Application of Alda-1 in preparation of medicine for preventing and / or treating myocardial hypertrophy

Abstract

The invention discloses application of Alda-1 in preparation of a medicine for preventing and / or treating myocardial hypertrophy, and belongs to the field of biological medicine. The invention relates to application of Alda-1 in preparation of medicines for preventing and / or treating myocardial hypertrophy and heart failure. Specifically, Alda-1 can inhibit the hypertrophy of primary myocardial cells, obviously reduce the surface area of the myocardial cells and reduce the expression of a hypertrophy gene ANF. The caudal vein injection of Alda-1 obviously inhibits myocardial hypertrophy of mice, reduces heart weight, and relieves hypertrophy-mediated cardiac dysfunction. The medicine further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an absorption carrier, a surfactant or a lubricant and the like. The invention provides a new choice for clinically preventing and treating myocardial hypertrophy and heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of Alda-1 in preparing a drug for preventing and / or treating myocardial hypertrophy. Background Art

[0002] Heart failure is a serious threat to humans, especially the elderly over 50 years old, with a high morbidity and mortality rate. Coronary heart disease, hypertension and senile degenerative valvular heart disease are the main causes of heart failure in the elderly. Due to changes in myocardial structure and function, these patients have significantly decreased ventricular pumping and filling functions. Myocardial hypertrophy is an intermediate process between ventricular remodeling and heart failure. How to prevent excessive myocardial hypertrophy to slow down the progression of heart failure is a clinical problem that needs to be solved urgently.

[0003] At present, the commonly used clinical treatment drugs focus on measures such as cardiotonic, vasodilation and diuresis, and these drugs all have limitations to varying degrees. For example, the cardiotonic drug digoxin can easily aggravate the patient's heart failure and cause serious adverse gastrointestinal reactions; ACEI-type vasodilation drugs are only suitable for patients with normal blood creatinine, blood potassium and blood pressure; and diuretics can easily cause the patient's blood to concentrate and activate the neuroendocrine system, further aggravating heart failure. Therefore, it is urgent to develop a drug that can effectively improve myocardial hypertrophy and is relatively safe. Summary of the invention

[0004] In view of the above technical problems, the present invention proposes a use of Alda-1 in preparing a drug for preventing and / or treating myocardial hypertrophy.

[0005] The technical solution adopted by the present invention is:

[0006] In one aspect, the present invention provides the use of Alda-1 in preparing a drug for preventing and / or treating myocardial hypertrophy.

[0007] Another aspect of the present invention provides the use of Alda-1 in the preparation of a drug for preventing and / or treating heart failure.

[0008] The present invention also proposes the application of Alda-1 in inhibiting primary myocardial cell hypertrophy, reducing the surface area of ​​myocardial cells and lowering the expression of hypertrophy gene ANF.

[0009] The chemical formula of Alda-1 is C 15 H 11 Cl 2 NO 3 , the molecular formula is:

[0010]

[0011] The above-mentioned medicine also includes a pharmaceutically acceptable carrier.

[0012] The above-mentioned pharmaceutically acceptable carrier is a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, an absorption carrier, a surfactant or a lubricant.

[0013] The above-mentioned medicine can be further prepared into tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

[0014] The beneficial technical effects of the present invention are as follows:

[0015] The present invention is based on the molecular mechanism of myocardial hypertrophy and targets lipid oxidation products to reveal the inhibitory effect of Alda-1 in myocardial cell hypertrophy and the therapeutic effect in myocardial hypertrophy in mice.

[0016] Alda-1 has a good effect in treating myocardial hypertrophy, as shown below:

[0017] Alda-1 inhibits the hypertrophy of primary cardiomyocytes, significantly reduces the surface area of ​​cardiomyocytes and reduces the expression of the hypertrophy gene ANF; tail vein injection of Alda-1 significantly inhibits myocardial hypertrophy in mice, reduces heart weight, and alleviates hypertrophy-mediated cardiac dysfunction.

[0018] The drug Alda-1 provided by the invention will provide a new option for clinical prevention and treatment of myocardial hypertrophy and heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the analysis of the surface area of ​​cardiomyocytes by phalloidin staining; A is a representative diagram, and B is a statistical diagram of the area of ​​cardiomyocytes;

[0020] Figure 2 Schematic diagram of PCR detection of ANF mRNA expression;

[0021] Figure 3 Schematic diagram of cardiac function detection by echocardiography in mice; A is a representative diagram of echocardiography, B is a statistical diagram of left ventricular posterior wall thickness (LVPWd) during diastole, C is a statistical diagram of left ventricular posterior wall thickness (LVPWs) during systole, and D is a statistical diagram of left ventricular mass (LVd Mass);

[0022] Figure 4 Schematic diagram of mouse heart mass analysis; wherein A shows the appearance of the heart, and B is a statistical graph of heart weight / body weight. DETAILED DESCRIPTION

[0023] Oxidative stress is a key cause of myocardial cell hypertrophy. The imbalance of oxidation and antioxidant systems in myocardial cells leads to the accumulation of lipid oxidation products, which can act as stimuli to induce the expression of fetal genes such as ANF and BNP in myocardial cells, thereby inducing an increase in cell volume and weight. Aldehyde dehydrogenase 2 (ALDH2) is an important antioxidant enzyme in cells, catalyzing the conversion of acetaldehyde into acetic acid and removing active aldehydes produced by lipid peroxidation. Alda-1 is a selective ALDH2 agonist that maintains cell homeostasis by increasing ALDH2 activity. Up to now, there are no reports on targeted antioxidants and anti-myocardial hypertrophy drugs related to the inhibition of lipid oxidation products. In order to meet the actual needs of clinical prevention and treatment of heart failure, the present invention is based on the mechanism of myocardial hypertrophy, takes the lipid oxidation products accumulated in myocardial cells as the target, and reveals the potential role of Alda-1 in the preparation of drugs for the prevention and / or treatment of myocardial hypertrophy.

[0024] In vitro and in vivo experiments have shown that Alda-1 can inhibit cardiomyocyte hypertrophy and improve cardiac function. The specific plan is as follows:

[0025] 1. The steps for extracting primary cardiomyocytes are as follows:

[0026] 20 SD rats within two days of birth were washed twice with 75% ethanol, each time for 5 seconds. Use sterile ophthalmic scissors to cut along the right side of the sternum, use sterile forceps to remove the heart, quickly place it in pre-cooled phosphate buffer, and wash it three times to remove blood cells and other impurities. Use sterile ophthalmic scissors to cut the heart into tissue blocks of approximately 0.5mm×0.5mm×0.5mm, and put it in a conical flask. Add 6mL of protease digestion solution to the flask, place it in a 37℃ water bath, and digest while shaking at a speed of 90-100r·min. -1 After 6 minutes of digestion, the supernatant was aspirated into a 50 mL centrifuge tube containing 5 mL of serum. The remaining tissues were digested multiple times, with 4 mL of protease digestion solution added each time, and each digestion lasted 2-3 minutes. The supernatant was collected after digestion until all tissues were completely digested. All supernatants were centrifuged at 1200 r·min -1 Centrifuge for 13 minutes, discard the supernatant, and gently blow off the precipitate with 20 mL of culture medium. Filter the cell suspension using a 200-mesh steel mesh, place the cell suspension in a culture dish for differential attachment, and take the cell suspension (cardiomyocytes) after 90 minutes at 1000 r / min. -1 Centrifuge for 6 minutes, and gently disperse the precipitate with culture medium. Adjust the cell density as needed for the next experiment.

[0027] 2. Analysis of cardiomyocyte surface area:

[0028] (1) Experimental procedure: Sterile glass slides were placed in a 24-well plate in advance, and the primary cardiomyocyte suspension was added to the 24-well plate (5000 / well), and the plate was placed in an incubator. After the cells adhered to the wall, the cells were treated with drugs. In order to induce lipid peroxidation in cardiomyocytes, RSL3 (an inhibitor of the antioxidant enzyme GPX4) was used to stimulate cardiomyocytes, and the final concentration of RSL3 was 0.125 μM. At the same time, an RSL3 and Alda-1 co-treatment group was set up, that is, 0.125 μM RSL3 and 0.05 μM Alda-1 were used to treat cardiomyocytes at the same time. After 24 hours of treatment, surface area detection and analysis were performed.

[0029] (2) Cell surface area detection and analysis: The cell culture supernatant in the well plate was discarded, the cells were fixed with 4% paraformaldehyde for 10 min, the paraformaldehyde was discarded, and the cells were washed once with phosphate buffer. 150 μL of phenotype-dependent cyclin working solution was added to each well and the cardiomyocytes were incubated for 30 min. The phenotype-dependent cyclin was discarded, the slide was taken out, and it was inverted on the mounting medium containing DAPI. The cell surface area was observed under a fluorescence microscope.

[0030] Figure 1 The figure is a schematic diagram of the analysis of the surface area of ​​cardiomyocytes by phalloidin staining; A is a representative figure and B is a statistical figure of the cardiomyocyte area. The figure compares and analyzes the inhibition of Alda-1 on the increase of cardiomyocyte surface area. Figure 1 As shown in Figure A, compared with the Control group, RSL3 treatment induced an increase in the surface area of ​​cardiomyocytes, while Alda-1 treatment inhibited the increase in the surface area. Figure 1 Center B reveals that the differences among the groups were statistically significant (*, p<0.05; **, p<0.01).

[0031] 3. Effect of Alda-1 on the expression of hypertrophy gene ANF:

[0032] Experimental operation: Take the primary cardiomyocyte suspension and add it to a 6-well plate (20,000 / well). After the cells adhere to the wall, they are treated with drugs. In order to induce lipid peroxidation in cardiomyocytes, RSL3 is used to stimulate cardiomyocytes, and the final concentration of RSL3 is 0.125μM. At the same time, RSL3 and Alda-1 co-treatment groups are set up, that is, 0.125μM RSL3 and 0.05μM Alda-1 are used to treat cardiomyocytes at the same time. After 24h of treatment, the expression of ANF is detected.

[0033] ANF ​​expression analysis: ① Extract RNA. Add 1mL TRIzol reagent to the well plate and transfer the cell lysate to a 1.5mL RNase-free EP tube. Let stand on ice for 5min. Add 200μL chloroform to each tube, mix thoroughly and centrifuge at 13000rpm for 15min at 4℃. Transfer the upper layer to a new EP tube and add an equal volume of isopropanol. Let stand on ice for 10min. Centrifuge at 13000rpm for 10min. Remove the supernatant. Wash the RNA precipitate once with 1ml 75% ethanol. Centrifuge at 12000rpm for 5min. Remove the supernatant and dissolve the RNA precipitate in 30-50μl deionized water. ② Reverse transcription. Take a 0.2mL EP tube, put in 1μg RNA sample, add 4μL gDNA wiper mix, and make up the system to 20μl with enzyme-free water. Heat at 42℃ for 2min. Add 4μL HisScript II super mix II, heat at 50℃ and 85℃ for 15min and 15s respectively to obtain cDNA. ③ Fluorescence quantitative PCR. Take 1μL cDNA, 0.5μL ANF upstream and downstream primers, 10μL mix and 8μL enzyme-free water and add them to PCR tubes respectively. Perform PCR amplification.

[0034] Figure 2 Schematic diagram of PCR detection of ANF mRNA expression. The figure shows the comparison between the control group, RSL3 treatment group and RSL3+Alda-1 treatment group, and analyzes the inhibition of ANF expression in cardiomyocytes by Alda-1. Figure 2 It can be seen that compared with the Control group, RSL3 treatment induced high expression of ANF in cardiomyocytes, while Alda-1 co-treatment inhibited its expression, and the difference was statistically significant (*, p<0.05; **, p<0.01).

[0035] 4. Construction of mouse aortic arch constriction (TAC) model: Mice were anesthetized with 0.3% sodium pentobarbital (40 mg / kg) and fixed in a supine position. The skin of the neck and chest was cut open, an incision was made at the second intercostal space at the edge of the left upper sternum, a chest retractor was inserted, the wound was widened, the thymus was separated, and the aortic arch and branches were exposed. Use a 6-0 line to thread the aortic arch, and after tying a knot, place a 26G pad needle next to the aorta and tie it tightly. Then remove the needle to form a lumen of aortic stenosis. The sham group was threaded without ligation. The wound was sutured with 4-0 sutures, and postoperative care was performed. After the TAC model was constructed in mice, Alda-1 (25 mg / kg, 150 μL / time) was injected into the tail vein once every 3 days for a total of 7 times. Analysis and testing were performed in the third week.

[0036] 5. Effect of Alda-1 on TAC-induced cardiac dysfunction:

[0037] (1) Experimental procedures: Mice were anesthetized with 0.3% sodium pentobarbital (40 mg / kg) and fixed in a supine position. A small animal ultrasound instrument was connected to detect cardiac function and record cardiac parameters, such as left ventricular ejection fraction (LVEF), left atrial diameter (LAD), left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), and interventricular septum thickness (IVS).

[0038] (2) Experimental results: Figure 3 Schematic diagram of cardiac function detection in mice by echocardiography; A is a representative diagram of echocardiography, B is a statistical diagram of left ventricular posterior wall thickness (LVPWd) during diastole, C is a statistical diagram of left ventricular posterior wall thickness (LVPWs) during systole, and D is a statistical diagram of left ventricular mass (LVd Mass); Alda-1 alleviates the abnormal cardiac function of TAC mice. Figure 3 It can be seen that compared with the sham group, the cardiac function of the TAC model group was abnormal, such as the left ventricular posterior wall thickness at diastole (LVPWd), the left ventricular posterior wall thickness at systole (LVPWs), and the left ventricular mass (LVd Mass) increased significantly, while Alda-1 treatment inhibited the increase of the left ventricular posterior wall thickness and reduced the left ventricular mass. The difference was statistically significant (*, p<0.05; **, p<0.01).

[0039] 6. Effects of Alda-1 on TAC-induced heart size and weight:

[0040] (1) Experimental procedure: Mice were anesthetized with 0.3% sodium pentobarbital (40 mg / kg), weighed first, and then fixed in a supine position. The sternum was cut open, the mouse heart was taken out, and the blood stains were removed. The heart weight was weighed.

[0041] (2) Experimental results: Figure 4 Schematic diagram of mouse heart mass analysis; wherein A shows the appearance of the heart, and B is a statistical graph of heart weight / body weight. Figure 4 The volume and weight of the hearts antagonized by Alda-1 treatment were analyzed. Figure 4 It can be seen that compared with the sham group, the heart size and weight of the TAC model group increased significantly, while Alda-1 treatment antagonized the increase in heart size and weight. The difference was statistically significant (**, p<0.01; ****, p<0.0001).

[0042] In summary, Alda-1 can inhibit the hypertrophy of primary cardiomyocytes, significantly reduce the surface area of ​​cardiomyocytes and reduce the expression of hypertrophy gene ANF. Tail vein injection of Alda-1 significantly inhibits myocardial hypertrophy in mice, reduces heart weight, and alleviates hypertrophy-mediated cardiac dysfunction. The drug Alda-1 provided by the present invention will provide a new option for the clinical prevention and treatment of myocardial hypertrophy and heart failure.

Claims

1. Application of Alda-1 in the preparation of drugs for preventing and / or treating myocardial hypertrophy.

2. Use of Alda-1 in the preparation of drugs for preventing and / or treating heart failure.

3. Application of Alda-1 in inhibiting primary cardiomyocyte hypertrophy, reducing cardiomyocyte surface area and lowering the expression of hypertrophy gene ANF.

4. The use according to claim 1 or 2, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable carrier is a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, an absorption carrier, a surfactant or a lubricant.

6. The use according to claim 4, characterized in that: The medicine is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

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