Application of exosome of enterococcus pluvialis Zz-2024

Abstract

The invention discloses an application of an exosome of enterococcus pluvialis Zz-2024, relates to the technical field of microorganisms, and particularly relates to an enterococcus pluvialis strain and an application of an exosome of the enterococcus pluvialis strain. The invention further discloses the application of the exosome of the enterococcus pluvialis Zz-2024 in the medicine for treating the drug-induced cardiomyopathy. The enterococcus pluvialis (Enterococcus pluvialis) Zz-2024 exosome disclosed by the invention can reverse myocardial fibroblast fibrosis caused by adriamycin chemotherapeutic drugs, so that the enterococcus pluvialis (Enterococcus pluvialis) Zz-2024 exosome has an effect of maintaining the stability of an extracellular matrix environment of myocardial cells. The enterococcus pluvialis Zz-2024 exosome can reduce inflammatory factor expression of dilated cardiomyopathy caused by drug toxicity, so that the enterococcus pluvialis Zz-2024 exosome has the effects of improving the inflammatory state of myocardial cells and recovering cardiac functions.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to an Enterococcus casseliflavus and application of exosomes thereof. BACKGROUND

[0002] Palmitate is often used as a high-fat reagent to simulate a high-fat environment and induce cells, especially cardiomyocytes, to build a high-fat cell model. As a high-fat inducer, palmitate induces cardiomyocytes to undergo senescence phenotype changes, and the expression of senescence-associated secretory phenotype (SASP) is up-regulated, which is manifested by a significant decrease in cell activity, an increase in apoptosis, cell cycle arrest, oxidative stress, and an increase in the expression of senescence markers such as SA-β-Gal. Senescent cells secrete inflammatory cytokines, chemokines, growth factors, and proteases, which are referred to as SASP-related factors. These changes cause an imbalance in cellular homeostasis, and the accumulation of myocardial cell damage, myocardial cell hypertrophy, and changes in cell structure and function lead to cardiac remodeling, which is manifested by left ventricular wall thickening, myocardial fibrosis, and reduced diastolic function. The myocardial contractility decreases, and the cardiac output decreases, which severely affects the pumping function of the heart and ultimately causes cardiovascular diseases and heart failure.

[0003] 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 intestines and exert various beneficial effects on the human body. By stimulating the immune system, Enterococcus can enhance the body's resistance and reduce the incidence of diseases. Enterococcus can secrete various digestive enzymes to help digest food and regulate lipid metabolism. Moreover, Enterococcus has strong acid resistance and can survive in the acidic environment of the stomach and reach the intestines smoothly, thereby exerting its effects in the intestines. This is a significant advantage compared to other acid-sensitive probiotics. After years of research and practical verification, Enterococcus has been proven to be a safe and effective probiotic suitable for people of different ages.

[0004] Currently, it has been disclosed that the exosomes of Enterococcus casseliflavus Zz-2024 can be used to alleviate the problems of cardiomyocytes caused by palmitate induction, such as improving the imbalance of myocardial fatty acid uptake and oxidation, avoiding lipid droplet accumulation, and reducing myocardial lipid toxicity caused by palmitate; and reducing the level of inflammation in cardiomyocytes under pathological conditions caused by palmitate.

[0005] However, drug-induced cardiomyopathy and dilated cardiomyopathy have different pathogenesis and etiology from the senescence of cardiomyocytes induced by palmitate. It is not possible to speculate on the effects of Enterococcus casseliflavus Zz-2024 exosomes on drug-induced cardiomyopathy and dilated cardiomyopathy. SUMMARY

[0006] The application aims to provide an application of the exosome of Enterococcus casseliflavus Zz-2024 different from the prior art.

[0007] The application aims to provide an application of the exosome of Enterococcus casseliflavus Zz-2024 different from the prior art.

[0008] Further, the exosome of Enterococcus casseliflavus Zz-2024 is used as an active ingredient for preparing a drug for treating drug-induced cardiomyopathy.

[0009] Further, the drug-induced cardiomyopathy is myocardial injury caused by an anthracycline chemotherapy drug.

[0010] Further, the drug-induced cardiomyopathy is dilated cardiomyopathy caused by an anthracycline chemotherapy drug.

[0011] Further, the exosome of Enterococcus casseliflavus Zz-2024 is used as an active ingredient for preparing a drug for reversing myocardial fibroblast fibrosis caused by an anthracycline chemotherapy drug.

[0012] Further, the exosome of Enterococcus casseliflavus Zz-2024 is used as an active ingredient for preparing a drug for reducing the inflammatory level of drug-induced cardiomyopathy.

[0013] Further, the exosome of Enterococcus casseliflavus Zz-2024 is used as an active ingredient for preparing a drug for reducing the inflammatory level of drug-induced cardiomyopathy.

[0014] Advantages:

[0015] The exosome of Enterococcus casseliflavus Zz-2024 can reverse myocardial fibroblast fibrosis caused by an anthracycline chemotherapy drug, thereby maintaining the stability of the extracellular matrix (ECM) environment of myocardial cells.

[0016] The exosome of Enterococcus casseliflavus Zz-2024 can reduce the expression of inflammatory factors of dilated cardiomyopathy caused by drug toxicity, thereby improving the inflammatory state of myocardial cells and restoring the function of the heart.

[0017] The exosome derived from Enterococcus casseliflavus has good biocompatibility with humans, and the biosafety test results of the exosome of Enterococcus casseliflavus Zz-2024 also show that the exosome of Enterococcus casseliflavus Zz-2024 has no toxic side effects. The exosome of Enterococcus casseliflavus Zz-2024 can protect the damage of adriamycin to myocardial fibroblasts in an in vitro cell model, and indirectly protect the expression of IL6 and other key inflammatory factors. The exosome of Enterococcus casseliflavus Zz-2024 has the effect of inhibiting myocardial cell inflammation caused by drugs.

[0018] The exosome of Enterococcus casseliflavus Zz-2024 can reverse the fibrosis of myocardial fibroblasts caused by adriamycin in an in vitro cell model, and inhibit the expression of type I collagen, thereby creating a favorable ECM microenvironment for myocardial cells. The exosome of Enterococcus casseliflavus Zz-2024 has the effects of regulating myocardial fibrosis metabolism and restoring myocardial function.

[0019] The exosome of Enterococcus casseliflavus Zz-2024 can improve the cardiac function indicators such as left ventricular ejection fraction (EF) of adriamycin-induced dilated cardiomyopathy model mice. In the treatment of dilated cardiomyopathy mice, it can inhibit myocardial inflammatory factors, reverse myocardial fibrosis and other experiments, and has excellent effects.

[0020] Enterococcus casseliflavus Zz-2024 is Enterococcus casseliflavus, belongs to the genus Enterococcus, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Beichen West Road, Haidian District, Beijing, China, and the Institute of Microbiology, Chinese Academy of Sciences, on July 10, 2024, with the preservation number of CGMCC No. 31249. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1is a transmission electron microscope observation image of the Enterococcus casseliflavus Zz-2024 exosome obtained in Example 1 (one of three);

[0022] Figure 2 is a transmission electron microscope observation image of the Enterococcus casseliflavus Zz-2024 exosome obtained in Example 1 (two of three);

[0023] Figure 3 is a transmission electron microscope observation image of the Enterococcus casseliflavus Zz-2024 exosome obtained in Example 1 (three of three);

[0024] Figure 4 is a nanometer particle size analysis chart of the Enterococcus casseliflavus Zz-2024 exosome obtained in Example 1;

[0025] Figure 5 is a safety comparison test result chart of the Enterococcus casseliflavus Zz-2024 exosome in Example 3;

[0026] Figure 6 is a confocal microscope observation chart of mouse myocardial cells and mouse fibroblasts treated with the Enterococcus casseliflavus Zz-2024 exosome for 24 hours in Example 3;

[0027] Figure 7 is an inhibition rate curve chart of mouse fibroblasts under different concentrations of doxorubicin in Example 4;

[0028] Figure 8 is a comparison chart of cell proliferation of mouse fibroblasts in each group under different concentrations of doxorubicin in Example 4;

[0029] Figure 9 is a comparison chart of the effect of 24h supernatant collected under different conditions on the viability of mouse HL-1 cells in each group in Example 5 detection method I;

[0030] Figure 10 is a comparison chart of the effect of 48h supernatant collected under different conditions on the viability of mouse HL-1 cells in each group in Example 5 detection method I;

[0031] Figure 11 is a comparison chart of the effect of 24h supernatant collected under different conditions on the viability of mouse HL-1 cells in group A and group B in Example 5 detection method I;

[0032] Figure 12 Figure 5 is a comparison chart of the effects of 48h supernatants collected under different conditions in the detection method I of Example 5 on the viability of HL-1 cells of mice in groups A and B;

[0033] Figure 13 Figure 6 is an agarose gel chart of IL-6 amplification products in the detection method II of Example 5, in which lane M: DNA marker (DL2000), 1-3: normal cells, 4-6: group A, 7-9: 0.5uM doxorubicin treatment group, 10-12: group B;

[0034] Figure 14 Figure 7 is a β-galactosidase staining effect chart of normal cell group in Example 6;

[0035] Figure 15 Figure 8 is a β-galactosidase staining effect chart of 0.5uM doxorubicin group in Example 6;

[0036] Figure 16 Figure 9 is a β-galactosidase staining effect chart of 0.5uM doxorubicin plus exosome group in Example 6;

[0037] Figure 17 Figure 10 is a comparison chart of echocardiography detection of mice in each experimental group in Example 7;

[0038] Figure 18 Figure 11 is a comparison chart of ejection fraction (EF) values of mice in each experimental group in Example 7;

[0039] Figure 19 Figure 12 is an immunofluorescence staining effect chart of myocardial tissue fibrosis related protein FABP4 / DLK1 of mice in each experimental group (normal group, model group, treatment group) in Example 8, in which FABP5-green fluorescence, DLK1-red fluorescence, and DAPI-blue fluorescence. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0041] Definitions and explanations:

[0042] Drug-induced cardiomyopathy: drug-induced cardiomyopathy refers to a lesion caused by drug toxicity to myocardial damage, leading to myocardial hypertrophy, cardiac enlargement or abnormal cardiac function.

[0043] Dilated cardiomyopathy (DCM): a type of cardiomyopathy characterized by enlargement of the left ventricle or both ventricles with systolic dysfunction.

[0044] Adriamycin chemotherapy drugs: anthracycline chemotherapy drugs, mainly including doxorubicin, epirubicin, pyran doxorubicin, liposome doxorubicin, etc. Doxorubicin and epirubicin, pyran doxorubicin, liposome doxorubicin have the same functional group (the same active and physical property core structure unit).

[0045] Left ventricular enlargement and hypertrophy are two different cardiac structural changes, although they are related to the left ventricle, but there are differences in causes and manifestations. Left ventricular enlargement usually refers to an increase in cardiac chamber volume, while left ventricular hypertrophy is an increase in myocardial wall thickness. Both can affect cardiac function, but the mechanisms and consequences are different.

[0046] LI6: interleukin 6 (IL-6), an interleukin encoded by the IL6 gene, can act as a pro-inflammatory cytokine and an anti-inflammatory myokine (the role of IL-6 as an anti-inflammatory myokine is mediated through its inhibitory effect on TNF-α and IL-1 and activation of IL-1ra and IL-10).

[0047] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0048] The "drug" in the present application includes but is not limited to injection, tablet, capsule, spray and patch, etc.

[0049] Example 1: The separation and preparation method of Enterococcus casseliflavus Zz-2024 exosome in the embodiment is as follows:

[0050] S1, after Enterococcus casseliflavus Zz-2024 was activated and recovered by MRS solid slant culture medium, it was inoculated into sterilized MRS liquid medium, and placed in a 37 ℃ constant temperature incubator for culture for 12h, to obtain the seed liquid of Enterococcus casseliflavus Zz-2024;

[0051] S2, inoculate into sterilized MRS liquid medium again at an amount of 1%, continue to culture to the late logarithmic growth phase, to obtain Enterococcus casseliflavus Zz-2024 bacterial liquid;

[0052] S3, centrifugal the Enterococcus casseliflavus Zz-2024 bacteria liquid and discard the supernatant, then add TY liquid medium, incubate at 30 DEG C for 6h;

[0053] S4, collection of exosomes: centrifugal the suspension liquid incubated for 6h at 3000g for 10min, discard the bacteria, collect the supernatant;

[0054] S5, differential centrifugation:

[0055] 5.1, 300g, 4 DEG C, centrifugal for 10min, discard the precipitate;

[0056] 5.2, 2000g, 4 DEG C, centrifugal for 10min, discard the precipitate;

[0057] 5.3, 10,000g, 4 DEG C, centrifugal for 30min, discard the precipitate, collect the supernatant, filter with pore size 0.45 μm filter;

[0058] 5.4, filter liquid 110000g, 4 DEG C, centrifugal for 70min, discard the supernatant;

[0059] 5.5, resuspend the precipitate with PBS, filter with pore size 0.22 μm membrane filter, filter liquid 110000g, 4 DEG C, centrifugal for 70min, discard the supernatant, obtain the preliminary Enterococcus casseliflavus Zz-2024 exosomes;

[0060] S6, add the preliminary Enterococcus casseliflavus Zz-2024 exosomes obtained in step S5 to PBS, then add iodixanol to prepare 3mL 40% iodixanol suspension containing outer membrane vesicles;

[0061] S7, prepare 5%, 10%, 20% iodixanol solutions with sterilized water and iodixanol solution; then add 3mL 40% iodixanol suspension containing exosomes prepared in step S6 into an ultracentrifuge tube, then sequentially add 3mL 20% iodixanol solution, 3mL 10% iodixanol solution and 3mL 5% iodixanol solution, then centrifugal at 100000g for 18h;

[0062] S8, after ultracentrifugation in step S7, divide the suspension in the ultracentrifuge tube into 12 layers, 1mL each, sequentially take 1mL from top to bottom, and ensure that the pipette gun head does not insert into the next layer each time; collect the 4th-6th layers, that is, obtain the Enterococcus casseliflavus Zz-2024 exosomes.

[0063] Enterococcus casseliflavus Zz-2024 was extracted from the intestinal feces of a newborn in Harbin in April 2023.

[0064] The separation, purification and identification method of Enterococcus casseliflavus Zz-2024:

[0065] 8.1, the intestinal feces of a newborn is added to sterile water according to 1:50, and gradient dilution is carried out;

[0066] 8.2, 100uL of appropriate gradient dilution liquid is added to solid MRS (containing CaCO3) culture medium, and plate coating is carried out, and culture is carried out at 37℃;

[0067] 8.3, select single colonies with obvious calcium fusion ring for three-zone streaking, culture at 37℃, and purify the strain;

[0068] 8.4, also select single colonies with obvious calcium fusion ring for three-zone streaking, culture at 37℃, and continue to purify;

[0069] 8.5, single colonies are selected from the purified plate and added to liquid MRS culture medium for expansion culture, and bacterial suspension is used for PCR sequencing and identification of species.

[0070] 8.6, after identification of the species, the bacterial suspension is mixed with 50% sterilized glycerol at 1:1, and stored at-80℃; in addition, the slope is inoculated, and the strain preservation center is preserved.

[0071] The culture of Enterococcus casseliflavus Zz-2024 in the application is not limited to the method and steps described in the embodiment.

[0072] The separation and acquisition method of the exosome of Enterococcus casseliflavus Zz-2024 in the application is not limited to the method and steps described in the embodiment.

[0073] Example 2: Transmission electron microscopy observation was carried out on the exosome of Enterococcus casseliflavus Zz-2024 obtained in Example 1, as shown in the electron microscope, a large number of vesicle-like enrichments in concave double-membrane structure can be directly observed, which confirms the morphology of the exosome extracted in Example 1. Figures 1-3

[0074] ​Nano particle size analysis was performed on the Enterococcus casseliflavus Zz-2024 exosomes obtained in Example 1, and it can be seen from the nano particle size results (as shown in Figure 4 Table 1) that the Enterococcus casseliflavus Zz-2024 exosomes are very pure, very uniform, and have a diameter of 80 nm to 120 nm.

[0075] Example 3: In this example, CCK8 experiment was used to detect the effect of exosomes on the viability of mouse myocardial cells and mouse fibroblasts.

[0076] Experimental grouping:

[0077] HL-1 cells (mouse myocardial cells) and mouse fibroblasts were cultured in high-glucose DMEM medium containing 10% fetal bovine serum. When the cells reached 80-90% confluence, they were passaged, and when the cells reached 50-60% confluence, PBS or exosomes were added to the culture medium for 72 hours. The treatments included the following: PBS (control group), 10 10 5 exosomes / ml (low-dose group), 10 6 exosomes / ml (medium-dose group), 10 7 exosomes / ml (high-dose group). At the same time, in order to better prove that the exosomes indeed entered the cells, the exosomes were dyed with fluorescent dye DIR and then added to the two types of cells. After incubation for 24 hours, confocal microscopy was performed and photographs were taken.

[0078] Detection method: The following operations were performed on the four groups of treatments of the two types of cells.

[0079] First step: cell inoculation

[0080] 1. Digest the cells: use trypsin or other enzymes to digest the cells and make them detach from the culture dish.

[0081] 2. Count: use a cell counting plate to count the cells.

[0082] 3. Inoculation: according to the experimental design, inoculate a certain number of cells into a 96-well plate, and the number of cells in each well should be consistent. Usually, a blank control group (only add culture medium, do not contain cells) and an experimental group (cells under different treatment conditions) are set up, and multiple replicates are set up for each group.

[0083] Second step: cell culture and treatment

[0084] 1. Place the 96-well plate inoculated with cells in the incubator for culture, so that they adhere and grow for 24 hours.

[0085] 2.24 hours later, the total volume was changed, and the above design was followed: the control group was added with PBS, and the experimental group was added with different doses of exosomes.

[0086] Step 3: CCK8 detection

[0087] 1. Before 24, 48, and 72 hours after treatment, 2 hours before, CCK8 reagent was added to each well, usually according to 1 / 10 of the volume of the culture medium.

[0088] 2. The 96-well plate was continued to be placed in the incubator for 2 hours.

[0089] Step 4: Determination of absorbance

[0090] The absorbance (OD value) at 450 nm wavelength was determined using an enzyme labeler.

[0091] Step 5: Data analysis

[0092] According to the determined OD value, the proliferation or inhibition of cells in each group was calculated.

[0093] Step 6: Exosome staining

[0094] According to the instructions, dilute the DIR dye to 4 μM, take 1 ml and add it to an equal volume of exosome solution containing 10 8 units. After 30 minutes at room temperature, centrifuge at 110,000 g at 4°C for 70 minutes. Discard the supernatant and wash twice with PBS liquid, each time at 110,000 g at 4°C for 70 minutes. Finally, resuspend the precipitate to 1 ml of sterile PBS solution and prepare for use.

[0095] Step 7: Confocal observation and photography

[0096] Inoculate two types of cells into a 6-well plate, and when they are 80% confluent, add 200 ul of labeled exosomes to each well; continue to culture for 24 hours, and then wash twice with PBS. Then, add 1 ml of normal culture medium, and observe and take pictures under a confocal microscope.

[0097] The experimental results are as follows Figure 5The results show that the exosomes of Enterococcus casseliflavus Zz-2024 have no effect on the viability of mouse myocardial cells (HL-1 cells) and mouse fibroblasts. The exosomes of Enterococcus casseliflavus Zz-2024 of any concentration have no significant difference in cell activity compared with the control group after being treated for 24 hours, 48 hours and 72 hours respectively. Therefore, the experimental results show that the exosomes of Enterococcus casseliflavus Zz-2024 are non-toxic and safe to cells. Observation under a confocal microscope (as shown in Figure 6 The results show that the exosomes of Enterococcus casseliflavus Zz-2024 have no effect on the viability of mouse myocardial cells (HL-1 cells) and mouse fibroblasts. The exosomes of Enterococcus casseliflavus Zz-2024 of any concentration have no significant difference in cell activity compared with the control group after being treated for 24 hours, 48 hours and 72 hours respectively. Therefore, the experimental results show that the exosomes of Enterococcus casseliflavus Zz-2024 are non-toxic and safe to cells. Observation under a confocal microscope (as shown in

[0098] Example 4: This example uses CCK8 experiment to detect the effect of exosomes on the viability of mouse fibroblasts damaged by doxorubicin.

[0099] Doxorubicin damages myocardial cells by free radicals and inhibits topoisomerase, which may cause acute and chronic cardiac damage. The clinical manifestations are arrhythmia, cardiomyopathy and even heart failure.

[0100] Free radical damage: Doxorubicin generates reactive oxygen species (ROS) through iron ion mediation, damages myocardial cell mitochondria function, and induces oxidative stress and apoptosis.

[0101] Inhibition of topoisomerase II leads to abnormal chromosome structure, interferes with myocardial cell DNA repair, and causes myocardial fibrosis.

[0102] Experimental grouping:

[0103] Mouse fibroblasts were cultured in high-glucose DMEM medium containing 10% fetal bovine serum. When the cells reached 80-90% confluence, they were passaged. When the cells reached 50-60% confluence, different concentrations of doxorubicin (0, 0.1, 0.5, 1.0, 2.0 uM) were added to the culture medium. After CCK8 detection for 24, 48 and 72 hours, a suitable stimulation concentration was determined as the subsequent experimental concentration. The subsequent treatments include the following: PBS (control group), doxorubicin treatment group 10 6 exosomes (low-dose group), 10 7 exosomes (medium-dose group), and 8 10

[0104] Detection method: the following operation is carried out on the cells.

[0105] First step: cell inoculation

[0106] 1. Digest the cells: use trypsin or other enzymes to digest the cells and make them detach from the culture dish.

[0107] 2. Counting: count the cells using a cell counting plate.

[0108] 3. Inoculation: according to the experimental design, inoculate a certain number of cells into a 96-well plate, and the number of cells in each well should be consistent. Usually set up blank control group (only add culture medium, no cells), experimental group (cells under different treatment conditions), and set up multiple replicates for each group.

[0109] Second step: cell culture and treatment

[0110] 1. Place the 96-well plate inoculated with cells into the incubator for culture, so that they can adhere and grow for 24 hours.

[0111] 2. After 24 hours, change the medium completely and perform the treatment according to the above design: add different concentrations of doxorubicin (0, 0.1, 0.5, 1.0, 2.0 uM) to the control group.

[0112] Third step: CCK8 detection

[0113] 1. Add CCK8 reagent to each well 2 hours before 24 and 48 hours after treatment, usually according to 1 / 10 of the volume of culture medium.

[0114] 2. Continue to incubate the 96-well plate in the incubator for 2 hours.

[0115] Fourth step: determination of absorbance

[0116] Use a microplate reader to measure the absorbance (OD value) at 450 nm wavelength.

[0117] Fifth step: data analysis

[0118] According to the measured OD value, calculate the proliferation or inhibition of cells in each group.

[0119] Sixth step: according to the calculation of inhibition rate, finally select 0.5 uM as the stimulation concentration for subsequent experiments. Repeat the first and second steps to inoculate the 96-well plate. According to the design, add: PBS (control group), 0.5 uM doxorubicin treatment group 0.5 uM doxorubicin plus 10 6 exosomes (low dose group), 0.5uM doxorubicin plus 10 7 Exosomes / ml (medium dose group), ⑤ 0.5uM doxorubicin plus 10 8 Exosomes / ml (high-dose group).

[0120] Step 7: Repeat steps 3 through 5.

[0121] The inhibitory rates of different concentrations of doxorubicin (0, 0.1, 0.5, 1.0, 2.0 uM) on mouse fibroblasts are as follows: Figure 7 As shown; experimental results are as follows Figure 7 As shown, since further experimental observation is needed after injury, a concentration of 0.5 μM with an inhibition rate of approximately 30% was selected as the concentration for subsequent experiments. Experimental results showed that *Enterococcus casseliflavus* Zz-2024 exosomes had a protective effect against doxorubicin-stimulated mouse fibroblast viability. Compared with the control group, the *Enterococcus casseliflavus* Zz-2024 exosomes of this invention, regardless of low, medium, or high concentrations, significantly increased cell viability after 48 hours of treatment (e.g., ...). Figure 8 As shown in the figure, the experimental results indicate that the exosomes of Enterococcus casseliflavus Zz-2024 have a repairing effect on doxorubicin-induced myocardial fibroblast damage.

[0122] Example 5: In this example, CCK8 and PCR methods were used to detect the effects of different simulated microenvironments on the viability and expression of inflammatory factors of mouse cardiomyocytes.

[0123] Experimental Groups:

[0124] HL-1 cells (mouse cardiomyocytes) were cultured in DMEM medium (containing 10% fetal bovine serum). Cells were passaged when they reached 80-90% confluence, and then treated with a drug in the medium for 24 hours when they reached 50-60% confluence. The drug treatment included the following: Mouse fibroblasts were treated with 0.5 uM doxorubicin; the supernatant was collected at 24 and 48 hours respectively; and stored at -20℃ for later use. Add 10 to mouse fibroblasts 7 Exosomes per ml; supernatant was collected at 24 and 48 hours respectively; stored at -20℃ for later use. 0.5 uM doxorubicin was added to mouse fibroblasts during treatment. 7 Exosomes were collected per ml; supernatant was collected at 24 and 48 hours; and stored at -20°C for later use. The above culture supernatant was added to the cardiomyocyte culture medium for 24 hours, and CCK8 assay was performed.

[0125] To further prove whether the pre-intervention of exosomes will produce different effects, two groups are set: (A group) 10 7 uM adriamycin, and then 0.5 uM adriamycin is added; the supernatant is collected at 24 and 48 hours, respectively; and it is stored at -20°C for standby use. (B group) 10 7 uM adriamycin is added to the mouse fibroblasts, and 0.5 uM adriamycin is added at the same time; the supernatant is collected at 24 and 48 hours, respectively; and it is stored at -20°C for standby use. The above culture supernatant is added to the myocardial cell culture medium for 24 hours. At the same time of CCK8 detection, the myocardial cells are collected, RNA is extracted, and inflammatory factor IL-6 is amplified.

[0126] Detection method I: using the above condition culture solution, the following operations are performed on HL-1 cells.

[0127] First step: cell inoculation

[0128] 1. Digest the cells: use trypsin or the like to digest the cells and make them detach from the culture dish.

[0129] 2. Count: count the cells using a cell counting plate.

[0130] 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. Generally, a blank control group (only medium, no cells) and an experimental group (cells under different treatment conditions) are set up, and multiple replicates are set up for each group.

[0131] Second step: cell culture and treatment

[0132] 1. Place the 96-well plate inoculated with cells into the incubator for culture, so that they can adhere and grow for 24 hours.

[0133] 2. After 24 hours, change the medium in full, and perform the treatment according to the above design: the control group is added with cell supernatant treated under different conditions (100 ul per well + 100 ul fresh culture medium).

[0134] Third step: CCK8 detection

[0135] 1. At 2 hours before and after 24 hours of treatment, CCK8 reagent is added to each well, usually according to 1 / 10 of the volume of the culture medium.

[0136] 2. Continue to place the 96-well plate into the incubator for 2 hours.

[0137] Fourth step: determination of absorbance

[0138] Determination of absorbance (OD value) at 450 nm wavelength using a microplate reader.

[0139] Step 5: Data Analysis

[0140] According to the measured OD value, calculate the proliferation or inhibition of each group of cells.

[0141] Detection Method II: Collect cells from groups A / B and perform the following operations.

[0142] Step 1: RNA Extraction

[0143] 1. Sample Preparation: Select healthy HL-1 cell samples from each experimental group to ensure that the number and quality of cells meet the experimental requirements.

[0144] 2. Cell Lysis: Lysate the above cells using lysis buffer (such as TRIzol or other commercial lysis reagents) to release RNA.

[0145] 3. RNA Isolation: Isolate RNA from the lysate by centrifugation or column purification method.

[0146] 4. Washing: Use appropriate washing buffer to remove protein and DNA contamination.

[0147] 5. RNA Purification: Purify RNA from the column through adsorption and elution steps.

[0148] 6. RNA Quantification: Determine RNA concentration and purity using ultraviolet spectrophotometer or fluorescence quantifier.

[0149] 7. RNA Quality Assessment: Assess RNA integrity by gel electrophoresis or bioanalyzer.

[0150] Step 2: Reverse Transcription

[0151] 1. Reverse Transcription Reaction: Mix purified RNA from each experimental group with reverse transcriptase, primers, and buffer to perform reverse transcription reaction.

[0152] 2. Reverse Transcription Conditions: Usually perform reverse transcription reaction at 42°C for 1 hour, then heat to 85°C to inactivate reverse transcriptase.

[0153] 3. cDNA Synthesis: Under the above conditions, reverse transcriptase transcribes RNA template into cDNA.

[0154] 4. cDNA Dilution: Dilute cDNA appropriately according to the needs of PCR reaction to obtain optimal amplification effect.

[0155] Step 3: Real-time PCR

[0156] 1. PCR reaction system: Prepare the PCR reaction system according to the instructions of the qPCR kit used, including cDNA template, primers, probe (if used), enzyme and buffer.

[0157] 2. Primer design: Design specific primers with the sequences shown in Table 1 below to ensure accurate amplification of the target gene.

[0158] Table 1

[0159] 3. PCR condition setting: Set 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), cycle 40 times.

[0160] Step 4: Data analysis

[0161] 1. Agarose gel preparation: Prepare a 2% agarose gel using 0.5X TBE buffer.

[0162] 2. Agarose gel electrophoresis: Take 10ul pcr product, add 2ul 5x loading buffer, mix and add to the gel well; Place the gel in the electrophoresis tank, add 0.5X TBE buffer to cover the gel surface; Cover the lid, connect the electrophoresis instrument, 100V electrophoresis for 20 minutes. Take a photo with UV transilluminator.

[0163] 3. Statistical analysis: Perform band intensity measurement and compare gene expression differences between different samples or treatment groups.

[0164] The experimental results are shown in Figures 9-12 , which demonstrate that Enterococcus casseliflavus Zz-2024 exosomes repair adriamycin-damaged mouse fibroblasts, indirectly affecting the influence of the in vitro microenvironment on the viability of cardiomyocytes. The conditioned medium containing exosomes for 48 hours showed a significant promoting effect on the proliferation of cardiomyocytes 24 hours after the addition of cardiomyocytes (as shown in Figure 10 ). Moreover, whether exosome intervention is performed first or simultaneously, exosomes show significant repair effects (as shown in Figure 11 , 12 ). Figure 13The results show that the exosomes of Enterococcus casseliflavus Zz-2024 can inhibit the inflammation of myocardial cells caused by drugs such as doxorubicin.

[0165] The results show that the exosomes of Enterococcus casseliflavus Zz-2024 can inhibit the inflammation of myocardial cells caused by drugs such as doxorubicin.

[0166] Example 6: Myocardial cell β-galactosidase staining experiment

[0167] In this example, β-galactosidase staining is used to observe the aging characteristics of mouse myocardial cells, and to detect the influence of the in vitro microenvironment involving Enterococcus casseliflavus Zz-2024 exosomes on the aging of mouse myocardial cells.

[0168] Experimental grouping:

[0169] HL-1 cells (mouse myocardial cells) were cultured in DMEM medium containing 10% fetal bovine serum. When the cells reached 80-90% confluence, they were passaged, and when the cells reached 50-60% confluence, they were treated with drugs for 24 hours. The drug treatment included the following: Normal culture of fibroblast cell supernatant, collected at 24 hours; -20°C storage for standby. 0.5 uM doxorubicin treatment of mouse fibroblasts; collect supernatant at 24 hours; -20°C storage for standby. 0.5 uM doxorubicin treatment of mouse fibroblasts with the addition of 10 7 μg / ml exosomes; collect supernatant at 24 hours; -20°C storage for standby. Add the above culture supernatant to the myocardial cell culture medium and treat for 48 hours, then perform β-galactosidase staining detection.

[0170] Detection method:

[0171] Incubate the HL-1 cells in each experimental group with the fixative in the kit at room temperature for 20 minutes, then wash slowly with PBS three times (pH 6). According to the dye ratio in the β-galactosidase kit (A-10ul, B-10ul, C-930ul, X-gal-50ul), add 1 ml of dye solution to each well, and incubate at 37°C overnight in the dark. Record the staining under a microscope.

[0172] Most normal cells are thought to have only a limited capacity for division, and after they can no longer divide, they enter a senescence state. At this time, the cells are still alive, but the expression profiles of genes and proteins of the cells have changed greatly. The senescent cells cannot be induced to divide again under some conventional stimulation, and the cell cycle distribution of the senescent cells is also special, different from some damage-induced cell dormancy and different from the case of cell growth contact inhibition. The senescent cells usually become larger in size and have high-enzyme-activity β-galactosidase expression at pH 6; after staining with the reagent of a β-galactosidase kit, the cells expressing β-galactosidase will turn blue.

[0173] The experimental results are shown in Table 1. Figures 14-16 The results show that the exosomes of Enterococcus casseliflavus Zz-2024 have an effect on the senescence of mouse myocardial cells. The exosomes of Enterococcus casseliflavus Zz-2024 of the present application can reduce the senescence of mouse myocardial cells caused by doxorubicin. Compared with the doxorubicin group, the intervention group of the exosomes significantly reduces the necrosis and senescence of myocardial cells, and greatly reduces the expression of β-galactosidase, and the blue area is obviously reduced after reagent staining. Therefore, the experimental results prove that the exosomes of Enterococcus casseliflavus Zz-2024 have the effect of regulating the microenvironment of myocardial cells, repairing the myocardial fibroblast damage caused by doxorubicin, thereby reducing the senescence of myocardial cells caused by doxorubicin.

[0174] Example 7: Improvement of tail vein injection of Enterococcus casseliflavus exosomes on cardiac function of mouse dilated cardiomyopathy model

[0175] In this example, a mouse dilated cardiomyopathy model is established by intraperitoneal injection of doxorubicin; and after modeling, Enterococcus casseliflavus Zz-2024 exosomes are injected through the tail vein, and the cardiac function indicators before and after treatment are compared two weeks later to verify the effect of Enterococcus casseliflavus Zz-2024 exosomes on repairing myocardial function damage in mice.

[0176] Detection method:

[0177] 1. Establishment of mouse dilated cardiomyopathy model

[0178] 1.1 BALB / C strain male experimental mice were selected as the modeling object.

[0179] 1.2 Intraperitoneal injection of mice with adriamycin at a dose of 5 mg / kg, once a week for 4 weeks.

[0180] 1.3 After stopping the drug for 1 week, observe whether the ejection fraction (EF value) of the mice decreases significantly (less than 60%) to determine whether the model is successfully established.

[0181] 2, Identification of the mouse dilated cardiomyopathy model

[0182] 2.1 Some successfully modeled mice were taken, fixed, paraffin-embedded, and then sliced for HE and MASSON staining to further determine the success of the modeling.

[0183] 3, Two weeks of tail vein injection as treatment

[0184] 3.1 Randomly take successfully modeled mice, and inject Enterococcus casseliflavus Zz-2024 exosomes (2 x 10 7单位 / mouse) into the tail vein; once every other day.

[0185] 3.2 After two weeks, small animal ultrasound detection was performed for comparative analysis.

[0186] EF less than 60% was used as the modeling standard. Heart-to-body ratio and HE staining were also performed to identify the model. Among the 15 surviving modeled mice, 11 had an EF less than 60%. The heart-to-body ratio of the model group was significantly higher than that of the normal control group; HE staining showed that the myocardial cells near the epicardium were arranged in disorder, the myocardial cells were twisted and hypertrophic, the intercellular space was increased, the extracellular matrix around the blood vessels was increased, and inflammatory cells were infiltrated (as shown in FIG. 2B). Figure 17

[0187] After modeling, two weeks of medium-dose exosome tail vein injection treatment, the EF value of all model mice was significantly increased (as shown in FIG. 3B). Figure 18 Figure 18 As shown in FIG. 4B, the myocardial motion amplitude of the same model mouse was significantly improved after treatment. It shows that the Enterococcus casseliflavus Zz-2024 exosomes of the application not only can repair myocardial damage and improve heart function, but also have certain heart targeting.

[0188] ​​In dilated cardiomyopathy (DCM), interleukin 6 (IL-6) as a key pro-inflammatory cytokine, can participate in the occurrence and progression of dilated cardiomyopathy through various mechanisms. It is manifested in 1. Myocardial fibrosis: IL-6 activates the JAK-STAT signaling pathway, stimulates cardiac fibroblasts to secrete collagen, leading to myocardial stiffness and decreased systolic function. 2. Myocardial cell damage: chronic IL-6 elevation promotes myocardial cell hypertrophy, apoptosis and autophagy imbalance, exacerbating ventricular remodeling. 3. Inflammatory cascade: IL-6 induces the release of other inflammatory factors (such as CRP), forming a positive feedback loop, continuously amplifying the inflammatory response. The experiments of this embodiment further confirm the results of the cell experiments. The exosomes of Enterococcus casseliflavus Zz-2024 can inhibit myocardial cell inflammation in dilated cardiomyopathy.

[0189] Example 8: Effect of tail vein injection of Enterococcus casseliflavus exosomes on expression of myocardial fibrosis-related proteins in myocardial tissue of mouse dilated cardiomyopathy model

[0190] In this example, doxorubicin was used to establish a mouse model of dilated cardiomyopathy by intraperitoneal injection; and after modeling, Enterococcus casseliflavus Zz-2024 exosomes were injected into the tail vein, and after two weeks, myocardial tissue was taken for FABP4 / DLK1 immunofluorescence double staining to compare the degree of myocardial fibrosis before and after treatment, confirming the effect of Enterococcus casseliflavus Zz-2024 exosomes on myocardial fibrosis in mice.

[0191] Detection method:

[0192] 1. De-waxing

[0193] Soak the slices in xylene 1 (15 min), xylene 2 (15 min), anhydrous ethanol 1 (5 min), anhydrous ethanol 2 (5 min), 95% ethanol (5 min), 85% ethanol (5 min), 75% ethanol (5 min), and finally rinse the slices with water.

[0194] 2. Antigen repair

[0195] Antigen repair usually uses 1x citric acid (PH 6.0) as a repair solution, high temperature and high pressure repair, put the slices in a pressure cooker, add appropriate amount of repair solution, close the pressure cooker, and count the time after it rises, count for 2 min, cool to room temperature, and the repair is complete. If the expression is weak, EDTA (PH 9.0) can be used as a repair solution for antigen repair.

[0196] 3. Endogenous enzyme blocking

[0197] Prepare 3% H2O2 solution in water, place sections in solution, incubate at room temperature for 20 min. If sections are fragile, reduce H2O2 concentration and incubation time as appropriate.

[0198] 4. Serum blocking

[0199] Block with serum of the same origin as the secondary antibody (typically 10% goat serum), incubate at 37°C for 30 min.

[0200] 5. Primary antibody incubation

[0201] Dilute primary antibody 1:200 (or other appropriate dilution) in antibody diluent, incubate overnight at 4°C or 2 h at 37°C.

[0202] 6. Secondary antibody incubation (HRP enzyme-labeled secondary antibody)

[0203] Prepare secondary antibody in PBST, incubate at 37°C for 1 h.

[0204] 7. Incubate TSA reagent

[0205] Add TYR-488 (if 488) to sections, incubate at 37°C for 30 min, wash in PBS 3x5 min.

[0206] 8. Antigen retrieval

[0207] Place slides in 1x citric acid, microwave at high for 5 min, cool to room temperature.

[0208] 9. Serum blocking (optional)

[0209] Block with serum of the same origin as the secondary antibody (typically 10% goat serum), incubate at 37°C for 30 min.

[0210] 10. Primary antibody incubation

[0211] Dilute primary antibody in antibody diluent, incubate overnight at 4°C or 2 h at 37°C.

[0212] 11. Secondary antibody incubation (HRP enzyme-labeled secondary antibody)

[0213] Prepare secondary antibody in PBST, incubate at 37°C for 1 h.

[0214] 12. Incubate TSA reagent

[0215] Add TYR-555 (IF-555) to sections, incubate at 37°C for 30 min, wash in PBS 3x5 min.

[0216] 13. DAPI nuclear stain

[0217] Add DAPI working solution drop to the tissue, incubate at room temperature for 5 min, then wash with PBS for 3 times, each time for 5 min. Spin dry the liquid, add anti-fluorescence sealing agent drop to the tissue, cover with cover glass. Put the prepared fluorescence slide to 4℃ dark storage.

[0218] 14. Take a photo under fluorescence microscope.

[0219] The experimental results are shown in Figure 19 It is proved that the Enterococcus casseliflavus Zz-2024 exosome has an effect on the fibrosis of myocardial fibroblasts of mice with dilated cardiomyopathy. The Enterococcus casseliflavus Zz-2024 exosome of the present application can reverse the fibrosis of myocardial fibroblasts of mice (mice with dilated cardiomyopathy) caused by adriamycin. Compared with the modeling group (simple adriamycin group), the expression of the protein DLK1 for inhibiting fibrosis in the exosome treatment group is significantly increased, and the expression of the protein FABP5 for promoting fibrosis is obviously reduced. Therefore, the experimental results prove that the Enterococcus casseliflavus Zz-2024 exosome has the effects of regulating metabolism, preventing the fibrosis process, repairing myocardial cell damage and recovering heart function on mice with dilated cardiomyopathy.

[0220] Myocardial fibroblasts expressing FABP5 (fatty acid binding protein 5) and DLK1 (Delta-like homolog protein 1) genes have significant differences in function, mainly reflected in metabolic regulation, fibrosis progression, cell differentiation and the role in signal pathways. Among them, in terms of fibrosis and tissue repair: - FABP5 has a pro-fibrotic effect: through lipid metabolism, it activates inflammation and fibrosis-related pathways (such as TGF-β), promotes the deposition of collagen (such as type I collagen) and extracellular matrix (ECM), leading to myocardial fibrosis. In clinical myocardial infarction or hypertensive heart disease, high expression of FABP5 may exacerbate cardiac remodeling. - DLK1 has an anti-fibrotic effect: it inhibits the transformation of fibroblasts into myofibroblasts, reduces ECM deposition and collagen production, and may play a role by blocking the TGF-β / Smad or Wnt / β-catenin pathway. And it is related to the regenerative potential. In heart injury models, DLK1 may participate in tissue repair rather than fibrosis by maintaining the "resting" state of fibroblasts or promoting stem cell properties. Therefore, the expression trend of DLK1 / FABP5 proteins in the myocardial tissue of the exosome treatment group further confirms that Enterococcus casseliflavus Zz-2024 exosomes have the function of regulating the in vitro microenvironment, preventing myocardial fibroblasts from fibrosis and other damage, and thus repairing the function of myocardial cells in mice with dilated cardiomyopathy.

Claims

1. Use of exosomes of Enterococcus casseliflavus Zz-2024, characterized in that, Use of exosomes of Enterococcus casseliflavus Zz-2024 in preparation of a drug for treating drug-induced cardiomyopathy.

2. The exosome of Enterococcus casseliflavus Zz-2024 for use according to claim 1, characterized in that, The exosomes of Enterococcus casseliflavus Zz-2024 are used as an active ingredient in preparation of a drug for treating drug-induced cardiomyopathy.

3. The exosome of Enterococcus casseliflavus Zz-2024 for use according to claim 1 or 2, characterized in that, The drug-induced cardiomyopathy is myocardial injury caused by anthracycline chemotherapy drugs.

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