Application of galangin-3-methyl ether in preparation of medicine for treating or preventing pathological cardiac hypertrophy

Abstract

The invention provides an application of galangin-3-methyl ether (G3-ME) in preparation of a medicine for treating or preventing pathological cardiac hypertrophy, a pharmaceutical composition of the galangin-3-methyl ether (G3-ME) and a treatment or prevention method of the galangin-3-methyl ether (G3-ME). The pathological cardiac hypertrophy comprises types caused by pressure load, hereditary or inflammatory factors. The active ingredient of the pharmaceutical composition is galangin 3-methyl ether, and the administration dosage is preferably 20-50 mg / kg. Through in-vitro and in-vivo researches, the application value of G3-ME in the medicine for preventing or treating pathological cardiac hypertrophy is disclosed for the first time, and a new way and means are provided for treatment of related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of galangin-3-methyl ether in preparing a drug for treating or preventing pathological myocardial hypertrophy. Background Art

[0002] The heart usually has a strong compensatory capacity. Physiological compensatory myocardial hypertrophy is the body's adaptive response to specific physiological stimuli and has no direct association with cardiac dysfunction. Pathological myocardial hypertrophy, on the other hand, is a structural change in the heart caused by disease or pathological conditions, often accompanied by risks such as arrhythmias and even heart failure. From a mechanical perspective, pathological myocardial hypertrophy can be secondary to a variety of hypertrophic stimuli and is the main clinical manifestation of patients with hereditary hypertrophic cardiomyopathy (HCM). Currently, there is no effective means to cure myocardial hypertrophy (especially HCM) in clinical practice. Therefore, exploring effective targets or therapeutic drugs to inhibit myocardial hypertrophy HCM has significant clinical therapeutic value.

[0003] Galangin 3-methyl ether (G3-ME) is a natural flavonoid extracted from the rhizome of Alpinia officinalis (Alpinia galanga). It is one of the main medicinal components of the traditional Chinese medicine Alpinia officinalis (Alpinia galanga). Its chemical formula is C16H12O5. Although galangin has been found to protect against myocardial ischemia-reperfusion injury, cardiac inflammation, and doxorubicin-induced cardiotoxicity, its mechanism of action and efficacy differ from those of its methylated derivative (G3-ME). Furthermore, the role of G3-ME in heart disease, particularly pathological myocardial hypertrophy, remains unclear. Currently, no studies have demonstrated the use of G3-ME in the treatment of any type of myocardial hypertrophy. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a use of galangin-3-methyl ether in the preparation of a drug for preventing or treating pathological myocardial hypertrophy.

[0005] The second aspect of the present invention aims to provide a pharmaceutical composition for treating or preventing pathological myocardial hypertrophy.

[0006] The third aspect of the present invention aims to provide a method for preventing or treating pathological myocardial hypertrophy.

[0007] To achieve the above purpose, the present invention is implemented through the following technical solutions

[0008] The first aspect of the present invention provides the use of galangin-3-methyl ether in the preparation of a drug for treating or preventing pathological myocardial hypertrophy.

[0009] In some embodiments of the present invention, the pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure load, hereditary myocardial hypertrophy, and myocardial hypertrophy caused by inflammatory factors.

[0010] In some embodiments of the present invention, the pathological myocardial hypertrophy includes myocardial hypertrophy caused by pressure load; in some embodiments of the present invention, the pathological myocardial hypertrophy includes hereditary myocardial hypertrophy; in some embodiments of the present invention, the pathological myocardial hypertrophy includes myocardial hypertrophy caused by inflammatory factors; in some embodiments of the present invention, the pathological myocardial hypertrophy includes myocardial hypertrophy caused by pressure load and hereditary myocardial hypertrophy; in some embodiments of the present invention, the pathological myocardial hypertrophy includes myocardial hypertrophy caused by pressure load and myocardial hypertrophy caused by inflammatory factors; in some embodiments of the present invention, the pathological myocardial hypertrophy includes hereditary myocardial hypertrophy and myocardial hypertrophy caused by inflammatory factors; in some embodiments of the present invention, the pathological myocardial hypertrophy includes myocardial hypertrophy caused by pressure load, hereditary myocardial hypertrophy and myocardial hypertrophy caused by inflammatory factors.

[0011] In some embodiments of the present invention, the pathological myocardial hypertrophy is manifested by any one or more of the following: increased myocardial cell area, increased heart or left ventricular weight, myocardial fibrosis, and increased expression of myocardial hypertrophy markers.

[0012] In some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the area of ​​myocardial cells; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the weight of the heart or left ventricle; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as myocardial fibrosis; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the expression of myocardial hypertrophy markers; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the area of ​​myocardial cells and an increase in the weight of the heart or left ventricle; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the area of ​​myocardial cells and myocardial fibrosis; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the area of ​​myocardial cells and an increase in the expression of myocardial hypertrophy markers; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as an increase in the weight of the heart or left ventricle and myocardial fibrosis. ; In some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as increased heart or left ventricular weight and increased expression of myocardial hypertrophy markers; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as myocardial fibrosis and increased expression of myocardial hypertrophy markers; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as increased myocardial cell area, increased heart or left ventricular weight and myocardial fibrosis; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as increased myocardial cell area, increased heart or left ventricular weight and increased expression of myocardial hypertrophy markers; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as increased myocardial cell area, increased heart or left ventricular weight and myocardial fibrosis; in some embodiments of the present invention, the symptoms of pathological myocardial hypertrophy are manifested as increased myocardial cell area, increased heart or left ventricular weight, myocardial fibrosis and increased expression of myocardial hypertrophy markers.

[0013] In some embodiments of the present invention, the cardiac hypertrophy markers include Nppa and / or Nppb.

[0014] In some embodiments of the present invention, the cardiac hypertrophy marker is Nppa; in some embodiments of the present invention, the cardiac hypertrophy marker is Nppb; in some embodiments of the present invention, the cardiac hypertrophy marker includes Nppa and Nppb.

[0015] In some embodiments of the present invention, the hereditary cardiac hypertrophy is caused by the Myh6 gene R404Q mutation or the Tnnt2 gene R109Q mutation;

[0016] In some embodiments of the present invention, the hereditary myocardial hypertrophy is caused by the Myh6 gene R404Q mutation; in some embodiments of the present invention, the hereditary myocardial hypertrophy is caused by the Tnnt2 gene R109Q mutation;

[0017] In some embodiments of the present invention, by administering G3-ME orally to the above two model mice, the symptoms of hereditary pathological myocardial hypertrophy can be significantly improved.

[0018] In some embodiments of the present invention, the galangin-3-methyl ether acts by inhibiting the hypertrophic phenotype of cardiomyocytes.

[0019] A second aspect of the present invention provides a pharmaceutical composition for treating or preventing pathological myocardial hypertrophy, wherein the active ingredient of the pharmaceutical composition is galangin 3-methyl ether.

[0020] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0021] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches.

[0022] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is a capsule; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a powder; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a tablet; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a granule; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a pill; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is an injection; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a syrup; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is an oral agent; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is an inhalant; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is an ointment; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a suppository; in some embodiments of the present invention, the dosage form of the pharmaceutical composition is a patch.

[0023] In some embodiments of the present invention, the dosage of galangin 3-methyl ether is 10-80 mg / kg; preferably, the dosage of galangin 3-methyl ether is 20-50 mg / kg.

[0024] In some embodiments of the present invention, the dosage of galangin 3-methyl ether is 10 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 20 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 30 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 40 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 50 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 60 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 70 mg / kg; in some embodiments of the present invention, the dosage of galangin 3-methyl ether is 80 mg / kg.

[0025] In some embodiments of the present invention, the pharmaceutical composition is used in the preparation of a drug for preventing or treating pathological myocardial hypertrophy and diseases caused by it.

[0026] A third aspect of the present invention provides a method for preventing or treating pathological cardiac hypertrophy, comprising administering a therapeutically effective amount of galangin-3-methyl ether to a patient in need thereof.

[0027] In some embodiments of the present invention, the dosage of galangin-3-methyl ether administered in the method is 10-80 mg / kg; preferably, the dosage of galangin-3-methyl ether administered in the method is 20-50 mg / kg.

[0028] In some embodiments of the present invention, the dosage of galangin-3-methyl ether administered in the method is 20 mg / kg; in some embodiments of the present invention, the dosage of galangin-3-methyl ether administered in the method is 50 mg / kg.

[0029] In some embodiments of the present invention, the pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure load, hereditary myocardial hypertrophy, and myocardial hypertrophy caused by inflammatory factors.

[0030] In some embodiments of the present invention, the hereditary cardiac hypertrophy is caused by the Myh6 gene R404Q mutation or the Tnnt2 gene R109Q mutation.

[0031] Beneficial effects:

[0032] The present invention not only uses animal-derived cell models to conduct in vitro studies on G3-ME, but also constructs an in vitro human cardiomyocyte hypertrophy model. After direct in vitro treatment with G3-ME, the model can significantly inhibit the hypertrophic phenotype of human embryonic stem cell-derived cardiomyocytes, significantly reduce the area of ​​cardiomyocytes, and reduce the expression level of myocardial hypertrophy markers. In in vivo experiments, oral administration of G3-ME can reduce Myh6 R404Q Mice and Tnnt2 R109Q The study also found that G3-ME reduced left ventricular and cardiac weight in mice, reduced cardiac volume and diastolic / systolic myocardial wall thickness, and effectively inhibited myocardial fibrosis. Through in vitro and in vivo studies, the present invention reveals for the first time the value of G3-ME in the prevention or treatment of pathological myocardial hypertrophy, providing a new approach and method for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :After direct treatment with different concentrations of G3-ME in vitro, rat myocardial cell line H9c2 ( Figure 1 a and b) and cardiomyocytes differentiated from human MYL2 Neo / w-H7 cell line ( Figure 1 c) Expression levels of cardiac hypertrophy markers Nppa and Nppb; * and # indicate p < 0.05.

[0034] Figure 2 :After direct treatment with G3-ME in vitro, rat myocardial cell line H9c2 ( Figure 2 a) and cardiomyocytes differentiated from human MYL2Neo / w-H7 cell line ( Figure 2 b) Cell area; * indicates p < 0.05.

[0035] Figure 3 :Use the drug G3-ME to inhibit Myh6 R404Q After oral administration of Myh6 R404Q Mouse cardiac ultrasound test results, including mouse M-mode and Doppler mode cardiac ultrasound images ( Figure 3 a) E / A peak ratio ( Figure 3 b) Left ventricular mass ( Figure 3 c) left ventricular anterior wall thickness during systole ( Figure 3 d) Diastolic left ventricular anterior wall thickness ( Figure 3 e) Left ventricular posterior wall thickness during systole ( Figure 3 f), left ventricular posterior wall thickness during diastole ( Figure 3 g), ejection fraction ( Figure 3 h); in the figure, * indicates p<0.05.

[0036] Figure 4 :Use the drug G3-ME to inhibit Tnnt2 R109QTnnt2 R109Q Mouse cardiac ultrasound test results, including mouse M-mode and Doppler mode cardiac ultrasound images ( Figure 4 a) Left ventricular anterior wall thickness during systole ( Figure 4 b) Diastolic left ventricular anterior wall thickness ( Figure 4 c) left ventricular posterior wall thickness during systole ( Figure 4 d) Diastolic left ventricular posterior wall thickness ( Figure 4 e); In the figure, * indicates p<0.05.

[0037] Figure 5 :Use the drug G3-ME to inhibit Myh6 R404Q Mice and Tnnt2 R109Q After oral administration to mice, Myh6 R404Q Nppa (a marker of myocardial hypertrophy in mouse models) Figure 5 a), Nppb( Figure 5 b) Expression levels of Tnnt2 R109Q Nppa (a marker of myocardial hypertrophy in mouse models) Figure 5 c) Nppb( Figure 5 d) Expression levels. In the figure, * indicates p < 0.05.

[0038] Figure 6 :Myh6 R404Q Mouse ( Figure 6 a) and Tnnt2 R109Q ( Figure 6 b) H&E, Masson, and WGA staining results of mouse myocardial tissue.

[0039] Specific implementation (embodiment)

[0040] In order to enable those skilled in the art to better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0041] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products or apparatuses.

[0042] In the present invention, 8-week-old wild-type mice (WT) and Myh6 gene R404Q point mutation mice (Myh6 R404Q ) and Tnnt2 gene R109Q point mutation mice (Tnnt2 R109Q ) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., among which the Myh6 gene R404Q point mutation mice (Myh6 R404Q ) are C57BL / 6JGpt-Myh6 em1Cin(R404Q) / Gpt, Myh6-p.R404Q,, Tnnt2 gene R109Q point mutation mice (Tnnt2 R109Q ) is C57BL / 6JGpt-Tnnt2em1Cin(R109Q) / Gpt, Tnnt2-p.R109Q.

[0043] G3-ME compound was purchased from MedChemExpress (HY-N4167, CAS No.: 6665-74-3).

[0044] Rat cardiomyocyte cell line H9c2 cells were purchased from ATCC cell bank (number CRL-1446). They were derived from embryonic rat heart tissue and were cultured in vitro to obtain an immortalized cell line with cardiomyocyte characteristics.

[0045] The human embryonic stem cell line MYL2 Neo / w-H7, wherein H7 cells were purchased from the cell bank #WA07 of WiCell Institute, and the stable human embryonic stem cell line MYL2Neo / w-H7 was obtained after editing using genome-directed modification technology (the construction method refers to LianX, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci US A. 2012; 109(27): E1848-E1857. doi: 0.1073 / pnas.1200250109; or see paragraph 0037 of the specification of patent CN108265029A for details: Editing human pluripotent stem cells (including the genome of human embryonic stem cells, introducing MYL2 driven Neo into the genome of human pluripotent stem cells and replacing the corresponding normal genes and Example 1).

[0046] Agonist CHIR-99021 (purchased from MedChemExpress, #HY-10182, CAS No.: 252917-06-9) and pathway inhibitor IWR-1 (purchased from MedChemExpress, #HY-12238, CAS No.: 1127442-82-3).

[0047] RPM1-1640 / B27-no insulin is an RPM1-1640 (Gibco) medium to which B-27 (Thermo Fisher Scientific, Cat# A1895601) without insulin is added, wherein the content of B-27 is 2%.

[0048] The following are some definitions used in this invention:

[0049] In the present invention, "cardiac hypertrophy markers" refer to biomolecules that are abnormally expressed during pathological myocardial hypertrophy and can be used to assess the occurrence, development, and treatment effects of myocardial hypertrophy. In the present invention, the markers include but are not limited to:

[0050] Nppa (atrial natriuretic peptide, ANP): secreted by atrial myocytes and upregulated in myocardial hypertrophy.

[0051] Nppb (brain natriuretic peptide, BNP): Secreted by ventricular myocytes, it increases significantly during myocardial hypertrophy. Other possible related markers include β-MHC (β-myosin heavy chain) and CTGF (connective tissue growth factor).

[0052] For the purposes of this invention, an "active ingredient" is a potent chemical component or biomolecule in a drug or biological product that directly exerts a pharmacological action and produces a therapeutic effect. The active ingredient is the core substance that enables the drug to exert its therapeutic effect. Its structure, concentration, and mechanism of action determine the drug's pharmacological activity and clinical efficacy.

[0053] In the present invention, "pharmaceutically acceptable carrier" refers to an inert ingredient used to carry, stabilize or promote the delivery of active ingredients in pharmaceutical preparations. It has no significant pharmacological activity itself, but can ensure the stability, safety and efficacy of the drug.

[0054] In the present invention, "administered dosage" refers to the amount of drug administered to a patient to achieve the desired therapeutic effect.

[0055] The present invention will be further analyzed below with reference to specific examples.

[0056] Example 1

[0057] Effects of direct treatment of animal-derived and human cardiomyocytes with G3-ME on the expression of cardiac hypertrophy markers in vitro

[0058] In the present invention, the effect of G3-ME on the expression of myocardial hypertrophy markers was detected using completely consistent experimental methods for animal-derived cardiomyocytes (rat cardiomyocyte cell line H9c2) and human-derived cardiomyocytes (cardiomyocytes differentiated from MYL2Neo / w-H7 cell line).

[0059] The cardiomyocyte differentiation steps of the MYL2 Neo / w-H7 cell line are as follows:

[0060] ① When the cell density reached 85%-90%, the medium was replaced with RPM1-1640 / B27-no insulin medium and 8 μM Wnt signaling pathway agonist CHIR-99021 was added for 2 consecutive days;

[0061] ②On the third day, the cells were replaced with fresh RPM1-1640 / B27-no insulin medium and cultured for 24 hours;

[0062] ③ On the 4th day, the cells were replaced with RPM1-1640 / B27-no insulin medium and 5 μM Wnt signaling pathway inhibitor IWR-1 was added for 2 consecutive days;

[0063] ④ On the 6th day, the cells were washed twice with DPBS and then replaced with RPMI1640 / B27-no insulin medium for 2 days. Beating cardiomyocytes were visible on the 7th day of differentiation.

[0064] ⑤ After day 8, continue culturing in RPM1-1640 / B27-no insulin medium for up to day 14, changing the medium daily;

[0065] ⑥ On the 14th day, the culture medium was changed to RPM1-1640 / B27-no insulin medium containing 100 μg / mL G418 antibiotic to select cardiomyocytes. The selection was continued for 7 days (the medium was changed and G418 was refreshed every day), and beating cell clusters were observed.

[0066] Through the above-mentioned directed differentiation and screening methods, a large number of high-purity human embryonic stem cell-derived cardiomyocytes can be obtained.

[0067] The specific operations are as follows: (1) Rat cardiomyocytes H9c2 were seeded into 12-well plates. After 24 hours of adherent culture, they were given PBS, angiotensin II (purchased from MedChemExpress, #HY-13948, CAS No.: 4474-91-3, 1 μM, for 24 hours) and different concentrations of G3-ME (2.5 μM, 5 μM, 10 μM, 20 μM) for pretreatment for 2 hours and Ang II (1 μM) for 24 hours to form PBS group, Ang II group, Ang II + 2.5 μM G3-ME group, Ang II + 5 μM G3-ME group, Ang II + 10 μM G3-ME group, and Ang II + 20 μM G3-ME group. (2) Myocardial cells differentiated from MYL2 Neo / w-H7 cell line were seeded into 12-well plates. After 24 hours of adherent culture, they were treated with PBS, angiotensin II (purchased from MedChemExpress, #HY-13948, CAS No.: 4474-91-3, 1 μM, for 24 hours), and 2.5 μM G3-ME pretreatment for 2 hours plus AngⅡ (1 μM) treatment for 24 hours, forming the PBS group, 2.5 μM G3-ME group, AngⅡ group, and AngⅡ+2.5 μM G3-ME group.

[0068] After treatment, the mRNA expression levels of cardiac hypertrophy markers (ANP, BNP) were detected using a unified real-time quantitative polymerase chain reaction (qRT-PCR) process. The specific steps are as follows:

[0069] ① Wash the treated cells twice with 4°C pre-cooled PBS, aspirate the liquid, add 1 mL of Trizol reagent to each well, let the cells stand at room temperature for 5 minutes after cell lysis to fully separate the nucleic acid-protein complex, transfer to a sterile EP tube, centrifuge at 4°C, 12,000 rpm for 15 minutes, and collect the supernatant;

[0070] ② Add 200 μL of chloroform to the supernatant, shake and mix, then centrifuge at 4°C for 15 minutes, and transfer the colorless aqueous phase to a new centrifuge tube;

[0071] ③ Add an equal volume of isopropanol to precipitate RNA. Centrifuge at low temperature for 15 minutes and discard the liquid. A clear white precipitate can be observed with the naked eye. Wash the precipitate with 1 mL of 75% alcohol and centrifuge. Invert the test tube to completely remove the residual alcohol.

[0072] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve it in a 55°C metal bath, and measure the concentration. The sample can be used directly for reverse transcription.

[0073] ⑤ The reverse transcription system (10 μL) contains 2 μL reverse transcriptase (5× Prime Script RT Master Mix), 1 μg RNA, and RNase-free ddH2O. The cDNA obtained by reverse transcription is diluted 5-fold with ddH2O for later use.

[0074] ⑥The qRT-PCR reaction system (10 μL) included 0.2 μL forward primer, 0.2 μL reverse primer, 1 μL cDNA, 5 μL SyBR Premix EX Taq (2×) and 3.6 μL ddH2O. 36b4 was used as the internal reference gene, and the 2-△△Ct relative quantitative analysis method was used for analysis.

[0075] In the experiment, six different batches of animal and human cardiomyocytes were used, with three replicates set up for each treatment. The data are presented as mean ± standard error (mean ± SEM). The primer sequences are shown in Table 1. The test results are shown in Table 1. Figure 1 This unified experimental method eliminates the interference of cell source differences on the detection system, ensuring the comparability of cross-species data and the reliability of conclusions.

[0076] Table 1. Primer sequences for quantitative real-time polymerase chain reaction (qRT-PCR) of hypertrophy markers

[0077]

[0078]

[0079] from Figure 1 It can be seen that AngⅡ treatment leads to a significant increase in the expression of myocardial hypertrophy markers. Direct treatment with 2.5μM, 5μM, 10μM and 20μM G3-ME in vitro can significantly and effectively reduce the expression of myocardial hypertrophy markers (p<0.05), which is significantly different from the AngⅡ group, indicating that G3-ME has a significant physiological effect of counteracting myocardial hypertrophy.

[0080] Example 2

[0081] Effects of G3-ME treatment on the area of ​​animal-derived and human cardiomyocytes in vitro

[0082] Cardiomyocytes differentiated from rat cardiomyocytes H9c2 and MYL2 Neo / w-H7 were seeded uniformly onto 24-well plates containing cell slides. After 24 hours of attachment, cells were pretreated with PBS, Ang II, or G3-ME (5 μM) for 2 hours followed by Ang II (1 μM) for 24 hours. These groups were designated as the Veh + PBS, G3-ME + PBS, Veh + Ang II, and G3-ME + Ang II treatment groups. Vehicle (abbreviated as Veh) refers to the solvent used to administer the drug at the same volume and concentration to eliminate the influence of the solvent itself on the experimental results. After 24 hours of treatment, cells in each group were immunostained for cardiac troponin (cTNT) by immunofluorescence.

[0083] Cardiac Troponin (cTNT) Immunofluorescence Staining Protocol

[0084] The specific operation of the immunofluorescence staining experiment is as follows:

[0085] ① Wash the treated cells twice with PBS (5 minutes each time), then fix the cells with 4% paraformaldehyde for 15 minutes, discard the fixative, and then wash them three times with PBS (5 minutes each time);

[0086] ② Take the cells from step ①, treat them with 0.2% Triton-X100 for 5 minutes, and then wash them three times with PBS (5 minutes each time);

[0087] ③ The cells in step ② were blocked with 5% BSA solution for 2 hours, and then washed with PBS three times (5 minutes each time);

[0088] ④ Add cTNT antibody (1:200, diluted in PBS) and incubate at 4°C overnight, remove the antibody, and wash three times with PBS (5 minutes each time);

[0089] ⑤ Add fluorescent anti-rabbit secondary antibody (1:1000, diluted in PBS) and incubate in the dark for 1 hour. After incubation, remove the secondary antibody and wash with PBS three times (5 minutes each time);

[0090] ⑥ Add DAPI (1:1000, diluted in PBS) and incubate for 10 minutes. After incubation, remove the staining solution and wash with PBS three times (5 minutes each time);

[0091] ⑦Finally, the slides were sealed with an anti-fluorescence quencher, and fluorescence images were captured using an inverted fluorescence microscope (RVL2-K, USA). The fluorescence staining area measurement function of the microscope was used to analyze the area of ​​myocardial cells.

[0092] Experimental data and analysis:

[0093] The rat myocardial cell line H9c2 was used for 6 different times, with 2 replicates set each time. The cell morphology of cTNT staining was randomly photographed and the area of ​​all myocardial cells was analyzed. The results are as follows Figure 2 As shown. The data shows:

[0094] 1 μM AngⅡ treatment significantly increased the area of ​​myocardial cells (Veh+AngⅡ-treated group vs Veh+PBS group: 1.58±0.14 vs 1.00±0.05);

[0095] Pretreatment with G3-ME for 2 hours followed by AngⅡ stimulation effectively inhibited the increase in cell area (G3-ME+AngⅡ group vs Veh+AngⅡ treatment group: 0.93±0.05 vs 1.58±0.14).

[0096] Data are presented as mean ± standard error (SEM). A total of 135 cells were counted in the Veh + PBS group, 160 cells in the G3-ME + PBS group, 97 cells in the Veh + Ang II group, and 153 cells in the G3-ME + Ang II group. * indicates p < 0.05. The results showed that G3-ME treatment significantly inhibited Ang II-induced proliferation of the rat cardiomyocyte H9c2 cell line in vitro.

[0097] Six different times of directed differentiation of cardiomyocytes were used, with two replicate wells set up each time. The cTNT-stained cell morphology images were randomly photographed and the area of ​​all cardiomyocytes was analyzed. The results are as follows: Figure 2 As shown. The data shows:

[0098] 1 μM AngⅡ treatment significantly increased the cardiomyocyte area (Veh+AngⅡ-treated group vs Veh+PBS group: 1.44±0.18 vs 1.00±0.32);

[0099] G3-ME pretreatment for 2 hours followed by AngⅡ stimulation can effectively inhibit the increase in cell area (G3-ME+AngⅡ group vs Veh+AngⅡ treatment group: 0.87±0.18 vs 1.44±0.18).

[0100] Data are presented as mean ± standard error (SEM). A total of 34 cells were counted in the Veh + PBS group, 39 cells in the G3-ME + PBS group, 40 cells in the Veh + Ang II group, and 44 cells in the G3-ME + Ang II group. * indicates p < 0.05. The results showed that G3-ME treatment significantly inhibited Ang II-induced cardiomyocyte differentiation in the MYL2Neo / w-H7 cell line in vitro.

[0101] Example 3

[0102] Effect of intragastric administration of G3-ME on Myh6 R404Q Effects of left ventricular mass and cardiac function in hypertrophic cardiomyopathy model mice

[0103] (1)Myh6 R404Q Heart testing in hypertrophic cardiomyopathy model mice:

[0104] 8-week-old SPF wild-type (WT) mice and Myh6 R404Q Mutant hypertrophic cardiomyopathy model mice were randomly divided into the following four groups according to body weight:

[0105] WT control group: wild-type mice, given an equal volume of normal saline by gavage daily;

[0106] Model control group: Myh6 R404Q Mice were gavaged daily with an equal volume of normal saline;

[0107] G3-ME low-dose group: Myh6 R404Q Mice were given G3-ME solution (20 mg / kg, prepared in 0.5% sodium carboxymethylcellulose solution) by oral gavage daily;

[0108] G3-ME high-dose group: Myh6 R404Q Mice were given G3-ME solution (concentration 50 mg / kg, prepared with 0.5% sodium carboxymethylcellulose solution) by oral gavage daily.

[0109] The mice in each group were housed in an SPF animal room with free access to food and water, and were given the drug for 30 consecutive days.

[0110] After administration, mice were anesthetized with isoflurane (maintenance concentration of 1.5%-2%), bled by eye, and sacrificed by cervical dislocation. The heart was rapidly isolated, rinsed with saline, and the surface moisture was blotted dry. The whole heart was weighed using an electronic balance. The left ventricular tissue was then isolated, and the left ventricular weight was accurately weighed and recorded.

[0111] Echocardiography

[0112] ①Myh6 R404Q After the mouse abdomen and chest were prepared, anesthesia was maintained with 1.5%-2% isoflurane gas, and the heart rate was controlled at 430-480 beats / min;

[0113] ② Using a high-resolution small animal ultrasound imaging system (Vevo3100LT, Canada), B-mode and M-mode ultrasound images were acquired in the parasternal long-axis and short-axis sections, and mitral valve blood flow ultrasound images were acquired in Doppler mode.

[0114] ③ Calculate the left ventricular weight using the instrument's built-in analysis software. Specific parameters include ventricular septum thickness, left ventricular posterior wall thickness, and left ventricular internal diameter. Calculate the left ventricular weight index using the formula.

[0115] like Figure 3 As shown in the figure, compared with the WT control group, the weight of the heart and left ventricle of the mice in the model control group increased significantly (*P<0.01); after oral administration of G3-ME, Myh6 R404Q Heart and left ventricular weights of mice decreased in a dose-dependent manner, with the 50 mg / kg group showing statistically significant differences (P<0.001). Echocardiographic examinations revealed that G3-ME treatment significantly improved left ventricular structural remodeling, consistent with the results of cardiac weighing.

[0116] The experimental results showed that oral administration of G3-ME could effectively inhibit Myh6 R404Q The heart and left ventricular weight of mutant mice increased, which had a significant intervention effect on the progression of myocardial hypertrophy in hereditary hypertrophic cardiomyopathy. Figure 3 .

[0117] (2) Effect of oral administration of G3-ME on Myh6 R404Q Effect of left ventricular wall thickness on hypertrophic cardiomyopathy in mice

[0118] Calculation of Myh6 using the Vevo3100 system R404Q The left ventricular anterior wall thickness at systole, the left ventricular anterior wall thickness at diastole, the left ventricular posterior wall thickness at systole, the left ventricular posterior wall thickness at diastole, and the E / A peak ratio of mice were measured. Figure 3 shown.

[0119] from Figure 3 As can be seen, Myh6 R404Q The left ventricular wall thickness of mice during diastole and systole was significantly higher than that of WT mice, and the Myh6 R404Q Left ventricular wall thickness in mice. R404Q The left ventricular diastolic function of mice decreased, which was manifested by a significant decrease in E peak. However, after oral administration of G3-ME, the E peak was significantly increased. Compared with the WT control group, the heart and left ventricular weight of mice in the model control group increased significantly (*P<0.01); after oral administration of G3-ME, Myh6 R404Q Heart and left ventricular weights of mice decreased in a dose-dependent manner, with the 50 mg / kg group showing statistically significant differences (P<0.001). Echocardiographic examinations revealed that G3-ME treatment significantly improved left ventricular structural remodeling, consistent with the results of cardiac weighing.

[0120] The experimental results showed that oral administration of G3-ME could effectively inhibit Myh6R404Q The heart and left ventricular weight of mutant mice increased, which had a significant intervention effect on the progression of myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0121] Example 4

[0122] Effect of oral administration of G3-ME on Tnnt2 R109Q Effects of mutant hypertrophic cardiomyopathy model on heart and left ventricular weight

[0123] (1)Tnnt2 R109Q Cardiac testing in mutant hypertrophic cardiomyopathy model mice:

[0124] 8-week-old SPF wild-type (WT) mice and Tnnt2 R109Q Mutant hypertrophic cardiomyopathy model mice were randomly divided into the following four groups according to body weight:

[0125] WT control group: wild-type mice, given an equal volume of normal saline by gavage daily;

[0126] WT+G3-ME group: wild-type mice, given G3-ME solution (concentration 20 mg / kg, prepared with 0.5% sodium carboxymethylcellulose solution) by oral gavage daily;

[0127] Tnnt2 R109Q Group: Tnnt2 R109Q Mice were gavaged daily with an equal volume of normal saline;

[0128] Tnnt2 R109Q +G3-ME group: Tnnt2 R109Q Mice were given G3-ME solution (20 mg / kg, prepared in 0.5% sodium carboxymethylcellulose solution) by oral gavage daily;

[0129] The mice in each group were housed in an SPF animal room with free access to food and water, and were given the drug for 30 consecutive days.

[0130] After administration, mice were anesthetized with isoflurane (maintenance concentration of 1.5%-2%), bled by eye, and sacrificed by cervical dislocation. The heart was rapidly isolated, rinsed with saline, and the surface moisture was blotted dry. The whole heart was weighed using an electronic balance. The left ventricular tissue was then isolated, and the left ventricular weight was accurately weighed and recorded.

[0131] Echocardiography

[0132] ①Tnnt2 R109Q After the mouse abdomen and chest were prepared, anesthesia was maintained with 1.5%-2% isoflurane gas, and the heart rate was controlled at 430-480 beats / min;

[0133] ② Using a high-resolution small animal ultrasound imaging system (Vevo3100LT, Canada), B-mode and M-mode ultrasound images were acquired in the parasternal long-axis and short-axis sections.

[0134] ③ Calculate the left ventricular weight using the instrument's built-in analysis software. Specific parameters include ventricular septum thickness, left ventricular posterior wall thickness, and left ventricular internal diameter. Calculate the left ventricular weight index using the formula.

[0135] (2) Effect of oral administration of G3-ME on Tnnt2 R109Q Effect of left ventricular wall thickness on hypertrophic cardiomyopathy in mice

[0136] Calculation of Tnnt2 by Vevo3100 system R109Q The left ventricular anterior wall thickness at systole, the left ventricular anterior wall thickness at diastole, the left ventricular posterior wall thickness at systole, the left ventricular posterior wall thickness at diastole, and the E / A peak ratio of mice were measured. Figure 4 shown.

[0137] from Figure 4 As can be seen, Tnnt2 R109Q The left ventricular wall thickness of mice during diastole and systole was significantly higher than that of WT mice, and G3-ME administration by gavage significantly reduced Tnnt2 R109Q Left ventricular wall thickness in mice. R109Q The left ventricular diastolic function of mice decreased, which was manifested by a significant decrease in E peak. However, after oral administration of G3-ME, the E peak was significantly increased. Compared with the WT control group, the heart and left ventricular weight of mice in the model control group increased significantly (*P<0.01); after oral administration of G3-ME, Tnnt2 R109Q The heart and left ventricular weights of the mice were significantly reduced (*P<0.001). Echocardiographic examination results showed that G3-ME treatment significantly improved left ventricular structural remodeling, consistent with the results of heart weighing.

[0138] The experimental results showed that oral administration of G3-ME could effectively inhibit Tnnt2 R109Q The heart and left ventricular weight of mutant mice increased, which had a significant intervention effect on the progression of myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0139] Example 5

[0140] Effects of intragastric administration of G3-ME on cardiac hypertrophy markers in mice with hypertrophic cardiomyopathy

[0141] In this example, the expression of WT and Myh6 in G3-ME was detected by oral administration. R404Q Mice and Tnnt2 R109QThe effect of G3-ME on the progression of myocardial hypertrophy in vivo was investigated by measuring the expression of myocardial hypertrophy markers in mouse left ventricular tissue. Mice were raised using the methods described in Examples 3 and 4, and then RNA was extracted from left ventricular tissue using the method described in Example 1. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure the expression of myocardial hypertrophy marker mRNA in cardiomyocytes.

[0142] The analysis results are as follows Figure 5 shown.

[0143] from Figure 5 As can be seen, Myh6 R404Q Mice and Tnnt2 R109Q The expression of Nppa and Nppb, the hypertrophic markers in the myocardial tissue of mice, was significantly higher than that in WT mice. G3-ME significantly inhibited the expression of Nppa and Nppb in Myh6 R404Q Mice and Tnnt2 R109Q This indicates that G3-ME has great potential for treating hereditary hypertrophic cardiomyopathy.

[0144] Example 6

[0145] HE, Masson and WGA staining of myocardial tissue

[0146] Myh6 was administered orally in Example 3 (G3-ME, dose 20 mg / kg, 50 mg / kg, administration period 1 month). R404Q Mice and Tnnt2 were administered orally in Example 4 (G3-ME, 20 mg / kg, 1 month administration period). R109Q Mice and wild-type (WT) control mice were subjected to myocardial tissue fibrosis and cell morphology detection.

[0147] The specific steps are as follows:

[0148] Tissue fixation and processing

[0149] After mice were sacrificed, they were immediately perfused systemically with 4% paraformaldehyde solution via the apex to remove residual blood from the myocardium. The hearts were then completely removed and fixed overnight in 4% paraformaldehyde. The following day, the fixed tissues were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin, and 5-μm serial sections were prepared.

[0150] Masson's trichrome staining

[0151] ① After dewaxing the sections, immerse them in solution A (hematoxylin staining solution) at room temperature overnight (about 15 hours);

[0152] ② Mix equal volumes of Solution B (acid fuchsin solution) and Solution C (phosphomolybdic acid solution), immerse the sections in the mixture for 1 minute, rinse with running water, and differentiate with 1% hydrochloric acid-alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) for 1 minute, until the nuclei appear gray-black and the background is light gray.

[0153] ③ After washing with running water, the sections were immersed in solution D (aniline blue solution) for 6 minutes, and the tissue turned bright red. After draining, they were immediately immersed in solution E (glacial acetic acid solution) for differentiation for 1-2 minutes, until the collagen fibers turned light red.

[0154] ④ Stain the sections directly in solution F (orange-yellow solution G) for 2-30 seconds without washing;

[0155] ⑤ Dehydrate in sequence using three cylinders of 1% glacial acetic acid (8 seconds per cylinder), three cylinders of anhydrous ethanol (5 seconds, 10 seconds, 30 seconds) and two cylinders of n-butanol (30 seconds, 2 minutes), then transparentize with two cylinders of xylene (5 minutes per cylinder) and seal with neutral gum.

[0156] The images were collected and observed under an optical microscope to analyze the degree of myocardial fibrosis (blue collagen fiber area) and the cross-sectional area of ​​myocardial cells.

[0157] HE staining ① The sections were dewaxed by placing them in xylene I and II for 10 minutes each, and then dehydrated to water by gradient dehydration in anhydrous ethanol I (5 minutes), anhydrous ethanol II (5 minutes), 95% ethanol (3 minutes), 85% ethanol (3 minutes), and 75% ethanol (3 minutes);

[0158] ② Immerse the sections in hematoxylin solution, stain at room temperature for 5-8 minutes, and rinse with tap water for 1-2 minutes;

[0159] ③ Place the slices in a 1% hydrochloric acid alcohol solution for 3-5 seconds, quickly wash with water, and then treat with a bluing solution (such as warm water or ammonia) for 1-2 minutes to make the cell nuclei blue;

[0160] ④ Stain the sections with eosin solution for 3-5 minutes, then rinse with tap water to remove excess stain.

[0161] ⑤ Dehydrate with 95% ethanol I (1 minute), 95% ethanol II (1 minute), anhydrous ethanol I (2 minutes), and anhydrous ethanol II (2 minutes) in sequence, make transparent with two cylinders of xylene (3-5 minutes each cylinder), and seal with neutral gum.

[0162] Observe and collect images under an optical microscope to analyze the morphological structure and pathological changes of tissue cells.

[0163] WGA staining

[0164] ① Wash the cell slides or tissue sections with PBS three times, 5 minutes each time, to remove residual culture medium and impurities;

[0165] ② Fix with 4% paraformaldehyde at room temperature for 15-20 minutes, wash with PBS three times, 5 minutes each time, and wash away the fixative;

[0166] ③ Permeabilize with 0.1% Triton X-100 (in PBS) at room temperature for 10-15 minutes (suitable for samples with poor cell membrane permeability), then wash with PBS three times, 5 minutes each time;

[0167] ④ Add appropriate amount of WGA staining working solution (diluted according to the instructions) and stain at room temperature in the dark for 30-45 minutes;

[0168] ⑤ Wash with PBS three times, 5 minutes each time, to remove unbound dye;

[0169] ⑥ If you need to counterstain the cell nucleus, use DAPI staining solution to stain at room temperature in the dark for 5-10 minutes, and then wash with PBS three times, 5 minutes each time;

[0170] ⑦ Seal the slides with anti-fluorescence quenching sealing medium.

[0171] Observe and collect images under a fluorescence microscope to analyze the cell membrane structure, cell boundaries and intercellular connections.

[0172] like Figure 6 Shown: Myh6 R404Q Mice and Tnnt2 R109Q Mouse model group: Compared with the WT control group, the myocardial cells showed significant hypertrophy (increase in cell cross-sectional area) and myocardial fibrosis (increase in blue area); in Myh6 R404Q In mice, the 50 mg / kg dose group of G3-ME gavage group significantly reduced Myh6 R404Q Collagen fibers were deposited in myocardial tissue and the area of ​​myocardial cells was reduced in a dose-dependent manner (P<0.05). R109Q The G3-ME gavage group in mice also significantly reduced Tnnt2 expression compared with the model group. R109Q Collagen fiber deposition in myocardial tissue.

Claims

1. Use of galangin-3-methyl ether in the preparation of drugs for treating or preventing pathological myocardial hypertrophy.

2. The use according to claim 1, characterized in that The pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure load, hereditary myocardial hypertrophy, and myocardial hypertrophy caused by inflammatory factors.

3. The use according to claim 1 or 2, characterized in that The pathological myocardial hypertrophy symptoms are manifested by any one or more of the following: enlarged myocardial cell area, increased heart or left ventricular weight, myocardial fibrosis, and elevated expression of myocardial hypertrophy markers.

4. The use according to claim 3, characterized in that The myocardial hypertrophy markers include Nppa and / or Nppb.

5. The use according to claim 2, characterized in that The hereditary myocardial hypertrophy is caused by the Myh6 gene R404Q mutation or the Tnnt2 gene R109Q mutation.

6. A pharmaceutical composition for treating or preventing pathological myocardial hypertrophy, characterized in that: The active ingredient of the pharmaceutical composition is galangin 3-methyl ether.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that The dosage form of the pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches.

9. The pharmaceutical composition according to claim 6, characterized in that The dosage of galangin 3-methyl ether is 10-80 mg / kg; preferably, the dosage of galangin 3-methyl ether is 20-50 mg / kg.

10. A method for preventing or treating pathological myocardial hypertrophy, characterized in that: It comprises administering a therapeutically effective amount of galangin 3-methyl ether to a patient in need thereof.

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