Application of empagliflozin in promoting protein lactylation and angiogenesis

Abstract

The invention discloses application of empagliflozin to promotion of protein lactylation and angiogenesis, and belongs to the field of biological medicine. The protein disclosed by the invention comprises IDH2, so that the lactylation of a K272 site on the IDH2 is further promoted; according to experimental verification, the empagliflozin can promote the lactylation of protein, especially the lactylation of IDH2-K272, and the empagliflozin can promote the lactylation of the protein; through further experimental verification, empagliflozin can promote angiogenesis after diabetic myocardial infarction and relieve cardiac function impairment; the invention provides a new application of the empagliflozin, provides reference for application expansion of the empagliflozin, provides reference for research on protein lactylation and a relationship between the protein lactylation and diseases, and has important significance for research on the protein lactylation.

Description

Technical Field

[0001] The present invention relates to the use of empagliflozin in promoting protein lactic acidification and angiogenesis, and belongs to the field of biomedicine. Background Art

[0002] Post-translational modification of proteins refers to the phenomenon that proteins undergo a series of chemical modification processes after RNA is translated into amino acid sequences. These modifications can affect the activity, specificity and interactions of proteins, thereby providing important regulatory means for the normal function of cells and environmental adaptation. Lactylation modification, as a newly discovered epigenetic modification, regulates protein function by adding lactyl-CoA groups to lysine residues and can promote specific gene transcription. Studies have shown that histone lactylation modification activates the endogenous "lactate clock" of cells and promotes the differentiation of macrophages into the M2 type with repair function.

[0003] Under hypoxic and inflammatory conditions, activation of the glycolytic pathway leads to an increase and release of lactate, which in turn triggers acidification of the extracellular environment. Protein lactylation is considered to be an important pathophysiological mechanism for the function of lactate, and is involved in regulating retinopathy, promoting tumor proliferation, regulating the nervous system, and regulating metabolism. In the environment of diabetic metabolic disorders, lactate is easily accumulated in the body. Studies have found that under diabetic conditions, lactate-mediated histone lactylation can promote the upregulation of fat mass and obesity-associated protein (FTO) expression, thereby promoting endothelial cell proliferation and enhancing angiogenesis in mouse and zebrafish models. It can be seen that in a high-sugar environment, lactylation may participate in the pathophysiological process of regulating the disease by promoting cell proliferation. In patients with diabetic myocardial infarction, angiogenesis is crucial for post-injury repair. Long-term abnormal blood glucose metabolism leads to lactate accumulation in the body, which increases the body's tolerance and action time to lactate, but the exact role of lactate in the heart is still unclear. Studies have shown that histone lactylation modification may remotely activate repair genes in monocytes early after myocardial infarction, while the regulatory role of non-histone lactylation on diabetic myocardial infarction remains unclear. In the field of vascular function regulation, the role of lactylation in endothelial cells and angiogenesis is unclear, and this modification shows different effects in different diseases by regulating protein function.

[0004] Therefore, protein lactylation and whether the selection of protein lactylation can be used as a new entry point for the treatment or diagnosis of related diseases, and how to promote protein lactylation, need further research and discussion.

[0005] Empagliflozin is a selective sodium glucose co-transporter type 2 inhibitor (SGLT-2 inhibitor), a new drug widely used in the treatment of diabetes in recent years. Studies have also found that empagliflozin helps reduce the incidence of myocardial infarction, the rate of re-hospitalization for heart failure, and cardiovascular mortality. These positive effects are usually evident in the early stages of empagliflozin treatment, but it is not clear whether empagliflozin is related to protein lactylation. Although SGLT-2 inhibitors have shown significant effects in the treatment of heart failure and improving myocardial structure and function after myocardial infarction, it is not clear whether they are also effective in angiogenesis. Summary of the invention

[0006] In view of some deficiencies in the prior art, the present invention provides the use of empagliflozin in promoting protein lactic acidification and angiogenesis.

[0007] In order to achieve the purpose of the above invention, the present invention adopts the following technical solutions:

[0008] The present invention first provides the use of empagliflozin in promoting protein lactic acidification and angiogenesis.

[0009] Furthermore, the protein lactylation includes IDH2.

[0010] Furthermore, the protein lactylation includes lactylation of the K272 site of IDH2.

[0011] Furthermore, the angiogenesis includes angiogenesis after myocardial infarction.

[0012] The present invention also provides a drug for promoting protein lactic acidification, wherein the drug comprises empagliflozin.

[0013] The present invention also provides a drug for promoting angiogenesis, wherein the drug includes empagliflozin.

[0014] The present invention also provides the use of the drug in preparing a drug for promoting the prognosis recovery of myocardial infarction; further, the myocardial infarction is diabetic myocardial infarction; the prognosis recovery includes promoting angiogenesis and / or alleviating cardiac function damage.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides the use of empagliflozin in promoting protein lactylation and angiogenesis, wherein the protein lactylation includes IDH2, and the protein lactylation includes lactylation of the K272 site of IDH2. The present invention has been experimentally verified to find that empagliflozin can promote IDH2-K272 lactylation.

[0017] Furthermore, the present invention has experimentally verified that exogenous use of empagliflozin has a promoting effect on myocardial injury and CMECs function in diabetic myocardial infarction mouse models and high-glucose hypoxia CMECs models, and further exploration has found that empagliflozin can promote protein lactic acidification and angiogenesis. The present invention broadens the application field of empagliflozin and also provides an experimental basis for exploring the relationship between lactic acidification and disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the quality inspection map of lactic acid modification in the infarct edge zone tissue of diabetic myocardial infarction mice.

[0019] Figure 2 The results of screening for IDH2 lactylation modification sites; A in the figure shows the identification of IDH2 lactylation modification sites by mass spectrometry, and B shows the comparison of IDH2 protein sequences between different species.

[0020] Figure 3 Figure 1 is a graph showing the effect of IDH2-K272 lactylation on CMECs' resistance to high-sugar environment; Figure A shows the effect of IDH2-K272 lactylation on CMECs' proliferation ability, where the left figure is the cell fluorescence detection result and the right figure is the cell proliferation data statistical result; Figure B shows the effect of IDH2-K272 lactylation on angiogenesis, where the left figure shows the changes in lumen formation under a microscope and the right figure is the lumen data statistical result.

[0021] Figure 4 The results show the effects of canagliflozin (CANA), empagliflozin (EMPA) and dapagliflozin (DAPA) on the lactic acid expression level of IDH2-K272. The left figure shows the protein electrophoresis results, and the right figure shows the statistical results of the expression level changes.

[0022] Figure 5 Figure 1 is the effect of empagliflozin on the lactic acidification of IDH2-K272; Figure A is the Western Blot experiment to detect the protein expression levels of eNOS and p-eNOS after adding DMSO and 1μM EMPA to HOD; Figure B is the Western Blot experiment to detect the changes in the expression level of IDH2-K272la in the low-glucose hypoxia group (NOD), high-glucose hypoxia group (HOD) and high-glucose hypoxia group (HOD+EMPA) with the addition of empagliflozin (EMPA) in CMECs intervened by IDH2 and its mutant plasmids (the left figure is the electrophoresis figure, and the right figure is the data statistical results); Figure C is the detection of lumen formation, Edu proliferation and NO generation ability of CMECs, n=6, scale bar is 100μm.

[0023] Figure 6The diagrams are the cardiac ultrasound results of each group of mice in Example 3, including cardiac ultrasound M-model diagrams, and statistical results of EF, FS, LVIDd, and LVIDs values.

[0024] Figure 7 The figures are the results of the arterial ring experiment of each group of mice in Example 3, including the microscopic results of the new blood vessel sprouts in the arterial ring experiment (upper figure) and the statistical results of the new blood vessel sprouts in the arterial ring experiment (lower figure).

[0025] Figure 8 The Masson staining results of myocardial tissue of each group of mice in Example 3 include a real shot of myocardial tissue sections (upper figure) and the statistical results of collagen content (lower figure). DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0027] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0028] In the examples, DMEM medium (this medium does not contain serum) and fetal bovine serum were purchased from Nanjing Vicente Biotechnology Co., Ltd.; ECM medium was purchased from Sciencell, USA; type II collagenase was purchased from Gibco, USA; type I rat tail collagen was purchased from Corning, USA; iron hematoxylin solution, bluing solution, differentiation solution and Ponceau fuchsin solution were all included in the Masson staining kit, and the Masson staining kit was purchased from Beijing Solebao Technology Co., Ltd.; canagliflozin (CANA), empagliflozin (EMNA), and dapagliflozin (DAPA) were purchased from MedChemExpress, USA. In the examples, Tubulin antibody (AC007, Abclonal, China) was used for Western Blot experiments, IDH2 antibody (A7190, Abclonal, China) was used for Western Blot experiments; IDH2-K272la (IDH2-K272 lactate) antibody was constructed by Hangzhou Jingjie Biotechnology Co., Ltd. and used for Western Blot experiments.

[0029] In the examples, the mutant plasmid IDH2-K272R and the non-mutant plasmid IDH2-WT were constructed based on the vector pHS-B-0080 (backbone pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-MCS-3flag, commonly available in the market).

[0030] In the examples, the complete culture medium is a serum-containing DMEM culture medium, which is prepared from serum-free DMEM culture medium and fetal bovine serum to form a DMEM culture medium containing 10% serum. The DMEM culture medium and fetal bovine serum are purchased from Nanjing Vicente Biotechnology Co., Ltd.

[0031] The method for preparing paraffin sections described in the examples is a conventional method in the art.

[0032] As a form of isocitrate dehydrogenase, isocitrate dehydrogenase 2 (IDH2) has multiple signal regulation functions. Studies have shown that IDH2 and its epigenetic modifications may be involved in regulating multiple processes such as oxidative stress, metabolism, and tumorigenesis. The function of IDH2 is regulated by post-translational modifications. When IDH2 is modified by SUMO1, its activity can be enhanced, prompting it to produce antioxidant metabolites to reduce cell damage. However, in some cases, IDH2 variants may cause the occurrence of certain diseases. For example, IDH mutants found in acute myeloid leukemia cells have additional functions that can produce a carcinogenic molecule called 2-hydroxyglutarate, which leads to immature proliferation of white blood cells and thus the occurrence of leukemia. Isocitrate dehydrogenase 2 (IDH2) shows significant differences in non-histone lactylation modifications. As a form of isocitrate dehydrogenase, IDH2 has multiple signal regulation functions. IDH2 and its epigenetic modification may be involved in regulating multiple processes such as oxidative stress, metabolism and tumorigenesis. Therefore, IDH2 is selected as an experimental subject in the present invention to verify whether empagliflozin can promote protein lactic acidification.

[0033] Embodiment 1:

[0034] Establishment of diabetic mouse model: 6-8 week old male C57BL / 6 (C57BL / 6, purchased from Jiangsu University Animal Center, public known public materials), mice adapted to the environment for 1 week, and were raised in an SPF environment. STZ (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., 40 mg / kg / day) was injected intraperitoneally into the mice for 5 consecutive days. After 2 weeks, the random blood glucose of mice was tested, and mice with a blood glucose level higher than 16.7 mmol / L for 3 consecutive times were identified as diabetic mice.

[0035] Establishment of myocardial infarction model: After normal mice or diabetic mice were anesthetized with 2% isoflurane, they were placed on the operating table, and the mouse skin was disinfected with 75% ethanol solution. An incision of about 1 cm long was made on the left midclavicular line, and the subcutaneous tissue and muscle were bluntly separated. The elbow vascular clamp was passed through the third and fourth intercostal spaces, and the heart was quickly pushed out with the left hand, so that the heart blocked the chest cavity opening at this time. The left anterior descending branch of the coronary artery was located 1-2mm below the left atrial appendage and ligated with 7-0 thread. After ligation, the ventricular wall below the ligation site turned dark red. The chest wall opening was expanded with a vascular clamp, and the heart was slowly pushed into the chest cavity. The gas in the chest cavity was squeezed out, and the chest wall skin incision was tied with a purse string knot to close the chest cavity. The electrocardiogram machine was connected to obtain a stable II limb lead, and the changes in the mouse electrocardiogram were closely observed. When the T wave was towering, the ST segment was elevated, and the QRS wave was widened, the myocardial infarction surgery model was successfully established. After the thoracotomy of the mice in the sham operation group, the coronary artery of the heart was only traversed without ligation, and other operations were the same. The experimental animal surgical model was raised in the Experimental Animal Center of Jiangsu University before and after construction. All animal experiments involved in the present invention were reviewed and approved by the Animal Care and Use Committee of Jiangsu University. In this embodiment, 4 groups of mice were obtained: non-diabetic myocardial infarction group (MI), diabetic myocardial infarction group (DMI), sham operation group (Sham), and diabetic sham operation group (diabetic sham, Dsham).

[0036] Tissue protein extraction: For tissue samples, the heart was removed after euthanasia of myocardial infarction mice, and the myocardial tissue of the infarction edge area (within 2 mm of the infarction edge) was collected. At the same time, the sham operation group also collected tissue from the same location. After washing the myocardial tissue with PBS, it was blotted with filter paper, and a pre-prepared protein lysis buffer (RIPA: protease inhibitor: phosphatase inhibitor = 100:1:1) was added to every 10 mg of myocardial tissue. Mix and chop on ice. Use a high-throughput tissue grinder to grind at 70 Hz for 100 seconds to thoroughly homogenize the myocardial tissue. Place the homogenized tissue on ice for 30 minutes for full lysis. For cell samples, after washing with PBS three times, 100 μL of the prepared protein lysis buffer was added to each well of the 6-well plate. After collecting the cells, centrifuge at 14000×g for 15 minutes to separate the supernatant. The protein concentration was determined by the BCA method. A 20 μL volume of BCA standard (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL) was prepared to calculate the standard curve. 5 μL of tissue protein sample was diluted to 20 μL and added to the sample well, and 3 replicate wells were tested. 200 μL of BCA working solution was added to each well, mixed and incubated at 37°C for 30 minutes, and then the absorbance at 562 nm was measured. The protein concentration was calculated after drawing the standard curve. 1 / 4 volume of 5× Loading buffer was added to the protein sample, and the protein was denatured by boiling water for 8 minutes after shaking. After denaturation, the sample was cooled to room temperature for subsequent experiments, and the remaining sample was stored at -20°C. After quantification of tissue protein in the infarct edge area, electrophoresis was performed and stained with Coomassie Brilliant Blue solution at room temperature for 3 hours to detect the quality of protein extraction.

[0037] Trypsin hydrolysis: According to the protein concentration of each group, take the same mass of protein for enzymatic hydrolysis and add it to the same volume. Slowly add 1 / 4 volume of trichloroacetic acid, mix well, and let the mixture stand at 4°C for 2 hours. Then centrifuge at 4500×g for 5 minutes. Wash the precipitate 3 times with acetone precooled at 4°C. After drying, add tetraethylammonium bromide to a final concentration of 200mM, mix well and perform ultrasonic disruption. Then add 1 / 50 volume of trypsin and digest overnight. Add dithiothreitol at a final concentration of 5mM the next day and reduce at 56°C for 30 minutes. Finally, add iodoacetamide at a final concentration of 11mM and incubate at 37°C in the dark for 15 minutes.

[0038] Lactated peptide enrichment: The peptide and lactated resin were dissolved in buffer, incubated with gentle rotation at 4°C for 12 hours, and then washed with buffer and deionized water. Finally, the peptide was eluted from the resin with eluent, and the obtained eluent was freeze-dried and desalted. Finally, the desalted sample was analyzed by liquid chromatography-tandem mass spectrometry.

[0039] Liquid chromatography tandem mass spectrometry analysis: First, the peptides were separated by NanoElute chromatography, then injected into the Capillary ion source for ionization, and then analyzed by the timsTOF Pro mass spectrometer. For secondary mass spectrometry data retrieval, the Mus musculus database containing 17,063 sequences was selected and searched using Maxquant software.

[0040] In order to further study the changes of lactic acid modification during the formation of diabetic myocardial infarction, on the 7th day after the successful establishment of the mouse myocardial infarction model, the infarct edge zone tissues of the Sham group, Dsham group, MI group and DMI group were separated, and the lactic acid modified protein group was studied. First, the tissue protein was extracted and the protein was quantified, followed by electrophoresis and Coomassie Brilliant Blue staining to detect the quality of the extracted protein. The results showed that the electrophoresis bands of the extracted tissue protein were clear, the protein was not degraded, and there was an obvious high-abundance protein ( Figure 1 ). Then, an equal amount of protein was enzymatically digested and peptides were enriched using a lactylated antibody, followed by mass spectrometry detection to study the lactylated proteome.

[0041] After mass spectrometry analysis, lactic acid modification sites were identified. Some lactic acid modification sites provided quantitative information and were expressed on multiple proteins. In order to determine the significant difference, a T test was performed on the relative quantitative value of each modification site. When P < 0.05, it indicated that there was a significant difference, and proteins and sites with significantly increased lactic acid modification levels were screened out. Further functional enrichment analysis of differentially lactic acid modified proteins and KEGG pathway enrichment analysis of differentially lactic acid modified proteins showed that IDH2 protein showed the most significant lactic acid modification.

[0042] Furthermore, through the mass spectrometry study of lactylation modification, this example found that the lactylation modification of IDH2 mainly occurred at its 272nd lysine (K272) ( Figure 2 A), and IDH2 lysine 272 is conserved among different species ( Figure 2 B).

[0043] In the present invention, IDH2 was selected as the experimental object for subsequent experiments.

[0044] Example 2: Culture of primary mouse myocardial microvascular endothelial cells (CMECs)

[0045] 1-3 day old suckling mice (C57BL / 6, purchased from the Animal Center of Jiangsu University, publicly known public materials) were euthanized with an overdose of carbon dioxide, disinfected with alcohol and transferred to a sterile environment for operation: the suckling mouse was placed in the left hand, and its sides were fixed with the thumb and index finger and the chest skin was tightened. Along the left clavicle midline of the suckling mouse, curved scissors were used to penetrate into the fourth to fifth intercostal space, and the ribs were cut close to the inside of the chest wall. The scissors were used to press both sides of the incision, and the heart was squeezed out with the thumb and index finger of the left hand. The heart was clamped with curved forceps, washed three times in a pre-cooled PBS solution and fat and connective tissue were removed, and then the heart was cut into pieces of about 1 mm 3 After the myocardial tissue block is washed, add a solution containing 0.2% type II collagenase prepared with DMEM culture medium to the washed myocardial tissue block, the volume of which is about 8-10 times that of the myocardial tissue block. After digestion on a 37°C shaker at 120rpm for 1 hour, add DMEM culture medium containing 10% serum to neutralize (the added DMEM culture medium containing serum is equal in volume to the type II collagenase solution to stop the digestion effect of the enzyme). After blowing and standing, use a sterile Pasteur pipette to filter the cell suspension (i.e., the cell liquid separated after digestion of the tissue) through a 200-mesh screen, transfer it to a 15mL centrifuge tube, centrifuge at room temperature at 900rpm for 10 minutes, and discard the supernatant. After resuspending the cells with DMEM culture medium containing 10% serum, culture them in a 37°C, 5% CO2 incubator for 1 hour for differential culture. In this process, myocardial fibroblasts are the first to adhere to the wall. After collecting the non-adherent cells, centrifuge again, resuspend them in ECM medium (endothelial cell medium (ECM) purchased from Sciencell, USA) and transfer them to a culture dish pre-coated with gelatin for further culture. The next day, wash away the non-adherent cells and replace with new DMEM medium containing 10% serum for further culture.

[0046] Cell passaging: When the cells grow to 70%-80% of the culture dish, pass them. After washing twice with sterile PBS, add 1mL of 0.25% trypsin for digestion. When the cell morphology changes from tightly attached to the wall to a transparent round shape under the microscope, discard the trypsin solution and add 2mL of ECM culture medium to neutralize. Gently blow the cells and mix them, transfer them to a sterile centrifuge tube, centrifuge at 900rpm for 5 minutes at room temperature, add ECM culture medium to resuspend the cells, and obtain primary mouse myocardial microvascular endothelial cells (CMECs). Subsequently, the cells will be inoculated in the culture plate according to the experimental requirements for the next round of culture or experimental operation.

[0047] Example 3: Effects of IDH2-K272 lactylation on CMECs proliferation, migration and angiogenesis

[0048] (1) Construction of cell model

[0049] The primary mouse myocardial microvascular endothelial cells (CMECs) obtained in Example 2 can be used for cell experiments after 2-3 generations of culture. First, glucose powder was added to the low-glucose ECM medium (normal glucose, NG) with a glucose concentration of 5.5mmol / L to a final concentration of 25mmol / L, and filtered through a filter to ensure sterility, and configured into a high-glucose medium (high glucose, HG). In order to simulate the environment of CMECs in the infarct marginal zone tissue, the cells were cultured in an incubator at 37°C, 5% CO2, and 95% N2 to achieve oxygen deprivation (oxygen deprivation, OD), and were divided into a low-glucose hypoxia (normal glucose oxygen deprivation, NOD) group and a high-glucose hypoxia (high glucose oxygen deprivation, HOD) group.

[0050] (2) Experiment on the effect of IDH2-K272 lactylation on CMECs proliferation and angiogenesis

[0051] In order to evaluate the effect of IDH2-K272 lactylation on CMECs function, this example adopts the plasmid transfection method, and the mutant plasmid IDH2-K272R (K272 site lysine is mutated to arginine (K272R)) is transferred into CMECs cells, so that the K272 site of IDH2 loses lactylation activity, and the non-mutant plasmid IDH2-WT (i.e., IDH2 site 272 is not mutated) is transferred as a control group. Both plasmids are constructed by Beijing Hesheng Gene Technology Co., Ltd. (the construction method is a well-known public method). Each group of cells was cultured in an incubator at 37°C, 5% CO2, and 95% N2 for 48 hours.

[0052] In this embodiment, several experimental groups are obtained:

[0053] The CMECs cells in the low-glucose hypoxia group transferred with IDH2-WT plasmid were recorded as WT+NOD; the CMECs cells in the high-glucose hypoxia group transferred with IDH2-WT plasmid were recorded as WT+HOD; the CMECs cells in the low-glucose hypoxia group transferred with IDH2-K272R plasmid were recorded as K272R+NOD; the CMECs cells in the high-glucose hypoxia group transferred with IDH2-K272R plasmid were recorded as K272R+HOD.

[0054] The effects of IDH2-K272 lactylation on the proliferation, migration and angiogenesis of CMECs were further investigated through Edu cell proliferation and tube formation experiments.

[0055] The Edu cell proliferation experiment process is as follows:

[0056] The EdU cell proliferation kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was used to detect the cell proliferation of CMECs cells after different group treatments.

[0057] First, the EdU preservation solution was diluted with DMEM medium to 2× working solution (20 μmol / L) and preheated in a 37°C incubator. Then, EdU 2× working solution was mixed with DMEM medium at a ratio of 1:1 and added to the cells after different grouping treatments, so that the final concentration of 2× working solution was 1× working solution (10 μmol / L). After incubation for 4 hours, wash with PBS solution. Next, fix with 4% paraformaldehyde solution for half an hour and wash with PBS solution. Then add 0.25% Triton X-100 and incubate at room temperature for 15 minutes. After washing, add freshly prepared Click reaction solution (provided in the EdU cell proliferation kit) and mix well. After incubation in the dark for 30 minutes, wash with PBS solution. Finally, add freshly prepared 1× Hoechst solution (provided in the EdU cell proliferation kit) to stain the nucleus and incubate at room temperature for 10 minutes. After thorough washing, take pictures under a fluorescence microscope for detection.

[0058] The process of tube formation experiment is as follows:

[0059] One day in advance, pre-cool the 96-well plate and 100 μL yellow pipette tip at 4°C, and move the packaged Matrigel matrix gel (purchased from Corning, USA) from -20°C to 4°C to thaw. Place the pre-cooled 96-well plate on ice, use the pre-cooled 100 μL yellow pipette tip to draw 75 μL of matrix gel, and slowly inject it into the bottom of the 96-well plate. The entire operation process is performed on ice. After equilibration at room temperature for 10 minutes, transfer to a 37°C incubator for 30 minutes to allow the matrix gel to solidify. While waiting for the matrix gel to solidify, the CMECs cells treated in different groups were digested with 0.25% trypsin solution, and the cell pellets were collected after neutralization with ECM culture medium and centrifugation, and 3×10 5 Resuspend the cells in ECM medium at a concentration of 10 cells / mL. Take 100 μL of cell suspension per well and gently add it to the solidified matrix gel in the 96-well plate along the well wall. Be careful to operate gently to avoid destroying the solidified matrix gel. Then, transfer the culture plate to a 37°C incubator and observe the lumen formation of CMECs under a microscope every hour.

[0060] The results of Edu cell proliferation and tube formation verification were as follows Figure 3 As shown, Figure 3 A is the result of detecting the proliferation ability of endothelial cells (CMECs) transferred with non-mutant plasmid IDH2-WT or mutant plasmid IDH2-K272R by Edu experiment. Figure 3B is the result of detecting angiogenesis ability through lumen formation experiment. Figure 3 In A, it can be observed that the high-sugar environment reduced the proliferation ability of CMECs under hypoxic conditions; and after the introduction of the mutant plasmid IDH2-K272R, the proliferation ability of CMECs further decreased.

[0061] In addition, from Figure 3 It can be observed in B that the high-glucose environment reduced the angiogenesis ability of CMECs under hypoxic conditions. After the mutant plasmid IDH2-K272R was transferred, the lumen formation ability of CMECs also decreased significantly, indicating that IDH2 lactylation plays an important role in regulating the angiogenesis ability of CMECs under high-glucose hypoxic environment.

[0062] These results illustrate the key role of IDH2-K272 lactylation in CMECs' resistance to the decreased proliferation ability and reduced angiogenesis caused by high glucose environment, and further illustrate that inhibiting IDH2-K272 lactylation reduces the proliferation ability and angiogenesis of CMECs in a high glucose environment. It also shows that IDH2-K272 lactylation can promote the proliferation ability and angiogenesis of CMECs in a high glucose environment.

[0063] In order to explore whether empagliflozin affects IDH2 lactation, three classic drugs were selected in the examples, namely canagliflozin (CANA), empagliflozin (EMPA) and dapagliflozin (DAPA), and their effects on the expression level of IDH2-K272 lactation were verified. DMSO, canagliflozin (CANA, 20 μM), empagliflozin (EMPA, 1 μM) and dapagliflozin (DAPA, 20 μM) were added to the high glucose hypoxia group (HOD), and the expression levels of IDH2 and IDH2-K272R proteins were detected, n=6; during the experiment, Tubulin antibody (AC007, Abclonal, China), IDH2 antibody (A7190, Abclonal, China), and IDH2-K272la (IDH2-K272 lactation) antibody were used for Western Blot experiments. The results showed that none of the three SGLT-2 inhibitors could affect the expression of IDH2 protein, but empagliflozin and dapagliflozin could promote the upregulation of IDH2 lactic acid levels, among which empagliflozin promoted the increase of IDH2 lactic acid modification most significantly ( Figure 4 ), therefore, the next study selected empagliflozin to further examine its effects on IDH2 lactic acidosis and cardiac function after diabetic myocardial infarction.

[0064] Further, in order to investigate the effect of empagliflozin on lactic acidification, in this embodiment, experiments were conducted by adding empagliflozin (purchased from MedChemExpress, USA, with a final concentration of 1 μM in each group) to the experimental group. The high-glucose hypoxia group CMECs cells with the mutant plasmid IDH2-K272R were transferred, and the experimental group with empagliflozin was added, recorded as HOD+EMPA; an equal volume of DMSO was added to the control group, recorded as HOD+DMSO. Each group of cells was cultured in an incubator at 37°C, 5% CO2, and 95% N2 for 24 hours. At the same time, the effect of empagliflozin on IDH2-K272 lactic acidification was investigated by Edu cell proliferation experiment and lumen formation experiment.

[0065] The results are as follows Figure 5 As shown, it is the effect of empagliflozin on the lactic acidification of IDH2-K272. Figure 5 A is the protein expression levels of eNOS and p-eNOS detected by Western Blot after adding DMSO and EMPA to the high-glucose hypoxia group into which the mutant plasmid IDH2-K272R was transferred. The changes in the activity level of eNOS in CMECs were reflected by calculating the protein expression level ratio of p-eNOS / eNOS. Figure 5 B is a Western Blot experiment to detect the changes in the lactic acid modification level of IDH2-K272 in CMECs transferred with non-mutant plasmid IDH2-WT or mutant plasmid IDH2-K272R under the conditions of low-glucose hypoxia group (NOD), high-glucose hypoxia group (HOD) and high-glucose hypoxia group with empagliflozin (EMPA) (HOD+EMPA); Figure 5 C is the detection of lumen formation, Edu proliferation and NO production ability of CMECs after adding DMSO and EMPA to the high-glucose hypoxia group transformed with mutant plasmid IDH2-K272R.

[0066] Among them, the detection method of NO generation ability described in this embodiment is as follows: CMECs cells in the high-glucose hypoxia group transferred with the mutant plasmid IDH2-K272R are added with DMSO and EMPA respectively, and then washed with PBS solution, and a fresh DAF-FM DA solution is prepared using DMEM medium without serum and phenol red, so that the final concentration of DAF-FM DA is 5 μmmol / L, and it is added to the culture plate to ensure that the cells in the culture plate can be covered. After incubating the culture plate in an incubator for 20 minutes, it is washed with PBS solution. Finally, the fluorescence intensity of the probe is observed under a fluorescence microscope to determine the activity of NO.

[0067] from Figure 5The protein expression level ratio of p-eNOS / eNOS in A represents the change of eNOS activity (eNOS activity is crucial to maintain the angiogenesis function of endothelial cells, and its phosphorylation is one of the core pathways to regulate eNOS activity). It can be observed that the eNOS activity of CMECs cells in the high-glucose hypoxia group with the addition of EMPA was significantly higher than that in the control group with the addition of DMSO, indicating that empagliflozin can restore the damage of eNOS activity caused by high-glucose hypoxia in CMECs cells; Figure 5 In B, it can be observed that under high-glucose hypoxia, the addition of empagliflozin to the CMECs cells in the high-glucose hypoxia group that were transferred with the non-mutant plasmid IDH2-WT can further promote the lactic acidation of IDH2-K272 (denoted as IDH2-K272la), but the addition of empagliflozin to the CMECs cells in the high-glucose hypoxia group that were transferred with the mutant plasmid IDH2-K272R could not restore the decrease in the lactic acidation level of IDH2-K272 caused by the site mutation, indicating that empagliflozin can promote the lactic acidation of IDH2, and the site that promotes lactic acidation is the lysine residue at position 272 of the IDH2 protein. In addition, as Figure 5 As shown in C, empagliflozin can promote endothelial cell proliferation, lumen formation and NO production, indicating that empagliflozin has a protective effect on CMECs under high glucose hypoxia.

[0068] Example 4: Empagliflozin promotes angiogenesis and alleviates cardiac function damage after diabetic myocardial infarction

[0069] The construction of the diabetic mouse model and the myocardial infarction model were the same as in Example 1.

[0070] Empagliflozin was administered orally to diabetic myocardial infarction mice (DMI) at a dose of 30 mg / kg / day. This group of mice was designated as DMI+EMPA.

[0071] (1) Mouse heart ultrasound detection

[0072] Mice in the MI, DMI, and DMI+EMPA groups were anesthetized with 2% isoflurane and placed on the testing table. After ensuring that the heart rate of the mice was maintained in the range of 400-550 beats per minute, cardiac ultrasound examination was performed. The position of the ultrasound probe was adjusted, the M-mode ultrasound position was determined, and the left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs) were measured. Three cardiac cycles were measured continuously, and the average value was calculated to evaluate the cardiac function of the mice.

[0073] Mouse echocardiography results Figure 6 As shown in the figure, it can be observed that the impaired cardiac function of mice in the DMI+EMPA group was restored. The changes in EF, FS, LVIDd and LVIDs values ​​of mice in the DMI+EMPA group compared with those of mice in the DMI group indicated that empagliflozin could restore the impaired EF and FS values ​​of mice with diabetic myocardial infarction and downregulate the increased LVIDd and LVIDs.

[0074] (2) Mouse arterial ring experiment

[0075] After euthanasia by excessive carbon dioxide in the MI, DMI, and DMI+EMPA groups, the mice were immersed in 75% alcohol for 10 minutes to reduce contamination. Then, the mice were fixed on the operating platform in a supine position, and an incision was made along the midline of the abdomen and extended to the neck. The subcutaneous tissue and muscle were removed, the sternum was cut, and the diaphragm and ribs were cut to expose the chest cavity. The lung tissue was removed, the esophagus was cut, and sterile PBS was injected into the apex of the heart with a 5mL sterile syringe to irrigate the blood vessels and remove residual blood. Sterile ophthalmic surgical instruments (including ophthalmic scissors, ophthalmic forceps, etc., purchased from Shanghai Medical Device Co., Ltd.) were used to remove the fat and connective tissue around the artery and completely expose the aorta. The aorta was transferred to sterile PBS for rinsing until the blood vessels were colorless and transparent, and then placed in a culture dish containing complete culture medium (complete culture medium is DMEM culture medium containing 10% fetal bovine serum), the arterial vascular adventitia was peeled off, and cut into rings of about 1 mm. Place the arterial ring in a culture plate containing serum-free Opti-MEM medium (purchased from Gibco, USA) and starve overnight. The next day, prepare type I rat tail collagen for plating, mix 200 μL type I rat tail collagen with 690 μL sterile ddH2O, stir quickly and add 10×PBS solution to make the final concentration of type I rat tail collagen 2 mg / mL. Add the collagen solution to a 96-well plate, 60 μL per well, avoiding bubbles. Place the arterial ring tissue in the well to ensure that it is embedded in the collagen solution, place it at room temperature for 15 minutes, and then place it in a 37°C incubator to solidify for 1 hour. After the collagen solution solidifies, add DMEM medium containing 2.5% FBS, and observe the budding of the arterial ring under a microscope on the 7th day.

[0076] The results of the arterial ring experiment are as follows Figure 7 As shown in the figure, it can be seen that the number of new blood vessel sprouts on the aortic ring in the DMI+EMPA group was significantly higher than that in the DMI group, indicating that empagliflozin can promote angiogenesis in diabetic myocardial infarction mice.

[0077] (3) Masson staining analysis

[0078] The myocardial tissues of mice in the MI, DMI, and DMI+EMPA groups were immersed in 4% paraformaldehyde solution and fixed in a 4°C refrigerator for 24-48 hours. After fixation, rinse with running water 3 times, 5 minutes each time. Then dehydrate, and soak the myocardial tissue in gradient ethanol solution (70%, 80%, 90%, 95%, 95%, 100%, 100%) and xylene in turn, soaking for 1 hour at each concentration. Subsequently, the tissue was immersed in a paraffin solution preheated to 55°C for 3 times, 30 minutes to 1 hour each time, to ensure that the paraffin completely replaced the xylene. For tissue embedding, the wax-soaked tissue was placed in a mold containing wax liquid, and the embedding machine was used to operate to ensure that the cut surface was parallel to the bottom. Move the embedded mold to a -10°C freezer to cool for half an hour. After removing the iron trough base, refrigerate the wax block in a 4°C refrigerator overnight. The paraffin sections were cut into about 4-6 microns, and carefully transferred to clean water at 38-40℃ using a brush. After the sections were unfolded, the fragments with complete shapes and no knife marks were selected and processed with adhesive slides. Each slide can hold 2 sections, and the sections were placed in the center of the slide. After the slides were heated in an oven at 65℃ for 2 hours, paraffin sections were made from the hearts of mice in each group, which could be stored at room temperature for later use.

[0079] The paraffin sections made from the hearts of mice in each group were placed in xylene twice for 5-7 minutes each time for dewaxing. Then, the sections were hydrated: sequentially passed through gradient ethanol solutions (100%, 100%, 95%, 85%, 80%, 75%) and PBS, each concentration for 5 minutes. Then the sections were immersed in distilled water for 2 times to prevent the slices from drying. Freshly prepared iron hematoxylin solution was dripped on the sections, left to stand for 10 minutes, then treated with differentiation solution for 10 seconds, then washed with double distilled water for 1 minute, returned to blue with blue solution for 5 minutes, then rinsed the surface liquid with running water, and washed with distilled water for 2 minutes. After washing, the sections were placed in a Ponceau fuchsin solution for staining for 12 minutes, and then washed with a 0.1% acetic acid solution for 1 minute. Next, the sections were washed with a phosphomolybdic acid solution for 2 minutes, then directly placed in an aniline blue solution for staining for 1 minute, rinsed with a 0.1% acetic acid solution, and finally observed under a microscope.

[0080] Masson staining results Figure 8 As shown in the figure, it can be seen that the collagen content in the DMI+EMPA group was significantly lower than that in the DMI group, indicating that empagliflozin can reduce the collagen content in the infarct edge zone of diabetic myocardial infarction mice, thereby alleviating myocardial fibrosis.

[0081] The above embodiments describe the basic principles and main features of the present invention and the advantages of the present invention. The present invention is not limited to the details of the above embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. The embodiments described are exemplary and not restrictive.

Claims

1. Use of empagliflozin in promoting protein lactic acidification and angiogenesis.

2. The use according to claim 1, characterized in that The protein lactylation includes the protein IDH2.

3. The use according to claim 1 or 2, characterized in that The protein lactylation includes the lactylation of the K272 site of IDH2.

4. The use according to claim 1, characterized in that The angiogenesis includes angiogenesis after myocardial infarction.

5. A drug for promoting protein lactic acidification, characterized in that: The drugs include empagliflozin.

6. A drug for promoting angiogenesis, characterized in that: The drugs include empagliflozin.

7. Use of the drug according to claim 5 or claim 6 in the preparation of a drug for promoting the recovery of the prognosis of myocardial infarction.

8. The use according to claim 7, characterized in that: The myocardial infarction is diabetic myocardial infarction.

9. The use according to claim 7, characterized in that: The prognosis restoration includes promoting angiogenesis and / or alleviating cardiac function damage.

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