Application of 2-methoxyestradiol in preparation of products for preventing and treating heart failure

Abstract

The invention provides application of 2-methoxyestradiol in preparation of products for preventing and treating heart failure, and belongs to the technical field of medicines. The invention provides the effect of 2-methoxyestradiol (2ME) for preventing and treating heart failure for the first time. According to the invention, an isoproterenol induced myocardial fibroblast proliferation and migration model and an in-vivo heart failure mouse model are constructed in vitro; according to the present invention, the cardiac muscle fibroblast migration rate, the cardiac function, the serum heart failure marker level, the cardiac muscle tissue collagen deposition key protein, the pathological analysis and the heart electrophysiological signal change on the heart failure mouse are detected, the anti-heart failure effect of the 2ME is researched, and the results show that the 2ME has significant treatment effect on the heart failure disease, and is superior to the positive control drug captopril;

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of 2-methoxyestradiol in the preparation of products for preventing and treating heart failure. Background Art

[0002] Heart failure (HF) clinically manifests as severe thinning of the ventricular walls, dilated ventricles, and impaired systolic and / or diastolic function. This can lead to insufficient blood perfusion to other organs, resulting in liver and kidney failure. When myocardial ischemia occurs, the body's neuroendocrine system is activated (such as the renin-angiotensin-aldosterone system), inducing ventricular remodeling, which includes both a compensatory phase (myocardial hypertrophy) and a decompensated phase (heart failure). During the compensatory phase, the surface area of myocardial cells increases, the number of cardiac fibroblasts significantly increases, and collagen is excessively deposited in the cardiac interstitium. Clinically, this manifests as a structural change in the heart with increased ventricular wall thickness, known as myocardial hypertrophy. During this phase, the heart attempts to maintain contractility by increasing muscle mass. During the decompensation stage, due to the increase in the number of myocardial fibroblasts and the increase in the surface area of myocardial cells, the oxygen consumption increases, while the ischemic myocardium is not supplied with enough oxygen, which eventually causes a large number of myocardial cells and fibroblasts to necrotize and apoptosis. At this time, cardiac function declines significantly, and the clinical manifestations are changes in cardiac structure with significantly thinned ventricular wall thickness and significantly dilated chambers, and a significantly reduced cardiac ejection fraction (<40%, while the normal cardiac ejection fraction is >50%). The entire process of ventricular remodeling is accompanied by excessive proliferation of cardiac interstitial fibroblasts, excessive deposition and abnormal distribution of collagen, resulting in myocardial fibrosis, which eventually leads to increased cardiac stiffness and affects the heart's contraction and relaxation functions. However, the current clinical treatment drugs for heart failure are very limited, and the treatment strategies to prevent HF cardiac remodeling and worsening of cardiac function are still limited. There is an urgent need to explore new and effective strategies for the prevention and treatment of heart failure.

[0003] 2-Methoxyestradiol (2ME) is a bioactive metabolite of 17β-estradiol with antitumor, anti-inflammatory, and vasoprotective properties. However, the role of 2ME in heart failure remains unknown. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of 2-methoxyestradiol in the preparation of products for preventing and treating heart failure.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of 2-methoxyestradiol in preparing a product for preventing and treating heart failure.

[0007] The present invention also provides the use of 2-methoxyestradiol in preparing a product for preventing and treating heart failure with reduced ejection fraction.

[0008] The present invention also provides the use of 2-methoxyestradiol in preparing a product for preventing and treating electrophysiological disorders caused by heart failure.

[0009] The present invention also provides the use of 2-methoxyestradiol in preparing a product for inhibiting the activation, proliferation or migration of cardiac fibroblasts.

[0010] The present invention also provides the use of 2-methoxyestradiol in preparing a product for inhibiting myocardial fibrosis.

[0011] Preferably, the product comprises a medicine.

[0012] The present invention also provides a medicine for preventing and treating heart failure, wherein the active ingredient of the medicine is 2-methoxyestradiol.

[0013] Preferably, the mass fraction of 2-methoxyestradiol in the drug is 1% to 99%.

[0014] Preferably, the medicine further comprises pharmaceutical excipients.

[0015] Preferably, the dosage form of the drug includes oral dosage form, inhalation dosage form, patch and injection.

[0016] Beneficial effects of the present invention:

[0017] The present invention constructs an in vitro isoproterenol-induced myocardial fibroblast proliferation and migration model and an in vivo heart failure mouse model, detects myocardial fibroblast migration rate, cardiac function, serum heart failure marker levels, key proteins of myocardial tissue collagen deposition, pathological analysis, and changes in cardiac electrophysiological signals in heart failure mice, to study the anti-heart failure effect of 2ME. The results show that 2ME has a significant therapeutic effect on heart failure and is superior to the positive control drug captopril. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the toxic effects of 2ME on cardiac fibroblasts, **P<0.01, ***P<0.001, vs. Control;

[0019] Figure 2 Effects of 2ME on ISO-induced cardiac fibroblast proliferation, ***P<0.001, vs. Control; ###P<0.001, vs. ISO;

[0020] Figure 3 Figure 2 shows the effect of 2ME on ISO-induced cardiac fibroblast migration, where A shows the photographic results at different times, B shows the statistical results of the effect of 2ME on cardiac fibroblast migration at 24 hours, and C shows the statistical results of the effect of 2ME on cardiac fibroblast migration at 48 hours.

[0021] Figure 4 The effect of 2ME on left ventricular ejection fraction (EF%) in heart failure mice;

[0022] Figure 5 The effect of 2ME on the fractional shortening (FS%) of left ventricle in mice with heart failure;

[0023] Figure 6 The graphs are the periodic changes of left ventricular ejection fraction (EF%) in mice of different groups;

[0024] Figure 7 The graphs show the periodic changes of left ventricular fractional shortening (FS%) in mice of different groups;

[0025] Figure 8 The effect of 2ME on diastolic left ventricular internal diameter (LVID; d; mm) in heart failure mice;

[0026] Figure 9 The effect of 2ME on systolic left ventricular internal diameter (LVID; s; mm) in heart failure mice;

[0027] Figure 10 This is a graph showing the periodic changes of left ventricular internal diameter (LVID; d; mm) during diastole in mice of different groups;

[0028] Figure 11 This is a graph showing the periodic changes of left ventricular internal diameter (LVID; s; mm) during systole in mice of different groups;

[0029] Figure 12 The effect of 2ME on diastolic left ventricular volume (LVVol; d; μL) in mice with heart failure;

[0030] Figure 13 The effect of 2ME on systolic left ventricular volume (LVVol; s; μL) in mice with heart failure;

[0031] Figure 14 The graphs show the periodic changes of diastolic left ventricular volume (LVVol; d; μL) in mice of different groups;

[0032] Figure 15 The graphs show the periodic changes of left ventricular volume (LVVol; s; μL) during systole in mice of different groups;

[0033] Figure 16 The effect of 2ME on diastolic left ventricular posterior wall thickness (LVPW; d; mm) in heart failure mice;

[0034] Figure 17 The effect of 2ME on systolic left ventricular posterior wall thickness (LVPW; s; mm) in heart failure mice;

[0035] Figure 18 This is a graph showing the periodic changes in left ventricular posterior wall thickness (LVPW; d; mm) in mice of different groups;

[0036] Figure 19 This is a graph showing the periodic changes in left ventricular posterior wall thickness (LVPW; s; mm) during systole in mice of different groups;

[0037] Figure 20 The effect of 2ME on diastolic interventricular septum thickness (IVS; d; mm) in mice with heart failure;

[0038] Figure 21 The effect of 2ME on systolic interventricular septum thickness (IVS; s; mm) in mice with heart failure;

[0039] Figure 22 This is a graph showing the periodic changes in diastolic interventricular septum thickness (IVS; d; mm) in mice of different groups;

[0040] Figure 23 This is a graph showing the periodic changes in the systolic interventricular septum thickness (IVS; s; mm) of mice in different groups;

[0041] Figure 24 is the effect of 2ME on left ventricular mass (LVmass; mg) in mice with heart failure;

[0042] Figure 25 This is a graph showing the periodic changes in left ventricular mass (LVmass; mg) in mice of different groups;

[0043] Figure 26 The results of the effect of 2ME on collagen production in myocardial tissue of heart failure mice, where A is the expression of α-SMA, Collagentype I, and Collagentype III proteins; B to D are the quantitative statistical analysis results of Collagentype I, α-SMA, and Collagentype III proteins respectively;

[0044] Figure 27 Figure 2 shows the effect of 2ME on heart failure marker proteins in myocardial tissue of heart failure mice, where A is the expression of BNP and ANP proteins, and B to C are the quantitative statistical analysis results of ANP and BNP proteins respectively;

[0045] Figure 28 The results of the detection of cardiac biochemical indicators in the serum of mice with heart failure after 2ME pretreatment are shown in Figure 1, where A is the serum NT-proBNP level of each group, B is the serum BNP level of each group, and C is the serum ANP level of each group;

[0046] Figure 29The results of the biochemical indexes of liver and kidney function in heart failure mice pretreated with 2ME are shown in Figure 1, where A is the serum urea nitrogen level of each group, B is the serum creatinine level of each group, and C is the serum aspartate aminotransferase level of each group;

[0047] Figure 30 The results show the effect of 2ME pretreatment on the pathological changes of myocardial tissue in mice with heart failure, where A is a representative image of hematoxylin-eosin staining, and B is a representative image of Masson staining, with a scale bar of 100 μm.

[0048] Figure 31 The effect of 2ME on ventricular action potential under spontaneous rhythm, where A is a schematic diagram of cardiac conduction velocity and action potential duration under spontaneous rhythm; B is ventricular conduction time under spontaneous rhythm; C is ventricular conduction velocity under spontaneous rhythm; D is cardiac depolarization time under spontaneous rhythm; E is action potential duration under spontaneous rhythm; F is action potential duration dispersion under spontaneous rhythm;

[0049] Figure 32 Figure 3 shows the effect of 2ME on ventricular action potential under 8 Hz stimulation, where A is a schematic diagram of cardiac conduction velocity and action potential duration under 8 Hz stimulation; B is ventricular conduction time under 8 Hz stimulation; C is ventricular conduction velocity under 8 Hz stimulation; D is cardiac depolarization time under 8 Hz stimulation; E is action potential duration under 8 Hz stimulation; F is action potential duration dispersion under 8 Hz stimulation;

[0050] Figure 33 Figure 3 shows the effect of 2ME on ventricular calcium signaling under spontaneous rhythm, where A is a schematic diagram of cardiac calcium signal conduction velocity and action potential duration under spontaneous rhythm; B is the ventricular calcium signal conduction time under spontaneous rhythm; C is the ventricular calcium signal conduction velocity under spontaneous rhythm; D is the depolarization time of cardiac calcium signal under spontaneous rhythm; E is the potential duration of cardiac calcium signal under spontaneous rhythm; F is the action potential duration dispersion of cardiac calcium signal under spontaneous rhythm;

[0051] Figure 34 Figure 3 shows the effect of 2ME on ventricular calcium signal under 8 Hz stimulation, where A is a schematic diagram of cardiac calcium signal conduction velocity and action potential duration under 8 Hz stimulation; B is the ventricular calcium signal conduction time under 8 Hz stimulation; C is the ventricular calcium signal conduction velocity under 8 Hz stimulation; D is the cardiac calcium signal depolarization time under 8 Hz stimulation; E is the cardiac calcium signal potential duration under 8 Hz stimulation; F is the action potential duration dispersion of cardiac calcium signal under 8 Hz stimulation;

[0052] Figures 4 and 5 、 Figures 8 and 9 、 Figures 12 and 13 、 Figures 16 and 17 、 Figures 20 and 21 、 Figure 24 、 Figures 26 to 29 and Figures 31 to 34 Medium, *P<0.05, **P<0.01, ***P<0.001, vs Sham; #P<0.05, ##P<0.01, ###P<0.001, vs HF; ^P<0.05, ^^P<0.01, ^^^P<0.001, vs Captopril. DETAILED DESCRIPTION

[0053] The present invention provides the use of 2-methoxyestradiol in preparing a product for preventing and treating heart failure.

[0054] The present invention also provides the use of 2-methoxyestradiol in preparing products for preventing and treating heart failure with reduced ejection fraction, preparing products for preventing and treating electrophysiological disorders caused by heart failure, or preparing products for inhibiting myocardial fibrosis.

[0055] The present invention also provides the use of 2-methoxyestradiol in preparing a product for inhibiting the activation, proliferation or migration of cardiac fibroblasts.

[0056] In the present invention, the product preferably includes a drug. The present invention does not specifically limit the specific source of 2-methoxyestradiol, and conventional commercially available products in the field can be used. The present invention proposes for the first time that 2ME has the effect of inhibiting the activation of myocardial fibroblasts and reducing myocardial fibrosis, and has the effect of improving the pathological process of heart failure. Echocardiography test results showed that compared with the Sham group, heart failure mice significantly showed left ventricular dysfunction and impaired cardiac function, reaching the level of heart failure. 2ME improved left ventricular ejection fraction (EF%) and fractional shortening (FS%) in a dose-dependent manner, and the therapeutic effect of the high-dose group was better than that of the Captopril group. In addition, 2ME significantly reduced the levels of heart failure markers NT-proBNP, BNP and ANP in the serum of HF mice, among which 2ME was better than the Captopril group in the effect of reducing NT-proBNP, while there was no significant difference in the regulation of BNP and ANP compared with the Captopril group. 2ME can reduce the deposition of α-SMA, type I collagen (Collagentype I) and type III collagen (Collagentype III) in the heart. These results show that 2ME can effectively improve the cardiac function of mice with heart failure. Pathological analysis further supports this conclusion. 2ME dose-dependently reduces the structural abnormalities of myocardial cells and inflammatory cell infiltration caused by HF, and significantly reduces the deposition of cardiac collagen fibers, improving pathological changes in the myocardium. 2ME can accelerate conduction time and depolarization time, shorten APD, and improve cardiac electrophysiological abnormalities. Compared with Captopril, the therapeutic effect of 2ME under spontaneous rhythm is more significant. 2ME has the effect of improving Ca 2+It can also reduce the conduction time and peak time, and relieve the electrophysiological disturbance caused by heart failure, and has anti-arrhythmic effect.

[0057] The present invention also provides a drug for preventing and treating heart failure, the active ingredient of which is 2-methoxyestradiol. In the drug of the present invention, 2-methoxyestradiol is the sole active ingredient, and the mass fraction of 2-methoxyestradiol in the drug is preferably 1% to 99%, more preferably 10% to 90%, and even more preferably 20% to 80%. In the present invention, the drug preferably also includes pharmaceutical excipients, and the pharmaceutical excipients preferably include disintegrants, binders, lubricants, suspending agents and / or solubilizers. The disintegrant preferably includes sodium carboxymethyl starch (CMS-Na), the binder preferably includes hydroxypropyl methylcellulose (HPMC), the lubricant preferably includes magnesium stearate, the solubilizer preferably includes polysorbate 80, the suspending agent preferably includes a polymer, and the polymer preferably includes cellulose or xanthan gum. In the present invention, the dosage form of the drug preferably includes oral dosage form, inhalation dosage form, patch and injection dosage form. In the present invention, the oral dosage form preferably includes tablets, solutions, suspensions or emulsions.

[0058] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] In the following examples, unless otherwise specified, all methods are conventional.

[0060] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0061] In the following examples, the experimental results were analyzed using GraphPad Prism 8.0 software, and all results are expressed as mean ± SD. Experimental data were analyzed using one-way ANOVA and Turkey method for pairwise comparisons between multiple groups. P < 0.05 indicated that the experimental data were statistically significant.

[0062] Example 1

[0063] Cell experiments

[0064] 1. Extraction of neonatal rat myocardial fibroblasts:

[0065] (1) Preparation: Prepare two pairs of 10cm ophthalmic surgical scissors and two pairs of 10cm elbow forceps, as well as the necessary consumables for sterilization, and prepare the necessary reagents. Place the Pasteurized dropper, surgical instruments, surgical board, and various necessary consumables in a clean bench and irradiate under ultraviolet light for at least 30 minutes.

[0066] (2) Treatment of suckling mice: Place the suckling mice in 75% alcohol for wiping and disinfection, and then place them in a clean bench for later use.

[0067] (3) Removal of the heart: pinch the skin on the back of the rat's neck with your left hand to fully expose the chest. Use ophthalmic straight scissors in your right hand to cut the skin and tear it apart completely. Cut the ribs upward along the lower left edge of the xiphoid process of the sternum, then cut the sternum horizontally in the middle of the incision. Use your left hand to push slightly to squeeze out the rat's heart. Use ophthalmic curved forceps to directly cut off the ventricular part from the middle of the heart and place it in a culture dish filled with 4°C pre-cooled D-Hank's.

[0068] (4) Heart tissue processing: Remove blood clots and fibrous tissue around the heart and cut the heart tissue into even pieces using ophthalmic curved scissors to facilitate subsequent digestion. Transfer the cut heart tissue into a 10 mL centrifuge tube, discard the D-Hank's solution, add trypsin solution, and place horizontally in a 4°C refrigerator overnight to initiate cell release.

[0069] (5) Cell digestion and centrifugation: The next day, transfer the chopped heart tissue to a new 10 mL centrifuge tube, discard D-Hank's, add type II collagenase to cover the tissue, and shake it evenly in a 37°C water bath for 5 minutes. Then take the supernatant and mix it with 10% culture medium in a 1:1 ratio to stop the digestion. Repeat the above steps of adding collagenase and stopping digestion until the tissue clumps turn white. After discarding the white reticular tissue, add collagenase for digestion again. After digestion is completed, centrifuge at 900 rpm for 5 minutes, then resuspend the cells, inoculate the cell suspension into a 10 cm culture dish or confocal microdish according to the required density, and culture in a 37°C incubator.

[0070] (6) Cell adhesion and separation: Using the differential adhesion method, neonatal rat cardiac fibroblasts (NRCFs) can completely adhere to the wall after the cell suspension is cultured at 37°C for 2 hours. They appear triangular or spindle-shaped under a microscope. After washing away other cells that are not attached to the wall, fresh culture medium is added and the cells are cultured in a 37°C incubator for subsequent experiments.

[0071] 2. Methylthiazolyldiphenyl-tetrazoliumbromide (MTT) assay for cell viability

[0072] Cells were seeded in a 96-well cell culture plate. 20 μL of MTT solution was added to each well of each group and incubated at 37°C for 4 hours. The supernatant was aspirated and discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the cells. The cells were shaken and mixed for 10 minutes. The absorbance (OD) was measured at a wavelength of 562 nm using a microplate reader. Cell viability = (OD value experimental group - OD value blank group) / OD value blank group, where the OD value experimental group is the absorbance of the experimental group and the OD value blank group is the absorbance of the blank group.

[0073] The results are as follows Figure 1 As shown, based on the cytotoxicity results of 2ME on cardiac fibroblasts, three concentrations of 1, 2, and 4 μM were selected for subsequent experiments.

[0074] 3. Establishment and treatment of isoproterenol (ISO) cardiac fibroblast injury model

[0075] Primary cardiac fibroblasts were randomly divided into a normal culture control group (Control group), an isoproterenol group (ISO group), and a 2-ME intervention group at different concentrations (1, 2, and 4 μM), with the ISO concentration being 20 μM. The cells were then cultured in a 37°C incubator for 48 hours to establish a cardiac fibroblast injury model. Cell viability was measured by MTT. Figure 2 As shown, compared with the ISO model group, the survival rate of myocardial fibroblasts in the 2ME (1, 2, 4 μM) pretreatment groups was significantly inhibited.

[0076] 4. Scratch test

[0077] (1) Cell culture: Isolate cardiac fibroblasts according to the above method and seed the cells at an appropriate density in a six-well plate, ensuring that the cell density of each group is consistent. Add culture medium and culture in an incubator until the cell confluence reaches more than 95%.

[0078] (2) Scratching: After the cells have grown fully, use a six-well plate cover as an auxiliary tool and use a sterilized 200 μL pipette tip to draw two vertical lines perpendicular to the well plate and the marked line to form several intersections as fixed detection points. Apply uniform force when scratching to avoid large differences in width that affect the accuracy of the result analysis.

[0079] (3) Washing: Discard the old culture medium and gently wash three times with PBS to ensure that the cells are rinsed clean.

[0080] (4) Drug addition: According to the experimental design, culture medium containing different concentrations of 2ME (1, 2, 4 μM) and ISO (20 μM) were added to the cells of different groups to create models.

[0081] (5) Photographing and observing: Use a microscope to take pictures of cells after scratching as a 0h control. Place the cells in a 37°C incubator and continue to culture. Take out the cells at 24h and 48h for photographing and observing. The results are as follows: Figure 3 Select the same location when taking pictures to ensure accurate comparison of changes in scratch width.

[0082] (6) Data Analysis: Image processing software was used to measure the changes in scratch width and calculate the cell migration rate. Statistical analysis was performed on the experimental data to compare the effects of different concentrations of 2ME on the migration ability of cardiac fibroblasts.

[0083] The results are as follows Figure 3 As shown in Figures B and C, compared with the normal control group, treatment with ISO solution after 24 hours and 48 hours can cause abnormal migration of cardiac fibroblasts; compared with the ISO group, the groups treated with different concentrations of 2ME significantly inhibited the migration distance.

[0084] Example 2

[0085] Animal experiments

[0086] 1. Grouping and drug administration of experimental mice

[0087] Male, SPF-qualified C57BL / 6 mice, aged 6-8 weeks, were acclimated for one week and then randomly divided according to body weight into control (Sham), heart failure (HF) group, 2ME low-dose group (2ME-L+HF), 2ME medium-dose group (2ME-M+HF), 2ME high-dose group (2ME-H+HF), and captopril group (Captopril+HF), with 12 mice in each group. Mice in the low-, medium-, and high-dose 2ME groups were administered 5, 10, and 20 mg / kg / day of 2ME for 1 week before model establishment and for 4 consecutive weeks thereafter. Mice in the captopril group were administered 20 mg / kg / day of 2ME via oral gavage for 1 week before model establishment and for 4 consecutive weeks thereafter. The sham and HF groups were administered the same volume of normal saline for 1 week before model establishment and for 4 consecutive weeks thereafter. Sham mice underwent sham surgery.

[0088] 2. Establishment of a mouse heart failure model

[0089] C57BL / 6 mice were anesthetized with isoflurane and maintained in a supine position on a workbench. The chest of the C57BL / 6 mouse was pre-haired and disinfected. A 1 cm incision was made in the skin along the 2nd and 3rd ribs on the left side of the chest using surgical scissors, and the muscle layer was bluntly separated using hemostats to expose the ribs to the operator's field of view. While the mouse was breathing normally, a small hole was made in the 2nd and 3rd intercostal muscles using curved forceps. The chest was then opened with hemostats, and the heart was squeezed with the left hand to expose the mouse heart. The left anterior descending coronary artery (LAD) was then ligated using a 5-0 suture needle. The heart was then quickly returned to the mouse body, and the ribs and skin were sutured. The mouse was then placed on a 37°C incubator and maintained as normal.

[0090] 3. Echocardiography of mice

[0091] At 1, 2, 3, and 4 weeks after modeling, cardiac function tests were performed on the experimental mice in each group under isoflurane anesthesia after the heart rate stabilized. The ultrasound probe was placed on the chest of the mouse, and the probe position and angle were adjusted to obtain clear two-dimensional ultrasound images. The left ventricular short-axis section images were recorded using M-mode ultrasound and processed by software to obtain indicators such as cardiac ejection fraction (EF%), cardiac short-axis shortening fraction (FS%), end-diastolic interventricular septal thickness (IVS; d), end-systolic interventricular septal thickness (IVS; s), end-diastolic left ventricular volume (LVvol; d), end-systolic left ventricular volume (LVvol; s), end-diastolic left ventricular internal diameter (LVID; d), end-systolic left ventricular internal diameter (LVID; s), end-diastolic left ventricular posterior wall thickness (LVPW; d), end-systolic left ventricular posterior wall thickness (LVPW; s), and left ventricular mass (LVMass).

[0092] The results are as follows Figures 4 to 25 shown.

[0093] Depend on Figures 4 to 7 As can be seen, the HF group mice exhibited severe left ventricular dysfunction, reaching the level of heart failure. Four weeks after modeling, ejection fraction and fractional shortening values in the HF group continued to decline. 2ME improved both ejection fraction and fractional shortening in a dose-dependent manner. The high-dose 2ME group showed superior therapeutic efficacy compared to the active drug group (captopril).

[0094] Depend on Figures 8 to 11 As can be seen, four weeks after modeling, the diastolic and systolic left ventricular internal diameters in the HF group continued to increase, and 2ME improved these values in a dose-dependent manner. The high-dose 2ME group had a better therapeutic effect than the active drug group (captopril).

[0095] Depend on Figures 12 to 15 As can be seen, four weeks after modeling, the diastolic and systolic left ventricular volumes in the HF group continued to increase, and 2ME improved these values in a dose-dependent manner. The high-dose 2ME group had a better therapeutic effect than the active drug group (captopril).

[0096] Depend on Figures 16 to 19 It can be seen that after two weeks of modeling, the LVPW;d (diastolic left ventricular posterior wall thickness) and LVPW;s (systolic left ventricular posterior wall thickness) values in the HF group continued to increase, and after three or four weeks, the LVPW;d and LVPW;s values decreased. 2ME improved the LVPW;d and LVPW;s values. The high-dose 2ME group had a better therapeutic effect than the active drug group (captopril). Figures 20 to 23 As can be seen, IVS;d (diastolic interventricular septal thickness) and IVS;s (systolic interventricular septal thickness) values in the HF group continued to increase after two weeks of modeling, and decreased after three or four weeks. 2ME improved these values. The therapeutic effect of the active drug group (captopril) was consistent with that of the medium-dose 2ME group.

[0097] Depend on Figure 24 and Figure 25 As can be seen, LVmass (left ventricular mass) values in the HF group continued to increase after two weeks of modeling, and decreased after three and four weeks. 2ME improved LVmass values at each stage. The positive drug group (captopril) showed similar therapeutic effects to the 2ME group.

[0098] 4. Animal serum and myocardial tissue sample processing

[0099] After the experiment, the mice were anesthetized and blood and heart samples were collected from the carotid artery. The heart was placed in normal saline and the residual blood was gently squeezed out and the heart was washed, and then the water was absorbed with filter paper. The blood samples collected in a 1.5 mL centrifuge tube without anticoagulant were centrifuged at 5000 rpm for 5 minutes twice, and the supernatant was collected as the serum sample. Part of the serum and heart tissue samples were stored in liquid nitrogen and quickly frozen for Elisa index detection and Western Blotting determination; 3 mouse heart tissue samples were randomly selected from each group and stored in 4% paraformaldehyde for fixation for pathological histological examination.

[0100] 5. Biochemical index detection

[0101] 5.1 Brain Natriuretic Peptide (BNP) Index Detection

[0102] Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a peptide hormone synthesized by the heart in response to ventricular wall dilation or stretch. It is a key hormone in regulating body fluids, sodium balance, and blood pressure. Myocardial tension is the primary factor stimulating BNP release, which is proportional to ventricular dilation and pressure overload. BNP is secreted in large quantities when cardiac volume increases and left ventricular pressure overload occurs. Myocardial ischemia, hormone regulation, and cytokine regulation are also important influencing factors.

[0103] (1) After equilibration at room temperature for 10 minutes, remove the required strips from the aluminum foil bag. Seal the remaining strips in a ziplock bag and return them to 4°C.

[0104] (2) Sample addition: Add 100 μL of sample or standard of different concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with sealing film and incubate at 37°C for 1 h.

[0105] (3) Add biotinylated antibody: Remove the ELISA plate, discard the liquid, and do not wash. Add the biotinylated antibody working solution directly to each well.

[0106] (4) Washing: Discard the liquid, add 300 μL of 1x washing solution to each well, let it stand for 1 min, shake off the washing solution, and pat dry on absorbent paper. Repeat this washing process 3 times.

[0107] (5) Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 min.

[0108] (6) Washing the plate: Discard the liquid and follow the washing method in step (4).

[0109] (7) Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0110] (8) Add stop solution: Take out the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm.

[0111] 5.2 N-terminal pro-B-type natriuretic peptide (NT-ProBNP) index detection

[0112] NT-ProBNP is the inactive N-terminal fragment of the prohormone BNP (proBNP), secreted primarily by the left ventricle when cardiomyocytes are subjected to increased volume and pressure overload. Compared with BNP, NT-ProBNP has a longer half-life (approximately 120 minutes) and is more stable in the blood, making it easier to detect. NT-ProBNP can reflect the severity of heart failure; higher plasma NT-ProBNP levels indicate more severe heart failure. This indicator is of great value in the diagnosis of heart failure.

[0113] The specific operation steps are the same as those in 6. Western Blot experiment below.

[0114] 5.3 Atrial natriuretic peptide (ANP) index detection

[0115] Atrial natriuretic peptide (ANP) is a polypeptide hormone synthesized and secreted by atrial myocytes and belongs to the natriuretic peptide family. ANP levels are often significantly elevated in heart failure and can therefore be used as a biomarker for heart failure.

[0116] The specific operation steps are the same as those in 6. Western Blot experiment below.

[0117] The results of the effect of 2ME on collagen production in myocardial tissue of heart failure mice are shown in Figure 26 , indicating that the high-dose 2ME group significantly reduced the level of cardiac collagen deposition.

[0118] The results of the effect of 2ME on heart failure marker proteins in myocardial tissue of heart failure mice are shown in Figure 27 , indicating that the therapeutic effect of the high-dose 2ME group was the most significant.

[0119] The results of 2ME pretreatment on the cardiac biochemical indexes in the serum of heart failure mice are shown in Figure 28 2ME increased serum NT-proBNP, BNP, and ANP levels in mice with heart failure. 2ME reduced these levels and improved cardiac function. Its therapeutic effect on NT-proBNP was superior to that of the positive control, captopril. However, its effect on BNP and ANP was not significantly different from that of captopril.

[0120] 5.4 Aspartate aminotransferase (AST) index detection

[0121] Aspartate aminotransferase (AST), also known as aspartate aminotransferase, is an important enzyme that is mainly found in the liver, heart, muscle and other tissues. Heart failure is often accompanied by insufficient blood supply to the liver, leading to decreased liver function. Increased AST levels not only reflect heart damage, but may also indicate insufficient blood supply and functional changes to the liver due to heart failure. In the assessment of heart failure, AST levels can be used in combination with other biomarkers (such as BNP, NT-proBNP, etc.) to help comprehensively assess patients' heart function and survival risks.

[0122] Prepare the AST / GOT assay reagents, including α-ketoglutarate, aspartic acid, and chromogen, according to the instructions. Dilute or mix as directed. Add the serum sample to a 96-well plate. Gently shake the plate on a shaker to ensure thorough mixing of the sample and reagents. After standing at room temperature for 15 minutes, measure the absorbance at 510 nm using a microplate reader.

[0123] Absolute OD value = OD value of test well - OD value of control well, substitute into the standard curve to obtain AST / GOT activity units.

[0124] 5.5 Creatinine (CRE) index detection

[0125] Creatinine is a product of muscle metabolism, produced by the conversion of creatine in muscle and mainly excreted through the kidneys. Therefore, creatinine levels are often used to assess renal function. In patients with heart failure, changes in creatinine can reflect the functional status of the heart and kidneys and have important clinical significance. When assessing fluid overload (such as edema caused by heart failure and worsening heart failure symptoms), creatinine levels, combined with other biomarkers (such as BNP and NT-proBNP), can more comprehensively assess the status of heart failure patients.

[0126] Prepare the CRE assay reagents according to the instructions, including enzyme solution A, enzyme solution B, CRE standard, and double-distilled water, and dilute or mix according to the instructions. Add the serum sample to a 96-well plate. Gently shake the 96-well plate on a shaker to ensure that the sample and reagents are thoroughly mixed. After standing in a 37°C incubator for 5 minutes, measure the absorbance value A1 at a wavelength of 546 nm using a microplate reader. After standing in a 37°C incubator for another 5 minutes, measure the absorbance value A2 at a wavelength of 546 nm using a microplate reader. Calculate ΔA according to the following formula and substitute it into the standard curve to obtain the CRE result.

[0127] △A=A2-K×A1,

[0128] 5.6 Urea nitrogen (BUN) index detection

[0129] Urea nitrogen refers to the nitrogen content of urea in the blood, which is mainly synthesized by the liver and excreted through the kidneys. In patients with heart failure, the level of urea nitrogen is often used to assess renal function and fluid status, and is of great significance for the diagnosis and management of heart failure. Heart failure can affect the heart's pumping function, leading to insufficient renal perfusion, which in turn affects the kidney's excretion function. An increase in urea nitrogen levels often indicates renal insufficiency and is an important sign of kidney damage. In the management of heart failure, the determination of urea nitrogen is often used in combination with other biomarkers (such as serum creatinine, BNP, NT-proBNP, etc.) to jointly assess the patient's cardiopulmonary function and renal function, providing a comprehensive basis for clinical diagnosis and treatment.

[0130] Prepare BUN assay reagents according to the instructions, including enzyme stock solution, phenol colorimetric reagent, alkaline sodium hypochlorite, BUN standard, and double-distilled water. Dilute or mix according to the instructions. Add the serum sample to a 96-well plate. Gently shake the 96-well plate on a shaker to ensure thorough mixing of the sample and reagents. After incubating in a 37°C water bath for 10 minutes, measure the absorbance at 640 nm using a microplate reader.

[0131] Absolute OD value = OD value of test well - OD value of control well, and substitute it into the standard curve to obtain BUN activity units.

[0132] The results of 2ME pretreatment on the biochemical indicators of liver and kidney function in heart failure mice are as follows Figure 29 As shown, creatinine and urea nitrogen levels were elevated in mice with heart failure. Both the 2ME group and the positive control captopril group reduced creatinine and urea nitrogen levels and improved renal function, with no significant difference in treatment effect. AST values were elevated in mice with heart failure. Both the 2ME group and the positive control captopril group reduced AST values and improved liver function, with 2ME being more effective than the positive control group.

[0133] 6. Western Blot experiment

[0134] BNP antibody was purchased from Abcam (Cambridge, UK), ANP antibody was purchased from Santa Cruz (Santa Cruz, MA, USA), GAPDH antibody was purchased from Bioworld (Beijing, China), and Collagen Type I, Collagen Type III, and α-SMA antibodies were purchased from Proteintech Group (Wuhan, China).

[0135] Experimental principle: The protein sample to be tested is separated by SDS-PAGE electrophoresis, then transferred to a solid support PVDF membrane, and then bound and detected by specific antibodies to achieve semi-quantitative analysis of the protein.

[0136] Experimental steps:

[0137] (1) Electrophoresis: Prepare separating gel and stacking gel by adjusting the gel concentration according to the size of the target protein. After solidification, fill it with 1× electrophoresis buffer. Pull out the comb vertically and add the marker (1.5 μL) and the pre-mixed protein sample in sequence. Set the electrophoresis instrument to 200 V and 300 mA for about 30 minutes to separate the proteins.

[0138] (2) Transfer: After electrophoresis, cut the gel area of the desired molecular weight according to the position indicated by the marker, and cut out a PVDF membrane of appropriate size and activate it in methanol. Then make a sandwich and place it in a transfer tank filled with electrophoresis solution. Set the electrophoresis instrument to 200V voltage and 300mA current for 30 minutes to transfer the protein from the gel to the PVDF membrane. After transfer, cut the band according to the marker position and mark it accordingly.

[0139] (3) Blocking: Place the PVDF membrane in an incubation box filled with 5% skim milk powder and incubate it on a shaker at room temperature for 1.5 h to prevent nonspecific binding and reduce background signals.

[0140] (4) Antibody binding: Use 5% skim milk powder to prepare a specific antibody for the target protein, add it dropwise to the PVDF membrane and incubate it in a refrigerator at 4°C overnight to allow it to specifically bind to the protein on the membrane.

[0141] (5) Washing: After incubation with the primary antibody at 4°C overnight, wash the PVDF membrane three times (10 min / time) with 1×PBST to remove unbound antibodies and other nonspecific binding substances to ensure the accuracy and specificity of the results.

[0142] (6) Secondary antibody binding: After washing the membrane, dilute the secondary antibody with PBST and incubate in a wet box at room temperature for 1.5 h.

[0143] (7) Washing: Wash the PVDF membrane three times (10 min / time) with 1×PBST to remove unbound secondary antibody and other nonspecific binding substances.

[0144] (8) Signal detection: By adding luminescent liquid, the antigen-antibody complex specifically bound to the membrane generates a detectable signal.

[0145] (9) Result analysis: Use professional Western Blot imaging equipment to record the signal on the PVDF membrane to evaluate the presence and expression level of the target protein, and use Image software to perform grayscale analysis of the target band.

[0146] 7. Cardiac histopathology

[0147] (1) Sample preparation

[0148] 1) Sampling: Myocardial tissue samples are obtained from experimental animals or patients. Ensure the sample is fresh and free from contamination and deterioration. The sample size should be moderate to facilitate subsequent processing and observation.

[0149] 2) Cleaning: Use saline or PBS to clean the sample to remove blood, fat, and other impurities on the surface.

[0150] (2) Tissue processing

[0151] 1) Fixation: Immerse the cleaned myocardial tissue sample in a fixative, such as 4% paraformaldehyde, for 48 hours to maintain the morphology and structure of the tissue.

[0152] 2) Dehydration: The fixed samples were dehydrated with a gradient of alcohols: 70% ethanol for 30 min, 80% ethanol for 30 min, 90% ethanol for 30 min, 95% ethanol for 30 min, anhydrous ethanol for 60 min (twice), xylene for 60 min (twice), and paraffin for 60 min (three times) to gradually remove moisture from the tissues and facilitate subsequent paraffin embedding.

[0153] 3) Wax immersion and embedding: The dehydrated sample is immersed in molten paraffin, allowing the paraffin to penetrate the tissue. The sample is then embedded in a paraffin block for easy sectioning.

[0154] 4) Sectioning: Use a microtome to cut the paraffin block into thin slices, usually 3-5 microns thick. The slices should be smooth and continuous to facilitate observation and diagnosis.

[0155] (3) Hematoxylin-eosin staining (HE)

[0156] 1) Sections were stained with hematoxylin and eosin to reveal the cellular structure and morphology of myocardial tissue. Hematoxylin stains the nuclei, while eosin stains the cytoplasm and connective tissue.

[0157] 2) Sealing and Photography: The sections were placed in 95%, 85%, and 75% ethanol, respectively, and immersed in xylene for 5 minutes each until transparent. The sections were then air-dried, sealed with neutral glue, and finally imaged and photographed using a microscope.

[0158] (4) Masson staining:

[0159] 1) Dewaxing to water: Follow the above steps.

[0160] 2) Staining: Use lapis lazuli blue stain to stain the sections for 2-3 minutes to ensure uniform staining. Gently rinse the sections with a small amount of distilled water to remove excess stain. Add Mayer's hematoxylin stain for 2-3 minutes to enhance the contrast of tissue structure. Rinse lightly with distilled water to remove floating color. Place the sections in acidic ethanol differentiation solution for a few seconds to fine-tune the depth of hematoxylin staining. Rinse the sections with distilled water for about 10 minutes to ensure complete removal of the differentiation solution. Add Ponceau fuchsin stain for 10 minutes to highlight specific tissue structures. Gently rinse the sections with distilled water to remove excess stain. Place the sections in phosphomolybdic acid solution for about 10 minutes to enhance the contrast of subsequent staining. After pouring off the phosphomolybdic acid solution, add aniline blue stain directly without washing and stain for 5 minutes to further enrich the tissue color.

[0161] 3) Seal the slides and take photos: Follow the above steps.

[0162] The results are as follows Figure 30 As shown in Figure 3, 2ME dose-dependently reduced HF-induced nuclear deformation, myocardial cell disorder, and inflammatory cell infiltration. Figure 30 A. 2ME in the dose-dependent manner reduced the increase of cardiac collagen fibers caused by HF and improved pathological changes such as Figure 30 B in.

[0163] 8. Electrophysiological index testing (the dosage of 2ME in the following tests is 20 mg / kg, marked as HF+2ME)

[0164] (1) Langendorff in vitro perfusion

[0165] The mice were weighed, and sodium heparin was injected intraperitoneally. After 15-20 minutes, they were anesthetized with isoflurane and killed by cervical dislocation.

[0166] Place the sacrificed mouse on the experimental table and secure it. Spray the chest and abdomen with alcohol for disinfection. Use surgical scissors to cut the chest skin, exposing the xiphoid process. Grasp the xiphoid process with hemostats, and cut the diaphragm to expose the thoracic cavity. Perform a thoracotomy by transecting the ribs bilaterally. Cut the thoracic cavity along the mid-ribcage of the left and right sides, and flip the thoracic cage upward to expose the heart. Pour pre-chilled KH buffer (95% O2, 5% CO2) containing oxygen into a glass culture dish.

[0167] Remove air bubbles from the syringe and perfusion needle in advance. With the help of microtweezers, quickly connect the aorta to the perfusion needle and secure it with an aortic clamp. Gently move the heart to determine the insertion depth of the perfusion needle, adjust it to the appropriate depth, tie it with 4G surgical sutures, and cut off the thread ends with corneal scissors. Gently push the syringe to retrogradely perfuse KH buffer containing saturated oxygen (95% O2, 5% CO2) into the coronary artery of the heart to restart the heart. To avoid damage caused by prolonged cardiac arrest, heart removal and mounting should be completed within 3-5 minutes.

[0168] Connect the perfusion needle connected to the heart to the perfusion system and start perfusion. When the heart is stable and no residual blood is pumped out, monitor and record the heart's electrical mapping data. Simulate lead II and attach ECG detection electrodes to the right atrium (G) and left ventricle (+), monitor and record the heart's pseudo GECG (pseudo ECG, this mode simulates the surface lead II, so it can also be called simulated ECG). Attach the pen electrodes MEAs to the atrium and ventricle respectively, and record the electrical signal conduction data. Analyze the collected electrical mapping data to confirm that the heart rate is within the normal range and the heart's electrical signal conduction is normal before proceeding to the next step. Use EMapScope software to analyze electrical mapping data.

[0169] (2) Stopping and fluorescent staining

[0170] Create a light-proof environment: draw the curtains and turn off all lights in the room.

[0171] Cardiopulmonary arrest: Add 300 μL of Lebbistatin to 100 mL of circulating KH buffer and completely arrest the heartbeat after 1-2 minutes.

[0172] Calcium indicator loading: 100 μL of calcium indicator Rhod-2AM was slowly injected from the 1-type dosing port and circulated for 15 minutes. Voltage-sensitive dye loading: 100 μL of voltage-sensitive dye RH237 was slowly injected from the 1-type dosing port and circulated for 15 minutes.

[0173] (3) Imaging and data acquisition

[0174] Start the cursor measurement data recording software OMapRecord and analysis software OMapScope on the computer.

[0175] Prepare the sample: Move the stained heart to the imaging perfusion chamber (i.e., in the bath) and adjust the perfusion cycle. Insert the pacing electrode approximately 1 mm deep into the apex of the heart to facilitate subsequent pacing stimulation. Place ECG electrodes on both sides of the heart (red "+" for the left ventricle and white "G" for the right atrium). Clip the ground electrode (black) to the perfusion needle to obtain a pseudo GECG. Using the software, set the ECG sampling rate to 0.8 kHz and continuously monitor and record the ECG throughout the experiment.

[0176] Spontaneous cardiac rhythm recording: Optical action potential (AP) and calcium (Ca) signals were recorded under sinus conditions.

[0177] Fixed-rate recordings: Optical AP and Ca signals were recorded using an apical stimulation electrode at a fixed frequency of 6 Hz.

[0178] Recording of effective refractory period in S1S2 stimulation mode: Use software to adjust to S1S2 stimulation mode to determine vERP (ventricular effective refractory period) while recording optical AP and Ca signals.

[0179] Stimulation mode-induced arrhythmia test: Use the software to adjust to the S1S1 stimulation mode, perform S1S1 stimulation at 5 times and 10 times the threshold current intensity, and record optical AP and Ca signals.

[0180] The results of the effects of 2ME pretreatment on cardiac electrophysiological signal changes in heart failure mice are as follows Figures 31 to 34 shown.

[0181] The effects of spontaneous rhythm on ventricular action potential are as follows: Figure 31 As shown, compared with the sham group, the HF group had delayed conduction time, delayed depolarization time, prolonged action potential duration, and repolarization dispersion. Compared with the HF group, the 2ME group had accelerated conduction time, depolarization time, and shortened action potential duration. Compared with the HF group, the captopril group had accelerated conduction time, slightly accelerated depolarization time, prolonged action potential duration, and repolarization dispersion. Under spontaneous rhythm, the 2ME group had a superior therapeutic effect compared with the captopril group.

[0182] The effect of 8HZ stimulation on ventricular action potential is as follows Figure 32 As shown in the figure, compared with the Sham group, the HF group had slower conduction velocity, delayed depolarization time, and prolonged action potential duration. Compared with the HF group, the 2ME group had faster depolarization time and shortened action potential duration.

[0183] Effects of spontaneous rhythm on ventricular calcium signaling Figure 33 As shown, compared with the Sham group, the HF group delayed Ca 2+ Conduction time, delaying Ca 2+ Compared with the HF group, the 2ME group accelerated the Ca 2+ Conduction time, Ca 2+ Peak time and shortened calcium transient duration.

[0184] Effects of 8 Hz stimulation on ventricular calcium signal Figure 34As shown, compared with the Sham group, the HF group delayed Ca 2+ Conduction time, delaying Ca 2+ Compared with the HF group, the 2ME group accelerated the Ca 2 + Conduction time, Ca 2+ Peak time and shortened calcium transient duration.

[0185] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

Application of 1.2-methoxyestradiol in the preparation of products for preventing and treating heart failure.

2. Application of 2-methoxyestradiol in the preparation of products for preventing and treating heart failure with reduced ejection fraction.

3. Application of 2-methoxyestradiol in the preparation of products for preventing and treating electrophysiological disorders caused by heart failure.

4. Use of 2-methoxyestradiol in the preparation of products for inhibiting cardiac fibroblast activation, proliferation or migration.

5. Application of 2-methoxyestradiol in the preparation of products for inhibiting myocardial fibrosis.

6. The use according to any one of claims 1 to 5, characterized in that: The products include pharmaceuticals.

7. A drug for preventing and treating heart failure, characterized in that: The active ingredient of the drug is 2-methoxyestradiol.

8. The drug according to claim 7, characterized in that The mass fraction of 2-methoxyestradiol in the medicine is 1% to 99%.

9. The drug according to claim 7, characterized in that The medicine also includes pharmaceutical excipients.

10. The drug according to claim 7, characterized in that The dosage forms of the drug include oral dosage forms, inhalation dosage forms, patches and injections.

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