Application of harbazoside in the preparation of drugs for the treatment or prevention of pathological myocardial hypertrophy

Abstract

This invention discloses the application of harbazoside in the preparation of drugs for the treatment or prevention of pathological myocardial hypertrophy, belonging to the field of biomedicine. Through in vitro and in vivo experiments, this invention demonstrates that harbazoside can significantly reduce the expression levels of myocardial hypertrophy markers, improve cardiomyocyte calcium homeostasis disorders, inhibit cardiomyocyte hypertrophy and myocardial fibrosis, and improve cardiac structural remodeling and cardiac function. It can be effectively applied in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy, especially in hypertrophic cardiomyopathy, improving left ventricular diastolic function. This provides a new feasible direction for the treatment of related clinical diseases and possesses high development value and application prospects.

Description

Technical Field

[0001] This invention relates to the application of harbazoside in the preparation of drugs for the treatment or prevention of pathological myocardial hypertrophy, and belongs to the field of biomedicine. Background Technology

[0002] Cardiovascular diseases are a prevalent and deadly category of diseases worldwide. Pathological myocardial hypertrophy, as a core pathological feature shared by these diseases, is closely related to disease prognosis and can further induce serious complications such as heart failure, arrhythmia, and even sudden death, posing a significant threat to patients' lives and health. Based on differences in pathogenesis, pathological myocardial hypertrophy can be divided into two main categories: primary myocardial hypertrophy and secondary myocardial hypertrophy. Secondary myocardial hypertrophy is mostly induced by underlying cardiovascular diseases such as hypertension, coronary heart disease, and valvular heart disease, while primary myocardial hypertrophy is mainly related to genetic factors, with hypertrophic cardiomyopathy (HCM) being its typical representative.

[0003] The treatment strategies for these two types of myocardial hypertrophy differ fundamentally. The core of treating secondary myocardial hypertrophy lies in controlling the primary disease (such as effectively controlling hypertension through ACEIs and ARBs). With the control of the primary disease, myocardial hypertrophy is usually reversed or delayed to some extent. However, the treatment of primary myocardial hypertrophy (HCM) faces far greater challenges. For a long time, drug treatment for HCM has mainly relied on non-specific symptom relief regimens, such as beta-blockers and calcium channel blockers, aimed at slowing the heart rate and prolonging diastolic filling time to improve symptoms, but these cannot fundamentally reverse the progression of myocardial hypertrophy or correct sarcomere dysfunction. For obstructive HCM patients who do not respond well to drug treatment, septal alcohol ablation or surgical resection is routinely chosen clinically, but these procedures are highly invasive and risky, and are not suitable for all patients. In recent years, innovative drugs targeting the fundamental pathological mechanisms of hemorrhage-induced myocardial hypertrophy (HCM), such as myosin inhibitors (e.g., Mavacamten), have emerged, bringing new breakthroughs in HCM treatment. However, the long-term safety, drug resistance, and applicable population of these drugs still have certain limitations. Therefore, in-depth exploration of effective targets for inhibiting myocardial hypertrophy and the development of HCM, and the development of corresponding specific therapeutic drugs, have significant clinical application value and practical significance for improving the clinical prognosis of patients with primary myocardial hypertrophy.

[0004] Harpagide (Harp) is a natural iridoid glycoside extracted from the dried root of Scrophularia ningpoensis, a plant in the Scrophulariaceae family. It is one of the most important medicinal components of Scrophularia ningpoensis, a traditional Chinese medicine. Its chemical formula is: C 15 H 24 O 10 Existing research reports confirm that harbazoside has anti-inflammatory, analgesic, and immunomodulatory effects, and has potential application value in the intervention of neurological diseases and osteoporosis.

[0005] In recent years, the cardioprotective effects of harbazoside have attracted attention. Existing studies have shown that harbazoside can reduce collagen deposition in the left ventricle of rats with heart failure after myocardial infarction, inhibit cardiomyocyte apoptosis, and improve cardiac dysfunction; it can also inhibit H2O2-induced H9c2 apoptosis in rat cardiomyocytes by inhibiting oxidative stress. These studies suggest that harbazoside has certain activity in ischemic myocardial injury, oxidative stress-induced cardiomyocyte damage, and vascular endothelial protection.

[0006] However, hypertrophic cardiomyopathy (HCM) differs fundamentally from other cardiac diseases studied in existing research in terms of etiology and core pathological mechanisms. Post-infarction heart failure and H2O2-induced cardiomyocyte damage are both acquired cardiac injuries, with their core pathology lying in cardiomyocyte necrosis, apoptosis, and ventricular remodeling caused by exogenous factors such as ischemia and oxidative stress. In contrast, HCM is a hereditary cardiomyopathy, with its fundamental cause being mutations in sarcomere protein genes (such as MYH7 and MYBPC3), leading to intrinsic abnormalities in cardiomyocyte structure and function, mainly manifested as asymmetrical left ventricular hypertrophy, disordered arrangement of cardiomyocytes, and diastolic dysfunction. Summary of the Invention

[0007] In view of the shortcomings of existing technologies in the treatment of pathological myocardial hypertrophy, the present invention aims to provide a new application of harbazoside in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy.

[0008] To achieve the above technical objectives, this invention, through in vitro experiments, directly applied Harp to cardiomyocytes of H9c2 rats and cardiomyocytes derived from the directed differentiation of human embryonic stem cells, demonstrating that Harp can significantly inhibit the hypertrophic phenotype of cardiomyocytes. In in vivo experiments, after administering Harp by gavage to mice with pathological myocardial hypertrophy induced by pressure load from aortic arch coarctation (TAC) and mice with hereditary hypertrophic cardiomyopathy induced by gene mutations, it was found that it could effectively alleviate the symptoms of pathological myocardial hypertrophy and improve cardiac function in mice. This invention reveals for the first time the application value of Harp in the preparation of anti-pathological myocardial hypertrophy drugs, providing a novel strategy for the treatment of pathological myocardial hypertrophy and hereditary hypertrophic cardiomyopathy.

[0009] On the one hand, the present invention provides harbazoside (C 15 H 24 O 10 Its application in the preparation of drugs for the prevention, treatment or adjuvant treatment of pathological myocardial hypertrophy.

[0010] In one embodiment of the present invention, the pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure overload, hereditary myocardial hypertrophy (hypertrophic cardiomyopathy), and myocardial hypertrophy caused by inflammatory factors; preferably hereditary myocardial hypertrophy (hypertrophic cardiomyopathy).

[0011] In one embodiment of the present invention, the pathological myocardial hypertrophy is characterized by one or more of the following: increased cardiomyocyte area, increased weight of the heart or left ventricle, myocardial fibrosis, and elevated expression of myocardial hypertrophy markers.

[0012] In one embodiment of the present invention, the myocardial hypertrophy markers include Nppa and / or Nppb.

[0013] In one embodiment of the present invention, the drug reduces cardiomyocyte area, improves left ventricular diastolic function, reduces left ventricular weight, improves myocardial fibrosis, and reduces the expression of myocardial hypertrophy markers.

[0014] In one embodiment of the present invention, the improvement of left ventricular diastolic function includes at least one of the following: (a) Increased diastolic diameter of the left ventricle; (b) Decreased diastolic thickness of the left ventricular anterior wall; (c) Decreased diastolic thickness of the left ventricular posterior wall; (d) Increased diastolic volume of the left ventricle.

[0015] In one embodiment of the present invention, the dosage form of the drug includes an oral dosage form, a parenteral dosage form, and / or a topical dosage form.

[0016] In one embodiment of the present invention, the dosage form of the drug is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

[0017] On the other hand, the present invention provides a pharmaceutical composition for the prevention, treatment or adjunctive treatment of pathological myocardial hypertrophy, the pharmaceutical composition containing harbazoside as an active ingredient and a pharmaceutically acceptable carrier.

[0018] In one embodiment of the present invention, the dosage form of the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0019] In one embodiment of the present invention, the pharmaceutically acceptable carrier is selected from at least one of starch, lactose, microcrystalline cellulose, and magnesium stearate.

[0020] Furthermore, the present invention also provides the use of the pharmaceutical composition in the preparation of a drug for treating, adjuvant treating or preventing pathological myocardial hypertrophy, characterized in that the pathological myocardial hypertrophy is selected from one or more of the following: myocardial hypertrophy caused by pressure overload, hereditary myocardial hypertrophy and myocardial hypertrophy caused by inflammatory factors; preferably hereditary myocardial hypertrophy.

[0021] In one embodiment of the present invention, the pharmaceutical composition reduces cardiomyocyte area, improves left ventricular diastolic function, reduces left ventricular weight, improves myocardial fibrosis, and reduces the expression of markers of myocardial hypertrophy.

[0022] Beneficial effects: This invention discovers a new medicinal use for harpaquinone, which can prevent, treat, or assist in the treatment of pathological myocardial hypertrophy and its related symptoms. Furthermore, harpaquinone is one of the most important medicinal components in the traditional Chinese medicine Scrophularia ningpoensis, making it safer for organisms and showing promising clinical application prospects.

[0023] In in vivo experiments, this invention uses a mouse model of myocardial hypertrophy induced by aortic arch coarctation surgery and the Myh6 gene R404Q mutation ( Myh6 R404Q Two mouse models of hypertrophic cardiomyopathy were developed, both exhibiting pathological myocardial hypertrophy, enlarged heart volume, and increased heart and left ventricular weight. Administration of Harp via gavage to these two mouse models significantly improved their pathological myocardial hypertrophy symptoms, significantly improved left ventricular and heart weight, reduced heart volume and diastolic / systolic myocardial wall thickness, and effectively inhibited myocardial fibrosis.

[0024] In in vitro experiments, Harp treatment of hypertrophic rat cardiomyocytes and human embryonic stem cell-induced cardiomyocytes significantly reduced cardiomyocyte area and decreased the expression levels of norepinephrine / angiotensin II-induced markers of myocardial hypertrophy.

[0025] This invention, through in vitro and in vivo studies, demonstrates that Harp has significant potential in treating pathological myocardial hypertrophy, ventricular remodeling, and hypertrophic cardiomyopathy, particularly in the development of novel drugs for hereditary hypertrophic cardiomyopathy, providing new approaches and methods for the treatment of related diseases. Simultaneously, this invention offers entirely new options and ideas for existing drugs against pathological myocardial hypertrophy and hereditary hypertrophic cardiomyopathy, broadening the range of drug choices in this field and positively promoting its development. Attached Figure Description

[0026] Figure 1: Validation experiment of cardiomyocyte differentiation from H9c2 rat cardiomyocyte line and human MYL2 Neo / w-H7 cell line using harpaquinone; Image (A) showing cell viability of rat H9c2 cardiomyocyte line after direct in vitro treatment with different concentrations of harpaquinone, and image (B) showing the expression levels of cardiomyocyte hypertrophy markers Nppa and Nppb; Flowchart of the experimental process for differentiating human MYL2 Neo / w-H7 cell line into cardiomyocytes; Image (D) showing cardiomyocyte viability of human MYL2 Neo / w-H7 cell line after direct in vitro treatment with different concentrations of harpaquinone, and image (C) showing the expression levels of cardiomyocyte hypertrophy markers. NPPA , NPPB , TNNT2 and MEF2c The expression level diagram (E) shows that... # indicates a statistically significant difference compared to the blank control group (P < 0.05); # indicates a statistically significant difference compared to the Ang II model group (P < 0.05); ns indicates no statistically significant difference between groups (P > 0.05). Figure 2 The experiment included: validation of Harp in TAC mice; a flowchart of the in vivo validation experiment (A); gross cardiac images of TAC mice after gavage administration of different concentrations of Harp (B); a statistical graph of the heart weight to tibia ratio (C); echocardiographic results of TAC mice after gavage administration of different concentrations of Harp, including M-mode and Doppler echocardiograms (D), left ventricular weight (E), diastolic thickness (LVAW;d) and systolic thickness (LVAW;s) of the left anterior wall, diastolic thickness (LVPW;d) and systolic thickness (LVPW;s) of the left ventricular posterior wall (F), and the E / A peak ratio (G); and the identification of myocardial hypertrophy markers in the cardiac tissue of TAC mice. Nppa , Nppb , β-Mhc Expression level diagram (H); in the diagram, express P <0.05, no statistically significant difference in ns; Figure 3 Harp validation experiment in TAC mice; H&E staining results (A, B), Masson staining results (C, D), and WGA staining results (E, F) of TAC mouse myocardial tissue; in the figures, express P <0.05, ns indicates no statistically significant difference; Figure 4 Harp in Myh6 R404Q In vivo validation experiment in mice; Myh6 R404QThe flowchart for the in vivo validation experiment in mice is shown in Figure A. TAC mice were administered Harp at different concentrations via gavage. Myh6 R404Q Gross image of mouse heart (B); Statistical graph of heart weight to tibia ratio (C); Effects of different concentrations of Harp on the heart. Myh6 R404Q The echocardiographic results of mice after gavage administration include M-mode and Doppler echocardiograms (D), left ventricular weight (E), ejection fraction EF and fractional shortening FS (F), left ventricular diastolic volume LVV;d and systolic volume LVV;s (G), left ventricular anterior wall diastolic thickness LVAW;d and systolic thickness LVAW;s, left ventricular posterior wall diastolic thickness LVPW;d and systolic thickness LVPW;s (H), left ventricular diastolic diameter LVD;d and systolic diameter LVD;s (I), and E / A peak ratio (J). In the figures, express P <0.05, no statistically significant difference in ns; Figure 5 Harp in Myh6 R404Q In vivo validation experiment in mice; Myh6 R404Q Images of mouse myocardial tissue H&E staining results (A, B), Masson staining results (C, D), and WGA staining results (E, F); Myh6 R404Q Markers of myocardial hypertrophy in mouse heart tissue Nppa , Nppb , β-Mhc The expression level diagram (G); in the diagram, express P <0.05, no statistically significant difference in ns. (See figure). express P <0.05, ns indicates no statistical difference. Detailed Implementation

[0027] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0028] The test methods involved in the following embodiments: 1. Cell viability (CCK-8 assay) The specific steps are as follows: (1) The cells to be tested were placed at 5×10 3 Inoculate into 96-well cell culture plates, add 100 μL of complete culture medium to each well, and incubate in a 37℃, 5% CO2 incubator until the preset intervention time; (2) Discard the original culture medium in the well and gently wash each well 1-2 times with sterile PBS to remove residual culture medium, cell debris and impurities, so as to avoid residual components affecting the test results; (3) Add 10 μL of freshly prepared CCK-8 working solution in the dark at a ratio of 10% of the culture medium volume per well, ensuring that the working solution completely covers the cells at the bottom of the well and that no bubbles are generated; (4) Place the 96-well plate with the sample added in a constant temperature incubator at 37°C and 5% CO2 and incubate in the dark for 1 to 4 hours. Adjust the incubation time according to the cell proliferation status and color development. (5) After incubation, gently shake the culture plate to mix the liquid in the wells to avoid generating bubbles, and set up blank wells, control group and experimental group calibration detection system; (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance (OD value) of each well at a wavelength of 450 nm, record and organize the data, and calculate relevant indicators such as cell survival rate and cell inhibition rate according to the formula.

[0029] 2. Quantitative real-time polymerase chain reaction (qRT-PCR) The specific steps are as follows: (1) After washing the cells twice with PBS pre-cooled at 4℃, after aspirating the liquid, add 1 mL of Trizol reagent to each well. After cell lysis, let stand at room temperature for 5 min to fully separate the nucleic acid-protein complex. Transfer to enzyme-free EP tubes and centrifuge at 4℃, 12,000 rpm for 15 min. Take the supernatant. (2) Add 200 μL of chloroform to the supernatant, shake to mix, centrifuge at 4°C for 15 min, and transfer the colorless aqueous phase to a new centrifuge tube. (3) Add an equal volume of isopropanol to precipitate the RNA. After centrifuging at low temperature for 15 min, discard the liquid. A 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. (4) Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve it in a metal bath at 55°C, and then measure the concentration. The sample is used directly for reverse transcription. (5) The reverse transcription system (10 μL) contains 2 μL of reverse transcriptase (5×Prime Script RT Master Mix), 1 μg of RNA and RNase-free ddH2O. The cDNA obtained by reverse transcription is diluted 5 times with ddH2O for later use. (6) The qRT-PCR reaction system (10 μL) includes 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. 36b4The internal reference gene was analyzed using the 2-ΔΔCt relative quantitative analysis method.

[0030] Table 1 Primer sequences for qRT-PCR

[0031] 3. Differentiation of cardiomyocytes in the MYL2 Neo / w-H7 cell line The specific steps are as follows: (1) When the cell density reaches 85%~90%, replace it with RPM1-1640 / B27-no insulin medium and add 8 μM Wnt signaling pathway agonist CHIR-99021, and culture continuously for 2 days; (2) On the 3rd day, replace with fresh RPM1-1640 / B27-no insulin medium and incubate for 24 h; (3) On the 4th day, replace with fresh RPM1-1640 / B27-no insulin medium, add 5 μM Wnt signaling pathway inhibitor IWR-1, and culture for 2 consecutive days; (4) After washing the cells twice with DPBS on day 6, replace them with fresh RPMI1640 / B27-no insulin medium and culture for 2 days. Beating cardiomyocytes can be seen on day 7 of differentiation. (5) Continue to culture in RPM1-1640 / B27-no insulin medium for 14 days after day 8, changing the medium daily; (6) On day 14, the medium was changed to RPM1-1640 / B27-no insulin containing 100 μg / mL G418 antibiotic to screen cardiomyocytes. The screening was carried out for 7 consecutive days (the medium was changed and G418 was updated daily). Beating cell clusters were observed, and a large number of high-purity human embryonic stem cell-derived cardiomyocytes were obtained.

[0032] 4. Echocardiography The specific steps are as follows: (1) After the mice were shaved on the abdomen and chest, they were anesthetized with 1.5%~2% isoflurane gas and the heart rate was controlled at 430~480 beats / min. (2) Using a high-resolution small animal ultrasound imaging system (Vevo3100LT, Canada), M-mode ultrasound images and Doppler ultrasound images were acquired in the parasternal long axis and short axis sections. (3) The weight of the left ventricle is calculated by the built-in analysis software of the instrument. Specific parameters include the thickness of the interventricular septum, the thickness of the posterior wall of the left ventricle, and the diameter of the left ventricle.

[0033] 5. Measurement of heart and left ventricular weight After administration, mice were anesthetized with isoflurane (1.5%–2% maintenance concentration), blood was collected through the eyeballs, and the mice were euthanized by cervical dislocation. The heart was quickly separated, rinsed with physiological saline, and the surface moisture was aspirated. The weight of the whole heart was measured using an electronic balance. Subsequently, the left ventricular tissue and the mouse tibia were separated, and the weight of the left ventricle and the length of the mouse tibia were accurately measured and recorded. 6. TAC surgery (aortic arch coarctation surgery) The specific steps are as follows: (1) Preoperative preparation and anesthesia: SPF-grade male C57BL / 6 mice (8-10 weeks old, weighing 22-25 g) were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After confirming that there was no tail retraction reflex, the mice were fixed supine on a constant temperature operating table (37℃). The neck and chest were prepared and the surgical area was disinfected with iodine. (2) Thoracotomy and aortic exposure: endotracheal intubation was performed and the animal was connected to a ventilator (respiratory rate 120 breaths / min, tidal volume 0.8 mL, inspiratory-to-expiratory ratio 1:2). A longitudinal incision was made in the 2nd to 3rd intercostal space above the sternum. The chest wall muscles were bluntly dissected, the chest cavity was opened, the aortic arch was exposed, and the fat and connective tissue around the aortic arch were separated to clearly expose the brachiocephalic artery, the left common carotid artery and the branches of the left subclavian artery. (3) Aortic coarctation procedure: 6-0 sterile silk suture is inserted below the aortic arch between the brachiocephalic artery and the left common carotid artery. A 27G blunt needle (about 0.4 mm in diameter) is attached parallel to the aortic arch. The aortic arch and the needle are tightly ligated together with silk suture. After ligation, the needle is slowly pulled out to form an aortic coarctation of about 0.4 mm in diameter. The sham surgery group only had the suture inserted but did not ligate. (4) Chest closure and postoperative care: After confirming that there is no active bleeding, the chest wall muscles and skin are sutured layer by layer with 4-0 silk thread, and the surgical area is disinfected again with iodine; after the operation, the mouse is placed on a warming pad to recover. After spontaneous breathing is restored, the endotracheal tube is removed, antibiotics are given to prevent infection, and the mouse is fed in a routine manner.

[0034] 7. HE staining The specific steps are as follows: (1) The slices were placed in xylene I and II for 10 min each for dewaxing, and then subjected to a gradient dehydration to water in anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 95% ethanol (3 min), 85% ethanol (3 min), and 75% ethanol (3 min); (2) Immerse the sections in hematoxylin staining solution and stain at room temperature for 5-8 min, then rinse with tap water for 1-2 min; (3) Place the slices in a 1% hydrochloric acid alcohol solution for 3-5 seconds to differentiate, wash quickly with water, and then treat with a blueing solution (such as warm water or ammonia) for 1-2 minutes to make the cell nuclei turn blue; (4) Stain the sections with eosin solution for 3-5 minutes, then rinse with tap water to remove excess solution; (5) Dehydrate with 95% ethanol I (1 min), 95% ethanol II (1 min), anhydrous ethanol I (2 min), and anhydrous ethanol II (2 min) in sequence, clear the xylene in both tanks (3~5 min each), and seal with neutral resin.

[0035] 8. Masson's trichrome staining The specific steps are as follows: (1) After dewaxing the sections to water, immerse them in solution A (hematoxylin staining solution) at room temperature overnight (about 15 hours). (2) Mix equal volumes of solution B (acidic fuchsin solution) and solution C (phosphomolybdic acid solution), immerse the slices in the mixture for 1 min, rinse with running water, and then differentiate with 1% hydrochloric acid alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) for 1 min until the cell nuclei are gray-black and the background is light gray. (3) After rinsing with running water, the slices are immersed in solution D (aniline blue solution) for 6 min. The tissue turns bright red. After draining, they are immediately immersed in solution E (glacial acetic acid solution) for 1-2 min to differentiate until the collagen fibers turn light red. (4) The sections are placed directly into solution F (orange yellow solution G) for staining for 2-30 seconds without washing with water; (5) The tablets were dehydrated by 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 min), then cleared by two cylinders of xylene (5 min per cylinder), and sealed with neutral resin.

[0036] 9. WGA staining The specific steps are as follows: (1) Wash cell smears or tissue sections with PBS three times for 5 min each time to remove residual culture medium and impurities; (2) Fix with 4% paraformaldehyde at room temperature for 15-20 min, wash with PBS 3 times for 5 min each time to remove fixative; (3) 0.1% Triton X-100 (prepared with PBS) was allowed to pass through at room temperature for 5 min, followed by washing with PBS 3 times for 5 min each time; (4) Place in sodium citrate antigen retrieval solution and boil at 100°C for 15 min. Let stand until room temperature, then wash 3 times with PBS for 5 min each time; (5) Draw circles with the histochemical pen, add an appropriate amount of WGA staining working solution (1:500, prepared with PBS) to the tissue, and incubate overnight at 4°C in the dark; (6) Wash with PBS 3 times, 5 min each time, to remove unbound dye; (7) Mount the slide with a DAPI-containing anti-fluorescence quenching mounting medium. The following are some definitions used in this invention: In this invention, "myocardial hypertrophy markers" refer to biomolecules that are abnormally expressed during pathological myocardial hypertrophy and can be used to assess the occurrence, development, and treatment efficacy of myocardial hypertrophy. In this invention, the markers include, but are not limited to: Nppa (Atrial natriuretic peptide, ANP): secreted by atrial myocytes, its expression is upregulated in myocardial hypertrophy.

[0037] Nppb (Brain natriuretic peptide, BNP): secreted by ventricular myocytes, and significantly elevated in cases of myocardial hypertrophy.

[0038] Other related markers include TNNT2 (Cardiomyocyte troponin T) β-Mhc (β-Myosin Heavy Chain) MEF2c (Myocyte Enhancer Factor 2C), etc.

[0039] The raw materials used in the following examples: In the following examples, 8-week-old wild-type mice (WT mice) and Myh6 gene R404Q point mutant mice were used. Myh6 R404Q All were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., including Myh6 gene R404Q point mutation mice ( Myh6 R404Q The value is C57BL / 6JGpt-Myh6 em1Cin(R404Q) / Gpt,Myh6-p.R404Q|Strain NO.T051403.

[0040] Harpagide compounds were purchased from MedChemExpress (HY-N0397, CAS No.:6926-08-5).

[0041] The rat cardiomyocyte H9c2 cell line was purchased from the ATCC cell bank (Cell Code CRL-1446). It was derived from embryonic rat heart tissue and was an immortalized cell line with cardiomyocyte characteristics obtained through in vitro passage culture.

[0042] The human embryonic stem cell line MYL2 Neo / w-H7, wherein H7 cells were purchased from the WiCell Institute Cell Bank #WA07, and a stable human embryonic stem cell line MYL2 Neo / w-H7 was obtained after editing using genome-directed editing technology (construction method refers to LianX, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem via temporal modulation of canonical Wntsignaling. Proc Natl Acad Sci US A. 2012;109(27):E1848-E1857. doi: 10.1073 / pnas.1200250109; or see section 0037 of the specification in patent CN108265029A: editing the genome of human pluripotent stem cells (including human embryonic stem cells, introducing MYL2 driven Neo into the genome of human pluripotent stem cells and replacing the corresponding normal genes).

[0043] Agonist CHIR-99021 (purchased from MedChemExpress, #HY-10182, CAS No.:252917-06-9). Pathway inhibitor IWR-1 (purchased from MedChemExpress, #HY-12238, CAS No.:1127442-82-3).

[0044] RPM1-1640 / B27-no insulin medium is RPM1-1640 (Gibco) medium supplemented with insulin-free B-27 (Thermo Fisher Scientific, Cat# A1895601), with the B-27 content being 2%.

[0045] Example 1: Effects of Harp treatment on the expression of myocardial hypertrophy markers in animal and human cardiomyocytes in vitro In this invention, the effects of Harp on the expression of myocardial hypertrophy markers were detected using completely consistent experimental methods for both animal-derived cardiomyocytes (rat cardiomyocyte line H9c2) and human-derived cardiomyocytes (cardiomyocytes differentiated from the MYL2Neo / w-H7 cell line). The specific procedures are as follows: 1. Harp toxicity test on cardiomyocytes The effects of different concentrations of harpaquinone (Harp) on the viability of two types of cardiomyocytes were detected using the CCK-8 assay. The results are as follows: Figure 1A and Figure 1 As shown in Figure D, Harp did not significantly inhibit the activity of either type of cardiomyocyte at concentrations ranging from 0 to 500 μM, further demonstrating that Harp has no significant toxic effects on cardiomyocytes within this concentration range.

[0046] 2. Effects of Harp on the expression of markers of myocardial hypertrophy: Cardiomyocytes differentiated from rat cardiomyocyte line H9c2 and MYL2 Neo / w-H7 were seeded into 12-well plates and cultured for 24 hours. Afterward, the cells were treated with PBS and angiotensin II (Ang II) for 24 hours, and pretreated with different concentrations of Harp for 2 hours followed by Ang II treatment for 24 hours, forming a control group and experimental groups (Table 2). After treatment, the mRNA expression levels of myocardial hypertrophy markers (ANP and BNP) were detected using qRT-PCR.

[0047] Table 2 Experimental Groups and Drug Treatments

[0048] In the experiment, six different batches of animal-derived and human cardiomyocytes were used, with three replicates per treatment. Data are expressed as mean ± standard error (mean ± SEM). Primer sequences are shown in Table 1, and detection results are as follows. Figure 1 As shown. This standardized experimental method eliminates the interference of differences in cell origin on the detection system, ensuring the comparability of cross-species data and the reliability of conclusions.

[0049] from Figure 1 As can be seen, in the rat cardiomyocyte cell line H9c2, Ang II treatment significantly increased the expression of myocardial hypertrophy markers, while direct in vitro treatment with 5 μM, 10 μM, and 20 μM Harp significantly decreased the expression of myocardial hypertrophy markers. P <0.05%, direct in vitro treatment of cardiomyocytes differentiated from the MYL2 Neo / w-H7 cell line with 10 μM Harp can also significantly and effectively reduce the expression of markers of myocardial hypertrophy. P <0.05). Therefore, the above results indicate that Harp has a significant physiological effect in combating myocardial hypertrophy.

[0050] Example 2: Effects of Harp intragastric administration on cardiac function in mice with TAC-induced pressure-overload-induced pathological myocardial hypertrophy. (1) Experimental grouping of TAC pressure-overload type pathological myocardial hypertrophy mice: Eight-week-old SPF-grade wild-type (WT) mice were selected and subjected to sham surgery and aortic coarctation surgery (TAC). They were then randomly divided into the following four groups: Sham+Veh control group: wild-type mice were administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage daily; TAC+Veh group: TAC mice were given an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage daily; TAC+Low Harp group: Harp solution (concentration 10 mg / kg, prepared with 0.5% sodium carboxymethyl cellulose solution) was administered by gavage daily. TAC+Mid Harp group: Harp solution (concentration 25 mg / kg, prepared with 0.5% sodium carboxymethyl cellulose solution) was administered by gavage daily. TAC+High Harp group: Harp solution (concentration 50 mg / kg, prepared with 0.5% sodium carboxymethyl cellulose solution) was administered by gavage daily. Mice in each group were housed in an SPF-grade animal facility with free access to food and water, and were administered the drugs continuously for 4 weeks.

[0051] (2) Effects of Harp gavage administration on heart weight and left ventricular wall thickness in TAC pressure-overloaded pathological myocardial hypertrophy mice The instrument's built-in analysis software was used to calculate the following parameters in TAC stress-overload-induced pathological myocardial hypertrophy mice and wild-type mice: systolic left ventricular anterior wall thickness, diastolic left ventricular anterior wall thickness, systolic left ventricular posterior wall thickness, diastolic left ventricular posterior wall thickness, and E / A peak ratio. The test results are as follows: Figure 2 As shown.

[0052] Compared with WT mice (Sham+Veh group), TAC stress-overload-induced pathological myocardial hypertrophy mice (TAC+Veh group) showed a significant increase in heart and left ventricular weight. P <0.05); After 4 weeks of Harp administration by gavage, the heart and left ventricular weights of mice in the TAC+High Harp group were significantly reduced ( P <0.05. The left ventricular wall thickness during both diastole and systole was significantly higher in the TAC+Veh group than in the Sham+Veh group. However, after 4 weeks of Harp administration, compared with the TAC+Veh group, the left ventricular anterior wall systolic thickness, left ventricular anterior wall diastolic thickness, left ventricular posterior wall systolic thickness, and left ventricular posterior wall diastolic thickness were all significantly decreased in the TAC+High Harp group. P <0.05). Echocardiographic results showed that Harp treatment significantly improved left ventricular structural remodeling. Experimental results indicate that Harp gavage administration effectively inhibited the increase in heart and left ventricular weight in TAC pressure-overload-induced pathological myocardial hypertrophy mice, and had a significant intervention effect on the progression of pressure-overload-induced pathological myocardial hypertrophy.

[0053] Further determination of myocardial hypertrophy markers Nppa , Nppb , β-Mhc The results showed that, compared with the Sham+Veh control group, the mRNA expression levels of myocardial hypertrophy markers in the myocardial tissue of mice in the TAC+Veh group were significantly increased. After gavage administration of different concentrations of Harp, the expression levels of the above markers in the myocardial tissue of mice in each administration group decreased in a dose-dependent manner, with the TAC+High Harp group showing the most significant reduction (P<0.05), which further verified the inhibitory effect of harbazoside on pathological myocardial hypertrophy in vivo.

[0054] Example 3: Harp gavage administration to patients with... Myh6 R404Q Effects on cardiac function in a mouse model of mutant hypertrophic cardiomyopathy (1) Myh6 R404Q Experimental grouping of mice with mutant hypertrophic cardiomyopathy model: Eight-week-old SPF-grade wild-type (WT) mice were selected and Myh6 R404Q Mice with a mutant hypertrophic cardiomyopathy model were randomly divided into the following four groups according to their body weight: WT+Veh control group (WT+Veh): wild-type mice were administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage daily; Myh6 R404Q Control group (R404Q+Veh): Myh6 R404Q Mice were administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage daily. Myh6 R404Q +Low Harp low-dose group (R404Q+Low Harp): Myh6 R404Q Mice were administered Harp solution (10 mg / kg, prepared with 0.5% sodium carboxymethyl cellulose solution) by gavage daily. Myh6 R404Q +High Harp high-dose group (R404Q+High Harp): Myh6 R404Q Mice were administered Harp solution (50 mg / kg, prepared with 0.5% sodium carboxymethyl cellulose solution) by gavage daily. Mice in each group were housed in an SPF-grade animal facility with free access to food and water, and were administered the drugs continuously for 8 weeks.

[0055] (2) Harp gavage administration to patients with... Myh6R404Q Effects of mutant hypertrophic cardiomyopathy model on heart weight and left ventricular wall thickness in mice: Calculated using the instrument's built-in analysis software Myh6 R404Q Mutant hypertrophic cardiomyopathy model mice and wild-type mice: interventricular septal thickness, left ventricular posterior wall thickness, and left ventricular diameter. like Figure 4 As shown, compared with the WT+Veh group, the R404Q+Veh group mice had significantly increased heart and left ventricular weights. P <0.05); both ejection fraction and shortening fraction increased significantly ( P <0.05), and the diastolic and systolic volumes of the left ventricle were significantly reduced ( P <0.05), the thickness of the anterior wall of the left ventricle during diastole and systole, and the thickness of the posterior wall of the left ventricle during diastole and systole were all significantly increased. P <0.05), and the diastolic and systolic diameters of the left ventricle both decreased significantly. P <0.05); the E / A peak ratio decreased significantly ( P <0.05); the above experimental results fully demonstrate Myh6 R404Q A mutant hypertrophic cardiomyopathy mouse model was successfully constructed.

[0056] After administration of Harp via gavage, compared with the R404Q+Veh group, Myh6 R404Q The weight of the heart and left ventricle in mice was significantly reduced. P <0.05); both ejection fraction and shortening fraction decreased significantly ( P <0.05), and the diastolic and systolic volumes of the left ventricle were significantly increased. P <0.05), the thickness of the left ventricular anterior wall during diastole and systole, and the thickness of the left ventricular posterior wall during diastole and systole were all significantly reduced. P <0.05), the left ventricular diastolic and systolic diameters were significantly increased, and the E / A peak ratio was significantly increased ( P <0.05). Echocardiographic results showed that Harp treatment significantly improved left ventricular structural remodeling, consistent with cardiac weighing results. Experimental results indicate that Harp gavage administration effectively inhibits... Myh6 R404Q The mutant mice exhibited increased heart and left ventricular weight, demonstrating a significant intervention effect on the progression of myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0057] Similarly, Harp gavage administration can effectively inhibit Myh6 R404Q Markers of myocardial hypertrophy in mutant mice Nppa , Nppb , β-Mhc The level of expression.

[0058] Example 4: TAC pressure-overload-induced pathological myocardial hypertrophy mice and Myh6 R404Q HE, Masson, and WGA staining of myocardial tissue from mutant mice TAC mice administered the drug via gavage in Example 2 and mice administered the drug via gavage in Example 3 Myh6 R404Q Mutant mice and wild-type (WT) control mice were used for myocardial fibrosis and cell morphology analysis. The specific steps are as follows: Immediately after mouse euthanasia, the entire body was perfused via the apex of the heart with 4% paraformaldehyde solution to remove residual blood from the myocardium. The heart was then completely removed and fixed overnight in 4% paraformaldehyde. The next day, the fixed tissue was dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare serial sections with a thickness of 5 μm. HE staining, Masson's trichrome staining, and WGA staining were then performed.

[0059] Hematoxylin and eosin (HE) stained sections were observed and images were acquired under an optical microscope to analyze the morphology and structure of tissue cells and pathological changes.

[0060] The sections were stained with Masson's trichrome, and images were observed and acquired under an optical microscope to analyze the degree of myocardial fibrosis (area of ​​blue collagen fibers) and the cross-sectional area of ​​myocardial cells.

[0061] WGA-stained sections were observed and images were acquired under a fluorescence microscope to analyze cell membrane structure, cell boundaries, and intercellular connections.

[0062] like Figure 3 , Figure 5 As shown, compared with the WT+Veh group, mice in the R404Q+Veh group and the TAC+Veh group showed significant cardiomyocyte hypertrophy (increased cell cross-sectional area) and myocardial fibrosis (increased area of ​​blue region), indicating successful model establishment. After oral administration of Harp, compared with the TAC+Veh group, Harp treatment significantly reduced collagen fiber deposition in the myocardial tissue of TAC mice. Myh6 R404Q In mice, Harp treatment significantly reduced [the risk of death] compared to the R404Q+Veh group after gavage administration. Myh6 R404Q Collagen fiber deposition in mouse myocardial tissue and a significant reduction in cardiomyocyte area ( P <0.05).

[0063] The results above demonstrate that Harp can effectively inhibit the progression of myocardial hypertrophy and fibrosis, and exhibits a clear cardioprotective effect in both stress overload and hereditary pathological models of myocardial hypertrophy. Combining evidence from heart weight, cardiac function parameters, and histopathological findings, Harp demonstrates significant anti-myocardial remodeling activity, providing a new strategy for the clinical treatment of hypertrophic and stress overload-induced ventricular remodeling.

[0064] In summary, intragastric administration of Harp effectively inhibited the progression of myocardial fibrosis and cardiomyocyte hypertrophy in two pathological myocardial hypertrophy models (stress overload and hereditary cardiomyopathy), suggesting its potential application in intervening in the early pathological progression of both pathological and hereditary myocardial hypertrophy. This invention, through in vitro and in vivo experiments, demonstrates the therapeutic effect of Harp on pathological myocardial hypertrophy, providing a new target and intervention strategy for drug development targeting both pathological and hereditary hypertrophic cardiomyopathy.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Harmazoside (C 15 H 24 O 10 Its application in the preparation of drugs for the treatment, adjuvant treatment, or prevention of pathological myocardial hypertrophy, characterized in that, The pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure overload, hereditary myocardial hypertrophy, and myocardial hypertrophy caused by inflammatory factors; preferably hereditary myocardial hypertrophy.

2. The application according to claim 1, characterized in that, The drug reduces cardiomyocyte area, improves left ventricular diastolic function, reduces left ventricular weight, improves myocardial fibrosis, and reduces the expression of markers of myocardial hypertrophy.

3. The application according to claim 2, characterized in that, The improvement in left ventricular diastolic function includes at least one of the following: (a) Increased diastolic diameter of the left ventricle; (b) Decreased diastolic thickness of the left ventricular anterior wall; (c) Decreased diastolic thickness of the left ventricular posterior wall; (d) Increased diastolic volume of the left ventricle.

4. The application according to claim 1, characterized in that, The dosage forms of the drug include oral dosage forms, parenteral dosage forms, and / or topical dosage forms.

5. The application according to claim 1, characterized in that, The dosage form of the drug is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

6. A pharmaceutical composition for treating or preventing pathological myocardial hypertrophy, characterized in that, The active ingredient of the pharmaceutical composition is harbazoside (C 15 H 24 O 10 ).

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

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of starch, lactose, microcrystalline cellulose, and magnesium stearate.

9. The use of the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a drug for treating, adjuvant treating, or preventing pathological myocardial hypertrophy, characterized in that, The pathological myocardial hypertrophy is selected from any one or more of the following: myocardial hypertrophy caused by pressure overload, hereditary myocardial hypertrophy, and myocardial hypertrophy caused by inflammatory factors; preferably hereditary myocardial hypertrophy.

10. The application according to claim 9, characterized in that, The pharmaceutical composition reduces cardiomyocyte area, improves left ventricular diastolic function, reduces left ventricular weight, improves myocardial fibrosis, and reduces the expression of markers of myocardial hypertrophy.

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