Use of the inhibitor rbl in the preparation of a medicament for the treatment of cardiovascular disease

Abstract

This invention discloses the application of an inhibitor of renin-angiotensin-aldosterone (RBL) in the preparation of drugs for treating cardiovascular diseases. Through experiments and analysis of RBL inhibitors in animal models, this invention reveals that RBL inhibitors can improve myocardial ischemia / reperfusion injury. This is the first discovery of RBL in cardiovascular disease. Further investigation into the mechanism suggests that RBL's ability to improve myocardial ischemia / reperfusion injury may be through regulating oxidative stress-mediated glucose and lipid metabolism, reducing the massive influx of glucose and lipids after reperfusion.

Description

Technical Field

[0001] This invention belongs to the field of clinical application of cardiovascular diseases, specifically, it relates to the application of an inhibitor RBL in the preparation of drugs for treating cardiovascular diseases. Background Technology

[0002] Cardiovascular disease (CVD) is a major risk factor for human health worldwide. According to the Global Burden of Disease study (GBD), as of 2015, the number of people suffering from CVD globally approached 430 million; in 2016, statistics showed that the number of people with ischemic heart disease in my country reached 11 million. Ischemic heart disease (IHD) is the leading cause of CVD-related deaths, and its incidence continues to rise annually with improved living standards and a faster pace of modern life. Therefore, IHD has become a major health burden globally.

[0003] Current treatment for ischemic heart disease primarily focuses on early restoration of coronary blood flow and salvage of damaged myocardium. Strategies to protect against ischemic myocardial injury include ischemic preconditioning and postconditioning. Clinically, intracoronary thrombolysis, balloon angioplasty for coronary artery stenosis, and coronary artery bypass grafting have been proven to effectively restore myocardial blood flow and salvage ischemic myocardium. However, it is crucial to recognize that reperfusion therapy is a double-edged sword. Acute reperfusion can lead to calcium overload, increased oxidative stress, and inflammatory infiltration in myocardial mitochondria, causing the mitochondrial permeability transition pore (mPTP) to open, resulting in further damage to the ischemic myocardium and irreversible myocardial necrosis, a condition known as ischemia / reperfusion injury (I / R injury). I / R injury can lead to poor prognosis and even death in patients with ischemic heart disease (IHD). However, there are few reported treatments for ischemia / reperfusion injury. Therefore, if a small molecule drug that can improve myocardial ischemia / reperfusion injury can be found, it is hoped that it can reduce the poor prognosis and patient mortality caused by reperfusion injury in clinical practice. Summary of the Invention

[0004] In view of this, the present invention addresses the problems existing in the prior art by providing an application of the inhibitor RBL in the preparation of drugs for treating cardiovascular diseases. Bengal erythrolactone RBL can broadly inhibit kinesin activity and can be used at the molecular level or clinically to alleviate and treat myocardial ischemia / reperfusion injury, thereby improving the poor prognosis or even death of patients with ischemic heart disease caused by ischemia / reperfusion injury.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] One aspect of the present invention provides the use of an inhibitor RBL in the preparation of a medicament for treating cardiovascular diseases.

[0007] Furthermore, the inhibitor RBL can improve myocardial ischemia / reperfusion injury.

[0008] Furthermore, the inhibitor RBL can significantly reduce the serum LDH level, a marker of myocardial infarction.

[0009] Furthermore, during ischemia, the inhibitor RBL can significantly reduce cardiomyocyte apoptosis following myocardial ischemia / reperfusion injury.

[0010] Furthermore, the inhibitor RBL can improve reperfusion arrhythmias.

[0011] Furthermore, the inhibitor RBL can significantly reduce the level of oxidative stress after myocardial ischemia / reperfusion injury.

[0012] In a second aspect, the present invention provides a medicament for treating cardiovascular diseases, including an inhibitor of RBL.

[0013] Kinesins are a class of motor proteins widely found in eukaryotes. They hydrolyze ATP, converting chemical energy into mechanical energy and directing the transport of various cargoes (such as vesicles and mitochondria) along microtubules to specific locations within the cell. Since their initial discovery in 1985, 45 members of 14 subfamilies have been identified in humans, forming the kinesin superfamily. Numerous studies have shown that kinesin dysfunction is closely related to the development and progression of diseases such as tumors, neurodegenerative diseases, and diabetes; however, its role in cardiovascular diseases has been less studied. Research indicates that upon restoration of blood flow after ischemia, a large influx of metabolic substrates and restoration of oxygen supply lead to the production of large amounts of reactive oxygen species (ROS), thereby activating apoptosis and necrosis pathways and causing irreversible myocardial damage. Therefore, it is generally accepted that the direct cause of myocardial reperfusion injury is ROS, and its initiating mechanism is energy metabolism disorder. Studies have shown that kinin KIF5B can mediate the membrane translocation of fatty acid transporter CD36 and glucose transporter GLUT4, thereby facilitating the entry of glucose and fatty acids into the cell. Therefore, it may be involved in the regulation of glucose and lipid metabolism and the induction of oxidative stress during reperfusion. Thus, inhibiting kinin activity may suppress metabolic disturbances and oxidative stress in the early stages of reperfusion, thereby improving ischemia / reperfusion injury.

[0014] Rose Bengal Lactone (RBL) is a kinin inhibitor with the following structural formula:

[0015]

[0016] The molecular formula is C 20 H4Cl4I4O5, with a molecular weight of 973.67, has been shown to competitively bind to microtubules, thereby reducing the affinity of kinin for microtubules, but without interfering with the affinity of kinin for ATP.

[0017] This invention, through experiments and analysis of RBL inhibitors in animal models, reveals that RBL inhibitors can improve myocardial ischemia / reperfusion injury. This is the first discovery of RBL in cardiovascular disease. Further investigation into the mechanism suggests that RBL's ability to improve myocardial ischemia / reperfusion injury may be through regulating oxidative stress-mediated glucose and lipid metabolism, reducing the massive influx of glucose and lipids after reperfusion. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1The transcriptional level of kinesin KIF5B in myocardial tissue of the Sham group, IS group and I / R group was detected by RT-PCR; *P<0.05, Sham vs. I / R; n=3, all values ​​are expressed as mean ± SEM;

[0020] Figure 2 The expression levels of kinesin KIF5B in myocardial tissue of the Sham group, IS group, I / R(30) group, and I / R(120) group (Sham: sham operation group; IS: ischemia 30 min; I / R(30): ischemia 30 min, reperfusion 30 min; I / R(120): ischemia 30 min, reperfusion 120 min) were detected by Western blot.

[0021] Figure 3 The test kits include TTC-Evans Blue double staining and AAR / LV ratio (Ratio of area at risk volume to left ventricle volume), INF / AAR ratio (Ratio of infarct volume to area at risk volume), and LDH detection kits to detect serum LDH levels (U / L).

[0022] # P<0.05, Sham vs.I / R+V; **P<0.01, I / R+V vs.IR+RBL; $ P<0.001, I / R+V vs. I / R+RBL; n=3, all values ​​are expressed as mean ± SEM;

[0023] Figure 4 This is a representative image (×400) of TUNEL staining on a heart section and the results of TUNEL staining; $ P<0.0001, Sham vs. I / R; $ P<0.0001, I / R+V vs. I / R+RBL; n=3, all values ​​are expressed as mean ± SEM;

[0024] Figure 5 It is a typical electrocardiogram for mouse arrhythmia scoring, premature ventricular complexes (PVC), ventricular tachycardia (VT), and ventricular fibrillation (VF).

[0025] Figure 6This includes statistical results on arrhythmia scores, the number of VT+VF episodes, the duration of VT+VF episodes, and heart rate in both groups. **P<0.01, Sham vs. I / R; *P<0.05, I / R+V vs. I / R+RBL; n=4, all values ​​are expressed as mean ± SEM;

[0026] Figure 7 These are representative images (×100) of DHE staining on cardiac sections and the results of DHE staining.

[0027] Figure 8 This is the level of lipid oxidation detected using an MDA assay kit. Detailed Implementation

[0028] The following will describe the implementation of the present invention in detail with reference to the embodiments, so that the process of how the present invention uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0029] Kinesins are a class of motor proteins widely found in eukaryotes. They hydrolyze ATP, converting chemical energy into mechanical energy and directing its movement along microtubules to transport various cargoes (such as vesicles and mitochondria) to specific locations within the cell. Since their initial discovery in 1985, 45 members of 14 subfamilies have been identified in humans, forming the kinesin superfamily. Numerous studies have shown that kinesin dysfunction is closely related to the development and progression of diseases such as tumors, neurodegenerative diseases, and diabetes; however, its role in cardiovascular diseases has been less studied. Research indicates that upon restoration of blood flow after ischemia, a large influx of metabolic substrates and restoration of oxygen supply lead to the production of large amounts of reactive oxygen species (ROS), thereby activating apoptosis and necrosis pathways and causing irreversible myocardial damage. Therefore, it is generally accepted that the direct cause of myocardial reperfusion injury is ROS, and its initiating mechanism is energy metabolism disorder. Studies have shown that kinin KIF5B can mediate the membrane translocation of fatty acid transporter CD36 and glucose transporter GLUT4, thereby facilitating the entry of glucose and fatty acids into the cell. Therefore, it may be involved in the regulation of glucose and lipid metabolism and the induction of oxidative stress during reperfusion. Thus, inhibiting kinin activity may suppress metabolic disturbances and oxidative stress in the early stages of reperfusion, thereby improving ischemia / reperfusion injury.

[0030] Rose Bengal Lactone (RBL) is a kinin inhibitor with the following structural formula:

[0031]

[0032] The molecular formula is C 20H4Cl4I4O5, with a molecular weight of 973.67, has been shown to competitively bind to microtubules, thereby reducing the affinity of kinin for microtubules, but without interfering with the affinity of kinin for ATP.

[0033] This invention, through experiments and analysis of RBL inhibitors in animal models, reveals that RBL inhibitors can improve myocardial ischemia / reperfusion injury. This is the first discovery of RBL in cardiovascular disease. Further investigation into the mechanism suggests that RBL's ability to improve myocardial ischemia / reperfusion injury may be through regulating oxidative stress-mediated glucose and lipid metabolism, reducing the massive influx of glucose and lipids after reperfusion.

[0034] [Experiment 1] To investigate the transcriptional and expression changes of kinesin KIF5B during different stages of myocardial ischemia / reperfusion.

[0035] The animals used were 8-12 week old male C57BL / 6J mice. The mice were randomly divided into three groups: sham operation group (Sham), ischemia group (IS), and ischemia / reperfusion group (I / R).

[0036] ①Establishment of the I / R model:

[0037] Mice were anesthetized preoperatively by intraperitoneal injection of sodium pentobarbital solution (60 mg / kg). Electrocardiograms were recorded after anesthesia, and mice were connected to a small animal ventilator with a tidal volume of 15 ml / kg and a respiratory rate of 70 breaths / min. The left anterior descending coronary artery was sutured to a tube with 7-0 sutures at a depth of approximately 1.5-2 mm and a width of 3-4 mm. Persistent ST-segment elevation and myocardial pallor distal to the ligation point indicated myocardial ischemia. After 30 minutes of ischemia, the suture was removed, and reperfusion was performed for 2 hours. At this point, ST-segment depression and reddening of the ischemic pallor indicated successful reperfusion. The sham-operated group underwent the same thoracotomy and treatment time as the I / R model, but without ischemia and reperfusion.

[0038] ②After the surgery, the tissue below the left ventricular ligation line was taken to extract protein and RNA for RT-PCR and Western Blot detection to detect the expression and transcription levels of KIF5B at each stage of the surgery.

[0039] Detection was performed using RT-PCR and Western Blot, as follows: Figure 1 and Figure 2 As shown, the transcriptional and expression levels of KIF5B tend to decrease significantly during reperfusion compared to ischemia, suggesting that KIF5B may play an important role during myocardial ischemia / reperfusion in mice.

[0040] [Experiment 2] Elucidating the role of RBL in inhibiting kinin in I / R from an animal experimental perspective.

[0041] (1) Animal experiments: The mice used were 8-12 week old male C57BL / 6J mice.

[0042] Mice will be randomly divided into three groups: sham operation group (Sham), ischemia / reperfusion group (I / R+V), and drug intervention group (I / R+RBL).

[0043] ① Establishment of the I / R model: The pretreatment is as described above, except that RBL treatment is performed. After 15-20 minutes of ischemia, RBL (1 mg / kg, dissolved in DMSO) is injected intraperitoneally. After 10-15 minutes, the connecting suture is removed, and perfusion is resumed for 2 hours.

[0044] ② Confirmation of infarct area: After the I / R model, the heart was removed and perfused with normal saline until no blood accumulation remained in the cavity. Then, the residual normal saline was rinsed off with 1% TTC solution. The sample was placed in a preheated dish containing 9 ml of 1% TTC solution and incubated in a 37°C water bath for 5 minutes. The sample was then frozen at -20°C until morphologically fixed, sectioned (operated on ice), and stained with 0.5% Evans blue. After staining, the sections were fixed in 4% paraformaldehyde and incubated overnight at 4°C before photographing and observation.

[0045] ③ Detect myocardial enzyme levels:

[0046] Determination of LDH levels in mouse serum: After the I / R model was established, blood samples were collected via the carotid artery and centrifuged at 3000 rpm for 10 min. Serum LDH levels were then measured using an LDH detection kit.

[0047] RBL is an inhibitor of kinesin. This study investigated whether RBL could improve myocardial ischemia-reperfusion injury and explored its potential clinical application value. C57BL / 6J mice underwent ligation of the left anterior descending coronary artery. 15-20 minutes after ischemia, RBL (1 mg / kg) or an equal volume of DMSO was injected intraperitoneally. Myocardial infarction area was measured 30 minutes after ischemia and 2 hours after reperfusion. TTC-Evans blue staining results showed that RBL significantly reduced ischemia / reperfusion-induced myocardial infarction. Furthermore, serum levels of LDH, a marker of myocardial infarction, were measured. Figure 3 As shown, the results indicate that RBL can significantly reduce serum LDH levels. All of the above results demonstrate that the inhibitor RBL can improve myocardial ischemia / reperfusion injury.

[0048] ④ Detection of cardiomyocyte apoptosis: Frozen sections of myocardial tissue were prepared, stained with the TUNEL kit, and the apoptosis of cardiomyocytes was detected by confocal microscopy.

[0049] Apoptosis is the main mode of cell death after reperfusion. Frozen sections of myocardial tissue were taken from each group, and TUNEL staining was used to calculate and compare the apoptosis status of cardiomyocytes in each group. Figure 4 As shown, the results indicate that RBL treatment during ischemia can significantly reduce cardiomyocyte apoptosis after I / R.

[0050] One of the common clinical manifestations of myocardial I / R injury is the occurrence of arrhythmias after reperfusion, especially ventricular arrhythmias. Therefore, electrocardiograms were recorded and saved during the operation, and ventricular arrhythmias within 30 minutes after reperfusion were evaluated according to the Lambeth Convention (II) after the experiment.

[0051] Post-reperfusion arrhythmias are an important indicator of reperfusion injury. Electrocardiograms of mice were recorded after reperfusion, and the incidence and duration of arrhythmia scores, PVCs, VTs, and VFs in each group were assessed according to Lambeth protocol at 30 minutes post-reperfusion. Figure 5 and Figure 6 As shown, the results indicate that RBL can improve reperfusion arrhythmias with significant statistical differences.

[0052] [Experiment 3] Mechanism of the kinin inhibitor RBL in improving myocardial ischemia / reperfusion injury

[0053] Animal experiments: Mice were randomly divided into three groups: sham operation group (Sham), ischemia / reperfusion control group (I / R+V), and drug intervention group (I / R+RBL). Two hours after reperfusion, blood samples and tissue proteins were collected to detect oxidative stress and metabolic-related indicators.

[0054] ① Lipid oxidation detection:

[0055] Blood was drawn from the carotid artery, serum was collected, and lipid oxidation levels were detected using an MDA assay kit.

[0056] ② ROS fluorescent probe-DHE staining:

[0057] Tissue was harvested from below the left ventricular ligation line and temporarily preserved in liquid nitrogen before being frozen sectioned. The sections were stained with dihydroethidium (DHE), photographed under a confocal microscope, and the ROS levels in the tissue were calculated and compared.

[0058] After treating mice in each group separately, myocardial tissue was collected, frozen sections were prepared, and the staining intensity of myocardial cells in each group was calculated and compared using a DHE kit. Figure 7 and Figure 8 As shown, statistical analysis revealed that RBL treatment can significantly reduce the level of oxidative stress after I / R.

[0059] The foregoing description illustrates and describes several preferred embodiments of the invention. However, as previously stated, it should be understood that the invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the invention should be within the protection scope of the appended claims.

Claims

1. The use of an inhibitor RBL, said RBL being: ###0001### in the manufacture of a medicament for the treatment of a cardiovascular disease, said cardiovascular disease being myocardial ischemia / reperfusion injury or reperfusion arrhythmia. ； ​

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