Medicine for treating or relieving heart failure

Abstract

The invention discloses a medicine for treating or relieving heart failure, the medicine comprises an estrogen compound, and the estrogen compound is preferably mestraanol. According to the invention, verapamil hydrochloride is used for inducing and establishing the chronic heart failure model of zebrafish, and direct exposure of the estrogen compound MESTRANOL is carried out on a zebrafish embryo with heart failure, so that the heart failure of zebrafish is relieved. The phenotype of zebrafish is observed and analyzed, and physiological data, gene expression quantity related to heart failure and key events causing heart failure in rat H9c2 myocardial cells, such as ROS level and cell apoptosis, are counted; the estrogen compound MESTRANOL is innovatively found and proved to be capable of relieving heart rate decline, venous sinus extravasated blood, pericardial cavity enlargement and heart cyclization abnormity caused by heart failure, and has more excellent drug effects compared with positive drugs E2 and G1, so that a foundation is laid for developing new drugs for treating heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, particularly to the field of cardiovascular system diseases, and more specifically, to a drug for treating or alleviating heart failure. Background Art

[0002] Currently, cardiovascular system diseases have become one of the greatest enemies threatening human health. They cause approximately 9.6 million male and 8.9 million female deaths globally each year, accounting for about one-third of all disease deaths. Among them, heart failure is the end stage of ischemic cardiovascular diseases and is a highly prevalent disease. Most heart failures are chronic processes, usually caused by long-term hypertension or cardiovascular diseases. However, acute heart failure may occur due to the sudden deterioration of chronic heart failure conditions, infections, or chemotherapy treatments. Weakened cardiac function usually impedes blood flow through the heart chambers, leading to congestion or fluid accumulation in the lungs and other tissues. As a reflex response, several physiological systems, including the neurohormonal, antidiuretic, and renin-angiotensin systems, are triggered under stress to the normal function of the heart to compensate for insufficient cardiac output. Heart failure is a multi-factorial disease associated with multiple bioenergetic metabolism abnormalities, including reduced energy metabolism, increased apoptosis, production of reactive oxygen species (ROS), calcium signal transduction dysfunction, impaired cardiac function, systemic inflammation, and activation of neurohormones. Its main clinicopathological features include cardiac enlargement, severe venous congestion, insufficient cardiac output, decreased heart rate, and slowed blood flow velocity. Currently, conventional drugs for treating heart failure include angiotensin-converting enzyme inhibitors (ACEIs), β-blockers, diuretics, corticosteroid receptor antagonists, sodium-glucose co-transporter inhibitors, etc. Despite the availability of many drugs, the morbidity and mortality of heart failure remain high. Therefore, how to effectively slow down the disease progression and reduce the morbidity and mortality of heart failure has become a global public health issue. Additionally, although current medical methods have also improved, the prognosis of heart failure patients remains poor.

[0003] Current research has found that there are gender differences in the clinical manifestations and rehabilitation of cardiovascular diseases. During the occurrence of heart failure in men, major vascular coronary artery diseases and myocardial infarction are the main causes, while during the occurrence of heart failure in women, coronary microvascular dysfunction, hypertension, and immune-inflammatory mechanisms play a greater role. Before menopause, compared with men, women have a lower probability of suffering from cardiovascular diseases, and this phenomenon is largely attributed to the effects of female hormones such as estrogen, which affect various processes in the heart and vascular system to reduce cardiovascular risks. Similarly, with the decline in estrogen levels during and after menopause, the cardiovascular risks of women suffering from various diseases increase, approaching or in some cases exceeding those of men of the same age. The cardiovascular mortality rate of women receiving estrogen therapy after menopause is 1 / 3 to 1 / 2 of that of women not receiving treatment. Although the biological effects of estrogen in men are not as clear as in women, more and more clinical evidence shows that endogenous estrogen 17β-estradiol can not only protect the cardiovascular health of female patients, but also that of male patients. For example, higher serum estrogen levels are associated with a lower risk of cardiovascular disease events in elderly men. In addition, in mammalian models with various heart defects, females have always shown lower mortality rates, lower disease severity phenotypes, and better functional recovery than males. However, the role and mechanism of estrogen in the heart have not been clearly studied.

[0004] Estrogen is an endogenous hormone with a variety of physiological effects and participates in reproductive and non-reproductive functions throughout embryonic development and adult life. 17β-Estradiol (E2) is the most effective natural estrogen in the human body, mainly secreted by the ovaries and is the main estrogen before menopause. Estrogen has many important effects on cardiovascular function and diseases, and can directly affect cardiovascular health and diseases through direct effects on vascular cells or myocardial cells or indirectly through systemic effects. However, natural estrogen is metabolized by the liver when taken orally and is rapidly metabolized by intramuscular injection, so its action time is short. Synthetic estrogens can change their chemical structure without losing their biological activity, slow down their metabolic process, and increase their absorption rate. Therefore, it is very important to find an estrogen compound with a long action time and high effectiveness. Mestranol (ZFE1) is a synthetic estrogen compound, and its role in cardiovascular diseases is currently unclear.

[0005] Drug discovery involves a complex iterative process of biochemical and cellular assays, which are ultimately validated in animal models. Commonly used mammalian heart failure models are usually costly and have long test cycles; moreover, the use of mammals is also increasingly restricted, requiring use only when absolutely necessary, such as in preclinical toxicity and safety assessments. Zebrafish have a short reproductive cycle and can generally lay eggs when they grow to 3 months old. They can be raised in 96-well plates or 384-well plates. The zebrafish heart is highly similar to the human heart in terms of structure, function, signaling pathways, and ion channels. Both are muscles designed to pump oxygenated blood through the body. The characteristics of the action potential of zebrafish cardiomyocytes are very similar to those of human cardiomyocytes. Its heart has a coronary vascular structure and exhibits similar functional characteristics to the mammalian heart, including blood flow direction, a high-pressure system driven by specialized endocardial muscle tissue, a heart rhythm regulated by an electrical system, and a heartbeat related to pacemaker activity. Due to the similar electrophysiological and kinetic properties of zebrafish to the mammalian heart, their response to drug treatment is very similar to that of humans, showing many advantages in human disease research and drug development. In addition, zebrafish have the ability to produce a large number of embryos, and the embryos are transparent and permeable to small molecules. Compounds can be directly added to the water environment of zebrafish and are easily absorbed, providing significant practical advantages in high-throughput chemical screening. Therefore, in recent years, zebrafish have gradually been used in different stages of the drug discovery process, becoming a useful and cost-effective alternative to some mammalian models. Especially in the field of cardiovascular system diseases, zebrafish have been used as a new type of cardiovascular system animal model to evaluate drug toxicity, efficacy, and drug screening. However, there is currently no report on using the zebrafish heart failure model to study the alleviating effect of estrogenic compounds on heart failure. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a drug for treating or alleviating heart failure, which can relieve the heart rate decline, venous sinus congestion, pericardial cavity enlargement, and abnormal cardiac looping caused by heart failure.

[0007] To solve the above technical problem, the drug for treating or alleviating heart failure of the present invention contains estrogenic compounds.

[0008] Another technical problem to be solved by the present invention is to provide the use of estrogenic compounds in the preparation of drugs for treating or alleviating heart failure.

[0009] The estrogenic compound is preferably mestranol (MESTRANOL, ZFE1).

[0010] The present invention establishes a zebrafish chronic heart failure model induced by verapamil hydrochloride (VPH), and directly exposes the zebrafish embryos with heart failure to the small molecule drug mestranol, which changes the phenotypes of the zebrafish with heart failure. By observing and analyzing the phenotypes of zebrafish and statistically analyzing the physiological data and the gene expression levels related to heart failure, the role of the estrogen compound mestranol in heart failure is studied, and it is confirmed that mestranol can relieve the heart rate decline, venous sinus congestion, pericardial cavity enlargement, and abnormal cardiac looping caused by heart failure. The heart failure-relieving effect of mestranol is also verified in rat H9c2 cardiomyocytes. Thus, the drug mestranol with a heart failure-relieving effect is innovatively discovered and screened, laying a foundation for the development of new drugs for the treatment of heart failure in the future. Brief Description of the Drawings

[0011] Figure 1 It is the establishment of a disease model of zebrafish heart failure induced by verapamil hydrochloride. Figure a shows the phenotype under the bright field of the microscope; VPH causes abnormal cardiac structure in zebrafish, including pericardial cavity enlargement, venous sinus congestion, and abnormal cardiac looping. The triangle indicates the pericardial cavity, and the arrow indicates the venous sinus. Figure b shows the cardiac morphology of zebrafish after cmlc2-EGFP under the fluorescence microscope. Scale bar: 200 μm.

[0012] Figure 2 It is that mestranol can relieve the abnormal cardiac looping and structural changes of zebrafish with heart failure. Figures a - d are the phenotype diagrams of different groups under the optical microscope; Figures e - h are the fluorescence images of the zebrafish heart labeled with cmlc2-EGFP; Figures i - l are the confocal fluorescence images of the zebrafish heart co-labeled with cmlc2-EGFP and flk-mCherry. Scale bar: 200 μm.

[0013] Figures 3 - 5 It is that mestranol can relieve the abnormal cardiac looping and systolic function of zebrafish with heart failure. Figure 3 Figure is the phenotype diagram of the zebrafish heart taken by a high-speed camera, showing the changes during ventricular diastole and systole; Figure 4 Figure is the statistical chart of the effect of mestranol on the abnormal cardiac looping angle of zebrafish; Figure 5 Figure is the statistical chart of the effect of mestranol on the abnormal fractional change in ventricular area of the zebrafish heart. (In the figure, **P < 0.01, ****P < 0.0001, ns: no statistical difference)

[0014] Figures 6 - 7 It is that mestranol can relieve the decrease in heart rate and stroke volume of zebrafish in the heart failure model group. Figure 6 Under the relief of mestranol, the heart rate of zebrafish increases significantly compared with the heart failure group.Figure 7 Under the alleviation of mestranol, the stroke volume of zebrafish heart increased significantly compared with the heart failure group. (***P < 0.001, ****P < 0.0001, ns: no statistical difference in the figure)

[0015] Figures 8 - 12 It is a comparison chart of the pharmacodynamic results of mestranol, E2, and G1 in alleviating zebrafish heart failure. Among them, Figure 8 It is a comparison chart of the effects of E2, G1, and mestranol in alleviating zebrafish heart failure after VPH modeling on the 2nd, 4th, and 6th dpf; Figure 9 , Figure 10 , Figure 11 , Figure 12 They are the comparison charts of heart rate, fractional change in right ventricular area, stroke volume, and ejection fraction of each group respectively. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in the figure)

[0016] Figures 13 - 14 It is the effect diagram of mestranol in alleviating the abnormal expression of genes related to VPH-induced zebrafish heart failure. Among them, Figure 13 It is the expression of five heart failure-related genes, Caspase-3, NLRP3, TNF-α, nppa, and nppb, in each group; Figure 14 It is the effect diagram of using whole-mount in situ hybridization technology to verify the abnormal expression patterns of the two heart failure marker genes, nppa and nppb, alleviated by mestranol in the VPH group. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in the figure)

[0017] Figures 15 - 17 It is the effect diagram of mestranol in alleviating the increase in ROS and the decrease in cell viability of VPH-induced H9c2 heart failure cells. Among them, Figure 15 It is the fluorescence microscope image of the ROS experiment on H9c2 cells; Figure 16 It is the statistical analysis chart of the detection of H9c2 cell viability; Figure 17 It is the statistical analysis chart of the detection of ROS in H9c2 cells. (*P < 0.05, **P < 0.01,

[0018] ***P < 0.001, ****P < 0.0001)

[0019] Figure 18This is the effect of MESTRANOL on alleviating the apoptosis of H9c2 heart failure cells induced by VPH. Figure A is a fluorescence microscope image of H9c2 cells subjected to Tunel apoptosis experiment and DAPI staining; Figure B is a statistical graph of the fluorescence intensity of apoptosis signals. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figure) DETAILED DESCRIPTION

[0020] In order to have a more specific understanding of the technical content, characteristics and effects of the present invention, the technical solution of the present invention is further described in detail in conjunction with the accompanying drawings and specific embodiments.

[0021] Example 1

[0022] 1. Experimental Subjects

[0023] The wild-type zebrafish used in this example is the AB strain, purchased from the National Zebrafish Resource Center in Wuhan, Hubei Province. The fluorescently labeled transgenic zebrafish used in the experiment were constructed by the laboratory of Shanghai Ocean University. All treatments of zebrafish in this example are bred and used for scientific research purposes, and are carried out in accordance with the relevant provisions of Shanghai Ocean University on animal ethics (IACUCSHOU-DW-2021-042). The laboratory has a well-equipped zebrafish fish room, and zebrafish are cultured in a circulation system treated with ultraviolet (UV) and aeration. The system can monitor the dissolved oxygen content, pH value and ammonia nitrogen content of the breeding water in real time and automatically adjust and circulate it. Probiotics are regularly added to the water to inhibit the growth of harmful bacteria. The breeding water temperature is 28.5°C, and the photoperiod is 14 hours of light and 10 hours of darkness. The laboratory provides a special fish tank. When the fish are matched, a female and a male zebrafish are placed in the fish tank. The next day, the plate is pulled out, and the eggs laid by the zebrafish are collected in a culture dish and placed in a 28.5°C constant temperature incubator for cultivation. The breeding water needs to be changed every morning and evening and the dead eggs that turn white need to be removed. Female zebrafish can lay eggs once a week. When the zebrafish embryo develops to 48hpf, the membrane breaks. Since the detached egg membrane will affect the embryo growth environment, the detached egg membrane needs to be sucked out in time, and the embryo is fed with paramecium after growing to 5dpf. When the zebrafish grows to 15dpf, it can be fed with brine shrimp in small amounts (collected from brine shrimp eggs incubated in 28°C sea salt water for 36h). At this time, it can be moved to the circulation system for cultivation. After three months, it reaches sexual maturity and can mate and lay eggs.

[0024] The H9c2 rat cardiomyocytes used in this example were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Experiments such as cell recovery, cell passage, and cell apoptosis were performed in accordance with standard cell experiment procedures.

[0025] 2. Reagents

[0026] Verapamil hydrochloride used in this example was purchased from Shanghai Xianding Biotechnology Co., Ltd., dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the remaining inorganic and organic reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0027] Preparation of E3 culture medium: Weigh 0.0133 g of KCl, 0.29 g of NaCl, 0.0365 g of CaCl2, and 0.0815 g of MgCl2·6H2O, and finally make up the volume to 1 L with ddH2O.

[0028] Preparation of PTU solution: PTU (1-Phenyl-2-thiourea) powder was dissolved in 0.3X Danieau culture medium at a concentration of 0.0045%.

[0029] Preparation of 0.3X Danieau solution: 17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 1.5 mM 2-(4-(2-Hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES) (pH 7.6), and make up the volume to 1 L with water.

[0030] Preparation of verapamil hydrochloride stock solution: Weigh verapamil hydrochloride powder and dissolve it in DMSO to prepare a solution with a concentration of 100 mM.

[0031] Preparation of mestranol stock solution: Weigh mestranol powder and dissolve it in DMSO to prepare a solution with a concentration of 5 mM.

[0032] Preparation of G1 stock solution: Weigh the estrogen receptor selective agonist G1 powder and dissolve it in DMSO to prepare a solution with a concentration of 5 mM.

[0033] Preparation of G15 stock solution: Weigh the estrogen receptor selective antagonist G15 powder and dissolve it in DMSO to prepare a solution with a concentration of 5 mM.

[0034] Preparation of E2 stock solution: Weigh natural estrogen E2 powder and dissolve it in DMSO to prepare a solution with a concentration of 5 mM.

[0035] III. Main instruments

[0036] Full-automatic pure water machine (MerckMillipo, Milli-QDirect8), -40 °C low-temperature refrigerator (Haier, DW-40L508), constant temperature incubator (Panasonic, MIR-154-PC), high-pressure steam sterilizer (SANYO, MLS-3780), 4 °C refrigerator (Haier, HYC-610), vortex oscillator (Kylin-Bell, vartex-6), -80 °C ultra-low temperature refrigerator (Panasonic, MDF-U53V), upright fluorescence microscope (Zeiss, AxioImager2), binocular stereomicroscope (Zeiss, Stemi305).

[0037] IV. Establishment of zebrafish heart failure model induced by verapamil hydrochloride (VPH) and observation of zebrafish heart phenotype

[0038] In this example, verapamil hydrochloride (VPH) was used to induce heart failure in zebrafish to establish a zebrafish heart failure model. During the experiment, the fluorescently labeled transgenic zebrafish line Tg(cmlc2:EGFP; flk:mCherry) was used to study the heart failure phenotype induced by VPH in zebrafish.

[0039] First, normal zebrafish embryos at 24 dpf were placed in a 10-cm-diameter petri dish, with 30 embryos in each dish as a group. A mixture of 30 mL of E3 culture medium and PTU solution at a ratio of 1:1 was added as a negative control. The prepared VPH stock solution was diluted into the E3 and PTU mixture to prepare an experimental group with a VPH concentration of 40 μM, and the final volume was also 30 mL, with the final DMSO concentration less than 0.1% (v / v%). Each group had three biological replicates and was cultured in a 28.5 °C constant temperature incubator. When the embryos hatched at 48 hpf the next day, the egg membranes needed to be aspirated. When the zebrafish embryos developed to 3 dpf, the zebrafish heart phenotype was observed under a fluorescence microscope, and abnormal hearts were found. According to the embryonic development observed under the bright field of the microscope (see Figure a in Figure 1 ), obvious pericardial cavity edema, venous sinus congestion, abnormal cardiac looping, and impaired cardiac contraction were observed in zebrafish. Abnormal cardiac structure was also clearly found in the fluorescence observation of zebrafish heart cmlc2-EGFP (see Figure b in Figure 1 ). These phenomena are similar to the pathophysiological characteristics observed in heart failure patients.

[0040] This experiment confirmed that the changes in the cardiac function and structure induced by verapamil hydrochloride in zebrafish can be easily identified with the naked eye, and its heart failure model can be used as a simple experimental animal model for heart failure to screen small molecule drugs for the treatment of heart failure.

[0041] V. Experiment on the effect of mestranol on zebrafish heart failure

[0042] To study the effect of mestranol on zebrafish heart failure, fluorescently labeled transgenic zebrafish line Tg(cmlc2:EGFP; flk:mCherry) embryos were used for the study. The experiment was divided into a control group, a mestranol exposure group, a VPH heart failure model group, and a VPH+mestranol co-exposure group. Drug exposure was also carried out when the zebrafish embryos developed to 24 hpf, with VPH and mestranol exposed simultaneously. The concentration of VPH was 40 μM, and the concentration of mestranol was 2 μM. After 3 dpf of drug intervention, the zebrafish were observed for hatching. Unhatched embryos were manually dechorionated under a microscope. First, the zebrafish were anesthetized with 0.04% tricaine (MS-222), then placed in 3% methylcellulose. Under the microscope, an egg pick was used to gently move the zebrafish so that its head faced left, and it was kept in a lateral position with the two eyes overlapping as much as possible and the head and tail at the same level to fully expose the heart and pericardial regions.

[0043] Bright-field and fluorescent photos of the zebrafish heart were taken with a fluorescence microscope (Figs. a-c in Figure 2 ). It was found that when the zebrafish embryos developed to 3 dpf, compared with the heart failure model group induced by VPH, as shown in Figs. a-d in Figure 2 , the heart abnormalities of the zebrafish in the VPH+mestranol co-exposure group were significantly alleviated, that is, the enlargement of the pericardial cavity disappeared, and the venous sinus congestion was restored. At the same time, the heart of the zebrafish embryos was further observed under a fluorescence microscope. As shown in Figs. e-l in Figure 2 , it was found that the heart of the zebrafish embryos in the heart failure model group induced by VPH was elongated, resulting in abnormal heart looping in the zebrafish. However, when mestranol was added, the abnormal heart looping was significantly restored and returned to the normal level.

[0044] The ventral heart green fluorescence (cmcl2-EGFP, see Figs. e-l in Figure 2 ), red-green double fluorescence (cmlc2:EGFP and flk:mCherr, see Figs. i-l in Figure 2 ) of the fluorescently labeled fish line were photographed with a two-photon confocal microscope, and the phenotypic maps were used to observe the changes in the zebrafish heart structure from multiple angles. It was found that mestranol alleviated the abnormal heart looping.

[0045] Furthermore, a high-speed camera was used to record the videos of the heartbeats of zebrafish in each group (see Figure 3), and based on the captured videos and confocal images, record and analyze relevant cardiac physiological data such as the blood flow and cardiac contractions of zebrafish, and use ImageJ software to measure the diameters during ventricular diastole and systole, the diastolic volume (DV) of the zebrafish ventricle, heart rate, and the cyclization angle. The results are as Figure 4 shown. Just as shown in the phenotypic diagram, compared with the control group, after zebrafish heart failure, the cardiac cyclization angle was significantly abnormal (P < 0.0001). When co-exposed with 2 μM mestranol, the cardiac cyclization angle was significantly restored (P < 0.0001), and single exposure to mestranol had no significant effect on cardiac cyclization (P > 0.05). In addition, the fractional change in ventricular area was statistically analyzed, and the results are as Figure 5 shown. Similarly, the fractional change in the ventricular area of zebrafish heart failure was significantly reduced, indicating that the systolic function of the zebrafish heart was significantly affected after heart failure. When 2 μM mestranol was added, compared with the VPH heart failure model group, the fractional change in area was significantly increased (P < 0.01), and the cardiac systolic function was significantly restored. Single exposure to mestranol had no significant effect on cardiac systolic function (P > 0.05).

[0046] Using the zebrafish heart beating videos, calculate and statistically analyze the heart rate and stroke volume of zebrafish in the VPH heart failure model group, mestranol exposure group, and VPH + mestranol co-exposure group to evaluate the alleviating effect of mestranol on zebrafish heart failure. The statistical results of heart rate are as Figure 6 shown. Compared with the control group, the heart rate of zebrafish in the VPH-induced heart failure model group was significantly decreased (P < 0.001), while the heart rate of zebrafish in the 2 μM mestranol single exposure group did not change significantly (P > 0.05), indicating that mestranol at this concentration itself does not affect the heart rate of zebrafish; compared with the VPH heart failure model group, when both VPH and mestranol were exposed, the heart rate was significantly restored (P < 0.001). In addition, the statistical results of cardiac stroke volume are as Figure 7 shown. It can be seen that compared with the control group, the stroke volume of the zebrafish heart in the VPH-induced heart failure model group was significantly reduced, seriously affecting the pumping function of the zebrafish heart; while the stroke volume of zebrafish in the mestranol single exposure group did not change significantly (P > 0.05), indicating that mestranol itself does not affect the pumping function of zebrafish at this concentration. Similar to the heart rate statistical results, compared with the VPH heart failure model group, co-exposure of 2 μM mestranol and VPH can significantly restore the stroke volume of the zebrafish heart (P < 0.0001).

[0047] VI. Pharmacodynamic comparison experiment of mestranol, E2, and G1 on alleviating zebrafish heart failure

[0048] To further verify the efficacy of mestranol, the positive drugs endogenous natural estrogen E2 and estrogen receptor selective agonist G1 were introduced to conduct a multi-time point efficacy comparison experiment. Fluorescently labeled transgenic zebrafish line Tg(cmlc2:EGFP; flk:mCherry) embryos were used in the experiment. The experiment was divided into five groups, namely the control group, the VPH heart failure model group, the VPH+mestranol co-exposure group, the VPH+G1 co-exposure group, and the VPH+E2 co-exposure group. The drug exposure was also carried out when the zebrafish embryos developed to 24 hpf. The concentration of VPH was 40 μM, the concentration of mestranol was 2 μM, the concentration of G1 was 20 nM, and the concentration of E2 was 400 nM. The method of zebrafish hatching was the same as above.

[0049] The experiment found that at 2 dpf, VPH caused venous sinus congestion in zebrafish, while mestranol alleviated the congestion well, and only G1 and E2 alleviated it to a certain extent. At 4 dpf, mestranol, G1, and E2 all alleviated the pericardial cavity enlargement caused by VPH and also improved the venous sinus congestion to a certain extent. However, at 6 dpf, the efficacy of G1 and E2 decreased significantly, and only the efficacy of mestranol remained stable and alleviated the venous sinus congestion. See Figure 8 as shown. It can be seen that compared with the endogenous natural estrogen E2, mestranol has a better ability to alleviate the venous sinus congestion and pericardial cavity enlargement caused by VPH. It can not only better alleviate the heart function decline caused by VPH, but also has a longer-lasting efficacy.

[0050] The heart beating videos of each group were taken by a high-speed camera, and various cardiac physiological indexes were statistically analyzed from them and found that (as Figures 9 - 12 shown), mestranol was superior to the endogenous natural estrogen E2 and estrogen receptor selective agonist G1 in terms of several cardiac function evaluation indexes such as heart rates, fractional area change (FAC) of the right ventricle, stroke volume, and ejection fraction (EF); at 6 dpf, the efficacy of E2 and G1 decreased sharply, and various cardiac physiological indexes also decreased, while the efficacy of mestranol remained stable and the cardiac physiological indexes were good. It can be seen that compared with the endogenous natural estrogen E2 and estrogen receptor selective agonist G1, the efficacy of mestranol is more excellent.

[0051] VII. Mechanism of action of mestranol

[0052] The experiment was conducted using transgenic zebrafish line Tg(cmlc2:EGFP; flk:mCherry) embryos labeled with fluorescence. The experiment was divided into five groups, namely the control group, the VPH heart failure model group, the VPH+MESTRANOL co-exposure group, the VPH+G1 co-exposure group, and the VPH+MESTRANOL+G15 co-exposure group. Drug exposure was also carried out when the zebrafish embryos developed to 24 hpf. The concentration of VPH was 40 μM, the concentration of MESTRANOL was 2 μM, the concentration of G1 was 20 nM, and the concentration of the estrogen receptor selective agonist G15 was 50 nM.

[0053] The qpcr experiment was carried out, and the results were as Figure 13 shown that the abnormal expressions of four heart failure-related genes, NLRP3, TNF-α, nppa, and nppb, in the heart failure model were alleviated by MESTRANOL.

[0054] The whole-mount in situ hybridization experiment was carried out, and the results were as Figure 14 shown that MESTRANOL could alleviate the abnormal expression patterns of the two heart failure marker genes, nppa and nppb, in the VPH group.

[0055] Thus, it was preliminarily confirmed that MESTRANOL played a role in alleviating heart failure by regulating the expressions and expression patterns of heart failure-related genes such as NLRP3, TNF-α, nppa, and nppb, as well as regulating the inflammatory response and cell apoptosis.

[0056] VIII. The alleviating effect of MESTRANOL on heart failure in H9c2 rat cardiomyocytes

[0057] The experiment was divided into: the control group (normal culture), the model group (induced by 80 μM VPH for 12 h), and 4 VPH+ZFE1 groups (after inducing H9c2 rat cardiomyocytes with VPH for 12 h, each group was added with ZFE1 at safe concentrations of 0.2 μM, 0.4 μM, 1 μM, and 2 μM respectively). After culturing in the incubator for 36 h, 10 μL of CCK-8 solution was added to each well, and it was placed in the 37 °C incubator for dark incubation for 1-2 h. The OD value at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay instrument, and the cell survival rate was obtained through conversion.

[0058] ROS staining experiments were performed on each group of cells. First, DCFH-DA (2,7-dichlorofluorescein diacetate) was diluted 1:2000 with serum-free cell culture medium to a final concentration of 10 μmol / L. The cell culture medium was removed, and an appropriate volume of diluted DCFH-DA was added (the added volume should be sufficient to cover the cells. Generally, for one well of a six-well plate, no less than 2.5 mL of diluted DCFH-DA is added). Incubate in a 37 °C cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. Finally, take pictures with a fluorescence microscope. The results are as Figures 15 - 17 shown. The green ROS signal increased in the cells of the model group, and the ROS signal in the VPH+ZFE1 group was significantly reduced compared with the model group. The cell viability in the VPH+ZFE1 group was significantly increased compared with the model group, and the best alleviating effect was achieved when the concentration of mestranol was 1 μM.

[0059] Tunel cell apoptosis experiment, preparation of cell slides: Prepare a cell suspension according to the above cell passage method. Place the cell slides in a 12-well plate, inoculate 2×10 5 cells per well, a total of 2 mL of cell suspension, and treat each group of cells with drugs according to the above method.

[0060] Tunel detection: First, wash each group of cells once with PBS, then fix the cells with 4% paraformaldehyde for 30 minutes, wash once with PBS, add PBS containing 0.3% Triton X-100, and incubate at room temperature for 5 minutes. Wash twice with PBS. Add 50 μl of Tunel detection solution to the sample, and incubate at 37 °C in the dark for 60 minutes. Finally, take out the slides, place them on a glass slide with anti-fluorescence quenching agent, and take pictures with a confocal microscope. The results are as Figure 18 shown. The red apoptotic signal increased in the cells of the model group, while the apoptotic signal in the VPH+ZFE1 group was significantly reduced compared with the model group.

[0061] The above embodiments are only feasible or preferred embodiments of the present invention, which are used to illustrate the present invention and are not intended to limit the scope of the patent application of the present invention. Therefore, all equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope covered by the patent of the present invention.

Claims

1. A drug for treating or alleviating heart failure, characterized in that, The drug contains estrogenic compounds.

2. The drug according to claim 1, characterized in that, The estrogenic compound is mestranol.

3. Use of an estrogenic compound in the preparation of a drug for treating or alleviating heart failure.

4. The use according to claim 3, characterized in that, The estrogenic compound is mestranol.

5. Method for constructing zebrafish heart failure model, characterized in that, Use verapamil hydrochloride to induce the establishment of a zebrafish heart failure model.

6. The method according to claim 5, wherein Expose zebrafish embryos to verapamil hydrochloride.

7. The method according to claim 6, characterized in that, After the formation of the zebrafish embryo heart and the start of blood circulation, soak the zebrafish embryos with verapamil hydrochloride. After observing abnormalities in the zebrafish heart, establish a zebrafish heart failure model.

8. The method according to any one of claims 5 to 7, characterized in that, The concentration of verapamil hydrochloride is 40 μM.

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