Use of quercetagetin in preparation of a drug for preventing and treating hypertrophic cardiomyopathy

Abstract

The application discloses application of Darutoside in preparation of a medicine for preventing and treating genetic hypertrophic cardiomyopathy. R109Q The application adopts Darutoside to directly treat hypertrophic cardiomyocytes in vitro, finds that Darutoside can significantly reduce the area of the cardiomyocytes, and reduce the increase of the expression of hypertrophic cardiomyopathy markers caused by angiotensin II; meanwhile, the Tnnt2 Mice are fed with the feed containing Darutoside, the weight of the left ventricle and the heart of the mice is reduced, the volume of the heart is reduced, the thickness of the myocardial wall in the diastolic and systolic phases is reduced, and the myocardial tissue fibrosis of the hypertrophic cardiomyopathy mice is effectively inhibited. The Darutoside can be used for preparing the medicine for preventing and treating the genetic hypertrophic cardiomyopathy, and provides a new approach and means for treating the hypertrophic cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of darutoside in the preparation of drugs for the prevention and treatment of hereditary hypertrophic cardiomyopathy. Background Technology

[0002] Hypertrophic cardiomyopathy (HCM) is a cardiomyopathy characterized by myocardial hypertrophy, primarily caused by pathogenic mutations in genes encoding sarcomere-related proteins, or of unknown etiology. It has an insidious onset and high incidence, and is a major cause of malignant arrhythmias and sudden cardiac death. The specific mechanisms of HCM are not yet fully understood. Although Mavacamten (MYK-461) can partially improve HCM caused by outflow tract obstruction by targeting and inhibiting cardiac myosin, there are currently no original, effective drugs for HCM in China. Currently, surgery remains the only option for symptom relief in severely ill patients, but the prognosis for surgical patients is often poor. Therefore, the development of drugs for the prevention and treatment of HCM is of great significance.

[0003] Darutoside is a diterpenoid compound extracted from the traditional Chinese medicine Siegesbeckia orientalis. Its chemical formula is: C 26 H 44 O8. Studies have shown that Darutoside can promote collagen synthesis during wound healing, accelerate wound healing, reduce inflammatory responses, and benefit tissue repair. Darutoside also has antioxidant activity, neutralizing free radicals, protecting cells from oxidative stress damage, and helping to maintain healthy skin. However, there are currently no reports of Darutoside being used in the preparation of drugs for the prevention and treatment of hypertrophic cardiomyopathy. Summary of the Invention

[0004] The purpose of this invention is to provide the use of darutoside in the preparation of medicaments for the prevention and / or treatment of hereditary hypertrophic cardiomyopathy.

[0005] On the one hand, this invention uses Darutoside to directly treat cardiomyocytes (hESC-CMs) obtained from 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, this invention feeds mice with hereditary hypertrophic cardiomyopathy caused by gene mutations with Darutoside-containing feed, and found that Darutoside significantly alleviates pathological myocardial hypertrophy and improves cardiac function in mice. The Darutoside described in this invention can be used to prepare drugs for the prevention and treatment of hereditary hypertrophic cardiomyopathy, providing a new approach and means for the treatment of hypertrophic cardiomyopathy.

[0006] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0007] Compared to current in vitro studies using animal-derived cell models for Darutoside research, this invention utilizes directed differentiation of human embryonic stem cells to obtain human cardiomyocytes (hESC-CMs) and the mature human cardiomyocyte line AC16, establishing an in vitro human cardiomyocyte hypertrophy model. This in vitro human cardiomyocyte hypertrophy model effectively avoids the mismatch between research results and human heart disease development caused by the significant differences between animal-derived and human cardiomyocytes. This invention, through direct in vitro treatment with Darutoside, has shown that it significantly inhibits the hypertrophic phenotype of both hESC-CMs and AC16 cells. Furthermore, a hypertrophic cardiomyopathy mouse model with the Tnnt2 gene R109Q mutation (Tnnt2) was successfully constructed using gene editing technology. R109Q This model is characterized by hereditary myocardial hypertrophy, increased cardiac area, and increased weight of the heart and left ventricle. (Tnnt2) R109Q Feeding mice with Darutoside significantly improved the symptoms of hereditary pathological myocardial hypertrophy.

[0008] This invention utilizes Darutoside to directly treat hypertrophic cardiomyocytes in vitro. It was found that Darutoside significantly reduces cardiomyocyte area and decreases the increase in angiotensin II-induced markers of cardiomyocyte hypertrophy. Tnnt2 cells were fed a diet containing Darutoside. R109Q In mice, it reduced the weight of the left ventricle and heart, decreased heart volume and myocardial wall thickness during diastole and systole, and effectively inhibited myocardial fibrosis in hypertrophic cardiomyopathy mice.

[0009] This invention, through in vitro and in vivo studies, has revealed that Darutoside possesses significant potential for treating hypertrophic cardiomyopathy (HCM) and can be developed as a novel anti-HCM drug, providing a new approach and method for treating HCM. Furthermore, this invention offers entirely new options and ideas for current anti-HCM drugs, broadening the selection range and contributing to the development of this technological field. Attached Figure Description

[0010] Figure 1 The figure shows the area of ​​cardiomyocytes after direct in vitro treatment with Darutoside; where ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0011] Figure 2The graph shows the expression levels of markers of myocardial hypertrophy in cardiomyocytes after direct in vitro treatment with Darutoside; where ns indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0012] Figure 3 WT and Tnnt2 after feeding with feed containing Darutoside R109Q Mouse heart weight / tibia length (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.

[0013] Figure 4 The results are shown in the echocardiogram of mice. (Top left) represents the thickness of the anterior wall of the left ventricle during systole, (bottom left) represents the thickness of the anterior wall of the left ventricle during diastole, (top right) represents the thickness of the posterior wall of the left ventricle during systole, and (bottom right) represents the thickness of the posterior wall of the left ventricle 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.

[0014] Figure 5 WT and Tnnt2 after feeding with feed containing Darutoside R109Q Expression levels of ANP and BNP, markers of myocardial hypertrophy, 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.

[0015] Figure 6 This is a diagram showing the results of Masson staining of mouse myocardial tissue; the blue area in the diagram represents the degree of fibrosis. Detailed Implementation

[0016] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] In the following examples, 8-week-old wild-type mice (WT) and Tnnt2 gene R109Q point mutant mice (Tnnt2) were used. R109Q All mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; normal mouse feed was purchased from Jiangsu Xietong Bioengineering Co., Ltd.; Darutoside was purchased from TargetMol (T3S1944).

[0020] Example 1

[0021] Effects of direct in vitro treatment with Darutoside on cardiomyocyte hypertrophy

[0022] First, this embodiment utilizes 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.

[0023] The MYL2 Neo / w The specific steps by which the H7 cell line differentiates into cardiomyocytes are as follows:

[0024] ① Place the cells in a 37℃, 5% CO2 incubator and culture MYL2 cells in mTeSR stem cell culture medium. Neo / w -H7 cells, when the cell density reaches 85%-90%, change the culture medium to RPMI1640 / B27-no insulin medium and add 8 μMWnt signaling pathway agonist CHIR-99021, and culture continuously for 2 days;

[0025] ②On the third day, the culture medium was replaced with a new RPMI1640 / B27-no insulin, and the cells were cultured for 24 hours;

[0026] 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. The medium was cultured for 2 consecutive days.

[0027] ④ After washing the cells twice with DPBS on day 6, replace the culture medium with RPMI1640 / B27-no insulin and culture for 2 days. On day 7 of differentiation, beating cardiomyocytes can be observed.

[0028] ⑤ After the 8th day, replace the culture medium with RPMI / B27-insulin medium and continue culturing for 14 days, changing the medium daily;

[0029] ⑥ On day 14, the culture medium was changed to RPMI / B27-insulin medium, and 50 μg / mL G418 antibiotic was added to screen cardiomyocytes. Screening was carried out for 7 consecutive days, with the medium and new G418 added every day.

[0030] This directed differentiation and screening method can yield a large number of high-purity human cardiomyocytes derived from human embryonic stem cells.

[0031] 1. Effect of Darutoside on cardiomyocyte area

[0032] Human cardiomyocytes (hESC-CMs) and AC16 cell lines obtained from directed differentiation of human embryonic stem cells were uniformly seeded into 24-well plates containing a cell spreader. After 24 h of cell adhesion and growth, the cells were treated with PBS, Ang II (10 μM), Darutoside (1 μM) + Ang II (10 μM), Darutoside (2.5 μM) + Ang II (10 μM), Darutoside (5 μM) + Ang II (10 μM), and Darutoside (10 μM) + Ang II (10 μM), respectively, to obtain PBS treatment group, Ang II treatment group, and different concentrations of Darutoside + Ang II treatment group. After 24 h of treatment, cardiac troponin (cTNT) immunofluorescence staining experiments were performed on each group.

[0033] The specific steps of the cardiac troponin (cTNT) immunofluorescence staining experiment are as follows:

[0034] ①After washing the cells twice with PBS (5 min each time), fix the cells with 4% paraformaldehyde for 20 min, discard the fixative, and then wash them three times with PBS (5 min each time).

[0035] ② Treat the cells obtained in ① with 0.2% Triton-X100 for 5 min, and then wash with PBS 3 times (5 min each time).

[0036] ③ Block the cells obtained in ② with 5% BSA solution for 2 h, and wash with PBS 3 times (5 min / time) after blocking.

[0037] ④ Incubate cTNT antibody (1:400, diluted with PBS) at 4ºC overnight, remove the antibody, and wash 3 times with PBS (5 min each time).

[0038] ⑤ Incubate with fluorescent anti-rabbit secondary antibody (1:500, diluted with PBS) for 1 h. After incubation, remove the secondary antibody and wash with PBS 3 times (5 min / time).

[0039] ⑥ Incubate with DAPI (5 μg / ml, diluted with PBS) for 20 min. After incubation, remove the DAPI staining solution and wash with PBS 3 times (5 min each time).

[0040] ⑦ Finally, the slides were stained with an anti-fluorescence quencher and mounted. Fluorescence images were then captured using a laser confocal microscope (Carl Zeiss LSM880, Germany). This model of fluorescence microscope has the function of measuring the area of ​​fluorescence staining, which can be used to display the area of ​​cardiomyocytes.

[0041] Cardiomyocytes obtained from six different time-directed differentiations were used, with two replicates per well. A cTNT-stained image of cell morphology was randomly taken, and the area of ​​all cardiomyocytes in the image was analyzed.

[0042] like Figure 1 As shown, treatment with 10 μM Ang II significantly increased the cell area of ​​both hESC-CMs and AC16 cardiomyocytes. Compared with the model group, treatment with different concentrations of Darutoside significantly reduced the cell area of ​​cardiomyocytes in all groups.

[0043] 2. Effects of Darutoside on the expression of myocardial hypertrophy markers in cells

[0044] Human cardiomyocytes (hESC-CMs) and AC16 cell lines obtained from directed differentiation of human embryonic stem cells were uniformly seeded into 12-well plates. After 24 h of adherent growth, the cells were treated with PBS, Ang II (10 μM), Darutoside (1 μM) + Ang II (10 μM), Darutoside (2.5 μM) + Ang II (10 μM), Darutoside (5 μM) + Ang II (10 μM), and Darutoside (10 μM) + Ang II (10 μM), respectively, to obtain PBS treatment group, Ang II treatment group, and different concentrations of Darutoside + Ang II treatment group. After 24 h of treatment, total RNA was extracted from the cells, and the expression of mRNA markers of myocardial hypertrophy in cardiomyocytes was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0045] ① After treatment, wash the cells twice with PBS pre-cooled to 4°C. After aspirating the PBS, add 1 mL of Trizol total RNA extraction reagent to each well. After cell lysis, incubate at room temperature for 5 min to allow complete separation of nucleic acid-protein complexes. After incubation, transfer all cells to sterile EP tubes and centrifuge at 4°C (12,000 rpm) for 5 min, and collect the supernatant.

[0046] ② Add 200 μL of chloroform to the supernatant obtained in step ①, vortex, centrifuge at 4ºC for 15 min, aspirate the colorless aqueous phase (RNA is in this layer), and transfer it to a new centrifuge tube.

[0047] ③ Add an equal amount of isopropanol to the liquid obtained in step ②, vortex to mix, and centrifuge at low temperature for 15 min. A clear RNA precipitate will be visible at the bottom of the tube. Discard the liquid, slowly add 1 mL of 75% ethanol along the tube wall to wash the RNA precipitate, centrifuge for 15 min, discard the liquid, and invert the test tube to remove excess ethanol.

[0048] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve the RNA in a 55℃ metal bath, then measure the RNA concentration, and directly perform reverse transcription on the sample.

[0049] ⑤ Reverse transcription: Prepare a 10 μL system including: 2 μL reverse transcriptase (5× Prime Script RT MacterMix), 1 μg RNA, and RNase-free ddH2O to bring the total to 10 μL. The resulting cDNA sample is diluted 10-fold with ddH2O and used directly for quantitative real-time polymerase chain reaction (qRT-PCR).

[0050] 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.

[0051] Six batches of differentiated cardiomyocytes from different groups were used in the experiment, and the in vitro treatment was repeated six times. For qRT-PCR detection, each sample was tested in triplicate. Data are expressed as mean ± standard error (mean ± SEM). β-ACTIN gene expression was used to detect the expression of internal reference genes. The main markers of myocardial hypertrophy include ANP, BNP, and TNNT2. The primer sequences for β-ACTIN, ANP, BNP, and TNNT2 are shown in Table 1. -△△Ct The results were analyzed using relative quantitative analysis, and the test results were as follows: Figure 2 As shown.

[0052] Table 1. Primer sequences for human cardiomyocyte hypertrophy markers

[0053]

[0054] from Figure 2 It can be seen that Ang II treatment significantly increased the expression of two cardiomyocyte hypertrophy markers in hESC-CMs and AC16, while Darutoside direct treatment in vitro effectively reduced the expression of cardiomyocyte hypertrophy markers, indicating that Darutoside has a physiological effect against cardiomyocyte hypertrophy.

[0055] Example 2

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

[0057] In the progression of hypertrophic cardiomyopathy, pathological thickening of the heart and increased ventricular wall thickness lead to abnormal increases in both heart and left ventricular weight. This example demonstrates the effects of these factors on wild-type mice (WT) and Tnnt2 mice. R109Q Mice were fed a diet containing Darutoside to investigate the effect of Darutoside on the progression of myocardial hypertrophy in vivo.

[0058] Eight-week-old wild-type mice (WT) and mice with hypertrophic cardiomyopathy caused by the R109Q point mutation in the Tnnt2 gene were selected. R109Q ), WT and Tnnt2 R109Q Mice were randomly divided into five groups. The five groups were then fed either a normal diet or a mixed diet containing different concentrations of Darutoside, resulting in the WT group, Tnnt2 group, and [other groups]. R109Q The groups were: a positive control group treated with metoprolol, a low-dose Darutoside group, and a high-dose Darutoside group.

[0059] in:

[0060] WT group: Wild-type mice (WT) were fed a normal diet;

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

[0062] Positive drug metoprolol treatment group: Tnnt2 R109Q Mice were fed diets containing metoprolol at a concentration of 0.05%.

[0063] Low-dose Darutoside treatment group: Tnnt2 R109Q Mice were fed a diet containing 0.02% Darutoside.

[0064] High-dose Darutoside treatment group: Tnnt2 R109Q Mice were fed a diet containing 0.1% Darutoside;

[0065] Mice were housed in an SPF-grade animal facility.

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

[0067] After four weeks of rearing, blood was collected from the mice via eyeballs. The mice were then euthanized by cervical dislocation, and the hearts were removed as quickly as possible. Surface moisture was absorbed, the hearts were weighed, and RNA was extracted from the left ventricle for later use. Additionally, left ventricular weight was determined using mouse echocardiography.

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

[0069] ① The mice were shaved in the abdomen and chest cavity, and then anesthetized with isoflurane gas at a flow rate of 1.5-2%. The heart rate of the mice was kept stable at 430-480 beats / min before testing.

[0070] ② Ultrasound examination was performed using the Vevo3100 high-resolution in vivo imaging system (Vevo3100LT, Canada). The heart was quickly located, and images and related parameters of the mouse heart's long and short axes were recorded in B-model and M-model. The left ventricular weight was calculated using the Vevo3100 system.

[0071] from Figure 3 The left and right images (heart weight / tibia length) show that, compared to the WT mouse heart, the Tnnt2... R109Q The weight of the mouse heart and left ventricle was abnormally increased, while feeding the mouse with a diet containing Darutoside significantly reduced Tnnt2. R109Q Mouse heart and left ventricle weight.

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

[0073] After feeding mice using the above method for 4 weeks, echocardiography was performed to detect whether feeding them a diet containing Darutoside improved the pathological hypertrophic phenotype of the heart in 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 Vevo3100 system.

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

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

[0076] This embodiment measures the WT and Tnnt2 of feed containing Darutoside. R109Q The expression of myocardial hypertrophy markers in mouse left ventricular tissue was used to investigate the effect of Darutoside on the progression of myocardial hypertrophy in vivo. Mice were fed using the above method, and then RNA was extracted from the left ventricular tissue. The expression of myocardial hypertrophy marker mRNA in cardiomyocytes was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0077] ① Take 50 mg of mouse left ventricular tissue sample into a sterile EP tube, add 1 mL of Trizol to each tube, and then use a tissue disruptor to disrupt the tissue. Centrifuge at 4ºC for 5 min and take the supernatant to obtain mouse myocardial tissue sample, i.e., mouse myocardial tissue supernatant.

[0078] ② Add 200 μL of chloroform to the supernatant obtained in step ①, vortex, centrifuge at 4ºC for 15 min, 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 liquid obtained in step ②, vortex to mix, and centrifuge at low temperature for 15 min. A clear RNA precipitate will be visible at the bottom of the tube. Discard the liquid, slowly add 1 mL of 75% ethanol along the tube wall to wash the RNA precipitate, centrifuge for 15 min, discard the liquid, and invert the test tube to remove excess ethanol.

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

[0081] ⑤ Reverse transcription: Prepare a 10 μL system including: 2 μL reverse transcriptase (5× Prime Script RT MacterMix), 1 μg RNA, and RNase-free ddH2O to bring the total to 10 μL. Dilute the resulting cDNA sample 10-fold with ddH2O and use it directly for qRT-PCR.

[0082] ⑥ qRT-PCR: Prepare a 10 μL reaction mixture: 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. Each sample was tested in triplicate. Hypertrophy markers in mouse myocardial tissue mainly include Anp and Bnp. The 18S gene was used to detect the expression of the internal reference gene. -△△CtThe results were analyzed using relative quantitative analysis. The primer sequences of the detected genes are shown in Table 2.

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

[0084]

[0085] from Figure 5 As can be seen from this, Tnnt2 R109Q The expression of hypertrophy markers such as Anp and Bnp in mouse myocardial tissue was significantly higher than that in WT mice. Darutoside significantly inhibited the expression of Anp and Bnp in Tnnt2. R109Q The expression of Darutoside in the mouse heart suggests that Darutoside has great potential for treating hereditary hypertrophic cardiomyopathy.

[0086] 3. Examination of Masson staining in myocardial tissue

[0087] Mice fed using the above method were euthanized by cervical dislocation, and then rapidly perfused with 4% paraformaldehyde solution to flush out residual blood from the myocardial tissue. The mouse hearts were removed and placed in 4% paraformaldehyde for fixation overnight. The fixed hearts were then dehydrated, cleared, and embedded in paraffin. After embedding, paraffin sections (5 μm) were prepared. Masson staining was then performed on the sections using a Masson trichrome staining kit (Sevier Biosciences, G1006), as follows:

[0088] ① Soak the slices in solution A at room temperature overnight (about 15 h).

[0089] ② Mix equal volumes of solution B and solution C (prepare fresh for immediate use), immerse the slices in the mixture of A and B for 1 min, rinse briefly with running water, and then differentiate for 1 min with 1% hydrochloric acid alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) until the cell nuclei are grayish-black and the background is almost colorless or light gray.

[0090] ③ Rinse briefly with running water, drain excess water from the sections, and immerse the sections in solution D for 6 minutes. At this point, the tissue will turn bright red. Drain the sections slightly (do not let them dry), and immediately immerse them in solution E for about 1 minute. This step is for differentiation. Differentiation continues until the collagen fibers turn light red and the fibers turn red, about 1-2 minutes.

[0091] ④ After slightly draining the E solution, immerse the slide directly in the F solution for staining for 2-30 seconds without rinsing with water.

[0092] ⑤ The slices were washed and differentiated in three consecutive tanks of 1% glacial acetic acid for about 8 seconds each. Then they were dehydrated in three consecutive tanks of anhydrous ethanol for about 5 seconds, 10 seconds, and 30 seconds respectively. Finally, they were dehydrated in two tanks of n-butanol for 30 seconds and 2 minutes respectively.

[0093] ⑥ Finally, the slides were cleared with xylene in two baths, 5 minutes each time, then sealed with neutral resin. After complete drying, the slides were photographed under an optical microscope, and the results were examined as follows. Figure 6 As shown.

[0094] from Figure 6 It can be seen that hypertrophic cardiomyopathy mice showed significantly increased myocardial fibrosis compared to normal mice, and the area of ​​cardiomyocytes was also significantly increased. Darutoside significantly inhibited Tnnt2. R109Q It modulates the progression of myocardial fibrosis in mice and effectively reduces the area of ​​cardiomyocytes. This suggests that Darutoside may have great potential for treating the early progression of hypertrophic cardiomyopathy.

[0095] In summary, this invention has demonstrated in in vitro experiments that Darutoside effectively inhibits the development and progression of hypertrophic cardiomyocytes in human cells; and in in vivo studies, Darutoside effectively inhibits the progression of hereditary pathological cardiomyopathy and the occurrence of myocardial fibrosis. Both in vitro and in vivo experiments show that Darutoside significantly improves disease progression in mice with hereditary hypertrophic cardiomyopathy, and it can be developed as a novel anti-hypertrophic cardiomyopathy drug, providing a new approach and method for treating hypertrophic cardiomyopathy.

[0096] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. 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 shall fall within the protection scope of the present invention.

Claims

1. Application of Siegesbeckia julibrissin in the preparation of drugs for the prevention and treatment of hereditary hypertrophic cardiomyopathy.

2. The application according to claim 1, characterized in that, Siegesbeckia glycosides reduce pathological myocardial hypertrophy or increased left ventricular weight.

3. The application according to claim 1, characterized in that, Siegesbeckia glycosides reduce the elevation of hypertrophy markers in myocardial tissue.

4. The application according to claim 1, characterized in that, The siegesine improves the increase in cardiomyocyte area and / or the increase in myocardial fibrosis level.

5. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition consisting of the active ingredient siegesine and pharmaceutically acceptable excipients.

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