Application of Auranofin in preparation of medicine for preventing and / or treating ischemic cardiomyopathy and heart failure

Abstract

The invention discloses application of Auranofin in preparation of a medicine for preventing and / or treating ischemic cardiomyopathy and heart failure, and belongs to the field of biological medicine. In-vivo and in-vitro experiments prove that the Auranofin can remarkably improve cardiac function indexes after myocardial ischemia reperfusion injury, so that the Auranofin has a definite pharmacological action in the aspects of preventing and treating ischemic cardiomyopathy and heart failure, a new candidate scheme is provided for the drug treatment of the diseases, and the application of the Auranofin in the treatment of ischemic cardiomyopathy and heart failure is developed. Meanwhile, the application prospect of the Auranofin in the field of cardiovascular diseases is expanded, and the Auranofin has important theoretical value and clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of Auranofin in the preparation of medicaments for the prevention and / or treatment of ischemic cardiomyopathy and heart failure. Background Technology

[0002] Ischemic cardiomyopathy (ICM) is left ventricular failure caused by coronary artery disease (CAD), accounting for a major proportion of heart failure (HF) cases worldwide. While timely revascularization (reperfusion) is a key treatment for improving prognosis, the reperfusion process itself can induce myocardial ischemia / reperfusion injury (MI / RI), further exacerbating myocardial dysfunction. Typical pathological manifestations of MI / RI include myocardial stunning, reperfusion arrhythmias, irreversible cell death, and microcirculatory disturbances (such as no-reflow). Studies have shown that reperfusion-related injury can lead to up to 50% of the final myocardial infarction area, with mechanisms involving the interaction of multiple pathways including oxidative stress, calcium overload, mitochondrial dynamics imbalance, and endoplasmic reticulum stress (ERS).

[0003] In recent years, the synergistic effect of regulating mitochondrial quality control and endoplasmic reticulum homeostasis has become a novel target for the treatment of MI / RI. Mitochondrial fusion proteins (MFN1 / 2) are not only key regulators of mitochondrial homeostasis, but also participate in ERS signaling through mitochondrial-endoplasmic reticulum contact sites (MAMs). Preclinical studies have shown that inhibiting ERS effector molecules (such as ATF6 and p-IRE1α) can significantly reduce reperfusion injury, but existing drugs cannot simultaneously target mitochondrial fusion and ERS pathways.

[0004] Auranofin is a clinically approved drug for the treatment of rheumatoid arthritis. Current research clearly shows that it primarily exerts its antioxidant effects by inhibiting thioredoxin reductase (TrxR), thereby alleviating the inflammatory response and tissue damage in rheumatoid arthritis. In recent years, with the continuous expansion of research on the pharmacological effects of auranofin, studies have found that it may have a broader regulatory effect on cellular homeostasis, specifically in two aspects: first, the regulation of mitochondrial dynamics, with preliminary studies suggesting that it may affect the expression level of mitochondrial fusion proteins (Mfn1 / 2), thereby altering the dynamic balance between mitochondrial fusion and division; second, the regulation of endoplasmic reticulum stress, with some experimental results showing that it may inhibit the ATF6 / p-IRE1α signaling pathway, thus affecting the endoplasmic reticulum stress response. However, to date, no relevant research has been reported on the role of auranofin in the specific pathological process of myocardial ischemia-reperfusion injury; whether it acts on myocardial tissue through the regulation of mitochondrial fusion and endoplasmic reticulum stress pathways, and the related molecular mechanisms, remain unknown and require further in-depth exploration. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Auranofin in the preparation of drugs for the prevention and / or treatment of ischemic cardiomyopathy and heart failure, thereby addressing the problems existing in the prior art. This invention experimentally demonstrates that Auranofin has a clear pharmacological effect in the prevention and treatment of ischemic cardiomyopathy and heart failure, providing a new candidate for the drug treatment of these diseases. It also expands the application prospects of Auranofin in the field of cardiovascular diseases, possessing significant theoretical value and clinical translational potential.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of Auranofin in the preparation of medicaments for the prevention and / or treatment of ischemic cardiomyopathy and heart failure.

[0007] Optionally, the drug may also include a pharmaceutically acceptable carrier or excipient.

[0008] Optionally, the dosage form of the drug includes oral dosage forms.

[0009] Optionally, the oral dosage form includes tablets, capsules, granules, pills, and oral liquids.

[0010] Optionally, Auranofin can improve ischemic cardiomyopathy and heart failure by promoting mitochondrial fusion and inhibiting endoplasmic reticulum stress.

[0011] The present invention also provides the use of Auranofin in the preparation of a kit for in vitro studies of myocardial ischemia-reperfusion injury.

[0012] The present invention also provides the use of Auranofin in the preparation of a medicament for reducing cardiomyocyte apoptosis and / or improving mitochondrial function.

[0013] The present invention also provides the use of Auranofin in the preparation of medicaments for inhibiting endoplasmic reticulum stress and / or promoting mitochondrial fusion.

[0014] The present invention discloses the following technical effects: This invention demonstrates through in vitro and in vivo experiments that Auranofin can significantly improve cardiac function parameters following myocardial ischemia-reperfusion injury (such as increasing left ventricular ejection fraction and fractional shortening), reduce myocardial infarction area, and effectively inhibit cardiomyocyte apoptosis. These effects indicate that Auranofin has a clear pharmacological effect in the prevention and treatment of ischemic cardiomyopathy and heart failure, providing a new candidate for the drug treatment of these diseases.

[0015] Furthermore, this invention reveals for the first time the core mechanism by which Auranofin alleviates reperfusion injury across multiple pathways by synergistically regulating the expression of mitochondrial fusion proteins (Mfn1 / 2), inhibiting key endoplasmic reticulum stress signaling molecules, and mitigating excessive activation of mitochondrial-endoplasmic reticulum structural interactions (MAMs). This mechanism not only overcomes the limitation of existing drugs in simultaneously targeting mitochondrial dynamics and endoplasmic reticulum stress but also expands the application prospects of Auranofin in the field of cardiovascular diseases, possessing significant theoretical value and clinical translational potential. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Ultrasound images of cardiac systolic and diastolic motion in four groups of mice; Figure 2 A comparison of left ventricular ejection fraction (A) and left ventricular fractional shortening (B) in four groups of mice; Figure 3 Cross-sectional views of TTC staining in the hearts of four groups of mice; Figure 4 The proportion of myocardial danger zone to left ventricular myocardial area in four groups of mice (A) and the proportion of myocardial infarction zone to left ventricular myocardial area (B); Figure 5 Microscopic image of TUNEL fluorescence staining in mouse heart tissue (A) and statistical comparison of the percentage of TUNEL-positive cell nuclei in four groups (B). Figure 6 Microscopic images of TUNEL fluorescence staining in primary rat cardiomyocytes (A), statistical charts comparing the percentage of TUNEL-positive cell nuclei in four groups (B), and statistical charts comparing the Bax / Bcl2 ratio in four groups (B). Figure 7 The images show the fluorescence detection of calcium ions in primary rat cardiomyocytes (A) and the quantitative graph of calcium ion concentration (B). Figure 8 To detect mitochondrial morphology in mouse cardiomyocytes using the MitoTracker Red CMXRos fluorescent probe; where A shows the mitochondrial fluorescence probe detection images of four groups of rat cardiomyocytes; B shows the statistical graph of mitochondrial division stained with Mitotracker in four groups; and C shows the statistical graph of ATP content in four groups of cardiomyocytes. Figure 9The results show the fluorescence staining of endoplasmic reticulum and mitochondria in primary rat cardiomyocytes; where A is the fluorescence staining localization map of endoplasmic reticulum and mitochondria in four groups of rat cardiomyocytes; and B is the co-localization statistical map of endoplasmic reticulum and mitochondria in rat cardiomyocytes. Figure 10 The results show the content of mitochondrial and endoplasmic reticulum-related proteins in mouse heart tissue; where A is a Western spectral representation of mitochondrial fission and endoplasmic reticulum stress-related proteins in the heart tissue of four groups of mice; B is a statistical graph of mitochondrial fission-related protein content in the heart tissue of four groups of mice; and C is a statistical graph of endoplasmic reticulum stress-related protein content in the heart tissue of four groups of mice. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The Auranofin (AF) described in this invention has the molecular formula C2. 12 H 34AuO9PS, MW: 678.5 g / mol, CAS: 34031-32-8, is a thioredoxin reductase inhibitor, a clinically approved antirheumatic drug known to exert its antioxidant effect by inhibiting thioredoxin reductase (TrxR). Its chemical structure is as follows: .

[0024] This invention, based on a mouse model of myocardial ischemia-reperfusion, uses intraperitoneal injection of auranofin (20 mg / kg) to assess changes in apoptosis and oxidative stress, mitochondrial fusion and division levels, endoplasmic reticulum stress, and related signaling molecules in mouse myocardial tissue. Furthermore, a rat neonatal mouse model of myocardial cell hypoxia-reoxygenation was established, with intervention performed after 4 hours of starvation. The study found that auranofin can reduce apoptosis and oxidative stress levels in mouse myocardial ischemia-reperfusion and rat neonatal mouse primary myocardial cells after hypoxia-reoxygenation, promote mitochondrial fusion, reduce endoplasmic reticulum stress, and improve mitochondrial structure and function, thereby improving ischemic cardiomyopathy and heart failure.

[0025] The present invention will be described in detail below with reference to specific embodiments: Example In this embodiment, the drug preparation was as follows: Auranofin powder (MedChemExpress, China) was purchased for in vitro and in vivo experiments. It was completely dissolved in 5% dimethyl sulfoxide (DMSO) to prepare a stock solution with a final concentration of 50 mg / mL, and then diluted with corn oil to a working concentration of 5-40 mg / mL. Mice were injected intraperitoneally with a dose of 20 mg / kg body weight one day before myocardial I / R surgery, and the same dose of Auranofin was injected again 1 hour before surgery. Samples were collected 24 hours post-surgery.

[0026] I. Establishment of a mouse model of ischemia-reperfusion injury (1) Feeding and grouping of laboratory animals Male C57BL / 6 mice, 8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; they were housed separately in cages under constant temperature (23-25℃) and constant humidity (55-70%). Animal experiments were divided into four groups: healthy control group (Vehicle group), normal drug group (Auranofin group), model group (I / R group), and treatment group (Auranofin+I / R group), with 6 mice in each group. In the Vehicle group and I / R group, C57BL / 6 mice were injected intraperitoneally with 0.2 mL of physiological saline per mouse. In the Auranofin group and Auranofin+I / R group, experimental mice were pretreated with Auranofin (20 mg / kg, diluted with physiological saline 0.2 mL per mouse, and injected intraperitoneally). Subsequently, myocardial ischemia-reperfusion (I / R) model was established in the I / R group and Auranofin+I / R group mice.

[0027] (2) Establishing a mouse model of ischemia-reperfusion injury Anesthesia and preoperative preparation: Before surgery, mice were treated with hair removal cream and their skin was prepared. After anesthesia with 1.5% isoflurane inhalation, the mice were placed in a supine position and their heads and limbs were fixed to the operating table with adhesive tape. Anesthesia was maintained with 1% isoflurane.

[0028] Surgical procedure: Place the mouse on the experimental table and disinfect the skin on the mouse's chest with 75% alcohol repeatedly. Make a 1-2 cm incision along the left midclavicular line from the 2nd to the 6th intercostal space using ophthalmic scissors. Suture the incision with 4-0 sutures and a purse-string suture without knotting. Use curved forceps and hemostats to bluntly dissect the subcutaneous tissue to expose the sternum. Use curved hemostats to bluntly dissect the left 4th and 5th intercostal spaces to expose the heart. Squeeze the heart out with the left hand and turn it to the left to expose the left atrial appendage. Small blood vessels and the left anterior descending branch can be seen in front of and below the left atrial appendage. Use 6-0 sutures to ligate the left anterior descending branch 1 mm in front of and below the left atrial appendage and tie a slipknot. Put the heart back into the heart chamber, suture the skin, put it back in the cage, and place it on a heating blanket. Time for 30 minutes and observe the mouse's condition. After 30 minutes, remove the slipknot from the mouse's heart to restore blood flow. The process should be slow, and the time for cardiac reperfusion should be recorded.

[0029] II. Establishment of a rat primary cardiomyocyte hypoxia-reoxygenation model (1) Extraction and culture of primary cardiomyocytes from Sprague-Dawley (SD) rats Neonatal rat cardiomyocytes (NRCMs) were extracted from 1-day-old SD rats and sterilized with 75% alcohol. The left ventricle was rapidly removed using pre-autoclaved ophthalmic scissors, cut into small pieces, and digested with 0.08% trypsin. The isolated cardiomyocytes were incubated for 24 h in DMEM / F12 supplemented with 15% FBS, and then incubated in serum-free DMEM / F12 containing appropriate chemicals for subsequent in vitro studies.

[0030] (2) Construction of a primary rat cardiomyocyte hypoxia-reoxygenation model For the construction of the in vitro hypoxia / reoxygenation (H / R) model, NRCM cells were incubated under hypoxic conditions (1% O2) for 6 h in a hypoxia buffer containing NaCl (118 mM), NaHCO3 (24 mM), NaH2PO4·2H2O (1 mM), anhydrous CaCl2 (2.5 mM), MgCl2·6H2O (1.2 mM), sodium lactate (20 mM), KCl (16 mM), 2-D-ribose, and 2-deoxyglucose (10 mM). Cells were then incubated in DMEM / F12 supplemented with 10% FBS and 1% penicillin / streptomycin, and reoxygenated under normal oxygen conditions (95% O2) for 24 h. Similar to the construction of the mouse ischemia-reperfusion injury model, Auranofin (200 nM) was added to the culture medium for 6 h before H / R injury.

[0031] III. Model Evaluation (1) Echocardiographic evaluation In the mouse ischemia-reperfusion injury model described above, 24 hours after reperfusion, the four groups of mice were anesthetized in a 1.5% isoflurane anesthesia box and fixed in a supine position on the operating table, with anesthesia maintained by 1% isoflurane. First, the parasternal long axis section was located, and the B-mode data was saved. The probe was rotated 90° to obtain the parasternal short axis section of the left ventricle of the mouse heart. The parasternal short axis section was marked by the section at the level of the left ventricular papillary muscle. The left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (LVFS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were recorded using M-mode ultrasound. The parasternal long axis section was marked by the section at the level of the left ventricular outflow tract, and the ventricular wall motion image was recorded using B-mode echocardiography.

[0032] The test results are as follows: Images of cardiac contraction and relaxation in four groups of mice recorded by M-mode echocardiography are shown below. Figure 1 As shown, the cardiac systolic and diastolic functions of mice were significantly reduced after I / R, while the cardiac systolic and diastolic functions of mice pretreated with Auranofin were significantly improved. Figure 2 The figures show a comparison of LVEF and LVFS in four groups of mice, indicating that pretreatment with Auranofin significantly increased LVEF and LVFS in the model mice, effectively alleviating the decline in cardiac function caused by I / R injury. These results demonstrate that pre-injection of Auranofin into mice undergoing myocardial ischemia-reperfusion improves cardiac function.

[0033] (2) 2,3,5-Triphenyltetrachloride (TTC) staining The above-mentioned four groups of mice were injected intraperitoneally with 0.2 mL of supersaturated tribromoethane solution. After anesthesia, the mice were fixed. In the two groups without modeling (Vehicle and Auranofin), the left anterior descending artery was ligated as in the I / R modeling. In the two groups with modeling (I / R and Auranofin+I / R), the heart was squeezed out again along the intercostal opening of the myocardial infarction modeling site, and the left anterior descending artery was ligated again at the previous ligation site. 0.9 mL of 1% Evans blue dye was injected at the apex of the heart, allowing it to flow through the bloodstream to the whole body, and the entire mouse body turned blue. The mouse heart was removed, washed with PBS to remove excess tissue and blood, and frozen at -20°C for 30 min. After freezing, the heart was placed in a mold, cut into four equal-thickness sections, and immersed in 1% TTC dye, and incubated in a water bath at 37°C for 30 min. The staining process was carried out in the dark. After staining, the heart sections were placed in 4% PFA fixative to terminate the reaction and fixed for 24 h. The sections were rinsed with physiological saline 3 times for 5 min to remove the filler material in the cardiac wall, and photographed. ImageJ software was used to measure the infarct area (white), the boundary area between the infarct and non-infarct areas (red), and the non-infarct area (blue) of each myocardial section. Finally, the percentage of the infarct area and the dangerous area to the total left ventricular area of ​​each section was calculated.

[0034] The test results are as follows: Cross-sectional images of TTC staining in the hearts of four groups of mice are shown below. Figure 3 As shown (where blue represents normal areas, red represents ischemic areas, and white represents infarct areas), it indicates that after I / R surgery, the myocardial infarction area in the I / R model group mice pretreated with Auranofin was significantly smaller than that in the Vehicle group. Figure 4 The figures show the proportion of myocardial danger zone and infarct area to the left ventricular myocardial area in four groups of mice. These results indicate that Auranofin pretreatment can partially salvage the myocardial infarction area caused by I / R injury.

[0035] (3) Tunel staining Mouse heart tissue was collected and fixed overnight by soaking in 20% sucrose, embedded in OCT, and sectioned using a cryostat at -20℃. Paraffin sections with intact tissue morphology were selected, baked at 50℃ for 20 min, fixed with methanol for 10 min, washed with PBS, and the tissue was circled on a slide using a histochemical pen. Wells were punched with 0.5% Triton X-100 solution for 15 min. After washing with PBS, TUNEL stain was prepared according to the manufacturer's instructions, working solution was added, and incubation was performed at 37℃ for 1 h. After washing with PBS, α-actinin antibody diluted in PBS (1:200 dilution) was added, and the slide was incubated overnight at 4℃. The next day, the slide was warmed, washed with PBS, and DAPI staining solution was added, incubating at room temperature for 10 min. After washing with PBS, the slide was mounted with anti-fluorescence quenching mounting medium, and all procedures were performed in the dark. Images were taken using an upright fluorescence microscope, and the three fluorescence channels were analyzed using ImageJ software. Primary cardiomyocytes were seeded in 24-well plates, washed with PBS, fixed with methanol for 10 min, and the remaining steps were the same as above.

[0036] The test results are as follows: Figure 5 and Figure 6 Microscopic images of TUNEL fluorescence staining in four groups of mouse heart tissue (primary cardiomyocytes) and a statistical comparison of the percentage of TUNEL-positive cell nuclei in the four groups were shown. The results indicated that cardiomyocyte apoptosis increased after I / R in mice (primary cardiomyocytes); while apoptosis was significantly reduced after pretreatment with Auranofin in mouse heart tissue (primary cardiomyocytes), indicating that pretreatment with Auranofin can reduce cardiomyocyte apoptosis after myocardial ischemia-reperfusion injury in mice.

[0037] (4) Detection of fluorescent calcium ions The intracellular calcium concentration in neonatal rat cardiomyocytes (NRCMs) was determined using the Fluo-4 AM calcium ion assay kit (S1061S, Beyotime, China). Cells were incubated with Fluo-4 in HBSS buffer at 37°C for 30 min according to a published method. The results are shown below. Figure 7 As shown, compared with the Vehicle group, the calcium ion level of primary cardiomyocytes increased after H / R, while pretreatment with Auranofin could reverse this situation and reduce the calcium ion level of cardiomyocytes.

[0038] (5) Mitochondrial probe detection Mitotracker Red CMXRos red fluorescent probe (Cell Signaling Technology) was used to detect mitochondrial morphology.

[0039] ① Reagent preparation: Prepare MitoTracker working solution in a clean bench. Dissolve 5 μL of MitoTracker stock solution (1.88 mmol / L) in 42 μL of DMSO to obtain a final stock solution concentration of 200 μmol / L. Store at -20℃ protected from light. When using, dilute with DMEM / F12 medium to a working solution of 200 nmol / L and calculate the total volume of working solution required.

[0040] ② Staining: After treatment, the cells (n=3) were washed once with PBS, 1 mL of working solution was added to each well, and the cells were placed in a cell culture incubator at 30℃ and stained in the dark for 30 min.

[0041] ③ Fixation and punching, nucleus staining and mounting: The procedure is the same as for TUNEL cell staining.

[0042] Images were taken using a Leica TCS SP8 confocal microscope. The red and blue fluorescence channels were adjusted, and the images were then overlaid. Five fields of view were randomly selected from each sample, and the mitochondrial division level was analyzed using Image Pro Plus. The number of dividing mitochondria was counted when at least 90% of the tubular mitochondria disintegrated into punctate mitochondria.

[0043] like Figure 8 As shown, the results indicate that mitochondrial division increased in mouse primary cardiomyocytes after I / R; however, pretreatment with Auranofin significantly reduced mitochondrial division in mouse myocardial tissue (primary cardiomyocytes) and increased ATP levels. This suggests that pretreatment with Auranofin can reduce mitochondrial division, increase mitochondrial fusion, and improve mitochondrial function and energy metabolism in mice following myocardial ischemia-reperfusion injury.

[0044] (6) Co-staining of endoplasmic reticulum and mitochondria Primary cardiomyocytes were stained with Sec61β-EGFP (endoplasmic reticulum marker, green) and Mito-DsRed (mitochondrial marker, red) adenoviruses, respectively. The transfection method for primary cardiomyocytes is as follows.

[0045] ① Pre-transfection treatment: 2 hours before transfection of primary cardiomyocytes, the culture medium was changed to serum-free and antibiotic-free DMEM / F12 medium.

[0046] ② Transfection: Based on the preliminary experiment, select an adenovirus concentration of 50 MOI. After calculating the concentration and the required cell volume, dissolve the adenovirus in pure DMEM / F12 medium and slowly add it to primary cardiomyocytes for transfection for 12 h. Then replace the whole medium and express for at least 36 h before proceeding with subsequent experiments.

[0047] ③ Observation: EGFP green fluorescence and DsRed red fluorescence can be seen under a microscope.

[0048] ④Nucleus staining: Cell nuclei were stained using Hoechst (C1028, Beyotime, China) live cell staining solution. The fluorescent staining solution (100X) was diluted with DMEM to a working concentration of 1X. 200 μL of working solution was added to each well. After incubation at room temperature for 10 min, the cells were washed 2-3 times with PBS for 3 min each time.

[0049] like Figure 9 As shown, the results indicate that the co-localization of mitochondria and endoplasmic reticulum in primary rat cardiomyocytes increased after H / R; while pretreatment with Auranofin significantly reduced the amount of mitochondria and endoplasmic reticulum in mouse cardiomyocytes. This suggests that pretreatment with Auranofin can reduce mitochondrial-endoplasmic reticulum interaction after myocardial ischemia-reperfusion injury.

[0050] (7) Protein blotting method Total protein was lysed from mouse heart tissue or primary rat neonatal cardiomyocytes in RIPA lysis buffer (Beyotime, China) containing a protease inhibitor cocktail (1:100, Thermo Fisher, USA). Equal volumes of protein (30 μg) were separated using 7.5% or 10% SDS-PAGE gels and then transferred to PVDF membranes (0.22 μm, Millipore, USA) and incubated overnight at 4°C with a primary antibody. Antibodies against the following proteins were used as primary antibodies: Mfn-1, Mfn-2, Drp-1, Pdzd8, Flag, Myc, α-actin (Proteintech, China); Mrpl2 (Santa Cruz, USA); ubiquitin (Ub, PTM-BIO, China); and GAPDH (Cell Signaling Technology, USA).

[0051] like Figure 10 As shown, the results indicate that after I / R, mice showed increased mitochondrial division, decreased fusion, and increased endoplasmic reticulum stress levels; while pretreatment with Auranofin reduced mitochondrial division, increased fusion, and decreased endoplasmic reticulum stress levels in mouse myocardial tissue. This suggests that pretreatment with Auranofin can improve mitochondrial and endoplasmic reticulum function after myocardial ischemia-reperfusion injury in mice.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of Auranofin in the preparation of a medicament for the prevention and / or treatment of ischemic cardiomyopathy and heart failure.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes oral dosage forms.

4. The application according to claim 3, characterized in that, The oral medications include tablets, capsules, granules, pills, and oral liquids.

5. The application according to claim 1, characterized in that, Auranofin improves ischemic cardiomyopathy and heart failure by promoting mitochondrial fusion and inhibiting endoplasmic reticulum stress.

6. The use of Auranofin in the preparation of a kit for in vitro studies of myocardial ischemia-reperfusion injury.

7. The use of Auranofin in the preparation of a medicament for reducing cardiomyocyte apoptosis and / or improving mitochondrial function.

8. The use of Auranofin in the preparation of a medicament for inhibiting endoplasmic reticulum stress and / or promoting mitochondrial fusion.

Images