Enterococcus pluvialis and application of exosome of enterococcus pluvialis

Abstract

The invention discloses application of enterococcus pluvialis and exosomes thereof, and relates to the field of application of enterococcus pluvialis and exosomes thereof. The enterococcus pluvialis disclosed by the invention is enterococcus pluvialis Zz-2024, is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 31249. The invention relates to an exosome produced by Enterococcus pluvialis Zz-2024, and an application of the exosome in a medicine for treating myocarditis, and particularly relates to an exosome produced by Enterococcus pluvialis Zz-2024 and an application of the exosome in a medicine for treating myocarditis. The enterococcus pluvialis exosome disclosed by the invention has excellent effects in experiments of inhibiting myocarditis factors, reducing lipid droplet aggregation, inhibiting myocardial cell senescence and the like, and breaks through the limitation that human stem cell source exosomes are adopted in related fields at present.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an Enterococcus fulvicus strain and application of its exosomes. Background Art

[0002] During myocardial metabolism, fatty acids are used as substrates to produce a large amount of adenosine triphosphate (ATP) through mitochondrial oxidative phosphorylation to provide energy, meet metabolic needs, and maintain normal cell function. More than 70% of myocardial metabolic substrates in adults come from fatty acids, while glucose, ketone bodies, and amino acids play a smaller role. Myocardial lipid metabolism disorders can lead to a variety of diseases, such as diabetic cardiomyopathy, heart failure, and inflammatory diseases. Therefore, the balance of lipid uptake and utilization is crucial for myocardial energy metabolism. Lipid droplets are a multifunctional organelle closely related to lipid metabolism. The neutral lipids stored in lipid droplets, especially triglycerides, can produce a large amount of fatty acids required for myocardial metabolism. Lipid droplets play an important role in myocardial metabolism. Excessive accumulation of lipid droplets may lead to the accumulation of toxic lipids (such as ceramide, diacylglycerol, etc.), which may activate inflammatory pathways.

[0003] Although both exosomes and secretory vesicles are membrane-enclosed structures released by cells, they differ significantly in their origin, composition, and function. Secretory vesicles are formed by organelles such as the Golgi apparatus in cells and are primarily responsible for transporting specific proteins, hormones, or other bioactive substances from inside cells to outside cells. These vesicles fuse with the cell membrane after reaching it, releasing their contents into the extracellular environment.

[0004] Exosomes are a special type of membrane-enclosed vesicles that are directly derived from the invagination and resealing of the cell membrane. The contents of exosomes are more complex, including proteins, lipids, RNA and even DNA fragments, which can reflect the physiological state of the source cells. Exosomes not only participate in intercellular communication, but also transmit genetic information and regulatory signals, affecting the function of receptor cells.

[0005] Compared with secretory vesicles, exosomes have many advantages. First, the contents of exosomes are richer and more diverse, and can transmit more types of information and regulatory factors. Second, because exosomes are derived from the cell membrane, they can more directly participate in cell-to-cell interactions, including long-distance cell communication. In addition, exosomes excel in stability and biocompatibility, giving them broad application prospects in drug delivery, biomarker detection, and disease treatment. However, the current sources of exosomes in the field of myocardial therapy are limited to human stem cells, and their sources also involve ethical issues.

[0006] Enterococcus pyogenes ( Enterococcus casseliflavus) belongs to the genus Enterococcus and is part of the normal flora in the intestines of humans and animals. It is a probiotic that plays an important role in maintaining the ecological balance of the intestinal flora of animals; it can stably exist in the intestine and play a variety of beneficial roles for the human body. By stimulating the immune system, enterococci can enhance the body's resistance and reduce the occurrence of diseases. Enterococci can secrete a variety of digestive enzymes to help digest food and regulate lipid metabolism. Not only that, enterococci have strong acid resistance and can survive in the environment of gastric acid and reach the intestine smoothly, thereby playing a role in the intestine. This has obvious advantages compared to some other acid-sensitive probiotics. After years of research and practical verification, enterococci have been proven to be a safe and effective probiotic suitable for people of different ages.

[0007] Currently, the research direction of Enterococcus pyogenes is mainly on its impact on intestinal and fecal flora. There are no reports on the application of Enterococcus pyogenes or Enterococcus pyogenes exosomes in other fields. Summary of the invention

[0008] In order to change the situation that the source of exosomes in the field of myocardial treatment is limited to human stem cells, the present invention provides a strain of Enterococcus fulvicus and the application of its exosomes.

[0009] The present invention is Enterococcus pyogenes. Enterococcus casseliflavus ) Zz-2024, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31249.

[0010] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) exosomes, which are produced by Enterococcus fulva ( Enterococcus casseliflavus )Exosomes produced by Zz-2024.

[0011] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus )Application of exosomes in the treatment of myocarditis.

[0012] Furthermore, Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are used as active ingredients in the preparation of drugs for the treatment of myocarditis.

[0013] Furthermore, Enterococcus pyogenes ( Enterococcus casseliflavus ) Application of exosomes of Zz-2024 in drugs to reduce the level of myocarditis.

[0014] Furthermore, Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are used as active ingredients in the preparation of drugs for reducing the level of myocarditis.

[0015] Furthermore, Enterococcus pyogenes ( Enterococcus casseliflavus)Zz-2024 exosomes are used as active ingredients in the preparation of drugs for reducing myocardial lipid droplets. Beneficial Effects

[0016] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) Exosomes are pure and uniform, with a diameter of 80nm~120nm.

[0017] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) Exosomes can reduce the inflammatory level of cardiomyocytes under pathological conditions, thereby maintaining the homeostasis of cardiomyocytes.

[0018] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) Exosomes can reduce the aggregation of lipid droplets in cardiomyocytes under pathological conditions, thereby improving myocardial fatty acid uptake and oxidative imbalance, avoiding lipid droplet accumulation, reducing the resulting myocardial lipotoxicity, and restoring cardiac function.

[0019] The present invention adopts Enterococcus pyogenes ( Enterococcus casseliflavus ) exosomes, because Enterococcus fulva is part of the normal flora in the intestines of humans and animals and is a probiotic; therefore, the exosomes secreted by it have good biocompatibility with humans. Enterococcus casseliflavus ) The biosafety test results of the exosomes of Zz-2024 also showed that Enterococcus fulva ( Enterococcus casseliflavus ) The exosomes of Zz-2024 have no toxic side effects. Enterococcus casseliflavus ) Zz-2024 exosomes can reduce the expression of CCL5 mRNA in mouse cardiomyocyte models, and can reduce the levels of CCL5, IL6, and NF-κB secreted by mouse cardiomyocytes. Enterococcus casseliflavus )Zz-2024 exosomes have the effect of inhibiting cardiomyocyte inflammation.

[0020] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) Zz-2024 exosomes can reduce the accumulation of lipid droplets in mouse cardiomyocytes, and Enterococcus pyogenes ( Enterococcus casseliflavus )The exosomes of Zz-2024 have the function of regulating lipid metabolism and restoring myocardial function.

[0021] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus ) Exosomes have shown excellent effects in experiments such as inhibiting myocardial inflammatory factors and reducing lipid droplet aggregation, breaking through the limitations of the current use of exosomes derived from human stem cells in related fields, and for the first time, Enterococcus flavus exosomes have been used in the treatment of myocardial diseases.

[0022] The present invention is Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 is Enterococcus fulva, belonging to the genus Enterococcus ( Enterococcus); deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is July 10, 2024, and the deposit number is CGMCC No.31249. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus ) Transmission electron microscopy observation of Zz-2024 exosomes (Part 1); Figure 2 The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus ) Transmission electron microscopy observation of Zz-2024 exosomes (part 2); Figure 3 The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus ) Transmission electron microscopy observation of Zz-2024 exosomes (Part 3); Figure 4 The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus )Zz-2024 exosome nanoparticle size analysis; Figure 5 is the lead yellow Enterococcus ( Enterococcus casseliflavus )Comparative safety test results of Zz-2024 exosomes; Figure 6 4 is a graph showing the experimental results of the expression of CCL5 mRNA levels in the mouse myocardial cell model in each experimental group in Example 4; Figure 7 Graph showing the experimental results of the expression of CCL5, IL6, and NF-κB cytokines in the myocardial cell model of each experimental group of mice in Example 5; Figure 8 This is a diagram showing the effect of lipid deposition in myocardial cells of mice in each experimental group in Example 6; Figure 9 3 is a diagram showing the effect of β-galactosidase staining experiment on myocardial cells of mice in each experimental group in Example 7. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0026] The "drugs" mentioned in the present invention include but are not limited to injections, tablets, capsules, sprays and patches.

[0027] Example 1: This example of Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 Exosome Isolation and Preparation Method: S1, Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 was activated and restored to vitality by passage in MRS solid slant medium, then inoculated into sterilized MRS liquid medium and cultured in a 37 ℃ constant temperature incubator for 12 h to obtain Enterococcus fulvei ( Enterococcus casseliflavus )Zz-2024 seed solution; S2, inoculated into the sterilized MRS liquid medium again at 1%, and continued to culture until the late logarithmic growth phase to obtain Enterococcus fulvei ( Enterococcus casseliflavus )Zz-2024 bacterial solution; S3, Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 bacterial culture was centrifuged and the supernatant was discarded, then added into TY liquid medium and incubated at 30°C for 6 h; S4. Collection of exosomes: The suspension after 6 h of incubation was centrifuged at 3000 g for 10 min, the cells were discarded, and the supernatant was collected; S5. Differential centrifugation: 5.1, 300g, 4℃, centrifuge for 10min, discard the precipitate; 5.2, centrifuge at 2000g, 4°C for 10 min and discard the precipitate; 5.3, centrifuge at 10,000g, 4°C for 30 min, discard the precipitate, collect the supernatant, and filter with a 0.45 μm filter; 5.4. Centrifuge the filtrate at 110,000 g, 4°C for 70 min and discard the supernatant; 5.5. Resuspend the precipitate with PBS, filter it with a 0.22 μm membrane filter, centrifuge the filtrate at 110000g, 4°C, for 70 min, discard the supernatant, and obtain preliminary Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes; S6, the preliminary Enterococcus pyogenes obtained in step S5 ( Enterococcus casseliflavus ) Zz-2024 exosomes were added to PBS, and then iodixanol was added to prepare a 3 mL suspension of 40% iodixanol containing outer membrane vesicles; S7, using sterile water and iodixanol solution to prepare iodixanol solutions with concentrations of 5%, 10%, and 20%; then adding 3 mL of the 40% iodixanol suspension containing exosomes prepared in step S6 to an ultracentrifuge tube, and then adding 3 mL of 20% iodixanol solution, 3 mL of 10% iodixanol solution, and 3 mL of 5% iodixanol solution in sequence, followed by centrifugation at 100,000 g for 18 h; S8. After ultracentrifugation in step S7, the suspension in the ultracentrifuge tube is divided into 12 layers, each layer is 1 mL, and 1 mL is successively drawn from top to bottom and it is ensured that the pipette tip does not insert into the next layer each time; the 4th to 6th layers are collected to obtain Enterococcus fulvidraco ( Enterococcus casseliflavus )Zz-2024 exosomes.

[0028] Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 was extracted from the intestinal feces of newborns in Harbin in April 2023.

[0029] Enterococcus pyogenes ( Enterococcus casseliflavus ) Isolation, purification and identification methods of Zz-2024: 8.1. Add the intestinal feces of the newborn to sterile water at a ratio of 1:50 and perform gradient dilution; 8.2. Take 100uL of appropriate gradient dilution solution and add it to solid MRS (containing CaCO 3 ) Culture concentrated, plated, and cultured at 37°C; 8.3. Select single colonies with obvious calcium melting circles for three-zone demarcation, culture at 37°C, and purify the strains; 8.4. Similarly, single colonies with obvious calcium melting circles were selected for three-zone demarcation, cultured at 37°C, and continued purification; 8.5. Pick a single colony from the purified plate and add it to liquid MRS medium for expansion culture. Perform PCR sequencing on the bacterial suspension to identify the species. 8.6. After species identification, mix the bacterial suspension with 50% sterile glycerol in a ratio of 1:1 and freeze at -80℃. In addition, inoculate the slant and send it to the strain collection center for preservation.

[0030] In the present invention, Enterococcus pyogenes ( Enterococcus casseliflavus )The cultivation of Zz-2024 is not limited to the methods and steps described in this embodiment.

[0031] In the present invention, Enterococcus pyogenes ( Enterococcus casseliflavus )The method for isolating and obtaining Zz-2024 exosomes is not limited to the methods and steps described in this embodiment.

[0032] Example 2: The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus )Zz-2024 exosomes were observed by transmission electron microscopy, such asFigures 1 to 3 As shown, a large number of vesicles with concave double membrane structures can be visually seen under the electron microscope, confirming the morphology of the exosomes extracted in Example 1.

[0033] The Enterococcus pyogenes obtained in Example 1 ( Enterococcus casseliflavus )Zz-2024 exosomes were subjected to nanoparticle size analysis. From the results of nanoparticle size (such as Figure 4 Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are very pure and uniform, with a diameter of 80nm~120nm.

[0034] Example 3: This example uses CCK8 experiment to detect the effect of exosomes on the viability of mouse cardiomyocytes and mouse fibroblasts.

[0035] Experimental groups: HL-1 cells (mouse cardiomyocytes) and mouse fibroblasts were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum). The cells were passaged when they reached 80-90% confluence, and when the cells reached 50-60% confluence, PBS or exosomes were added to the culture medium for 48 hours. The treatments included the following: ① PBS (control group), ② 10 4 / ml exosomes (low-dose group), ③10 5 / ml exosomes (medium dose group), ④10 6 / ml exosomes (high dose group).

[0036] Detection method: The following operations were performed on 4 groups of treatments of two types of cells.

[0037] Step 1: Cell seeding 1. Digest cells: Use trypsin or other proteases to digest the cells and remove them from the culture dish.

[0038] 2. Counting: Use a cell counting chamber to count the cells.

[0039] 3. Inoculation: According to the experimental design, a certain number of cells are inoculated into a 96-well plate, and the number of cells in each well should be kept consistent. Usually, a blank control group (only culture medium is added, without cells) and an experimental group (cells under different treatment conditions) are set up, and multiple replicates are set up for each group.

[0040] Step 2: Cell culture and treatment 1. Place the 96-well plate seeded with cells in an incubator and allow them to adhere and grow for 24 hours.

[0041] 2. After 24 hours, the medium was completely replaced and the treatment was performed according to the above design: PBS was added to the control group and different doses of exosomes were added to the experimental group. Step 3: CCK8 detection

[0042] 1. Add CCK8 reagent to each well 2 hours before 24, 48, and 72 hours after treatment, usually at 1 / 10 of the culture medium volume.

[0043] 2. Place the 96-well plate in the incubator and incubate for 2 hours.

[0044] Step 4: Determine absorbance The absorbance (OD value) at a wavelength of 450 nm was measured using an enzyme reader.

[0045] Step 5: Data Analysis The proliferation or inhibition of cells in each group was calculated based on the measured OD values.

[0046] The experimental results are as follows Figure 5 As shown. Enterococcus casseliflavus )Zz-2024 exosomes have no effect on the viability of mouse cardiomyocytes and mouse fibroblasts. Enterococcus casseliflavus )Zz-2024 exosomes showed no significant difference in cell activity compared with the control group at low, medium and high concentrations after treatment for 24, 48 and 72 hours, so the experimental results indicate that Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are non-toxic and safe to cells.

[0047] Example 4: In this example, real-time quantitative PCR was used to detect the effect of exosomes on the expression of CCL5 mRNA in mouse cardiomyocytes.

[0048] Experimental groups: HL-1 cells (mouse cardiomyocytes) were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum). The cells were passaged when they reached 80-90% confluence, and when the cells reached 50-60% confluence, they were treated with drugs in the culture medium for 24 hours. The drug treatments included the following: ①200 μmol / L palmitate (modeling group), ②200 μmol / L palmitate + 100 nmol / L thapsigargin (Tg) (modeling + Tg group), ③200 μmol / L palmitate + 5 mmol / L 4-phenylbutyric acid (modeling + 4-PBA), ④200 μmol / L palmitate + 10 6 5. Exosomes / ml (modeling + low-dose exosomes group) and 6. 200 μmol / L palmitate + 10 8 / ml exosomes (modeling + high-dose exosome group).

[0049] Detection method: The following operations were performed on the above five groups of HL-1 cells.

[0050] Step 1: RNA extraction 1. Sample preparation: Select healthy HL-1 cell samples from the above experimental groups to ensure that the cell quantity and quality meet the experimental requirements.

[0051] 2. Lyse cells: Use a lysis buffer (such as TRIzol or other commercial lysis reagents) to lyse the above cells and release RNA.

[0052] 3. Isolate RNA: Isolate RNA from the lysate by centrifugation or column purification.

[0053] 4. Washing: Use appropriate washing buffer to remove protein and DNA contamination.

[0054] 5. RNA purification: RNA is purified from the column through adsorption and elution steps.

[0055] 6. RNA quantification: Use a UV spectrophotometer or a fluorescence quantifier to determine RNA concentration and purity.

[0056] 7. RNA quality assessment: Assess RNA integrity by gel electrophoresis or bioanalyzer.

[0057] Step 2: Reverse transcription 1. Reverse transcription reaction: Mix the purified RNA from each experimental group with reverse transcriptase, primers and buffer to perform reverse transcription reaction.

[0058] 2. Reverse transcription conditions: The reverse transcription reaction is usually carried out at 42°C for 1 hour and then heated to 85°C to inactivate the reverse transcriptase.

[0059] 3.cDNA synthesis: Reverse transcriptase transcribes the RNA template into cDNA.

[0060] 4. cDNA dilution: According to the needs of the PCR reaction, dilute the cDNA appropriately to obtain the best amplification effect.

[0061] Step 3: Real-time PCR 1. PCR reaction system: Prepare the PCR reaction system including cDNA template, primers, probe (if used), enzyme and buffer according to the instructions of the qPCR kit used.

[0062] 2. Primer design: Design specific primers, the primer sequences are shown in Table 1 below, to ensure accurate amplification of the target gene.

[0063] Table 1

[0064] 3. PCR condition setting: Set up the PCR amplification program, including initial denaturation, annealing, and extension steps. Initial denaturation: 95°C, 10 minutes; cycle: 95°C, 15 seconds (denaturation); 60°C, 1 minute (annealing and extension), 40 cycles.

[0065] 4. Data collection: Use a real-time PCR instrument to collect fluorescence signals and monitor the PCR amplification process in real time.

[0066] 5. Melting curve analysis: Melting curve analysis was performed to confirm the specificity of the amplified product.

[0067] 6. Quantitative analysis: Use a standard curve or relative quantitative method (such as the ΔΔCT method) to quantify the expression level of the target gene.

[0068] Step 4: Data Analysis 1. Data export: Export raw data from the PCR instrument.

[0069] 2. Data normalization: Use the internal reference gene (GAPDH) to normalize the data.

[0070] 3. Statistical analysis: Perform necessary statistical analysis to compare the differences in gene expression between different samples or treatment groups.

[0071] The experimental results are as follows Figure 6 As shown. Enterococcus casseliflavus )Zz-2024 exosomes have an effect on the expression of CCL5 mRNA in mouse cardiomyocytes. Enterococcus casseliflavus )Zz-2024 exosomes can reduce the expression of CCL5 mRNA in the mouse cardiomyocyte model, and both low-dose and high-dose exosomes have significant effects. The CCL5 gene, also known as chemokine (CC motif) ligand 5, is a small molecule cytokine and a member of the CC-type chemokine family. CC-type chemokines are a class of signaling proteins that can attract white blood cells (such as monocytes, lymphocytes, etc.) to sites of inflammation or infection. During the inflammatory process, CCL5 promotes the chemotaxis of immune cells by binding to its receptors. The level of CCL5 gene expression can reflect the degree of inflammation. Therefore, the experimental results show that Enterococcus flavus ( Enterococcus casseliflavus )Zz-2024 exosomes have the effect of inhibiting cardiomyocyte inflammation.

[0072] Example 5: This example uses ELISA to detect Enterococcus pyogenes ( Enterococcus casseliflavus)Effects of Zz-2024 exosomes on the levels of secretory proteins CCL5, IL6, and NF-κB in the culture medium of mouse cardiomyocytes.

[0073] Experimental groups: HL-1 cells (mouse cardiomyocytes) were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum). The cells were passaged when they reached 80-90% confluence, and when the cells reached 50-60% confluence, they were treated with drugs in the culture medium for 24 hours. The drug treatments included the following: ①200 μmol / L palmitate (modeling group), ②200 μmol / L palmitate + 100 nmol / L thapsigargin (Tg) (modeling + Tg group), ③200 μmol / L palmitate + 5 mmol / L 4-phenylbutyric acid (modeling + 4-PBA), ④200 μmol / L palmitate + 10 6 5. Exosomes / ml (modeling + low-dose exosomes group) and 6. 200 μmol / L palmitate + 10 8 / ml exosomes (modeling + high-dose exosome group).

[0074] Detection method: The following operations were performed on the above five groups of HL-1 cells.

[0075] Step 1: Prepare HL-1 cell culture supernatant samples 1. Cell culture: The HL-1 cells in the above experimental groups were cultured in high-glucose DMEM medium to the logarithmic growth phase.

[0076] 2. Collecting the supernatant: After replacing the culture medium with fresh high-glucose DMEM, continue to culture the cells in each experimental group for 24 hours to collect the culture supernatant containing secreted proteins.

[0077] 3. Centrifugation: The collected cell supernatant of each experimental group was centrifuged at 3000 g for 20 minutes to remove cell debris and macromolecular complexes.

[0078] 4. Sample preservation: Collect the supernatant of each experimental group after centrifugation into new sterile tubes and freeze them for subsequent experiments.

[0079] Step 2: Prepare ELISA reagents 1. Reagent preparation: Prepare sufficient ELISA reagents, including washing buffer, enzyme-labeled antibody, color developer, stop solution and standards.

[0080] 2. Room temperature balance: Take out the well strips coated with CCL5, IL6, and NF-κB proteins according to the number of samples, and prepare them according to 2 replicates. Put the strips into the rack and balance them to room temperature before use.

[0081] Step 3: Addition of samples and standards 1. Determine the sample volume: Take 40 μl of the cell culture supernatant sample stock solution of each experimental group as recommended in the instructions.

[0082] 2. Add samples and standards: Add samples of each experimental group and a series of concentrations of standards to the corresponding wells of the ELISA plate.

[0083] 3. Add enzyme-labeled antibody: Follow the instructions and add biotin-labeled antibody.

[0084] 4. Incubation: Incubate the ELISA plate at room temperature for 30 minutes to allow the antigen in each experimental group sample to bind to the coated antibody.

[0085] Step 4: Color reaction 1. Washing: Wash with plate washing buffer to remove unbound enzyme-labeled antibodies.

[0086] 2. Addition of substrate: Add color developer to make the enzyme catalyze the substrate to produce color change.

[0087] 3. Color development: Incubate at room temperature for 15 minutes until the color change reaches sufficient intensity.

[0088] Step 5: Determine absorbance 1. Add stop solution: Add stop solution to each well to terminate the reaction.

[0089] 2. Absorbance measurement: Use an ELISA reader to measure the absorbance of each well (usually at a wavelength of 450 nm).

[0090] Step 6: Data Analysis 1. Preparation of standard curve: Use the absorbance value of the standard to prepare the standard curve.

[0091] 2. Sample concentration calculation: Calculate the protein concentration in the sample based on the standard curve.

[0092] 3. Data analysis: Conduct statistical analysis on the experimental data and draw conclusions.

[0093] The experimental results are as follows Figure 7 As shown. Enterococcus casseliflavus )Zz-2024 exosomes have an effect on the content of secretory proteins CCL5, IL6, and NF-κB in the supernatant of mouse cardiomyocytes. The exosomes of Enterococcus pyogenes of the present invention can reduce the content of CCL5, IL6, and NF-κB secreted by the mouse cardiomyocyte model. Compared with the modeling group and the Tg group, both high and low doses of exosomes can significantly reduce the content of three inflammatory factors CCL5, IL6, and NF-κB in the supernatant. Therefore, the experimental results once again prove that Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes have the effect of inhibiting cardiomyocyte inflammation.

[0094] Example 6: Cardiomyocyte lipid droplet staining experiment In this example, BODIPY 493 / 503 staining was used to observe lipid droplets in mouse cardiomyocytes and detect Enterococcus pyogenes ( Enterococcus casseliflavus )Effects of Zz-2024 exosomes on lipid droplet aggregation in mouse cardiomyocytes.

[0095] Experimental groups: HL-1 cells (mouse cardiomyocytes) were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum). The cells were passaged when they reached 80-90% confluence, and when the cells reached 50-60% confluence, they were treated with drugs in the culture medium for 24 hours. The drug treatments included the following: ① normal HL-1 cell group, ② 200 μmol / L palmitate (modeling group), ③ 200 μmol / L palmitate + 100 nmol / L thapsigargin (Tg) (modeling + agonist group), ④ 200 μmol / L palmitate + 5 mmol / L 4-phenylbutyric acid (modeling + inhibitor group), ⑤ 200 μmol / L palmitate + 10 6 10 / ml exosomes (modeling + low-dose exosomes group) and ⑥200 μmol / L palmitate + 10 8 / ml exosomes (modeling + high-dose exosome group).

[0096] Detection method: HL-1 cells in each experimental group were incubated with 4% paraformaldehyde for 20-30 minutes and then slowly rinsed three times with PBS. BODIPY 493 / 503 dye was mixed with PBS to a final dye concentration of 1 μg / mL, and the cells were incubated at 37°C in the dark for 30 minutes. After carefully rinsing three times with PBS buffer, the stained lipid droplets were recorded under a fluorescence microscope.

[0097] The experimental results are as follows Figure 8 As shown. Enterococcus casseliflavus )Zz-2024 exosomes have an effect on lipid droplet aggregation in mouse cardiomyocytes. Enterococcus casseliflavus )Zz-2024 exosomes can reduce the accumulation of lipid droplets in mouse cardiomyocytes. Compared with the modeling group and Tg group (modeling + agonist group), both high and low doses of exosomes can significantly reduce the content of lipid droplets in cardiomyocytes. Therefore, the experimental results prove that Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes have the function of regulating lipid metabolism and restoring myocardial function.

[0098] Example 7: Myocardial cell β-galactosidase staining experiment In this example, β-galactosidase staining was used to observe the aging characteristics of mouse cardiomyocytes and detect Enterococcus pyogenes ( Enterococcus casseliflavus )Effects of Zz-2024 exosomes on aging of mouse cardiomyocytes.

[0099] Experimental groups: HL-1 cells (mouse cardiomyocytes) were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum). The cells were passaged when they reached 80-90% confluence, and when the cells reached 50-60% confluence, they were treated with drugs in the culture medium for 24 hours. The drug treatments included the following: ① normal HL-1 cell group, ② 200 μmol / L palmitate (modeling group), ③ 200 μmol / L palmitate + 100 nmol / L thapsigargin (Tg) (modeling + agonist group), ④ 200 μmol / L palmitate + 5 mmol / L 4-phenylbutyric acid (modeling + inhibitor group), ⑤ 200 μmol / L palmitate + 10 6 10 / ml exosomes (modeling + low-dose exosomes group) and ⑥200 μmol / L palmitate + 10 8 / ml exosomes (modeling + high-dose exosome group).

[0100] Detection method: HL-1 cells in each experimental group were incubated with the fixative in the kit at room temperature for 20 minutes, and then slowly rinsed three times with PBS (pH 6). 1 ml of dye solution (A-10ul, B-10ul, C-930ul, X-gal-50ul) was added to each well according to the ratio of the β-galactosidase kit and incubated overnight at 37°C in the dark. The staining was recorded under a microscope.

[0101] Most normal cells are believed to have limited ability to divide, and enter a state of senescence when they cannot divide. At this time, the cells are still alive, but the expression profiles of the cells' genes and proteins have changed greatly. Senescent cells can no longer induce cell division under some conventional stimuli, and the cell cycle distribution of senescent cells is also quite special, which is different from some damage-induced cell dormancy and contact inhibition of cell growth. Senescent cells usually become larger in size and express β-galactosidase with high enzyme activity at pH 6; cells expressing β-galactosidase will turn blue after staining with the reagent of the β-galactosidase kit.

[0102] The experimental results are as follows Figure 9 As shown. Enterococcus casseliflavus )Zz-2024 exosomes have an effect on the aging of mouse cardiomyocytes. Enterococcus casseliflavus)Zz-2024 exosomes can reduce the aging of mouse cardiomyocytes. Compared with the modeling group and Tg group (modeling + agonist group), both high and low doses of exosomes can significantly reduce the aging of cardiomyocytes and significantly reduce the expression of β-galactosidase. The blue area after reagent staining is significantly reduced. Therefore, the experimental results prove that Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes have the function of regulating lipid metabolism and preventing cardiomyocyte aging.

Claims

1. A strain of Enterococcus pyogenes, which is Enterococcus pyogenes ( Enterococcus casseliflavus ) Zz-2024, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31249.

2. Exosomes of Enterococcus pyogenes, which are obtained from the strain Enterococcus pyogenes ( Enterococcus casseliflavus )Exosomes produced by Zz-2024.

3. Use of the Enterococcus leucoderma exosomes according to claim 2 in drugs for treating myocarditis.

4. The use of Enterococcus leucoderma exosomes in a drug for treating myocarditis according to claim 3, characterized in that: Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are used as active ingredients in the preparation of drugs for the treatment of myocarditis.

5. The use of Enterococcus leucoderma exosomes in a drug for treating myocarditis according to claim 3, characterized in that: Enterococcus pyogenes ( Enterococcus casseliflavus ) Application of exosomes of Zz-2024 in drugs to reduce the level of myocarditis.

6. The use of Enterococcus leucoderma exosomes in a drug for treating myocarditis according to claim 5, characterized in that: Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are used as active ingredients in the preparation of drugs for reducing the level of myocarditis.

7. The use of Enterococcus leucoderma exosomes in a drug for treating myocarditis according to claim 3, characterized in that: Enterococcus pyogenes ( Enterococcus casseliflavus )Zz-2024 exosomes are used as active ingredients in the preparation of drugs for reducing myocardial lipid droplets.

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