Use of HY-Q67280 in the preparation of a medicament for preventing and / or treating myocardial ischemia / reperfusion injury

Abstract

The application discloses application of HY-Q67280 in preparation of a medicine for preventing and / or treating myocardial ischemia / reperfusion injury, and belongs to the technical field of biological medicine. The application discloses application of HY-Q67280 in preparation of a medicine for preventing and / or treating myocardial ischemia / reperfusion injury, and HY-Q67280 can activate E3 ubiquitin ligase MARCH9, MARCH9 degrades by targeting FXR1, inhibits Drp1-dependent mitochondrial dysfunction, oxidative stress and ferroptosis cell death, reduces the apoptosis and mitochondrial oxidative stress level of a mouse myocardial ischemia / reperfusion and a rat neonatal rat primary myocardial cell after hypoxia / reoxygenation, and reduces the mitochondrial division and improves the mitochondrial function, so that the myocardial ischemia / reperfusion injury is protected.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of HY-Q67280 in the preparation of drugs for the prevention and / or treatment of myocardial ischemia / reperfusion injury. Background Technology

[0002] Ischemic heart disease remains a leading cause of high morbidity and mortality worldwide. Although timely intervention with coronary reperfusion is effective in salvaging ischemic myocardium, myocardial ischemia / reperfusion (MI / R) injury remains a significant cause of death in a large number of patients.

[0003] Cell death, particularly the death of cardiomyocytes (CMs), is a key aspect of the pathophysiology and pathology of cardiovascular disease. Ferraphobia is an iron-driven pattern of cell death characterized by iron accumulation and excessive lipid peroxidation. Ferraphobia is closely associated with mitochondrial function, and studies have shown that mitochondrial dysfunction and damage promote oxidative stress, thereby inducing ferrophobia. Key morphological features include membrane contraction, increased membrane density, and a decrease or disappearance of mitochondrial cristae. During ferrophobia, the intracellular redox balance is disrupted, with decreased levels of antioxidants such as glutathione (GSH) and glutathione peroxidase 4 (GPX4), while levels of oxidative substances such as ferrous ions and lipid reactive oxygen species (ROS) begin to rise. Ferraphobia is closely associated with a variety of heart diseases. To date, despite decades of in-depth research, no specific targeted drugs for MI / R damage have entered the clinical field. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of HY-Q67280 in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia / reperfusion injury.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the use of HY-Q67280 in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia / reperfusion injury.

[0006] Furthermore, the drug exerts its effect in preventing and / or treating myocardial ischemia / reperfusion injury by reducing myocardial ischemia / reperfusion and myocardial cell apoptosis after hypoxia / reoxygenation and reducing mitochondrial oxidative stress levels.

[0007] Furthermore, the drug exerts its effect in preventing and / or treating myocardial ischemia / reperfusion injury by activating the E3 ubiquitin ligase MARCH9 and improving mitochondrial function and status after hypoxia / reoxygenation of cardiomyocytes.

[0008] Furthermore, the HY-Q67280 is an E3 ubiquitin ligase MARCH9 activator, with the chemical formula C3.23 H 26 N4O3, structural formula: .

[0009] The present invention also provides a medicament for preventing and / or treating myocardial ischemia / reperfusion injury, wherein the active ingredient of the medicament includes HY-Q67280.

[0010] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses the application of HY-Q67280 in the preparation of drugs for the prevention and / or treatment of myocardial ischemia / reperfusion injury. A mouse model of myocardial ischemia / reperfusion was established, and HY-Q67280 was injected intraperitoneally to intervene. Changes in apoptosis and oxidative stress, energy metabolism, and related signaling molecules in mouse myocardial tissue were evaluated. A rat hypoxia / reoxygenation model of neonatal mouse cardiomyocytes was also constructed. After starvation for 4 hours, HY-Q67280 was injected to intervene. The results showed that HY-Q67280 can activate the E3 ubiquitin ligase MARCH9. MARCH9 inhibits Drp1-dependent mitochondrial dysfunction, oxidative stress, and ferroptosis by targeting FXR1 degradation. It reduces the levels of apoptosis and mitochondrial oxidative stress in mouse myocardial ischemia / reperfusion and rat neonatal mouse primary cardiomyocytes after hypoxia / reoxygenation, and reduces mitochondrial division to improve mitochondrial function, thereby protecting against myocardial ischemia / reperfusion injury.

[0011] This invention found that HY-Q67280 can reduce myocardial ischemia / reperfusion and myocardial cell apoptosis after hypoxia / reoxygenation, reduce mitochondrial oxidative stress level, and also found that HY-Q67280 can activate E3 ubiquitin ligase MARCH9, improve mitochondrial function and state after myocardial cell hypoxia / reoxygenation, increase mitochondrial membrane potential, increase ATP production, and reduce mitochondrial division. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The changes in protein levels of MARCH9, FXR1, and Drp1 in NRCM at different time points using HY-Q67280 (20 μM) and comparative statistical graphs. Figure 1 The figure shows the changes in MARCH9, FXR1, and Drp1 protein levels in NRCM under different concentrations of HY-Q67280, along with comparative statistical graphs. Figure 1 In the middle, I represents the mRNA content of MARCH9 after treatment with HY-Q67280 (20 μM) for 24 h; Figure 2The results of cardiac function testing in mice in each experimental group in Example 2 are shown. A is a cardiac systolic and diastolic motion image, and B and C are comparison graphs of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), respectively. Figure 3 The results of myocardial infarction area detection in mice of each experimental group in Example 2 are shown. A is a cross-sectional view of TTC staining of the heart, and B and C are the proportions of myocardial danger area and infarction area to the left ventricular myocardial area, respectively. Figure 4 The images show DHE fluorescence staining micrographs and fluorescence intensity comparison charts of mouse heart tissues from each experimental group in Example 2. In the image, A is a DHE fluorescence staining micrograph and B is a fluorescence intensity comparison chart. Figure 5 The images shown are TUNEL fluorescence staining micrographs of mouse heart tissues from each experimental group in Example 2, and a statistical comparison of the percentage of TUNEL-positive cell nuclei. In the image, A is a TUNEL fluorescence staining micrograph, and B is a statistical comparison of the percentage of TUNEL-positive cell nuclei. Figure 6 The images show microscopic images and fluorescence intensity statistics of NRCMs from each experimental group in Example 2, where A is a microscopic image of NRCMs stained with Mitosox fluorescence and B is a statistical graph of fluorescence intensity. Figure 7 The images shown are Mitotracker fluorescence staining micrographs and mitochondrial division statistics of NRCMs in each experimental group in Example 2. In the image, A is a Mitotracker fluorescence staining micrograph and B is a mitochondrial division statistics. Figure 8 The images show TMRE fluorescence staining micrographs and fluorescence intensity statistics of NRCMs in each experimental group in Example 2. In the image, A is a TMRE fluorescence staining micrograph and B is a fluorescence intensity statistics chart. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Mitochondria are organelles with a double-membrane structure, consisting of the outer mitochondrial membrane (MOM) and the inner mitochondrial membrane (MIM), separated by the intermembrane space (IMS). Mitochondriasis aids in quality control because it splits a damaged mitochondria into two: one functional and the other dysfunctional. The dysfunctional mitochondria is then cleared through mitophagy, maintaining mitochondrial homeostasis. During mitochondrial splitting, the GTPase dynamin-associated protein 1 (Drp1), also known as dynamin-1-like protein (DNM1L), is a key mediator. Drp1 interacts with multiple protein receptors in the MIM or MOM, including mitochondrial fission factor (Mff), mitochondrial fission protein 1 (Fis1), mitochondrial kinetic protein 49 (MiD49), and mitochondrial kinetic protein 51 (MiD51). Multiple Drp1 molecules are recruited to individual mitochondria and bind to receptors, forming a ring-finger structure tightly surrounding the mitochondria. Subsequently, through its GTPase activity, Drp1 molecules hydrolyze GTP, leading to increased permeability of the MIM and MOM and initiating mitochondrial splitting. Following mitochondrial division, Drp1 relocates to the cytoplasm. Notably, the function of Drp1 is regulated by post-translational modifications (such as ubiquitination and phosphorylation) and metabolic signaling.

[0015] HY-Q67280 is an activator of the E3 ubiquitin ligase MARCH9. MARCH9 participates in regulating various cellular processes, including immune responses, cell signaling, and protein transport, by catalyzing the ubiquitination of substrate proteins. However, the role of HY-Q67280 in ischemic cardiomyopathy has not yet been reported.

[0016] This invention provides the application of HY-Q67280 in the preparation of drugs for the prevention and / or treatment of myocardial ischemia / reperfusion injury. HY-Q67280 exerts its preventive and / or therapeutic effects on myocardial ischemia / reperfusion injury by reducing apoptosis and mitochondrial oxidative stress levels following myocardial ischemia / reperfusion and hypoxia / reoxygenation. Furthermore, it enhances mitochondrial function and status in cardiomyocytes after hypoxia / reoxygenation by activating the E3 ubiquitin ligase MARCH9, thereby also contributing to the prevention and / or treatment of myocardial ischemia / reperfusion injury.

[0017] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0018] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0019] Example 1 Preparation of HY-Q67280: The synthetic route for compound 5 is as follows: ; The preparation process of compound 5 is as follows: (1) Synthesis of compound 5b: Compound 5a (10 g, 37.736 mmol) was dissolved in dichloromethane (DCM, 100 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 21 g, 56.604 mmol) and N,N-diisopropylethylamine (DIPEA, 14.6 g, 113.208 mmol) were added. The mixture was stirred at room temperature for 10 min, and then S1 (2) was added. 67 g (45.283 mmol), stirred at room temperature for 2 h, LCMS monitoring showed the reaction was complete, water / DCM (50 mL / 100 mL) extraction was added, the organic phase was collected, washed successively with distilled water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated to half the original volume, petroleum ether (100 mL) was added, the precipitated solid was collected by filtration to give compound 5b (10 g, off-white solid, yield 47.7%); wherein, the structural formula of S1 is ; The LCMS monitoring results are as follows: MS(ESI): m / z 307.09 [M+1] + LCMS:YX007C-102-AaX; (2) Synthesis of compound 5: Compound 5b (13g) was dissolved in ethyl acetate (EA, 60mL), and HCl / EA (60mL, volume ratio 1:1) was added under an ice-water bath (0-5℃). The ice-water bath was removed, and the mixture was stirred at 10-15℃ for 1h. The reaction of the starting material was monitored by LCMS until it was complete. The organic phase was dried by rotary evaporation to obtain compound 5 (10g, off-white solid, yield 20%). The LCMS monitoring results are as follows: MS(ESI): m / z 207.12 [M+1] + ; The synthesis route of HY-Q67280 is as follows: ; The specific preparation process of HY-Q67280 is as follows: (3) Synthesis of compound 6: Compound 4 (1 g, 10.204 mmol) was dissolved in dimethylformamide (DMF, 10 mL), and DIPEA (2.6 g, 20.4 mmol) and HATU (11.6 g, 30.6 mmol) were added at 10-15 °C. The mixture was stirred in an ice bath for 10 min, and then compound 5 (2.7 g, 11.22 mmol) was added. The mixture was heated to room temperature and stirred for 1.5 h. The reaction of the starting material was monitored by LCMS until it was complete. The mixture was extracted with water / EA, washed with NaCl, dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography was used to obtain compound 6 (1.2 g, off-white solid, yield 41%).

[0020] The LCMS monitoring results are as follows: MS(ESI): m / z 287.17 [M+1] + ; Iodine fumigation (PE:EA 1:2) for TLC detection; (4) Synthesis of compound 7: Compound 6 (500 mg, 1.748 mmol) was dissolved in anhydrous DMF (5 ml), and DIPEA (177 mg, 1.748 mmol), CuI (66 mg, 0.349 mmol), and Pd(pph3)2Cl2 (123 mg, 0.175 mmol) were added at 0-5 °C. Then S2 (460 mg, 2.098 mmol) was added, the temperature was raised to 30 °C, and the mixture was stirred for 1 h. The reaction of the starting material was monitored by LCMS until complete. The mixture was extracted with water / EA, the organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and column chromatography was used to obtain compound 7 (300 mg, off-white solid, yield 46%). The structural formula of S2 is as follows: ; The LCMS monitoring results are as follows: MS(ESI): m / z 378.21 [M+1] + YX007C-109-AAX(DMSO); (5) Synthesis of compound HY-Q67280: Compound 7 (250 mg, 0.663 mmol) was dissolved in a mixed solvent of dioxane and HCl (the ratio of dioxane to HCl was 2.5 mL: 2.5 mL). Sodium nitrite (NaNO2, 70 mg, 0.995 mmol) was added at 0-5 °C. The mixture was then heated to room temperature and stirred for 2 h. The reaction was monitored by LCMS until the starting material was completely reacted. The mixture was extracted with water / EA, the precipitated solid was filtered, and the solid was dried under reduced pressure to obtain compound HY-Q67280 (120 mg, off-white solid, yield 44%). The LCMS monitoring results are as follows: MS(ESI): m / z 407.20 [M+1] + , LCMS: YX050A-20-P1G1, NMR: YX007C-111-C (DMSO).

[0021] Example 2 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 (HY-Q67280 group), model group (I / R group), and treatment group (HY-Q67280+I / R group), with 6 mice in each group. Among them, C57BL / 6 mice were injected intraperitoneally with 0.2 mL of corn oil per mouse in the Vehicle group and I / R group. Mice in the HY-Q67280 group and HY-Q67280+I / R group were pretreated with HY-Q67280 (0.2 mL of corn oil containing HY-Q67280 per mouse was injected intraperitoneally, and the injection dose of HY-Q67280 was 10 mg / kg). Subsequently, myocardial ischemia / reperfusion (I / R) model was established in the I / R group and HY-Q67280+I / R group mice.

[0022] 2. Construction of a mouse ischemia / reperfusion injury model Anesthesia and preoperative preparation: Before surgery, mice were treated with hair removal cream and anesthetized with 1.5% isoflurane. 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. 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. A thin blood vessel 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 the 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 blood flow reperfusion should be recorded.

[0023] 3. Extraction and culture of primary cardiomyocytes from Sprague-Dawley (SD) rats Neonatal rat cardiomyocytes (NRCMs) were extracted from 1-day-old SD rats, sterilized with 75% alcohol, and the left ventricle was quickly removed with autoclaved ophthalmic scissors, cut into small pieces, and digested with 0.08% trypsin. The isolated cardiomyocytes were incubated in DMEM / F12 medium containing 15% FBS (fetal bovine serum) for 24 hours, and then incubated in serum-free DMEM / F12 medium for subsequent in vitro studies.

[0024] 4. Construction of a rat primary cardiomyocyte hypoxia / reoxygenation (H / R) model NRCM cells were incubated for 6 h under hypoxic conditions (1 vol.% O2) in a hypoxic buffer containing NaCl, NaHCO3, NaH2PO4·2H2O, anhydrous CaCl2, MgCl2·6H2O, sodium lactate, KCl, 2-deoxy-D-ribose, and 2-deoxyglucose. The cells were then incubated in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin and reoxygenated for 24 h under normal oxygen conditions (95 vol.% O2). Following the experimental groupings described above, HY-Q67280 (20 µM) was added to the medium and cultured for 24 h before H / R damage.

[0025] 5. Exploration of drug delivery dosage and time in in vitro culture of HY-Q67280 After 24 h of NRCM extraction and culture, the cells were starved in serum-free DMEM / F12 medium for 4 h. After starvation, the NRCM cells were cultured in serum-free DMEM / F12 medium containing different concentrations of HY-Q67280 (0, 10 µM, 20 µM, 50 µM) for 36 h. Proteins and RNA were extracted from each group using protein lysis buffer RIPA or Trizol containing phosphatase inhibitors. Alternatively, after starvation, the NRCM cells were cultured in serum-free DMEM / F12 medium containing HY-Q67280 (20 µM) at different time points (0, 12 h, 24 h, 36 h). Proteins were extracted from each group using protein lysis buffer RIPA containing phosphatase inhibitors.

[0026] 6. RNA and protein extraction (1) After washing NRCMs with PBS, digest them with 1 mL of Trizol into a 1.5 mL enzyme-free sterile centrifuge tube, lyse them on ice for 15 min, add 200 µL of chloroform, cap the tube, invert it 10 times, and let it stand for 10 min. After that, centrifuge at 4 °C and 13300 rpm for 15 min, and aspirate the clear liquid from the top layer into a 1.5 mL centrifuge tube containing 500 µL of isopropanol. Invert it 10 times and place it in a -20 °C freezer for 3 h. After that, centrifuge it at 4 °C and 13300 rpm for 15 min. A white precipitate, i.e., RNA, will be visible. Discard the liquid and add 75% ethanol. Invert it 10 times to wash the RNA. Repeat this process once more. After that, discard the 75% ethanol and add 15 µL of enzyme-free sterile water to dissolve the RNA. Then, determine the concentration and quantify the RNA by reverse transcription to obtain cDNA. The primers used are: Forward-GCCTAATCCGCTGGATCAGTG Reverse-CGATAGCAGCAATCTGGACCTT, real-time quantitative detection of MARCH9 content at different concentrations; (2) 10 mg of mouse left ventricular heart tissue or NRCMs were lysed in a 1.5 mL centrifuge tube on ice for 10 min using lysis buffer RIPA (containing 1× phosphatase inhibitor cocktail). The tissue was then minced with scissors and homogenized with a grinder. The homogenate was then sonicated and centrifuged at 4 °C for 15 min at 13300 rpm. The protein concentration was determined using the BCA method, and then quantified to 3 µg / µL with protein loading buffer. The protein was denatured by boiling at 100 °C. The levels of MARCH9, FXR1, and Drp1 proteins were detected by Western blotting. The loading amount was 30 µg. After electrophoresis for 3 h (60 V), the sample was transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, and incubated with the corresponding primary antibody overnight at 4 °C. The primary antibody was recovered the next day, washed 3 times with 1× TBST, and then incubated with the corresponding secondary antibody at room temperature for 1 h. After the secondary antibody was removed, the sample was washed 4 times with 1× TBST and the bands were exposed.

[0027] The test results are as follows: Figure 1 The changes in protein levels of MARCH9, FXR1, and Drp1 in NRCM at different time points using HY-Q67280 (20 μM) and comparative statistical graphs. Figure 1 The figure shows the changes in MARCH9, FXR1, and Drp1 protein levels in NRCM under different concentrations of HY-Q67280, along with comparative statistical graphs. Figure 1 In the figure, I represents the mRNA content of MARCH9 after treatment with HY-Q67280 (20 μM) for 24 h. Figure 1The results showed that HY-Q67280 was dose- and time-dependent, increasing the MARCH9 protein level with increasing dose and time, while inhibiting the protein levels of FXR1 and Drp1 in NRCMs, without altering the MARCH9 mRNA level.

[0028] 7. Echocardiographic evaluation Twenty-four hours after reperfusion in a mouse ischemia / reperfusion injury model, cardiac ultrasound was performed on mice in each group. The method was as follows: After anesthetizing the four groups of mice in a 1.5% isoflurane anesthesia box, they were fixed in a supine position on the operating table and anesthetized with 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.

[0029] 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 2 As shown in section A, the cardiac contractile function of mice after I / R was significantly reduced, while the cardiac contractile function of model mice pretreated with HY-Q67280 was significantly improved. Figure 2 Parts B and C show comparisons of LVEF and LVFS in four groups of mice, respectively. The results indicate that HY-Q67280 pretreatment 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 HY-Q67280 (10 mg / kg) into mice with myocardial ischemia / reperfusion improved cardiac function.

[0030] 8.2,3,5-Triphenyltetrachloride (TTC) staining Four groups of mice were anesthetized and fixed after intraperitoneal injection of 0.2 mL of tribromoethane supersaturated solution. The two groups without modeling (Vehicle and HY-Q67280) had the left anterior descending artery ligated as in the I / R modeling. The two groups with modeling (I / R and HY-Q67280+I / R) had their hearts squeezed out again at 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 mice were observed to turn blue all over. The mouse heart was removed, washed with PBS, and the sutures in the tissue were removed. The heart was then filled with a gel and frozen at -20°C for 30 min. After freezing, the heart was placed in a mold and cut into four thin slices of equal thickness. The slices were then incubated in 1% TTC dye at 37°C for 30 min. After freezing, the heart slices were placed in 4% tissue fixative overnight and photographed the next day.

[0031] 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 in section A (where blue represents normal areas, red represents ischemic areas, and white represents infarct areas), compared with the Vehicle group, the infarct area in the I / R group was significantly larger, while the myocardial infarction area in the model group mice pretreated with HY-Q67280 was significantly smaller. Figure 3 Parts B and C show the proportions of the myocardial danger zone and infarct area to the left ventricular myocardial area in the four groups of mice, respectively. These results indicate that HY-Q67280 pretreatment can partially salvage the myocardial infarction area caused by I / R injury.

[0032] 9. DHE staining Four groups of mouse hearts were harvested and soaked in 20% sucrose until they sank to the bottom. They were then embedded in OCT, frozen at -20°C, sectioned, baked at 50°C for 20 min, fixed with methanol for 10 min, washed with PBS, and the tissues were circled on the slides using a drawing tool. Wells were punched with 0.5% Triton X-100 solution for 15 min, washed with PBS, and DHE dye diluted 100-fold with PBS. Each tissue was incubated at 37°C for 30 min, washed with PBS, and then mounted with anti-fluorescence quenching mounting medium. All procedures were performed in the dark. Fluorescence intensity was captured using a fluorescence microscope and analyzed using ImageJ software. Primary cardiomyocytes were seeded in 24-well plates, washed with PBS, and fixed with methanol for 10 min; the remaining steps were the same as above.

[0033] The test results are as follows: Figure 4The images show DHE fluorescence staining microscopic images of four groups of mouse heart tissues and a statistical comparison of the corresponding fluorescence intensities. The results show that the oxidative stress level and reactive oxygen species content in the myocardial tissue of mice increased after I / R. However, after HY-Q67280 pretreatment, the oxidative stress level of mouse myocardium decreased significantly, indicating that HY-Q67280 pretreatment can reduce oxidative stress after myocardial ischemia / reperfusion injury in mice.

[0034] 10. TUNEL staining After harvesting heart tissue from four groups of mice, the tissue was soaked in 20% sucrose until it sank to the bottom, embedded in OCT, frozen at -20℃, sectioned, baked at 50℃ for 20 min, fixed with methanol for 10 min, washed with PBS, and the tissue was circled on the slide with a group drawing pen. Holes were punched with 0.1% Triton X-100 solution at 4℃ for 2 min, washed with PBS, incubated with TUNEL dye at 37℃ for 60 min, washed with PBS, stained with DAPI for 5 min, washed with PBS, and α-actinin antibody diluted 100-fold with PBS, 30 μL per tissue, incubated overnight at 4℃. The next day, after 30 min of warming, 488 fluorescent secondary antibody diluted 100-fold with PBS, 30 μL per tissue, incubated at 37℃ for 30 min, washed with PBS, and mounted with anti-fluorescence quenching mounting medium. All procedures were performed in the dark. Fluorescence intensity was photographed using a fluorescence microscope and analyzed using ImageJ software.

[0035] The test results are as follows: Figure 5 Microscopic images of TUNEL fluorescence staining in the heart tissues of four groups of mice and a statistical comparison of the percentage of TUNEL-positive cell nuclei in the four groups showed that cardiomyocyte apoptosis increased after I / R in mice; while HY-Q67280 pretreatment significantly reduced cardiomyocyte apoptosis in mouse heart tissues, indicating that HY-Q67280 pretreatment can reduce cardiomyocyte apoptosis after myocardial ischemia / reperfusion injury in mice.

[0036] 11. Mitosox staining Primary cardiomyocytes were seeded in 24-well plates. The culture medium was aspirated, and the cells were washed 1-2 times with PBS. Mitosox staining solution (1×) was added, and the cells were gently shaken to ensure that the dye evenly covered all cells. Generally, the volume of each well in a 24-well plate is 250 μL. The cells were incubated at 37°C in the dark for 30-45 min. After incubation, the cells were washed 2-3 times with PBS, and then detection buffer was added for observation under a fluorescence microscope.

[0037] The test results are as follows: Figure 6Microscopic images of four groups of primary cardiomyocytes stained with Mitosox fluorescence and statistical graphs of fluorescence intensity were shown. The results indicated that the mitochondrial oxidative stress level of primary cardiomyocytes increased after H / R; while the mitochondrial oxidative stress level of primary cardiomyocytes decreased after HY-Q67280 pretreatment, indicating that HY-Q67280 pretreatment can reduce mitochondrial oxidative stress in primary cardiomyocytes after hypoxia-reoxygenation.

[0038] 12. Mitotracker staining Primary cardiomyocytes were seeded in 24-well plates. The culture medium was aspirated, and the cells were washed 1-2 times with PBS. Mitotracker staining solution (0.2µM) was added, and the cells were gently shaken to ensure that the dye evenly covered all cells. Generally, the volume of each well in a 24-well plate was 250μL. The cells were incubated at 37°C in the dark for 30-45 minutes. After incubation, the staining solution was discarded, and Hoechst (1×) staining solution was added. The cells were incubated at 37°C in the dark for 10 minutes. After incubation, the cells were washed 2-3 times with PBS, and then detection buffer was added for observation under a fluorescence microscope.

[0039] The test results are as follows: Figure 7 Images of four groups of primary cardiomyocytes stained with Mitotracker fluorescence and statistical diagrams of mitochondrial division were shown. The results indicated that the degree of mitochondrial division in primary cardiomyocytes increased after H / R treatment; while the degree of mitochondrial division in primary cardiomyocytes decreased after HY-Q67280 pretreatment, indicating that HY-Q67280 pretreatment can reduce the degree of mitochondrial division in primary cardiomyocytes after hypoxia-reoxygenation.

[0040] 13.TMRE staining Primary cardiomyocytes were seeded in 24-well plates. The culture medium was aspirated, and the cells were washed 1-2 times with PBS. TMRE staining solution (1×) was added, and the cells were gently shaken to ensure that the dye evenly covered all cells. Generally, the volume of each well in a 24-well plate is 250 μL. The cells were incubated at 37°C in the dark for 30-45 min. After incubation, the cells were washed 2-3 times with PBS, and then detection buffer was added for observation under a fluorescence microscope.

[0041] The test results are as follows: Figure 8 Microscopic images of TMRE fluorescence staining of four groups of primary cardiomyocytes and statistical graphs of fluorescence intensity were shown. The results indicated that the mitochondrial membrane potential of primary cardiomyocytes decreased after H / R; while the mitochondrial membrane potential of primary cardiomyocytes increased after HY-Q67280 pretreatment, indicating that HY-Q67280 pretreatment can improve the mitochondrial membrane potential of primary cardiomyocytes after hypoxia-reoxygenation.

[0042] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of HY-Q67280 in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia / reperfusion injury, characterized in that, The chemical formula of HY-Q67280 is C 23 H 26 N4O3, and the structural formula is: .

2. Use according to claim 1, characterized in that, The drug exerts its preventive and / or therapeutic effects on myocardial ischemia / reperfusion injury by reducing myocardial ischemia / reperfusion and myocardial cell apoptosis after hypoxia / reoxygenation, and by lowering mitochondrial oxidative stress levels.

3. Use according to claim 1, characterized in that, The drug exerts its effect in preventing and / or treating myocardial ischemia / reperfusion injury by activating the E3 ubiquitin ligase MARCH9 and improving mitochondrial function and status after myocardial cell hypoxia / reoxygenation.

4. A drug for preventing and / or treating myocardial ischemia / reperfusion injury, characterized in that, The active ingredient of the drug includes HY-Q67280; The chemical formula of HY-Q67280 is C 23 H 26 N4O3, structural formula: .

Images