Use of (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in the preparation of a drug against dilated cardiomyopathy

Abstract

The application provides an application of (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in preparation of an anti-dilated cardiomyopathy drug, belongs to the technical field of anti-dilated cardiomyopathy drugs, and discloses that the compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one has an anti-dilated cardiomyopathy effect for the first time, and provides an experimental basis and a theoretical basis for development of a new drug for preventing or treating dilated cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of anti-dilated cardiomyopathy drug technology, specifically relating to the use of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in the preparation of drugs for the treatment or prevention of dilated cardiomyopathy. Background Technology

[0002] Dilated cardiomyopathy (DCM), a heterogeneous cardiomyopathy, is characterized by ventricular enlargement and decreased myocardial contractility, presenting with a wide variety of clinical manifestations. Patients often experience complications such as left ventricular failure, arrhythmias, and thromboembolism. Globally, the incidence of DCM is on the rise, with an annual incidence of approximately 1 in 2500. As a cardiovascular disease that seriously endangers public health, DCM poses a severe threat to patients' health and lives.

[0003] Zebrafish, a model organism, shares a high degree of similarity with mammals in heart structure and function. Both mammals have hearts composed of atria and ventricles, and their diastolic and systolic mechanisms are highly conserved. Zebrafish genes are highly homologous to humans; approximately 87% of human genes have corresponding homologous genes in zebrafish, including many key genes related to diabetic cerebrovascular disease (DCM), such as cardiac sarcomin genes (MYH7, TNNT2). The transparent nature of zebrafish embryos allows for the specific labeling of cardiac cells using fluorescent protein labeling technology. Combined with microscopy imaging techniques and image processing software, precise analysis of cardiac function parameters such as pericardial area, sinus venosus-bulbar (SV-BA) distance, stroke volume, fractional shortening, and ejection fraction can be achieved. Furthermore, the high-throughput characteristics of zebrafish make them ideal for large-scale drug screening and gene function studies. Large numbers of embryos or juveniles can be cultured and processed simultaneously, rapidly screening for drugs that affect cardiac development and function, thereby accelerating the discovery of new therapeutic targets and drugs and providing more potential drug options for the clinical treatment of DCM. These significant advantages enable the zebrafish model to play an important role in the study of the pathogenesis of DCM and drug screening, providing a powerful tool for drug research in heart disease.

[0004] The etiology of dilated cardiomyopathy (DCM) is complex, involving multiple factors including genetics, infection and autoimmunity, alcohol, and drugs. Among these, gene mutations and genetic factors are key intrinsic causes of DCM. Numerous studies have shown that various gene mutations are closely related to the pathogenesis of DCM, especially mutations in genes such as Titin (TTN), LaminAC (LMNA), MYH7, MYH6, and TNNT2. In addition to these gene mutation factors, recent studies have also found that microRNA-16-5p (miR-16-5p) plays an important regulatory role in the development and progression of DCM. In normal cardiomyocytes, miR-16-5p expression is at a relatively stable level, and it can regulate the expression of a series of genes related to cell survival and apoptosis. However, the application of miR-16-5p in constructing a zebrafish model of dilated cardiomyopathy has not yet been reported.

[0005] Currently, dilated cardiomyopathy is mostly treated symptomatically, such as with angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers, and aldosterone receptor antagonists to alleviate symptoms and improve cardiac function, but these methods cannot completely cure the disease. Some medications can produce significant side effects during treatment. For example, while positive inotropic drugs can enhance myocardial contractility, they may increase myocardial oxygen consumption, thereby inducing arrhythmias. Long-term use may also increase the burden on the heart, adversely affecting patient prognosis. Therefore, screening for innovative drugs to treat dilated cardiomyopathy has become an urgent clinical need.

[0006] Marine natural products, due to their structural diversity and significant bioactivity, have shown great potential in the treatment of cardiovascular diseases. For example, active ingredients derived from marine organisms, such as polyunsaturated fatty acids, peptides, and polysaccharides, have been shown to possess anti-inflammatory, antioxidant, and anti-fibrotic effects, and can improve myocardial function. However, the development of marine drugs targeting dilated cardiomyopathy (DCM) still faces many challenges. Therefore, screening marine natural products with anti-dilated cardiomyopathy activity is of great significance, providing a theoretical basis and experimental evidence for the development of new anti-dilated drugs.

[0007] The compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one, with a molecular weight of 206.1, has the structure shown in Formula I. This compound has been reported in existing technical literature, such as in "Study on the Active Secondary Metabolites of Two Fungi from the Beibu Gulf" (Feng Ting, Guangxi University for Nationalities, 2024) and "Discovery, synthesis, biological evaluation and molecular docking study of (R)-5-methylmellein and its analogs as selective monoamine oxidase A inhibitors" (C Huang et al., 2019). Currently, there are no reports on the anti-dilated cardiomyopathy activity of this compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides the application of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in the preparation of drugs for the prevention or treatment of dilated cardiomyopathy. This study utilizes a zebrafish dilated cardiomyopathy model with miR-16-5p overexpression in cardiomyocytes, and uses pericardial area, SV-BA distance, and cardiac function as evaluation indicators to screen for the protective activity of the compound.

[0009] The technical solution of the present invention is as follows:

[0010] The use of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in the preparation of drugs for the prevention or treatment of dilated cardiomyopathy, the compound having the structure shown in Formula I:

[0011]

[0012] Formula I.

[0013] According to a preferred embodiment of the invention, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0014] More preferably, the excipient is at least one of a sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, or lubricant.

[0015] According to a preferred embodiment of the present invention, the dosage form of the drug is a capsule, pill, tablet, oral liquid, granule, tincture, or injection.

[0016] A drug for the prevention or treatment of dilated cardiomyopathy, the active ingredient comprising the compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one having the structure shown in Formula I.

[0017] According to a preferred embodiment of the invention, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0018] More preferably, the excipient is at least one of a sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, or lubricant.

[0019] According to a preferred embodiment of the present invention, the dosage form of the drug is a capsule, pill, tablet, oral liquid, granule, tincture, or injection.

[0020] The beneficial effects of the present invention include at least the following:

[0021] This invention is the first experimental discovery that the compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one can alleviate... Tg (cmlc2: miR-16-5p, EGFP) Pericardial edema in zebrafish reduces the SV-BA distance and improves cardiac function, demonstrating a significant effect against dilated cardiomyopathy. This provides a theoretical basis and experimental evidence for the development of drugs against dilated cardiomyopathy. Attached Figure Description

[0022] Figure 1 The image shows the recombinant plasmid Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40.

[0023] Figure 2 Identification diagram of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid;

[0024] In the figure: A is the Sanger sequencing result of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid, where the blue box is the partial promoter sequence of cmlc2, the red box is the miR-16-5p sequence, and the green box is the partial DNA sequence of IRES2; B is the electrophoresis band of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid after double enzyme digestion.

[0025] Figure 3 Electrophoresis and sequencing images of F1 generation zebrafish overexpressing miR-16-5p;

[0026] In the figure: A is a fluorescence micrograph of 48 hpf miR-16-5p overexpressing zebrafish; B is an agarose gel electrophoresis image of PCR products, where 1 is the DNA marker (from bottom to top: 250, 1000, 5000, 7500, 10000, 15000 bp), 2 is the template-free negative control, 3 is the plasmid template positive control, and 4 is the PCR amplification product of the F1 genomic DNA fragment; C is the PCR sequencing results, where the blue box is the partial promoter sequence of cmlc2, the green box is the miR-16-5p sequence, and the black box is the partial DNA sequence of IRES2.

[0027] Figure 4 Electrophoresis and sequencing images of F2 generation zebrafish overexpressing miR-16-5p;

[0028] In the figure: A is an agarose gel electrophoresis image of PCR products; B is a graph of PCR sequencing results.

[0029] Figure 5 A graph showing the morphology and statistics of the zebrafish heart;

[0030] In the figure: A is a morphological diagram of each group of zebrafish; B is a statistical diagram of the pericardial area of ​​zebrafish; C is a statistical diagram of the SV-BA distance of zebrafish.

[0031] Compared with the blank control group, ** p<0.01, *** p<0.001, **** p<0.0001.

[0032] Figure 6 A statistical chart of cardiac function in zebrafish;

[0033] In the figure: A is a statistical chart of zebrafish stroke volume; B is a statistical chart of zebrafish ejection fraction; C is a statistical chart of zebrafish short axis shortening rate.

[0034] Compared with the blank control group, * p<0.05, ** p<0.01, **** p < 0.0001.

[0035] Figure 7 A pathological diagram of the zebrafish heart.

[0036] Figure 8 Figure showing the effect of clinical drugs on the heart morphology of zebrafish with dilated cardiomyopathy;

[0037] In the figure: A shows the morphology of zebrafish in each group, with the red dashed box indicating the heart region and the orange arrow indicating pericardial edema; B shows the statistical chart of the pericardial area of ​​zebrafish; C shows the statistical chart of the SV-BA distance of zebrafish; compared with the blank control group,** p<0.01, **** p < 0.0001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0038] Figure 9 The effects of clinical drugs on cardiac function in zebrafish with dilated cardiomyopathy;

[0039] In the figure: A is a statistical chart of stroke volume in each group of zebrafish; B is a statistical chart of ejection fraction in zebrafish; C is a statistical chart of short axis shortening rate in zebrafish; compared with the blank control group, *** p<0.001, **** p < 0.0001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0040] Figure 10 The diagram shows the safety evaluation of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in zebrafish.

[0041] Figure 11 Figure showing the effect of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on the morphology of the heart in zebrafish with dilated cardiomyopathy.

[0042] In the figure: A is a morphological diagram of zebrafish; B is a statistical diagram of pericardial area of ​​zebrafish; C is a statistical diagram of SV-BA distance of zebrafish; compared with the blank control group, *** p<0.001, **** p < 0.0001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0043] Figure 12 The effect of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on cardiac function in zebrafish with dilated cardiomyopathy;

[0044] In the figure: A is a statistical chart of zebrafish stroke volume; B is a statistical chart of zebrafish ejection fraction; C is a statistical chart of zebrafish short axis shortening rate; compared with the blank control group, * p<0.05, ** p<0.01; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0045] Figure 13 The figure shows the effect of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on the cardiac pathological tissue of zebrafish with dilated cardiomyopathy. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0047] Unless otherwise specified in the examples, the procedures were performed under standard conditions; reagents or instruments used without a specified manufacturer were all commercially available products.

[0048] The inventors utilized a zebrafish dilated cardiomyopathy model based on the previously constructed miR-16-5p overexpression in cardiomyocytes. Tg (cmlc2: miR-16-5p, EGFP) The activity of various trace compounds against dilated cardiomyopathy was screened and evaluated, and the compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one was found to have significant anti-dilated cardiomyopathy effects.

[0049] 1. Materials and Instruments

[0050] 1.1 Experimental Reagents

[0051] PCS-tp plasmid (Yao Shunyu Biotechnology); TAE Buffer (Shanghai Sangon Biotech, catalog number B548101-0500), DL2000 Plus DNA Marker (Nanjing Novizan Biotechnology Co., Ltd., catalog number MD101-01), UltraGelRed (Nanjing Novizan Biotechnology Co., Ltd., catalog number GR501-01), 2 × Rapid Taq Master Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number P222-01), Agarose (Yisheng, catalog number 10208ES60), FastDigest NotI (Thermo Fisher Scientific, catalog number FD0594), Potassium chloride (Shanghai Sangon Biotech, catalog number A610440-0500), Calcium chloride dihydrate (Shanghai Sangon Biotech, catalog number A610050-0500), Magnesium chloride hexahydrate (Shanghai Sangon Biotech, catalog number A601336-0500), Methylene blue (Sigma-Aldrich) Aldrich (catalog number M9140), 1-phenyl-2-thiourea (Sigma-Aldrich, catalog number P7629), MS-222 (Sigma-Aldrich, catalog number A5040), 10 × DNA Loading Buffer (Nanjing Novizan Biotechnology Co., Ltd., catalog number P022-01), YSYBuffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., catalog number K-101-100), 1kb DNA Marker (Beijing Bio-Lab Technology Co., Ltd., catalog number M20024); PCR Cleaning Kit (Axygen, catalog number AP-PCR-50), mMESSAGE mMACHINE™ SP6 Transcription Kit (Thermo Fisher Scientific, catalog number AM1340); Restriction endonucleases ApaI (NEB, catalog number R0114L) and XhoI (NEB, catalog number R0146M); Dapagliflozin (AbMole, catalog number 461432-26-8); Empagliflozin (AbMole, catalog number 98418-47-4).

[0052] 1.2 Instruments and equipment used in the experiment

[0053] Zebrafish embryo microinjection instrument IM-300 (Neimo, Japan), Bori New XP gene amplification instrument TC-XP-G (Hangzhou / Bori), zebrafish rearing system (Beijing Aisheng Technology Co., Ltd.), horizontal electrophoresis apparatus HE-120 (Tanon), Tanon 1600 series multifunctional gel image analysis system Tanon 1600 (Tanon), fluorescence microscope MZX81 (Mingmei), electric thermostatic drying oven DH6-903385-III (Xinmiao), benchtop centrifuge Sorvall Legend Micro 17 (Thermo Fisher), low temperature centrifuge 5428000295 (Eppendorf), Ausen Nano-100 micro spectrophotometer Nano-100 (Hangzhou Ausen), benchtop centrifuge (Sorvall Legend Micro 17, Thermo Fisher); 20L constant temperature water bath (DWB20-P, SCILOGEX); SZX16 stereo fluorescence microscope (Olympus, Japan); AXIO-V16 fluorescence microscope (Carl Zeiss Optics GmbH, Germany); Light Cycler@96 real-time fluorescence quantitative PCR instrument (Roche, Switzerland).

[0054] 1.3 Laboratory Animals

[0055] The wild-type AB strain zebrafish (referred to as AB strain zebrafish) and heart-marked green fluorescent transgenic zebrafish used in this experiment Tg (cmlc2: EGFP) Zebrafish can be purchased from the National Zebrafish Resource Center or from commercially available products. Adult zebrafish are kept in separate tanks by sex within the zebrafish breeding system, with a constant water temperature of 28°C and a light-to-dark ratio of 14:10. Zebrafish are fed twice daily: brine shrimp in the morning and solid feed in the afternoon. For experimental egg collection, male and female zebrafish are placed on opposite sides of the spawning tank (separated by a partition, which is removed at 8:00 AM the following day), with a male-to-female ratio of 2:2. The following morning, the collected zebrafish embryos are sterilized in zebrafish culture water containing methylene blue, and then incubated in a 28°C constant temperature and light-controlled incubator. The embryo culture water is changed regularly to ensure normal embryo development and the smooth progress of the experiment.

[0056] 2 Experimental Methods

[0057] 2.1 Construction of a zebrafish model of dilated cardiomyopathy with miR-16-5p overexpression

[0058] 2.1.1 Design and Obtaining of Tol2 Recombinant Plasmid

[0059] The Cmlc2 promoter can drive the specific high expression of exogenous genes in the heart region. IRES2 (Internal Ribosome Entry Site 2) is an internal ribosome entry site that can recruit ribosomes and initiate protein translation without relying on traditional 5′ cap structures. By linking multiple genes to IRES, simultaneous expression of multiple genes on the same mRNA molecule can be achieved, which is significant for the expression of genes with complex structures or specific regulatory needs. Enhanced Green Fluorescent Protein (EGFP), as a reporter gene, has stronger fluorescence intensity and a wider range of applications for studying biological processes such as gene expression, protein localization, and cell differentiation. The Tol2 transposon system is a mobile genetic element consisting of a Tol2 transposon helper plasmid and a Tol2 transposase synthesis plasmid. The Tol2 transposon helper plasmid has specific inverted repeat sequences (Tol2-L and Tol2-R) flanking it, which are the sites for transposase recognition and action, enabling stable gene delivery and expression. After the Tol2 recombinant plasmid and transposase mRNA are co-transferred into the host cell, the foreign gene can be integrated into the genome in a "cut-paste" manner.

[0060] The self-designed Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid uses the Tol2 helper plasmid as a framework, into which the cmlc2 promoter, miR-16-5p, IRES2, and EGFP sequences are sequentially inserted. Figure 1 This transgenic expression vector can simultaneously express miR-16-5p and EGFP under the same promoter cmlc2.

[0061] The sequence of miR-16-5p is shown in SEQ ID NO.1.

[0062] The recombinant plasmid map of Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 is shown below. Figure 1 As shown, the sequence is SEQ ID NO.2, where the yellow highlighted text indicates the cmlc2 promoter sequence, the red highlighted text indicates the miR-16-5p sequence, the cyan highlighted text indicates the IRES2 sequence, and the green highlighted text indicates the EGFP sequence.

[0063] Based on SEQ ID NO.2, the company synthesized the cmlc2-miR-16-5p-IRES2-EGFP tandem sequence in vitro using artificial double-stranded DNA synthesis technology. This tandem sequence was then inserted into the helper plasmid of the Tol2 transposon system using homologous recombination technology, thus constructing the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid. The constructed Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid was sequence verified using Sanger sequencing and double enzyme digestion techniques.

[0064] 2.1.2 Synthesis of Tol2 transposase mRNA

[0065] The original plasmid PCS-tp (Yao Shun Yu Biotechnology) was linearized by digestion with a single NotI restriction site. The linearized DNA was then purified using a nuclease-free PCR cleaning kit. Using the linearized PCS-tp as a template, transcription was performed using the mMESSAGE mMACHINE™ SP6 Ultra transcription kit to obtain capped Tol2 transposase mRNA. After in vitro transcription, the DNA template was removed, and the mRNA was purified. The Tol2 transposase mRNA concentration was finally determined using a micro-spectrophotometer.

[0066] 2.1.3 Zebrafish Embryo Microinjection

[0067] Zebrafish fertilized eggs of the AB line were collected using standard methods and transferred to the grooves of an agarose gelatin plate. Excess moisture was removed while preventing the eggs from drying out. The injection time and pressure of the microinjector were adjusted. The Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid was mixed with Tol2 transposase mRNA at a final concentration of 60 ng / μL and 200 ng / μL, and then microinjected into the single-cell stage fertilized eggs of zebrafish at a volume of 1 nL per embryo. The injection solution contained 0.05% phenol red solution as an indicator. The injection process was performed as quickly as possible to minimize mechanical damage to the embryo.

[0068] 2.1.4 Detection of EGFP reporter gene expression in F0 generation zebrafish

[0069] After injection, zebrafish were placed in zebrafish culture water containing 0.003% phenylthiourea (PTU) (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) to inhibit melanin production and facilitate fluorescence observation. Zebrafish were raised to 48 hpf and 72 hpf, and their fluorescence was observed under an inverted fluorescence microscope. Zebrafish exhibiting cardiac fluorescence were selected; these were designated as the F0 generation and cultured in a constant-temperature incubator until adulthood.

[0070] 2.1.5 Screening and Identification of miR-16-5p-F1 Transgenic Zebrafish

[0071] filter:

[0072] After raising the F0 generation zebrafish selected in the previous step to adulthood, they are crossed with AB generation zebrafish. The hearts of the offspring zebrafish embryos are observed under a fluorescence microscope to see if they have EGFP green fluorescence. If the hearts of the offspring zebrafish embryos show green fluorescence, then the zebrafish is a transgenic zebrafish, namely the F1 generation transgenic zebrafish.

[0073] Identification:

[0074] (1) Extraction of zebrafish F1 genome from miR-16-5p overexpression

[0075] Five F1 embryos were collected to prepare genomic DNA templates. 10 μL of YSYBuffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., catalog number K-101-100) was added one by one to the wall of a PCR tube containing zebrafish embryos (care should be taken not to let the pipette tip touch the tissue). After addition, the PCR tubes were rapidly centrifuged to ensure the solution remained at the bottom of the tube. The PCR tubes were then placed in a PCR instrument to prepare the genomic template for subsequent PCR amplification. The PCR program is shown in Table 1.

[0076] Table 1. Reaction procedures for genome extraction

[0077]

[0078] (2) PCR amplification of genomic DNA fragments

[0079] The prepared template genomic DNA was amplified. The reaction components are shown in Table 2, the reaction conditions are shown in Table 3, and the PCR primer sequences are shown in Table 4.

[0080] Table 2 PCR reaction system for template genomic DNA amplification

[0081]

[0082] Table 3. PCR reaction conditions for template genomic DNA amplification

[0083]

[0084] Table 4. PCR primer sequences for genotyping of transgenic zebrafish

[0085]

[0086] (3) Electrophoretic detection

[0087] Mix an appropriate amount of PCR amplification product with the loading buffer, add the mixed sample to the wells of a 1% agarose gel electrophoresis, and perform electrophoresis at an appropriate voltage to separate DNA fragments in the gel according to their size.

[0088] (4) Sequencing verification

[0089] The PCR products were sent to General Electric for Sanger sequencing. Primer-F and Primer-R were selected for sequencing, and their sequences are shown in Table 4.

[0090] 2.1.6 Screening and Identification of miR-16-5p-F2 Transgenic Zebrafish

[0091] filter:

[0092] The F1 generation zebrafish selected in the previous step were raised to adulthood and then self-crossed. The hearts of the offspring zebrafish embryos were observed under a fluorescence microscope to determine if they exhibited EGFP green fluorescence. If the offspring zebrafish embryos showed green fluorescence in their hearts, then these zebrafish were transgenic zebrafish, specifically F2 generation transgenic zebrafish, which are transgenic zebrafish with overexpression of miR-16-5p in their cardiomyocytes. Tg (cmlc2: miR-16-5p, EGFP) .

[0093] Identification:

[0094] (1) Extraction of zebrafish F2 genome from miR-16-5p overexpression

[0095] Five F2 generation embryos were collected to prepare genomic DNA templates. 10 μL of YSYBuffer was added one by one to the wall of each PCR tube containing zebrafish embryos. After addition, the PCR tubes were rapidly centrifuged to ensure the solution remained at the bottom. The PCR tubes were then placed in a PCR instrument to prepare the genomic template for subsequent PCR amplification. The PCR program is shown in Table 1.

[0096] (2) PCR amplification of genomic DNA fragments

[0097] The prepared template genomic DNA was amplified. The reaction components are shown in Table 2, the reaction conditions are shown in Table 3, and the PCR primer sequences are shown in Table 4.

[0098] (3) Electrophoretic detection

[0099] Mix an appropriate amount of PCR amplification product with loading buffer, add the mixed sample to the wells of a 1% agarose gel for electrophoresis, and perform electrophoresis at an appropriate voltage to separate DNA fragments according to their size in the gel. From left to right, the first lane is the DNA marker, the second lane is the DNA Loading Buffer (template-free negative control), and the third lane is the F2 genomic DNA fragment PCR amplification product.

[0100] (4) Sequencing verification

[0101] The PCR products were sent to General Electric for Sanger sequencing. Primer-F and Primer-R were selected for sequencing, and their sequences are shown in Table 4.

[0102] 2.2 Observation of the disease progression of miR-16-5p overexpression in zebrafish cardiomyopathy

[0103] 2.2.1 Morphological observation of zebrafish hearts

[0104] The development of 2-day and 3-day zebrafish embryos and larvae was observed using stereofluorescence microscopy. Morphology of each group of zebrafish was photographed and recorded, and abnormalities such as pericardial edema and increased SV-BA distance were observed. Pericardial area and SV-BA distance (increased SV-BA distance reflects zebrafish cardiac enlargement) were measured using Image-ProPlus 6.0 software.

[0105] 2.2.2 Cardiac function testing

[0106] Patients with dilated cardiomyopathy present with arrhythmias, ventricular dilation, and impaired cardiac systolic function. Stroke volume, fractional shortening, and ejection fraction in zebrafish can reflect ventricular dilation and cardiac systolic function. Zebrafish with 2 dpf and 3 dpf were fixed with 4% methylcellulose. After fixation, heartbeat videos of the zebrafish in a prone position were recorded under an inverted fluorescence microscope, and images of end-diastolic and end-systolic ventricular activity were extracted. The long and short axis lengths of the zebrafish at end-diastolic and end-systolic ...

[0107]

[0108]

[0109]

[0110] 2.2.3 H&E staining

[0111] Randomly select 10 records with 2 DPF and 3 DPF. Tg (cmlc2: EGFP) and Tg (cmlc2: miR-16-5p, EGFP) Zebrafish were stained with hematoxylin and eosin (HE). After washing three times with phosphate-buffered saline (PBS), the fish were anesthetized and fixed with 4% paraformaldehyde fixative. They were then dehydrated in a gradient of ethanol and immersed in xylene until clear, followed by paraffin embedding and sectioning. The sections were dewaxed sequentially in xylene and ethanol until water was removed, stained with hematoxylin and eosin, dehydrated, and mounted. The tissue sections were observed under a stereofluorescence microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan), and cardiac tissue was photographed and recorded.

[0112] 2.3 Validation of the applicability of miR-16-5p overexpression in zebrafish with dilated cardiomyopathy using clinical drugs.

[0113] To further confirm the successful construction of a zebrafish model of dilated cardiomyopathy induced by miR-16-5p overexpression, the inventors introduced the clinically approved positive control drugs dapagliflozin and empagliflozin for validation. Both dapagliflozin and empagliflozin are sodium-glucose cotransporter 2 (SGLT2) inhibitors, and their mechanisms of action against dilated cardiomyopathy are similar. By inhibiting glucose reabsorption in the proximal convoluted tubules of the kidneys, they increase urinary glucose excretion and produce osmotic diuresis, reducing cardiac preload and alleviating edema symptoms in patients with dilated cardiomyopathy. They also promote the shift of cardiomyocytes from primarily glucose metabolism to primarily fatty acid metabolism, optimizing myocardial energy supply, enhancing myocardial contractile function, and helping to improve myocardial function in patients with dilated cardiomyopathy. Furthermore, they have anti-inflammatory and antioxidant effects, reducing oxidative damage and inflammatory responses in myocardial cells, protecting them, and delaying the progression of dilated cardiomyopathy.

[0114] 2.3.1 Drug Grouping

[0115] Selected 48 hpf with normal development Tg (cmlc2: EGFP) and Tg (cmlc2: miR-16-5p, EGFP) Zebrafish were placed in 24-well plates, with 15 juveniles per well. A blank control group was set up based on the preliminary experiment. Tg (cmlc2: EGFP) +zebrafish culture water), model group ( Tg (cmlc2: miR-16-5p, EGFP) +zebrafish culture water), drug treatment group ( Tg (cmlc2: miR-16-5p, EGFP)+dapagliflozin / empagliflozin), with 3 replicate wells in each group, and incubated in a light incubator for 24 h.

[0116] 2.3.2 Effects of clinical drugs on cardiac morphology in zebrafish with dilated cardiomyopathy

[0117] Twenty-four hours after drug administration, zebrafish were anesthetized with an anesthetic (0.3% tricaine) and then fixed with 4% methylcellulose. After anesthesia, the cardiac morphology of each group of zebrafish was photographed from the side under a stereofluorescence microscope, and the pericardial area and SV-BA distance were measured using Image-Pro Plus 6.0 software.

[0118] 2.3.3 Effects of clinical drugs on cardiac function in zebrafish with dilated cardiomyopathy

[0119] Twenty-four hours after drug administration, zebrafish were fixed with 4% methylcellulose. After fixation, heartbeat videos of zebrafish in a prone position were recorded under an inverted fluorescence microscope, and images of end-diastolic and end-systolic ventricles were extracted. The long and short axis lengths of the zebrafish at end-diastolic and end-systolic ventricles were measured using Image-Pro Plus 6.0 software, and stroke volume, ejection fraction, and short axis shortening rate were calculated.

[0120] 2.4 Safety evaluation of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one

[0121] 2.4.1 Compound Sources

[0122] The compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one (structure shown in Formula I) used in this experiment was provided by Professor Wang Cong's research group at Guangxi University for Nationalities. This compound has been reported in the prior art and can be obtained by fermentation of the marine fungus Neopestalotiopsis sp. HN-1-6 followed by extraction with ethyl acetate (see "Study on Active Secondary Metabolites of Two Fungi from Beibu Gulf", Feng Ting, Guangxi University for Nationalities, 2024). It can also be obtained through other routes.

[0123] 2.4.2 Grouping of compounds

[0124] Select 2 DPFs with normal development Tg (cmlc2: EGFP) Zebrafish were placed in a 24-well plate, with 10 juveniles per well. A blank control group was set up ( Tg (cmlc2: EGFP) + Zebrafish culture water), compound treatment group ( Tg (cmlc2: EGFP)+ 10 / 20 / 40 / 80 μM (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one), with 3 replicate wells per group, and incubated in a light incubator for 24 h.

[0125] 2.4.3 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on zebrafish

[0126] Twenty-four hours after drug administration, zebrafish were anesthetized with an anesthetic (0.3% tricaine) and then fixed with 4% methylcellulose. After anesthesia, zebrafish were photographed from the side under a stereofluorescence microscope to record their morphology and to calculate the deformity rate and mortality rate.

[0127] 2.5 Screening for compounds against dilated cardiomyopathy using a zebrafish model with miR-16-5p overexpression in cardiomyocytes.

[0128] 2.5.1 Grouping of compounds

[0129] Select 2 DPFs with normal development Tg (cmlc2: EGFP) and 2 dpf Tg (cmlc2: miR-16-5p, EGFP) Zebrafish were placed in a 24-well plate, with 10 juveniles per well. A blank control group was set up ( Tg (cmlc2: EGFP) + Zebrafish culture water), model group ( Tg (cmlc2: miR-16-5p, EGFP) + Zebrafish culture water), positive control group ( Tg (cmlc2: miR-16-5p, EGFP) + 5 μM empagliflozin), low, medium, and high concentration compound treatment groups ( Tg (cmlc2: miR-16-5p, EGFP) + 5 / 10 / 20 μM (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one), with 3 replicate wells per group, and incubated in a light incubator for 24 h.

[0130] 2.5.2 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on cardiac morphology in zebrafish with dilated cardiomyopathy

[0131] Same as 2.3.2.

[0132] 2.5.3 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on cardiac function in zebrafish with dilated cardiomyopathy

[0133] Same as 2.3.3.

[0134] 2.5.4 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on the cardiac pathological tissue of zebrafish with dilated cardiomyopathy

[0135] 10 normally developed 3 dpf genes were randomly selected. Tg (cmlc2: EGFP), 3dpf Tg (cmlc2: miR-16- 5p, EGFP) and 3 dpf treated with 20 µM compound Tg (cmlc2: miR-16-5p, EGFP) Zebrafish were stained with hematoxylin and eosin (HE). After washing three times with phosphate-buffered saline (PBS), the fish were anesthetized and fixed with 4% paraformaldehyde fixative. They were then dehydrated in a gradient of ethanol and immersed in xylene until clear, followed by paraffin embedding and sectioning. The sections were dewaxed sequentially in xylene and ethanol until water was removed, stained with hematoxylin and eosin, dehydrated, and mounted. The tissue sections were observed under a stereofluorescence microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan), and cardiac tissue was photographed and recorded.

[0136] 2.6 Data Analysis

[0137] Experimental data are expressed as mean ± SD, and statistical difference analysis was performed using the t-test. p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference. Statistical analysis was performed using GraphPad Prism 8.0 and Image-Pro Plus 6.0 software.

[0138] 3 Experimental Results

[0139] 3.1 Qualification of the Tol2 transpose system

[0140] 3.1.1 Identification of Tol2 recombinant plasmid

[0141] The Sanger sequencing results of the To12-cmlc2-miR-16-5p-IRES2-EGFP-UTRSv40 recombinant plasmid are as follows: Figure 2 As shown in Figure A, the sequence is consistent with the expected base sequence; ApaI-XhoI double digestion identification is as follows. Figure 2 As shown in Figure B, the size after enzyme digestion is consistent with the expected 1416 bp and 3884 bp. These results indicate that the To12-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid has been successfully constructed.

[0142] 3.1.2 Identification of Tol2 transposase mRNA

[0143] The final concentration of Tol2 transposase mRNA was determined to be 2154 ng / µL, with an absorbance ratio of 260 / 280 of 1.91.

[0144] 3.1.3 Screening and identification results of zebrafish overexpressing miR-16-5p-F1

[0145] (1) Fluorescence screening results

[0146] like Figure 3 As shown in Figure A, green fluorescence expression was observed in the heart region of zebrafish that had developed for 48 hours under an inverted fluorescence microscope, indicating that the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid was successfully injected into zebrafish and expressed in cardiomyocytes.

[0147] (2) Results of agarose gel electrophoresis

[0148] like Figure 3 As shown in Figure B, by comparing with DNA markers of known molecular weights (250, 1000, 5000, 7500, 10000, 15000 bp from bottom to top), the F1 progeny genome in lane 4 is consistent with the positive control band of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid template in lane 3, showing a clear target band of the expected size (907 bp). This band represents a DNA fragment between the partial sequence of the cmlc2 promoter inserted into the zebrafish genome and the partial sequence of EGFP. This preliminarily suggests the possible presence of transgenic elements in the F1 generation zebrafish genome, but further sequencing verification is needed.

[0149] (3) PCR sequencing verification results

[0150] like Figure 3 As shown in Figure C, analysis of the peak diagram obtained from the F1 generation Sanger sequencing revealed that the key elements of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid (cmlc2 promoter sequence, miR-16-5p sequence, and IRES2 partial DNA sequence) were completely consistent with the expected target gene sequence. This confirms that the F1 generation zebrafish successfully inherited the target gene, achieving stable transgene transfer.

[0151] 3.1.4 Screening and identification results of zebrafish overexpressing miR-16-5p-F2

[0152] (1) Results of agarose gel electrophoresis

[0153] like Figure 4 As shown in Figure A, by comparing with DNA markers of known molecular weight (250, 500, 750, 1000, 1500 bp from bottom to top), a clear target band of the expected size (907 bp) appeared in the F2 generation genome (third lane), which preliminarily indicates that transgenic elements may exist in the F2 generation zebrafish genome.

[0154] (2) PCR sequencing verification results

[0155] like Figure 4 As shown in Figure B, analysis of the peak diagram obtained from the F2 generation Sanger sequencing revealed that the key elements of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid (cmlc2 promoter sequence, miR-16-5p sequence, and partial IRES2 DNA sequence) were completely consistent with the expected target gene sequence. This confirms that the F2 generation zebrafish successfully inherited the target gene, achieving stable transgene transfer. The F2 generation zebrafish are miR-16-5p overexpressing zebrafish. Tg (cmlc2: miR-16-5p, EGFP) .

[0156] 3.2 Disease progression study in a zebrafish model with miR-16-5p overexpression

[0157] 3.2.1 Effects of miR-16-5p overexpression on zebrafish heart morphology

[0158] Changes in the size of the pericardium in zebrafish can directly reflect abnormalities in the zebrafish heart; zebrafish cardiac dilatation is caused by the opening of the S-ring of the heart, leading to an increase in the distance between the bulboarteriovenous sinus (SV-BA). Therefore, pericardial area and SV-BA distance can directly reflect whether abnormalities have occurred in the zebrafish heart. Figure 5 As shown, compared with heart-specific fluorescent transgenic zebrafish Tg (cmlc2: EGFP) In comparison, miR-16-5p overexpression in zebrafish Tg (cmlc2: miR-16-5p, EGFP) When the pericardium develops to 2-4 days postpartum (dpf), the pericardial area increases significantly, and the SV-BA distance becomes noticeably longer. When it develops to 5-7 days postpartum (dpf), Tg (cmlc2: miR-16- 5p, EGFP) The pericardial area and SV-BA distance of zebrafish were not significantly different from those of normal zebrafish. This indicates that miR-16-5p overexpression can lead to abnormal cardiac morphology in zebrafish.

[0159] 3.2.2 Effects of miR-16-5p overexpression on cardiac function in zebrafish

[0160] Patients with dilated cardiomyopathy clinically present with ventricular dilation and impaired cardiac contractility. Stroke volume, ejection fraction, and fractional shortening in zebrafish reflect ventricular dilation and cardiac contractility, and these indicators can be used to evaluate the impact of miR-16-5p overexpression on cardiac function in zebrafish with dilated cardiomyopathy. Figure 6 As shown, compared with heart-specific fluorescent transgenic zebrafish Tg (cmlc2: EGFP) In comparison, miR-16-5p overexpression in zebrafish Tg (cmlc2: miR-16-5p, EGFP) When development reaches 2-3 days post-exposure (dpf), stroke volume, ejection fraction, and fractional shortening decrease significantly. When development reaches 4-5 days post-exposure (dpf), Tg (cmlc2: miR-16-5p, EGFP) Zebrafish showed decreased stroke volume, ejection fraction, and fractional shortening compared to normal zebrafish, but these differences were not statistically significant. At 6-7 days post-exposure (dpf), stroke volume and ejection fraction decreased further compared to normal zebrafish. This indicates that miR-16-5p overexpression can lead to cardiac damage and weakened cardiac function in zebrafish.

[0161] In patients with dilated cardiomyopathy, the ventricular walls are thinned, the ventricular cavity is enlarged, cardiomyocytes are disorganized, and myocardial fibrosis is prevalent. HE staining allows for microscopic observation of these morphological changes to diagnose cardiomyopathy. HE staining results are as follows: Figure 7 As shown, groups 2 and 3 have dpf. Tg (cmlc2: EGFP) The zebrafish hearts had normal atrium and ventricle sizes, normal intercellular spaces, and tightly packed cardiomyocytes. (Groups 2 and 3, dpf) Tg (cmlc2: miR-16-5p, EGFP) The zebrafish showed significant enlargement of the atria and ventricles, larger intercellular spaces, and disordered arrangement of myocardial cells, consistent with typical pathological features of DCM.

[0162] 3.3 Validation of the applicability of miR-16-5p overexpression in zebrafish with dilated cardiomyopathy using clinical drugs.

[0163] 3.3.1 Effects of clinical drugs on cardiac morphology in zebrafish with dilated cardiomyopathy

[0164] like Figure 8 As shown, 24 hours after administration, compared with the blank control group ( Tg (cmlc2: EGFP) Compared to zebrafish culture water, the model group (+zebrafish culture water) Tg (cmlc2: miR-16-5p, EGFP)(Zebrafish culture water) Significant pericardial edema and increased SV-BA distance were observed in the zebrafish, indicating successful establishment of the zebrafish dilated cardiomyopathy model. Compared with the model group, pericardial edema was reduced in the low- and medium-concentration (1.25, 2.5 μM) dapagliflozin and medium- and high-concentration (5, 10 μM) empagliflozin (EMPA) groups; no significant change in pericardial area was observed in the high-concentration (5 μM) dapagliflozin and low-concentration (2.5 μM) empagliflozin groups; and the SV-BA distance was significantly reduced in the dapagliflozin (1.25, 2.5, 5 μM) and empagliflozin (2.5, 5, 10 μM) groups. This indicates that dapagliflozin (1.25, 2.5 μM) and empagliflozin (5, 10 μM) have a relieving effect. Tg (cmlc2: miR-16-5p, EGFP) Effects of zebrafish pericardial edema on reducing SV-BA distance.

[0165] 3.3.2 Effects of clinical drugs on cardiac function in zebrafish with dilated cardiomyopathy

[0166] like Figure 9 As shown, 24 hours after administration, compared with the blank control group ( Tg (cmlc2: EGFP) Compared to zebrafish culture water, the model group (+zebrafish culture water) Tg (cmlc2: miR-16-5p, EGFP) (+Zebrafish culture water) The zebrafish showed a significant decrease in stroke volume, ejection fraction, and short axis shortening, indicating that the zebrafish dilated cardiomyopathy model was successfully established. Compared with the model group, zebrafish stroke volume increased in the dapagliflozin (1.25, 2.5, 5 μM) and empagliflozin (2.5, 5, 10 μM) administration groups; ejection fraction increased in the low and high concentration (1.25, 5 μM) dapagliflozin and low and medium concentration (2.5, 5 μM) empagliflozin administration groups; fractional shortening increased in the low and high concentration (1.25, 5 μM) dapagliflozin and low concentration (2.5 μM) empagliflozin administration groups; ejection fraction showed no significant change in the medium concentration (2.5 μM) dapagliflozin and high concentration (10 μM) empagliflozin administration groups; fractional shortening showed no significant change in the medium concentration (2.5 μM) dapagliflozin and medium and high concentration (5, 10 μM) empagliflozin administration groups. This indicates that dapagliflozin (1.25, 5 μM) and empagliflozin (5 μM) have effects on... Tg (cmlc2: miR-16-5p, EGFP) Zebrafish have a restorative effect on cardiac function.

[0167] 3.4 Safety evaluation of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in zebrafish

[0168] like Figure 10 As shown, compared with the blank control group Tg (cmlc2: EGFP)Compared to zebrafish, zebrafish exhibited normal morphology with zero deformity and mortality rates when compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one was present at concentrations of 10, 20, and 40 μM. At a concentration of 80 μM, a small number of zebrafish showed morphological abnormalities, including spinal curvature, with a deformity rate of 30% and zero mortality. This indicates that compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one has high safety at concentrations of 10, 20, and 40 μM.

[0169] 3.5 Screening for the anti-dilated cardiomyopathy activity of compounds using a zebrafish model with miR-16-5p overexpression in cardiomyocytes.

[0170] 3.5.1 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on the morphology of the heart in zebrafish overexpressing miR-16-5p in cardiomyocytes

[0171] like Figure 11 As shown, compared with the blank control group Tg (cmlc2: EGFP) In comparison, the model group Tg (cmlc2: miR-16- 5p, EGFP) The pericardial area and SV-BA distance in zebrafish were significantly increased, indicating that miR-16-5p overexpression leads to cardiac damage in zebrafish. (Compared to the model group) Tg (cmlc2: miR-16-5p, EGFP) In contrast, the pericardial area and SV-BA distance of zebrafish in the positive control group were significantly reduced, indicating that empagliflozin has a relieving effect at a concentration of 5 μM. Tg (cmlc2: miR-16-5p, EGFP) Effects of zebrafish pericardial edema on reducing SV-BA distance. (Compared with model group) Tg (cmlc2: miR-16-5p, EGFP) In comparison, the pericardial area of ​​zebrafish treated with medium and high concentrations of the compound was significantly reduced, while the pericardial area of ​​zebrafish treated with low concentrations showed no significant change; the SV-BA distance of zebrafish treated with low, medium, and high concentrations of the compound was significantly reduced. This indicates that compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one has a mitigating effect at concentrations of 10 and 20 μM. Tg (cmlc2: miR-16-5p, EGFP) Effects of zebrafish pericardial edema on reducing SV-BA distance.

[0172] 3.5.2 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on cardiac function in zebrafish with miR-16-5p overexpression in cardiomyocytes

[0173] like Figure 12 As shown, compared with the blank control group Tg (cmlc2: EGFP) In comparison, the model group Tg (cmlc2: miR-16- 5p, EGFP) The zebrafish showed a significant decrease in stroke volume, ejection fraction, and fractional shortening, indicating that miR-16-5p overexpression leads to decreased cardiac function in zebrafish. (Compared to the model group) Tg (cmlc2: miR-16-5p, EGFP) In contrast, the positive control group of zebrafish showed significantly increased stroke volume, ejection fraction, and fractional shortening, indicating that empagliflozin at a concentration of 5 μM has a relieving effect. Tg (cmlc2: miR-16-5p, EGFP) The effect of zebrafish on decreased cardiac function. (Compared to the model group) Tg (cmlc2: miR-16-5p, EGFP) In contrast, when the compound concentrations were 5, 10, and 20 μM, the stroke volume, ejection fraction, and fractional shortening of zebrafish were significantly increased. This indicates that the compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one has a mitigating effect at concentrations of 5, 10, and 20 μM. Tg (cmlc2: miR-16-5p, EGFP) The effect of zebrafish on decreased cardiac function.

[0174] 3.5.3 Effects of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one on the pathological tissue of zebrafish with miR-16-5p overexpression in cardiomyocytes

[0175] HE staining results are as follows Figure 13 As shown, Tg(cmlc2:EGFP) The zebrafish in this group had normal atrium and ventricle size, normal intercellular spaces, tightly packed myocardial cells, small vacuolar area (orange arrow), and thick ventricular walls (black arrow). Tg(cmlc2: miR-16-5p,EGFP) The zebrafish showed significantly enlarged atria and ventricles, disordered arrangement of myocardial cells, severe vacuolation, and thinner ventricular walls. Tg(cmlc2:miR-16-5p,EGFP) After zebrafish were given 20 μM of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one, the myocardial cells were more neatly arranged and the degree of vacuolation was reduced, showing a significant therapeutic effect.

[0176] This invention successfully constructed a transgenic zebrafish model that specifically overexpresses miR-16-5p in the myocardium. Tg (cmlc2: miR-16-5p, EGFP) The stability of transgene integration was confirmed through fluorescence screening and PCR verification. Tg (cmlc2: miR-16-5p, EGFP)Zebrafish exhibited typical dilated cardiomyopathy phenotypes at 2-3 days post-exposure (dpf), including pericardial edema, increased SV-BA distance, and significantly decreased cardiac function, highly consistent with the ventricular dilation and systolic dysfunction characteristics of human dilated cardiomyopathy patients. Histological analysis revealed ultrastructural abnormalities such as disordered cardiomyocyte arrangement, vacuolation, and loss of mitochondrial cristae, further validating the pathological relevance of this zebrafish model. However, symptoms gradually lessened after 4 dpf, presumably related to early death in individuals with severe phenotypes, resulting in milder symptoms in surviving individuals after 4 dpf, consistent with the progressive course of human dilated cardiomyopathy. Zebrafish are among the few vertebrates with complete cardiac regeneration capabilities; their cardiomyocytes can re-enter the cell cycle after injury, such as by activating the Notch signaling pathway, promoting myocardial tissue repair. Therefore, the severe symptoms at 2-3 dpf may stem from myocardial structural damage and dysfunction directly caused by miR-16-5p overexpression, while the symptom relief after 4 dpf is a result of the combined effects of early death in severely phenotypical individuals and the zebrafish's cardiac regenerative capacity. Therefore, transgenic zebrafish at 2-3 dpf were ultimately selected as the zebrafish model for dilated cardiomyopathy.

[0177] This invention utilizes Tg (cmlc2: miR-16-5p, EGFP) Using a zebrafish disease model, pericardial area, SV-BA distance, and cardiac function as indicators, the anti-dilated cardiomyopathy activity of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one was preliminarily evaluated. This invention is the first to discover that compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one has an anti-dilated cardiomyopathy effect. Compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one can improve pericardial edema and increased SV-BA distance caused by dilated cardiomyopathy; compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one can improve... Tg (cmlc2: miR-16-5p, EGFP) Zebrafish stroke volume, ejection fraction, and fractional shortening improve the pathological structure of cardiac tissue. This invention provides a theoretical basis and experimental evidence for the development of new drugs to treat dilated cardiomyopathy.

Claims

1. The use of compound (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one in the preparation of drugs for the prevention or treatment of dilated cardiomyopathy, the compound having the structure shown in Formula I: Formula I.

2. The application as described in claim 1, characterized in that, The drug contains one or more pharmaceutically acceptable carriers or excipients.

3. The application as described in claim 2, characterized in that, The excipient is at least one of the following: a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, or a lubricant.

4. The application as described in claim 1, characterized in that, The dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

Images