Use of valproic acid in constructing an animal model of dilated cardiomyopathy, and a method for constructing an animal model of dilated cardiomyopathy and a method for screening drugs

Abstract

This invention relates to the use of valproic acid in constructing animal models of dilated cardiomyopathy, and to a method for constructing such an animal model and screening drugs, belonging to the field of pharmaceutical technology. This invention involves administering 50-100 μM valproic acid to zebrafish farms. The dilated cardiomyopathy animal model constructed by this invention complements existing models. In drug screening applications, this complementarity effectively avoids the missed screening of candidate active compounds due to the limitations of a single model mechanism, significantly improving the comprehensiveness and success rate of screening. Simultaneously, the phenotype of the drug screening model of this invention is highly consistent with clinical findings, the model construction cycle is short and low-cost, and it can achieve dynamic visualization and be applied to high-throughput drug screening.

Description

Technical Field

[0001] This invention relates to the use of valproic acid in constructing animal models of dilated cardiomyopathy, as well as a method for constructing an animal model of dilated cardiomyopathy and a method for screening drugs, belonging to the field of pharmaceutical technology. Background Technology

[0002] Dilated cardiomyopathy (DCM) is a myocardial disease characterized by ventricular dilation and systolic dysfunction. Its pathological features mainly include cardiomyocyte hypertrophy, interstitial fibrosis, and ventricular remodeling. Clinically, it manifests as progressive heart failure, malignant arrhythmias, and an increased risk of sudden cardiac death, making it a significant cause of heart failure and heart transplantation. The pathogenesis of DCM is highly heterogeneous, with complex etiologies including both genetic and acquired factors. Approximately 40% of cases are related to genetic factors (such as mutations in genes like TTN and LMNA), while various acquired factors, including infection, toxins, cancer treatment, endocrine disorders, pregnancy, tachyarrhythmias, and immune-mediated diseases, pose significant challenges to the study of DCM's pathological mechanisms and clinical treatment. The selection and efficacy of clinical drugs for this disease are very limited; therefore, there is an urgent need to screen for novel drugs for the treatment of dilated cardiomyopathy.

[0003] Animal models can simulate the complex pathophysiological processes of human diseases, serving as an irreplaceable platform for evaluating the efficacy and safety of candidate drugs. Zebrafish models, with their advantages of small size, ease of rearing, rapid growth, in vitro fertilization, and transparency, have become important model organisms in pharmaceutical research, demonstrating unique advantages, especially in the field of cardiac diseases. Zebrafish share approximately 87% homology with the human genome, exhibiting a highly conserved genetic background. Furthermore, the zebrafish cardiovascular system consists of atria, ventricles, sinus venosus, and bulbus arteriosus connected in series, with valves between the atria and ventricles. Their heart rate (120-180 bpm) is close to that of humans, and their heart development, contractile structure, and electrophysiological characteristics are highly conserved. Juvenile zebrafish are completely transparent, allowing for direct, real-time monitoring of cardiac phenotype, function, and hemodynamic changes in a living organism. Combined with their large egg production and small size, this enables truly high-throughput drug screening.

[0004] Currently, the most commonly used models for dilated cardiomyopathy (DCM) in zebrafish are genetic mutation models, such as TTN and MYH7 gene mutations. These models have long construction cycles and high costs. A few drug-induced models exist, such as those using doxorubicin and terfenadine (Zebrafish Model Reveals Early Electrocardiographic and Molecular Signatures of Doxorubicin-Induced Cardiotoxicity, Cardiovascular Toxicology, 2026, 26(4): 39; Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine, Korean Circulation Journal, 2017, 47(6): 960-969). Dilated cardiomyopathy requires animal models to exhibit phenotypes consistent with the core clinical pathological features of DCM, such as ventricular enlargement, ventricular wall thinning, and myocardial contractile dysfunction, after the basic cardiac structure has formed. However, the etiology of DCM is complex, and the types and pathogenic mechanisms of existing models are relatively simple, making it difficult to comprehensively cover the complex pathological features of DCM caused by multiple etiologies, thus easily leading to the omission of effective drugs. Therefore, developing an animal model that has a short construction cycle, low cost, and can simulate DCM from a new pathological mechanism perspective is of great significance for drug screening and pathological research of DCM.

[0005] Valproic acid is a broad-spectrum antiepileptic drug. There are existing literature reports on the cardiotoxicity of maternal exposure to valproic acid in embryos (Sodium valproate-induced congenital cardiac abnormalities in mice areas associated with the inhibition of histone deacetylase, Journal of Biomedical Science, 2010, 17(1):16). Some studies have also shown its cardiotoxicity in zebrafish (Heartmalformation is an early response to valproic acid in developing zebrafish, Naunyn-Schmiedeberg's Archives of Pharmacology, 2020, 393:2387–2409). These studies mainly focus on toxicity assessment from the beginning of embryonic development to early development, and the observation indicators are mostly general, non-specific cardiac developmental toxicity or teratogenic endpoints such as changes in heart rate, pericardial edema, and abnormal cardiac circumduction. None of these studies addressed, nor provided any technical insights, into applying valproic acid at a specific stage after the formation of the basic cardiac structure to specifically induce a dilated cardiomyopathy model that conforms to the core clinical characteristics.

[0006] Currently, there are no reports in this field regarding the construction of animal models of dilated cardiomyopathy using valproic acid. Therefore, how to control the broad, non-specific effects of valproic acid on the heart through specific intervention conditions and transform it into an animal model that can stably simulate the core pathological process of dilated cardiomyopathy remains an unsolved technical challenge in this field. Research on the application of valproic acid in the construction of zebrafish dilated cardiomyopathy models has significant practical value. Summary of the Invention

[0007] This invention relates to the use of valproic acid in constructing animal models of dilated cardiomyopathy, and a method for constructing an animal model of dilated cardiomyopathy or for screening drugs.

[0008] This invention provides the use of valproic acid in constructing animal models of dilated cardiomyopathy.

[0009] The animal mentioned is a zebrafish.

[0010] The dosage of valproic acid is 50-100 μM.

[0011] Preferably, the dosage of valproic acid is 50 μM.

[0012] This invention provides a method for constructing an animal model of dilated cardiomyopathy, which involves administering 50-100 μM valproic acid to zebrafish farms.

[0013] Specifically, zebrafish embryos 48 hours after fertilization are placed in an embryo culture medium containing 50-100 μmol / L valproic acid and cultured at a constant temperature of 28±0.5℃ for 24 hours to obtain a zebrafish model of dilated cardiomyopathy. Preferably, the concentration of valproic acid is 50 μmol / L.

[0014] The method for processing zebrafish embryos is as follows:

[0015] The fertilized embryos were placed in embryo culture medium and cultured at a constant temperature of 28±0.5℃. After 24 hours, the zebrafish embryos were transferred to embryo culture medium containing 0.2mM phenylthionine (PTU) and cultured at a constant temperature of 28±0.5℃ for another 24 hours.

[0016] The present invention also provides the use of animal models constructed by the described method in screening drugs for the prevention and / or treatment of dilated cardiomyopathy.

[0017] This invention provides a method for screening candidate drugs for the prevention and / or treatment of dilated cardiomyopathy, wherein the candidate drugs are applied to an animal model of dilated cardiomyopathy constructed by the method described above.

[0018] The candidate drug mentioned is rutin.

[0019] This invention provides the use of rutin in the preparation of medicaments for the prevention and / or treatment of dilated cardiomyopathy.

[0020] The animal model of dilated cardiomyopathy constructed in this invention is based on valproic acid inducing pathological remodeling in the heart within a specific developmental time window and concentration range, highly consistent with clinical dilated cardiomyopathy (DCM). This model complements existing models, filling the gap in models for specific pathological types. In drug screening applications, this complementarity effectively avoids the missed screening of candidate active compounds due to the limitations of single-model mechanisms, significantly improving the comprehensiveness and success rate of screening. Furthermore, the phenotype of the drug screening model in this invention is highly consistent with clinical findings, the model construction cycle is short and low-cost, and it enables dynamic visualization and application in high-throughput drug screening. Attached Figure Description

[0021] Figure 1 Phenotypic diagram of zebrafish treated with valproic acid;

[0022] Figure 2 Valproic acid treatment of zebrafish heart rate and pericardial area (where * indicates statistical significance compared with the control group (p<0.05)).

[0023] Figure 3 Valproic acid-induced cardiac injury in zebrafish (where ac represents statistical results of pericardial area, SV-BA distance, and ventricular size in zebrafish; d represents the phenotype of zebrafish with green fluorescent heart markers; eg represents statistical results of heart rate, ventricular shortening fraction, and blood flow velocity in zebrafish; h represents the statistical results of blood flow velocity in zebrafish after treatment with 100 μM valproic acid. * indicates statistical significance compared with the control group (p<0.05)).

[0024] Figure 4 Zebrafish ventricular pathological section;

[0025] Figure 5 Results of acridine orange staining in zebrafish hearts;

[0026] Figure 6 Effects of carvedilol on heart rate and pericardial area in zebrafish treated with valproic acid (where * indicates statistical significance compared to the model group (p<0.05)).

[0027] Figure 7 Effects of rutin on valproic acid-induced heart phenotype in DCM zebrafish (* indicates statistical significance compared to the model group (p<0.05)).

[0028] Figure 8 Effect of rutin on the cardiac phenotype of zebrafish induced by terfenadine (* indicates statistical significance compared with the model group (p<0.05)). Detailed Implementation

[0029] Example 1: Animal Model Construction Method of the Present Invention

[0030] (1) Zebrafish embryos 48 hours after fertilization (48hpf) were placed in embryo culture medium containing 1~200μmol / L valproic acid and cultured at a constant temperature of 28±0.5℃ for 24 hours to obtain a zebrafish model with dilated cardiomyopathy.

[0031] (2) If a transparent DCM zebrafish model is needed for observation, step (1) can be replaced by: placing the fertilized embryo in embryo culture medium and culturing it at a constant temperature of 28±0.5℃ for 24 hours (24 hpf). After that, the zebrafish embryo is transferred to an embryo culture medium containing 0.2 mM phenylthionine (PTU) and cultured at a constant temperature of 28±0.5℃ for another 24 hours. The zebrafish embryos cultured to 48 hpf are placed in an embryo culture medium containing 1~200 μmol / L valproic acid and cultured at a constant temperature of 28±0.5℃ for 24 hours to obtain a completely transparent zebrafish model with dilated cardiomyopathy.

[0032] Example 2: Drug screening and indicator evaluation of the present invention

[0033] (1) Zebrafish embryos 48 hours after fertilization (48 hpf) were placed in embryo culture medium containing 1~200 μmol / L valproic acid and various concentrations of the test compound, and cultured at a constant temperature of 28±0.5℃ for 24 hours to obtain DCM zebrafish treated with the test compound.

[0034] (2) If it is necessary to obtain transparent DCM zebrafish after embryo treatment, step (1) can be replaced by: placing the fertilized embryo in embryo culture medium and culturing it at a constant temperature of 28±0.5℃ for 24 hours (24 hpf). After that, the zebrafish embryo is transferred to an embryo culture medium containing 0.2 mM phenylthionine (PTU) and cultured at a constant temperature of 28±0.5℃ for another 24 hours. The zebrafish embryos cultured to 48 hpf are placed in embryo culture medium containing 1~200 μmol / L valproic acid and various concentrations of the test compound and cultured at a constant temperature of 28±0.5℃ for 24 hours to obtain transparent DCM zebrafish treated with the test compound.

[0035] (3) The zebrafish treated with drugs in step (1) or step (2) were anesthetized with 0.03% tricaine, the zebrafish were adjusted to the side position, and 3% carboxymethyl cellulose was added for fixation. The zebrafish heart rate, pericardial area, arterial bulb-venous sinus distance, blood flow velocity and ventricular shortening fraction were counted under a microscope.

[0036] (4) Using blank zebrafish cultured in embryo culture medium containing the same solvent ratio and model zebrafish cultured in embryo culture medium containing 1-200 μmol / L valproic acid as controls, the heart rate, pericardial area, bulbo-venous sinus distance, ventricular size, and ventricular shortening fraction of zebrafish treated with the test compound were compared. If, compared with the model group zebrafish, the pericardial area, bulbo-venous sinus distance, and ventricular size of zebrafish treated with the test compound were significantly reduced (p<0.05), and the heart rate and ventricular shortening fraction were significantly increased (p<0.05), it indicates that the test compound has a cardioprotective effect.

[0037] Example 3: Valproic acid modeling and concentration screening

[0038] 1. Reagent preparation:

[0039] Embryo culture medium: Weigh 0.29g NaCl, 0.036g CaCl2, 0.012g KCl, and 0.08g MgSO4·7H2O, and dilute to 1 L with deionized water.

[0040] Valproic acid gradient concentration stock solution: Take 28.8 mg of valproic acid (Shanghai Maclean, catalog number P83099-100 mL) and dilute to 1 mL with dimethyl sulfoxide (Diamond, catalog number A00231-0250) to prepare a 200 mM valproic acid stock solution. Then, dilute successively with dimethyl sulfoxide to prepare valproic acid stock solutions with concentrations of 100 mM, 50 mM, 10 mM, 5 mM, and 1 mM.

[0041] Valproic acid gradient concentration working solutions: Add 0.1% volume of valproic acid gradient concentration stock solution to the embryo culture medium to prepare valproic acid working solutions with concentrations of 200 μM, 100 μM, 50 μM, 10 μM, 5 μM and 1 μM.

[0042] 2. Laboratory animals:

[0043] Healthy adult AB strain zebrafish were raised in an independent zebrafish aquarium with a constant temperature of 28±0.5℃ and a photoperiod of 14 hours of light followed by 10 hours of darkness. The zebrafish were fed brine shrimp twice daily. Healthy adult zebrafish were transferred in pairs to spawning tanks, with two pairs of males and females per tank, separated by a partition. The partition was removed the following morning, and spawning began. Eggs were collected 30 minutes later and transferred to embryo culture medium, then cultured in a constant temperature incubator at 28±0.5℃. Water was changed regularly, and dead embryos were removed.

[0044] 3. Compound treatment

[0045] The experiment consisted of one solvent control group and six valproic acid treatment groups. Zebrafish embryos cultured for 48 hours were transferred to 6-well plates, 20 embryos per well, with one replicate per group. The solvent control group received 3 mL of embryo culture medium containing 0.1% dimethyl sulfoxide (DMSO). The valproic acid treatment groups received 3 mL of valproic acid working solution at concentrations of 200 μM, 100 μM, 50 μM, 10 μM, 5 μM, and 1 μM, respectively. The plates were incubated at 28 ± 0.5 °C for 24 hours.

[0046] 4. Phenotypic observation and data statistics

[0047] Zebrafish were anesthetized with a 0.03% tricaine solution and fixed onto a glass slide with 3% carboxymethyl cellulose. The zebrafish were positioned laterally, and their cardiac phenotype was observed under a stereomicroscope. The number of heartbeats per minute was counted, and the heart rate was recorded. The zebrafish were photographed under the microscope, and the pericardial area was measured and analyzed using DenioScope software.

[0048] 5. Experimental Results

[0049] Experimental results showed that treatment with 1-10 μM valproic acid had no significant effect on the heart rate and pericardial area of ​​zebrafish. However, increasing the concentration led to enlarged hearts and bent bodies in zebrafish, and at a concentration of 200 μM valproic acid, the fish were essentially dead. Figure 1 Statistical results showed that treatment with 50-100 μM valproic acid significantly reduced the heart rate and significantly increased the pericardial area in zebrafish. Figure 2 Therefore, the results indicate that valproic acid at concentrations of 50-100 μM can cause significant cardiac damage in zebrafish. However, at a concentration of 100 μM valproic acid, some zebrafish samples exhibited hypocardia or even cardiac arrest. To further improve safety and make the statistical data of cardiac indicators more representative, a concentration of 50 μM valproic acid was preferred for constructing the DCM zebrafish model. At a concentration of 50 μM, no cases of hypocardia or cardiac arrest were observed in the zebrafish samples.

[0050] Example 4: Observation of fluorescence in the heart of transgenic zebrafish

[0051] 1. Reagent preparation:

[0052] Same as Example 3.

[0053] 2. Laboratory animals:

[0054] Healthy adult Tg(cmlc2:eGFP) zebrafish were cultured in an independent zebrafish aquaculture system with a constant temperature of 28±0.5℃ and a photoperiod of 14 hours of light followed by 10 hours of darkness. The zebrafish were fed brine shrimp twice daily. Healthy adult zebrafish were transferred in pairs to spawning tanks, with two pairs of males and females per tank, separated by a partition. The partition was removed the following morning, and spawning began. Eggs were collected 30 minutes later and transferred to embryo culture medium, then cultured in a constant temperature incubator at 28±0.5℃. Water was changed regularly, and dead embryos were removed.

[0055] 3. Compound treatment:

[0056] Same as Example 3.

[0057] 4. Phenotypic observation and data statistics:

[0058] Zebrafish were anesthetized with 0.03% tricaine solution and fixed on a glass slide with 3% carboxymethyl cellulose. The zebrafish were positioned laterally, and their cardiac phenotype was observed under 488nm excitation light using a fluorescence microscope. The number of heartbeats per minute was counted, and the heart rate was recorded. The zebrafish were photographed under the microscope, and the pericardial area, ventricular size, bulbo-venous sinus distance, and blood flow velocity were measured and statistically analyzed using DenioScope software. The diastolic and systolic diameters of the zebrafish were measured separately, and the ventricular shortening fraction was calculated using the following formula: Ventricular shortening fraction (%) = (Diastolic diameter - Systolic diameter) / Diastolic diameter × 100%.

[0059] 5. Experimental Results:

[0060] Experimental results showed that treatment with 1-10 μM valproic acid had no significant effect on zebrafish heart rate, pericardial area, ventricular size, or SV-BA distance. Treatment with 50-100 μM valproic acid significantly increased pericardial area, ventricular size, and SV-BA distance in zebrafish. Figure 3 The results (ad) indicate that valproic acid treatment caused a phenotype of cardiac enlargement. Heart rate and blood flow velocity were significantly reduced, and the fractional shortening was decreased, showing a significant difference at a concentration of 100 μM. Figure 3 (eh), indicating that valproic acid treatment successfully induced a phenotype of cardiac dysfunction in zebrafish.

[0061] Example 5: Detection of pathological sections and apoptosis in zebrafish after valproic acid treatment.

[0062] 1. Reagent preparation:

[0063] Embryo culture medium: Weigh 0.29g NaCl, 0.036g CaCl2, 0.012g KCl, and 0.08g MgSO4·7H2O, and dilute to 1 L with deionized water.

[0064] PTU culture medium: Weigh 30.4 mg PTU (SIGMA, catalog number P76299-10G) and add it to 1 L of embryo culture medium, mix thoroughly to dissolve.

[0065] Valproic acid gradient concentration stock solution: Take 14.4 mg of valproic acid (Shanghai Maclean, catalog number P83099-100 mL) and dilute to 1 mL with dimethyl sulfoxide (Diamond, catalog number A00231-0250) to prepare a 100 mM valproic acid stock solution. Then dilute with an equal volume of dimethyl sulfoxide to a concentration of 50 mM valproic acid stock solution.

[0066] Valproic acid gradient concentration working solutions: Add 0.1% volume of valproic acid gradient concentration stock solution to PTU culture medium to prepare valproic acid working solutions with concentrations of 100 μM and 50 μM respectively.

[0067] Acridine orange staining solution: Weigh 1 mg of acridine orange and add it to 100 mL of PTU culture medium. Shake well to prepare a 10 mg / L acridine orange solution.

[0068] 2. Laboratory animals:

[0069] Same as Example 3.

[0070] 3. Compound treatment:

[0071] The experiment included one solvent control group and two valproic acid treatment groups. Zebrafish embryos cultured for 48 hours were transferred to 6-well plates, 20 embryos per well, with one replicate per group. The solvent control group received 3 mL of embryo culture medium containing 0.1% dimethyl sulfoxide (DMSO), while the valproic acid treatment groups received 3 mL of 100 μM and 50 μM valproic acid working solution, respectively. The plates were incubated at 28 ± 0.5 °C for 24 hours.

[0072] 4. Paraffin sectioning and HE staining:

[0073] Ten zebrafish were harvested from each group, euthanized by anesthesia with 0.3% tricaine, and rapidly transferred to 4% paraformaldehyde for fixation. After 24 hours of fixation, they were dehydrated using a gradient of different concentrations of ethanol and then transferred to xylene for clearing. They were subsequently transferred to an equal volume mixture of xylene and paraffin for paraffin infiltration and embedding. Sections were cut into 5 μm thick sections using a paraffin microtome, stained with hematoxylin and eosin (HE), and photographed under a microscope for observation.

[0074] 5. Acridine orange staining

[0075] Ten zebrafish were harvested from each group and transferred to 12-well plates, with two replicates per group. The culture medium was aspirated, and 2 mL of acridine orange staining solution was added to each well. The plates were incubated at 28±0.5℃ in the dark for 50 min. After staining, the background was washed away with PTU culture medium three times, 20 min each time. The green fluorescence under 488 nm excitation light was observed using a fluorescence microscope.

[0076] 6. Experimental Results:

[0077] (1) Pathological sections

[0078] Paraffin sections and HE staining results comparing the solvent control group and the 50 μM valproic acid treatment group showed that, under 50 μM valproic acid treatment, the zebrafish ventricles were significantly enlarged and the ventricular walls were thinned. Figure 4 This is consistent with the characteristics of ventricular dilation.

[0079] (2) Acridine orange staining

[0080] Acridine orange emits green fluorescence upon binding to nucleic acids. During apoptosis, it forms highly concentrated and fragmented chromatin fragments, resulting in even denser fluorescence after staining. Treatment with 50 μM and 100 μM valproic acid significantly increased green fluorescence in the zebrafish heart region, forming numerous dense fluorescent particles. Figure 5 This indicates that valproic acid treatment led to an increase in apoptosis in zebrafish heart cells.

[0081] Example 6: Validation of the model using carvedilol, a positive drug for dilated cardiomyopathy.

[0082] 1. Reagent preparation:

[0083] Embryo culture medium: Weigh 0.29g NaCl, 0.036g CaCl2, 0.012g KCl, and 0.08g MgSO4·7H2O, and dilute to 1 L with deionized water.

[0084] PTU culture medium: Weigh 30.4 mg PTU (SIGMA, catalog number P76299-10G) and add it to 1 L of embryo culture medium, mix thoroughly to dissolve.

[0085] Valproic acid stock solution: Take 14.4 mg of valproic acid (Shanghai Maclean, catalog number P83099-100 mL) and dilute to 1 mL with dimethyl sulfoxide (Diamond, catalog number A00231-0250) to prepare 100 mM valproic acid stock solution.

[0086] Valproic acid working solution: Add 0.05% volume of valproic acid stock solution to PTU culture medium to prepare a 50 μM valproic acid working solution.

[0087] Carvedilol stock solution: Weigh 24.36 mg of carvedilol (Shanghai Dibai, product number K949750-2g), dissolve it in 1 mL of dimethyl sulfoxide, and prepare a carvedilol stock solution with a concentration of 60 mM.

[0088] Carvedilol working solutions: Add 0.05% volume of carvedilol stock solution to valproic acid working solution to prepare carvedilol working solutions with concentrations of 5μM, 10μM, 20μM, and 30μM.

[0089] 2. Laboratory animals:

[0090] Experimental animal example 3.

[0091] 3. Compound treatment:

[0092] The experiment included one solvent control group, one model group, and four carvedilol treatment groups. Zebrafish embryos cultured for 48 hours were transferred to 6-well plates, 20 embryos per well, with one replicate per group. The solvent control group received 3 mL of PTU medium containing 0.1% (v / v) dimethyl sulfoxide (DMSO). The valproic acid treatment groups received 3 mL of 50 μM valproic acid working solution and 0.05% (v / v) DMSO. The carvedilol treatment groups received 3 mL of 5 μM, 10 μM, 20 μM, and 30 μM carvedilol working solutions, respectively. The plates were incubated at 28 ± 0.5 °C for 24 hours.

[0093] 4. Phenotypic observation and data statistics

[0094] Zebrafish were anesthetized with a 0.03% tricaine solution and fixed onto a glass slide with 3% carboxymethyl cellulose. The zebrafish were positioned laterally, and their cardiac phenotype was observed under a stereomicroscope. The number of heartbeats per minute was counted, and the heart rate was recorded. The zebrafish were photographed under the microscope, and the pericardial area was measured and analyzed using DenioScope software.

[0095] 5. Experimental Results

[0096] Carvedilol is a beta-blocker and one of the main treatments for dilated cardiomyopathy (DCM). Experimental results showed that 5-10 μM carvedilol had no significant effect on heart rate and pericardial area in valproic acid-induced DCM zebrafish. However, when the carvedilol concentration increased to 20 μM and 30 μM, valproic acid-induced DCM zebrafish heart rate significantly increased, and pericardial area significantly decreased. Figure 6 Therefore, carvedilol can significantly alleviate the valproic acid-induced decrease in heart rate and pericardial edema phenotype in zebrafish, reducing cardiac damage, indicating that carvedilol has a cardioprotective effect, and also demonstrating the feasibility of drug screening in the valproic acid-induced DCM zebrafish model.

[0097] Example 7: Application of valproic acid-induced DCM in zebrafish for drug screening in dilated cardiomyopathy

[0098] 1. Reagent preparation:

[0099] Embryo culture medium: Weigh 0.29g NaCl, 0.036g CaCl2, 0.012g KCl, and 0.08g MgSO4·7H2O, and dilute to 1 L with deionized water.

[0100] PTU culture medium: Weigh 30.4 mg PTU (SIGMA, catalog number P76299-10G) and add it to 1 L of embryo culture medium, mix thoroughly to dissolve.

[0101] Valproic acid stock solution: Take 14.4 mg of valproic acid (Shanghai Maclean, catalog number P83099-100 mL) and dilute to 1 mL with dimethyl sulfoxide (Diamond, catalog number A00231-0250) to prepare 100 mM valproic acid stock solution.

[0102] Valproic acid working solution: Add 0.05% volume of valproic acid stock solution to PTU culture medium to prepare a 50 μM valproic acid working solution.

[0103] Terfenadine stock solution: Weigh 9.4 mg of terfenadine (Shanghai Maclean, catalog number T856786-250 mg), dissolve it in 1 mL of dimethyl sulfoxide, and prepare a terfenadine stock solution with a concentration of 20 mM.

[0104] Terfenadine working solution: Add 0.05% volume of terfenadine stock solution to PTU culture medium to prepare a 10 μM terfenadine working solution.

[0105] Rutin stock solution: Weigh 250.0 mg of rutin (Shanghai Maclean, product number R835820-5g), dissolve it in 1 mL of dimethyl sulfoxide, and prepare a rutin stock solution with a concentration of 250.0 mg / mL.

[0106] Rutin working solution: Add 0.05% volume of rutin stock solution to valproic acid working solution or terfenadine working solution to prepare rutin working solutions with concentrations of 12.5 mg / L, 25 mg / L, 50 mg / L and 125 mg / L respectively.

[0107] 2. Laboratory animals:

[0108] The experimental animals were the same as in Example 3.

[0109] 3. Compound treatment:

[0110] The experiment included one solvent control group, two model groups, and four rutin treatment groups for each model. Zebrafish embryos cultured for 48 hours were transferred to 6-well plates, 20 embryos per well, with one replicate per group. The solvent control group received 3 mL of PTU medium containing 0.1% (v / v) dimethyl sulfoxide (DMSO). The valproic acid model group received 3 mL of 50 μM valproic acid working solution and 0.05% (v / v) DMSO. The terfenadine model group received 3 mL of 10 μM terfenadine working solution and 0.05% (v / v) DMSO. The rutin treatment groups received 3 mL of rutin working solution at concentrations of 12.5 mg / L, 25 mg / L, 50 mg / L, and 125 mg / L, respectively. The plates were incubated at 28 ± 0.5 °C for 24 hours.

[0111] 4. Phenotypic observation and data statistics:

[0112] Zebrafish were anesthetized with 0.03% tricaine solution and fixed on a glass slide with 3% carboxymethyl cellulose. The zebrafish were positioned laterally, and their cardiac phenotype was observed under 488nm excitation light using a fluorescence microscope. The number of heartbeats per minute was counted, and the heart rate was recorded. The zebrafish were photographed under the microscope, and the pericardial area, ventricular size, and bulbovenous sinus distance were measured and statistically analyzed using DenioScope software. The diastolic and systolic ventricular diameters were measured separately, and the ventricular shortening fraction was calculated using the following formula: Ventricular shortening fraction (%) = (Diastolic ventricular diameter - Systolic ventricular diameter) / Diastolic ventricular diameter × 100%.

[0113] 5. Experimental Results:

[0114] Experimental results showed that treatment with 12.5-50 mg / L rutin could reduce the pericardial area, ventricular size, and SV-BA distance in valproic acid-induced DCM zebrafish. Figure 7 ac), and increases zebrafish heart rate and ventricular shortening fraction ( Figure 7 The study found that rutin significantly reduced valproic acid-induced cardiac damage in DCM zebrafish, alleviated cardiac enlargement, and relieved cardiac dysfunction, demonstrating a certain cardioprotective effect.

[0115] However, in the terfenadine-induced zebrafish model, different concentrations of rutin did not show statistically significant improvements in pericardial area, heart rate, and ventricular shortening fraction in DCM zebrafish, and only some effect was observed in reducing ventricular size and SV-BA distance. Figure 8 ).

[0116] The results of the comparative experiment show that: (1) the candidate drug rutin showed a significant protective effect in the valproic acid-induced model of the present invention, which confirms the effectiveness and sensitivity of the model of the present invention in drug screening; (2) rutin showed different pharmacodynamic responses in two DCM models induced by different mechanisms, which experimentally verified that the valproic acid model of the present invention and the existing terfenadine model are different in simulating the pathological mechanism of DCM. The two are not simply substitutes, but have important complementary value.

[0117] Therefore, this invention not only provides a novel high-throughput DCM drug screening model, but can also be used in conjunction with existing models to evaluate candidate drugs from multiple perspectives and mechanisms, thereby effectively reducing the risk of "false negative" results in drug screening and improving the efficiency of discovering novel anti-DCM drugs.

Claims

1. The use of valproic acid in constructing an animal model of dilated cardiomyopathy; the animal is zebrafish.

2. Use according to claim 1, characterized in that: The dosage of valproic acid is 50-100 μM.

3. Use according to claim 2, characterized in that: The dosage of valproic acid is 50 μM.

4. A method for constructing an animal model of dilated cardiomyopathy, characterized by: It is valproic acid administered at a dosage of 50-100 μM in zebrafish farming.

5. The method of claim 4, wherein the method is performed on a non-human animal. The method involves placing zebrafish embryos, 48 ​​hours after fertilization, in an embryo culture medium containing 50-100 μmol / L valproic acid and culturing them at a constant temperature of 28±0.5℃ for 24 hours to obtain a zebrafish model of dilated cardiomyopathy.

6. The method of claim 5, wherein the method is performed on a non-human animal. The concentration of valproic acid is 50 μmol / L.

7. The method of claim 5 or 6, wherein the method is characterized by: The method for processing zebrafish embryos is as follows: The fertilized embryos were placed in embryo culture medium and cultured at a constant temperature of 28±0.5℃. After 24 hours, the zebrafish embryos were transferred to embryo culture medium containing 0.2mM phenylthionine PTU and cultured at a constant temperature of 28±0.5℃ for another 24 hours.

8. Use of the animal model constructed by the construction method according to any one of claims 4-7 in screening drugs for the prevention and / or treatment of dilated cardiomyopathy.

9. A method of screening for a candidate drug for preventing or / and treating dilated cardiomyopathy, characterized by: The candidate drug is administered to an animal model of dilated cardiomyopathy constructed by the construction method according to any one of claims 4-7.

10. The method for screening candidate drugs for the prevention and / or treatment of dilated cardiomyopathy according to claim 9, characterized in that: The candidate drug is rutin.

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