New application of tamibarotene in medicine

Abstract

The invention discloses application of tamibarotene in preparation of a medicine for preventing and treating myocardial infarction, the tamibarotene can be directly combined to an NACHT structural domain of NLRP3 in a targeting manner, the ATP enzyme activity of the NLRP3 is inhibited, the assembly of NLRP3 inflammasome is inhibited, the activation of the NLRP3 inflammasome is effectively prevented, and the tamibarotene has a remarkable prevention and treatment effect on the myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a new application of Tamibarotene in medicine. Background Art

[0002] Myocardial infarction (AMI) is a serious disease that affects life due to insufficient blood supply to the corresponding myocardial area caused by acute obstruction of the coronary artery, resulting in myocardial tissue necrosis. The disease is mainly caused by insufficient blood supply, myocardial hypoxia and excessive myocardial oxygen consumption due to various reasons such as coronary atherosclerosis, arrhythmia, respiratory failure, etc. Myocardial infarction is an important cause of human death. In recent years, the mortality rate caused by myocardial infarction has continued to increase. At present, the treatment of myocardial infarction mainly focuses on reperfusion therapy such as percutaneous coronary intervention, coronary artery bypass grafting and drug thrombolysis. However, this can only improve the blood supply of myocardial tissue in the myocardial infarction area, but cannot improve the long-term prognosis of myocardial infarction patients. Although rapid restoration of ischemic myocardial blood flow is an important means of clinical treatment, myocardial ischemia will activate inflammatory response, induce ischemic myocardial scar formation and further apoptosis of myocardial cells, thereby increasing the area of ​​ischemic myocardial tissue, aggravating cardiac dysfunction and ventricular remodeling, inducing heart failure, and increasing the risk of long-term death in patients with myocardial infarction. Although heart transplantation can completely solve severe heart failure caused by heart damage, the lack of donors limits the clinical application of this treatment. Therefore, exploring a new, safe and effective drug for the prevention and treatment of myocardial ischemic injury remains a clinical need.

[0003] The NLRP3 inflammasome is an important component of the host's innate immune system. It is a high-molecular protein complex that can be activated by various pathogens or cell stress stimuli. It consists of three parts: Nod-like receptor family pyrin domain-containing protein 3 (NLRP3), ASC and Pro-caspase-1, of which NLRP3 is the core protein and plays a crucial regulatory role in the activation of the NLRP3 inflammasome. The NLRP3 inflammasome can be activated by a variety of danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), including some particulate matter, extracellular ATP, nigericin, pore-forming toxins, viruses, bacteria, fungi and protozoan pathogens. When cells respond to external danger signals, the NLRP3 inflammasome is activated and participates in innate immune defense.

[0004] A large number of studies have reported that abnormal activation of NLRP3 inflammasome plays an important role in the pathological process of myocardial infarction. Inflammatory cell infiltration and high expression of NLRP3 inflammasome-related proteins were found in the myocardial tissue of patients who died of myocardial infarction, indicating that NLRP3 inflammasome regulates myocardial infarction injury, while abnormally activated NLRP3 inflammasome induces myocardial cell fibrosis, myocardial cell apoptosis and ventricular remodeling, promoting myocardial infarction injury. Studies have shown that inhibiting myocardial NLRP3 inflammasome activation can reduce myocardial inflammatory response, improve cardiac dysfunction and remodeling, and improve the prognosis of myocardial infarction.

[0005] The chemical structural formula of Tamibarotene is formula (I), and its molecular formula is C 22 H 25 NO3, with a molecular weight of 351.45 and a CAS number of 94497-51-5, is a selective retinoic acid receptor α (RARα) agonist, mainly used for the treatment of relapsed or refractory cases of acute promyelocytic leukemia (APL). However, there is no clear report on whether Tamibarotene has the effect of preventing and treating myocardial infarction.

[0006] Summary of the invention

[0007] Based on this, the object of the present invention is to provide a new application of Tamibarotene in medicine, which can be embodied in that Tamibarotene is used for preventing and treating myocardial infarction.

[0008] The technical solutions for achieving the above-mentioned purpose include the following.

[0009] Application of Tamibarotene in preparing medicine for preventing and treating myocardial infarction.

[0010] In some embodiments, the use includes Tamibarotene improving cardiac function in myocardial infarction.

[0011] In some embodiments, the use includes that Tamibarotene inhibits the infiltration of inflammatory cells and the degree of fibrosis in myocardial infarction tissue.

[0012] In some embodiments, the use includes that tamibarotene inhibits NLRP3 inflammasome activation in myocardial tissue of myocardial infarction.

[0013] In some embodiments, the dosage form of the drug is an injection or an oral agent, and the oral agent can be a tablet, capsule, granule, pill, syrup, granule (sugar-free type), powder, ointment, tincture, or aqueous solution (such as emulsion, solution, suspension).

[0014] In some embodiments, the applicable object or subject of the application is a mammal.

[0015] In some embodiments, the mammal is a human.

[0016] The inventors of the present invention have found in their studies that Tamibarotene can improve the cardiac function of mice with myocardial infarction, inhibit the infiltration of inflammatory cells and the degree of fibrosis in myocardial infarction tissue, and inhibit the activation of NLRP3 inflammasomes in myocardial infarction tissue. It can be seen that Tamibarotene has a significant preventive and therapeutic effect on myocardial infarction. Moreover, further studies have found that Tamibarotene can directly target and bind to the NACHT domain of NLRP3, inhibit the ATPase activity of NLRP3, inhibit the assembly of NLRP3 inflammasomes, and effectively prevent the activation of NLRP3 inflammasomes. This may be an important reason why Tamibarotene has the effect of treating myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the animal experiment flow chart and grouping treatment table; Figure 1 B is a representative cardiac ultrasound examination image; Figure 1 CJ are quantitative analysis statistical charts of the cardiac ultrasound indicators EF, FS, LVIDs, LVIDd, LVAWs, LVAWd, LVPWs, and LVPWd.

[0018] Figure 2 A is a representative LVTTP image within each group; Figure 2 B represents the speckle tracking analysis of cardiac global longitudinal strain (GLS) measured by parasternal long axis (PSLAX) B-mode echocardiography; Figure 2 CE represents the radial strain statistics of the anterior wall of the left ventricle.

[0019] Figure 3 A is a representative image of H&E staining of heart tissue; Figure 3 B, F are representative images and statistical graphs of immunohistochemical analysis of F4 / 80 in heart tissue; Figure 3 C is a representative image of Masson staining of heart tissue; Figure 3 D, G are representative images and statistical graphs of immunohistochemistry of Collagen Ⅰ in cardiac tissue; Figure 3 E, H are representative and statistical graphs of cardiac tissue apoptosis detection.

[0020] Figure 4 A is a statistical graph of ELISA detection of IL-1β in mouse serum; Figure 4 B is a representative picture of Western blotting analysis of myocardial tissue protein NLRP3 inflammasome-related proteins; Figure 4 C, D are representative images and statistical graphs of ASC spots in myocardial tissue.

[0021] Figure 5A is the chemical structure of tamibarotene; Figure 5 BF are representative graphs and statistical graphs of Western blotting analysis of IL-1β and Caspase-1 levels in supernatants and expression levels of IL-1β, Caspase-1, NLRP3, ASC, and β-actin in lysates; Figure 5 G is a statistical graph of ELISA detection of IL-1β in cell culture supernatant; Figure 5 H is the statistical graph of the percentage of lactate dehydrogenase released by macrophages.

[0022] Figure 6 A, B are representative images and statistical graphs of immunofluorescence detection of ASC spots in macrophages; Figure 6 C, D are representative images and statistical graphs of immunoblot analysis of ASC oligomerization in macrophages; Figure 6 E is a representative diagram of NLRP3 oligomerization detected by non-denaturing gel electrophoresis; Figure 6 F is a representative image of inflammasome assembly detected by co-immunoprecipitation; Figure 6 G is a representative figure of the binding of NLRP3 and NEK7 detected by co-immunoprecipitation.

[0023] Figure 7 A is a representative immunoblot of macrophage proteins after pyrolysis; Figure 7 B is a representative immunoblot of macrophage lysate proteins after enzymatic treatment; Figure 7 C is a diagram showing the structural domains of NLRP3; Figure 7 D is a representative immunoblot of proteins of Flag-LRR, Flag-NACHT, and Flag-PYD domains in plasmid-transfected HEK-293T cells after enzymatic treatment; Figure 7 E is a statistical graph of NLRP3 ATPase activity detection; Figure 7 F Representative image of surface plasmon resonance detection.

[0024] Figure 8 A and B are 2D and 3D model diagrams of molecular docking; Figure 8 C is the detection graph of RMSD; Figure 8 D is a diagram showing the various structural domains of NACHT of NLRP3; Figure 8 E is the energy value display diagram of each molecular docking site; Figure 8 F is a statistical graph of ATPase activity detection of purified protein; Figure 8 G is a representative immunoblot of proteins in HEK-293T cells transfected with site mutant plasmids after enzymatic treatment. DETAILED DESCRIPTION

[0025] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0028] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0029] The "applicable subject", "applicable person", "subject" or "patient" of the present invention refers to a human or a non-human animal such as a mammal. "Applicable person" may include any mammal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, sheep, and cattle. A human subject may also be referred to as a patient, such as a person or patient at risk of myocardial infarction.

[0030] In some embodiments of the present invention, an "effective amount" or "therapeutically effective amount" of tamibarotene is administered to a patient at potential risk of suffering from myocardial infarction for the prevention of myocardial infarction or the treatment of myocardial infarction. As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to affect a desired biological effect, including a clinical outcome, such as a beneficial effect. As such, the "effective amount" depends on the context in which it is applied. The effective amount may vary according to factors known in the art, such as the disease state, age, sex, and weight of the individual being treated. Several separate doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. In addition, according to the present invention, tamibarotene may be administered to achieve a therapeutic amount as needed in a conventional manner, or according to the present invention, the tamibarotene effect on myocardial infarction exhibits a significant time and dose dependence.

[0031] As used herein, the term "prevention" and variations thereof and other grammatical equivalents include preventing the development, occurrence, hindering or avoiding myocardial infarction or its symptoms and reducing the occurrence of symptoms. Prevention can be complete (i.e., no detectable symptoms) or partial, so that the observed symptoms are less than the symptoms that would occur without treatment. The term also includes a preventive benefit. For the disease or condition to be prevented, tamibarotene can be administered to a population with test indicators that they may suffer from myocardial infarction in the future, even if a diagnosis of the disease may not have been made.

[0032] Some embodiments of the present invention relate to the use of Tamibarotene in the preparation of a medicament for preventing and treating myocardial infarction.

[0033] In some embodiments, the drug is administered orally. For example, the drug may be in the form of tablets, capsules, granules, injections, pills, syrups, powders, ointments, or aqueous solutions (such as emulsions, solutions, suspensions) or tinctures.

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Tamibarotene was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.

[0036] C57BL / 6 male mice were purchased from Guangdong Experimental Animal Center.

[0037] H&E staining solution was purchased from Biotech Biotechnology Co., Ltd.

[0038] Masson staining solution was purchased from Biotech Biotechnology Co., Ltd.

[0039] Lipopolysaccharide (LPS) was purchased from Merck.

[0040] Nigericin was purchased from MedChemExpress.

[0041] The ELISA kit for detecting IL-1β inflammatory factor was purchased from ThermoFisher.

[0042] The lactate dehydrogenase (LDH) detection kit was purchased from Biotime Biotechnology Co., Ltd.

[0043] Anti-NLRP3 and ASC antibodies were purchased from Cell Signaling Technology.

[0044] Anti-Caspase-1 was purchased from AdipoGen.

[0045] Anti-IL-1β was purchased from Invitrogen.

[0046] DSS cross-linking reagent was purchased from Aladdin.

[0047] J774A.1 mouse macrophages and HEK293T cells were purchased from Guangzhou Geneo Biotechnology Co., Ltd.

[0048] Protein A / G plus-agarose was purchased from Santa Cruz Biotechnology.

[0049] ATPase activity detection kit was purchased from Promega.

[0050] Pronase E was purchased from MedChemExpress.

[0051] The present invention is further described in detail below with reference to specific embodiments.

[0052] Example 1 Tamibarotene can improve the cardiac function of mice with myocardial infarction

[0053] C57BL / 6 male mice aged 6-8 weeks were weighed, grouped and numbered, and divided into 5 groups, with 6 mice in each group, namely: Sham group (negative control group), Sham + Tamibarotene 20 mg / kg (negative control + Tamibarotene 20 mg / kg), MI group (myocardial infarction group), MI + Tamibarotene 10 mg / kg (myocardial infarction + Tamibarotene 10 mg / kg), MI + Tamibarotene 20 mg / kg (myocardial infarction + Tamibarotene 20 mg / kg).

[0054] The steps for constructing the myocardial infarction model are as follows: (1) anesthetize mice with 90 mg / kg of 3% sodium pentobarbital injected intraperitoneally, shave the mouse chest and armpit hair with a mouse shaver to fully expose the surgical area, and disinfect the surgical area with iodine and 75% ethanol; (2) set the various parameters of the small animal ventilator, the respiratory rate is 110 bpm, insert the endotracheal tube along the glottis into the mouse trachea, connect the ventilator, observe the mouse's respiratory condition, and the chest rise and fall consistent with the ventilator frequency indicates that the intubation is successful, and then proceed to the subsequent steps; (3) The mouse is placed in the right lateral decubitus position, and the chest cavity is opened with ophthalmic scissors under the left forelimb armpit and microscissors between the third and fourth ribs. Fully expose the heart, gently pick up a small amount of pericardium with straight forceps and tear open a little pericardium under the left atrial appendage to fully expose the area where the left anterior descending coronary artery (LAD) is located; (4) Under the microscope, find the direction and location of the LAD, use a needle holder to hold a 7-0 suture with a needle, insert the needle 2 mm below the lower edge of the left atrial appendage, and pass the suture through the LAD to completely block the blood flow of the LAD; (5) After ligation, completely suture the chest opening with 6-0 sutures to close the chest cavity, and suture each layer of muscle and skin from the inside to the outside; (6) Pay close attention to the condition of the mice after the operation. After the mice wake up naturally, remove them from the ventilator and the endotracheal tube, and feed them normally.

[0055] The mice in the drug group were intragastrically injected with 10mg / kg and 20mg / kg of tamibarotene for 7 consecutive days. One hour after drug administration on the 7th day, the myocardial infarction model was established, and the cardiac function of the mice was detected by ultrasound 24 hours after ligation. Figure 1 and Figure 2 .

[0056] Figure 1 A is the mouse grouping and experimental flow chart; Figure 1 B is a representative image of the mouse heart detected by ultrasound; Figure 1CJ is an ultrasound index of cardiac function in mice, including cardiac ejection fraction (EjectionFraction), fractional shortening (FractionalShortening), left ventricular anterior wall thickness (LVAWs), left ventricular end-diastolic thickness (LVAWd), left ventricular posterior wall thickness (LVPWs), left ventricular posterior wall thickness (LVPWd), left ventricular end-diastolic internal diameter (LVIDd), and left ventricular end-systolic internal diameter (LVIDs). Compared with the negative control group, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular anterior wall thickness (LVAWs), left ventricular end-diastolic thickness (LVAWd), and left ventricular posterior wall thickness (LVPWs) in the myocardial infarction group were significantly reduced. However, compared with the myocardial infarction group, although there was no significant difference in left ventricular posterior wall thickness (LVPWd) in diastole, the above other indicators in the tamilbarotene-treated group were significantly increased. This shows that tamibarotene has excellent therapeutic effect on myocardial infarction injury and has a significant improvement effect on cardiac function after myocardial infarction.

[0057] Synchronous contraction of the anterior and posterior walls of the left ventricle is essential for left ventricular function, and asynchrony is associated with the pathological state of myocardial infarction. Synchrony is assessed by measuring systolic to peak strain on parasternal long-axis images using two-dimensional speckle tracking echocardiography. Figure 2 AThe results showed that the contraction of the left ventricular anterior wall and the left ventricular posterior wall of the mice in the myocardial infarction group was not synchronized, and tamibarotene could significantly reduce the delayed ventricular wall contraction and improve radial strain asymmetry after myocardial infarction in mice. Therefore, this study suggests that tamibarotene improves cardiac function after myocardial infarction by reducing ventricular contraction asynchrony. Left ventricular strain is also an important parameter for evaluating myocardial deformation and reflects the left ventricular contractile function. Global longitudinal strain (GLS) measures the longitudinal contractile capacity of the left ventricle, and a lower negative value indicates poor ventricular remodeling. Figure 2 B The results showed that compared with the myocardial infarction group, tamibarotene could significantly improve GLS after myocardial infarction. Segmental strain analysis is used to measure specific left ventricular regions and evaluate cardiac function. Left ventricular radial strain is also an indicator of myocardial deformation. The lower the value, the more likely it is that the heart is insufficiency. Figure 2 The results of segmental analysis of CE showed that compared with the myocardial infarction group, tamibarotene significantly improved the radial strain of the middle segment of LVAW and the longitudinal strain of the apex segment of LVAW after myocardial infarction ( Figure 2 CE).

[0058] Example 2 Tamibarotene can inhibit the inflammatory cell infiltration and fibrosis degree of myocardial infarction tissue in mice

[0059] 6-8 week old C57BL / 6 male mice were weighed, grouped and numbered, and divided into 5 groups, with 6 mice in each group, namely: Sham group (negative control group), Sham + Tamibarotene 20 mg / kg (negative control + Tamibarotene 20 mg / kg), MI group (myocardial infarction group), MI + Tamibarotene 10 mg / kg (myocardial infarction + Tamibarotene 10 mg / kg), MI + Tamibarotene 20 mg / kg (myocardial infarction + Tamibarotene 20 mg / kg). The mice in the drug-treated groups were intragastrically injected with 10 mg / kg and 20 mg / kg of Tamibarotene for 7 consecutive days. On the 7th day, the left coronary artery of the mice was permanently ligated 1 hour after the drug administration. The myocardial tissue of the mice was collected 24 hours after ligation, and paraffin sections and frozen sections were prepared. The infiltration of inflammatory cells was evaluated by H&E staining and F4 / 80 immunohistochemistry. The results are shown in Figure 3 A, B, F; Myocardial fibrosis was evaluated by Masson staining and Collagen I immunohistochemistry. Figure 3 C, D, G; Cardiomyocyte apoptosis was evaluated by TUNEL staining. Figure 3 E, H.

[0060] Figure 3 A The results showed that hematoxylin and eosin staining showed extensive myocardial necrosis and obvious inflammatory cell infiltration in the myocardium of the myocardial infarction group, while tamibarotene could significantly reduce necrosis and inflammatory cell infiltration in a dose-dependent manner; Figure 3 The results of B and F showed that a large number of macrophages infiltrated the myocardial tissue in the myocardial infarction group, and the administration of tamibarotene could significantly improve the infiltration of macrophages; Figure 3 The results of C, D, and G showed that collagen deposition and fibrosis occurred in the myocardial tissue of the myocardial infarction group, and the administration of tamibarotene could significantly improve the degree of myocardial fibrosis. Figure 3 The results of E and H showed that a large number of myocardial cells in the myocardial infarction group underwent ischemic apoptosis, while treatment with tamibarotene could significantly reduce the number of apoptotic myocardial cells.

[0061] Example 3 Tamibarotene can inhibit the activation of NLRP3 inflammasome in myocardial tissue of myocardial infarction

[0062] C57BL / 6 male mice aged 6-8 weeks were weighed, grouped and numbered, and divided into 5 groups, with 6 mice in each group, namely: Sham group (negative control group), Sham+Tamibarotene 20 mg / kg (negative control + Tamibarotene 20 mg / kg), MI group (myocardial infarction group), MI+Tamibarotene 10 mg / kg (myocardial infarction + Tamibarotene 10 mg / kg), MI+Tamibarotene 20 mg / kg (myocardial infarction + Tamibarotene 20 mg / kg). The mice in the drug-treated group were intragastrically injected with 10 mg / kg and 20 mg / kg of Tamibarotene for 7 consecutive days. On the 7th day, the left coronary artery of the mice was permanently ligated 1 hour after the drug administration. The myocardial tissue and serum of the mice were collected 24 hours after the ligation, and the release of inflammatory factors in the serum was detected by ELISA. The results are shown in Figure 4 A; Mouse myocardial tissue proteins were extracted, and the expression levels of NLRP3 inflammasome proteins in myocardial tissue were detected and analyzed by conventional Western blotting methods. The results are shown in Figure 4 B; Immunofluorescence was used to detect the formation of ASC spots in myocardial tissue. The results are shown in Figure 4 C, D.

[0063] Figure 4 A The results showed that the release level of inflammatory factors in the serum of the myocardial infarction group increased, while Tamibarotene could significantly reduce the level of inflammatory factors in the serum in a dose-dependent manner; Figure 4 B The results showed that the expression of NLRP3 inflammasome-related proteins in the myocardial tissue of the myocardial infarction group increased significantly, and the treatment with tamibarotene could significantly inhibit the expression level of related proteins; Figure 4 The results of C and D showed that a large number of ASC spots were formed in the cardiomyocytes of the myocardial infarction group, and treatment with tamibarotene could significantly reduce the number of ASC spots formed.

[0064] Example 4 Study on Tamibarotene Targeting NLRP3 Inflammasome

[0065] 4.1 Inhibitory effect of tamibarotene on NLRP3 inflammasome

[0066] (1) On the first day, J774A.1 cells were seeded into 35 mm diameter cell culture dishes, with 6.3 × 10 5 cells;

[0067] (2) After 24 hours of cell growth and proliferation in the cell culture dish, most of the cells were spindle-shaped, and a few were round. They were in good condition and could be processed later. The supernatant was discarded, and 1 ml of DMEM medium containing LPS (1 μg / ml) and 10% serum was added to each dish for 5 hours, and then different concentrations of Tamibarotene were added for 1 hour, and then Nigericin (10 μM) was added for 1 hour;

[0068] (3) After the treatment in step (2), the cell supernatant and cell lysate were collected, and the supernatant protein and cell lysate protein were extracted according to conventional methods. Western blotting analysis was performed using anti-IL-1β antibody, anti-Caspase-1 antibody, anti-NLRP3 antibody and anti-ASC antibody. The results are shown in Figure 5 The supernatant cell culture fluid was collected, and the concentration of inflammatory factor IL-1β was detected using IL-1β ELISA kit, and the half inhibitory concentration of drug inhibition of IL-1β was calculated. The results are shown in Figure 5 G. The LDH detection kit was used to detect the LDH level of macrophages. The results are shown in Figure 5 H.

[0069] Figure 5 A is the chemical structure of tamibarotene; Figure 5 BF results showed that under the combined effects of LPS pretreatment and the second signal Nigericin, NLRP3 inflammasome was activated, Caspase-1 and IL-1β matured and secreted into the supernatant. The addition of different concentrations of Tamibarotene could effectively inhibit the maturation and secretion of Caspase-1 and IL-1β, and this inhibitory effect was dose-dependent. Figure 5 G The results showed that Tamibarotene could inhibit the release of IL-1β stimulated by LPS and Nigericin, and this inhibitory effect was dose-dependent, with a half-maximal inhibitory concentration (IC50) of 13.6μM; Figure 5 The results showed that tamibarotene could inhibit the release level of LDH, and this inhibitory effect was dose-dependent, indicating that tamibarotene could improve cell pyroptosis induced by NLRP3 inflammasome activation.

[0070] 4.2 Study on the mechanism of action of tamibarotene in inhibiting NLRP3 inflammasome activation

[0071] (1) On the first day, J774A.1 cells were seeded into 35 mm diameter cell culture dishes, with 6.3 × 10 5 cells;

[0072] (2) After 24 hours of cell growth and proliferation in the cell culture dish, most of the cells were spindle-shaped, and a few were round. They were in good condition and could be processed later. The supernatant was discarded, and 1 ml of DMEM medium containing LPS (1 μg / ml) and 10% serum was added to each dish for 5 hours, and then different concentrations of Tamibarotene were added for 1 hour, and then Nigericin (10 μM) was added for 1 hour;

[0073] (3) After treatment in step (2), the formation of ASC spots was detected by immunofluorescence. The results are shown in Figure 6 AB; Collect cell lysate, collect cell precipitate after centrifugation, and use DSS cross-linking reagent to detect the oligomerization level of ASC. The results are shown in Figure 6 CD; cell lysate was collected and the oligomerization level of NLRP3 was detected by non-denaturing gel electrophoresis. The results are shown in Figure 6 E; Collect cell lysate and use protein co-precipitation experiment to detect NLRP3 inflammasome assembly. Figure 6 FG.

[0074] Figure 6 AB results showed that under the combined effects of LPS pretreatment and the second signal Nigericin, ASC spots were formed in large quantities, which are markers of NLRP3 inflammasome activation. The addition of different concentrations of tamibarotene can effectively inhibit the formation of ASC spots. Figure 6 CD results showed that under the combined effects of LPS pretreatment and the second signal Nigericin, ASC oligomerization increased, while Tamibarotene could inhibit the oligomerization level of ASC; Figure 6 E The results showed that tamibarotene can also inhibit the oligomerization level of NLRP3, which is the initial step of NLRP3 inflammasome activation. NLRP3 oligomerization can induce the assembly of inflammasomes. Figure 6 The FG results showed that tamibarotene could inhibit the assembly of NLRP3 inflammasome, but did not affect the binding of NLRP3 to NEK7, indicating that the effect of tamibarotene in inhibiting the activation of NLRP3 inflammasome was unrelated to NEK7, further indicating that its target was in the upstream stage of inflammasome assembly.

[0075] 4.3 Tamibarotene directly binds to the NACHT domain of NLRP3

[0076] (1) On the first day, J774A.1 cells were seeded into 35 mm diameter cell culture dishes, with 6.3 × 10 5 cells;

[0077] (2) After 24 hours of cell growth and proliferation in the cell culture dish, most of the cells were spindle-shaped, and a few were round. They were in good condition and could be processed later. The supernatant was discarded, and 1 ml of DMEM medium containing LPS (1 μg / ml) and 10% serum was added to each dish for 5 hours, and then different concentrations of Tamibarotene were added for 1 hour, and then Nigericin (10 μM) was added for 1 hour;

[0078] (3) After the treatment in step (2), the cell lysate proteins were collected and the binding target of tamibarotene was preliminarily explored using the drug affinity reaction target stability technology and cell thermal shift experiment. The results are shown in Figure 7 AB;

[0079] (4) HEK293T cells were inoculated into 35 mm diameter cell culture dishes, with 6.3 × 10 5 cells;

[0080] (5) After the cells grew and proliferated in the cell culture dish for 24 hours, the cells were in good condition for subsequent treatment, the supernatant was discarded, and the DNA plasmids of different truncated fragments of NLRP3 protein were transfected into the cells. Cell proteins were extracted 48 hours after transfection, and the drug affinity reaction targeted stability technology was used to clarify the binding region of tamibarotene and NLRP3. The results are shown in Figure 7 CD;

[0081] (6) The effect of tamibarotene on the ATPase activity of NLRP3 was detected using an ATPase assay kit. The results are shown in Figure 7 E;

[0082] (7) Surface plasmon resonance technology was used to detect the affinity of tamibarotene to the NACHT domain protein of NLRP3. The results are shown in Figure 7 F.

[0083] Figure 7 AB results showed that tamibarotene could enhance the enzymatic and thermal stability of NLRP3, but had no effect on the enzymatic and thermal stability of ASC, which preliminarily indicated that the target of tamibarotene might be NLRP3 protein; Figure 7 CD results showed that NLRP3 is composed of three domains: PYD, NACHT, and LRR, and tamibarotene can enhance the enzymatic stability of the NACHT domain, indicating that tamibarotene may directly bind to the NACHT domain of NLRP3, which has the ATPase activity regulatory site of NLRP3 and affects the oligomerization of NLRP3; Figure 7 E The results showed that tamibarotene could inhibit the ATPase activity of NLRP3; Figure 7F The results showed that tamibarotene had a strong affinity with the NACHT domain protein of NLRP3, with a KD value of 19 μM, which clearly indicated that tamibarotene directly bound to the NACHT domain of NLRP3, thereby inhibiting the activation of NLRP3 inflammasome.

[0084] 4.4 Exploration of the binding site of tamibarotene to the NACHT domain of NLRP3

[0085] Molecular docking simulation was used to calculate the binding conformation of tamibarotene and NLRP3, and the docking box was found. The results are shown in Figure 8 AB; The best binding conformation was selected for RMSD measurement to calculate the stability of the binding between Tamibarotene and NLRP3 protein. The results are shown in Figure 8 C; and according to the high and low energy values ​​of the docking sites, the docking sites were selected to construct site mutants, the mutant proteins were purified, and the ATPase activity detection kit was used to detect the effect of Tamibarotene on the ATPase activity of the site mutant proteins. The results are shown in Figure 8 DF; and the mutant plasmid was transfected into HEK293T cells, cell lysate proteins were extracted, and drug affinity reaction targeted stability technology was used to verify the binding site of tamibarotene with the NACHT domain of NLRP3. The results are shown in Figure 8 G.

[0086] Figure 8 AB results showed that molecular docking predicted that tamibarotene could bind to NLRP3, and the docking sites in the docking box were basically on the NACHT domain of NLRP3; Figure 8 C The results showed that the stability of Tamibarotene binding to NLRP3 protein was good; Figure 8 DF results showed that the NACHT domain of NLRP3 was formed by the combination of NBD, HD1, WHD, and HD2. The sites with the largest energy values ​​for molecular docking were IIe417 and Phe410. When mutants of the two sites were constructed and proteins were purified, the inhibitory effect of tamibarotene on the ATPase active site of NLRP3 disappeared. Figure 8 G The results showed that tamibarotene had no effect on the enzymatic stability of the mutant, indicating that tamibarotene directly binds to the IIe417 site and Phe410 site of the NACHT domain of NLRP3 and inhibits the activation of inflammasomes.

[0087] In summary, we found for the first time that tamibarotene can improve the cardiac function of mice with myocardial infarction, inhibit the infiltration of inflammatory cells and the degree of fibrosis in myocardial infarction tissue, and inhibit the activation of NLRP3 inflammasome in myocardial infarction myocardial tissue. It can be seen that tamibarotene has a significant preventive and therapeutic effect on myocardial infarction. Further research found that tamibarotene can directly target and bind to the NACHT domain of NLRP3, inhibit the ATPase activity of NLRP3, inhibit the assembly of NLRP3 inflammasome, and effectively prevent the activation of NLRP3 inflammasome. Inhibiting the activation of NLRP3 inflammasome is an important target for the prevention and treatment of myocardial infarction, which may be an important reason why tamibarotene has the effect of preventing and treating myocardial infarction.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of tamibarotene in the preparation of drugs for preventing and treating myocardial infarction.

2. The use according to claim 1, wherein: The application includes that tamibarotene inhibits the inflammatory cell infiltration and fibrosis degree of myocardial infarction tissue.

3. The use according to claim 1, wherein: The application includes Tamibarotene improving cardiac function in myocardial infarction.

4. The use according to any one of claims 1 to 3, wherein: The application includes that tamibarotene inhibits the activation of NLRP3 inflammasome in myocardial tissue of myocardial infarction.

5. The use according to any one of claims 1 to 3, wherein: The dosage form of the drug is injection or oral preparation.

6. The use according to claim 5, wherein: The oral preparation is a tablet, capsule, granule, pill, syrup, powder, paste, emulsion, solution or suspension.

7. The use according to claim 1, wherein: The applicable object or subject of the application is a mammal.

8. The use according to claim 1, wherein: The mammal is a human.

9. The use according to claim 1, wherein: The mammal is a mouse or a rat.

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