Use of darutoside in preparing drug for preventing and treating hereditary hypertrophic cardiomyopathy

Abstract

Use of darutoside in preparing a drug for preventing and treating hereditary hypertrophic cardiomyopathy. The darutoside is used for directly treating hypertrophic cardiomyocytes in vitro. It is found that the darutoside significantly reduces the area of cardiomyocytes, and reduces the increase of myocardial hypertrophy marker expression caused by angiotensin II; meanwhile, feed containing the darutoside is applied to feed Tnnt2R109Q mice, such that the weight of the left ventricle and heart of the mice is reduced, the heart volume and the thickness of the myocardial wall during diastole and systole are reduced, and myocardial tissue fibrosis of the mice with hypertrophic cardiomyopathy is effectively inhibited. The darutoside can be used for preparing a drug for preventing and treating hereditary hypertrophic cardiomyopathy and provides a new way and means for treating hypertrophic cardiomyopathy.

Description

Application of darutoside in the preparation of medicine for preventing and treating hereditary hypertrophic cardiomyopathy Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of darutoside in the preparation of medicines for preventing and treating hereditary hypertrophic cardiomyopathy. Background Art

[0002] Hypertrophic cardiomyopathy (HCM) is a cardiomyopathy characterized by myocardial hypertrophy, mainly caused by pathogenic variants in genes encoding sarcomere-related proteins or of unknown etiology. It has an insidious onset and a high incidence rate, and is the main cause of malignant arrhythmias and sudden cardiac death. The specific mechanism of hypertrophic cardiomyopathy is still unclear. Although Mavacamten (MYK-461) can partially improve hypertrophic cardiomyopathy caused by outflow tract obstruction by targeted inhibition of cardiac myosin, there is no original specific drug for hypertrophic cardiomyopathy in China. At present, surgical treatment is still the only option for relieving symptoms in critically ill patients, but the prognosis of surgical patients is often poor. Therefore, the development of drugs for the prevention and treatment of hypertrophic cardiomyopathy is of great significance.

[0003] Darutoside is a diterpene compound extracted from the Chinese herbal medicine Darutoside, and its chemical formula is: 26 H 44 O8. Studies have shown that darutoside can promote collagen synthesis during wound healing, accelerate wound healing, reduce inflammatory responses, and facilitate tissue repair. Darutoside also possesses antioxidant activity, neutralizing free radicals and protecting cells from oxidative stress, helping to maintain skin health. However, there are currently no reports on the use of darutoside in the preparation of drugs for the prevention and treatment of hypertrophic cardiomyopathy.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide the use of darutoside in the preparation of a medicament for preventing and / or treating hereditary hypertrophic cardiomyopathy.

[0006] On the one hand, the present invention uses Darutoside to directly treat cardiomyocytes (hESC-CMs) obtained by directed differentiation of human embryonic stem cells and the human cardiomyocyte cell line AC16 in vitro, and found that Darutoside treatment in vitro can significantly inhibit the hypertrophic phenotype of cardiomyocytes. On the other hand, the present invention uses feed containing Darutoside to feed mice with hereditary hypertrophic cardiomyopathy caused by gene mutations, and found that Darutoside significantly alleviates pathological myocardial hypertrophy in mice and improves cardiac function. The Darutoside described in the present invention can be used to prepare drugs for preventing and treating hereditary hypertrophic cardiomyopathy, providing a new approach and means for treating hypertrophic cardiomyopathy.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] Compared with the current in vitro research on Darutoside using animal-derived cell models, the present invention uses the directed differentiation of human embryonic stem cells to obtain human cardiomyocytes hESC-CMs and mature human cardiomyocyte cell line AC16, and establishes an in vitro human cardiomyocyte hypertrophy model. The in vitro human cardiomyocyte hypertrophy model can effectively avoid the situation where the research results do not match the development of human heart disease due to the huge differences between animal-derived cardiomyocytes and human cardiomyocytes. The present invention found that Darutoside can significantly inhibit the hypertrophic phenotype of both hESC-CMs and AC16 cells through direct in vitro treatment. In addition, a hypertrophic cardiomyopathy mouse model with the R109Q mutation of the Tnnt2 gene (Tnnt2 R109Q ), which is characterized by hereditary cardiac hypertrophy, increased heart area, and increased heart and left ventricular weight. R109Q After mice were fed a diet containing Darutoside, the symptoms of hereditary pathological cardiac hypertrophy were significantly improved.

[0009] The present invention uses Darutoside to directly treat hypertrophic cardiomyocytes in vitro and finds that Darutoside significantly reduces the area of ​​cardiomyocytes and reduces the increase in the expression of cardiac hypertrophy markers caused by angiotensin II. R109Q It can reduce the weight of the left ventricle and heart, the volume of the heart, and the thickness of the myocardial wall during diastole and systole, and effectively inhibit the myocardial fibrosis in mice with hypertrophic cardiomyopathy.

[0010] Through in vitro and in vivo studies, the present invention demonstrates that Darutoside has significant potential for treating hypertrophic cardiomyopathy and can be developed as a new anti-hypertrophic cardiomyopathy drug, providing a novel approach and method for treating hypertrophic cardiomyopathy. Furthermore, the present invention provides a new alternative and approach to current anti-hypertrophic cardiomyopathy drugs, broadening the scope of available anti-hypertrophic cardiomyopathy drugs and contributing to the advancement of this technical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the area of ​​cardiomyocytes after direct treatment with Darutoside in vitro; ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0012] Figure 2 shows the expression levels of cardiac hypertrophy markers in cardiomyocytes after direct treatment with Darutoside in vitro; ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0013] Figure 3 shows the difference between WT and Tnnt2 after feeding with Darutoside-containing diet. R109Q Mouse heart (left) and left ventricular weight (right); in the figure, ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0014] Figure 4 shows the results of cardiac ultrasound detection in mice, (first from the left) is the thickness of the left ventricular anterior wall during systole, (second from the left) is the thickness of the left ventricular anterior wall during diastole, (third from the left) is the thickness of the left ventricular posterior wall during systole, and (fourth from the left) is the thickness of the left ventricular posterior wall during diastole; in the figure, ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0015] Figure 5 shows the difference between WT and Tnnt2 after feeding with Darutoside-containing diet. R109Q Expression levels of myocardial hypertrophy markers ANP, BNP, and TNNT2 in mouse hearts. In the figure, ns indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0016] FIG6 is a diagram showing the results of Masson staining of mouse myocardial tissue; the blue area in the diagram indicates the degree of fibrosis. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] In the following examples, 8-week-old wild-type mice (WT) and Tnnt2 gene R109Q point mutation mice (Tnnt2 R109Q ) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; normal mouse feed was purchased from Jiangsu Collaborative Bioengineering Co., Ltd.; Darutoside was purchased from TargetMol (T3S1944).

[0021] Example 1

[0022] Effects of direct treatment with darutoside on cardiomyocyte hypertrophy in vitro

[0023] First, this example uses the human embryonic stem cell line MYL2 Neo / w -H7 differentiated cardiomyocytes, and then a large number of high-purity human cardiomyocytes were obtained through in vitro screening.

[0024] MYL2 Neo / w The specific steps for differentiating the H7 cell line into cardiomyocytes are as follows:

[0025] ① Place the cells in a 37°C, 5% CO2 incubator and culture them with mTeSR stem cell culture medium. Neo / w For H7 cells, when the cell density reached 85%-90%, the culture medium was changed to RPMI1640 / B27-no insulin medium and 8 μM Wnt signaling pathway agonist CHIR-99021 was added and cultured for 2 consecutive days;

[0026] ②On the third day, the culture medium was replaced with fresh RPMI1640 / B27-no insulin and the cells were cultured for 24 hours;

[0027] ③ On the 4th day, the culture medium was changed to RPMI1640 / B27-no insulin, and 5 μM Wnt signaling pathway inhibitor IWR-1 was added and cultured for 2 consecutive days;

[0028] ④ On the 6th day, the cells were washed twice with DPBS and the culture medium was replaced with RPMI1640 / B27-no insulin. The cells were cultured for 2 days. Beating cardiomyocytes could be observed starting on the 7th day of differentiation.

[0029] ⑤ After the 8th day, the culture medium was replaced with RPMI / B27-insulin medium and culture was continued for 14 days, with the medium changed every day;

[0030] ⑥ On day 14, the culture medium was replaced with RPMI / B27-insulin medium, and 50 μg / mL G418 antibiotic was added to select cardiomyocytes. The selection was continued for 7 days, and the medium and G418 were replaced every day.

[0031] This directed differentiation and screening method can be used to obtain a large number of highly pure human cardiomyocytes derived from human embryonic stem cells.

[0032] 1. Effect of Darutoside on Cardiomyocyte Area

[0033] Human cardiomyocytes (hESC-CMs) derived from human embryonic stem cell differentiation and AC16 cell lines were evenly seeded into 24-well plates containing a cell slide. After 24 hours of adherent growth, the cells were treated with PBS, AngⅡ (10μM), darutoside (1μM) + AngⅡ (10μM), darutoside (2.5μM) + AngⅡ (10μM), darutoside (5μM) + AngⅡ (10μM), and darutoside (10μM) + AngⅡ (10μM). These groups were treated with PBS, AngⅡ, and different concentrations of darutoside + AngⅡ. After 24 hours of treatment, cardiac troponin (cTNT) immunofluorescence staining was performed in each group.

[0034] The specific steps of the cardiac troponin (cTNT) immunofluorescence staining experiment are as follows:

[0035] ① After washing the treated cells twice with PBS (5 min / time), the cells were fixed with 4% paraformaldehyde for 20 min. After discarding the fixative, the cells were washed three times with PBS (5 min / time).

[0036] ② Treat the cells obtained in ① with 0.2% Triton-X100 for 5 minutes, and then wash with PBS three times (5 minutes / time).

[0037] ③ The cells obtained in ② were blocked with 5% BSA solution for 2 h, and then washed with PBS for 3 times (5 min / time).

[0038] ④ Incubate with cTNT antibody (1:400, diluted in PBS) at 4°C overnight, remove the antibody, and wash three times with PBS (5 min / time).

[0039] ⑤ Incubate with fluorescent anti-rabbit secondary antibody (1:500, diluted in PBS) for 1 hour. After incubation, remove the secondary antibody and wash with PBS three times (5 minutes each time).

[0040] ⑥ Incubate with DAPI (5 μg / ml, diluted in PBS) for 20 min. After incubation, remove the DAPI staining solution and wash with PBS three times (5 min / time).

[0041] ⑦ Finally, the slides were stained with an anti-fluorescence quencher and sealed, and fluorescence images were captured using a laser confocal microscope (Carl Zeiss LSM880, Germany). This model of fluorescence microscope has a function for measuring the area of ​​fluorescent staining, which can be used to display the area of ​​myocardial cells.

[0042] Cardiomyocytes obtained by directed differentiation for 6 different times were used, with 2 replicates each time. A cTNT-stained cell morphology image was randomly photographed, and the area of ​​all cardiomyocytes in the image was analyzed.

[0043] As shown in Figure 1, after treatment with 10 μM AngⅡ, the cell area of ​​both hESC-CMs and AC16 cardiomyocytes increased significantly. Compared with the model group, the cardiomyocyte area of ​​each group was significantly reduced after treatment with different concentrations of Darutoside.

[0044] 2. Effect of Darutoside on the Expression of Cardiac Hypertrophy Markers in Cells

[0045] Human cardiomyocytes (hESC-CMs) derived from directed differentiation of human embryonic stem cells and AC16 cell lines were evenly seeded into 12-well plates. After 24 hours of adherent growth, the cells were treated with PBS, AngⅡ (10μM), Darutoside (1μM) + AngⅡ (10μM), Darutoside (2.5μM) + AngⅡ (10μM), Darutoside (5μM) + AngⅡ (10μM), and Darutoside (10μM) + AngⅡ (10μM), respectively. These groups were treated with PBS, AngⅡ, and different concentrations of Darutoside + AngⅡ. After 24 hours of treatment, total RNA was extracted from the cells, and the expression of cardiac hypertrophy marker mRNA in the cardiomyocytes was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0046] ① Wash the treated cells twice with 4°C pre-chilled PBS. After aspirating the PBS, add 1 mL of Trizol®, a total RNA extraction reagent, to each well. After cell lysis, incubate at room temperature for 5 minutes to completely dissociate the nucleic acid-protein complex. After incubation, transfer the tube to a sterile EP tube and centrifuge at 4°C (12,000 rpm) for 5 minutes. Collect the supernatant.

[0047] ② Add 200 μL of chloroform to the supernatant obtained in step ①, shake and centrifuge at 4°C for 15 minutes, aspirate the colorless aqueous phase (RNA is in this layer), and transfer it to a new centrifuge tube.

[0048] ③ Add an equal amount of isopropanol to the solution obtained in step ②, vortex to mix, and centrifuge at low temperature for 15 minutes. A clear RNA precipitate will be visible at the bottom of the tube. Discard the solution and slowly add 1 mL of 75% alcohol along the tube wall to wash the RNA precipitate. Centrifuge for 15 minutes, discard the solution, and invert the tube to remove excess alcohol.

[0049] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve the RNA in a 55°C metal bath, then measure the RNA concentration and directly perform reverse transcription on the sample.

[0050] ⑤ Reverse transcription: Prepare a 10 μL system with 2 μL of reverse transcriptase (5× Prime Script RT Mix), 1 μg of RNA, and RNase-free ddH₂O to make up to 10 μL. The resulting cDNA sample was diluted 10-fold with ddH₂O and used directly for quantitative real-time polymerase chain reaction (qRT-PCR).

[0051] ⑥qRT-PCR: Prepare a 10 μL reaction system: 0.2 μL PCR Forward Primer, 0.2 μL PCR Reverse Primer, 1 μL cDNA, 5 μL SyBR Premix EX Taq (2×), and 3.6 μL ddH2O.

[0052] The experiment used 6 different batches of differentiated cardiomyocytes and repeated 6 in vitro treatments. For qRT-PCR detection, 3 replicates were set for each sample. Data are expressed as mean ± standard error (mean ± SEM). β-ACTIN gene was used to detect the expression of internal reference genes. Myocardial hypertrophy markers mainly include ANP, BNP, and TNNT2. Among them, the primer sequences of β-ACTIN, ANP, BNP and TNNT2 are shown in Table 1. -△△Ct The results were analyzed by relative quantitative analysis, and the test results are shown in Figure 2.

[0053] Table 1. Primer sequences for human cardiomyocyte hypertrophy markers

[0054] As can be seen from Figure 2, AngⅡ treatment led to a significant increase in the expression of cardiac hypertrophy markers in both hESC-CMs and AC16 cardiomyocytes. Darutoside directly treated in vitro effectively reduced the expression of cardiac hypertrophy markers, indicating that Darutoside has a physiological effect against cardiac hypertrophy.

[0055] Example 2

[0056] Effects of feeding diet containing Darutoside on mice with hypertrophic cardiomyopathy

[0057] During the progression of hypertrophic cardiomyopathy, the heart becomes pathologically thickened and the ventricular wall thickness increases, leading to abnormal increases in heart weight and left ventricular weight. R109Q Mice were fed a diet containing Darutoside to investigate the effect of Darutoside on the progression of cardiac hypertrophy in vivo.

[0058] Eight-week-old wild-type mice (WT) and mice with hypertrophic cardiomyopathy caused by the R109Q point mutation of the Tnnt2 gene (Tnnt2 R109Q ), WT and Tnnt2 R109Q The mice were randomly divided into five groups. Then the five groups of mice were fed with normal feed and mixed feed containing different concentrations of Darutoside to obtain the WT group, Tnnt2 R109Q group, positive drug metoprolol treatment group, low-dose Darutoside treatment group and high-dose Darutoside treatment group.

[0059] in:

[0060] WT group: wild-type mice (WT) fed with normal chow;

[0061] Tnnt2 R109Q Group: Tnnt2 R109Q Mice were fed a normal diet;

[0062] Positive drug metoprolol treatment group: Tnnt2 R109Q Mice were fed a diet containing metoprolol, in which the drug concentration was 0.05%;

[0063] Low-dose Darutoside-treated group: Tnnt2 R109Q Mice were fed a diet containing Darutoside at a drug concentration of 0.02%;

[0064] High-dose Darutoside-treated group: Tnnt2 R109Q Mice were fed a diet containing Darutoside at a drug concentration of 0.1%;

[0065] Mice were housed in an SPF animal room.

[0066] 1. Effects on heart and left ventricular weight in mice with hypertrophic cardiomyopathy

[0067] After four weeks of feeding, mice were bled via the eyeballs and then sacrificed by cervical dislocation. The hearts were quickly removed and dried, and the hearts were weighed. RNA was also extracted from the left ventricle for later use. In addition, the mice were observed using echocardiography to measure the weight of the left ventricle.

[0068] The steps for a cardiac echocardiogram are as follows:

[0069] ① Prepare the hair on the mouse's abdomen and chest, then anesthetize the mouse with 1.5-2% isoflurane gas flow rate, control the mouse's heart rate to be stable within 430-480 beats / min, and perform the test.

[0070] ② Ultrasound examination was performed using the Vevo3100 high-resolution in vivo imaging system (Vevo3100LT, Canada). The heart was quickly located and images of the long and short axes of the mouse heart, along with relevant parameters, were recorded in both the B-model and M-model. Left ventricular mass was calculated using the Vevo3100 system.

[0071] As can be seen from the left graph (heart weight / tibia length) and right graph (left ventricular weight) in Figure 3, compared with the WT mouse heart, Tnnt2 R109Q The weight of the heart and left ventricle of mice increased abnormally, while feeding the diet containing Darutoside significantly reduced Tnnt2 R109Q Mouse heart and left ventricular weights.

[0072] 2. Effect on left ventricular wall thickness in mice with hypertrophic cardiomyopathy

[0073] After four weeks of feeding, mice were subjected to echocardiography to determine whether feeding a diet containing darutoside improved the pathological hypertrophic phenotype of the hearts of mice with hypertrophic cardiomyopathy. Systolic left ventricular anterior wall thickness (LVAW;s), diastolic left ventricular anterior wall thickness (LVAW;d), systolic left ventricular posterior wall thickness (LVPW;s), and diastolic left ventricular posterior wall thickness (LVPW;d) were calculated using the Vevo 3100 system.

[0074] As can be seen from Figure 4, Tnnt2 R109QThe left ventricular wall thickness of mice during diastole and systole was significantly higher than that of WT mice, and feeding the diet containing Darutoside significantly reduced Tnnt2 R109Q This indicates that Darutoside has a beneficial effect in counteracting the progression of pathological myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0075] 3. Effects on myocardial hypertrophy markers in mice with hypertrophic cardiomyopathy

[0076] In this example, the WT and Tnnt2 mice fed with Darutoside-containing diet were detected. R109Q The effect of Darutoside on the progression of myocardial hypertrophy was investigated by measuring the expression of myocardial hypertrophy markers in the left ventricular tissue of mice. Mice were raised using the above method, and RNA was extracted from the left ventricle of the heart. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of myocardial hypertrophy marker mRNA in cardiomyocytes. The specific steps are as follows:

[0077] ① Take 50 mg of mouse left ventricular tissue sample and place it in a sterile EP tube. Add 1 mL of Trizol to each tube and then use a tissue disruptor to disrupt the tissue. Centrifuge at 4°C for 5 minutes and collect the supernatant to obtain the mouse myocardial tissue sample, i.e., the mouse myocardial tissue supernatant.

[0078] ② Add 200 μL of chloroform to the supernatant obtained in step ①, shake and centrifuge at 4°C for 15 minutes, aspirate the colorless aqueous phase (RNA is in this layer), and transfer it to a new centrifuge tube.

[0079] ③ Add an equal amount of isopropanol to the solution obtained in step ②, vortex to mix, and centrifuge at low temperature for 15 minutes. A clear RNA precipitate will be visible at the bottom of the tube. Discard the solution and slowly add 1 mL of 75% alcohol along the tube wall to wash the RNA precipitate. Centrifuge for 15 minutes, discard the solution, and invert the tube to remove excess alcohol.

[0080] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve the RNA in a 55°C metal bath, then measure the RNA concentration and directly perform reverse transcription on the sample.

[0081] ⑤ Reverse transcription: Prepare a 10 μL system with 2 μL of reverse transcriptase (5× Prime Script RT Mix), 1 μg of RNA, and RNase-free ddH2O to make up to 10 μL. Reverse transcriptase yields a cDNA sample, which is diluted 10-fold with ddH2O and used directly for qRT-PCR.

[0082] ⑥qRT-PCR: Prepare 10 μL reaction system: 0.2 μL PCR Forward Primer, 0.2 μL PCR Reverse Primer, 1 μL cDNA, 5 μL SyBR Premix EX Taq (2×), 3.6 μL ddH2O. Set up 3 replicates for each sample. The main markers of mouse myocardial hypertrophy include Anp and Bnp. The 18S gene is used to detect the expression of the internal reference gene. -△△Ct The results were analyzed by relative quantitative analysis. The sequences of the detected gene primers are shown in Table 2.

[0083] Table 2. Primer sequences for mouse myocardial hypertrophy markers

[0084] As can be seen from Figure 5, Tnnt2 R109Q The expression of hypertrophy markers such as Anp and Bnp in myocardial tissue of mice was significantly higher than that in WT mice. Darutoside significantly inhibited the expression of Anp and Bnp in Tnnt2 R109Q This indicates that Darutoside has great potential for the treatment of hereditary hypertrophic cardiomyopathy.

[0085] 3. Masson staining of myocardial tissue

[0086] After the mice raised using the above method were killed by cervical dislocation, they were quickly perfused with 4% paraformaldehyde solution to flush out the residual blood in the myocardial tissue. The mouse heart was removed and placed in 4% paraformaldehyde and fixed overnight. The fixed heart was dehydrated, transparent and paraffin-embedded. After embedding, paraffin sections (5 μm) were made. The sections were then stained with Masson's trichrome staining kit (Sevier Bio, G1006) as follows:

[0087] ① Soak the slices in solution A at room temperature overnight (about 15 hours).

[0088] ② Mix equal volumes of Solution B and Solution C (prepare for immediate use), immerse the sections in the mixture of Solution A and Solution B for 1 minute, rinse briefly with running water, and then differentiate in 1% hydrochloric acid alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) for 1 minute until the cell nuclei appear gray-black and the background is almost colorless or light gray.

[0089] ③ Rinse briefly with running water, drain any excess water from the sections, and immerse the sections in Solution D for 6 minutes, until the tissue appears bright red. Slightly drain the sections (do not allow them to dry out) and immediately immerse them in Solution E for approximately 1 minute. This step is the differentiation step, which should proceed until the collagen fibers appear light red and then red. This step takes approximately 1-2 minutes.

[0090] ④ After draining liquid E slightly, the slices were directly stained with liquid F for 2-30 seconds without washing.

[0091] ⑤ Rinse and differentiate the sections in three consecutive cylinders of 1% glacial acetic acid, each for approximately 8 seconds. Dehydrate the sections in three consecutive cylinders of anhydrous ethanol for approximately 5 seconds, 10 seconds, and 30 seconds, respectively. Dehydrate the sections in two cylinders of n-butanol for 30 seconds and 2 minutes, respectively.

[0092] ⑥Finally, the slides were transparentized in two cylinders of xylene for 5 minutes each time, sealed with neutral gum, and photographed under an optical microscope after being completely dried. The results are shown in Figure 6.

[0093] As can be seen from Figure 6, HCM mice showed significantly increased myocardial fibrosis compared to normal mice, and the area of ​​myocardial cells was also significantly increased. Darutoside significantly inhibited Tnnt2 R109Q Darutoside inhibited the progression of myocardial fibrosis in mice and effectively reduced the area of ​​cardiomyocytes, suggesting that it may have great potential in treating the early progression of hypertrophic cardiomyopathy.

[0094] In summary, the present invention found in vitro that Darutoside effectively prevents the development of human cardiomyocyte hypertrophy; in vivo studies found that Darutoside effectively inhibits the progression of hereditary pathological cardiac hypertrophy and effectively inhibits the occurrence of myocardial fibrosis. Both in vitro and in vivo experiments demonstrated that Darutoside can significantly improve disease progression in mice with hereditary hypertrophic cardiomyopathy and could be developed as a new anti-hypertrophic cardiomyopathy drug, providing a new approach and means for the treatment of hypertrophic cardiomyopathy.

[0095] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. Application of darutoside in the preparation of drugs for preventing and treating hereditary hypertrophic cardiomyopathy.

2. The use according to claim 1, characterized in that The darutoside reduces pathological cardiac hypertrophy, heart weight or increased left ventricular weight.

3. The use according to claim 1, characterized in that The darutoside reduces the elevation of hypertrophy markers in myocardial tissue.

4. The use according to claim 1, characterized in that The darutoside improves the enlargement of myocardial cell area and / or the increase of myocardial fibrosis level.

5. The use according to claim 1, characterized in that The medicine is a pharmaceutical composition consisting of an active ingredient, darutoside, and pharmaceutically acceptable excipients.

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