Drug for treating or alleviating heart failure

Abstract

Disclosed is a drug for treating or alleviating heart failure, which comprises an estrogenic compound. The estrogenic compound is preferably mestranol. By means of induction with verapamil hydrochloride, a zebrafish model of chronic heart failure is established. Zebrafish embryos with heart failure are directly exposed to the estrogenic compound MESTRANOL, resulting in the alleviation of heart failure in the zebrafish. By means of observation and analysis of zebrafish phenotypes and statistical analysis of physiological data and expression levels of heart failure-related genes, as well as key events causing heart failure in rat H9c2 cardiomyocytes such as ROS level and apoptosis, it is innovatively discovered and confirmed that the estrogenic compound MESTRANOL can alleviate decreased heart rate, venous sinus congestion, pericardial cavity enlargement, and abnormal cardiac looping caused by heart failure, and has superior efficacy to positive drugs E2 and G1, thereby laying a foundation for developing new drugs for treating heart failure.

Description

A drug used to treat or relieve heart failure Technical Field

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

[0002] Cardiovascular disease has become one of the greatest threats to human health. It causes approximately 9.6 million deaths in men and 8.9 million deaths in women worldwide each year, accounting for approximately one-third of all deaths from disease. Heart failure, the end-stage of ischemic cardiovascular disease, is a highly prevalent condition. Most heart failures are chronic, often caused by long-standing hypertension or cardiovascular disease. However, acute heart failure can occur due to sudden exacerbation of chronic heart failure, infection, or chemotherapy. Reduced cardiac function often impairs 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 to compensate for insufficient cardiac output in response to stress on normal cardiac function. Heart failure is a multifactorial disease associated with multiple abnormalities in bioenergetic metabolism, including decreased energy metabolism, increased apoptosis, production of reactive oxygen species (ROS), dysfunctional calcium signaling, impaired cardiac function, systemic inflammation, and neurohormonal activation. Its main clinical pathological features include heart enlargement, severe venous congestion, insufficient cardiac output, decreased heart rate and slowed blood flow. Currently, conventional drugs used to treat heart failure include angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers, diuretics, corticosteroid receptor antagonists, sodium-glucose cotransporter inhibitors, etc. Despite the availability of many drugs, the morbidity and mortality of heart failure remain high. Therefore, how to effectively slow down the progression of the disease and reduce the morbidity and mortality of heart failure has become a global public health issue. In addition, although current medical methods have also improved, the prognosis of patients with heart failure remains poor.

[0003] Current research has revealed gender differences in the clinical presentation and recovery of cardiovascular disease. While large vessel coronary artery disease and myocardial infarction are the primary causes of heart failure in men, microvascular dysfunction in the middle coronary arteries, hypertension, and immune-inflammatory mechanisms play a greater role in the development of heart failure in women. Before menopause, women have a lower incidence of cardiovascular disease than men. This phenomenon is largely attributed to the effects of female hormones, such as estrogen, which influence various processes in the heart and vascular system to reduce cardiovascular risk. Similarly, as estrogen levels decline during and after menopause, women's cardiovascular risk for a variety of conditions increases, approaching or, in some cases, exceeding that of men of the same age. The cardiovascular mortality rate in postmenopausal women who receive estrogen therapy is one-third to one-half that of untreated women. Although the biological effects of estrogen in men are less well-defined than in women, accumulating clinical evidence suggests that the endogenous estrogen 17β-estradiol can protect cardiovascular health not only in women but also in men. For example, higher serum estrogen levels are associated with a lower risk of cardiovascular disease events in older men. In addition, in various mammalian models of heart defects, females consistently show lower mortality and less severe disease phenotypes, as well as better functional recovery than males. However, the role of estrogen in the heart and its mechanisms remain largely unknown.

[0004] Estrogens are endogenous hormones with diverse physiological effects, participating in both reproductive and non-reproductive functions throughout embryonic development and adult life. 17β-estradiol (E2) is the most potent natural estrogen in the human body, primarily produced by the ovaries and serving as the predominant estrogen before menopause. Estrogens play numerous important roles in cardiovascular function and disease, influencing cardiovascular health and disease through direct effects on vascular or cardiac cells or indirectly through systemic effects. However, natural estrogens are metabolized by the liver after oral administration and rapidly metabolized after intramuscular injection, resulting in a short duration of action. Synthetic estrogens, on the other hand, can alter their chemical structure without losing their biological activity, slowing their metabolic process and increasing their absorption rate. Therefore, it is crucial to identify an estrogen compound with a long and potent duration of action. MESTRANOL (ZFE1) is a synthetic estrogen compound whose role in cardiovascular disease is currently unclear.

[0005] Drug discovery involves a complex, iterative process of biochemical and cellular analyses, culminating in validation in animal models. Commonly used mammalian heart failure models are often expensive and require lengthy testing cycles. Furthermore, their use is increasingly restricted to situations where absolutely necessary, such as in preclinical toxicity and safety assessments. Zebrafish have a short reproductive cycle, typically spawning at three months of age, and can be maintained in either 96-well or 384-well plates. The zebrafish heart closely resembles the human heart in structure, function, signaling pathways, and ion channels. Both are muscles designed to transport oxygen and blood throughout the body. The action potential characteristics of zebrafish cardiomyocytes closely resemble those of human cardiomyocytes. The zebrafish heart possesses a coronary vascular structure and displays similar functional characteristics to mammalian hearts, including blood flow direction, a high-voltage system driven by specialized endocardial muscle tissue, cardiac rhythm regulated by an electrical system, and heartbeats associated with pacemaker activity. Because zebrafish share similar electrophysiological and kinetic properties to mammalian hearts, their responses to drug treatment closely resemble those of humans, offering numerous advantages for human disease research and drug development. Furthermore, zebrafish have the ability to produce large numbers of embryos, and their embryos are transparent and permeable to small molecules. Compounds can be added directly to the zebrafish's water environment and readily absorbed, offering significant practical advantages in high-throughput chemical screening. Consequently, in recent years, zebrafish have been increasingly used at various stages of the drug discovery process, becoming a useful and cost-effective alternative to some mammalian models. This is particularly true in the field of cardiovascular disease, where zebrafish have been used as a novel cardiovascular animal model for assessing drug toxicity, efficacy, and drug screening. However, there are currently no reports of using the zebrafish heart failure model to study the mitigating effects of estrogen-like compounds on heart failure.

[0006] Summary of the Invention

[0007] 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 alleviate the decreased heart rate, venous sinus congestion, pericardial cavity enlargement and cardiac cyclization abnormalities caused by heart failure.

[0008] To solve the above technical problems, the drug for treating or alleviating heart failure of the present invention contains an estrogen compound.

[0009] The second technical problem to be solved by the present invention is to provide a use of an estrogen compound in the preparation of a drug for treating or alleviating heart failure.

[0010] The estrogen compound is preferably mestranol (MESTRANOL, ZFE1).

[0011] The present invention establishes a zebrafish chronic heart failure model by inducing verapamil hydrochloride (VPH), and directly exposes zebrafish embryos with heart failure to the small molecule drug MESTRANOL, thereby changing the phenotype of the zebrafish with heart failure. By observing and analyzing the zebrafish phenotype and statistically analyzing physiological data and the expression levels of genes related to heart failure, the effect of the estrogen compound MESTRANOL on heart failure is studied, confirming that MESTRANOL can alleviate the symptoms of heart failure, such as decreased heart rate, venous sinus congestion, pericardial enlargement, and abnormal cardiac cyclization. The heart failure-alleviating effect of MESTRANOL is also verified in rat H9c2 cardiomyocytes. Thus, the drug MESTRANOL with a heart failure-alleviating effect is innovatively discovered and screened, laying a foundation for the future development of new drugs for the treatment of heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the establishment of a zebrafish model of heart failure induced by verapamil hydrochloride. Panel a shows the phenotype under brightfield microscopy; VPH causes structural abnormalities in the zebrafish heart, including pericardial enlargement, venous sinus congestion, and abnormal cardiac ringing. The triangle indicates the pericardial cavity, and the arrowhead indicates the venous sinus. Panel b shows the zebrafish heart morphology under a fluorescence microscope after cmlc2-EGFP expression. Scale bar: 200 μm.

[0013] Figure 2 shows that MESTRANOL can alleviate abnormal cardiac circularization and structural changes in zebrafish with heart failure. Panels a–d show phenotypic images of different groups under light microscopy; Panels e–h show fluorescence images of zebrafish hearts labeled with cmlc2-EGFP; Panels i–l show confocal fluorescence images of zebrafish hearts co-labeled with cmlc2-EGFP and flk-mCherry. Scale bar: 200 μm.

[0014] Figures 3-5 demonstrate that MESTRANOL can alleviate abnormalities in cardiac circularization and contractile function in zebrafish with heart failure. Figure 3 shows a zebrafish cardiac phenotype captured by a high-speed camera, demonstrating changes in ventricular diastole and systole. Figure 4 shows the statistical effect of MESTRANOL on abnormalities in cardiac circularization angles in zebrafish. Figure 5 shows the statistical effect of MESTRANOL on abnormalities in ventricular fractional area change in zebrafish hearts. (**P<0.01, ****P<0.0001, ns: not statistically significant)

[0015] Figures 6 and 7 show that MESTRANOL can alleviate the decrease in heart rate and stroke volume in zebrafish in the heart failure model group. Figure 6: Under the treatment of MESTRANOL, the zebrafish heart rate increased significantly compared to the heart failure group. Figure 7: Under the treatment of MESTRANOL, the zebrafish stroke volume increased significantly compared to the heart failure group. (***P<0.001, ****P<0.0001, ns: not statistically significant)

[0016] Figures 8 to 12 are comparative diagrams of the efficacy of MESTRANOL, E2, and G1 in alleviating heart failure in zebrafish. Figure 8 compares the efficacy of E2, G1, and MESTRANOL in alleviating heart failure in zebrafish after VPH modeling at 2, 4, and 6 dpf. Figures 9, 10, 11, and 12 compare heart rate, right ventricular area fractional change, stroke volume, and ejection fraction, respectively, in each group. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figures)

[0017] Figures 13 and 14 illustrate the effect of MESTRANOL on alleviating abnormal expression of heart failure-related genes induced by VPH in zebrafish. Figure 13 shows the expression of five heart failure-related genes, Caspase-3, NLRP3, TNF-α, nppa, and nppb, in each group; Figure 14 demonstrates the effect of MESTRANOL on alleviating abnormal expression patterns of two heart failure marker genes, nppa and nppb, in the VPH group using whole-mount in situ hybridization. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figures)

[0018] Figures 15-17 show the effect of MESTRANOL on alleviating VPH-induced ROS elevation and decreased cell viability in H9c2 heart failure cells. Figure 15 is a fluorescence microscopy image of a ROS assay in H9c2 cells; Figure 16 is a statistical analysis of H9c2 cell viability assays; and Figure 17 is a statistical analysis of ROS assays in H9c2 cells. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figures)

[0019] Figure 18 shows the effect of MESTRANOL on alleviating VPH-induced apoptosis in H9c2 heart failure cells. Panel A shows fluorescence microscopy images of H9c2 cells subjected to a Tunel apoptosis assay and DAPI staining; Panel B shows the fluorescence intensity of apoptosis signals. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figures) 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 with reference to the accompanying drawings and specific embodiments.

[0021] Example 1

[0022] 1. Experimental Subjects

[0023] The wild-type zebrafish used in this embodiment is the AB strain, purchased from the National Zebrafish Resource Center in Wuhan, Hubei Province. The fluorescently labeled transgenic zebrafish used in the experiment was constructed by the laboratory of Shanghai Ocean University. All treatments of zebrafish in this embodiment are bred and used for scientific research purposes, and are carried out in accordance with the relevant regulations 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 that has been treated with ultraviolet (UV) and aeration. The system can monitor the dissolved oxygen content, pH value and ammonia nitrogen content of the aquaculture 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 aquaculture water temperature is 28.5°C, and the photoperiod is 14 hours of light and 10 hours of darkness. The laboratory provides a dedicated fish tank. When breeding, a male and female zebrafish are placed in the fish tank. The next day, 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 have turned white are removed. Female zebrafish can lay eggs once a week. When the zebrafish embryo develops to 48hpf, the membrane breaks. Because the shed egg membrane will affect the embryonic growth environment, it is necessary to suck out the shed egg membrane in time. After the embryo grows to 5dpf, it is started to feed Paramecium. When the zebrafish grows to 15dpf, it can be fed a small amount of brine shrimp (collected from brine shrimp eggs incubated in 28°C sea salt water for 36h). At this time, it can be moved to the described circulation system for cultivation. After three months, it reaches sexual maturity and can be mated and spawned.

[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 according to 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 Biotechnology (Shanghai) Co., Ltd., and the remaining inorganic and organic reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

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

[0028] Preparation of PTU solution: PTU (1-Phenyl-2-thiourea, 1-phenyl-2-sulfur, Sigma P7629) 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 hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH 7.6), and make up 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] 3. Main instruments

[0036] Fully automatic water purification machine (Merck Millipo, Milli-Q Direct 8), -40℃ low-temperature refrigerator (Haier, DW-40L508), constant temperature incubator (Panasonic, MIR-154-PC), high-pressure steam sterilizer (SANYO, MLS-3780), 4℃ refrigerator (Haier, HYC-610), vortex oscillator (Kylin-Bell, vartex-6), -80℃ ultra-low temperature refrigerator (Panasonic, MDF-U53V), upright fluorescence microscope (Zeiss, AxioImager2), binocular stereo microscope (Zeiss, Stemi305).

[0037] 4. Establishment of a Verapamil Hydrochloride (VPH)-Induced Zebrafish Heart Failure Model and Observation of Zebrafish Cardiac Phenotypes

[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, a fluorescently labeled transgenic zebrafish line Tg (cmlc2:EGFP; flk:mCherry) was used to study the VPH-induced heart failure phenotype in zebrafish.

[0039] First, 24dpf normally developed zebrafish embryos were placed in a 10cm diameter culture dish, with each dish as a group of 30 embryos. 30mL of E3 culture medium: PTU solution in a 1:1 mixture was added as a negative control. The prepared VPH mother solution was diluted into the E3 and PTU mixture to make a concentration for the experimental group containing 40μMVPH, with the final volume also being 30mL, and the final DMSO concentration being less than 0.1% (v / v%). Three biological replicates were performed in each group and cultured in a 28.5℃ constant temperature incubator. The next day, when the embryos developed to 48hpf, the membranes were broken and the egg membranes needed to be sucked out. When the zebrafish embryos developed to 3dpf, the zebrafish heart phenotype was observed under a fluorescence microscope, and abnormalities were found in the heart. Embryonic development observed under a brightfield microscope (see Figure 1, Panel a) showed significant pericardial edema, venous sinus congestion, abnormal cardiac ringing, and impaired cardiac contraction in the zebrafish. Abnormal cardiac structure was also evident in cmlc2-EGFP fluorescence observation of the zebrafish heart (see Figure 1, Panel b). These phenomena are similar to the pathophysiological features observed in patients with heart failure.

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

[0041] 5. Experimental study on the effect of MESTRANOL on zebrafish heart failure

[0042] To investigate the effects of mestranolide on zebrafish heart failure, fluorescently labeled transgenic zebrafish (cmlc2:EGFP; flk:mCherry) embryos were used. Experimental groups included a control group, a mestranolide-induced pulmonary embolism (VPH) heart failure model, and a VPH+mestranolide co-exposure group. Drug exposure was also initiated at 24 hpf, with simultaneous VPH and mestranolide exposure at concentrations of 40 μM VPH and 2 μM MESTRANOL. Three days after drug treatment, zebrafish were observed for decapitation, and unhatched embryos were manually decapitated under a microscope. Zebrafish were anesthetized with 0.04% tricaine (MS-222) and placed in 3% methylcellulose. Under a microscope, the fish were gently manipulated with an egg poker to position their heads to the left and maintain a lateral recumbent position, with their eyes overlapping as much as possible and their head and tail aligned, to fully expose the heart and pericardium.

[0043] Brightfield, or fluorescent, images of zebrafish hearts were taken using a fluorescence microscope (Figures 2a-c). When zebrafish embryos reached 3 dpf, compared with the VPH-induced heart failure model group, the heart abnormalities in the group co-exposed to VPH and MESTRANOL were significantly alleviated, as shown in Figures 2a-d. Pericardial enlargement disappeared, and venous sinus congestion was restored. Further observation of the zebrafish embryos under a fluorescence microscope, as shown in Figures 2e-l, revealed that the hearts of the zebrafish embryos in the VPH-induced heart failure model group were elongated, resulting in abnormal circularization. However, the addition of MESTRANOL significantly reversed the abnormal cardiac circularization and returned to normal levels.

[0044] Two-photon confocal microscopy was used to capture phenotypes of the zebrafish ventral heart, including green fluorescence (cmcl2-EGFP, see Figure 2 e to l), and red-green dual fluorescence (cmlc2:EGFP and flk:mCherr, see Figure 2 i to l), of fluorescently labeled fish lines. Changes in zebrafish heart structure were observed from multiple angles, and it was found that MESTRANOL alleviated cardiac circularization abnormalities.

[0045] High-speed video cameras were used to capture heartbeats in each group (see Figure 3). Based on the captured videos and confocal images, relevant cardiac physiological data, such as blood flow and cardiac contraction, were recorded and analyzed. ImageJ software was used to measure ventricular diameters during diastole and systole, ventricular diastolic volume (DV), heart rate, and circularization angle. The results, shown in Figure 4, show that, as demonstrated in the phenotype graph, cardiac circularization angles were significantly abnormal in zebrafish after heart failure compared to the control group (P < 0.0001). Co-exposure to 2 μM MESTRANOL significantly restored this circularization angle (P < 0.0001). MESTRANOL exposure alone had no significant effect on cardiac circularization (P > 0.05). In addition, the fractional change in ventricular area was calculated. The results are shown in Figure 5. The fractional change in ventricular area was significantly reduced in zebrafish hearts with heart failure, indicating that the contractile function of the heart was significantly affected. When 2 μM MESTRANOL was added, the fractional change in area was significantly increased compared to the VPH heart failure model group (P < 0.01), indicating a significant recovery of cardiac contractile function. MESTRANOL alone had no significant effect on cardiac contractile function (P > 0.05).

[0046] Using zebrafish heartbeat videos, heart rate and stroke volume were calculated and statistically analyzed for the VPH heart failure model group, the MESTRANOL-exposed group, and the VPH+MESTRANOL co-exposure group to assess the mitigation of heart failure in zebrafish. The heart rate statistical results are shown in Figure 6. Compared with the control group, the heart rate of zebrafish in the VPH-induced heart failure model group decreased significantly (P<0.001), while the heart rate of zebrafish exposed to 2μM MESTRANOL alone did not change significantly (P>0.05), indicating that MESTRANOL alone at this concentration does not affect the heart rate of zebrafish. Compared with the VPH heart failure model group, the heart rate of zebrafish exposed to both VPH and MESTRANOL significantly recovered (P<0.001). In addition, the statistical results of cardiac output are shown in Figure 7. It can be seen that compared with the control group, the stroke volume of the zebrafish hearts in the VPH-induced heart failure model group was significantly reduced, seriously affecting the zebrafish heart's pumping function. However, the stroke volume of the zebrafish in the group exposed to mestrol alone did not change significantly (P>0.05), indicating that mestrol itself does not affect the zebrafish's pumping function at this concentration. Similar to the heart rate statistical results, co-exposure to 2μM mestrol and vph significantly restored the zebrafish heart's stroke volume compared with the vph heart failure model group (P<0.0001).

[0047] VI. Comparative study of the efficacy of MESTRANOL, E2, and G1 in alleviating heart failure in zebrafish

[0048] To further validate the efficacy of MESTRANOL, endogenous natural estrogen E2 and estrogen receptor selective agonist G1 were introduced as positive drugs for multi-time point efficacy comparison experiments. The experiment used fluorescently labeled transgenic zebrafish Tg (cmlc2:EGFP; flk:mCherry) embryos for research. The experiment was divided into five groups: control group, VPH heart failure model group, VPH+MESTRANOL co-exposure group, VPH+G1 co-exposure group, and VPH+E2 co-exposure group. Drug exposure was also performed when the zebrafish embryos developed to 24 hpf, with a VPH concentration of 40 μM, a MESTRANOL concentration of 2 μM, a G1 concentration of 20 nM, and an E2 concentration of 400 nM. The zebrafish decapsulation method was the same as above.

[0049] The experiment found that at 2 dpf, VPH caused venous sinus congestion in zebrafish, and MESTRANOL effectively alleviated the congestion, while G1 and E2 only alleviated it to a certain extent. At 4 dpf, MESTRANOL, G1, and E2 all alleviated the pericardial swelling caused by VPH and also had a certain improvement in venous sinus congestion. However, at 6 dpf, the efficacy of G1 and E2 decreased significantly, and only MESTRANOL remained stable, relieving venous sinus congestion. See Figure 8. It can be seen that MESTRANOL has a better ability to relieve venous sinus congestion and pericardial swelling caused by VPH than the endogenous natural estrogen E2. It can not only better alleviate the heart function decline caused by VPH, but also has a longer-lasting effect.

[0050] High-speed cameras were used to record heartbeat videos of each group, and various cardiac physiological indicators were analyzed (as shown in Figures 9-12). The results showed that MESTRANOL outperformed endogenous natural estrogen E2 and the estrogen receptor selective agonist G1 in terms of heart rate, right ventricular area fractional change (FAC), stroke volume, and ejection fraction (EF). At 6 days postpartum, the efficacy of E2 and G1 decreased dramatically, along with various cardiac physiological indicators, while the efficacy of MESTRANOL remained stable, with good cardiac physiological indicators. This indicates that MESTRANOL is superior to endogenous natural estrogen E2 and the estrogen receptor selective agonist G1 in terms of cardiac function.

[0051] VII. Mechanism of Action of MESTRANOL

[0052] The experiment used fluorescently labeled transgenic zebrafish (cmlc2:EGFP; flk:mCherry) embryos. Five groups were divided into: a control group, a VPH heart failure model group, a VPH+MESTRANOL co-exposure group, a VPH+G1 co-exposure group, and a VPH+MESTRANOL+G15 co-exposure group. Drug exposure was also performed at 24 hpf in zebrafish embryos, with VPH concentrations of 40 μM, MESTRANOL concentrations of 2 μM, G1 concentrations of 20 nM, and the estrogen receptor selective agonist G15 concentration of 50 nM.

[0053] qPCR experiments were performed, and the results are shown in Figure 13 . The abnormal expression of four heart failure-related genes, NLRP3, TNF-α, NPPA, and NPPB, in the heart failure model was alleviated by MESTRANOL.

[0054] Whole-mount in situ hybridization experiments were performed, and the results are shown in FIG14 . MESTRANOL can alleviate the abnormal expression patterns of nppa and nppb, two heart failure marker genes, in the VPH group.

[0055] This preliminarily confirmed that MESTRANOL alleviates heart failure by regulating the expression and expression patterns of heart failure-related genes such as NLRP3, TNF-α, nppa, nppb, as well as regulating inflammatory response and cell apoptosis.

[0056] 8. Alleviating Effects of MESTRANOL on Cardiomyocytes of H9c2 Rats with Heart Failure

[0057] The experiment was divided into a control group (normal culture), a model group (80μM VPH induction for 12 hours), and four VPH+ZFE1 groups (H9c2 rat cardiomyocytes were induced with VPH for 12 hours, and then ZFE1 was added to each group at safe concentrations of 0.2μM, 0.4μM, 1μM, and 2μM, respectively). After 36 hours of incubation in an incubator, 10μL of CCK-8 solution was added to each well and incubated in a 37°C incubator in the dark for 1-2 hours. The OD value at a wavelength of 450nm was measured using a microplate reader, and the cell viability was calculated after conversion.

[0058] ROS staining was performed on each cell group. First, DCFH-DA (2,7-dichlorofluorescein diacetate) was diluted 1:2000 in 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 volume added should be sufficient to fully cover the cells; typically, at least 2.5 mL of diluted DCFH-DA was added to one well of a six-well plate). The cells were incubated in a 37°C cell culture incubator for 20 minutes. The cells were washed three times with serum-free cell culture medium to fully remove any DCFH-DA that had not entered the cells. Finally, images were taken using a fluorescence microscope. The results, as shown in Figures 15-17, show an increase in green ROS signals in the model group, a significant decrease in ROS signals in the VPH+ZFE1 group, and a significant increase in cell viability in the VPH+ZFE1 group compared to the model group. The best mitigating effect was achieved at a MESTRANOL concentration of 1 μM.

[0059] Tunel cell apoptosis experiment, preparation of cell slides: prepare cell suspension according to the above cell passage method, place the cell slides into 12-well plates, and inoculate 2×10 5 cells, a total of 2 mL of cell suspension, and each group of cells was treated with drugs according to the above method.

[0060] Tunel assay: First, wash each cell group once with PBS, then fix the cells with 4% paraformaldehyde for 30 minutes, wash once with PBS, add PBS containing 0.3% TritonX-100, and incubate at room temperature for 5 minutes. Wash twice with PBS. Add 50μl of Tunel assay solution to the sample and incubate at 37°C in the dark for 60 minutes. Finally, remove the slide, place it on a slide with anti-fluorescence quencher, and photograph it using a confocal microscope. The results are shown in Figure 18. The red apoptotic signal increased in the model group cells, while the apoptotic signal in the VPH+ZFE1 group was significantly reduced compared to the model group.

[0061] The above embodiments are merely feasible or preferred embodiments of the present invention and are intended to illustrate the present invention rather than to limit the scope of the patent application of the present invention. Therefore, all equivalent changes and modifications made within the scope of the patent application of the present invention should fall within the scope covered by the patent application of the present invention.

Claims

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

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

3. Use of estrogen compounds in the preparation of drugs for treating or alleviating heart failure.

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

5. A method for constructing a zebrafish heart failure model, characterized in that: Verapamil hydrochloride was used to induce a zebrafish heart failure model.

6. The method according to claim 5, characterized in that Zebrafish embryos were exposed to verapamil hydrochloride.

7. The method according to claim 6, characterized in that After the zebrafish embryo's heart was formed and blood circulation began, the zebrafish embryo was immersed in verapamil hydrochloride. After abnormalities were observed in the zebrafish heart, the zebrafish heart failure model was established.

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

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