Lispiraceae exosome as well as preparation method and application of Lispiraceae exosome

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hispidobium exosome as well as a preparation method and application of the hispidobium exosome. The application is the application of the hispidobium exosome in preparation of medicines for treating myocardial infarction. The hispidobium exosome provided by the invention has a heart protection effect and can effectively relieve myocardial infarction, meanwhile, the preparation method is simple and easy to control, in addition, the hispidobium source is wide, and the sustainability of exosome preparation is ensured. Therefore, the trichospirillum exosome is expected to become a safe and effective new strategy for treating myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Lachnospiraceae exosome, a preparation method thereof, and an application thereof. Background Art

[0002] Myocardial infarction is a necrotic lesion caused by acute occlusion of the coronary artery, resulting in persistent myocardial ischemia and hypoxia. This disease is common in the middle-aged and elderly populations, and its clinical manifestations include severe chest pain, arrhythmia, heart failure, and even sudden death. Its pathological mechanisms include atherosclerotic plaque rupture, thrombosis, vascular endothelial dysfunction, and oxidative stress. At present, clinical treatments mainly adopt percutaneous coronary intervention (PCI) and thrombolytic therapy, but still face many problems such as reperfusion injury, difficult myocardial fibrosis repair, and long-term decline in cardiac function. Although stem cell therapy and gene editing technology have opened up new paths for myocardial repair, the safety, targeting, and long-term efficacy of these technologies still need to be further verified.

[0003] Exosomes, as membranous vesicles secreted by various types of cells, are rich in bioactive molecules such as proteins, lipids, mRNA, and non-coding RNA. By mediating intercellular signal transduction, they participate in immune regulation, tissue repair, and disease processes. Their mechanisms of action include membrane fusion, endocytosis, and ligand-receptor interactions, and they can regulate the gene expression and functional phenotypes of target cells. In recent years, microbial-derived exosomes have shown unique potential in the field of cardiovascular diseases due to their low immunogenicity, high biosafety, stability, and ability to cross biological barriers. For example, it has been reported that mesenchymal stem cell exosomes have the effects of promoting angiogenesis and assisting myocardial repair, and can be applied to the treatment of myocardial infarction. However, the culture conditions of these cells are harsh, increasing production costs and technical difficulties, which limits their large-scale clinical application.

[0004] Lachnospiraceae belongs to the phylum Firmicutes, family Lachnospiraceae, and is a type of Gram-positive anaerobic bacterium. These bacteria are widely colonized in the intestines of humans and mammals and are one of the core members of the gut microbiota. Lachnospiraceae ferments dietary fiber to produce short-chain fatty acids (such as butyric acid), which can not only provide energy for intestinal epithelial cells, but also strengthen the integrity of the intestinal barrier, inhibit the colonization of pathogenic bacteria, and regulate the host immune homeostasis (such as inducing the differentiation of regulatory T cells). In recent years, Lachnospiraceae has been regarded as a potential "next-generation probiotic", and its strain-specific functions and interaction mechanisms with the host have become research hotspots, showing broad research and application prospects.

[0005] However, up to now, there have been no reports on the use of probiotic exosomes, especially Lachnospiraceae exosomes, for protecting heart health. Summary of the Invention

[0006] Technical Problem

[0007] The present invention aims to explore the application of Lachnospiraceae exosomes in the treatment or alleviation of myocardial infarction and provide an effective method for extracting Lachnospiraceae exosomes.

[0008] Technical Solution

[0009] In the first aspect of the present invention, there is provided an application of Lachnospiraceae exosomes in the preparation of a medicament for treating myocardial infarction.

[0010] In some embodiments, the dosage form of the medicament is any one or more of injection, capsule, tablet, oral liquid, and granule.

[0011] In some embodiments, the medicament comprises Lachnospiraceae-derived exosomes and a pharmaceutically acceptable carrier.

[0012] In some embodiments, the Lachnospiraceae exosomes are prepared by ultracentrifugation.

[0013] In some embodiments, the ultracentrifugation method comprises the following specific steps: S1. Centrifuge the bacterial culture solution at 10,000 g for 15 - 25 min, and collect the supernatant; S2. Filter the supernatant to obtain a filtrate; S3. Centrifuge the filtrate at 150,000 g for 1 - 3 h, and collect the precipitate; S4. Mix the precipitate evenly with ice-cold PBS, and perform density gradient centrifugation at 180,000 g for 1 - 3 h to collect the exosome gradient layer; S5. After mixing the exosome gradient layer evenly with ice-cold PBS solution, centrifuge at 180,000 g for 60 - 120 min, and collect the precipitate to obtain Lachnospiraceae exosomes.

[0014] In some embodiments, the ultracentrifugation process is carried out under the condition of 4 ± 0.5 °C.

[0015] In some embodiments, in step S2, the supernatant is sequentially filtered through a 0.22 μm filter membrane and a 100 kDa ultrafiltration membrane to obtain a filtrate.

[0016] In some embodiments, in step S4, the precipitate is subjected to density gradient centrifugation in a sucrose solution with a mass concentration gradient of 10% - 55%.

[0017] In some embodiments, the mass concentration gradients in step S4 are 55%, 40%, 30%, and 10% in sequence.

[0018] In the second aspect of the present invention, there is provided a Lachnospiraceae exosome, which is used for the preparation of a medicament for treating myocardial infarction.

[0019] Technical Effect

[0020] (1) The preparation method of Lachnospiraceae exosomes in the present invention is simple, easy to control, and has good repeatability. The quality of the extracted exosomes is reliable.

[0021] (2) The Lachnospiraceae exosomes provided by the present invention can reduce the myocardial infarction area, inhibit cardiomyocyte apoptosis, alleviate myocardial fibrosis, inhibit adverse ventricular remodeling, and reduce inflammatory infiltration. These effects contribute to maintaining the cardiac structure and function, can effectively relieve cardiac dysfunction and adverse ventricular remodeling, and help treat or relieve myocardial infarction.

[0022] (3) As one of the core members of the gut microbiota, Lachnospiraceae has a wide source, and the raw materials for preparing its exosomes are easy to obtain. At the same time, exosomes have low immunogenicity, stability, and tolerance. This makes Lachnospiraceae exosomes have potential advantages in clinical applications and are expected to become a safe and effective new strategy for treating myocardial infarction.

[0023] (4) Compared with live bacteria, exosomes have obvious advantages. Exosomes have stronger stability, higher safety, and more convenient storage. In addition, the composition of exosomes is clear, the mechanism of action is clear, and the targeting is precise, enabling them to better adapt to complex pathological environments, which endows them with the potential for multi-functional treatment and has great application prospects in the field of disease treatment. Description of the Drawings

[0024] Figure 1 Shown is the flow chart for extracting Lachnospiraceae exosomes;

[0025] Figure 2 Shown are the scanning electron micrographs of Lachnospiraceae and its exosomes;

[0026] Figure 3 Shown is the scanning electron micrograph of Lachnospiraceae exosomes;

[0027] Figure 4 Shown is the transmission electron micrograph of Lachnospiraceae exosomes;

[0028] Figure 5 Shown is the particle size distribution map of Lachnospiraceae exosomes;

[0029] Figure 6 Shown is the flow chart of the mouse animal experiment;

[0030] Figure 7 Shown are the Masson staining map of the mouse myocardium after myocardial infarction and the statistical chart of the change in the proportion of the myocardial infarction area in the mouse;

[0031] Figure 8 Shown are the TUNEL staining map and the statistical chart of the change in the proportion of apoptotic cardiomyocytes;

[0032] Figure 9 Shown are the immunofluorescence map of collagen III and the statistical chart of the change in the proportion of the fibrotic area;

[0033] Figure 10 Shown are the immunofluorescence image of platelet endothelial cell adhesion molecule (CD31) and the statistical chart of angiogenesis analysis;

[0034] Figure 11 Shown are the immunohistochemical image of neutrophils and the statistical chart of neutrophil infiltration analysis. Detailed implementation manners

[0035] For facilitating the understanding of the technical solution of the application, the terms and expressions involved in the present application are generally described and defined as follows first.

[0036] The term "comprise", "include" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0037] In each group of comparative experiments provided in the present application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent to enable comparability.

[0038] The reagents and instrument devices used in the embodiments of the present invention can be obtained from the market without detailed description.

[0039] The following further describes a Lachnospiraceae exosome and its preparation method and application provided by the present application.

[0040] Example 1: Extraction of Lachnospiraceae exosomes

[0041] 1.1 Extract Lachnospiraceae exosomes

[0042] Culture Lachnospiraceae (DSM 24430, DSMZ) at 37 °C under anaerobic conditions (80% N2, 10% CO2, 10% H2) in brain heart infusion medium (BHI) supplemented with 5% fetal bovine serum (FBS), 0.01% L-cysteine, 1% corn starch and 0.5% konjac powder.

[0043] The extraction process is as Figure 1 shown and specifically includes the following steps:

[0044] S1. Centrifuge the bacterial culture solution at 4 °C and 10,000 g for 20 min, and collect the supernatant;

[0045] S2. After the supernatant is filtered through a 0.22 μm filter membrane to remove residual bacterial cell debris, it is then concentrated using a 100 kDa ultrafiltration membrane;

[0046] S3. Centrifuge at 4 °C and 150,000 g for 2 h at high speed;

[0047] S4. Then perform density gradient centrifugation using a sucrose solution with a mass concentration gradient of 10% - 55%, and finally complete purification by centrifuging at 4 °C and 180,000 g for 2 h at high speed; specifically, the sucrose solution is divided into several gradient solutions of 55%, 40%, 30%, and 10%. After stacking the concentration gradient, centrifuge at high speed once (2 h).

[0048] S5. After high-speed centrifugation, the exosomes will migrate to the gradient solution layer with the same buoyant density as them. Precisely aspirate the exosome gradient layer into a centrifuge tube, add ice-cold PBS solution and mix evenly, then centrifuge at 180,000 g for 90 min in a high-speed freezing centrifuge to obtain Lachnospiraceae exosomes;

[0049] S6. Resuspend the precipitate with 3 mL of ice-cold PBS solution and filter through a 0.22 μm filter membrane, then aliquot the filtrate.

[0050] 1.2 Identification

[0051] Observe Lachnospiraceae and its exosomes using a scanning electron microscope (SEM) or a transmission electron microscope (TEM); detect the particle size distribution using nanoparticle tracking analysis technology (NTA).

[0052] Figure 2 are the SEM images of Lachnospiraceae and its exosomes. It can be seen from Figure 2 that Lachnospiraceae can secrete exosomes. The yellow scale bar in the figure is 200 nm, and the white scale bar is 1 μm.

[0053] Figure 3 are the SEM images of the extracted Lachnospiraceae exosomes. It can be seen from Figure 3 that the extracted exosomes have a typical round appearance. The scale bar in the figure is 0.5 μm.

[0054] Figure 4 are the TEM images of the extracted Lachnospiraceae exosomes, further clarifying that the extracted exosomes have a round appearance, clear contour, and complete structure. The scale bar in the figure is 100 nm.

[0055] Figure 5 are the particle size distribution diagrams of the extracted Lachnospiraceae exosomes. It can be seen from Figure 5 that the average particle size of the extracted exosomes is 124 nm, further corroborating the size distribution of the nanovesicles.

[0056] Example 2: Application of Lachnospiraceae exosomes in mouse heart protection

[0057] 2.1 Experimental purpose

[0058] The extracted Lachnospiraceae exosomes were used as the experimental group drug for intervening in myocardial infarction to observe the cardioprotective effect of Lachnospiraceae exosomes.

[0059] 2.2 Experimental protocol

[0060] 1) Experimental subjects

[0061] Mice (C57BL / 6J) were divided into three groups: PBS, Lachnospiraceae exosomes (L-EVs), and Lachnospiraceae (Lach), with 6 male mice in each group.

[0062] 2) Animal treatment protocol

[0063] The treatment steps for mice were as Figure 6 shown below, specifically as follows:

[0064] Gavage administration: Pre-protection was carried out for 21 days with Lachnospiraceae exosomes (L-EVs) or Lachnospiraceae (live bacteria), and PBS was used as a control. The gavage dose of L-EVs was 100 μg / mouse, the gavage dose of Lach was 1×10 9 CFU / mouse, and the gavage dose of PBS was 0.2 mL / mouse. Among them, L-EVs and Lach were dissolved in 0.2 mL of PBS. Mice were gavaged once every 3 days for a total of 7 times.

[0065] Coronary artery ligation: Permanent ligation of the left anterior descending coronary artery was performed on day 21. The specific operation was as follows: After the mice were slightly anesthetized with 1.0% -

[0066] 1.5% isoflurane gas, the heart was exposed using thoracotomy, and the left coronary artery was quickly ligated with 7-0 suture. After the heart was repositioned in the chest cavity, the air was quickly discharged, and the chest wall was promptly closed.

[0067] Heart detection: Finally, on day 28, the mice were dissected to obtain the heart, and paraffin sections were prepared for Masson staining, TUNEL staining (manufacturer: Sevier; product number: G1504), immunofluorescence staining of type III collagen (collagen III), immunofluorescence staining of platelet-endothelial cell adhesion molecule (CD31), and immunohistochemical staining of neutrophils. At the same time, the changes in the proportion of myocardial infarction area, the proportion of cardiomyocytes, the level of type III collagen (collagen III) in the heart sections, the level of platelet-endothelial cell adhesion molecule (CD31), and the level of neutrophils were analyzed.

[0068] 2.3 Experimental results

[0069] Figure 7are the Masson staining diagrams of mouse heart sections under different treatments and the statistical charts of the changes in the proportion of myocardial infarction area in mice. From Figure 7 it can be seen that the exosomes of Lachnospiraceae (L-EVs) can reduce the infarction area in mice with myocardial infarction, significantly reducing it by about 53% compared with the PBS control group. The scale bar in the figure is 500 μm.

[0070] Figure 8 are the TUNEL staining diagrams of mouse heart sections under different treatments and the statistical charts of the changes in the proportion of apoptotic cardiomyocytes. From Figure 8 it can be seen that the exosomes of Lachnospiraceae can reduce the apoptosis of cardiomyocytes in mice with myocardial infarction, significantly reducing it by about 67% compared with the PBS control group, and also significantly less than that in the Lachnospiraceae control group. The scale bar in the figure is 50 μm.

[0071] Figure 9 are the immunofluorescence diagrams of type III collagen (collagen III) in mouse heart sections under different treatments and the statistical charts of the changes in the proportion of fibrosis area. From Figure 9 it can be seen that the exosomes of Lachnospiraceae can reduce the fibrosis area in mice with myocardial infarction, significantly reducing it by about 30% compared with the PBS control group, and being comparable to the Lachnospiraceae control group. The yellow scale bar in the figure is 100 μm. The scale bar in the figure is 50 μm.

[0072] Figure 10 are the immunofluorescence diagrams of platelet-endothelial cell adhesion molecule (CD31) in mouse heart sections under different treatments and the statistical charts of angiogenesis analysis. From Figure 10 it can be seen that the exosomes of Lachnospiraceae can reduce the adverse ventricular remodeling in mice with myocardial infarction, and the CD31 level is significantly reduced by about 76% compared with the PBS control group, being comparable to the Lachnospiraceae control group. The scale bar in the figure is 50 μm.

[0073] Figure 11 are the immunohistochemical diagrams of neutrophils in mouse heart sections under different treatments and the statistical charts of neutrophil infiltration analysis. From Figure 11 it can be seen that the exosomes of Lachnospiraceae can reduce the neutrophil infiltration in mice with myocardial infarction, significantly reducing it by about 25% compared with the PBS control group, being comparable to the Lachnospiraceae control group. The scale bar in the figure is 50 μm.

[0074] In summary, the exosomes of Lachnospiraceae have a significant protective effect on mice with myocardial infarction. It can effectively reduce the myocardial infarction area, inhibit cardiomyocyte apoptosis, alleviate myocardial fibrosis, relieve adverse ventricular remodeling, and at the same time reduce the level of neutrophil infiltration, playing a positive role in improving cardiac function after myocardial infarction. The effect of the exosomes of Lachnospiraceae is comparable to or better than that of the Lachnospiraceae control group, indicating that the key cardiac protection is exerted by the exosomes rather than the whole Lachnospiraceae.

[0075] In the above specific embodiments, the purpose, technical solution and beneficial effects of the present application have been further described in detail. It should be understood that the above are only specific embodiments of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solution of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Lachnospira exosomes in the preparation of drugs for the treatment of myocardial infarction.

2. The use according to claim 1, characterized in that: The dosage form of the drug is any one or more of injection, capsule, tablet, oral liquid, and granule.

3. The use according to claim 1, characterized in that: The drug comprises exosomes derived from Lachnospira and a pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that: The Lachnospira exosomes are prepared by ultracentrifugation.

5. The use according to claim 4, characterized in that: The ultracentrifugation method comprises the following specific steps: S1. Centrifuge the bacterial culture at 10,000 g for 15-25 min and collect the supernatant; S2, filtering the supernatant to obtain a filtrate; S3, centrifuging the filtrate at 150,000g for 1-3h, collecting the precipitate; S4. Mix the precipitate with ice PBS solution evenly, and centrifuge at 180,000g for 1-3 hours to collect the exosome gradient layer. S5. After the exosome gradient layer is evenly mixed with the ice PBS solution, the mixture is centrifuged at 180,000 g for 60 to 120 min, and the precipitate is collected to obtain Lachnospira exosomes.

6. The use according to claim 5, characterized in that: The ultracentrifugation process was carried out at 4±0.5°C.

7. The use according to claim 5, characterized in that: The supernatant in step S2 is filtered through a 0.22 μm filter membrane and a 100 kDa ultrafiltration membrane in sequence to obtain a filtrate.

8. The use according to claim 5, characterized in that: The precipitate in step S4 is subjected to density gradient centrifugation in a sucrose solution with a mass concentration gradient of 10% to 55%.

9. A Lachnospira exosome, characterized in that The Lachnospira exosomes are used for preparing a medicine for treating myocardial infarction.

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