Construction method and application of animal model for cardiomyopathy induced by circulating system

Abstract

The invention relates to the technical field of medical animal model construction, in particular to a construction method and application of a cardiomyopathy animal model induced by a circulating system. According to the invention, the Abeta25-35 is injected into the adventitia, so that the animal model of the cardiomyopathy induced by the Abeta25-35 through the tissue fluid circulatory system of the adventitia is constructed in the SD rat for the first time. In the model, A beta 25-35 is subjected to myocardial perfusion through lower limb vascular adventitia to induce myocardial diffuse fibrosis and ejection fraction reduction of SD rats, which are in accordance with epidemiological research data. The model can be used for subsequently researching the pathogenic mechanism of A beta25-35 induced cardiomyopathy and a novel treatment method taking vascular adventitia tissue fluid transmission as a way, and also provides a brand new view angle for researching the pathogenic mechanism of AD or cerebrovascular diseases and a diagnosis and treatment method.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing medical animal models, and particularly to a method for constructing an animal model of cardiomyopathy induced by the circulatory system and its application. Background Art

[0002] β-amyloid not only plays an important role in the pathogenic process of cognitive impairment diseases (such as Alzheimer's disease, AD), but since 2016, a large amount of epidemiological data has confirmed that the level of β-amyloid in plasma is closely related to abnormal cardiac function. In human specimens of AD patients, Aβ40 and Aβ42 are deposited in cardiomyocytes and the interstitial space of cardiac tissue. Most AD patients are accompanied by varying degrees of abnormal cardiac function. In addition, studies have shown that the accumulation of Aβ40 in blood, blood vessel walls, and myocardial tissue is closely related to the incidence of abnormal cardiac function, coronary heart disease, and heart failure. However, current epidemiological data can only prove the correlation between the level of circulating Aβ40 and abnormal cardiac function, and cannot prove that Aβ40 can directly damage cardiac function through cardiac perfusion of the circulatory system. There is currently no reported animal model of cardiac lesions induced by injecting Aβ40 through the circulatory system.

[0003] In the mammalian circulatory system, in addition to blood circulation and lymphatic circulation, Starling proposed in 1896 that interstitial fluid, which is derived from plasma and enters the extracellular matrix after capillary filtration, can flow continuously in the matrix. Studies have shown that in addition to free diffusion, interstitial fluid can also be transported over long distances within organs or tissues, such as the perivascular tissues of the brain or thymus, and the interstitial space of tumor tissues. In 2018, Benias et al. found that the space filled with interstitial fluid is supported by a complex structure of collagen fiber bundles, which mainly exists in the submucosa of the nasal sinuses, gastrointestinal tract, and bladder, the dermis, and various fibrous connective tissues (such as around bronchi, around arteries, and fascia).

[0004] Constructing an animal model of cardiomyopathy can well study the pathogenesis of cardiomyopathy and can also be used to screen drugs for the treatment of cardiomyopathy. Based on the current lack of an animal model of cardiac lesions induced by injecting Aβ through the circulatory system, the present invention intends to select the Aβ25-35 polypeptide and construct an Aβ-induced cardiomyopathy rat model by periadventitial injection. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing an animal model of cardiomyopathy induced by the circulatory system and its application to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing an animal model of cardiomyopathy induced by the circulatory system, comprising the following steps:

[0008] Take SD rats and subcutaneously inject Aβ25-35 solution every 2 days for a total of 4 injections to obtain the cardiomyopathy animal model.

[0009] Preferably, the position of the subcutaneous injection is the outer membrane of the lower limb blood vessels.

[0010] Preferably, the injection volume of the Aβ25-35 solution each time is 400 μL.

[0011] Preferably, the concentration of Aβ25-35 in the Aβ25-35 solution is 1 mg / mL.

[0012] Aβ25-35 is a key peptide segment in Aβ40 or Aβ42, which can promote the aggregation of Aβ40 or Aβ42 and has similar physicochemical properties to Aβ40 or Aβ42.

[0013] The present invention provides the application of the cardiomyopathy animal model obtained by the above construction method in studying the pathogenic mechanism of Aβ-induced cardiomyopathy.

[0014] The present invention provides the application of the cardiomyopathy animal model obtained by the above construction method in screening drugs for preventing and / or treating cardiomyopathy.

[0015] The present invention provides the application of the cardiomyopathy animal model obtained by the above construction method in evaluating the safety of drugs for preventing and / or treating cardiomyopathy.

[0016] The present invention provides the application of the cardiomyopathy animal model obtained by the above construction method in evaluating the efficacy of drugs for preventing and / or treating cardiomyopathy.

[0017] The present invention discloses the following technical effects:

[0018] The present invention constructs an animal model of "cardiomyopathy induced by Aβ25-35 through the interstitial fluid circulation system of the outer blood vessel membrane" in SD rats for the first time by injecting Aβ25-35 through the outer blood vessel membrane. In this model, Aβ25-35 is perfused into the myocardium through the outer membrane of the lower limb blood vessels, inducing diffuse myocardial fibrosis in SD rats and a decrease in ejection fraction, which is consistent with the epidemiological research data. The construction method is simple, and the Aβ25-35 fragment can be directly purchased as a commercialized powder and configured into a working solution. The method of injecting through the outer blood vessel membrane is simple and mature and easy to operate. The construction method provided by the present invention has a relatively short model construction period. A decrease in ejection fraction appears about 30 days after the first injection, and diffuse fibrosis appears 60 days later. This model can subsequently study the pathogenic mechanism of Aβ25-35-induced cardiomyopathy and new treatment methods through the interstitial fluid transmission of the outer blood vessel membrane, and at the same time provide a new perspective for studying the pathogenic mechanism and diagnosis and treatment methods of AD or cerebrovascular diseases. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0020] Figure 1 are the echocardiogram detection results; where A is the echocardiogram (left ventricular long axis) of the heart of an SD rat injected with Aβ25-35 in the adventitia; B is the echocardiogram (left ventricular long axis) of the heart of an SD rat injected with physiological saline in the adventitia; C is the echocardiogram (left ventricular long axis) of the heart of an SD rat injected with Aβ25-35 intravenously; D is the echocardiogram (left ventricular long axis) of the heart of an SD rat injected with physiological saline intravenously; E is the change curve of the left ventricular ejection fraction (EF%) of the heart of an SD rat injected with Aβ25-35 or physiological saline (Saline) in the adventitia; F is the change curve of the left ventricular ejection fraction (EF%) of the heart of an SD rat injected with Aβ25-35 or physiological saline (Saline) intravenously;

[0021] Figure 2 are the results of Masson staining and the myocardial fibrosis score results; where A1 and A2 are the Masson staining diagrams of the heart of an SD rat injected with Aβ25-35 in the adventitia; B1 and B2 are the Masson staining diagrams of the heart of an SD rat injected with physiological saline in the adventitia; C1 and C2 are the Masson staining diagrams of the heart of an SD rat injected with Aβ25-35 intravenously; D1 and D2 are the Masson staining diagrams of the heart of an SD rat injected with physiological saline intravenously; Interstitia is the myocardial interstitium; Perivascular is around the blood vessels; E is the myocardial fibrosis score of SD rats with different treatments; Adventitial infusion is injection in the adventitia, Intravenous infusion is intravenous injection, Saline is the control group for adventitial injection or intravenous injection, scale bar = 250μm. Detailed implementation manners

[0022] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0023] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0026] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0027] Unless otherwise required, the materials used in the present invention are all routinely purchased by those skilled in the art, and the methods used in the present invention are all well-known methods to those skilled in the art.

[0028] Example 1 Construction of an animal model of heart disease (cardiomyopathy) induced by Aβ protein through the circulatory system

[0029] 1. Preparation of Aβ25-35 working solution:

[0030] Add Aβ25-35 powder to normal saline to prepare a working solution with a concentration of 1 mg / mL, and assist dissolution with ultrasound.

[0031] 2. Treat the Aβ25-35 working solution at 37 °C for 5 - 7 days.

[0032] 3. Perivascular injection: Aβ25-35 (1 mg / mL) was injected into the adventitia of the lower limb blood vessels of SD rats (250 - 300 g), 400 μL each time, once every 3 days (i.e., once every 2 days), for a total of 4 injections, denoted as the Aβ25-35 perivascular induction group. The perivascular injection control group was injected with an equal volume of normal saline. The perivascular injection site was the subcutaneous tissue space of the rat ankle. After injecting the Aβ25-35 solution at this site, the Aβ25-35 fragment could flow centripetally along the adventitia of the lower limb blood vessels and thus enter the heart.

[0033] 4. Intravascular injection: The procedure was the same as that in step "3. Perivascular injection", except that the injection site was intravascular, i.e., intravenous injection, denoted as the Aβ25-35 intravascular induction group. At the same time, an intravascular injection control group was set up, and the intravascular injection control group was injected with an equal volume of normal saline.

[0034] 5. At 30 days and 60 days after the first injection, parameters such as left ventricular ejection fraction were detected by echocardiogram. At 30 days after injection, the results of echocardiogram were as shown in A - D in Figure 1 , and the change curve of ejection fraction was as shown in E and F in Figure 1 . The results showed that the left ventricular ejection fraction of SD rats in the Aβ25-35 perivascular induction group decreased, with a significant difference compared with that of rats in the perivascular injection control group; although the left ventricular ejection fraction of SD rats in the Aβ25-35 intravascular induction group showed a downward trend, there was no significant difference compared with that of rats in the intravascular injection control group. Thus, it can be seen that injecting Aβ25-35 into the adventitia of the lower limb blood vessels of SD rats can induce cardiac function injury (decrease in ejection fraction).

[0035] 5. At 60 days after the first injection, the hearts of SD rats were harvested, paraffin-embedded, and longitudinally cut into 5 slices along the largest plane of the heart, and Masson staining was performed to analyze myocardial fibrosis.

[0036] The steps of Masson staining are as follows:

[0037] 1). Dewax the sections routinely to water; 2). Stain with the prepared Weigert iron hematoxylin staining solution for 5-10 min; 3). Wash thoroughly with water, and if overstained, differentiate with hydrochloric acid alcohol; 4). Blue with Masson bluing solution for 3-5 min, then wash with water; 5). Wash with distilled water for 1 min; 6). Stain with ponceau fuchsin staining solution for 5-10 min; 7). During the above operation process, prepare a weak acid working solution according to the volume ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 min; 8). Wash with 1% phosphomolybdic acid solution for 1-2 min; 9). Wash with the prepared weak acid working solution for 1 min; 10). Without washing with water, directly put it into aniline blue staining solution and stain for 1-2 min; 11). Wash with the prepared weak acid working solution for 1 min; 12). Rapidly dehydrate with 95% (V / V) ethanol; 13). Dehydrate with absolute ethanol 3 times, 5-10 s each time; 14). Clear with xylene 3 times, 1-2 min each time; 15). Mount with neutral balsam.

[0038] After that, the degree of myocardial fibrosis was scored, and the scoring criteria for the degree of fibrosis were as follows:

[0039] 0: Normal fibroblasts, without over-proliferation;

[0040] 1: Scattered myocardial interstitial fibroblasts with a small amount of proliferation;

[0041] 2: Medium-range proliferation of myocardial interstitial fibroblasts and perivascular fibroblasts;

[0042] 3: Large-range proliferation of myocardial interstitial fibroblasts and perivascular fibroblasts.

[0043] The results of myocardial fibrosis analysis were as Figure 2 shown. The results showed that Aβ25-35-induced myocardial fibrosis was mainly concentrated around blood vessels and in the myocardial interstitium, manifested as over-proliferation of fibroblasts, belonging to diffuse fibrosis. Among them, Figure 2 A1 in showed proliferation of myocardial interstitial fibroblasts in Aβ25-35-injected SD rats with epicardial injection; Figure 2 A2 in showed proliferation of perivascular fibroblasts in Aβ25-35-injected SD rats with epicardial injection; Figure 2 B1 in showed normal myocardial interstitial fibroblasts in SD rats injected with normal saline epicardially; Figure 2 B2 in showed normal perivascular fibroblasts in SD rats injected with normal saline epicardially; Figure 2 C1 in showed only less proliferation of myocardial interstitial fibroblasts in Aβ25-35-injected SD rats with intracardiac injection; Figure 2 C2 in showed only less proliferation of perivascular fibroblasts in Aβ25-35-injected SD rats with intracardiac injection; Figure 2In D1, the myocardial interstitial fibroblasts of the rats injected with normal saline intravascularly are normal; Figure 2 In D2, the perivascular fibroblasts of the rats injected with normal saline intravascularly are normal. The myocardial fibrosis score of the SD rats in the Aβ25-35 epicardial induction group is high, and there is a significant difference from that of the epicardial control group; similarly, although the myocardial fibrosis score of the SD rats in the Aβ25-35 intravascular induction group increases, there is no significant difference from that of the rats in the intravascular injection control group. Thus, it can be seen that the method of intravascular injection cannot successfully establish an animal model of heart disease (cardiomyopathy) induced by Aβ protein through the circulatory system in the short term; the method provided by the present invention (epicardial injection) can successfully establish an animal model of heart disease (cardiomyopathy) induced by Aβ protein through the circulatory system, and the time for establishing the model is shorter. The ejection fraction decreases about 30 days after the first injection, and diffuse fibrosis appears 60 days later.

[0044] The above-described embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an animal model of cardiomyopathy induced by the circulatory system, characterized in that Comprising the following steps: Taking SD rats, subcutaneously injecting Aβ25-35 solution every 2 days, for a total of 4 injections, to obtain the cardiomyopathy animal model.

2. The construction method according to claim 1, characterized in that The position of the subcutaneous injection is the outer membrane of the lower limb blood vessels.

3. The construction method according to claim 1, characterized in that The injection volume of the Aβ25-35 solution each time is 400 μL.

4. The construction method according to claim 1, characterized in that, The concentration of Aβ25-35 in the Aβ25-35 solution is 1 mg / mL.

5. Application of the cardiomyopathy animal model obtained by the construction method according to any one of claims 1-4 in studying the pathogenic mechanism of Aβ-induced cardiomyopathy.

6. Application of the cardiomyopathy animal model obtained by the construction method according to any one of claims 1-4 in screening drugs for preventing and / or treating cardiomyopathy.

7. Application of the cardiomyopathy animal model obtained by the construction method according to any one of claims 1-4 in evaluating the safety of drugs for preventing and / or treating cardiomyopathy.

8. Application of the cardiomyopathy animal model obtained by the construction method according to any one of claims 1-4 in evaluating the efficacy of drugs for preventing and / or treating cardiomyopathy.

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