Application of exosome in preparation of medicine for treating myocardial injury

Abstract

The invention discloses application of an exosome in preparation of a medicine for treating myocardial injury, and belongs to the technical field of medicines. The exosome is derived from spleen tissues. The exosome can target the heart, is effectively enriched in ischemic myocardium, can significantly improve myocardial cell energy metabolism by enriching mitochondrial oxidative phosphorylation pathway key proteins, and provides an efficient and high-specificity solution for damage treatment of myocardium.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to the use of exosomes in the preparation of drugs for treating myocardial injury. Background Art

[0002] Acute myocardial infarction (AMI) is a severe cardiovascular disease caused by acute coronary artery occlusion, leading to myocardial ischemia, hypoxia, and necrosis. It is characterized by high morbidity, mortality, and disability. Although early reperfusion therapies (such as percutaneous coronary intervention and thrombolytic therapy) can partially restore blood flow, the necrotic myocardium cannot regenerate, and the oxidative stress, inflammatory response, and ventricular remodeling triggered by reperfusion may lead to heart failure, seriously compromising patient prognosis.

[0003] Mitochondrial oxidative phosphorylation dysfunction is a core mechanism of cardiomyocyte apoptosis after acute myocardial infarction (AMI). Ischemia-reperfusion injury leads to downregulation of key mitochondrial electron transport chain (ETC) proteins (such as COX5A, ATP5A1, and SDHB), reduced ATP synthesis, and a massive accumulation of reactive oxygen species (ROS), ultimately triggering cardiomyocyte apoptosis. While therapeutic strategies such as antioxidants and energy metabolism regulators can alleviate these issues, they lack targeted delivery methods and are unable to effectively restore mitochondrial structure and function.

[0004] Exosome therapy has become a research hotspot due to its low immunogenicity and molecular regulatory capabilities. However, the mechanisms by which exosomes improve myocardial energy metabolism disorders have yet to be systematically elucidated. Traditional exosomes derived from mesenchymal stem cells, cardiac progenitor cells, and other sources have the following limitations: they cannot target the heart, are easily cleared by the mononuclear phagocytic system (MPS), and have a low accumulation rate (<5%) in the infarcted area. To improve the targeting and delivery efficiency of exosomes, artificial modification (such as conjugation with targeting peptides) is required, which increases production costs and safety risks. Summary of the Invention

[0005] Based on this, the main purpose of this application is to provide the use of exosomes derived from spleen tissue in the preparation of a drug for treating myocardial injury. The exosomes can target the heart and effectively accumulate in ischemic myocardium. By enriching key proteins in the mitochondrial oxidative phosphorylation pathway, they can significantly improve myocardial cell energy metabolism, providing an efficient and highly specific solution for the treatment of damaged myocardium.

[0006] In a first aspect of the present application, there is provided a use of exosomes in the preparation of a drug for treating myocardial injury, wherein the exosomes are derived from spleen tissue.

[0007] In some embodiments, the spleen tissue is derived from a mammal; optionally, the mammal comprises at least one of a rodent, a cow, a pig, a sheep, a dog, a cat, and a horse; optionally, the rodent comprises at least one of a mouse and a rabbit; optionally, the mouse comprises at least one of a mouse, a rat, a hamster, and a guinea pig.

[0008] In some embodiments, the mammal is a mammal having myocardial infarction; optionally, the mammal is a mammal having acute myocardial infarction.

[0009] In some embodiments, the method for preparing the exosome comprises the following steps:

[0010] dissociating the spleen tissue to obtain a spleen tissue solution;

[0011] differential centrifugation of the spleen tissue solution to prepare the exosome.

[0012] In some embodiments, the step of dissociating the spleen tissue to obtain a spleen tissue solution comprises: incubating the spleen tissue with a collagenase-containing buffer solution, filtering, and collecting the spleen tissue solution.

[0013] In some embodiments, the method comprises one or more of the following features:

[0014] (1) the collagenase comprises a type I collagenase;

[0015] (2) the buffer solution comprises a phosphate buffer;

[0016] (3) the concentration of the collagenase in the collagenase-containing buffer solution is 0.05wt%-0.5wt%;

[0017] (4) the temperature of the incubation is 30°C-40°C;

[0018] (5) the time of the incubation is 20min-60min;

[0019] (6) the filtering uses a filter membrane with a pore size of 50μm-100μm;

[0020] (7) the mass-to-volume ratio of the spleen tissue to the collagenase-containing buffer solution is (5-50)mg:1mL.

[0021] In some embodiments, the step of differential centrifugation of the spleen tissue solution comprises: centrifuging the spleen tissue solution at 1°C-10°C, 200xg-400xg for 5min-20min to obtain a first supernatant;

[0022] The first supernatant was centrifuged at 1°C-10°C, 2000×g-4000×g for 10 min-30 min, and the second supernatant was collected;

[0023] The second supernatant was centrifuged at 1°C-10°C, 8000×g-12000×g for 20 min-40 min, and the third supernatant was collected;

[0024] The third supernatant is centrifuged at 1° C.-10° C. and 100,000×g-150,000×g for 1 h-4 h, and the precipitate is collected to obtain the exosomes.

[0025] In some embodiments, the step of performing differential centrifugation on the spleen tissue solution comprises: centrifuging the spleen tissue solution at 3.5° C.-4.5° C. and 280×g-320×g for 9 min-11 min, collecting a first supernatant;

[0026] The first supernatant was centrifuged at 3.5°C-4.5°C and 2900×g-3100×g for 19 min-21 min, and the second supernatant was collected;

[0027] The second supernatant was centrifuged at 3.5°C-4.5°C, 9000×g-11000×g for 29 min-31 min, and the third supernatant was collected;

[0028] The third supernatant was centrifuged at 3.5° C.-4.5° C. and 115,000×g-125,000×g for 110 min-130 min, and the precipitate was collected to obtain the exosomes.

[0029] In some embodiments, the myocardial injury comprises at least one of acute myocardial infarction, ischemic myocardial injury, coronary heart disease, coronary artery syndrome, and hypoxia-related heart disease.

[0030] In some embodiments, the drug for treating myocardial injury is a drug targeting the heart.

[0031] In some embodiments, the drug for treating myocardial injury is a drug that improves myocardial cell energy metabolism by enriching key proteins in the mitochondrial oxidative phosphorylation pathway.

[0032] In some embodiments, the treatment of myocardial injury includes at least one of reducing myocardial infarct size, increasing left ventricular ejection fraction, increasing left ventricular fractional shortening, increasing survival time, upregulating mitochondrial pyruvate transporter 1 expression, and improving energy metabolism in hypoxic myocardial cells.

[0033] The second aspect of the present application provides the use of exosomes in the preparation of a drug for treating myocardial injury, wherein the expression level of mitochondrial pyruvate transporter 1 in the exosomes is 6 ng / mL-50 ng / mL; optionally 18 ng / mL-50 ng / mL.

[0034] The third aspect of the present application provides the use of exosomes in targeting cardiac tissue for non-diagnostic and therapeutic purposes, wherein the exosomes are the exosomes described in the first aspect or the second aspect.

[0035] In a fourth aspect, the present application provides a drug for treating myocardial injury, comprising exosomes; the exosomes are the exosomes described in the first aspect or the second aspect.

[0036] Beneficial effects of this application:

[0037] This application provides the use of exosomes derived from spleen tissue in the preparation of a drug for treating myocardial injury. These exosomes can target the heart and effectively accumulate in ischemic myocardium. They can significantly improve myocardial cell energy metabolism by enriching key proteins in the mitochondrial oxidative phosphorylation pathway, providing a highly efficient and specific solution for treating myocardial injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following briefly introduces the drawings required for the embodiments or the prior art description. The drawings are only used to illustrate the preferred embodiments and are not considered to limit the present application. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0039] Figure 1 For the identification of spleen exosomes, A is the protein blotting result, B is the TEM result, C and D are the nanoflow cytometry results; Sham: sham-operated mice; MI: acute myocardial infarction mice; MI-EVs: spleen exosomes of acute myocardial infarction mice in Example 1; Sham-EVs: spleen exosomes of sham-operated mice in Example 2; Tissue is spleen tissue; Tissue-EVs are spleen tissue exosomes.

[0040] Figure 2 These are the results of in vivo animal tracing, where A shows the retention of DiR-traced spleen exosomes at different time points after myocardial infarction in mice; B shows the retention of DiR-traced spleen exosomes in different organs after myocardial infarction.

[0041] Figure 3The effects of spleen exosomes on myocardial injury in acute myocardial infarction mice, wherein A is the TTC staining detection of myocardial infarction area results, B-C is the results of cardiac ultrasound detection, and D is the survival curve; * indicates P<0.05 compared with the Sham+PBS group, n=6; # indicates P<0.05 compared with the MI+PBS group, n=6.

[0042] Figure 4 The mitochondrial pyruvate transporter 1 expression results of spleen exosomes. Among them, A is the expression up-regulated protein of the plasma of acute myocardial infarction mice for 3 days relative to the sham operation mice fed for 3 days (Plasma UP), and the intersection analysis results of the expression up-regulated protein of the exosomes of Example 1 relative to the exosomes of Example 2 (Spleen-EV UP); B is the MPC1 expression amount of the exosomes of Examples 1-2 detected by ELISA, * indicates P<0.05 compared with the Sham-EVs group, N=6; C is the effect of the exosomes of Examples 1-2 and MPC1 inhibitor on the ATP / ADP ratio of OGD-treated myocardial cells, * indicates P<0.05 compared with the OGD+Sham-EVs group, N=6, # indicates P<0.05 compared with the OGD+MI-EVs group, N=6. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present application more clear, and the disclosure of the present application more thorough and comprehensive, the technical scheme of the present application will be described in detail below with reference to the specific embodiments of the present application and the corresponding drawings. The described embodiments are only a part of the embodiments of the present application, not all the embodiments.

[0044] The embodiments of the present application will be described in detail below with reference to the drawings. The present embodiment is implemented on the premise of the technical scheme of the present application, and gives a detailed implementation manner and specific operation process, but the protection scope of the present application is not limited to the following embodiments.

[0045] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments of the present application, and are not intended to limit the present application.

[0046] Terminology

[0047] Unless otherwise stated or contradictory, the terms or phrases used in the present application have the following meanings:

[0048] In the present application, "a plurality of", "a plurality of", etc. are used without special limitation, which means more than two or equal to two in quantity. For example, "one or more", "at least one" means one or more than two.

[0049] In this application, "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0050] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0051] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" is broadly allowed to include numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0052] In this application, unless otherwise specified, temperature parameters may be either constant temperature or fluctuating within a certain temperature range. It should be understood that constant temperature processing allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0053] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0054] In this application, temperature parameters, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. The room temperature referred to in this application refers to 0°C-40°C, preferably 10°C-35°C, and more preferably 20°C-30°C.

[0055] In this application, "exosomes" are a type of tiny vesicles secreted by cells, typically with a diameter between 30 and 500 nm. They are secreted from cells to the extracellular space or have a membrane structure composed of a lipid bilayer present within the cell. Exosomes are primarily derived from intracellular multivesicular bodies (MVBs). When the MVBs fuse with the cell membrane, the exosomes are released into the extracellular environment.

[0056] In this application, "treatment" can refer to therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (mitigate) the targeted pathological condition or disease. Subjects in need of treatment include subjects who already have a condition, as well as subjects who have a tendency to have a disease, or subjects in need of disease prevention. The therapeutic effect can refer to the eradication or improvement of symptoms or the underlying disease being treated. The preventive effect can include delaying, preventing or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping or reversing the progression of a disease or condition, or any combination thereof. The subject can be a mammal, including humans and non-human mammals.

[0057] In a first aspect of the present application, an exosome is provided, wherein the exosome is derived from spleen tissue.

[0058] Exosomes derived from spleen tissue can target the heart and effectively accumulate in ischemic myocardium. They have excellent heart-targeting delivery efficiency. They can significantly improve myocardial cell energy metabolism by enriching key proteins in the mitochondrial oxidative phosphorylation pathway, providing an efficient and highly specific solution for the treatment of damaged myocardium.

[0059] In some specific examples, the spleen tissue is derived from a mammal; optionally, the mammal includes at least one of a rodent, a cow, a pig, a sheep, a dog, a cat and a horse; optionally, the rodent includes at least one of a mouse and a rabbit; optionally, the mouse includes at least one of a mouse, a rat, a hamster and a guinea pig.

[0060] It is understandable that spleen tissues of different animals have similar functions and structures, and the present application is not limited to spleen tissues of mice.

[0061] In some embodiments, the mammal is a mammal suffering from myocardial infarction; optionally, a mammal suffering from acute myocardial infarction.

[0062] In some embodiments, the method for preparing exosomes comprises the following steps:

[0063] The spleen tissue was dissociated and the spleen tissue solution was collected;

[0064] The spleen tissue solution was subjected to differential centrifugation to prepare the exosomes.

[0065] In some specific examples, the steps of dissociating the spleen tissue and collecting the spleen tissue solution include: incubating the spleen tissue with a buffer solution containing collagenase, filtering, and collecting the spleen tissue solution.

[0066] In some embodiments, the collagenase comprises type I collagenase.

[0067] In some specific examples, the buffer solution includes phosphate buffer.

[0068] In some specific examples, the concentration of collagenase in the collagenase-containing buffer solution is 0.05wt%-0.5wt%, for example, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, etc.

[0069] In some specific examples, the incubation temperature is 30°C-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0070] In some specific examples, the incubation time is 20 min-60 min, for example, 20 min, 30 min, 40 min, 50 min, 60 min, etc.

[0071] In some specific examples, the filtration uses a filter membrane of 50 μm-100 μm, for example, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, etc.

[0072] In some specific examples, the mass volume ratio of the spleen tissue to the collagenase-containing buffer solution is (5-50) mg:1 mL, for example, 5 mg:1 mL, 10 mg:1 mL, 15 mg:1 mL, 20 mg:1 mL, 25 mg:1 mL, 30 mg:1 mL, 35 mg:1 mL, 40 mg:1 mL, 45 mg:1 mL, 50 mg:1 mL, etc.

[0073] In some specific examples, the step of performing differential centrifugation on the spleen tissue solution includes: centrifuging the spleen tissue solution at 1°C-10°C (e.g., 1°C, 2°C, 4°C, 6°C, 8°C, 10°C) and 200×g-400×g (e.g., 200×g, 250×g, 300×g, 350×g, 400×g) for 5 min-20 min (e.g., 5 min, 10 min, 15 min, 20 min), and collecting a first supernatant;

[0074] centrifuging the first supernatant at 1°C-10°C (e.g., 1°C, 2°C, 4°C, 6°C, 8°C, 10°C) and 2000×g-4000×g (e.g., 2000×g, 2500×g, 3000×g, 3500×g, 4000×g) for 10 min-30 min (e.g., 10 min, 15 min, 20 min, 25 min, 30 min), and collecting the second supernatant;

[0075] centrifuging the second supernatant at 1°C-10°C (e.g., 1°C, 2°C, 4°C, 6°C, 8°C, 10°C) and 8000×g-12000×g (e.g., 8000×g, 9000×g, 10000×g, 11000×g, 12000×g) for 20 min-40 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min), and collecting the third supernatant;

[0076] The third supernatant is centrifuged at 1°C-10°C (e.g., 1°C, 2°C, 4°C, 6°C, 8°C, 10°C) and 100,000×g-150,000×g (e.g., 100,000×g, 110,000×g, 120,000×g, 130,000×g, 1400,000×g, 150,000×g) for 1 h-4 h (1 h, 2 h, 3 h, 4 h), and the precipitate is collected to obtain the exosomes.

[0077] In some specific examples, after collecting the precipitate, the step of washing the precipitate with a buffer solution and centrifuging the precipitate at 1°C-10°C (e.g., 1°C, 2°C, 4°C, 6°C, 8°C, 10°C) and 100,000×g-150,000×g (e.g., 100,000×g, 110,000×g, 120,000×g, 130,000×g, 1400,000×g, 150,000×g) for 1h-4h (1h, 2h, 3h, 4h).

[0078] In some specific examples, the step of performing differential centrifugation on the spleen tissue solution includes: centrifuging the spleen tissue solution at 3.5° C.-4.5° C. and 280×g-320×g for 9 min-11 min, collecting a first supernatant;

[0079] The first supernatant was centrifuged at 3.5°C-4.5°C and 2900×g-3100×g for 19 min-21 min, and the second supernatant was collected;

[0080] The second supernatant was centrifuged at 3.5°C-4.5°C, 9000×g-11000×g for 29 min-31 min, and the third supernatant was collected;

[0081] The third supernatant was centrifuged at 3.5° C.-4.5° C. and 115,000×g-125,000×g for 110 min-130 min, and the precipitate was collected to obtain the exosomes.

[0082] In some embodiments, the myocardial injury comprises at least one of acute myocardial infarction, ischemic myocardial injury, coronary heart disease, coronary artery syndrome, and hypoxia-related heart disease.

[0083] In some embodiments, the drug for treating myocardial injury is a drug targeting the heart.

[0084] In some embodiments, the drug for treating myocardial injury is a drug for improving energy metabolism of myocardial cells by enriching key proteins in the mitochondrial oxidative phosphorylation pathway.

[0085] In some embodiments, the treatment of myocardial injury comprises at least one of reducing the size of a cardiac infarction, increasing left ventricular ejection fraction, increasing left ventricular fractional shortening, increasing survival time, up-regulating mitochondrial pyruvate transporter 1 expression, and improving energy metabolism of hypoxic myocardial cells.

[0086] In a second aspect of the present application, use of exosomes in the preparation of a drug for treating myocardial injury is provided, wherein the expression amount of mitochondrial pyruvate transporter 1 in the exosomes is 6 ng / mL-50 ng / mL, optionally 18 ng / mL-50 ng / mL, such as 6 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc.

[0087] The expression of mitochondrial pyruvate transporter 1 is down-regulated in the heart of patients and rodents with heart failure, up-regulated in surviving myocardium after ischemia-reperfusion in mice, and the reduction of mitochondrial pyruvate transporter 1 exacerbates ischemic myocardial injury, indicating that mitochondrial pyruvate transporter 1 can be a new target for intervention in ischemia-related heart disease.

[0088] The above exosomes can effectively express mitochondrial pyruvate transporter 1, solve mitochondrial dysfunction, significantly improve energy metabolism of myocardial cells by enriching key proteins in the mitochondrial oxidative phosphorylation pathway, and provide an efficient and specific solution for treating injured myocardium. In addition, the above exosomes can be targeted to the heart and effectively enriched in ischemic myocardium, and have excellent delivery efficiency for targeting the heart.

[0089] In a third aspect of the present application, use of exosomes in non-diagnostic and therapeutic purposes for targeting cardiac tissue is provided, wherein the exosomes are the exosomes of the first aspect or the second aspect.

[0090] In a fourth aspect of the present application, a drug for treating myocardial injury is provided, comprising exosomes, wherein the exosomes are the exosomes of the first aspect or the second aspect.

[0091] In a specific example, the drug comprises an excipient.

[0092] It can be understood that the solid dosage form includes powder, tablet, capsule, pill or granule; the semi-solid dosage form includes ointment, gel ointment or plaster; and the liquid dosage form includes solution, syrup, emulsion or tincture.

[0093] In addition to exosomes, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils. In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, a suspension may contain a suspending agent, specifically ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or combinations thereof.

[0094] In addition to exosomes, semi-solid dosage forms may include conventional matrices and excipients. Depending on the specific dosage form, the corresponding matrix or excipient can be selected. For example, common matrices for ointments include vaseline, lard, lanolin, etc., and common matrices and excipients for pastes include glycerin, liquid paraffin, etc.; other active ingredients can be appropriately added to extracts; common matrices for licking agents include starch, syrup, vegetable oil, etc.

[0095] In addition to exosomes, solid dosage forms may contain conventional inert excipients such as: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, pellets, capsules, pills, or granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and release of the exosomes or compounds in such compositions can be delayed in a specific portion of the digestive tract. Examples of possible encapsulating components include polymeric substances and waxes. If desired, the exosomes can also be microencapsulated with one or more of the above-mentioned excipients.

[0096] In addition to exosomes, the drugs of the present application may also include drugs for treating myocardial injury, such as aspirin, P2Y12 receptor inhibitors, heparin, amiodarone, lidocaine, etc.

[0097] The drugs of the present application can be administered to mammals such as mice, livestock, and humans via a variety of routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardial, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. Oral administration is preferred.

[0098] Unless otherwise specified in this application, the raw materials used in the following experiments can be purchased from the market.

[0099] The following are specific examples.

[0100] Example 1

[0101] 1. Preparation of Mouse Acute Myocardial Infarction (AMI) Model

[0102] C57BL / 6 mice aged 6 to 8 weeks were used and anesthesia was induced with 5% isoflurane. The mice were fixed on the operating table in a supine position, the precordial area was depilated, and the area was disinfected with 75% alcohol cotton balls. Under the irradiation of a cold light source, the tongue of the mouse was fixed with a tongue clamp, and then an endotracheal tube was used to perform oral tracheal intubation, and the small animal ventilator was connected for assisted ventilation. Anesthesia was maintained at a concentration of 2% isoflurane, and the ventilator parameters were set to a tidal volume of 2.0 ml / time and a respiratory rate of 100 times / min for assisted ventilation. Before surgery, the electrocardiogram of the mouse was recorded. Next, a sterile blade was used to incise the skin at the second intercostal space on the left side of the sternum, and hemostats were used to bluntly separate the subcutaneous tissue, pectoralis major and pectoralis minor muscles, and the intercostal space was bluntly expanded to expose the heart. An 8-0 suture needle was used to pass through the 1 / 3 myocardial layer 1-2 mm below the lower edge of the left atrial appendage, and the left anterior descending coronary artery was ligated. At this point, ST segment elevation can be observed on the electrocardiogram, and the myocardium turns white after ligation, contractility is weakened, and the ST segment elevation exceeds 1 / 2 R wave for 5 minutes to confirm the success of the model. Within 24 hours after surgery, the animal's physiological indicators are continuously monitored to ensure that they have returned to normal.

[0103] 2. Preparation of exosomes:

[0104] A) The spleen of a mouse with acute myocardial infarction (AMI) 3 days after myocardial infarction (MI) prepared in step 1 was isolated and lavaged with cold phosphate-buffered saline (PBS, pH 7.2-7.4). The spleen was then minced into small pieces and transferred to a centrifuge tube containing 0.1 wt% collagenase type I in PBS (pH 7.2-7.4). The weight-to-volume ratio of mouse spleen to PBS containing 0.1 wt% collagenase type I was 10 mg:1 mL.

[0105] B) Incubate on a shaker at 37°C for 30 minutes, gently pipetting the tissue up and down twice every 10 minutes. After dissociation is complete, filter the dissociation solution through a 70 μm filter to remove large tissue fragments and collect the spleen tissue solution.

[0106] C) Centrifuge the spleen tissue solution at 300 × g for 10 min at 4°C and collect the first supernatant (this pellet contains cell debris and nuclei);

[0107] D) Transfer the first supernatant to a new tube and centrifuge at 3000 × g for 20 min at 4°C to collect the second supernatant (this pellet contains apoptotic bodies and microsomes).

[0108] E) Transfer the second supernatant to a new tube and centrifuge at 10,000 × g for 30 min at 4°C to collect the third supernatant (this pellet contains large EVs and plasma membranes);

[0109] F) Transfer the third supernatant to a new tube and ultracentrifuge at 120,000 × g for 2 h at 4°C to collect the precipitate.

[0110] G) The pellet was then washed with PBS and ultracentrifuged at 120,000 × g at 4°C for 2 h to obtain exosomes (denoted as MI-EVs).

[0111] Example 2

[0112] The same procedures as in Example 1 were used except that spleen cells from sham-operated mice were used. Exosomes were designated as Sham-EVs. The preparation process of sham-operated mice was the same as in Example 1 except that the left anterior descending coronary artery was not ligated.

[0113] Test Case

[0114] Performance characterization of the exosomes of Example 1-2

[0115] 1. Exosome Particle Size Distribution and Particle Concentration Measurement

[0116] A) Standard polystyrene nanoparticles (250 nm) were loaded into the nanoflow cytometer.

[0117] B) The protein concentration of the exosome sample was determined using a BCA protein assay kit. Based on the BCA protein assay results, the exosome sample was diluted with PBS (to a concentration of 1 ng / μL-10 ng / μL), then loaded into the nanoflow cytometer and the side scatter intensity was measured.

[0118] C) Exosome concentration was calculated based on the ratio of SSI to particle concentration in standard polystyrene nanoparticles. Additionally, standard silica nanoparticles with a gradient size range (68 nm, 91 nm, 113 nm, 155 nm) were loaded into the nanoflow cytometer to generate a standard curve.

[0119] See the results Figure 1C. It can be seen that the diameter of the isolated exosomes ranges from 30 nm to 150 nm, and the diameter of the isolated particles is mainly distributed in the range of 50 nm to 80 nm.

[0120] 2. Transmission electron microscopy characterization of exosomes

[0121] A) Exosomes (2 μg / μL-6 μg / μL) were fixed with 2% electron microscopy (EM)-grade paraformaldehyde for 2 hours at room temperature. 10 μL of the exosome solution was placed on a copper grid and incubated at room temperature for 10 minutes. The grid was then rinsed with sterile distilled water and the excess liquid was blotted off with absorbent paper.

[0122] B) Copper grids were stained with 2% uranyl acetate for 1 minute, excess liquid was blotted with filter paper, and then dried under an incandescent lamp for 2 minutes.

[0123] C) Transmission electron microscopy observation of the prepared sample at 80 kV.

[0124] D) TEM is used to determine whether the extracted exosomes have vesicular characteristics. Exosome samples must be fresh or stored at 4°C for a short period of time.

[0125] See the results Figure 1 As shown in Figure B, the exosomes in Examples 1-2 all showed the presence of cup-shaped vesicles, and these vesicles had a clear lipid bilayer structure, confirming that the exosomes were successfully isolated from the spleen tissue.

[0126] 3. Western Blot

[0127] A) Protein lysis was performed by adding 100 μL of RIPA lysis buffer to the exosome pellet. The pellet was vortexed, rocked at room temperature for 20 minutes, and then vortexed again.

[0128] B) Briefly centrifuge at 1000 × g for 30–60 s to collect the sample, transfer to a new 1.5 mL microcentrifuge tube, and store at -20°C to -80°C until analysis.

[0129] C) For immunoblotting, samples were mixed with 5x loading buffer to a 1x concentration and prepared for gel electrophoresis.

[0130] D) If tissue or cell experiments are required, lyse the tissue or cells using a strong lysis buffer containing protease inhibitors. Then, centrifuge at 10,000 × g for 10 minutes to discard any remaining extracellular matrix, whole cells, or intact cell debris.

[0131] E) Boil the sample buffer containing exosomes or tissue homogenate at 95°C for 5-10 minutes. Load equal amounts of protein per lane onto a 10% SDS-PAGE gel. An equal amount of tissue homogenate protein was loaded as a control.

[0132] F) Gel electrophoresis was performed at 80 V for approximately 2 hours using standard electrophoresis buffer. Western blot analysis was performed to determine the expression of exosomal or cellular proteins in the purified lysates and to compare the relative abundance of proteins in each fraction.

[0133] See the results Figure 1 A, Western blotting detected exosome markers TSG101, CD63, and CD9 (present on the exosome membrane), while GM130 (a Golgi matrix protein used as a negative control) was not detected, indicating that the exosomes we isolated were of high purity and met the needs of subsequent experiments.

[0134] 4. Enrichment test of spleen exosomes in the heart

[0135] 4.1 Preparation of DiR-labeled spleen exosomes

[0136] A) Preparation of dye working solution: Dilute the exosome fluorescent dye (DiR) with 1X PBS to a concentration of 100 μM. It is recommended to prepare the dye working solution freshly to ensure activity.

[0137] B) DiR staining step: Add the dye working solution to the exosome suspension in Example 1. The recommended addition ratio is 10:1 between the volume of the exosome suspension and the volume of the dye working solution.

[0138] C) After adding the dye working solution, seal the tube and mix thoroughly using a vortex shaker for 1 minute. Then, incubate at 37°C for 30 minutes, protecting from light.

[0139] D) Washing and Purification: Washing and centrifugation are performed to remove unbound DiR dye and ensure the purity of the exosomes. DiR-labeled exosomes should be stored at -80°C.

[0140] 4.2 Heart enrichment experiment

[0141] DiR-labeled spleen exosomes were injected into mice with acute myocardial infarction via the tail vein, and the distribution of exosomes in various important organs in the body was monitored in real time (0h, 6h, 12h, 24h, 48h) using a small animal in vivo imaging system. The results are as follows Figure 2 As shown, it can be seen that the spleen exosomes of Example 1 mainly reside in the heart, indicating that the spleen exosomes can target and reach the heart of mice with acute myocardial infarction.

[0142] 5. Myocardial infarction area test, ultrasound detection and survival time test of mice by multi-point in situ injection

[0143] 5.1 Grouping and intervention

[0144] The mice were randomly divided into:

[0145] ① sham + PBS group: sham-operated mice were injected with PBS equivalent to the amount of exosomes in the myocardium.

[0146] ② MI + PBS group: mice were ligated with left anterior descending coronary artery, i.e. acute myocardial infarction mice were injected with PBS equivalent to the amount of exosomes in the myocardium.

[0147] ③ MI + sham-EVs group: mice were ligated with left anterior descending coronary artery, i.e. acute myocardial infarction mice were injected with exosomes of Example 2 (1.5 µg / µL protein concentration) in the myocardium.

[0148] ④ MI + MI-EVs group: mice were ligated with left anterior descending coronary artery, i.e. acute myocardial infarction mice were injected with exosomes of Example 1 (1.5 µg / µL protein concentration) in the myocardium.

[0149] Intervention process: The mouse modeling or sham operation process was as described in Examples 1-2. After the mouse was ligated with left anterior descending coronary artery or sham operation, a microsyringe (100 µL) was immediately used to inject the spleen exosomes or equivalent PBS into the myocardium at 3-5 equidistant sites on the surface of the heart, 5-10 µL per site, the protein concentration of the spleen exosomes was 1.5 µg / µL, a total of 20 µL was injected. After injection, the chest cavity was sutured layer by layer. After modeling, the mice were normally fed 6 h later.

[0150] 5.2 Ultrasound examination

[0151] The experimental mice were anesthetized on the 1st, 3rd, 7th, 14th and 28th day after modeling and intervention, and the heating platform was turned on to maintain the body temperature of the mice at about 37°C. The mouse abdomen without chest hair was placed on the operation table with the abdomen facing up, and the limbs were smeared with conductive medium, then the limbs were fixed in the corresponding position with tape. To ensure that the apex of the mouse's heart is level with the bottom, the operation table needs to be adjusted so that the mouse's head is slightly higher than the tail. At the same time, about 2 centimeters thick conductive glue is applied on the chest. When measuring, the ultrasound probe needs to be buried in the conductive glue.

[0152] A) Acquisition of parasternal long-axis B-mode ultrasound images. At this time, the structures visible include the left ventricle, the aorta, and the mitral valve leaflets. The probe should be positioned so that its notch is facing the mouse's head and rotated counterclockwise by 30° to 45°.

[0153] B) Acquisition of parasternal long-axis M-mode ultrasound images. The probe should also be positioned so that its notch is facing the mouse's head and rotated counterclockwise by 30° to 45°. When the anatomical structures are clear and the axis angle is correct, M-mode ultrasound measurements can be performed to more deeply assess the cardiac function of the left ventricle.

[0154] C) Acquisition of parasternal short axis B-mode ultrasound images. The probe is positioned by rotating 90° clockwise from the parasternal long axis view to the short axis view. At this point, the probe notch should be adjusted from facing the mouse head to facing the mouse left side to obtain a complete circular view of the left ventricle. The image quality can be optimized by rotating or tilting the probe.

[0155] D) Acquisition of parasternal short axis M-mode ultrasound images. The probe is positioned in the same way as for B-mode ultrasound, by rotating 90° clockwise from the parasternal long axis view to the short axis view, and the probe notch is facing the mouse left side to obtain a complete view of the left ventricle. At this point, the M-mode cursor should be placed in the parasternal short axis view to obtain the best left ventricular internal diameter measurements. These measurements will provide important data for ejection fraction, fractional shortening, and left ventricular volume calculations. The results of the ultrasound examination are shown in Figure 3 B and C.

[0156] 5.3 TTC staining to detect the area of cardiac infarction

[0157] The hearts were taken for TTC staining to detect the area of cardiac infarction at 24h after the acute myocardial infarction mouse model (modeling process as in Example 1) was sacrificed, the specific process is as follows:

[0158] A) Sample collection: The mouse was sacrificed quickly after anesthesia, and its heart tissue was collected within 10 minutes.

[0159] B) Freezing treatment: The collected heart was placed in a -80°C freezer for freezing treatment for 15 minutes.

[0160] C) Sample sectioning: After the heart tissue was taken out, it was cut into 5 pieces, each with a thickness of 1.5 millimeters.

[0161] D) Sample staining: The cut heart tissue was soaked in a 37°C TTC solution for 30 minutes in the dark.

[0162] E) Observation and recording: After staining, normal tissue appears red, while ischemic tissue is white.

[0163] 5.4 Survival time observation

[0164] The mice modeled and intervened in Section 5.1 were observed every 24 hours to see if they died, and the ratio of the number of mice that did not die each day to the total number of mice was the survival rate of the mice, and a total of 28 days of observation was conducted.

[0165] The results of the area of cardiac infarction are shown in Figure 3 A, the results of the ultrasound examination are shown in Figure 3 B and C, and the results of the survival time are shown in Figure 3D. It can be seen that the intervention of spleen exosomes in mice with myocardial infarction significantly reduced the myocardial infarction area of ​​mice with acute myocardial infarction, significantly improved the left ventricular ejection fraction and left ventricular short axis shortening fraction of mice with acute myocardial infarction, and significantly increased the survival time of mice with acute myocardial infarction.

[0166] 6. Expression of mitochondrial pyruvate transporter 1 in spleen exosomes

[0167] 6.1 Preparation of Cellular Oxygen and Glucose Deprivation (OGD) Model

[0168] A hypoxic model was established for H9C2 cells using a three-gas incubator (1% O₂, 5% CO₂, 94% N₂). One-day-old H9C2 cells were washed twice with phosphate-buffered saline (PBS, pH 7.2-7.4) and then incubated with sugar- and serum-free DMEM. The appropriate reagents and drugs were added according to the experimental group, and the cells were then cultured in the three-gas incubator.

[0169] 6.2 Expression of mitochondrial pyruvate transporter 1 in spleen exosomes

[0170] The expression of upregulated proteins (Plasma UP) in the plasma of acute myocardial infarction mice (modeling process see Example 1) 3 days after myocardial infarction was compared with that in sham-operated mice (preparation process see Example 2) after 3 days of feeding, and the expression of upregulated proteins (Spleen-EV UP) in the exosomes of Example 1 compared with that of the exosomes of Example 2 were analyzed. Figure 4 A. The intersection of spleen exosome proteomics differential proteins and plasma proteomics differential proteins revealed that MPC1 was significantly upregulated.

[0171] The expression of MPC1 in the exosomes of Example 1-2 was detected by ELISA. Figure 4 B. It can be seen that the expression of MPC1 in the exosomes of the acute myocardial infarction mice in Example 1 was significantly higher than that in the exosomes of the sham-operated mice in Example 2.

[0172] 6.3 ATP / ATP ratio detection

[0173] 24 h before the establishment of the OGD model, the cell culture medium was replaced with 100 nM GW604714x or 2.5 μM UK5099 for 12 h.

[0174] Figure 4 Group C:

[0175] ①NC+PBS group: cells were treated with an equal amount of PBS in normoxia and normoglucose medium.

[0176] ②OGD+PBS group: cells were treated with an equal amount of PBS in glucose-oxygen deprivation medium.

[0177] ③OGD+Sham-EVs group: cells were treated with the exosomes of Example 2 (15 μg / mL protein concentration) in glucose-oxygen deprivation medium.

[0178] ④OGD+MI-EVs group: cells were treated with the exosomes of Example 1 (15 μg / mL protein concentration) in glucose-oxygen deprivation medium.

[0179] ⑤OGD+GW604714x+MI-EVs: After the cells were treated with the GW604714x inhibitor, the exosomes of Example 1 (15 μg / mL protein concentration) were added to the glucose-oxygen deprivation medium.

[0180] ⑥OGD+UK5099+MI-EVs group: cells were treated with UK5099 inhibitor and then with the exosomes of Example 1 (15 μg / mL protein concentration) in glucose-oxygen deprivation medium.

[0181] Changes in ATP / ADP were measured using the ATP / ATP ratio assay kit (luminescence assay) as follows: 100 μL of cell suspension was seeded into a white 96-well plate and then incubated overnight in a 37°C, 5% CO2 incubator. Cells were grouped as described above and incubated in a tri-gas incubator for 12 hours. The culture medium was carefully removed from the wells to prevent cell detachment. 90 μL of ATP working solution was added to each well of the seeded white 96-well plate and shaken on a microplate shaker for 2 minutes. The plate with ATP working solution was placed in a microplate reader set at 25°C for 10 minutes to measure luminescence (data 1). ADP working solution was prepared and measured before addition (data 2). Immediately after the measurement, 5 μL of ADP working solution was added to each well and shaken on a microplate shaker for 2 minutes. The plate with ADP working solution was placed in a microplate reader set at 25°C for 8 minutes to measure luminescence (data 3). Calculate the ADP / ATP ratio based on the luminescence values ​​obtained (Data 1, Data 2, and Data 3). ADP / ATP ratio = (Data 3 - Data 2) / Data 1.

[0182] It can be seen that MPC1 inhibitors (GW604714X and UK5099) can block the exosomes derived from the spleen tissue of mice with acute myocardial infarction in Example 1 from improving the production of ATP in hypoxic cardiomyocytes, indicating that MPC1 in spleen exosomes is a key effector molecule.

[0183] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0184] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. The use of exosomes in the preparation of a drug for treating myocardial injury, characterized in that: The exosomes are derived from spleen tissue.

2. The use according to claim 1, characterized in that The spleen tissue is derived from a mammal; optionally, the mammal includes at least one of a rodent, a cow, a pig, a sheep, a dog, a cat, and a horse; Optionally, the rodent includes at least one of a mouse and a rabbit; Optionally, the rat includes at least one of a mouse, a rat, a hamster and a guinea pig.

3. The use according to claim 2, characterized in that The mammal is a mammal suffering from myocardial infarction; optionally, the mammal is a mammal suffering from acute myocardial infarction.

4. The use according to any one of claims 1 to 3, characterized in that The method for preparing exosomes comprises the following steps: The spleen tissue was dissociated and the spleen tissue solution was collected; performing differential centrifugation on the spleen tissue solution to prepare the exosomes; Optionally, the step of performing differential centrifugation on the spleen tissue solution includes: centrifuging the spleen tissue solution at 1° C.-10° C. and 200×g-400×g for 5 min-20 min, and collecting a first supernatant; The first supernatant was centrifuged at 1°C-10°C, 2000×g-4000×g for 10 min-30 min, and the second supernatant was collected; The second supernatant was centrifuged at 1°C-10°C, 8000×g-12000×g for 20 min-40 min, and the third supernatant was collected; Centrifuging the third supernatant at 1° C.-10° C. and 100,000×g-150,000×g for 1 h-4 h, collecting the precipitate to obtain the exosomes; Optionally, the step of performing differential centrifugation on the spleen tissue solution includes: centrifuging the spleen tissue solution at 3.5° C.-4.5° C. and 280×g-320×g for 9 min-11 min, and collecting a first supernatant; The first supernatant was centrifuged at 3.5°C-4.5°C and 2900×g-3100×g for 19 min-21 min, and the second supernatant was collected; The second supernatant was centrifuged at 3.5°C-4.5°C, 9000×g-11000×g for 29 min-31 min, and the third supernatant was collected; The third supernatant was centrifuged at 3.5° C.-4.5° C. and 115,000×g-125,000×g for 110 min-130 min, and the precipitate was collected to obtain the exosomes.

5. The use according to claim 4, characterized in that The steps of dissociating the spleen tissue and collecting the spleen tissue solution include: incubating the spleen tissue with a buffer solution containing collagenase, filtering, and collecting the spleen tissue solution.

6. The use according to claim 5, characterized in that Include one or more of the following characteristics: (1) The collagenase includes type I collagenase; (2) The buffer solution includes phosphate buffer; (3) In the collagenase-containing buffer solution, the concentration of collagenase is 0.05wt%-0.5wt%; (4) The incubation temperature is 30°C-40°C; (5) The incubation time is 20 min to 60 min; (6) The filtration uses a 50 μm-100 μm filter membrane; (7) The mass-to-volume ratio of the spleen tissue to the collagenase-containing buffer solution is (5-50) mg:1 mL.

7. The use according to any one of claims 1 to 3, characterized in that The myocardial injury includes at least one of acute myocardial infarction, ischemic myocardial injury, coronary heart disease, coronary artery syndrome and hypoxia-related heart disease; Optionally, the drug for treating myocardial injury is a drug targeting the heart; Optionally, the drug for treating myocardial injury is a drug that improves myocardial cell energy metabolism by enriching key proteins in the mitochondrial oxidative phosphorylation pathway; Optionally, the treatment of myocardial injury includes at least one of reducing the area of ​​myocardial infarction, increasing left ventricular ejection fraction, increasing left ventricular fractional shortening, increasing survival time, upregulating mitochondrial pyruvate transporter 1 expression, and improving energy metabolism of hypoxic myocardial cells.

8. Use of exosomes in the preparation of a drug for treating myocardial injury, characterized in that: The expression level of mitochondrial pyruvate transporter 1 in the exosomes is 6 ng / mL-50 ng / mL; optionally 18 ng / mL-50 ng / mL.

9. The use of exosomes in targeting cardiac tissue for non-diagnostic purposes, characterized in that: The exosomes are the exosomes according to any one of claims 1 to 6 or the exosomes according to claim 8.

10. A drug for treating myocardial damage, characterized in that: Including exosomes; the exosomes are the exosomes according to any one of claims 1 to 6 or the exosomes according to claim 8.

Images