Application of Alismatol B Acetate in the Prevention or Treatment of Hypertrophic Cardiomyopathy

Abstract

This invention discloses the application of alismazone B acetate in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy and its related symptoms. This invention is the first to propose the use of alismazone B acetate (AB23a) for the prevention or treatment of hypertrophic cardiomyopathy. The invention utilizes AB23a to directly treat cardiomyocytes obtained from directed differentiation of human embryonic stem cells in vitro, finding that AB23a treatment significantly inhibits the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes. Feeding mice with hereditary hypertrophic cardiomyopathy caused by gene mutations to a diet containing AB23a showed that AB23a significantly alleviated pathological myocardial hypertrophy and improved cardiac function in mice. AB23a can be used to prepare drugs against hereditary hypertrophic cardiomyopathy, providing a new approach and method for treating hypertrophic cardiomyopathy. AB23a is the most important medicinal component of the traditional Chinese medicine Alisma plantago-aquatica, is safe for organisms, and has good clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of alismazone B acetate in the prevention or treatment of hypertrophic cardiomyopathy. Background Technology

[0002] Hypertrophic cardiomyopathy (HBC) is an autosomal dominant genetic disorder caused by mutations in genes encoding sarcomere-related proteins. It has an insidious onset and high incidence, and is a major cause of malignant arrhythmias and sudden cardiac death. The specific mechanisms of HBC are not yet fully understood. Although Mavacamten (MYK-461) can partially improve HBC caused by outflow tract obstruction by targeting and inhibiting cardiac myosin, there are currently no original, effective drugs for HBC 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, there is a need to develop a new drug for the treatment of HBC.

[0003] Alisol-B 23-acetate (AB23a) is a triterpenoid compound extracted from the tuber of Alisma plantago-aquatica, a plant in the Alismataceae family. It is the most important medicinal component of the traditional Chinese medicine Alisma plantago-aquatica, and its chemical formula is: C 32 H 50 O5. Studies have shown that AB23a can promote the regeneration of damaged liver by activating farnesol receptors; AB23a can inhibit IgE / Ag-mediated activation and allergic reactions of basophilic leukemia cells, exhibiting good in vitro and in vivo anti-inflammatory effects. However, there are currently no reports on the role of AB23a in the preparation of drugs for the treatment of hereditary hypertrophic cardiomyopathy. Summary of the Invention

[0004] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides the application of alismazone B acetate (AB23a) in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy. This invention utilizes AB23a to directly treat cardiomyocytes obtained from the directed differentiation of human embryonic stem cells in vitro, finding that AB23a treatment significantly inhibits the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes. Feeding mice with hereditary hypertrophic cardiomyopathy caused by gene mutations to a diet containing AB23a showed that AB23a significantly alleviated pathological myocardial hypertrophy and improved cardiac function in mice. This invention is the first to propose that AB23a can be used to prepare drugs against hypertrophic cardiomyopathy, providing a new approach and method for treating hypertrophic cardiomyopathy.

[0005] The present invention also provides a pharmaceutical composition for the prevention or treatment of hypertrophic cardiomyopathy and the symptoms thereof.

[0006] Technical solution: In order to achieve the above technical objectives, the present invention provides the use of Alismatol B acetate in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy and its related symptoms.

[0007] The hypertrophic cardiomyopathy mentioned above includes diseases caused by inherited hypertrophic cardiomyopathy and hereditary hypertrophic cardiomyopathy.

[0008] The hypertrophic cardiomyopathy includes pathological myocardial hypertrophy, increased heart weight or left ventricular weight.

[0009] The hypertrophic cardiomyopathy includes a significant increase in the expression of hypertrophy markers in myocardial tissue.

[0010] The hypertrophic cardiomyopathy includes an increase in cardiomyocyte area and / or an elevated level of myocardial fibrosis caused by hereditary hypertrophic cardiomyopathy.

[0011] Among them, the area of ​​myocardial cells from patients with hypertrophic myocarditis is increased, and the level of myocardial hypertrophy markers is elevated.

[0012] The use of Alismatol B acetate in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy and its related symptoms by inhibiting the hypertrophic phenotype of cardiomyocytes.

[0013] The present invention relates to a pharmaceutical composition for the prevention or treatment of hypertrophic cardiomyopathy and its associated symptoms, wherein the pharmaceutical composition contains alismazone B acetate as an active ingredient and a pharmaceutically acceptable carrier.

[0014] The dosage form of the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0015] The use of the pharmaceutical composition described in this invention in the preparation of medicaments for the prevention or treatment of hypertrophic cardiomyopathy and its associated symptoms.

[0016] This invention discovers a novel medicinal use for alismosiderin B acetate (AB23a), which can combat hypertrophic cardiomyopathy and the symptoms caused by hypertrophic cardiomyopathy. Furthermore, alismosiderin B acetate is the most important medicinal component of the traditional Chinese medicine Alisma plantago-aquatica, making it safer for organisms and showing promising clinical application prospects.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] Compared to current in vitro studies using animal-derived cell models for AB23a, this invention utilizes directed differentiation of human embryonic stem cells to obtain human cardiomyocytes and establishes 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 cardiomyocytes and human cardiomyocytes. This invention, through direct in vitro treatment with AB23a, has shown that it can significantly inhibit the hypertrophic phenotype of human embryonic stem cells-cardiomyocytes. Furthermore, this invention utilizes a hypertrophic cardiomyopathy mouse model with the Myh6 gene R404Q mutation (Myh6... R404Q This model is characterized by hereditary myocardial hypertrophy, increased cardiac area, and increased weight of the heart and left ventricle. Myh6 R404Q Feeding mice with a diet containing AB23a significantly improved the symptoms of hereditary pathological myocardial hypertrophy.

[0019] This invention utilizes AB23a to directly treat hypertrophic human embryonic stem cells induced with cardiomyocytes, significantly reducing cardiomyocyte area and decreasing the expression of norepinephrine / angiotensin II-induced markers of myocardial hypertrophy. Myh6 cells were fed a diet containing AB23a. R404Q 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.

[0020] This invention, through in vitro and in vivo studies, has revealed that AB23a possesses significant potential for treating hypertrophic cardiomyopathy and can be developed as a novel anti-hypertrophic cardiomyopathy drug, providing a new approach and method for its treatment. Furthermore, this invention offers entirely new options and ideas for current anti-hypertrophic cardiomyopathy drugs, broadening the selection scope and contributing to the development of this technological field. Attached Figure Description

[0021] Figure 1 The figure shows the area of ​​human cardiomyocytes after direct in vitro treatment with AB23a; *** indicates p<0.001.

[0022] Figure 2 This figure shows the expression levels of myocardial hypertrophy markers (left: BNP, middle: ANP, right: TNNT2) in human cardiomyocytes after direct in vitro treatment with AB23a; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

[0023] Figure 3 After feeding WT and Myh6 with feed containing AB23a R404QMouse heart (left) and left ventricular weight (right); in the figure, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

[0024] Figure 4 The images show the results of echocardiography in mice. (Left 1) shows the thickness of the anterior wall of the left ventricle during systole, (Left 2) shows the thickness of the anterior wall of the left ventricle during diastole, (Left 3) shows the thickness of the posterior wall of the left ventricle during systole, and (Left 4) shows the thickness of the posterior wall of the left ventricle during diastole. In the figures, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

[0025] Figure 5 After feeding WT and Myh6 with feed containing AB23a R404Q Expression levels of ANP, BNP, and TNNT2, markers of myocardial hypertrophy in mouse hearts. In the figure, ns indicates p>0.05; ** indicates p<0.01; **** indicates p<0.0001.

[0026] 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

[0027] The present invention will now be specifically illustrated through embodiments. In this invention, the embodiments described below are for better explanation and are not intended to limit the scope of the invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope.

[0028] In the following examples, 8-week-old wild-type mice (WT) and Myh6 gene R404Q point mutant mice (Myh6) were used. R404Q All were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., including Myh6 gene R404Q point mutant mice (Myh6). R404Q (C57BL / 6JGpt-Myh6) em1Cin(R404Q) / Gpt, Myh6-p.R404Q|Strain NO.T051403. Normal mouse diet was purchased from Jiangsu Xiehe Biotechnology Co., Ltd. Compound AB23a was purchased from MedChemExpress (HY-N0805, CAS No.:26575-95-1).

[0029] Human embryonic stem cell line MYL2 Neo / w -H7, where H7 cells were purchased from the WiCell Institute Cell Bank #WA07, and were edited using site-directed genome editing technology to obtain the stable human embryonic stem cell line MYL2. Neo / w-H7 (Construction method refers to LianX, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells 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 drivedNeo into the genome of human pluripotent stem cells and replacing the corresponding normal gene and Example 1).

[0030] Agonist CHIR-99021 (purchased from MedChemExpress, #HY-10182, CAS No.:252917-06-9).

[0031] IWR-1 pathway inhibitor (purchased from MedChemExpress, #HY-12238, CAS No.:1127442-82-3).

[0032] RPM1-1640 / B27-no insulin is RPM1-1640 (Gibco) medium supplemented with insulin-free B-27 (Thermo Fisher Scientific, Cat#A1895601), with the B-27 content being 2%.

[0033] Example 1

[0034] Detection of directed differentiation of human embryonic stem cells into cardiomyocytes and the effect of direct in vitro treatment of AB23a on cardiomyocyte area.

[0035] 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. The MYL2... Neo / w The specific steps for the H7 cell line to differentiate into cardiomyocytes are as follows:

[0036] ①MYL2 Neo / wWhen the H7 cell density reaches 85%-90%, replace the culture medium with RPM1-1640 / B27-noinsulin medium and add 8 μM Wnt signaling pathway agonist CHIR-99021, and culture continuously for 2 days.

[0037] ②On the third day, the culture medium was changed to a new RPM1-1640 / B27-no insulin medium, and the cells were cultured for 24 hours;

[0038] ③ On the 4th day, the culture medium was changed to RPM1-1640 / B27-no insulin medium, and 5 μM of Wnt signaling pathway inhibitor IWR-1 was added. The medium was cultured for 2 consecutive days.

[0039] 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.

[0040] ⑤ After day 8, change the culture medium to RPM1-1640 / B27-no insulin medium and continue culturing for 14 days, changing the medium daily;

[0041] ⑥ On day 14, the culture medium was changed to RPM1-1640 / B27-no insulin medium, and 50 μg / mL of G418 antibiotic was added to screen cardiomyocytes. Screening was carried out for 7 consecutive days, with the medium and new G418 added every day. Beating cell clusters were observed.

[0042] This directed differentiation and screening method can yield a large number of high-purity human cardiomyocytes derived from human embryonic stem cells.

[0043] Human cardiomyocytes obtained from directed differentiation of human embryonic stem cells were uniformly seeded into 24-well plates containing a cell crawling sheet. After 24 h of cell adhesion and growth, the cells were treated with PBS, Ang II (purchased from MedChemExpress, #HY-13948, CAS No.:4474-91-3, 10 μM, 24 h), AB23a (2.5 μM, pretreatment 2 h) + Ang II (10 μM, 24 h), and AB23a (5 μM, pretreatment 2 h) + Ang II (10 μM, 24 h). The cells were then treated with MET (metoprolol tartrate, positive control, purchased from MedChemExpress, #HY-17503B, CAS No.:56392-17-7; 5 μM, pretreatment 2 h) + Ang II (10 μM, 24 h), resulting in PBS treatment group, Ang II treatment group, different concentrations of AB23a + Ang II treatment group, and MET + Ang II positive control group. After 24 hours of treatment, each group underwent an immunofluorescence staining experiment for cardiac troponin (cTNT).

[0044] The specific steps of the cardiac troponin (cTNT) immunofluorescence staining experiment are as follows:

[0045] ① After 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).

[0046] 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).

[0047] ③ Block the cells obtained in ② with 5% BSA solution for 2 hours. After blocking, wash with PBS 3 times (5 min / time).

[0048] ④ Incubate cTNT antibody (1:400, diluted with PBS) at 4°C overnight, remove the antibody, and wash with PBS 3 times (5 min / time).

[0049] ⑤ Incubate with fluorescent anti-rabbit secondary antibody (1:500, diluted with PBS) for 1 hour. After incubation, remove the secondary antibody and wash with PBS 3 times (5 min each time).

[0050] 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).

[0051] 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.

[0052] Cardiomyocytes obtained from six directed differentiations at different time points were used, with two replicates per well. A cTNT-stained image of cell morphology was randomly captured, and the area of ​​all cardiomyocytes in the image was analyzed. The results are as follows: Figure 1 As shown. From Figure 1 It can be seen that treatment of cardiomyocytes with 10 μM Ang II significantly increased cell area (Ang II vs PBS: 4432.1±175.9 vs 3306.13±119.9), while pretreatment of cardiomyocytes with AB23a for 3 h followed by Ang II stimulation effectively reduced the Ang II-induced increase in cell area (AB23a+Ang II vs Ang II: 2849.4±89 vs 4432.1±175.9). Data are expressed as mean ± standard error (mean±SEM). 170 cardiomyocytes were counted in the PBS group, 138 in the NE group, and 181 in the AB23a+Ang II group. *** indicates p<0.001. Therefore, direct in vitro treatment of human cardiomyocytes with AB23a can significantly inhibit the Ang II-induced increase in cardiomyocyte area, with no significant difference compared to the blank control group and the positive control group.

[0053] Example 2

[0054] Effects of AB23a direct in vitro treatment of cardiomyocytes on the expression of myocardial hypertrophy markers in cells

[0055] Human cardiomyocytes were obtained through directed differentiation according to the method described in Example 1. The cells were evenly seeded into 12-well plates. After 24 hours of adherent growth, the cells were treated with PBS, Ang II (10 μM, 24 h), AB23a (1 μM, pretreatment for 2 h) + Ang II (10 μM, 24 h), AB23a (2.5 μM, pretreatment for 2 h) + Ang II (10 μM, 24 h), AB23a (5 μM, pretreatment for 2 h) + Ang II (10 μM, 24 h), and AB23a (10 μM, pretreatment for 2 h) + Ang II (10 μM, 24 h), respectively, resulting in PBS-treated groups, Ang II-treated groups, and groups treated with different concentrations of AB23a + Ang II. After 24 hours of treatment in each group, total RNA was extracted from the cells, and the expression of mRNA markers of myocardial hypertrophy in cardiomyocytes was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0056] 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.

[0057] ② Add 200 μL of chloroform to the supernatant obtained in step ①, vortex, centrifuge at 4°C for 15 min, aspirate the colorless aqueous phase (RNA is in this layer), and transfer it to a new centrifuge tube.

[0058] ③ Add an equal volume of isopropanol to the liquid 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 liquid, slowly add 1 mL of 75% ethanol along the tube wall to wash the RNA precipitate, centrifuge for 15 minutes, discard the liquid, and invert the test tube to remove excess ethanol.

[0059] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve the RNA in a 55°C metal bath, and then measure the RNA concentration. The sample is then directly reverse transcribed.

[0060] ⑤ Reverse transcription: Prepare a 10 μL system including: 2 μL reverse transcriptase (5× Prime ScriptRT Macter Mix), 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).

[0061] ⑥qRT-PCR: Prepare a 10μL reaction system: 0.2μL PCRForward Primer, 0.2μL PCRReverse Primer, 1μL cDNA, 5μL SyBRPremix EX Taq (2×), and 3.6μL ddH2O.

[0062] 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. (The last sentence appears to be incomplete and possibly refers to a 2-...) △△Ct The results were analyzed using relative quantitative analysis, and the test results were as follows: Figure 2 As shown.

[0063] Table 1. Primer sequences for human cardiomyocyte hypertrophy markers

[0064]

[0065]

[0066] from Figure 2 As can be seen, Ang II treatment significantly increased the expression of myocardial hypertrophy markers. Direct treatment with 1 μM, 2.5 μM, 5 μM and 10 μM AB23a in vitro could significantly and effectively reduce the expression of myocardial hypertrophy markers (p<0.05), showing significant differences compared with the model group, indicating that AB23a has a significant physiological effect against myocardial hypertrophy.

[0067] Example 3

[0068] Effects of feeding mice with AB23a-containing diet on heart and left ventricular weight

[0069] In the progression of hypertrophic cardiomyopathy, pathological thickening of the heart and increased ventricular wall thickness lead to abnormal increases in both heart weight and left ventricular weight. This embodiment measures WT and Myh6 after feeding the patient with a diet containing AB23a. R404Q The effects of AB23a on the progression of myocardial hypertrophy in vivo were investigated by measuring the weight of the mouse heart and left ventricle.

[0070] Eight-week-old wild-type mice (WT) and Myh6 gene R404Q point mutation-induced hypertrophic cardiomyopathy mice (Myh6) were selected. R404Q ), and will WT and Myh6 R404Q Mice were randomly divided into five groups. The five groups were then fed normal diets and diets containing different concentrations of AB23a, respectively, resulting in the WT group, Myh6 group, and [other groups]. R404Q The groups were divided into a low-dose AB23a treatment group, a medium-dose AB23a treatment group, and a high-dose AB23a treatment group, and all groups had free access to food.

[0071] Among them, the WT group: wild-type mice (WT) were fed normal diet;

[0072] Myh6 R404Q Group: Myh6 R404Q Mice were fed a normal diet;

[0073] Low-dose AB23a treatment group: Myh6 R404Q Mice were fed a diet containing AB23a at a concentration of 0.025% (0.25g of AB23a per kg of feed).

[0074] Medium-dose AB23a treatment group: Myh6 R404QMice were fed a diet containing AB23a at a concentration of 0.05% (0.50g of AB23a per kg of feed).

[0075] High-dose AB23a treatment group: Myh6 R404Q Mice were fed a diet containing AB23a at a concentration of 0.1% (1g of AB23a per 1kg of feed);

[0076] Positive drug treatment group: Myh6 R404Q Mice were fed a diet containing MET at a concentration of 0.025% (0.25g MET per kg of feed).

[0077] Mice were housed in an SPF-grade animal facility for 3 months.

[0078] After three months of rearing, blood was collected from the eyes of mice. 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. The test results are shown below. Figure 3 The left image is shown. Additionally, left ventricular weight in mice was determined using echocardiography. The steps for echocardiography are as follows:

[0079] 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.

[0080] ②Using the Vevo3100 high-resolution in vivo imaging system (Vevo3100LT, Canada) for ultrasound examination. The heart was quickly located, and images and related parameters of the mouse heart's long and short axes were recorded in the B-model and M-model, respectively.

[0081] ③ The left ventricular weight was calculated using the Vevo3100 system, and the results are as follows: Figure 3 The image on the right.

[0082] from Figure 3 It can be seen that, compared with the heart of WT mice, Myh6 R404Q The weight of the mouse heart and left ventricle was abnormally increased, while feeding the mouse with a diet containing AB23a significantly reduced Myh6. R404Q The reduction in heart and left ventricular weight significantly inhibited cardiac hypertrophy in mice.

[0083] Example 4

[0084] Effect of feeding mice with AB23a-containing diet on left ventricular wall thickness in hypertrophic cardiomyopathy mice

[0085] Mice were fed the method described in Example 3 for 3 months, and then echocardiography was performed to detect whether feeding them a diet containing AB23a improved the pathological hypertrophic phenotype of the heart in mice with hypertrophic cardiomyopathy. The echocardiography procedure was the same as in Example 3. Finally, the thickness of the left ventricular anterior wall during systole, diastole, systole, and diastole was calculated using a Vevo 3100 system. The test results are as follows: Figure 4 As shown (from left to right).

[0086] from Figure 4 As can be seen from this, Myh6 R404Q 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 AB23a significantly reduced Myh6. R404Q Left ventricular wall thickness in mice. This indicates that AB23a has a beneficial effect in combating the progression of pathological myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0087] Example 5

[0088] Effects of feeding diets containing AB23a on markers of myocardial hypertrophy

[0089] This embodiment measures the WT and Myh6 levels of individuals fed with feed containing AB23a. R404Q The expression of myocardial hypertrophy markers in mouse left ventricular tissue was used to investigate the effect of AB23a on the progression of myocardial hypertrophy in vivo. Mice were fed using the method described in Example 3, and then RNA was extracted from the left ventricle of the heart. 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:

[0090] 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 4°C for 5 min and take the supernatant to obtain mouse myocardial tissue sample, i.e., mouse myocardial tissue supernatant.

[0091] ② Add 200 μL of chloroform to the supernatant obtained in step ①, vortex, centrifuge at 4°C for 15 min, aspirate the colorless aqueous phase (RNA is in this layer), and transfer it to a new centrifuge tube.

[0092] ③ Add an equal volume of isopropanol to the liquid 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 liquid, slowly add 1 mL of 75% ethanol along the tube wall to wash the RNA precipitate, centrifuge for 15 minutes, discard the liquid, and invert the test tube to remove excess ethanol.

[0093] ④ Add 50 μL of sterile DEPC water to the RNA precipitate, dissolve the RNA in a 55°C metal bath, and then measure the RNA concentration. The sample is then directly reverse transcribed.

[0094] ⑤ Reverse transcription: Prepare a 10 μL system including: 2 μL reverse transcriptase (5× Prime ScriptRT Macter Mix), 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.

[0095] 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 SyBRPremix EX Taq (2×), and 3.6 μL ddHO. Each sample was tested in triplicate. Hypertrophy markers in mouse myocardial tissue mainly include Anp, Bnp, and Tnnt2. The 18S gene was used to detect the expression of the internal reference gene, with a 2- △△Ct The results were analyzed using relative quantitative analysis. The results are shown in Table 2.

[0096] Table 2. Primer sequences for hypertrophy markers in mouse myocardial tissue

[0097]

[0098] from Figure 5 As can be seen from this, Myh6 R404Q The expression of hypertrophy markers such as Anp, Bnp, and Tnnt2 in mouse myocardial tissue was significantly higher than that in WT mice. AB23a significantly inhibited the expression of Anp, Bnp, and Tnnt2 in Myh6 mice. R404Q The expression of AB23a in the mouse heart suggests that AB23a has great potential for treating hereditary hypertrophic cardiomyopathy.

[0099] Example 6

[0100] Masson staining of myocardial tissue

[0101] In Example 3, mice that had been fed for 3 months 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 then 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 (Saiwell Biotech, G1006), as follows:

[0102] ① Soak the slices in solution A at room temperature overnight (about 15 hours).

[0103] ② Mix equal volumes of solution B and solution C (prepare fresh for 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 gray-black and the background is almost colorless or light gray.

[0104] 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, or the fibers turn red, about 1-2 minutes.

[0105] ④ After slightly draining the E solution, immerse the slide directly in the F solution for 2-30 seconds without washing with water.

[0106] ⑤ The slices were rinsed 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.

[0107] 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.

[0108] 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. AB23a significantly inhibited Myh6... R404Q The progression of myocardial fibrosis in mice was observed, and the area of ​​cardiomyocytes was effectively reduced. This suggests that AB23a may have great potential for treating the early progression of hypertrophic cardiomyopathy.

[0109] In summary, this invention has demonstrated in in vitro experiments that AB23a effectively inhibits the development and progression of hypertrophic cardiomyocytes in human cells; and in in vivo studies, it has shown that AB23a effectively inhibits the progression of hereditary pathological cardiomyopathy and the occurrence of myocardial fibrosis. Both in vitro and in vivo experiments indicate that AB23a significantly improves disease progression in mice with hereditary hypertrophic cardiomyopathy, making it an effective target for the treatment of hypertrophic cardiomyopathy. It can be developed as a novel anti-hypertrophic cardiomyopathy drug or drug target, providing a new approach and method for the detection and treatment of hypertrophic cardiomyopathy.

Claims

1. Application of Alismatol B Acetate in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy and its related symptoms.

2. Use according to claim 1, characterized in that, The hypertrophic cardiomyopathy includes conditions caused by inherited hypertrophic cardiomyopathy and hereditary hypertrophic cardiomyopathy.

3. Use according to claim 1, characterized in that, Hypertrophic cardiomyopathy includes pathological myocardial hypertrophy, increased heart weight or left ventricular weight.

4. Use according to claim 1, characterized in that, The hypertrophic cardiomyopathy is characterized by a significant increase in the expression of hypertrophy markers in myocardial tissue.

5. The use according to claim 1, characterized in that, The hypertrophic cardiomyopathy includes an increase in cardiomyocyte area and / or an elevated level of myocardial fibrosis due to hereditary hypertrophic cardiomyopathy.

6. Use according to claim 1, characterized in that, The patient with hypertrophic cardiomyopathy had an increased area of ​​myocardial cells and elevated levels of myocardial hypertrophy markers.

7. Use according to claim 1, characterized in that, The use of Alismatol B acetate in the preparation of drugs for the prevention or treatment of hypertrophic cardiomyopathy and its related symptoms by inhibiting the hypertrophic phenotype of cardiomyocytes.

8. Use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of hypertrophic cardiomyopathy and its associated symptoms, said pharmaceutical composition comprising alismosiderin B acetate as an active ingredient and a pharmaceutically acceptable carrier.

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