2-DG patch, preparation method and application

Abstract

The invention discloses a 2-DG patch, a preparation method and application, and belongs to the technical field of 2-DG patches, the preparation method comprises the following steps: S1, mixing 4% of chitosan and 2% of gelatin according to a weight ratio of 1: 1 to form a patch substrate; s2, adding 1% of 2-DG, fully and uniformly mixing the 2-DG with the patch substrate to form a mixed solution, and centrifuging the mixed solution to remove bubbles and impurities so as to obtain a centrifugate; s3, adding the centrifugate into a culture dish or a pore plate, and drying the culture dish or the pore plate in a drying oven to obtain a dried substance; and S4, soaking the dried substance in a NaOH solution for 30 minutes for deacidification, and washing the dried substance to be neutral by using deionized water to form the 2-DG patch. The 2-DG patch disclosed by the invention can be pasted on the surface of infarcted myocardium, and the slow release of the 2-DG can regulate the polarization of macrophages in a myocardial infarction region, relieve the inflammatory microenvironment in the myocardial infarction region, improve the cardiac function after myocardial infarction, reduce the infarct area and promote heart repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of 2-DG patches, and in particular relates to a 2-DG patch, a preparation method and an application thereof. Background Art

[0002] Cardiovascular disease is still the number one killer threatening human health today, among which ischemic heart disease, especially acute myocardial infarction, is the main cause of death. After acute myocardial infarction occurs, the infarct area suffers from ischemia and hypoxia, nutrients are depleted, glycolysis occurs in the infarct area, acid metabolites increase, and there is a continuous accumulation of oxygen free radicals, necrotic cells and fragmented extracellular matrix. Severe aseptic inflammation occurs in the myocardial infarction area, and various immune cells are activated, thus forming a harsh inflammatory microenvironment in the infarct area, severely damaging heart function and hindering the process of cardiac repair after myocardial infarction. Therefore, it is of great significance to find a therapeutic strategy that can regulate the inflammatory microenvironment after myocardial infarction and promote cardiac repair.

[0003] In recent years, 2-DG has been used in a variety of diseases as a glycolysis inhibitor. In previous studies, we designed a chitosan / gelatin-based 2-DG composite patch and attached it to the surface of the infarcted myocardium. The slow release of 2-DG can regulate the polarization of macrophages in the infarcted area, increase the residence and survival of transplanted stem cells, and ultimately promote the therapeutic effect of mesenchymal stem cells on myocardial infarction. In this study, this patch was directly attached to the myocardial infarction area to study its regulation of the inflammatory microenvironment of myocardial infarction and its effect on cardiac repair in myocardial infarction. Summary of the invention

[0004] The present invention overcomes the deficiencies of the prior art and provides a 2-DG patch, a preparation method and an application thereof to solve the problems existing in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a 2-DG patch,

[0006] The 2-DG patch includes 4% chitosan and 2% gelatin, and the weight ratio of the chitosan to the gelatin is 1:1;

[0007] The preparation method comprises the following steps:

[0008] S1. Mix 4% chitosan and 2% gelatin in a weight ratio of 1:1 to form a patch matrix;

[0009] S2, adding 1% 2-DG, and mixing it with the patch matrix to form a mixed solution, and then centrifuging the mixed solution to remove bubbles and impurities to obtain a centrifuged solution;

[0010] S3, adding the centrifuged liquid into a culture dish or a well plate, placing it in an oven for drying to obtain a dried product;

[0011] S4. Soak the dried product in NaOH solution for 30 minutes for deacidification, and then use deionized water to wash the dried product until it is neutral to form a 2-DG patch.

[0012] In a preferred embodiment of the present invention, the chitosan and the gelatin are both dissolved in 1% acetic acid before use.

[0013] In a preferred embodiment of the present invention, in step S2, the centrifugal conditions for the mixed solution are: 3000-4000 rpm and centrifugation for 15 minutes.

[0014] In a preferred embodiment of the present invention, in step S3, the centrifuged liquid is dried in an oven at 50-60° C. for 24 hours.

[0015] In a preferred embodiment of the present invention, in step S4, before the dried object is washed with deionized water, the dried object is washed with an ethanol solution for several times, each time for 10 minutes.

[0016] In a preferred embodiment of the present invention, the concentration of the ethanol solution is 80%, and the number of cleaning times is three times.

[0017] In a preferred embodiment of the present invention, the concentration of the NaOH solution is 2%.

[0018] The invention also discloses an application of a 2-DG patch, wherein the 2-DG patch with a diameter of 3-4 mm is used for treating myocardial infarction.

[0019] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0020] The 2-DG patch of the present invention can be directly attached to the surface of the infarcted myocardium. The slow release of 2-DG can regulate the polarization of macrophages in the myocardial infarction area, alleviate the inflammatory microenvironment in the myocardial infarction area, improve cardiac function after myocardial infarction, reduce the infarction area, and promote heart repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0022] Figure 1 This is a graph showing the detection of inflammatory factor expression after RNA and protein extraction from the heart in a preferred embodiment of the present invention;

[0023] Figure 2 This is a schematic diagram of flow cytometry detection of macrophages after preparing single cell suspension in the heart in a preferred embodiment of the present invention;

[0024] Figure 3 This is a schematic diagram of the ultrasonic detection results of cardiac function after myocardial infarction in a preferred embodiment of the present invention;

[0025] Figure 4 This is a schematic diagram of reducing infarct area using the DG patch in the preferred embodiment of the present invention; DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0027] The main materials and sources used in this example are as follows:

[0028] C57BL / 6J mice (provided by Zhaoyan (Suzhou) New Drug Research Center); small animal ventilator (Shanghai Olcott Biotechnology Co., Ltd., Shanghai); surgical instruments (Six Six Vision Company, Suzhou); suture needles and threads (Shanghai Pudong Jinhuan Medical Supplies Co., Ltd., Shanghai); small animal ultrasound imaging system (Visua lSon i cs Vevo 2100); 2-deoxy-D-glucose (2-DG, Bio-Technology Co., Ltd., Shanghai); chitosan (Sinopharm Chemical Reagent Co., Ltd., Shanghai); gelatin (Sinopharm Chemical Reagent Co., Ltd., Shanghai)

[0029] This embodiment provides a method for preparing a 2-DG patch, wherein the 2-DG patch includes 4% chitosan and 2% gelatin, and the weight ratio of chitosan to gelatin is 1:1;

[0030] The preparation method comprises the following steps:

[0031] S1. 4% chitosan and 2% gelatin were mixed in a weight ratio of 1:1 to form a patch matrix. Before use, chitosan and gelatin were dissolved in 1% acetic acid;

[0032] S2, adding 1% 2-DG, and mixing it with the patch matrix to form a mixed solution, centrifuging the mixed solution at 3000-4000 rpm for 15 minutes to remove bubbles and impurities, and obtaining a centrifuged solution;

[0033] S3, adding the centrifuged liquid into a culture dish or well plate, placing it in an oven for drying, and drying the centrifuged liquid in the oven at 50-60° C. for 24 hours to obtain a dried product;

[0034] S4. Soak the dried product in a 2% NaOH solution for 30 minutes for deacidification, and then use deionized water to wash the dried product until it is neutral to form a 2-DG patch.

[0035] In step S4 of this embodiment, before the dried object is washed with deionized water, the dried object is washed with ethanol solution for several times, each time for 10 minutes. In this embodiment, the concentration of the ethanol solution is 80%, and the number of washing times is three times.

[0036] Establishment of myocardial infarction model in mice

[0037] C57BL / 6J male mice weighing about 25g were selected as experimental subjects. The myocardial infarction model was established by ligating the left anterior descending coronary artery. After intraperitoneal injection of anesthesia, the mice were intubated through the mouth and connected to an air ventilator. The respiratory rate was 110 times / min, the tidal volume was 2.5ml, and the inspiration-expiration ratio was 1:1.3. The mice were placed in the right lateral position. A longitudinal incision was made on the left chest to cut the outer skin, the pectoralis major muscle was peeled off, and a transverse incision was made between the third and fourth intercostal spaces to expose the heart. The pericardium was torn with forceps. The approximate course of the left coronary artery could be seen with the help of an operating microscope. The anterior descending coronary artery (LAD) was ligated together with a small amount of myocardial tissue at about 1.5mm below the lower edge of the left auricle. The needle was inserted to a depth of about 1mm and the width was controlled within 3mm. The chest was closed layer by layer.

[0038] After ligation, the area from the ligation site to the apex of the heart turned white visibly. One week later, left ventricular tissue was obtained for cardiac tissue staining, and obvious fibrosis was seen, proving that the myocardial infarction model was successfully established.

[0039] In the sham operation group, the needle was only passed under the LAD without tying a knot, and the rest was the same as in the model group.

[0040] In the 2-DG group, a 2-DG patch was immediately attached to the myocardial infarction area after myocardial infarction was established. In the simple patch group, a patch without 2-DG was attached to the myocardial infarction area. All mice were fed in the same way, and then cardiac function and myocardial infarction area were tested after myocardial infarction.

[0041] Results 1: 2-DG patch reduces the expression of inflammatory factors in the myocardial infarction area

[0042] On the third day after surgery, the heart was taken to extract RNA and protein, and the expression of inflammatory factors was detected. Figure 1 , **P<0.01.

[0043] Results 2: 2-DG patch modulates macrophage phenotype

[0044] On the third day after surgery, the heart was removed to prepare single cell suspension, and the macrophage phenotype was detected by flow cytometry. Figure 2 , ***P<0.001.

[0045] Result 3: 2-DG patch improves cardiac function in patients with myocardial infarction

[0046] Cardiac ultrasound was used to detect cardiac function after myocardial infarction. Mice were anesthetized (same method as above), depilated and placed in left lateral decubitus position. The probe of cardiac ultrasound diagnostic instrument was placed on the anterior wall of the heart. A two-dimensional short-axis view of the left ventricle was taken at the level of the papillary muscle. M-mode scan was recorded at the same time. The left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) were measured for three consecutive cardiac cycles. The experimental results are shown in Figure 3 , *P<0.05, **P<0.01.

[0047] Results 4: 2-DG patch reduces infarct size and inhibits adverse ventricular remodeling

[0048] Heart weight and Masson staining: Mice were killed 28 days after surgery, and their body weight and heart weight were measured; heart weight / body weight was calculated. Masson staining: Mice were killed 28 days after surgery, and left ventricular tissue was taken for heart tissue staining to observe the therapeutic effect. The routine Masson staining steps were followed, and the images were observed and photographed under an ordinary optical microscope. Image analysis software Image J was used to analyze the area of ​​each part and calculate the myocardial infarction area / heart area. The experimental results are shown in Figure 4 , **P<0.01.

[0049] In summary, in this embodiment, mice are divided into two groups: a control group and an experimental group. The control group was treated with normal saline, and the experimental group was topically applied with a 2-DG patch. The results showed that the degree of pro-inflammatory polarization of macrophages in the infarcted area of ​​the experimental group of mice was significantly reduced, and the level of oxidative stress was also significantly reduced. The cardiac function was significantly better than that of the control group, and the infarct area was significantly reduced. In addition, the number of new blood vessels in the cardiomyocytes of the experimental group of mice increased significantly. In order to further explore its mechanism, we performed Western blot analysis on macrophages. The results showed that there is a close relationship between glycolysis and macrophage polarization. These results indicate that the 2-DG patch may improve the inflammatory microenvironment after myocardial infarction by regulating macrophage polarization, reducing oxidative stress, and inhibiting glycolysis, thereby promoting cardiac repair.

[0050] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a 2-DG patch, characterized in that: The 2-DG patch includes 4% chitosan and 2% gelatin, and the weight ratio of the chitosan to the gelatin is 1:1; The preparation method comprises the following steps: S1. Mix 4% chitosan and 2% gelatin in a weight ratio of 1:1 to form a patch matrix; S2, adding 1% 2-DG, and mixing it with the patch matrix to form a mixed solution, and then centrifuging the mixed solution to remove bubbles and impurities to obtain a centrifuged solution; S3, adding the centrifuged liquid into a culture dish or a well plate, placing it in an oven for drying to obtain a dried product; S4. Soak the dried product in NaOH solution for 30 minutes for deacidification, and then use deionized water to wash the dried product until it is neutral to form a 2-DG patch.

2. The method for preparing a 2-DG patch according to claim 1, characterized in that: The chitosan and the gelatin were dissolved in 1% acetic acid before use.

3. The method for preparing a 2-DG patch according to claim 1, characterized in that: In step S2, the mixed solution is centrifuged at 3000-4000 rpm for 15 minutes.

4. The method for preparing a 2-DG patch according to claim 1, characterized in that: In step S3, the centrifuged liquid is dried in an oven at 50-60° C. for 24 hours.

5. The method for preparing a 2-DG patch according to claim 1, characterized in that: In step S4, before the dried object is washed with deionized water, the dried object is washed with an ethanol solution for several times, each time for 10 minutes.

6. The method for preparing a 2-DG patch according to claim 5, characterized in that: The concentration of the ethanol solution is 80%, and the number of washing times is three times.

7. The method for preparing a 2-DG patch according to claim 1, characterized in that: The concentration of the NaOH solution is 2%.

8. An application of a 2-DG patch, using the 2-DG patch according to any one of claims 1 to 7, characterized in that: A 2-DG patch with a diameter of 3-4 mm is used for the treatment of myocardial infarction.

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