Establishment method of heart failure mouse model with supernormal ejection fraction

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a method for establishing a heart failure mouse model with an exceptional ejection fraction, which adopts an adenine-containing diet to continuously feed a mouse for 25-30 days. A C57BL / 6J mouse is continuously fed for 25-30 days by adopting adenine-containing diet, the heart function of the mouse is detected by using a vevo2100 small animal ultrasonic image system, and the phenomena of ejection fraction increase, cardiac output reduction, left ventricular diameter in systolic and diastolic periods, left ventricular volume reduction in systolic and diastolic periods, slight heart fibrosis and the like of the mouse are found. It is proved that adenine diet can induce the heart failure with the supernormal left ventricular ejection fraction of the mouse, and a feasible direction is provided for research of the heart failure.

Description

Technical Field

[0001] The invention relates to a method for establishing a heart failure mouse model with abnormal ejection fraction, and belongs to the technical field of animal model construction. Background Art

[0002] The prevalence of heart failure (HF) is a global problem, and the number of HF patients has been increasing rapidly in recent years. HF is defined as a clinical syndrome whose symptoms and / or signs are caused by structural and / or functional cardiac abnormalities and confirmed by elevated natriuretic peptide levels and / or objective evidence of pulmonary or systemic congestion. In 2021, a universal definition and classification of ejection fraction (EF) was proposed. Ejection fraction refers to the percentage of stroke volume to ventricular end-diastolic volume (i.e., cardiac preload). It is one of the important indicators for determining the type of heart failure. The calculation formula is: EF = (EDV-ES) × 100% / EDV, where EF is the ejection fraction; EDV is the ventricular end-diastolic volume; ES is the ventricular end-systolic volume. In daily practice, heart failure is usually classified according to the left ventricular ejection fraction (LVEF). The definition of normal LVEF varies according to guidelines or medical / scientific societies. For example, the British Society of Echocardiography defines a normal LVEF as ≥55%. The American Society of Echocardiography and the European Society of Cardiovascular Imaging define a normal LVEF as 52% to 72% in men and 54% to 74% in women.

[0003] In recent years, the definition of HF has been revised from "HF with reduced EF, preserved EF, and intermediate (or borderline) EF" to "HF with reduced EF, preserved EF, and mildly reduced EF", and HF with improved EF has been introduced. The definitions of this classification are as follows: (1) HF with reduced EF (HFrEF): HF with LVEF ≤ 40%; (2) HF with mildly reduced EF (HFmrEF): LVEF 41% to 49%; (3) HF with preserved EF (HFpEF): HF with LVEF ≥ 50%; (4) HF with improved EF: HF with baseline LVEF ≤ 40%, LVEF increased by ≥ 10 points from baseline, and LVEF > 40% at the second measurement.

[0004] A new type of heart failure, HF with supra-normal ejection fraction (HFsnEF), has recently been proposed, which is characterized by a supra-normal LVEF (LVEF>65%). Some studies have reported that patients with supra-normal LVEF may have an increased risk of long-term major adverse cardiovascular events and a U-shaped mortality pattern. Currently, the prognosis of HFsnEF is not fully determined, but it seems to be associated with an increased risk of long-term major adverse cardiovascular events. Patients with LVEF>60% have reportedly smaller ventricular internal diameters (increased relative wall thickness, smaller left ventricular end-diastolic volume, and smaller left ventricular end-systolic volume). Patients with higher LVEF have a lower stroke volume index (the stroke volume index is the ratio of stroke volume (SV) to body surface area (BSA), expressed in mL / m 2 ), which is usually associated with smaller heart chambers and left ventricular concentricity due to shortening and thickening of the chiasmatic fibers and increased contraction rate of myocytes at the endocardial level, thereby increasing the ejection fraction. These structural changes are associated with abnormal left ventricular diastolic function and impaired diastolic function.

[0005] Heart failure with abnormal ejection fraction is a newly discovered type of heart failure. There is no animal model that can be used for basic research. Therefore, how to establish an animal model of heart failure with abnormal ejection fraction is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0006] Aiming at the fact that there is no animal model available in the prior art for studying heart failure with abnormal ejection fraction, the present invention provides a method for establishing a heart failure mouse model with abnormal ejection fraction.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: a method for establishing a heart failure mouse model with abnormal ejection fraction, using an adenine-containing diet to continuously feed the mice for 25 to 30 days.

[0008] On the basis of the above technical solution, the present invention can also make the following improvements:

[0009] Furthermore, the adenine-containing diet is an animal feed containing adenine.

[0010] Furthermore, in the animal feed containing adenine, the mass fraction of adenine is 0.15%-0.20%.

[0011] Furthermore, in the animal feed containing adenine, the mass fraction of adenine is 0.20%.

[0012] Furthermore, 5 g to 10 g of animal feed containing adenine is fed daily.

[0013] Furthermore, the feeding days are 28 days.

[0014] Furthermore, the animal feed is rodent feed.

[0015] Further, the rodent feed comprises:

[0016] 20% casein;

[0017] 39.75% corn starch;

[0018] 13.2% corn dextrin;

[0019] 10% sucrose;

[0020] 5% cellulose;

[0021] 7% soybean oil;

[0022] 3.5% complex minerals;

[0023] 1% multivitamin.

[0024] Furthermore, the mouse is a C57BL / 6J wild-type male mouse.

[0025] The beneficial effects of the present invention are as follows: the present invention adopts adenine-containing diet to continuously feed C57BL / 6J mice for 25 to 30 days, and uses the vevo2100 small animal ultrasound imaging system to detect the cardiac function of the mice. It is found that compared with the control group, the adenine-fed mice have an increased ejection fraction (increased from 45.6702±8.90 to 71.94±8.56), a decreased cardiac output (decreased from 13307.5±9109.55 to 2994.34±1188.75), and a decreased left ventricular internal diameter during systole (decreased from 2.97±0 .22 decreased to 1.68±0.33), diastolic left ventricular internal diameter decreased (from 3.87±2.77 to 0.17±0.24), systolic left ventricular volume decreased (from 34.04±6.41 to 5.79±2.96), diastolic left ventricular volume decreased (from 62.73±28.02 to 7.52±5.48), and mild cardiac fibrosis, which prove that adenine diet can induce heart failure with abnormal left ventricular ejection fraction in mice, providing a practical direction for the study of heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The cardiac M-mode ultrasound images of mice in the embodiment and the comparative example were obtained by using the vevo2100 small animal ultrasound imaging system to detect the cardiac M-mode ultrasound images of mice in the embodiment and the comparative example;

[0027] Figure 2 The ejection fraction is calculated based on the test results of the vevo2100 small animal ultrasound imaging system;

[0028] Figure 3The cardiac output is calculated based on the test results of the vevo2100 small animal ultrasound imaging system;

[0029] Figure 4 The changes of the systolic and diastolic interventricular septum thickness, systolic and diastolic left ventricular internal diameter, and systolic and diastolic left ventricular volume in the mice of the embodiment after feeding for 29 days;

[0030] Figure 5 The photos of the heart tissues of mice after Masson staining in the embodiment and comparative example;

[0031] Figure 6 The following is the body weight change of mice in Example. DETAILED DESCRIPTION

[0032] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0033] Embodiment and comparative example:

[0034] (1) Animal preparation:

[0035] Eight-week-old C57BL / 6J wild-type male mice weighing 18 g to 20 g were selected from Weitonglihua Company and raised in the animal room of Beijing Shijitan Hospital with free access to sterile food. The temperature of the breeding environment was 22°C to 25°C and the humidity was 40% to 60%.

[0036] (2) Group modeling:

[0037] The mice were divided into a control group (comparative example) and a model group (example), with 8 mice in each group.

[0038] The mice in the control group were fed with normal rodent diet for 28 days.

[0039] The mice in the model group were fed with a feed containing 0.2% adenine synthesized by Medison for 28 days. Adenine was purchased from Sigma, USA.

[0040] (3) Index detection:

[0041] Use the vevo2100 small animal ultrasound imaging system to detect mouse cardiac function and collect cardiac images. Figure 1, This figure mainly shows the ventricular wave group of the hearts of mice in each group (which can show the movement curves of the ventricular wall and ventricular septum). By measuring the amplitude, slope and other parameters of the curve, the ventricular wall thickness, ventricular cavity size, EF, FS and other cardiac function indicators can be accurately calculated, which is an important basis for quantitative evaluation of cardiac function; according to the test results, the ejection fraction, cardiac output, ventricular septal thickness during systole (IVS; s), interventricular septal thickness during diastole (IVS; d), left ventricular diameter during systole (LVID; s), left ventricular diameter during systole (LVID; d), left ventricular volume during systole (Lv vol; s), and left ventricular volume during systole (Lvvol; d) were calculated.

[0042] (4) Masson staining to observe cardiac fibrosis in mice:

[0043] The paraffin sections were dewaxed and hydrated, then dyed with mordant at 37°C for 1 hour, rinsed with tap water until the yellow color disappeared; dyed with lapis lazuli blue solution for 5 minutes, rinsed with tap water, dyed with fuchsin solution for 10 minutes, and rinsed with tap water; rinsed quickly in 70%, 80%, 90%, and 95% alcohol for 30 seconds in sequence, and placed in 100% alcohol to terminate the color separation; dehydrated with 100% alcohol 3 times, 5 minutes each time; made transparent with xylene 2 times, 5 minutes each time; sealed with neutral gum, observed the results under an optical microscope and took pictures.

[0044] Test result analysis:

[0045] The weight of mice was measured on day 29, and it was found that the weight of mice in the model group was significantly reduced (see Figure 6 ).

[0046] Cardiac function tests revealed that the model group mice had significant myocardial hypertrophy and stenosis, increased ejection fraction, and decreased cardiac output (see Figure 1-3 ), increased systolic and diastolic ventricular septal thickness, decreased systolic and diastolic left ventricular internal diameters, and decreased systolic and diastolic left ventricular volumes (see Figure 4 ),in, Figure 2-Figure 4The results are expressed as mean ± standard deviation, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0047] Masson staining revealed that the heart of the model group mice had mild fibrosis (see Figure 5 ). The embodiment of the present invention illustrates that the heart failure model with abnormal ejection fraction is successfully constructed, and this model provides a practical research method for studying the pathogenesis of human heart failure with abnormal ejection fraction and exploring new treatment methods.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a mouse model of heart failure with abnormal ejection fraction, characterized in that: The mice were fed with an adenine-containing diet for 25 to 30 days.

2. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 1, characterized in that: The adenine-containing diet is an animal feed containing adenine.

3. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 2, characterized in that: In the animal feed containing adenine, the mass fraction of adenine is 0.15%-0.20%.

4. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 3, characterized in that: In the animal feed containing adenine, the mass fraction of adenine is 0.20%.

5. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 4, characterized in that: Feed animal feed containing adenine 5g-10g per day.

6. The method for establishing a heart failure mouse model with abnormal ejection fraction according to any one of claims 1 to 5, characterized in that: The feeding days are 28 days.

7. The method for establishing a heart failure mouse model with abnormal ejection fraction according to any one of claims 2 to 5, characterized in that: The animal feed is rodent feed.

8. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 7, characterized in that: The rodent feed comprises, by mass fraction, 20% casein, 39.75% corn starch, 13.2% corn dextrin, 10% sucrose, 5% cellulose, 7% soybean oil, 3.5% complex minerals, and 1% complex vitamins.

9. The method for establishing a heart failure mouse model with abnormal ejection fraction according to claim 6, characterized in that: The mice are C57BL / 6J wild-type male mice.

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