cervical dislocation after anesthesia, and blood and tissue samples were collected rapidly. Blood was collected from the orbital cavity using a 1 mL EP tube containing sodium citrate and centrifuged at 3000 r / min for 10 min. The plasma was separated and stored at -80℃. Blood lipids and platelet-activating factor were measured using a microplate reader with a kit. Heart and brain tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe the pathological changes in heart / brain tissues.

[0072] Model evaluation includes behavioral observation, biochemical testing and histopathological analysis: behavioral observation mainly records the mice's coat color, activity level, diet, mental state, changes in body shape and tongue appearance; scores are given according to the self-made "Evaluation Scale for Phlegm-Blood Stasis Intertwined Animal Model (Table 1)", with a score range of 0-2 points, and the average score is taken after independent evaluation by two raters.

[0073] 5. Statistical methods

[0074] The experimental data were analyzed using Graphpad Prism software and presented as follows: The results indicate that the t-test was used for comparisons between two groups, and the one-way AVONA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0075] II. Results

[0076] 1. Phlegm-Blood Stasis Intertwined Mouse Behavioral Scale Scoring

[0077] During the modeling process, from day 14 to 21 of high-fat feeding, mice in the phlegm-stasis syndrome group showed dull fur, obesity, reduced activity, and decreased food intake; while mice in the normal control group had normal diets, frequent exploratory behavior, and quick reactions. From day 5 to 7 after epinephrine hydrochloride injection, the animals in the phlegm-stasis syndrome group exhibited dark brown fur, lethargy, sluggishness, huddling together, and a purplish-dark tongue, consistent with the clinical characteristics of "phlegm-dampness accumulation combined with blood stasis obstructing the collaterals" in Traditional Chinese Medicine.

[0078] like Figure 1 As shown in the figure, the behavioral scores indicated that the overall score of the phlegm-blood stasis group was significantly higher than that of the normal control group, indicating that the phlegm-blood stasis syndrome was significantly formed.

[0079] 2. Blood lipids and metabolic indicators

[0080] like Figures 2a-2dAs shown, compared with the normal group, the serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) levels of mice in the phlegm-stasis combination group were significantly increased, while the high-density lipoprotein (HDL-C) level was decreased, with statistically significant differences (p<0.05). Further comparison of the disease model groups revealed that, in both the myocardial infarction and cerebral infarction groups, the degree of dyslipidemia was aggravated after the addition of the phlegm-stasis combination factor. The myocardial infarction group showed a more significant increase in TC and LDL-C (p<0.05), suggesting an increased risk of accumulation and arteriosclerosis. The cerebral infarction group showed increased TG and a significant decrease in HDL-C, reflecting impaired plasma lipoprotein metabolism and increased blood viscosity. These results indicate that the mice exhibited significant lipid metabolism disorders and dyslipidemia, consistent with the pathological basis of "internal generation of phlegm and dampness."

[0081] 3. Platelet activation and blood stasis related indicators

[0082] like Figure 3 As shown, the plasma thromboxane B (TXB) content in mice in the phlegm-stasis intermingling group was significantly higher than that in the normal group (p<0.05), indicating enhanced platelet activation, increased blood viscosity, and impaired vascular endothelial function. This is consistent with the "impaired blood flow and obstruction of meridians" manifestation in the pathological process of "blood stasis" in traditional Chinese medicine. The increase was more pronounced in the myocardial infarction group and the cerebral infarction group with phlegm-stasis intermingling (p<0.05), indicating that the phlegm-stasis internal environment aggravates the blood stasis of ischemic injury.

[0083] 4. Brain HE staining results

[0084] like Figures 4-5 As shown, the brain tissue cells of mice in the normal group were neatly and tightly arranged, and stained evenly. The cell nucleoli were clear and intact, and appeared blue-purple under the action of hematoxylin staining solution. The brain tissue of mice in the phlegm-blood stasis cerebral infarction group showed large-area vacuolar changes, sparse cells, disordered arrangement, morphological variations, increased eosinophilicity of cytoplasm, and obvious nucleolus condensation, all of which indicated cell necrosis.

[0085] 5. Results of HE staining of the heart

[0086] like Figures 6-7 As shown, in the normal group of mice, HE staining of myocardium showed a light pink color, with cells arranged neatly and rod-shaped nuclei in the center, without inflammatory cells or fibrosis; in the group of mice with phlegm and blood stasis causing myocardial infarction, eosinophilic cells in the infarct area were enhanced, nuclei disappeared, accompanied by inflammatory cell infiltration and interstitial edema, peripheral myocardial hypertrophy, and later granulation tissue and collagen proliferation, resulting in structural disorder.

[0087] In summary, the results indicate that a high-fat diet combined with subcutaneous injection of epinephrine hydrochloride successfully induced a dual pathological state of phlegm-dampness and blood stasis in mice, exhibiting typical characteristics of phlegm-stasis syndrome. Further, by combining this with coronary artery ligation or cerebral artery embolization surgery, a composite model exhibiting both phlegm-stasis syndrome and ischemic cardiovascular and cerebrovascular lesions can be stably replicated in animals. This model not only matches clinical phlegm-stasis syndrome in terms of general symptoms, hematology, and pathology, but it is also reproducible, providing a reliable experimental basis for the study of the "different diseases, same syndrome" mechanism.

[0088] This invention belongs to the field of experimental animal modeling and TCM syndrome modeling. Using C57BL / 6 mice as subjects, a state of phlegm-blood stasis was induced by high-fat feeding combined with subcutaneous injection of epinephrine hydrochloride. Myocardial infarction and cerebral infarction models were established using the left anterior descending coronary artery ligation method and the middle cerebral artery suture occlusion method, respectively, forming a complex disease model with characteristics of both phlegm-dampness accumulation and blood stasis obstruction. A corresponding evaluation scale for the phlegm-blood stasis animal model was developed for the quantitative and standardized assessment of the model status. This invention is simple to operate, highly reproducible, and produces stable model characteristics. It can be used simultaneously to study the common molecular mechanisms of myocardial infarction and cerebral infarction under the phlegm-blood stasis syndrome and the effects of TCM intervention.

[0089] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for constructing an animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis, characterized in that, Includes the following steps: Healthy mice were selected and induced to have a state of phlegm and blood stasis by high-fat diet combined with subcutaneous injection of epinephrine hydrochloride for 28 days. On day 21, administer 1 mg / kg of epinephrine hydrochloride subcutaneously daily for 7 consecutive days. A myocardial infarction model was established by ligation of the left anterior descending coronary artery, and a cerebral infarction model was established by suture occlusion of the middle cerebral artery.

2. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claim 1, characterized in that, Also includes: On day 21, 1 mg / kg of epinephrine hydrochloride was injected subcutaneously into the back daily for 7 consecutive days to induce a state of phlegm and blood stasis.

3. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claim 1 or 2, characterized in that, The concentration of the epinephrine hydrochloride was 0.54 mg / mL.

4. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claim 3, characterized in that, The mice used were SPF-grade male C57BL / 6J mice, weighing 19-20g, kept in a normal temperature and humidity environment, and started the experiment after 7 days of acclimatization feeding.

5. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claim 4, characterized in that, The mice were randomly divided into a normal group, a phlegm-blood stasis group, a myocardial infarction group, a cerebral infarction group, a phlegm-blood stasis myocardial infarction group, and a phlegm-blood stasis cerebral infarction group.

6. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claims 1, 2, 4 or 5, characterized in that, When using the left anterior descending coronary artery ligation method, the ligation point is 2-3 mm away from the root of the left atrial appendage.

7. The method for constructing an animal model of myocardial infarction or cerebral infarction of the phlegm-blood stasis type as described in claims 1, 2, 4 or 5, characterized in that, The middle cerebral artery suture method was used, with the suture marking point reaching the bifurcation point. The occlusion time was 60 minutes, and the sample was taken 24 hours after reperfusion.

8. An animal model of myocardial infarction or cerebral infarction caused by phlegm and blood stasis, characterized in that, The animal model of myocardial infarction or cerebral infarction with phlegm and blood stasis as described in claims 1-7 was constructed.

9. The application of an animal model of phlegm-blood stasis type myocardial infarction or cerebral infarction in the preparation of drugs for preventing phlegm-blood stasis type myocardial infarction or cerebral infarction, characterized in that, Use the animal model of myocardial infarction or cerebral infarction with phlegm and blood stasis as described in claim 8.

10. The application of an animal model of phlegm-blood stasis type myocardial infarction or cerebral infarction in the preparation of drugs for treating phlegm-blood stasis type myocardial infarction or cerebral infarction, characterized in that, Use the animal model of myocardial infarction or cerebral infarction with phlegm and blood stasis as described in claim 8.

Images