Use of atrophin d2 in preparation of a drug for alleviating and / or treating myocardial infarction and its related diseases

Abstract

The application discloses application of a resolution D2 in preparation of a medicine for relieving and / or treating myocardial infarction and related diseases, and belongs to the technical field of medicines. The application finds that the resolution D2 has the effects of improving myocardial infarction and reducing myocardial remodeling after the infarction, and the resolution D2 can be used as an active ingredient to prepare the medicine for relieving and / or treating myocardial infarction and related diseases. The application finds the function of the resolution D2 in improving myocardial remodeling after myocardial infarction, and provides a target for developing a new medicine for relieving and / or treating myocardial infarction and related diseases.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, and application of a small molecule substance, resolution D2, in preparation of a medicine for relieving and / or treating myocardial infarction and related diseases. BACKGROUND

[0002] Myocardial infarction (MI for short) refers to persistent ischemia and hypoxia of myocardial cells caused by acute reduction or interruption of coronary artery blood flow, which eventually leads to irreversible necrosis of myocardial cells and deterioration of heart function. Myocardial infarction is one of the main causes of heart failure in clinical practice, and has characteristics such as high morbidity, high disability rate and high mortality, which leads to heavy disease burden, economic burden and social burden. Pathological myocardial remodeling after myocardial infarction is the main pathological process of myocardial infarction, which can lead to malignant arrhythmia, heart failure and even sudden cardiac death. Therefore, finding new intervention targets for pathological myocardial remodeling will help improve the prognosis of patients with myocardial infarction.

[0003] Resolution D2 (RvD2 for short) is one of the members of specific pro-resolution lipid mediators, and belongs to a small molecule substance, which is generated by metabolism of polyunsaturated fatty acid docosahexaenoic acid (DHA for short). The substrate DHA is metabolized by 12 / 15-LOX to generate an intermediate product 7-epoxy hydrogen-17-HDHA, and then the intermediate product is metabolized by 5-LOX to generate 7-epoxy-17-HDHA, and further to synthesize resolution D2. Studies have shown that resolution D2 can inhibit the generation of inflammasomes in vitro, reduce the secretion of inflammatory factors, and show significant anti-inflammatory effects in the inflammatory response. However, the role of resolution D2 in myocardial infarction and myocardial remodeling still needs to be further explored.

[0004] REFERENCES

[0005] [1] Thygesen K, Alpert JS, White HD; Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. J Am Coll Cardiol. 2007; 50(22): 2173-95.

[0006] [2] Likosky DS, Van Parys J, Zhou W, et. al. Association Between Medicare Expenditure Growth and Mortality Rates in Patients With Acute Myocardial Infarction: A Comparison From 1999 Through 2014. JAMA Cardiol. 2018; 3(2): 114-122.

[0007] [3] Wang F, Yu Y, Mubarik S, et. al. Global Burden of Ischemic Heart Disease and Attributable Risk Factors, 1990-2017: A Secondary Analysis Based on the Global Burden of Disease Study 2017. Clin Epidemiol. 2021; 13: 859-870.

[0008] [4] Haugaa KH, Smedsrud MK, Steen T, et. al. Mechanical dispersion assessed by myocardial strain in patients after myocardial infarction for risk prediction of ventricular arrhythmia. JACC Cardiovasc Imaging. 2010; 3(3): 247-56.

[0009] [5] Bahit MC, Kochar A, Granger CB. Post-Myocardial Infarction Heart Failure. JACC Heart Fail. 2018; 6(3): 179-186.

[0010] [6] Serhan C N, Levy B D. Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators. J Clin Invest, 2018, 128(7): 2657-2669.

[0011] [7] Cao L, Wang Y, Wang Y, et.al. Resolvin D2 suppresses NLRP3 inflammasome by promoting autophagy in macrophages. Exp Ther Med. 2021; 22(5): 1222. SUMMARY

[0012] At present, there are still deficiencies in the clinical treatment of pathological myocardial remodeling after myocardial infarction, and novel treatment methods / drugs are still needed to reduce pathological myocardial remodeling after infarction and improve the prognosis of myocardial infarction patients. The present application finds that resolvin D2 can improve cardiac function after myocardial infarction and reduce myocardial remodeling after infarction. The purpose of the present application is to provide a new application of resolvin D2 in the preparation of drugs for relieving and / or treating myocardial infarction and related diseases.

[0013] The purpose of the present application is achieved by the following technical solutions:

[0014] The present application takes C57BL / 6 mice as experimental objects, and intervenes by intraperitoneal injection of resolvin D2 (2 μg / kg, once a day, solvent is normal saline), constructs a mouse myocardial infarction model by permanent ligation of the left anterior descending branch of the coronary artery, and observes the effect of resolvin D2 on myocardial infarction and myocardial remodeling after infarction. The results show that resolvin D2 can significantly improve the left ventricular function after infarction and reduce myocardial remodeling after infarction. The present application provides a theoretical basis and clinical basis for studying new strategies for relieving and / or treating myocardial infarction and related diseases.

[0015] The present application provides the application of resolvin D2 in the preparation of drugs for relieving and / or treating myocardial infarction and related diseases. In the application, resolvin D2 is used as an active ingredient to prepare drugs for relieving and / or treating myocardial infarction and related diseases.

[0016] The present application also provides a drug for relieving and / or treating myocardial infarction and related diseases. The drug comprises resolvin D2 and can also comprise a pharmaceutically acceptable carrier of resolvin D2.

[0017] The myocardial infarction and related diseases include but are not limited to acute ST segment elevation myocardial infarction, acute non-ST segment elevation myocardial infarction, ischemic cardiomyopathy, myocardial infarction with heart failure, etc.

[0018] The advantages and beneficial effects of the present application are:

[0019] (1) The present application finds a new function of resolvin D2, i.e. resolvin D2 has the effect of improving myocardial infarction and reducing myocardial remodeling after infarction.

[0020] (2) Based on the improvement of myocardial remodeling after myocardial infarction by Regulin D2, it provides a target for developing new drugs for relieving and / or treating myocardial infarction and related diseases.

[0021] (3) With Regulin D2 as the active ingredient, it can be used for preparing new drugs for relieving and / or treating myocardial infarction and related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the result graph of Regulin D2 improving cardiac function after myocardial infarction. A: ultrasonic image representative graph; B: left ventricular end-diastolic diameter (LVEDd); C: left ventricular end-systolic diameter (LVEDs); D: left ventricular ejection fraction (LVEF); E: left ventricular ejection fraction (LVFS). # indicates that the difference is statistically significant. MI: myocardial infarction; d: days (Day); Vehicle: placebo; RvD2: Regulin D2.

[0023] Figure 2 is the result graph of Regulin D2 reducing myocardial remodeling after infarction. A: HE staining shows myocardial infarction area (left ventricular papillary muscle level + apical level); B: Masson staining shows myocardial infarction area (left ventricular papillary muscle level + apical level); C: myocardial infarction area statistical result; D: PSR staining shows the area of myocardial fibrosis after infarction; E: statistical result of myocardial fibrosis area after infarction. # indicates that the difference is statistically significant. MI: myocardial infarction; Vehicle: placebo; RvD2: Regulin D2; Papillary level: papillary muscle level; Apical level: apical level. DETAILED DESCRIPTION

[0024] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application.

[0025] If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0026] Experimental animals and feeding: C57BL / 6 mice, male, 8 weeks old, purchased from Jiangsu Jizhu Pharmaceutical Biotechnology Co., Ltd. Feeding in the standardization experimental animal center (SPE level) of Wuhan University People's Hospital, feeding conditions: temperature between 22-24℃, humidity between 40-70%, light and dark alternating lighting time for 12h, free water and food.

[0027] Example 1 Regulin D2 improves cardiac function after myocardial infarction

[0028] 1. RvD2 acquisition

[0029] RvD2 was purchased from Cayman Chemical Company, USA.

[0030] 2. MI model acquisition and RvD2 intervention

[0031] (1) 3% sodium pentobarbital 90 mg / kg intraperitoneal injection anesthesia, use mouse hair clipper to shave the mouse chest and axillary hair (expose the operation area), use iodine and 75% ethanol to disinfect the operation area.

[0032] (2) Tracheal intubation: after anesthesia, clamp the toe to check for no response, then proceed with the MI operation. Turn on the external light source, microscope switch, and set the parameters (respiratory frequency 110 bpm) of the respirator. Insert the tracheal tube into the trachea along the glottis, remove the mouse and connect it to the respirator. Observe the mouse's breathing condition, and if the thoracic fluctuation is consistent with the respirator frequency, it indicates successful intubation, and the MI operation can proceed.

[0033] (3) The mouse adopts a right lateral position, and the left forelimb axillary is cut with an ophthalmic scissors. The thoracic cavity is opened between the third and fourth ribs to fully expose the heart with a microscopical scissors. A small amount of pericardium is gently clamped with a microscopical straight forceps and torn a little below the left auricle to fully expose the left anterior descending branch (LAD) of the coronary artery or the region where it is located.

[0034] (4) Coronary artery ligation: find the LAD trajectory or possible location under the microscope, hold the 7-0 needle suture with a needle holder, and insert the needle 2 mm below the left auricle. The suture passes through the LAD to completely block the blood flow of the LAD, and the distal myocardial ischemia of the blood vessel can be observed.

[0035] (5) Close the chest: after ligation is completed, the thoracic opening is completely sutured with a 6-0 suture (ensure no gap and no misplacement) to close the thoracic cavity. Suture each layer of muscle and skin layer by layer from the inside out.

[0036] (6) RvD2 intervention: immediately after the myocardial infarction operation, give RvD2 2 μg / kg once a day for 28 days of continuous intervention until the end of the experiment. The solvent is normal saline, which is injected intraperitoneally.

[0037] (7) Postoperative management: closely observe the mouse's condition after the operation, such as respiratory abnormalities, etc. After the mouse wakes up naturally, remove it from the respirator and remove the tracheal intubation, and raise it normally. Take the heart tissue at 28 days after the myocardial infarction operation and freeze it at -80°C for future use, and store it in 10% formalin.

[0038] (8) Grouping: the experiment follows the principles of randomization and blinding and is divided into two groups: myocardial infarction group (MI) + placebo (Vehicle), MI + RvD2 (RvD2).

[0039] 3. Assessment of cardiac function

[0040] (1) Left ventricular function was assessed by echocardiography at pre-AMI, 3, 7, and 28 days post-AMI.

[0041] (2) Mice were anesthetized with 1.5% isoflurane and echocardiography was performed using a Vevo 6 (VisualSonics, Suzhou, China). Short axis views were used to measure heart rate (HR), left ventricular end diastolic dimension (LVEDD), left ventricular end systolic dimension (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS).

[0042] Cardiac ultrasound results are shown in FIG. 6A. Figure 1 Echocardiography showed that Resolvin D2 improved cardiac function after myocardial infarction (A-E). Figure 1

[0043] Example 2. Resolvin D2 reduces infarct size and improves myocardial remodeling after myocardial infarction

[0044] 1. Preparation of paraffin sections

[0045] (1) The mouse heart tissue was removed from the 10% formalin solution, trimmed in a fume hood, and the trimmed heart tissue and corresponding labels were placed in a dehydration box.

[0046] (2) Dehydration and wax immersion: The dehydration box was placed in a dehydration machine for dehydration and wax immersion. 75% ethanol (4 h)→ 85% ethanol (2 h)→ 90% ethanol (2 h)→ 95% ethanol (1 h)→ absolute ethanol I (30 min)→ absolute ethanol II (30 min)→ alcohol benzene (5-10 min)→ xylene I (5-10 min)→ xylene II (5-10 min)→ 65°C melted paraffin I (1 h)→ 65°C melted paraffin II (1 h)→ 65°C melted paraffin III (1 h).

[0047] (3) Heart tissue embedding: The melted paraffin was first injected into the embedding frame, and the heart tissue was removed from the dehydration box while the paraffin was not yet solidified. The heart tissue was placed in the embedding frame with the bottom of the heart facing down, and the corresponding label was attached. The embedding frame was placed horizontally on a -20°C freezing table to cool. After the paraffin solidified, the wax block was removed from the embedding frame.

[0048] (4) Heart tissue sectioning: The wax block was trimmed and fixed on a paraffin microtome, and the section thickness was 4-5 μm. The microtome handle was rotated to continuously section the tissue, and the sections were floated in warm water and flattened. The sections were picked up with a glass slide, and the sections were placed in the center of the glass slide. The glass slide was placed in a 60°C oven for baking and storage.

[0049] 2. Hematoxylin-eosin staining (HE)​

[0050] The paraffin specimen block was cut into 5 μm thick sections and the following operations were performed in order:

[0051] (1) Xylene (I) 15 min;

[0052] (2) Xylene (II) 15 min;

[0053] (3) Xylene: anhydrous ethanol = 1:1 12 min;

[0054] (4) 100% ethanol (I) 5 min;

[0055] (5) 100% ethanol (II) 5 min;

[0056] (6) 80% ethanol 5 min;

[0057] (7) Distilled water 5 min;

[0058] (8) Hematoxylin solution staining 5 min;

[0059] (9) Running water for 1-3 s to wash off hematoxylin solution;

[0060] (10) 1% hydrochloric acid ethanol 1-3 s;

[0061] (11) Washed with water for 10-30 s;

[0062] (12) Distilled water for 1-2 s;

[0063] (13) 0.5% eosin solution staining 1-3 min;

[0064] (14) Distilled water for 1-2 s;

[0065] (15) 80% ethanol for 1-2 s;

[0066] (16) 95% ethanol (I) 2-3 s;

[0067] (17) 95% ethanol (II) 3-5 s;

[0068] (18) Anhydrous ethanol (I) 5-10 min;

[0069] (19) Anhydrous ethanol (II) 5-10 min;

[0070] (20) Xylene (I) 2 min;

[0071] (21) Xylene (II) 2 min;

[0072] (22) Xylene (III) 2 min;

[0073] (23) Neutral gum fixation;

[0074] After the slide is dried, take a photo record using an optical microscope and calculate the myocardial infarction area using Image J software.

[0075] 2. Masson staining

[0076] (1) Paraffin section deparaffinization to water: sequentially place the section in xylene I 20 min-xylene II 20 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-75% alcohol 5 min, tap water wash.

[0077] (2) Soak the section in Masson A liquid overnight, tap water wash.

[0078] (3) Put the section into the dyeing liquid of the equal ratio mixture of Masson B liquid and Masson C liquid, immerse and dye for 1 min, tap water wash, 1% hydrochloric acid alcohol differentiation, tap water wash.

[0079] (4) Put the section into Masson D liquid and immerse and dye for 6 min, tap water rinse.

[0080] (5) Immerse and dye in Masson E liquid for 1 min.

[0081] (6) Without water washing, slightly drain and directly into Masson F liquid for 2-30 s.

[0082] (7) Rinse and differentiate the section with 1% glacial acetic acid, dehydrate with two cylinders of anhydrous ethanol.

[0083] (8) Transparent mounting: place the section into the third cylinder of anhydrous ethanol for 5 min, transparent with xylene for 5 min, neutral gum mounting.

[0084] (9) Microscope examination, image acquisition and analysis (use Image J software to calculate the myocardial infarction area).

[0085] 3. Picrosirius red (PSR) staining

[0086] (1) Place the paraffin section in xylene solution for 5 min, repeat the same for 3 times.

[0087] (2) Sequentially pass through 100%, 90%, and 70% ethanol for 1 min each time.

[0088] (3) Rinse the specimen with running water for 10 min.

[0089] (4) After rinsing, place the specimen in pure water for 1 min, and then place it in 0.2% phosphomolybdic acid for 1-5 min.

[0090] (5) The section was placed in a moist staining box, and a few drops of 0.1% picrosirius picric acid solution were added to allow the solution to fully contact the sample, and the sample was dyed for 90 min.

[0091] (6) The picrosirius picric acid solution was fully shaken off, and the section was immersed in pure water several times.

[0092] (7) Finally, the section was subjected to 70%, 95% ethanol for 30 s each, and 100% ethanol for 30 s, for a total of 3 times.

[0093] (8) Xylene was used for 2 min, repeated 3 times, and the section was mounted.

[0094] (9) After staining, the section was photographed under a light microscope, and the area of myocardial fibrosis was calculated using Image-Pro Plus.

[0095] The myocardial infarction area and the fibrosis area are shown in Figure 2 . Resolvin D2 reduces the myocardial infarction area and alleviates post-infarction myocardial fibrosis, indicating that resolvin D2 alleviates pathological myocardial remodeling after myocardial infarction Figure 2 A-E).

[0096] The above results show that in the acute myocardial infarction model, resolvin D2 can significantly reduce the infarction area, alleviate pathological myocardial remodeling, and improve cardiac function. Resolvin D2 can be used as an active ingredient for preparing a drug for relieving and / or treating myocardial infarction and related diseases.

Claims

1. Use of atrogin-2 for the preparation of a medicament for the alleviation and / or treatment of myocardial infarction, characterized in that: The medicine is a medicine for improving heart function after myocardial infarction, reducing myocardial infarction area and reducing pathological myocardial remodeling.

2. Use according to claim 1, characterized in that: The medicine for relieving and / or treating myocardial infarction is prepared by taking the resolvin D2 as the only active ingredient.

3. Use according to claim 1 or 2, characterized in that: The myocardial infarction includes acute ST segment elevation myocardial infarction and acute non-ST segment elevation myocardial infarction.

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