Use of alisol-b23-acetate in prevention or treatment of hypertrophic cardiomyopathy

Abstract

Disclosed in the present invention is a use of alisol-B23-acetate (AB23a) in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy and diseases caused by the hypertrophic cardiomyopathy. The present invention provides for the first time a use of AB23a in the prevention or treatment of hypertrophic cardiomyopathy. According to the present invention, use of AB23a in direct in vitro treatment of cardiomyocytes derived from directed differentiation of human embryonic stem cells revealed that AB23a-based in-vitro treatment can significantly inhibit hypertrophic phenotypes of the human embryonic stem cells-cardiomyocytes; using an AB23a-containing feed to feed mice with hereditary hypertrophic cardiomyopathy caused by a gene mutation revealed that AB23a prominently relieves pathological myocardial hypertrophy of the mice and improves the cardiac function; AB23a can be used for preparing a drug for resisting hereditary hypertrophic cardiomyopathy, offering a novel way and means for treating hypertrophic cardiomyopathy; and AB23a is the main medicinal ingredient in the traditional Chinese medicinal herb-Rhizoma Alismatis, is safe to organisms, and has good clinical application prospects.

Description

Application of alismatol B acetate in preventing or treating hypertrophic cardiomyopathy Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of alismatol B acetate in preventing or treating hypertrophic cardiomyopathy. Background Art

[0002] Hypertrophic cardiomyopathy is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere-related proteins. 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, there is a need to develop a new drug for the treatment of hypertrophic cardiomyopathy.

[0003] Alisol-B 23-acetate (AB23a) is a triterpenoid compound extracted from the tuber of Alisma orientalis, a plant of the Alismataceae family. It is the main medicinal ingredient in Alisma orientalis, a traditional Chinese medicine. Its chemical formula is: 32 H 50 O5. Studies have shown that AB23a can promote regeneration of damaged livers by activating the farnesoid receptor. AB23a can also inhibit IgE / Ag-mediated activation of basophilic leukemia cells and allergic reactions, demonstrating promising anti-inflammatory effects in vitro and in vivo. However, there are currently no reports on the role of AB23a in the development of therapeutics for hereditary hypertrophic cardiomyopathy. Summary of the Invention

[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides the use of alismatol B acetate (AB23a) in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy; the present invention uses AB23a to directly treat cardiomyocytes obtained by directed differentiation of human embryonic stem cells in vitro, and finds that AB23a treatment in vitro can significantly inhibit the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes; feed containing AB23a is used to feed mice with hereditary hypertrophic cardiomyopathy caused by gene mutations, and finds that AB23a significantly alleviates pathological myocardial hypertrophy in mice and improves cardiac function; the present invention proposes for the first time that AB23a can be used to prepare drugs against hypertrophic cardiomyopathy, providing a new approach and means for treating hypertrophic cardiomyopathy.

[0005] The present invention also provides a pharmaceutical composition for preventing or treating hypertrophic cardiomyopathy and diseases caused by it.

[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 a medicament for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it.

[0007] The hypertrophic cardiomyopathy includes diseases caused by hereditary 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 hypertrophic markers in myocardial tissue.

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

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

[0012] The alismatol B acetate is used in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it by inhibiting the hypertrophic phenotype of myocardial cells.

[0013] The pharmaceutical composition for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it of the present invention contains alismatol B acetate as an active ingredient and a pharmaceutically acceptable carrier.

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

[0015] The pharmaceutical composition of the present invention is used in preparing medicine for preventing or treating hypertrophic cardiomyopathy and diseases caused by it.

[0016] The present invention discovers a new medical use for alismatol B acetate (AB23a), which can treat hypertrophic cardiomyopathy and its associated symptoms. Furthermore, alismatol B acetate, the main medicinal component of the traditional Chinese medicine Alisma orientalis, is safer for living organisms and has promising clinical applications.

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

[0018] Compared with the current in vitro research on AB23a using animal-derived cell models, the present invention uses human embryonic stem cell directed differentiation to obtain human cardiomyocytes 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 AB23a can significantly inhibit the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes through direct in vitro treatment. In addition, the present invention uses a mouse model of hypertrophic cardiomyopathy with the Myh6 gene R404Q mutation (Myh6 R404Q ), which is characterized by hereditary cardiac hypertrophy, increased heart area, and increased heart and left ventricular weight. R404Q After mice were fed a diet containing AB23a, the symptoms of hereditary pathological cardiac hypertrophy were significantly improved.

[0019] The present invention uses AB23a to directly treat hypertrophic cardiomyocytes obtained by induction of human embryonic stem cells in vitro, significantly reducing the area of ​​cardiomyocytes and reducing the increase in the expression of myocardial hypertrophy markers caused by norepinephrine / angiotensin II. R404Q 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.

[0020] Through in vitro and in vivo studies, the present invention demonstrates that AB23a 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 novel 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

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

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

[0023] Figure 3 shows the expression of WT and Myh6 after feeding with AB23a-containing diet. R404Q Mouse 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 shows the results of cardiac ultrasound detection in mice, where (first from the left) is the left ventricular anterior wall thickness during systole, (second from the left) is the left ventricular anterior wall thickness during diastole, (third from the left) is the left ventricular posterior wall thickness during systole, and (fourth from the left) is the left ventricular posterior wall thickness during diastole. In the figure, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

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

[0026] 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

[0027] The present invention is specifically described below by way of examples. In the present invention, the following examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0028] In the following examples, 8-week-old wild-type mice (WT) and Myh6 gene R404Q point mutation mice (Myh6 R404Q ) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., among which the Myh6 gene R404Q point mutation mice (Myh6 R404Q ) is C57BL / 6J Gpt-Myh6em1Cin(R404Q) / Gpt, Myh6-p.R404Q|Strain NO.T051403. Normal mouse feed was purchased from Jiangsu Collaborative Bioengineering Co., Ltd. AB23a compound 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 edited using genome-directed modification technology to obtain a stable human embryonic stem cell line MYL2. Neo / w-H7 (the construction method refers to Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci US A. 2012; 109(27): E1848-E1857. doi: 10.1073 / pnas.1200250109; or see patent CN108265029A, paragraph 0037: Editing human pluripotent stem cells (including the genome of human embryonic stem cells, introducing MYL2 driven Neo into the genome of human pluripotent stem cells and replacing the corresponding normal genes and Example 1).

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

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

[0032] RPM1-1640 / B27-no insulin is an RPM1-1640 (Gibco) medium to which B-27 (Thermo Fisher Scientific, Cat# A1895601) without insulin is added, wherein the content of B-27 is 2%.

[0033] Example 1

[0034] Directed differentiation of human embryonic stem cells into cardiomyocytes and detection of the effect of direct treatment with AB23a in vitro on cardiomyocyte area

[0035] In this example, human embryonic stem cell line MYL2 was used Neo / w -H7 differentiated cardiomyocytes, and then a large number of high-purity human cardiomyocytes were obtained through in vitro screening. Neo / w The specific steps for differentiating the H7 cell line into cardiomyocytes are as follows:

[0036] ①MYL2 Neo / wWhen the H7 cell density reached 85%-90%, the culture medium was replaced with RPM1-1640 / B27-no insulin medium and 8 μM Wnt signaling pathway agonist CHIR-99021 was added, and the cells were cultured for 2 consecutive days;

[0037] ② On the third day, the culture medium was replaced with fresh RPM1-1640 / B27-no insulin medium and the cells were cultured for 24 h;

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

[0039] 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.

[0040] ⑤ After the 8th day, the culture medium was replaced with RPM1-1640 / B27-no insulin medium and culture was continued for 14 days, with the medium changed every day;

[0041] ⑥ On day 14, the culture medium was replaced with RPMl-1640 / B27-no insulin medium, and 50 μg / mL of G418 antibiotic was added to select cardiomyocytes. The selection was continued for 7 consecutive days, with the medium and G418 replaced daily. Beating cell clusters were observed.

[0042] 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.

[0043] Human cardiomyocytes derived from human embryonic stem cell-directed differentiation were evenly seeded into a 24-well plate containing a cell slide. After 24 h of adherent growth, the cells were treated with PBS, AngⅡ (purchased from MedChemExpress, #HY-13948, CAS No.: 4474-91-3, 10 μM, 24 h), AB23a (2.5 μM, pretreatment for 2 h) + AngⅡ (10 μM, 24 h), AB23a (5 μM, pretreatment for 2 h) + AngⅡ (10 μM, 24 h), and MET (metoprolol tartrate, a positive drug, purchased from MedChemExpress, #HY-17503B, CAS No.: 56392-17-7; 5 μM, pretreatment for 2 h) + AngⅡ (10 μM, 24 h), respectively. PBS-treated group, AngⅡ-treated group, different concentrations of AB23a + AngⅡ-treated group, and positive drug MET + AngⅡ-treated group were obtained. After 24 hours of treatment, each group was subjected to cardiac troponin (cTNT) immunofluorescence staining experiments.

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

[0045] 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).

[0046] ② The cells obtained in ① were treated with 0.2% Triton-X100 for 5 minutes, and then washed three times with PBS (5 minutes each time).

[0047] 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).

[0048] 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).

[0049] 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).

[0050] 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).

[0051] 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.

[0052] Cardiomyocytes obtained by directed differentiation for six different times, with two replicates per well, were randomly photographed using cTNT-stained cell morphology images. The area of ​​all cardiomyocytes in the images was analyzed, as shown in Figure 1. As shown in Figure 1, 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). Pretreatment of cardiomyocytes with AB23a for 3 hours 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 presented as mean ± standard error (SEM). A total of 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, AB23a directly treated human cardiomyocytes in vitro and could significantly inhibit the AngⅡ-induced cardiomyocyte area enlargement effect, with no significant difference compared with the blank control group and the positive drug control group.

[0053] Example 2

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

[0055] Human cardiomyocytes were obtained by directed differentiation according to the method in Example 1. The cells were evenly seeded into 12-well plates. After the cells adhered to the wall for 24 hours, they were treated with PBS, AngⅡ (10μM, 24 hours), AB23a (1μM, pretreatment 2 hours) + AngⅡ (10μM, 24 hours), AB23a (2.5μM, pretreatment 2 hours) + AngⅡ (10μM, 24 hours), AB23a (5μM, pretreatment 2 hours) + AngⅡ (10μM, 24 hours), and AB23a (10μM, pretreatment 2 hours) + AngⅡ (10μM, 24 hours) to obtain PBS treatment group, AngⅡ treatment group and different concentrations of AB23a + AngⅡ treatment group. After 24 hours of treatment in each group, total RNA was extracted from the cells, and the expression of myocardial hypertrophy marker mRNA in cardiomyocytes was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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.

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

[0064] As can be seen from Figure 2, AngⅡ treatment led to a significant increase in the expression of myocardial hypertrophy markers. Direct in vitro treatment with 1μM, 2.5μM, 5μM and 10μM AB23a could significantly and effectively reduce the expression of myocardial hypertrophy markers (p<0.05), which was significantly different from the model group, indicating that AB23a has a significant physiological effect against myocardial hypertrophy.

[0065] Example 3

[0066] Effects of feeding a diet containing AB23a on heart and left ventricular weight in mice

[0067] During the progression of hypertrophic cardiomyopathy, the heart becomes pathologically thickened and the ventricular wall thickness increases, resulting in abnormal increases in heart weight and left ventricular weight. R404Q The effect of AB23a on the progression of cardiac hypertrophy in vivo was investigated by measuring the heart and left ventricular weights of mice.

[0068] Eight-week-old wild-type mice (WT) and mice with hypertrophic cardiomyopathy caused by the R404Q point mutation of the Myh6 gene (Myh6 R404Q ), and WT and Myh6 R404Q The mice were randomly divided into five groups. Then the five groups of mice were fed with normal feed and feed containing different concentrations of AB23a to obtain the WT group, Myh6 R404Q The rats in the three groups were treated with AB23a at a low dose, a medium dose and a high dose, and all groups had free access to food.

[0069] Among them, WT group: wild-type mice (WT) were fed with normal diet;

[0070] Myh6 R404Q Group: Myh6 R404Q Mice were fed a normal diet;

[0071] Low-dose AB23a treatment group: Myh6 R404Q Mice were fed a diet containing AB23a at a drug concentration of 0.025% (0.25 g AB23a per 1 kg of diet);

[0072] Medium-dose AB23a-treated group: Myh6 R404Q Mice were fed a diet containing AB23a at a drug concentration of 0.05% (0.50 g AB23a per kg of diet);

[0073] High-dose AB23a treatment group: Myh6 R404Q Mice were fed a diet containing AB23a at a drug concentration of 0.1% (1 g AB23a per 1 kg of diet);

[0074] Positive drug treatment group: Myh6 R404Q Mice were fed a diet containing MET at a drug concentration of 0.025% (0.25 g MET per 1 kg of diet);

[0075] The mice were housed in an SPF animal room for 3 months.

[0076] After three months of feeding, mice were bled via eyeballs and then sacrificed by cervical dislocation. The hearts were quickly removed, their surface moisture blotted off, and their weights were measured. RNA was then extracted from the left ventricle for later use. The test results are shown in the left panel of Figure 3. In addition, the mice were observed using echocardiography to measure the weight of their left ventricles. The steps for echocardiography were as follows:

[0077] 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.

[0078] ②Using the Vevo3100 high-resolution in vivo imaging system (Vevo3100LT, Canada), ultrasound detection was performed to quickly locate the heart and record the long-axis and short-axis images and related parameters of the mouse heart in both the B-model and M-model.

[0079] ③ The left ventricular mass was calculated using the Vevo3100 system. The test results are shown in the right figure of Figure 3.

[0080] As shown in Figure 3, compared with the WT mouse heart, Myh6 R404Q The weight of the heart and left ventricle of mice increased abnormally, while feeding the diet containing AB23a significantly reduced Myh6 R404Q The heart and left ventricular weight of mice were significantly reduced, and cardiac hypertrophy was significantly inhibited in mice.

[0081] Example 4

[0082] Effects of feeding AB23a-containing diet on left ventricular wall thickness in mice with hypertrophic cardiomyopathy

[0083] Mice were reared for three months using the method described in Example 3, and then subjected to echocardiography to determine whether feeding the AB23a-containing diet improved the pathological hypertrophic phenotype of the hearts of mice with hypertrophic cardiomyopathy. The echocardiographic procedure was similar to that described in Example 3. Finally, the systolic, diastolic, systolic, and diastolic left ventricular anterior wall thicknesses (LVAWTs) were calculated using the Vevo 3100 system. The results are shown in Figure 4 (from left to right).

[0084] As can be seen from Figure 4, Myh6 R404Q The left ventricular wall thickness of mice during diastole and systole was significantly higher than that of WT mice, and feeding the diet containing AB23a significantly reduced Myh6 R404Q This suggests that AB23a has a beneficial effect in counteracting the progression of pathological myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.

[0085] Example 5

[0086] Effects of feeding a diet containing AB23a on cardiac hypertrophy markers

[0087] In this example, the WT and Myh6 mice fed with AB23a-containing feed were detected. R404Q The effect of AB23a on the progression of myocardial hypertrophy in vivo was investigated by measuring the expression of myocardial hypertrophy markers in mouse left ventricular tissue. Mice were raised using the method described in Example 3, and RNA was extracted from the left ventricle. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure the expression of myocardial hypertrophy marker mRNA in cardiomyocytes. The specific steps are as follows:

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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 ddH O. Set up three replicate wells for each sample. The hypertrophy markers of mouse myocardial tissue mainly include Anp, Bnp and Tnnt2. The 18S gene is used to detect the expression of the internal reference gene. -△△Ct The results were analyzed by relative quantitative analysis. The analysis results are shown in Table 5.

[0094] Table 3. Primer sequences for mouse myocardial hypertrophy markers

[0095] As can be seen from Figure 5, Myh6 R404Q The expression of hypertrophy markers such as Anp, Bnp and Tnnt2 in myocardial tissue of mice was significantly higher than that in WT mice. AB23a significantly inhibited the expression of Anp, Bnp and Tnnt2 in Myh6 R404Q This indicates that AB23a has great potential for treating hereditary hypertrophic cardiomyopathy.

[0096] Example 6

[0097] Masson staining of myocardial tissue

[0098] In Example 3, mice raised for 3 months were sacrificed by cervical dislocation and then rapidly perfused with a 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:

[0099] ① Soak the slices in solution A at room temperature overnight (about 15 hours).

[0100] 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.

[0101] 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.

[0102] ④ After draining liquid E slightly, the slices were directly stained with liquid F for 2-30 seconds without washing.

[0103] 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.

[0104] 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.

[0105] 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, while AB23a significantly inhibited Myh6 R404Q The results showed that AB23a can inhibit the progression of myocardial fibrosis in mice and effectively reduce the area of ​​cardiomyocytes, which suggests that AB23a may have great potential in treating the early stages of hypertrophic cardiomyopathy.

[0106] In summary, the present invention found in vitro that AB23a effectively prevents the development and progression of human cardiomyocyte hypertrophy; in vivo studies, AB23a effectively inhibits the progression of hereditary pathological cardiac hypertrophy and the development of myocardial fibrosis. Both in vitro and in vivo experiments demonstrated that AB23a significantly improved disease progression in mice with hereditary hypertrophic cardiomyopathy, making it an effective target for the treatment of hypertrophic cardiomyopathy and a promising candidate for development as a new anti-hypertrophic cardiomyopathy drug or drug target, providing a novel approach and method for the detection and treatment of hypertrophic cardiomyopathy.

Claims

1. Use of alismatol B acetate in the preparation of a medicament for preventing or treating hypertrophic cardiomyopathy and diseases caused by it.

2. The use according to claim 1, characterized in that The hypertrophic cardiomyopathy includes diseases caused by hereditary hypertrophic cardiomyopathy and hereditary hypertrophic cardiomyopathy.

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

4. The use according to claim 1, characterized in that The hypertrophic cardiomyopathy includes a significant increase in the expression of hypertrophic markers in myocardial tissue.

5. The use according to claim 1, characterized in that The hypertrophic cardiomyopathy includes an increase in the area of ​​myocardial cells and / or an increase in the level of myocardial fibrosis caused by hereditary hypertrophic cardiomyopathy.

6. The use according to claim 1, characterized in that The area of ​​the patient-derived myocardial cells with hypertrophic cardiomyopathy is increased, and the level of myocardial hypertrophy markers is elevated.

7. The use according to claim 1, characterized in that The alismatol B acetate is used in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it by inhibiting the hypertrophic phenotype of myocardial cells.

8. A pharmaceutical composition for preventing or treating hypertrophic cardiomyopathy and its induced diseases, characterized in that: The pharmaceutical composition contains alismatol B acetate as an active ingredient and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

10. Use of the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing or treating hypertrophic cardiomyopathy and diseases caused by it.

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