Application of dandelion exosome in preparation of medicine for treating mastitis

Abstract

The invention belongs to the field of Chinese herbal medicine application, and particularly relates to application of dandelion exosomes in preparation of medicine for treating mastitis. According to the invention, a Chinese herbal medicine plant dandelion is taken as an object, an exosome vesicle structure is successfully separated and prepared, and the exosome is taken as a prodrug and is applied to treatment of mastitis. Compared with the prior art, the plant exosome vesicle provided by the invention can well avoid activity loss of effective components of traditional Chinese medicines, improves the treatment effect, weakens the body injury and safety problems caused by traditional medicines, and has a wide application prospect in treatment of mastitis.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese herbal medicines, and in particular relates to the application of dandelion exosomes in preparing a medicine for treating mastitis. Background Art

[0002] According to the latest data report from international research institutions, mastitis is a common disease among women worldwide, especially among lactating women. According to the World Health Organization, the incidence of mastitis among women of childbearing age is about 10% to 20%, among which the incidence is higher among primiparas.

[0003] With the development of traditional Chinese medicine, Chinese herbal medicine plays an important role in improving inflammatory response, including dandelion. Dandelion tastes bitter and sweet, and is cold in nature. It has the effects of clearing away heat and detoxifying, reducing swelling and dispersing knots, and diuresis and relieving stranguria. It is often used clinically to treat furuncle swelling, breast abscess, scrofula, red eyes, sore throat, lung abscess, intestinal abscess, damp-heat jaundice, hot stranguria and other diseases. Dandelion also has a broad-spectrum anti-pathogenic microbial effect, and has a certain inhibitory effect on a variety of pathogens such as pneumococcus, meningococcus, diphtheria bacillus, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhi, etc., and is known as the "natural antibiotic" in traditional Chinese medicine. However, how to improve the efficacy, solubility and safety of the active ingredients in traditional Chinese medicine based on traditional processing has always been a hot topic of concern.

[0004] Patent CN112063575A provides a method for extracting dandelion exosomes and its application. The application uses dandelion exosomes to prepare drugs for treating colitis, and preliminarily proves that dandelion exosomes have anti-inflammatory activity; Patent CN118236413A discloses a dandelion extract that can neutralize bacterial toxins and its application. The application adds dandelion extract to a pharmaceutical preparation, which can be used to neutralize bacterial toxins and exert anti-inflammatory and anti-infective effects. At present, there are no reports on the preparation of drugs for treating mastitis in the relevant research on the application of dandelion exosomes. Summary of the invention

[0005] Based on the problem that the prior art lacks the application of dandelion exosomes in the preparation of drugs for treating mastitis, the purpose of the present invention is to provide the application of dandelion exosomes in the preparation of drugs for treating mastitis. The present application isolates dandelion exosomes (Dan-Exo), which can carry bioactive substances, are enriched in inflamed tissues and significantly inhibit the malignant progression of inflammation.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The technical solution of the present invention provides the use of dandelion exosomes (Dan-Exo) in the preparation of a drug for treating mastitis.

[0008] Furthermore, the dandelion exosomes (Dan-Exo) have a particle size of about 150 to 250 nm, are spherical under an electron microscope, and have a double-layer membrane structure.

[0009] Furthermore, the drug for treating mastitis is an external-use drug, and the drug for treating mastitis is in the form of a smear or a spray, and contains dandelion exosomes.

[0010] Furthermore, the drug for treating mastitis (including Dan-Exo drug) is applied topically and is taken up and absorbed by mammary epithelial cells and accumulated in mammary tissue, thereby inhibiting the malignant progression of inflammation.

[0011] Furthermore, in the drug for treating mastitis (including Dan-Exo drug), Dan-Exo serves as an inflammatory cell apoptosis inhibitor, an inflammatory factor inhibitor, and an inflammatory tissue cell recovery agent; the concentration of dandelion exosomes in the ointment and spray for treating mastitis is 20 to 50 μg / mL.

[0012] Furthermore, the present application provides a method for preparing the dandelion exosomes, comprising the following steps:

[0013] (1) crushing fresh dandelions to obtain crushed liquid;

[0014] (2) After filtering the above-mentioned disrupted liquid, Dan-Exo is separated and collected by ultra-high-speed centrifugation.

[0015] Furthermore, the fresh dandelions in step (1) need to be washed with sterile water before being crushed, and mixed with pre-cooled PBS buffer, the mixing ratio of dandelion: PBS buffer = 1 g: (1-1.1) mL; the concentration of PBS buffer is 0.0067 M; pH = 7.4-7.6.

[0016] Furthermore, the filtration in step (2) refers to filtering using a 0.45 μm filter membrane to obtain a filtrate;

[0017] Furthermore, the ultra-high speed centrifugation method in step (2) comprises the following steps:

[0018] a) centrifuging the filtrate at 4°C, 2000-3000 g for 15-25 min, and collecting the supernatant I;

[0019] b) centrifuging supernatant I at 4°C, 10,000-20,000 g for 30-45 min, and collecting supernatant II;

[0020] c) centrifuging the supernatant II at 4°C, 100,000-120,000 g for 90-120 min, and collecting the precipitate;

[0021] d) Resuspend the precipitate in pre-cooled PBS, repeat 3 to 5 times, filter through a 0.22 μm filter membrane, and the filtrate is the solution containing dandelion exosomes.

[0022] Furthermore, the Dan-Exo is soluble in physiological saline and can exist at 4°C for 1 month.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The Dan-Exo extraction process is simple and less polluting; the raw material dandelion is a common plant in nature and is very easy to obtain in large quantities.

[0025] (2) Dan-Exo treatment is an innovation based on traditional Chinese medicine treatment. It has sufficient safety, stability of effective drug ingredients, low toxicity, and solubility, and has good application prospects.

[0026] (3) The dandelion exosomes provided in the present application can be absorbed by mastitis cell tissues and enriched in inflammatory cell tissues, significantly inhibiting the malignant progression of the inflammatory site, causing little damage to the body, and can effectively reduce the apoptosis of inflammatory cells, inhibit the production of inflammatory factors, and promote the healing and recovery of inflammatory cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The isolation and characterization of dandelion exosomes (Dan-Exo) in Examples 1 to 2; the symbols in the figure indicate: (A) Schematic diagram of the isolation process of Dan-Exo from fresh dandelion; (B) Dan-Exo particle size analysis; (C) Transmission electron microscopy detection of Dan-Exo morphology; (D) Uptake of PKH67-labeled Dan-Exo by mouse mammary epithelial cells;

[0028] Figure 2 The results of the experiment of Dan-Exo inhibiting mouse mammary epithelial cell apoptosis in Example 3; the marks in the figure indicate: (A) fluorescence signal diagram in flow cytometer; (B) statistical analysis results of cell apoptosis ratio;

[0029] Figure 3 The construction of mouse mastitis model in Example 4;

[0030] Figure 4 The results of the experiment of Dan-Exo promoting the healing of inflammatory tissue in Example 5; the symbols in the figure indicate: (A) ROS detection results under electron microscope; (B) ROS level statistical results; (C) Tunel microscope photography detection results; (D) cell apoptosis level statistical results;

[0031] Figure 5 Results of the concentration experiment of Dan-Exo inhibiting inflammatory factors in Example 6; the markings in the figure indicate: (A) Statistical analysis results of the MPO detection experiment; (B) Statistical analysis results of the TNF-α factor in the serum inflammatory factor detection test; (C) Statistical analysis results of the IL-1β factor in the serum inflammatory factor detection test; (D) Statistical analysis results of the IL-6 factor in the serum inflammatory factor detection test;

[0032] In the above statistical analysis figures, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001, indicates 0.0001 < p < 0.001. Detailed implementation manners

[0033] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0034] For all raw materials of the present invention, there is no special limitation on their sources, and those purchased on the market or prepared according to the conventional methods well-known to those skilled in the art are all acceptable. The concentration of the PBS buffer solution used in all examples of this application is: 0.0067 M, and the pH is 7.4.

[0035] All instruments used need to be sterilized before use and are ready for use.

[0036] Example 1 Preparation and morphological characterization of Dan-Exo

[0037] (1) Preparation of Dan-Exo, referring to Figure 1 A, specifically including the following steps:

[0038] Take 500 g of fresh dandelion, thoroughly wash it with sterile water, chop it and add 500 mL of PBS (pre-cooled at 4°C), and juice it with a spiral juicer (voltage / frequency / power: 220 V, 50 HZ, 1.2 KW). Collect the juice and filter it through a 0.45 μm filter membrane, and collect the filtrate;

[0039] Place the filtrate in a 50 mL centrifuge tube, centrifuge at 2000 g at 4°C for 15 min, and collect the supernatant I;

[0040] Place the supernatant I in a 50 mL centrifuge tube, centrifuge at 10000 g at 4°C for 30 min, and collect the supernatant II;

[0041] Place supernatant II in an ultracentrifuge tube, centrifuge at 4°C, 120,000 g for 90 min, and collect the precipitate; resuspend each precipitate in 1 mL of pre-cooled PBS, repeat 3 times, filter through a 0.22 μm filter membrane, and store the filtrate, i.e., the solution containing Dan-Exo, at 4°C for later use.

[0042] (2) Particle size analysis of Dan-Exo: The particle size of Dan-Exo prepared in (1) was measured. The results are shown in Figure 1 B, it can be seen that the particle size of Dan-Exo is around 150-250nm.

[0043] (3) Morphological characterization of Dan-Exo under electron microscope Figure 1 As shown in C, Dan-Exo can be seen to have a double-layer membrane structure.

[0044] Example 2 Uptake of Dan-Exo by Mouse Mammary Epithelial Cells

[0045] (1) PKH26 staining experimental steps: 60 μL of exosome Dan-Exo diluted in PBS was added to 0.5 mL of diluent C (Sigma-Aldrich); 4 μL of PKH26 dye was added to the above 0.5 mL of diluent C to obtain a mixed solution, and the mixed solution was incubated for 4 min to allow the exosome Dan-Exo to fully combine with the dye PKH26;

[0046] To bind excess dye, 1 mL of 0.5% BSA / PBS was added; the PKH26-labeled exosomes were washed at 100,000 g for 1 h to obtain exosome precipitates, which were diluted in 100 μL PBS and used for subsequent uptake experiments.

[0047] (2) Uptake of Dan-Exo by mouse mammary epithelial cells:

[0048] Treatment system of experimental group: 1 μL PKH26-labeled Dan-Exo was added to HCC1833 cells;

[0049] The blank control group consisted of HCC1833 cells only without exosome treatment.

[0050] The operation of the ingestion experiment is as follows:

[0051] The cells in the control group and experimental group were cultured in 4 mL of DMEM complete medium for 24 h, fixed with paraformaldehyde (a conventional fixation method in the art) for 15-20 min, and washed 3 times with PBS; 400 μL of DAPI staining solution (just enough to cover the bottom of the dish) was added, and cultured at room temperature for 3-5 min to allow the stain to fully bind to the cells; after the culture was completed, the cells were washed 2-3 times with PBS to remove unbound dye, each time for 3-5 min, and finally observed under a microscope.

[0052] The results of fluorescence microscopy are as follows Figure 1 As shown in D, the exosomes stained with PKH26 appear red ( Figure 1 D Left), cells stained with DAPI appear green ( Figure 1 D), the blue fluorescence and red fluorescence parts of the cells after uptake are almost overlapping. Therefore, according to the position and color of the fluorescence in the figure, it can be judged that the exosomes Dan-Exo can be taken up and absorbed by mouse mammary epithelial cells.

[0053] Example 3 Effect of Dan-Exo on apoptosis of mouse mammary epithelial cells

[0054] The cell apoptosis experiment uses flow cytometry to detect the number and proportion of cells in different cell cycles, and then determine the effect of dandelion exosomes on the cell cycle of cancer cells. This experiment is divided into Normal group, LPS group (lipopolysaccharide, LPS) and EXO+LPS group.

[0055] Experimental groups: LPS group (mouse mammary epithelial cells + 100 ng / mL LPS), EXO+LPS group (mouse mammary epithelial cells + 100 ng / mL LPS + Dan-Exo);

[0056] The Normal group served as the control group and was treated with PBS instead of LPS and Dan-Exo.

[0057] The specific steps of the cell apoptosis experiment are as follows:

[0058] ① Preparation of cell suspension: Mouse mammary epithelial cells in the logarithmic growth phase were digested with trypsin and centrifuged, then resuspended in DMEM complete medium. After cell counting, 3×10 4 The cells were evenly plated in 6-well plates according to the above group settings, where the final concentration of LPS in the LPS group was 100 ng / mL; the final concentration of LPS in the EXO+LPS group was 100 ng / mL, and the final concentration of Dan-Exo was 20-50 μg / ml; the Normal group was treated with an equal volume of PBS buffer;

[0059] The treated 6-well plate was placed in a 37°C carbon dioxide incubator for 24 h to allow the cells to adhere to the wall. The cell suspension was collected after trypsin digestion, centrifuged and the supernatant was discarded. The cells were washed twice with pre-cooled PBS buffer and resuspended with 1× Binding Buffer to a density of 1×10 cells / ml. 6 cells to obtain a cell suspension, and transfer 100 μL of the cell suspension to a 5 mL centrifuge tube for later use;

[0060] ②Cell staining: Add 5μL FITC Annexin V and 5μL PI dye solution to each centrifuge tube, incubate at 25℃ in the dark for 15min. After incubation, add 400μL 1×Binding Buffer to each tube, filter the cell suspension into the flow tube with a 100-mesh screen, and detect on an Agilent flow cytometer within 2h.

[0061] The results of flow cytometry were as follows Figure 2 As shown in A, the statistical results are plotted on Figure 2 In B, it can be seen that there are more normal cells and fewer apoptotic cells in the Normal group, while the proportion of apoptotic cells in the LPS-treated group increased compared with that in the Normal group, and the increase in apoptotic cells in the LPS+EXO-treated group was smaller than that in the Normal group, indicating that Dan-Exo can inhibit LPS-induced apoptosis of mouse mammary epithelial cells.

[0062] In summary, the results of this example confirm that Dan-Exo has an inhibitory effect on LPS-induced mouse mammary epithelial cell apoptosis, suggesting that Dan-Exo has a good anti-inflammatory effect.

[0063] Example 4 Establishment of mouse mastitis model

[0064] Fifteen mice were randomly divided into three groups according to the standard of 5 mice / group, namely: Normal group (blank control group), LPS group (i.e. Mastitis group), and dandelion exosome treatment + LPS group (i.e. Mastitis + Exo group). The Normal group was not treated.

[0065] The LPS treatment operation is as follows: LPS is diluted with DMSO to 1 mg / mL, and 30uL LPS + 20uL PBS is injected into the mammary gland on one side of each mouse to establish the model. The model is successfully established after 3 consecutive days of injection.

[0066] The treatment procedure of dandelion exosomes was as follows: after successful modeling, mice were treated with dandelion exosomes twice a day for 7 consecutive days, and the healing condition was observed; the LPS group was smeared with an equal volume of PBS buffer.

[0067] The results are as follows Figure 3As shown, the mice in the Normal group were in normal condition and did not suffer from mastitis; the mice in the Mastitis group suffered from mastitis; and the mice in the Exo+Mastitis group suffered from mastitis and were treated with exosomes, and the symptoms of mastitis were alleviated. This indicates that the dandelion exosomes provided by the present invention have a certain inhibitory effect on mastitis in the in vivo smear treatment, and Dan-Exo can promote the healing time of the LPS-induced mouse mastitis model.

[0068] Example 5 Breast Histopathological Analysis

[0069] In this example, a pathological analysis was performed on the mouse mastitis model constructed in Example 4, and the effect of exosome smear treatment on the mastitis model was verified by ROS reactive oxygen species experiment and Tunel staining experiment.

[0070] (1) ROS detection experiment

[0071] Reactive oxygen species (ROS) are products of normal aerobic metabolism in the body. They are a general term for a class of substances that are composed of oxygen, contain oxygen and are active in nature. They include superoxide anions (O 2- ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH - ), ozone (O 3 ) and singlet oxygen ( 1 O 2 ), etc., because they contain unpaired electrons, they have high chemical reactivity. Reactive oxygen is the main molecule produced when the body is under oxidative stress, and has always been considered an important factor in the occurrence, development and recurrence of tumors. Research on ROS in recent years has found that ROS can achieve therapeutic purposes by accelerating the death of tumor cells. At present, drugs aimed at increasing the level of ROS in tumor cells are gradually being used in clinical practice. Under normal circumstances, ROS, as a natural byproduct of oxygen metabolism, is at a low level in the body. As a "redox messenger", it participates in intracellular signal transmission and regulation, and plays an important role in the cell cycle, gene expression and maintenance of the body's internal environment homeostasis.

[0072] The specific steps are as follows:

[0073] 1) One mouse was selected from each group for the experiment. The mammary gland tissue of the mouse was removed and then placed in 4% formaldehyde solution (fixative) for 48 hours;

[0074] 2) Rinse the fixed tissue blocks with sterile water to remove residual fixative;

[0075] 3) Dehydrate the tissue blocks with 70%, 80%, 90%, 95% and 100% ethanol in sequence;

[0076] 4) After dehydration, make the dehydrated tissue blocks transparent;

[0077] 5) Embed the tissue block with wax, then slice and spread it;

[0078] 6) Place the slides in xylene I for 10 min and xylene II for 10 min to wash out the paraffin for staining;

[0079] 7) Place the slides in anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 95% alcohol for 3 minutes, 90% alcohol for 3 minutes, 80% alcohol for 3 minutes, and 75% alcohol for 3 minutes, and rinse briefly with distilled water 2 to 3 times. Do not rinse the slides directly with distilled water to prevent them from falling off;

[0080] 8) Place the slide in hematoxylin stain for 3 minutes;

[0081] 9) Put the slide into hydrochloric acid alcohol, then take it out, so that the color that should not be colored is removed and the color that should be colored becomes clearer and more moderate;

[0082] 10) Wash with distilled water 2-3 times, turn blue, observe under microscope that the nucleus is blue and the background is colorless;

[0083] 11) Place the slide in eosin staining solution and stain for 3 minutes;

[0084] 12) Dehydrate the slides in 95% alcohol I for 30 seconds, 95% alcohol II for 30 seconds, anhydrous ethanol I for 3 minutes, and anhydrous ethanol II for 3 minutes;

[0085] 13) The slides were sequentially treated with xylene I for 5 min, xylene II for 5 min, and neutral gum for sealing. The pathological histological changes of breast tissue were observed under a microscope and photographed for preservation.

[0086] Hematoxylin (H) is a basic dye that can stain the cell nucleus and intracellular ribosomes into blue-purple. The structures stained by basic dyes are basophilic.

[0087] Eosin (Eosin, E) is an acidic dye that can dye the cytoplasm red or light red. The structure stained by the acidic dye is eosinophilic. Before staining, the paraffin in the slice must be removed with xylene, and then the slice is stained through high-concentration to low-concentration alcohol, and finally into distilled water.

[0088] Figure 4 The results of the pathological analysis experiment are as follows: Figure 4 The corresponding statistical analysis results are shown in A. Figure 4As shown in B. This indicates that the ROS content in mastitis tissue after LPS treatment is high, while the ROS level is significantly reduced after dandelion exosome smear treatment, indicating that dandelion exosomes can eliminate ROS produced at the inflammatory site and promote the healing of inflammatory tissue.

[0089] (2) Tunel experiment

[0090] The TUNEL staining method determines the number of cells in apoptotic state by detecting the staining of broken DNA fragments. It is actually a research method based on the combination of molecular biology and morphology. It performs in situ staining of intact single apoptotic cell nuclei or apoptotic bodies, and can accurately reflect the most typical biochemical and morphological characteristics of cell apoptosis. It can be used for apoptosis determination of paraffin sections, frozen sections, cultured cells and cells separated from tissues, and can detect extremely small amounts of apoptotic cells. Its sensitivity is much higher than that of general histochemical and biochemical determination methods, and therefore it has been widely used in the study of cell apoptosis.

[0091] When cells undergo apoptosis, they activate some DNA endonucleases, which can cause double-strand breaks or single-strand breaks in chromosomal DNA to produce a large number of sticky 3'-OH ends. Under the action of terminal deoxyribonucleotide transferase (TdT), derivatives formed by deoxyribonucleotides and fluorescein, peroxidase, alkaline phosphatase or biotin can be labeled to the 3'-end of DNA, thereby allowing apoptotic cells to be detected by fluorescence microscopy or flow cytometry.

[0092] The specific experimental steps are as follows:

[0093] ① Tumor tissue sections: The mastitis tissues collected above were embedded in paraffin, and the embedded paraffin sections were dewaxed to water: the sections were sequentially placed in xylene I for 15 min-xylene II for 15 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-85% alcohol for 5 min-75% alcohol for 5 min-distilled water for washing;

[0094] ② Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 8.0) and perform antigen repair in a microwave oven. Set the microwave oven parameters as follows: medium heat for 8 minutes, stop the fire for 8 minutes, and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the sections should not be dried. After natural cooling, place the tissue sections in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0095] ③ BSA blocking: After the tissue sections are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), add 3% BSA in the circle, evenly cover the tissue, and block at room temperature for 30 minutes;

[0096] ④Add primary antibody (red): Gently shake off the blocking solution, add Ki67 primary antibody (PBS) on the slices, and incubate the slices flat in a light-proof humid box at 4°C overnight;

[0097] ⑤ DAPI counterstaining of cell nuclei (blue): Place tissue sections in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 min each time; After the tissue sections are slightly dried, add DAPI staining solution in the circle and incubate at room temperature away from light for 10 min;

[0098] ⑥ Sealing: Place the tissue sections in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time; after the sections are slightly dried, seal them with anti-fluorescence quenching sealing media;

[0099] ⑦ Microscopic examination and photography: The sections were observed and images were collected under a Nikon inverted fluorescence microscope ( Figure 4 C), the corresponding statistical analysis results are as follows Figure 4 As shown in D.

[0100] The results showed that the apoptosis rate in mastitis tissues after LPS treatment was significantly increased, while the amount of apoptotic cells in mastitis tissues after dandelion exosomes smear treatment was significantly decreased, indicating that dandelion exosomes can significantly inhibit apoptosis in inflammatory sites. In summary, Dan-Exo can promote the healing of inflammatory tissues.

[0101] Example 6 Dan-Exo inhibits the concentration of inflammatory factors

[0102] In this example, myeloperoxidase (MPO) activity assay and serum cytokine assay were used to verify that Dan-Exo can inhibit the concentration of inflammatory factors. The active breast tissues in the two experiments were from the mouse mastitis model constructed in Example 4. The preparation of active breast tissues was as follows: the mice treated in the above groups were killed, the mice were dissected, the breast tissues were removed, and the breast tissues were simply cleaned. 100 mg was weighed, ground in a mortar, dissolved in PBS, centrifuged briefly, and the supernatant was collected to obtain active breast tissues.

[0103] (1) Myeloperoxidase (MPO) activity assay: The activity of breast tissue was detected using an MPO kit. The specific operation is as follows:

[0104] 1) Add 100 μL of sample diluent to the blank well, preventing bubbles from forming during the addition process, and add 100 μL of standard or sample to be tested to each of the remaining wells, and place at 37°C for 2 h;

[0105] 2) Aspirate the supernatant, add 100 μL of detection solution A to each well, react at 37°C for 1 hour, then discard the liquid in the well, wash 3 times and spin dry;

[0106] 3) Add 100 μL of detection solution B to each well, cover the plate with the membrane, treat at 37°C for 1 hour, aspirate the supernatant, wash 3 times and spin dry;

[0107] 4) Add 90 μL of substrate solution to each well, cover the plate with a film, and develop color at 37°C in the dark;

[0108] 5) Add 50 μL of stop solution, and the color changes from blue to yellow;

[0109] 6) Measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme reader;

[0110] 7) Calculate and draw a standard curve, and then calculate the concentration of the sample to be tested based on the standard curve.

[0111] The experimental results are plotted on Figure 5 In A, it can be seen that the concentration of MPO in mastitis cells is higher than that in normal cells, indicating that LPS treatment will increase the concentration of MPO; after dandelion exosome smear treatment, the MPO content is significantly reduced and close to the normal value, indicating that dandelion exosomes can significantly reduce the concentration of MPO and restore the healing of breast inflammation tissue.

[0112] (2) Serum cytokine determination: ELISA kits were used to detect the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6. The specific steps are as follows;

[0113] 1) Dilute the primary antibody at a ratio of 200:1, then add 100 μL to each well, repeat 3 times for each group, incubate at 4°C overnight, wash the plate 3 times, and tap the plate;

[0114] 2) Add 200 μL of sample diluent to each well, then incubate at room temperature for 1 h, discard the solution, wash the plate 3 times, and tap the plate;

[0115] 3) Add 100 μL of the sample to be tested to each well, then incubate at room temperature for 2 h, wash the plate three times with washing solution, shake gently and tap the plate;

[0116] 4) Add 100 μL of secondary antibody to each well, incubate at room temperature for 1 h, wash the plate three times with washing solution, and then tap the plate;

[0117] 5) Add 100 μL / well of horseradish peroxidase, incubate at room temperature for 30 min, wash the plate with washing solution, and then tap the plate;

[0118] 6) Add 100 μL of TMB to each well, then incubate in the dark for 15 min. The positive wells will show blue.

[0119] 7) Add 100 μL / well of stop solution;

[0120] 8) Measure the OD value at a wavelength of 450 nm using an enzyme-labeled instrument.

[0121] The contents of inflammatory cytokines TNF-α, IL-1β and IL-6 were calculated according to the OD values, and the results were shown in Figure 5 B. Figure 5 C. Figure 5 In D, it can be seen that in mastitis cells, the concentrations of several inflammatory cytokines are higher than those in normal cells, indicating that LPS treatment will increase the concentration of inflammatory cytokines; after dandelion exosome smear treatment, the contents of several inflammatory factors are significantly reduced and close to normal values, indicating that dandelion exosomes can significantly inhibit the concentration of inflammatory factors and restore the healing of breast inflammation tissue.

[0122] In summary, MPO detection confirmed that Dan-Exo had a certain anti-inflammatory effect on the LPS-induced mouse mastitis model; serum inflammatory factor detection confirmed that Dan-Exo could significantly inhibit the concentrations of TNF-α, IL-1β, and IL-6 in the serum of the LPS-induced mouse mastitis model.

[0123] The above examples confirm that the dandelion exosomes Dan-Exo prepared in the present application can be taken up by mouse mammary epithelial cells and slow down the apoptosis of mastitis cells; through the construction of a mouse mastitis model and the application of dandelion exosomes for treatment, it was found that dandelion exosomes can promote the healing of inflammatory tissue; through MPO activity assay experiments and inflammatory cytokine concentration assay experiments, it was confirmed that dandelion exosomes have an inhibitory effect on myeloperoxidase and serum inflammatory factors, thereby promoting the recovery of inflammatory tissue.

[0124] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of dandelion exosomes in the preparation of drugs for the treatment of mastitis.

2. The use according to claim 1, characterized in that: The particle size of the dandelion exosomes is 150 to 250 nm, is spherical under an electron microscope, and has a double-layer membrane structure.

3. The use according to claim 1, characterized in that: The medicine for treating mastitis is an external medicine.

4. The use according to claim 1 or 3, characterized in that: The medicine for treating mastitis is in the form of a smear or a spray, and contains dandelion exosomes.

5. The use according to claim 1 or 3, characterized in that: In the drug for treating mastitis, dandelion exosomes are used as an inhibitor of inflammatory cell apoptosis, an inhibitor of inflammatory factors, and an agent for restoring inflammatory tissue cells; the concentration of dandelion exosomes in the ointment and spray for treating mastitis is 20 to 50 μg / mL.

6. The use according to claim 1, characterized in that: The method for preparing dandelion exosomes comprises the following steps: (1) crushing fresh dandelions to obtain crushed liquid; (2) After filtering the above-mentioned crushed liquid, the dandelion exosomes were separated and collected by ultrahigh-speed centrifugation.

7. The use according to claim 6, characterized in that: In the step (1) of the preparation method, the fresh dandelions need to be washed with sterile water and mixed with pre-cooled PBS buffer before being crushed.

8. The use according to claim 6, characterized in that: In step (1) of the preparation method, the mixing ratio is dandelion: PBS buffer = 1 g: (1-1.1) mL; the concentration of PBS buffer is 5-15 mM; and the pH is 7.4-7.

6.

9. The use according to claim 6, characterized in that: The filtration described in step (2) of the preparation method refers to filtering using a 0.45 μm filter membrane to obtain a filtrate.

10. The use according to claim 6, characterized in that: The ultra-high speed centrifugation method in step (2) of the preparation method comprises the following steps: a) centrifuging the filtrate at 4°C, 2000-3000 g for 15-25 min, and collecting the supernatant I; b) centrifuging supernatant I at 4°C, 10,000-20,000 g for 30-45 min, and collecting supernatant II; c) centrifuging the supernatant II at 4°C, 100,000-120,000 g for 90-120 min, and collecting the precipitate; d) Resuspend the precipitate in pre-cooled PBS, repeat 3 to 5 times, filter through a 0.22 μm filter membrane, and the filtrate is the solution containing dandelion exosomes.

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