Method for constructing atrial fibrillation small animal model through succinic acid induction and application

Abstract

The invention discloses a method for constructing an atrial fibrillation small animal model through succinic acid induction and application, and the method comprises the following steps: in an animal model construction period, enabling a small animal to ingest an aqueous solution containing succinic acid or all pharmaceutically acceptable salts of succinic acid every day, and finishing the modeling period to obtain the animal model. Compared with a traditional angiotensin AngII administration method, the atrial fibrillation small animal model has the advantages that the cost of a medicine for constructing the animal model is lower, the economic benefit is higher, and the atrial fibrillation induction effect is better; the model represents the most common atrial fibrillation type in clinic, and compared with an acute transient atrial fibrillation model, the research and application range is wider; compared with tail vein injection, intraperitoneal injection, subcutaneous pump burying and other modes, the non-creative model has the advantages that damage to small animals is smaller, and the requirement for the operation technology is low.

Description

Technical Field

[0001] This invention belongs to the field of medical animal experiments, specifically relating to a method and application of succinic acid-induced construction of a small animal model of atrial fibrillation. Background Technology

[0002] Atrial fibrillation (AF) is one of the most common cardiac arrhythmias in clinical practice, with a continuously rising global prevalence. Currently, it affects approximately 2% of the general population, and the prevalence in people over 65 years of age is as high as 5%-10%. The National Health Commission of China predicts that by 2050, the number of people with atrial fibrillation in my country will reach 9 million, becoming one of the largest epidemics and public health challenges. Since the physiological and pathological mechanisms of the occurrence, development, and maintenance of atrial fibrillation are not yet fully understood, establishing animal models of atrial fibrillation to study its mechanisms is of positive significance for precise drug-targeted therapy of atrial fibrillation. Furthermore, mediators of inflammatory responses can alter atrial electrophysiology and structural matrix, thereby increasing susceptibility to atrial fibrillation. Establishing stable atrial fibrillation inflammatory models not only helps to understand the complex pathophysiological processes and dynamic changes of atrial fibrillation-related inflammation but also helps to identify specific anti-inflammatory strategies for preventing atrial fibrillation.

[0003] Drug induction is one of the main methods for constructing animal models of atrial fibrillation. Li H et al. constructed a mouse atrial fibrillation model by subcutaneously pumping angiotensin II into mice (Li H, Feng Z, Li B, et al. GSTP1 inhibits sangiotensin II-induced atrial fibrillation by regulating ferroptosis). Europace . 2025;27(5):euaf083.). Qian Cheng established a rat model of atrial fibrillation with aseptic pericarditis by uniformly spreading sterile talc powder on the surface of the rat atrium (Qian Cheng. Blocking TRPV4 to inhibit atrial fibrosis and atrial fibrillation in a rat model of aseptic pericarditis [D]. Wuhan: Huazhong University of Science and Technology, 2017.). Chen Chunlin et al. established an SD rat model of atrial fibrillation by injecting a mixture of acetylcholine and calcium chloride into the tail vein of rats (Chen Chunlin, Gong Tiantian, Tang Yiqun et al. Establishment of an SD rat model of atrial fibrillation [J]. Experimental Animal Science, 2009, 26(3): 1-4.). CN116897885B, A method for constructing a small animal model of atrial fibrillation and its application, discloses a C57 / B6 mouse model of atrial fibrillation constructed by intraperitoneal injection of dexamethasone.

[0004] However, the above-mentioned drug induction methods have the following drawbacks: I. When angiotensin II is used to establish an animal model, the atrial fibrillation induction effect is poor and there is no stable inflammatory phenotype.

[0005] II. When talc powder or angiotensin II is used to establish an animal model, the animal needs to be subjected to a traumatic operation, which can easily cause the death of the model animal, and the modeling operation is difficult and costly.

[0006] III. The acetylcholine-calcium chloride model represents an acute and transient state, and it is difficult to simulate the key pathological characteristics such as chronic electrical remodeling, structural remodeling (such as fibrosis) and inflammatory response commonly seen in human atrial fibrillation. In addition, the injection speed and dose require precise control. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a method for constructing a small animal model of atrial fibrillation induced by succinic acid and an application thereof.

[0008] The technical solution adopted by the present application is as follows: The present application provides a method for constructing a small animal model of atrial fibrillation induced by succinic acid, which comprises using succinic acid or a pharmaceutically acceptable salt thereof as an inducer during the construction period of the animal model, and making the small animal ingest a water solution containing 4% by mass concentration of the inducer every day. After the construction period, the animal model is obtained.

[0009] Preferably, the inducer is sodium succinate.

[0010] Preferably, the ingestion of the water solution containing 4% by mass concentration of the inducer is 100-120 mL / kg / d.

[0011] Preferably, the construction period of the animal model is 4 weeks.

[0012] Preferably, the small animal is a murine, more preferably an SD rat.

[0013] In one specific embodiment of the present application, before the modeling starts, the small animal needs to be subjected to weight, electrocardiogram, echocardiogram detection, so as to select a sufficient number of animals from a large number of animals to meet the needs of the modeling group and the control group for constructing the model.

[0014] In another specific embodiment of the present application, the process of constructing the animal model further comprises detecting the weight, electrocardiogram, echocardiogram and atrial fibrillation susceptibility of the small animals in the modeling group and the control group after the modeling ends. When the detection results between the small animals in the modeling group and the control group present a significant difference, the animal model is successfully constructed.

[0015] The present application also provides an application of the method for constructing a small animal model of atrial fibrillation induced by succinic acid.

[0016] Preferably, the application can be the establishment of an animal model related to atrial fibrillation.

[0017] Preferably, the animal model can use large animals (such as sheep, dogs, rabbits, pigs, etc.), or small animals (such as rats, mice).

[0018] Compared with the prior art, the present application has the following beneficial effects: First, the succinic acid used in the present application has higher economic benefits than angiotensin II (AngII), lower modeling cost, better atrial fibrillation induction effect, and stable inflammatory characteristics.

[0019] Second, the present application uses a non-invasive drug delivery method, which causes less damage to small animals, has lower technical requirements than invasive operations, and has a higher success rate of modeling.

[0020] Third, the present application uses succinic acid to induce the construction of a small animal model of atrial fibrillation, which represents the most common type of atrial fibrillation in clinical practice, and has a wider range of research and application compared with acute and transient atrial fibrillation models. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The rat animal model construction process.

[0022] Figure 2 The results of the influence of succinic acid induction on the atrial fibrillation induction rate and duration of rats are shown in the figure; where A represents the atrial fibrillation induction probability of rats (AF induction probability), B represents the average atrial fibrillation duration of rats (AF duration); where Control is the control group, and Succinic acid is the modeling group; where ***P<0.001, **P<0.01.

[0023] Figure 3 The atrial schematic diagram of the rat heart evaluated after succinic acid induction is shown in the figure; where Control is the control group, and Succinic acid is the modeling group.

[0024] Figure 4 The rat atrial analysis diagram after succinic acid induction is shown in the figure; where A is the heart structure slice diagram after HE staining; B is the local fibrosis performance diagram after Masson staining; C is the immune cell distribution diagram after myeloperoxidase (MPO) staining; D is the collagen volume fraction analysis diagram; E is the count of positive cells, i.e. neutrophils and monocytes; where Control is the control group, and Succinic acid is the modeling group; where **P<0.01.

[0025] Figure 5 Figure 1 shows the results of q-PCR analysis of inflammatory factors in primary atrial myocytes of lactating rats in vitro. A represents the relative expression of Ccl3 (macrophage inflammatory protein 1-α), B represents the relative expression of Cxcl2 (chemokine CXC ligand 2), C represents the relative expression of IL-1β (interleukin 1β), and D represents the relative expression of IL-6 (interleukin 6). Control group represents the control group, and succinic acid group represents the model group. ***P<0.0001, ***P<0.001, **P<0.01. Detailed Implementation

[0026] Invention Concept: Succinic acid is a common natural organic acid and an important intermediate in the tricarboxylic acid cycle, participating in cellular energy metabolism. Previous studies have demonstrated that succinic acid plays a crucial role in the development and progression of heart disease. The applicant previously conducted metabolomics analysis of 305 atrial fibrillation (AF) blood samples, combined with mGWAS Mendelian randomization analysis, revealing for the first time that succinic acid is a risk factor for AF. Animal experiments confirmed the role of succinic acid in inducing AF susceptibility and atrial remodeling. After four weeks of succinic acid-fed water intake, an AF susceptibility model was induced in rats, which was subsequently induced by isolated cardiac electrical stimulation. This invention provides a stable rat AF model with easy induction and good reproducibility, offering a theoretical basis for exploring the pathological mechanisms of AF and evaluating the mechanisms and targets of drug action on AF.

[0027] The present invention will be further described below with reference to specific embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0028] It should be noted that, unless otherwise specified, the reagents and experimental methods used in this invention are all conventional reagents and experimental methods in the field.

[0029] The experimental materials, reagents, and manufacturers used in this invention are as follows: SD rats: purchased from Shanghai Slack Biotechnology Co., Ltd.

[0030] Sodium succinate granules: Product No. 6106-21-4, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0031] Example 1: Construction of a rat atrial fibrillation model according to Figure 1 The illustrated construction process constructs a rat animal model. The specific animal model construction process is as follows: 1.1 Adaptive Feeding of SD Rats Several male 6-week-old SD rats (weighing approximately 200g-250g) were randomly divided into a modeling group and a control group. After 3 days of acclimatization, the formal experiment began. Before the experiment, the rats were weighed, and echocardiography and electrocardiogram tests were performed to ensure that both groups of rats met the requirements for the modeling experiment.

[0032] 1.2. Construction of an atrial fibrillation model in SD rats Rats in the model group were given 100-120 mL / kg / day of an aqueous solution containing 4% succinic acid for 4 weeks; rats in the control group were given an aqueous solution without other substances. An atrial fibrillation model was established in SD rats after the drug administration was completed.

[0033] 1.3 Identification of the SD rat model 1.3.1 After drug administration, the susceptibility to atrial fibrillation and the duration of atrial fibrillation in the control group and the model group were measured. The measurement data were statistically analyzed, and the results are shown in [the table below]. Figure 2 .

[0034] Figure 2 The results showed that after succinic acid treatment, there were significant differences in the atrial fibrillation induction rate and duration between the model group and the control group (P<0.05), with the atrial fibrillation induction rate and duration being significantly higher in the model group than in the control group. Succinic acid induction can effectively establish a rat model of atrial fibrillation.

[0035] 1.3.2. Echocardiography was used to assess atrial size. After measurement, atrial tissue from rats in the model group and control group was collected for HE and Masson staining, and atrial structure and the proportion of fibrotic area (collagen volume fraction) were analyzed. Immune cells (including neutrophils and monocytes) were observed and counted. The results are shown in […]. Figure 3 , Figure 4 .

[0036] Figure 3 As can be seen, under echocardiographic assessment, the atria of the rats in the model group (right figure) were significantly enlarged; Figure 4 As can be seen from A, the myocardial distribution in the model group rats was more disordered; Figure 4As can be seen from Figure B, the fibrosis of the cardiomyocytes in the model group rats (red area in Figure B) is obvious (blue area in Figure B), and the proportion of fibrotic tissue is large, while the fibrosis of the cardiomyocytes in the control group rats is not obvious, and the proportion of fibrotic tissue is small. Figure 4 D is Figure 4 The analysis results of B show that the atrial fibrosis area in the model group rats was significantly higher than that in the control group rats (P<0.05). Figure 4 C indicates that the infiltration of immune cells (including neutrophils and monocytes) in the atrial tissue of the model group rats was significantly increased compared with that of the control group rats. Figure 4 E is Figure 4 The analysis results for C indicate a more severe inflammatory response (P<0.05). Figure 3 and Figure 4 The results showed that, compared with the control group rats, the atrial tissue structure, fibrosis and inflammation characteristics of the rats in the model group were more obvious after feeding them with succinic acid.

[0037] 1.3.3. Primary atrial myocardial cells from lactating rats were isolated for in vitro experiments. Atrial myocardial cells were randomly divided into two equal groups. The model group was given complete culture medium supplemented with 1 mM succinic acid, while the control group was given normal complete culture medium. Cell samples from both groups were collected after 72 hours, RNA was extracted, and the expression levels of inflammatory factor-related RNA were analyzed using PCR. The results are shown in [Figure number missing]. Figure 5 .

[0038] Figure 5 The results showed that the expression levels of inflammatory factor-related RNA in the atrial myocytes of the model group were significantly higher than those in the control group (P < 0.05). Figure 4 and Figure 5 The results are sufficient to demonstrate that succinic acid induction is an effective way to construct an inflammatory model of atrial fibrillation.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a small animal model of atrial fibrillation induced by succinic acid, characterized in that: The method includes: during the animal model construction period, using succinic acid or its pharmaceutically acceptable salt as an inducer, ingesting an aqueous solution containing 4% by mass of the inducer into small animals daily, and obtaining the animal model at the end of the modeling period.

2. The method according to claim 1, characterized in that: The inducing agent is sodium succinate.

3. The method according to claim 1, characterized in that: The intake of an aqueous solution containing a 4% mass concentration of the inducer is 100-120 mL / kg / day.

4. The method according to claim 1, characterized in that: The construction cycle is 4 weeks.

5. The method according to claim 1, characterized in that: The method also includes performing electrocardiogram and echocardiogram tests on the small animal before modeling begins.

6. The method according to claim 1, characterized in that: The method also includes performing electrocardiogram, echocardiogram, and atrial fibrillation susceptibility tests on the model group animals and the control group animals after modeling is completed; when the test results between the model group animals and the control group animals show significant differences, the animal model is successfully constructed.

7. The method according to claim 1, characterized in that: The small animals mentioned are selected from rodents.

8. The method according to claim 7, characterized in that: The selected rodent was the SD rat.

9. The application of the method as described in any one of claims 1 to 8 in constructing an animal model of atrial fibrillation.

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