Medicine for treating coronary artery calcification or myocardial ischemia or infarction

Abstract

The invention relates to the technical field of biological medicine, in particular to application of an Orai1 protein inhibitor in preparation of a medicine for treating coronary artery calcification. By using the Orai1 protein inhibitor, coronary artery calcification can be accurately treated, the occurrence proportion of serious coronary artery calcification is reduced, and economic and social burdens brought by surgical treatment are relieved. Besides, the Orai1 protein inhibitor can also improve and inhibit acute myocardial ischemia symptoms caused by factors such as coronary vessel and myocardial cell calcification, calcium overload and the like, the myocardial infarction area is reduced, and death caused by acute myocardial ischemia is reduced.

Description

Technical Field

[0002] The present invention relates to the field of biomedicine technology, and more specifically, to the use of an Orai1 protein inhibitor in preparing a drug for treating coronary artery calcification or myocardial ischemia or infarction. Background Art

[0004] Coronary artery calcification refers to the process of calcium salt deposition in the coronary artery wall, which leads to hardening and narrowing of the wall, and eventually leads to stenosis and insufficient blood supply of the coronary artery, which is prone to clinical serious consequences such as angina pectoris and acute myocardial infarction. Epidemiological studies have shown that the incidence of coronary artery calcification increases with age, with the highest incidence in people aged 60 to 80, about 80%, and the incidence rate in men is higher than that in women in the same age group.

[0005] At present, the main method for treating coronary artery calcification is cardiovascular intervention. Among them, for mild to moderate calcified lesions, percutaneous coronary intervention is routinely used as the main treatment method; for moderate to severe calcified lesions and lesions that cannot be passed by balloons, coronary artery plaque atherectomy is used as the main treatment method.

[0006] The main clinical drug treatments for coronary calcification include: drugs that can alleviate the process of coronary atherosclerosis, such as simvastatin tablets, atorvastatin calcium tablets, ezetimibe tablets, etc., and drugs that can inhibit platelet aggregation, such as aspirin enteric-coated tablets, clopidogrel bisulfate tablets, etc. However, these drugs are all treatments for the causes of coronary calcification, not for the targets of coronary calcification. There is an urgent need for a precise therapeutic drug targeting the targets of coronary calcification. Therefore, exploring and excavating new prevention and treatment strategies is of great clinical significance for the control of vascular calcification.

[0007] Acute myocardial infarction (AMI), also known as myocardial infarction, is a life-threatening disease caused by acute blockage of the coronary arteries, which leads to insufficient blood supply to the corresponding myocardial area and myocardial necrosis. The disease is mainly caused by multiple factors such as insufficient blood supply, myocardial hypoxia and excessive myocardial oxygen consumption caused by various reasons such as coronary atherosclerosis, arrhythmia, respiratory failure, etc. Globally, the incidence and mortality of acute myocardial infarction are high, and it is more common in middle-aged and elderly people and high-risk groups such as those with heart disease, smoking, diabetes and hyperlipidemia.

[0008] The main symptoms of acute myocardial infarction include clinically obvious chest pain or a feeling of tightness and other symptoms of cardiac ischemia that last for more than 30 minutes, and patients often feel like they are dying. In the early stages of the pain attack, patients may also experience symptoms of heart failure such as chest tightness, shortness of breath, and coughing, and in severe cases, symptoms of shock may occur. Acute myocardial infarction is a non-communicable disease that is mainly caused by primary diseases such as cardiovascular diseases.

[0009] The main goal of treating acute myocardial infarction is to immediately clear the coronary arteries and slow down the progression of myocardial ischemia and myocardial infarction. Common treatments include drug therapy, such as the use of antiplatelet drugs, anticoagulants, lipid-lowering drugs, etc., and interventional and surgical treatments such as percutaneous coronary intervention and emergency coronary artery bypass grafting. The main way to prevent myocardial infarction is to control risk factors for heart disease such as hypertension, diabetes, and hyperlipidemia, and maintain a reasonable lifestyle, such as quitting smoking, exercising moderately, and eating a balanced diet. The prognosis of acute myocardial infarction varies depending on many factors, such as the timeliness of treatment and the extent of ischemic myocardium.

[0010] With reference to the acute myocardial infarction (AMI) treatment guidelines revised by the American College of Cardiology and the American Heart Association (ACC / AHA) in 1999, and combined with the specific situation in my country, it is divided into two categories according to the principle of clinical practicality: ST-segment elevation and non-ST-segment elevation. Among them, for the treatment of AMI patients with ST-segment elevation or left bundle branch block, in addition to general treatment and reperfusion therapy, drug treatment mainly includes 1. Nitrates: Commonly used nitrates include nitroglycerin, isosorbide dinitrate and 5-mononitrol. 2. Antiplatelet therapy: Coronary plaque rupture induces local thrombosis, which is the main cause of AMI. Platelet activation plays a very important role in acute thrombosis. Antiplatelet therapy has become a routine treatment for AMI and should be used before thrombolysis. Aspirin and ticlopidine or clopidogrel are currently commonly used antiplatelet drugs in clinical practice. 3. Anticoagulant therapy: Thrombin is the key link in converting fibrinogen into fibrin and ultimately forming thrombus, so inhibiting thrombin is crucial. The main drugs are ordinary heparin and low molecular weight heparin. 4. β-blockers: β-blockers reduce myocardial oxygen consumption by slowing heart rate, lowering systemic blood pressure and weakening myocardial contractility. They have a positive effect on improving the imbalance of oxygen supply and demand in the ischemic area, reducing the area of ​​myocardial infarction and reducing the mortality rate in the acute phase. The commonly used β-blocker is metoprolol. 5. Angiotensin converting enzyme inhibitors (ACEI): The main mechanism of action of ACEI is to reduce the incidence and mortality of filling heart failure by affecting myocardial remodeling and reducing ventricular overexpansion. 6. Calcium antagonists: Calcium antagonists are not used as first-line drugs in the treatment of AMI. Clinical trials have shown that whether it is early or late AMI, Q wave or non-Q wave myocardial infarction, or whether β-blockers are used in combination, the administration of fast-acting nifedipine cannot reduce the reinfarction rate and mortality rate, and is even harmful to some patients. Therefore, calcium antagonists are considered to be inappropriate drugs in the routine treatment of AMI. 7. Digitalis preparations: Digitalis preparations are generally not used within 24 hours of AMI. There has been controversy over whether it is beneficial to routinely take digitalis preparations 24 hours after AMI in patients with left heart failure. 8. Other symptomatic treatments.

[0011] The current treatment methods are mainly symptomatic treatment for myocardial ischemia and infarction, and prevention of inducing factors or treatment to slow down the progression of the disease. There is no precise treatment for the multiple effects of platelets, coronary artery smooth muscle cells and myocardial cell calcium overload targets. Therefore, it is of great clinical significance to explore new prevention and treatment strategies and provide new potential therapeutic drugs for the treatment of myocardial ischemia and infarction. Summary of the invention

[0013] In order to solve the above technical problems, the present invention provides a medicine for treating coronary artery calcification or myocardial ischemia or infarction.

[0014] The present invention provides the use of an Orai1 protein inhibitor in preparing a medicine for treating coronary artery calcification.

[0015] The present invention provides the use of an Orai1 protein inhibitor in preparing a medicine for treating myocardial ischemia or infarction.

[0016] Preferably, the Orai1 protein inhibitor is selected from synta66, MRS1845 or a combination thereof.

[0017] Preferably, the Orai1 protein inhibitor is used as the sole active ingredient in the preparation of the drug.

[0018] Preferably, the Orai1 protein inhibitor is used together with other active ingredients in the preparation of the medicine.

[0019] Preferably, the drug is selected from one or more of oral preparations or injections.

[0020] Preferably, the oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes.

[0021] Preferably, the injection is administered by one or more of subcutaneous administration, intramuscular administration or intravenous administration.

[0022] The present invention provides the use of Orai1 gene in regulating coronary artery calcification or myocardial ischemia or infarction.

[0023] The present invention also provides a method for inhibiting phosphate-induced calcification and calcium store-operated calcium influx in coronary artery smooth muscle cells, wherein the coronary artery smooth muscle cells are treated with an Orai1 protein inhibitor.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a new therapeutic target for vascular calcification. Orai1 protein inhibitors can be used to accurately treat coronary artery calcification, reduce the incidence of severe coronary artery calcification, and alleviate the economic and social burdens of surgical treatment.

[0026] The present invention provides a potential target and inhibitor for the treatment of myocardial ischemia and infarction, which is expected to provide precise treatment for the occurrence and development of myocardial ischemia and infarction in the future. The inhibitor targeting Orai1 can more effectively inhibit the occurrence and development of myocardial ischemia and infarction, alleviate or cure myocardial ischemia and infarction, reduce the incidence of severe myocardial ischemia and infarction, reduce the economic and social burden of surgical treatment, and reduce patient deaths caused by myocardial ischemia and infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 is a schematic diagram of the effect of phosphate on calcification of rat coronary artery smooth muscle cells according to an embodiment of the present invention;

[0030] Figure 2 is a schematic diagram of changes in Orai1 / STIM1 protein expression and its mediated store-operated calcium influx in phosphate-induced rat coronary artery smooth muscle calcification according to an embodiment of the present invention;

[0031] Figure 3 is a schematic diagram of the effect of inhibiting the expression of Orai1 protein on phosphate-induced rat coronary artery smooth muscle calcification according to an embodiment of the present invention;

[0032] Figure 4 is a schematic diagram of the effect of Synta66 on inhibiting Orai1 protein on phosphate-induced rat coronary artery smooth muscle calcification according to an embodiment of the present invention;

[0033] Figure 5 is a schematic diagram of the effect of MRS1845 on inhibiting the effect of Orai1 protein on phosphate-induced rat coronary artery smooth muscle calcification according to an embodiment of the present invention;

[0034] Figure 6 is a schematic diagram of the effect of Synta66 and MRS1845 inhibiting Orai1 protein on coronary artery calcification in rats according to an embodiment of the present invention;

[0035] Figure 7 is a schematic diagram of the effect of the Orai1 channel inhibitor on rat myocardial and coronary artery calcification according to an embodiment of the present invention;

[0036] Figure 8 is a schematic diagram of the effect of the Orai1 channel inhibitor on serum markers of myocardial ischemia in calcified rats according to an embodiment of the present invention;

[0037] Fig. 9 is a schematic diagram of the effect of the Orai1 channel inhibitor on the myocardial ischemic area of ​​SD rats according to an embodiment of the present invention;

[0038] Fig.10 Schematic diagram of the effect of the Orai1 channel inhibitor on the ischemic area of ​​the myocardium in calcified rats according to an embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be noted that the following embodiments do not limit the scope of protection claimed in the present invention and are only illustrative embodiments. The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0041] Current treatments for coronary artery calcification

[0042] Drug treatment: The formation of vascular calcification is a chronic inflammatory process in which activated macrophages promote osteoblast differentiation of vascular smooth muscle cells (VSMC) by producing proinflammatory cytokines such as interleukin 6 (IL-6), recombinant human interleukin-1β (human ILbete protein, IL-1β), tumor necrosis factor-α (TNF-α) and tumor suppressor factor (oncostatin M, OSM) in the intima and medial layers of the arterial wall. This process may be mainly mediated by the NF-κB signaling pathway. Vitamin K has been shown to exert anti-inflammatory effects by antagonizing NF-κB signaling, indicating that vitamin K can prevent vascular calcification through anti-inflammatory mechanisms. Matrix Gla protein (MGP) has the effect of inhibiting soft tissue calcification and helps prevent intimal calcification. Vitamin K enhances the activity of MGP by promoting γ-carboxylation of MGP, thereby slowing down the progression of vascular calcification. Other studies have shown that endothelial-to-mesenchymal transition (EndoMT) drives endothelial-promoted vascular calcification in diabetes, and glycogen synthase kinase-3β (GSK-3β) inhibition induces β-catenin, directing osteoblast-like cells to the endothelial lineage, thereby reducing vascular calcification in MGP deficiency.

[0043] Interventional treatment: Traditional percutaneous coronary intervention (PCI) technology is a common way to treat calcified lesions in clinical practice, but with the development of intravascular imaging technology, IVUS and OCT are completely changing the treatment of CAC. Currently, surgeries for treating calcified lesions can be divided into two categories: ablation and balloon-based techniques. The former includes rotational atherectomy (RA), orbital atherectomy (OA) and excimer laser, which are designed to ablate calcification and promote stent expansion. The latter includes cutting balloons, ultra-high pressure balloons, lithotripsy balloons, etc. Balloon-based techniques do not make calcification disappear, but are designed to increase plaque elasticity or split regional calcification to allow stent expansion. The information obtained from intravascular imaging will enable the operator to choose the most appropriate technology to treat calcified lesions. When the degree of calcification is moderate, the operator can use balloon-based techniques for lesion preparation, and increasingly use lithotripsy balloons for lesion preparation. In contrast, higher levels of calcification may still require more aggressive lesion preparation using ablative techniques such as RA or OA.

[0044] It should be noted that the Orai1 protein inhibitor used in the present invention is not limited to Synta66, MRS1845, and Orai1-specific small interfering RNA, and any substance that can inhibit the Orai1 protein is included in the protection scope of the present invention.

[0045] Example 1 Extraction and cultivation of primary rat coronary smooth muscle cells

[0046] Step S1: Take out 50 mL of fetal bovine serum (FBS) and penicillin-streptomycin mixed solution (P / S) from a -80°C refrigerator in advance and thaw at 4°C;

[0047] Step S2: The biosafety cabinet in the cell operation room was sterilized by ultraviolet for 20 min. After sterilization, the DMEM medium, FBS and P / S were taken out from the 4°C refrigerator and sterilized by spraying the surface with 75% alcohol;

[0048] Step S3: Prepare complete culture medium (20% DMEM culture medium), DMEM high glucose culture medium + 20% FBS + 1% P / S according to the following formula in a biosafety cabinet; mix well by pipetting, seal, and store in a refrigerator at 4°C for later use;

[0049] Step S4: Prepare sterile surgical instruments in advance, place the sterilized surgical instruments, culture dishes, and centrifuge tubes in a biosafety cabinet, and irradiate with ultraviolet light for 20 minutes;

[0050] Step S5: Kill clean male Sprague-Dawley (SD) rats weighing about 200 g with CO2, spray the rat's chest and abdomen with alcohol, carefully open the chest and abdomen, take out the heart, soak it in PBS, wash it three times, and then put it on a stereo microscope to fix the heart, separate the coronary artery under the microscope, and carefully peel off the myocardial tissue around the coronary artery under the stereo microscope with sterilized ophthalmic scissors and ophthalmic forceps;

[0051] Step S6: After the myocardial tissue is peeled off, the coronary artery is taken out and washed 3 times with PBS. After spraying with alcohol, it is taken to the clean bench and washed again with PBS 3 times. The coronary artery is placed in complete culture medium and cut into pieces with sterilized ophthalmic scissors and added to a 6-well plate. The culture medium is discarded to allow the tissue pieces to adhere to the wall. After 1 hour, complete culture medium is added;

[0052] Step S7: Culture in a 37°C, 5% CO2 incubator, observe the adhesion after 3 days, change the culture medium every two days, and culture in bottles after 10 days.

[0053] Example 2 Detection of the role of Orai1 / STIM1 in coronary artery smooth muscle cell calcification and store-operated calcium influx

[0054] In this example, the primary cultured rat vascular smooth muscle cells obtained in Example 1 were subjected to phosphate calcification treatment for 0 days, 5 days, 7 days, and 10 days. The cells after phosphate calcification treatment were subjected to Alizarin red staining, protein blotting experiments, and calcium imaging experiments. The results are as follows: Figure 1 , Figure 2 shown.

[0055] The steps of phosphate calcification treatment are as follows:

[0056] Step S101: Preparation of 2% calcification solution: accurately weigh a certain amount of Na2HPO4 and NaH2PO4 respectively in DMEM culture medium, and make the final concentration of both to be 3mmol / L according to the ratio of 1:1, filter and sterilize, add 2% FBS and 1% P / S, mix by pipetting, tighten the bottle cap, seal, and store in a refrigerator at 4°C for later use;

[0057] Step S102: Plant the cells in a 12-well plate, and when the cell density reaches 70%, establish a calcification model;

[0058] Step S103: discard the complete culture medium, wash once with sterile PBS, add 2% DMEM culture medium to the control group, add 2% calcification culture medium to the experimental group, place in a 37°C, 5% CO2 incubator, change the medium every two days, and observe cell morphology changes every day.

[0059] The steps of alizarin red blood cell staining are as follows:

[0060] Step S201: before staining the cells, fix them with 4% paraformaldehyde for 45 min and discard the fixative;

[0061] Step S202: add 1 mL of Alizarin Red and dye at room temperature for 30 min, discard the dye solution, and wash three times with acidic PBS to wash away the floating color;

[0062] Step S203: placing the well under a stereo microscope to take a picture, and after the picture is taken, adding 10% acetic acid solution to the well;

[0063] Step S204: aspirate the supernatant and measure the absorbance at 405 nm for relative quantitative analysis of changes in calcium salt deposition.

[0064] The steps of the Western blot experiment are as follows:

[0065] Step S301: add an appropriate amount of RIPA buffer lysis solution (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other protease inhibitors) to each group of primary cultured cells, and place them on ice for lysis for 20 minutes;

[0066] Step S302: centrifuging the lysate at 12000 rpm and 4°C for 20 min, and discarding the precipitate;

[0067] Step S303: adding loading buffer, boiling the sample, loading the sample, performing SDS-PAGE gel electrophoresis, and then transferring the SDS-PAGE gel protein to the PVDF membrane using a wet transfer method;

[0068] Step S304: incubate the PVDF membrane transferred with the target protein with primary antibodies of various proteins (1:250) at 4°C overnight;

[0069] Step S305: Incubate with horseradish peroxidase (HRP) labeled secondary antibody at room temperature for 1 hour, and detect protein content by luminescent agent ECL. The optical density of each blot is normalized with the corresponding internal reference β-tubulin.

[0070] The calcium imaging experiment steps are as follows:

[0071] Step S401: Incubate each group of cells with a calcium fluorescent indicator Fluo-8 AM (10 μM) at 37°C for 40 min. Fluo-8 is the brightest visible light excitation wavelength for Ca 2+ Fluorescent probe, Fluo-8 AM is an acetyl methyl ester derivative of Fluo-8, which is cell membrane permeable and can easily enter cells with simple incubation;

[0072] Step S402: After taking out the cell slide, carefully wash it three times with calcium-free buffer (OPSS (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 5 mM HEPES, pH 7.4)), and place it on a microscope of a fluorescent calcium imaging system;

[0073] Step S403: Setting the fluorescence microscope to 488 nm excitation light and 514 nm emission light, and collecting fluorescence signals using a calcium imaging recording system;

[0074] Step S404: After stimulating the cells with thapsigargin (2 μM) for 20 minutes, 2 mM calcium ions were added to the extracellular fluid to induce store-operated calcium influx, and the changes in intracellular calcium concentration were measured. The differences in the increase of intracellular calcium in each group were compared to determine the changes in the intensity of store-operated calcium influx.

[0075] in, Figure 1 Schematic diagram of the effect of phosphate on calcification of rat coronary artery smooth muscle cells. Figure 1 A is the image of rat vascular smooth muscle cells under a microscope after being treated with phosphate calcification for different periods of time and stained with Alizarin red. Figure 1 B is a schematic diagram of the changes in calcium salt deposition in rat vascular smooth muscle cells after phosphate calcification treatment for different days, obtained by using the absorbance of calcium salts. The ordinate OD value (optical density) represents the absorbance of a substance at a specific wavelength. In analytical chemistry, a chemical substance can absorb light of a certain wavelength, and the absorbance of light is proportional to the concentration of the chemical substance. Therefore, the absorbance can be used to determine the concentration of a substance. Figure 1 C is the content of OPN, Runx2 and β-tubulin proteins detected by the luminescent agent ECL. Figure 1 D is the histogram of the relative expression of OPN relative to its internal reference β-tubulin protein. Figure 1 E is the histogram of the relative expression of Runx2 relative to its internal reference β-tubulin protein. Figure 2 Schematic diagram of the changes in Orai1 / STIM1 protein expression and its mediated store-operated calcium influx in phosphate-induced rat coronary artery smooth muscle calcification. Figure 2 A is the content of Orai1, Orai2, Orai3, STIM1, and β-tubulin proteins detected by the luminescent agent ECL. Figure 2 B is the histogram of the relative expression of Orai1 relative to its internal reference β-tubulin protein. Figure 2 C is the histogram of the relative expression level of Orai2 relative to its internal reference β-tubulin protein. Figure 2 D is the histogram of the relative expression of Orai3 relative to its internal reference β-tubulin protein. Figure 2 E is the histogram of the relative expression of STIM1 relative to its internal reference β-tubulin protein. Figure 2 F is a schematic diagram of the calcium store-operated calcium entry curve of rat vascular smooth muscle cells after phosphate calcification treatment for different days. Figure 2 G is the relative value of calcium signal of rat vascular smooth muscle cells after phosphate calcification treatment for different days.

[0076] like Figure 1As shown in AB, the degree of calcification shown by Alizarin red staining increases with time; Figure 1 As shown in CE, the expression levels of OPN and Runx2, marker proteins of cell calcification, were significantly increased. Figure 2 As shown in AE, after 10 days of cell calcification treatment, the expression of Orai1 and STIM1 proteins was significantly enhanced, while the expression of Orai2 and Orai3 proteins was significantly reduced. Figure 2 As shown in FG, calcification also caused a significant enhancement in store-operated calcium influx.

[0077] Example 3 Detection of the effects of Orai1 siRNA, Synta66, and MRS1845 on calcification and store-operated calcium influx in coronary artery smooth muscle cells

[0078] In this example, Orai1 specific small interfering RNA (Orai1 siRNA) was used to inhibit the expression of Orai1 in cells, and Alizarin red staining, protein imprinting experiment, and calcium imaging experiment were performed on them respectively. The rat vascular smooth muscle cells in this example were the primary cultured rat vascular smooth muscle cells obtained in Example 1, and the phosphate calcification treatment, Alizarin red staining, protein imprinting experiment, and calcium imaging experiment steps were the same as those in Example 2. The results are shown in FIG. Figure 3 shown.

[0079] in, Figure 3 Schematic diagram of the effect of inhibiting Orai1 protein expression on phosphate-induced rat coronary artery smooth muscle calcification. Figure 3 A is the content of Orai1 and β-tubulin proteins detected by the luminescent agent ECL using Orai1 siRNA and Scrambled siRNA respectively. Figure 3 B is the histogram of the relative expression levels of Orai1 using Orai1 siRNA and Scrambled siRNA respectively relative to its internal reference β-tubulin protein. Figure 3 C is the image under the microscope of rat vascular smooth muscle cells treated with Orai1 siRNA and Scrambled siRNA for different periods of time and stained with Alizarin red. Figure 3 D is a schematic diagram of the changes in calcium salt deposition in rat vascular smooth muscle cells using Orai1 siRNA and Scrambled siRNA respectively, obtained using the absorbance of calcium salt. Figure 3 E is the content of OPN, Runx2 and β-tubulin proteins detected by the luminescent agent ECL using Orai1 siRNA and Scrambled siRNA respectively. Figure 3 F is the histogram of the relative expression of OPN relative to its internal reference β-tubulin protein. Figure 3 G is the histogram of the relative expression of Runx2 relative to its internal reference β-tubulin protein. Figure 3 H is a schematic diagram of the store-operated calcium entry curve of rat vascular smooth muscle cells without phosphate calcification treatment. Figure 3 I Relative value of calcium signal in rat vascular smooth muscle cells without phosphate calcification treatment. Figure 3 J is a schematic diagram of the store-operated calcium entry curve of rat vascular smooth muscle cells after phosphate calcification treatment for 10 days. Figure 3 Relative values ​​of calcium signals in K rat vascular smooth muscle cells treated with phosphate calcification for 10 days.

[0080] like Figure 3 As shown in AB, Orai1 siRNA significantly inhibited the expression of Orai1 protein in primary cultured rat coronary artery smooth muscle. More importantly, Figure 3 As shown in CG, the results of Alizarin red staining and OPN and Runx2 marker protein expression also showed that Orai1 siRNA transfection also significantly inhibited phosphate-induced coronary artery smooth muscle cell calcification. Figure 3 As shown by HK, the results of intracellular calcium imaging experiments showed that Orai1 siRNA transfection also significantly inhibited calcium store-operated calcium influx in both calcified solution-treated and control cells.

[0081] Next, this example uses two Orai1 protein specific inhibitors Synta66 and MRS1845 to treat cells, and performs Alizarin red staining, protein blotting, and calcium imaging experiments on them. Similarly, the rat vascular smooth muscle cells in this example use the primary cultured rat vascular smooth muscle cells obtained in Example 1, and the phosphate calcification treatment, Alizarin red staining, protein blotting, and calcium imaging experimental steps are the same as those in Example 2. The results are shown in FIG. Figure 4 and Figure 5 shown.

[0082] in, Figure 4 Schematic diagram of the effect of Synta66 on inhibiting Orai1 protein on phosphate-induced rat coronary artery smooth muscle calcification. Figure 4 A is the image under the microscope of rat vascular smooth muscle cells treated with Synta66 and without Synta66 after phosphate calcification for different time periods and stained with Alizarin red. Figure 4 B is a schematic diagram of the changes in calcium salt deposition in rat vascular smooth muscle cells using Synta66 and not using Synta66, obtained using the absorbance of calcium salt. Figure 4 C is the content of OPN, Runx2 and β-tubulin proteins detected by the luminescent agent ECL with and without Synta66. Figure 4 D is the histogram of the relative expression of OPN relative to its internal reference β-tubulin protein. Figure 4 E is the histogram of the relative expression of Runx2 relative to its internal reference β-tubulin protein. Figure 4 F is a schematic diagram of the calcium store-operated calcium entry curves of rat vascular smooth muscle cells treated with Synta66 and without Synta66 after 10 days of phosphate calcification. Figure 4 G Relative values ​​of calcium signals in rat vascular smooth muscle cells treated with Synta66 and without Synta66 after 10 days of phosphate calcification.

[0083] Figure 5 Schematic diagram of the effect of MRS1845 on inhibiting Orai1 protein on phosphate-induced rat coronary artery smooth muscle calcification. Figure 5 A is the image of rat vascular smooth muscle cells treated with MRS1845 and without MRS1845 under a microscope after phosphate calcification treatment for different periods of time and stained with Alizarin red. Figure 5 B is a schematic diagram of the changes in calcium salt deposition in rat vascular smooth muscle cells using MRS1845 and not using MRS1845, obtained using the absorbance of calcium salt. Figure 5 C is the content of OPN, Runx2 and β-tubulin proteins detected by the luminescent agent ECL with and without MRS1845. Figure 5 D is the histogram of the relative expression of OPN relative to its internal reference β-tubulin protein. Figure 5 E is the histogram of the relative expression of Runx2 relative to its internal reference β-tubulin protein. Figure 5 F is a schematic diagram of the calcium store-operated calcium entry curves of rat vascular smooth muscle cells treated with MRS1845 and without MRS1845 after 10 days of phosphate calcification. Figure 5 G Relative values ​​of calcium signals in rat vascular smooth muscle cells treated with MRS1845 and without MRS1845 after 10 days of phosphate calcification.

[0084] like Figure 4 , Figure 5 As shown, treatment of cells with Orai1 protein-specific inhibitors Synta66 and MRS1845 had similar effects to those with Orai1 siRNA, and also significantly inhibited phosphate-induced coronary artery smooth muscle cell calcification and calcium store-operated calcium influx.

[0085] Example 4 Detection of the effects of Synta66 and MRS1845 on coronary artery calcification in a rat model of coronary artery calcification

[0086] In this example, a rat coronary artery calcification animal model was prepared, and the operation steps are as follows:

[0087] Step S201: Select clean-grade male Sprague-Dawley (SD) rats, 8 weeks old, about 220 g, all rats are fed with regular feed and purified water, and feed, drinking water and bedding are changed regularly. Each group of animals is randomly assigned in the experiment.

[0088] Step S201: Forty male SD rats aged 8 weeks were randomly divided into four groups: a calcification group (VC group), a control group (CON group), a calcification + MRS1845 group (VC+MRS1845 group), and a calcification + Synta66 group (VC+Synta66 group). In the VC group, nicotine was dissolved in peanut oil (25 mg / kg) and intragastrically administered once at 8:00 am and 6:00 pm on the first day, and no intragastrically administered was continued in the later period; vitamin D3 was dissolved in anhydrous ethanol (300,000 U / kg) and intramuscularly injected once a day for 7 consecutive days, and then the rats were allowed to eat and drink freely, and the model was formed in 21 days. ; CON group: the same amount of anhydrous ethanol was injected intramuscularly at the same time, and peanut oil was gavaged; VC+MRS1845 group: MRS1845 30mg / kg was given by intraperitoneal injection on the basis of the same method as the calcification group, once every other day for 21 days; VC+Synta66 group: Synta66 30mg / kg was given by intraperitoneal injection on the basis of the calcification model, once every other day for 21 days. All rats were fed with regular feed and purified water, and feed, drinking water and bedding were changed regularly.

[0089] The rats obtained by the above steps were subjected to Alizarin red staining and immunohistochemistry experiments. The results are as follows: Figure 6 .

[0090] The steps for Alizarin Red animal blood vessel staining are as follows:

[0091] Step S501: After the animal modeling is completed, the rats are killed by CO2, the entire coronary artery is removed, and fixed with 4% paraformaldehyde overnight;

[0092] Step S502: On the next day, the coronary artery is stained with Alizarin Red (0.003% Alizarin Red solution), the solvent is 1% KOH, and the staining solution is left overnight.

[0093] Step S503: The next day, the tissue was rinsed twice with 2% KOH solution and placed under a stereo microscope for photography.

[0094] The immunohistochemistry experimental steps are as follows:

[0095] Step S601: fix the arterial tissues of each group with 4% paraformaldehyde at 4°C for 24 hours and then embed them in wax blocks;

[0096] Step S602: The pathological slicer cuts the blood vessel tissue longitudinally into thin slices with a thickness of 6 μm and sticks them on a glass slide for later use;

[0097] Step S603: dewaxing and slicing the tissue, blocking with 0.3% H2O2, washing with water and PBS 3 times (5 min interval each time), and blocking at room temperature for 30 min;

[0098] Step S604: Incubate with primary antibody at 4°C overnight, remove on the next day and wash three times with PBS;

[0099] Step S605: adding horseradish peroxidase-labeled secondary antibody (at a ratio of 1:100), incubating at room temperature for 1 hour, washing 3 times with PBS, adding ABC complex dropwise, incubating at room temperature for 1 hour, washing 3 times, and developing with DAB-H2O2;

[0100] Step S606: After washing, staining the cell nucleus with hematoxylin, dehydration, transparency and sealing, an Olympus BX51 upright microscope was used to take pictures and analyze.

[0101] in, Figure 6 Schematic diagram of the effect of Synta66 and MRS1845 inhibiting Orai1 protein on coronary artery calcification in rats. Figure 6 A is a microscope image of rat coronary artery stained with Alizarin red. Figure 6 B is the immunohistochemical microscope images of the coronary arteries of the four groups of rats. Figure 6 C is the histogram of the average cell area of ​​Orai1 protein. Figure 6 D is the histogram of the mean cell area of ​​STIM1 protein. Figure 6 E is the immunohistochemical microscope images of the coronary arteries of the four groups of rats. Figure 6 F is the average cell area histogram of Runx2 protein. Figure 6 As shown in A, the coronary arteries of rats in the model group showed typical calcification, and Alizarin red staining was significantly enhanced. More importantly, Alizarin red staining of the coronary arteries of rats in the Synta66 and MRS1845 treatment groups was significantly weakened. Figure 6 As shown in BD, the expression of Orai1 and STIM1 in coronary artery smooth muscle of rats in the model group was significantly increased. Figure 6 As shown in EF, the calcification marker protein Runx2 was significantly increased in the model group, but not in the Synta66 and MRS1845 treatment groups.

[0102] It was concluded from Experimental Examples 2-4 that the expression of Orai1 and STIM1 proteins increased significantly during the calcification of coronary artery smooth muscle, and the calcium store-operated calcium influx mediated by them was significantly enhanced. Inhibition of Orai1-mediated calcium store-operated calcium influx can significantly inhibit phosphate-induced coronary artery calcification and nicotine-induced coronary artery calcification in animals.

[0103] According to the disclosure of the present invention, coronary artery smooth muscle cells can be treated with an Orai1 protein inhibitor to inhibit phosphate-induced coronary artery smooth muscle cell calcification and calcium store-operated calcium influx.

[0104] The present invention provides a new therapeutic target for vascular calcification. By using Orai1 inhibitors to achieve precise treatment of the occurrence and development of coronary artery calcification, it is possible to more effectively inhibit the occurrence and development of coronary artery calcification, alleviate or cure coronary artery calcification, reduce the incidence of severe coronary artery calcification, and alleviate the economic and social burdens brought by surgical treatment.

[0105] Current treatment options for acute myocardial infarction (AMI)

[0106] The current treatment methods are mainly symptomatic treatment for myocardial ischemia and infarction and prevention of inducing factors or treatment to slow down the progression of the disease, but there is no precise treatment for the multiple effects of platelets, coronary smooth muscle cells and myocardial calcium overload targets at the same time. Therefore, the purpose of the present invention is to find the common targets on platelets, coronary smooth muscle cells and myocardial cells that cause myocardial ischemia and infarction from the source. Thereby providing more effective potential treatment methods for clinical treatment, providing new drug screening targets for drug discovery, and providing new potential therapeutic drugs for the treatment of myocardial ischemia and infarction.

[0107] Example 5

[0108] (1.1) Experimental animals

[0109] Clean grade male Sprague-Dawley (SD) rats, 8 weeks old, about 220 g, all rats were fed with regular feed and purified water, and feed, drinking water and bedding were changed regularly. Animals in each group were randomly assigned in the experiment.

[0110] (1.2) Construction of animal model of coronary artery calcification

[0111] Forty male SD rats aged 8 weeks were randomly divided into four groups: calcification group (VC group), control group (CON group), calcification + Synta66 group (VC + Synta66 group). VC group: VD3 dissolved in anhydrous ethanol (300,000 U / kg) was intramuscularly injected on the first day, and nicotine dissolved in peanut oil (25 mg / kg) was gavaged once each. CON group: The same amount of anhydrous ethanol was injected intramuscularly and peanut oil was gavaged at the same time. VC + Synta66 group: Synta66 30 mg / kg was given on the basis of the calcification model; VC + Nifedipine group: Nifedipine 0.2 mg / kg was given on the basis of the calcification model. The above three drugs were all injected intraperitoneally, once every other day, for 21 days (7 times). All rats were fed with regular feed and purified water, and feed, drinking water and bedding were changed regularly.

[0112] (1.3) Construction of animal model of acute myocardial ischemia

[0113] (a) Construction of acute myocardial ischemia model in normal rats: Forty male 8-week-old SD rats were randomly divided into five groups: Control group, ischemia group, ischemia+Synta66 group, and ischemia+Nifedipine group. All rats were fed with conventional feed and purified water, and feed, drinking water and bedding were changed regularly. Subsequent experiments were conducted after one week of adaptation. The ischemia+Synta66 group was intraperitoneally injected with 10 mg / kg Synta66, the ischemia+Nifedipine group was intraperitoneally injected with 0.2 mg / kg Nifedipine, and the Control group and the ischemia group were injected with the same dose of solvent. At the same time, each group was intraperitoneally injected with 80 mg / kg isoproterenol hydrochloride. After 24 hours, each group was intraperitoneally injected with the same dose of isoproterenol hydrochloride again, and the Control group was injected with the same dose of solvent for dissolving isoproterenol hydrochloride. After the modeling, blood and tissue were collected from the abdominal vein for subsequent experiments.

[0114] (b) Construction of acute myocardial ischemia model in calcified rats: The rats were divided into an ischemia control group (Control) and a coronary artery calcification group (VC), and then the rats with coronary artery calcification model were randomly divided into four groups: calcification ischemia group (VC+MI), calcification ischemia + Synta66 group (VC+MI+Synta66), calcification ischemia + Nifedipine group (VC+MI+Nifedipine); among them, VC+MI+Synta66 group: Synta66 (10 mg / kg) was given on the basis of the calcification model; VC+MI+Nifedipine group: Nifedipine (0.2 mg / kg) was given on the basis of the calcification model. The above three drugs were all injected intraperitoneally, once every other day, for 21 days (7 times). All rats were fed with regular feed and purified water, and feed, drinking water and bedding were changed regularly. After the coronary artery calcification model was established, each group was intraperitoneally injected with 80 mg / kg isoproterenol hydrochloride. 24 hours later, each group was intraperitoneally injected with the same dose of isoproterenol hydrochloride again. After the model was established, blood was collected from the abdominal vein, and blood vessels and heart tissues were taken for subsequent experiments.

[0115] (1.4) Alizarin red staining of tissue sections

[0116] Before staining, the tissue was first dehydrated and embedded in paraffin. After sectioning, the sections were dewaxed to water, and the sections were placed in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, and anhydrous ethanol II for 5 minutes, and rinsed with running water; dyed with 0.003% alizarin red for 5 minutes, rinsed with running water, and then placed in an oven to dry; finally, the sections were placed in clean xylene for 5 minutes to make them transparent, and sealed with neutral gum; and photographed under a microscope.

[0117] (1.5) Electrocardiogram test

[0118] After anesthetizing the rats with myocardial infarction with anesthetics, the rats were fixed on a splint with the abdomen facing upwards, and the leads were fixed on the rats' subcutaneous limbs, and an anti-interference wire was clamped on the splint; after the leads were connected, the multi-channel physiological signal recording software (BL-420S physiological signal recording system, Chengdu Taimeng Software Co., Ltd., China) was turned on to record the electrocardiogram and detect the ST segment elevation; after the detection, a section containing three ST segment elevations was selected, the values ​​of the lowest points on both sides of the P wave were read and the average value was calculated, the value of the starting point of the ST segment was read, the average value of both sides of the P wave was subtracted from the value of the starting point of the ST segment, and the values ​​of the three ST segment elevations were read and the average value was the ST segment elevation value of this experiment.

[0119] (1.6) TTC staining of myocardial tissue

[0120] After the rats were anesthetized, the heart was quickly removed, washed twice with physiological buffer (PBS), and placed in a -20℃ refrigerator for 30 minutes; the heart was removed and evenly cut into three slices. After the heart slices returned to room temperature, they were placed in PBS containing 1% TTC (2,3,5-Triphenyltetrazolium chloride) and the tissue was placed in a 37℃ constant temperature box to avoid light. After 20 minutes, the heart was removed and laid flat on the table for photography. Image-J software was used to measure and analyze the area of ​​stained and unstained areas. The red area was the non-infarct area, and the white area was the infarct area. The larger the infarct area / non-infarct area, the more severe the infarction.

[0121] (1.7) Enzyme-linked immunosorbent assay

[0122] Blood drawn from the inferior vena cava was placed in a tube without anticoagulant for 1.5 h and centrifuged at 2000 rpm for 10 min at room temperature. The supernatant was diluted with an appropriate proportion of phosphate buffer to optimize the detection. According to the instructions, the concentrations of lactate dehydrogenase (LDH) and troponin I (cTnI) in rat plasma were detected using an ELISA kit (JL13014, Jiangsu Jianglai Biotechnology Co., Ltd., China), and a standard curve was established by linear regression for calculation.

[0123] (1.8) Serum calcium and alkaline phosphatase (ALP) testing

[0124] Serum calcium test: Calcium ions in the sample combine with methylthymol blue (MTB) in an alkaline solution to form a blue complex; the calcium content in the sample can be calculated by colorimetric comparison with the calcium standard treated in the same way.

[0125] (a) Serum operation steps

[0126] (b) Calculation formula: Calcium content in sample = (A 测定 -A 空白 ) / (A 标准 -A 空白 )x C 标准 x N

[0127] Serum alkaline phosphatase test: Alkaline phosphatase decomposes disodium phenyl phosphate to produce free phenol and phosphoric acid. Phenol reacts with 4-aminoantipyrine in an alkaline solution and is oxidized by potassium ferrocyanide to form a red quinone derivative. The enzyme activity can be determined by the depth of the red color.

[0128] (a) Sample preparation: Serum samples were diluted 5 or 10 times with saline before use.

[0129] (b) Operation table

[0130] (c) Calculation formula: Liquid sample ALP activity = (A 测定 -A 空白 ) / (A 标准 -A 空白 )x C 标准 x 100xN

[0131] (1.9) Statistical analysis

[0132] All values ​​are expressed as mean ± standard error. Comparisons between two groups were analyzed by t-test. Comparisons between multiple groups (>2 groups) were analyzed by one-way analysis of variance. Two-sided P values ​​< 0.05 were considered statistically significant.

[0133] (2) Results

[0134] (2.1) Orai1 channel inhibitor significantly inhibits myocardial and coronary artery calcification in rats

[0135] The experiment used SD rats to prepare a coronary artery calcification model. After the model was established, the rats were killed and the heart and coronary artery tissues were removed for alizarin red staining. The results showed that the alizarin red staining of the myocardial tissue of the calcification group animals was significantly enhanced, indicating that the tissue had calcified ( Figure 1 A). Serum calcium concentration (Ca 2+ ) and alkaline phosphatase (ALP) concentrations also showed that the blood Ca 2+ and ALP concentrations were significantly increased. More importantly, both increases were significantly inhibited by intraperitoneal injection of the Orai1 inhibitor Synta66 ( Figure 1 BC). The results of Alizarin red staining of coronary artery tissue also showed that the Alizarin red staining of coronary artery tissue in the calcification group was significantly enhanced, indicating that significant calcification occurred in the coronary artery tissue and could be significantly inhibited by intraperitoneal injection of Synta66. However, intraperitoneal injection of Nifedipine, a calcium channel blocker and positive control drug for the treatment of myocardial ischemia, did not significantly inhibit coronary artery calcification. There was a significant difference between the Synta66 and Nifedipine groups ( Figure 1 DE).

[0136] (2.2) Orai1 channel inhibitor significantly reduces serum marker concentrations of calcified rats with acute myocardial ischemia

[0137] Orai1 protein inhibitors are selected from Synta66, MRS1845, Orai1 specific small interfering RNA or their combination. The experiment used SD rats, coronary artery calcification and acute myocardial ischemia model rats, which were killed after modeling and venous blood was taken for Elisa detection. The results showed that compared with the normal rat (Control) myocardial ischemia model, the serum concentrations of myocardial infarction markers lactate dehydrogenase (LDH) and troponin I (cTnI) in the calcification group myocardial ischemia rats were significantly increased. More importantly, the increase of both was significantly inhibited by intraperitoneal injection of the Orai1 inhibitor Synta66 ( Figure 2 ). Moreover, it can also be significantly inhibited by intraperitoneal injection of Nifedipine, a calcium channel blocker for the treatment of myocardial ischemia and a positive control drug ( Figure 2 ), and there was no significant difference between the Synta66 and Nifedipine groups.

[0138] (2.3) Orai1 channel inhibitor significantly inhibited the acute myocardial ischemia area in normal and calcified rats

[0139] Orai1 protein inhibitors are selected from Synta66, MRS1845, Orai1 specific small interfering RNA or their combination. The experiment used SD rats, coronary artery calcification and acute myocardial ischemia model rats, which were killed after modeling, and the hearts were removed for TTC staining. The results showed that in normal SD rats, myocardial infarction group showed obvious myocardial tissue infarction. More importantly, intraperitoneal injection of Synta66 significantly reduced the infarct area, while intraperitoneal injection of calcium channel blocker and positive control drug Nifedipine for the treatment of myocardial ischemia did not significantly reduce the infarct area. There was a significant difference between the Synta66 and Nifedipine groups ( Figure 3 ); In calcified SD rats, the myocardial infarction group also showed obvious myocardial tissue infarction. More importantly, intraperitoneal injection of Synta66 significantly reduced the infarct area. At the same time, intraperitoneal injection of Nifedipine also significantly reduced the infarct area. However, Synta66 showed a stronger effect than Nifedipine, and there was a significant difference between the two groups ( Figure 4 ).

[0140] (3) Conclusion

[0141] The above results show that the Orai1 channel inhibitor Synta66 can significantly inhibit acute myocardial infarction and show a stronger effect in the calcification model. Orai1 protein inhibitors can be used to prepare drugs for the treatment of myocardial ischemia or infarction.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of Orai1 protein inhibitors in the preparation of drugs for the treatment of coronary artery calcification.

2. Application of Orai1 protein inhibitors in the preparation of drugs for treating myocardial ischemia or infarction.

3. The use according to claim 1 or 2, characterized in that: The Orai1 protein inhibitor is selected from Synta66, MRS1845, Orai1-specific small interfering RNA or a combination thereof.

4. The use according to claim 1 or 2, characterized in that: The Orai1 protein inhibitor is used as the sole active ingredient in the preparation of the medicine.

5. The use according to claim 1 or 2, characterized in that: The Orai1 protein inhibitor is used together with other active ingredients in preparing the medicine.

6. The use according to claim 1 or 2, characterized in that: The drug is selected from one or more of oral preparations or injections.

7. The use according to claim 6, characterized in that: The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes.

8. The use according to claim 6, characterized in that: The injection is administered by one or more of subcutaneous administration, intramuscular administration or intravenous administration.

9. Use of the Orai1 gene in regulating coronary artery calcification or myocardial ischemia or infarction.

10. A method for inhibiting phosphate-induced coronary artery smooth muscle cell calcification and store-operated calcium influx, characterized in that: The coronary artery smooth muscle cells were treated with an Orai1 protein inhibitor.

Images