A kind of platycodon grandiflorum exosome and its application in preparing drugs for treating lung diseases

Abstract

The present invention provides a platycodon grandiflorum exosome and its application in the preparation of drugs for treating lung diseases. Through the method of the present invention, platycodon grandiflorum exosomes meeting the standards and having stable quality can be obtained. Moreover, these exosomes can be efficiently taken up by A549 cells and RAW264.7 cells in the lungs and have the biological activities of inhibiting the proliferation of lung tumor cells and anti-lung inflammation, demonstrating that platycodon grandiflorum exosomes can provide ideas for the research and development of new drugs in aspects such as drug targeting and anti-tumor in the clinical treatment of lung diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and more particularly, to a platycodon grandiflorum exosome and its application in the preparation of drugs for treating lung diseases. Background Art

[0002] Exosomes are a class of vesicles that encapsulate substances such as miRNAs, lncRNAs, DNAs, and proteins, and have an efficient "information communication" function of transmitting functional substances and a natural tendency to parental cells. They are sized 30 - 150 nm, mainly distributed in various body fluids, and have functions such as cell communication, cell migration, promoting angiogenesis, and anti-tumor immunity. They are closely related to the occurrence and progression of various diseases, and can not only be used as biological indicators for diagnosing various diseases, but also as a treatment means, and are one of the "troika" in the field of liquid biopsy.

[0003] Since exosomes are widely present in body fluids, current research on exosomes mainly focuses on animal studies. In fact, not only mammalian cells, but also plant cells can secrete vesicles, and even earlier than mammalian vesicles. Plant-derived EVs (PEVs) refer to various nano-sized membrane vesicles actively released by plant cells, which play a role in information and material transmission in intercellular and interspecies communication. Although plant vesicles were discovered earlier, compared with mammalian and human-derived vesicles, the research is not deep enough, especially in the biomedical field, most of which are in the laboratory research stage. In recent years, more and more studies have confirmed that as an important medium for cell communication, PEVs play an important role in the efficacy of medicinal plants. Medicinal plant-derived vesicles MPEVs have more prominent biological activities than ordinary plant-derived EVs, and show unique advantages as therapeutic agents and drug carriers, and thus have very broad application prospects. In addition, some studies have shown that exosome-like vesicles secreted by edible plants do not cause immune responses in the human body. Based on the above advantages, the research and application of medicinal plant exosome-like vesicles in diseases have great potential, and exploring the uniqueness of the structure and function of MPEVs is the work that needs to be deeply carried out in this field in the future.

[0004] At present, the main methods for exosome extraction include ultracentrifugation, ultrafiltration, size exclusion chromatography, exosome precipitation, immunomagnetic beads, chromatography, etc., among which ultracentrifugation is the most widely used. For plant exosomes, due to the geographical distribution and seasonal differences of plants, it is necessary to systematically evaluate whether the PEVs obtained in different regions and seasons are stable and consistent. Especially when used as therapeutic agents or drug carriers in the biomedical field, the safety, effectiveness, stability, and controllability of the products are the golden standards that must be met. Moreover, at present, many PEVs are derived from fresh juice extraction, and how to preserve the extracted PEVs is a limiting factor determining whether they are suitable for large-scale processing. There is still a lack of research in this regard, and some reports even have contradictory findings. Therefore, it is necessary to construct a system with a stable extraction process and a mature identification method to obtain plant vesicles with stable yields and reliable quality.

[0005] Platycodon grandiflorum is the dried root of Platycodon grandiflorum, a plant of the Campanulaceae family, and is mainly used to treat symptoms such as sore throat, lung abscess with expectoration, chest fullness and pain in the hypochondrium. As the "boat of the lungs", Platycodon grandiflorum is widely used in traditional compound prescriptions. Platycodon grandiflorum is bitter, pungent, and neutral, and belongs to the lung meridian. This medicine is pungent and dispersing, bitter and purging, and is good at dispersing the lung qi. Its nature is dispersing and ascending, and it is the main medicine for carrying other drugs upward. Modern research shows that Platycodon grandiflorum has a wide range of pharmacological activities such as expectorant, antitussive, anti-inflammatory, anti-tumor, and improving human immunity. It is praised as the "agent for guiding the flow of qi" in "Pearl Sac", and "Compendium of Materia Medica Seeking Truth" states: "Platycodon grandiflorum is a product that disperses and raises the lung qi and can be the boat of various drugs." As a guiding drug, Platycodon grandiflorum guides the medicinal power of drugs in the compound prescription upward to treat upper-jiao diseases and is the first choice for the "guiding messenger" of the heart and lung meridians, playing an important role and position in traditional compound prescriptions.

[0006] Therefore, it is of great significance to study an extraction method for Platycodon grandiflorum-derived exosomes and the application of Platycodon grandiflorum-derived exosomes. Summary of the Invention

[0007] Aiming at the problems of the existing technology, the objectives of the present invention are as follows:

[0008] One objective of the present invention is to provide a simple and efficient extraction method for Platycodon grandiflorum exosomes, which requires obtaining Platycodon grandiflorum exosomes that meet the standards and have stable quality.

[0009] Another objective of the present invention is to provide the application of Platycodon grandiflorum exosomes, specifically the application in the preparation of drugs for treating lung diseases.

[0010] The third objective of the present invention is to provide proteins related to the association between Platycodon grandiflorum exosomes and lung diseases.

[0011] Aiming at the above invention objectives, the technical solution of the present invention is:

[0012] A kind of platycodon grandiflorum exosome is obtained by the following extraction method, which includes the following specific steps: Fresh platycodon grandiflorum is sliced and juiced, and the juice is collected. It is centrifuged at 2,000g - 3,000g for 20min - 30min, and then centrifuged at 10,000g - 15,000g for 60min - 90min twice. The supernatant of the second time is collected and centrifuged at 150,000g - 180,000g for 60min - 90min. The precipitate is collected, resuspended with PBS and placed in a centrifuge tube to obtain the crude extract of platycodon grandiflorum exosome after resuspension; Different concentrations of gradient sucrose solutions are added successively from the bottom in the centrifuge tube. Finally, the crude extract of platycodon grandiflorum exosome after resuspension is added to the resuspension solution at the 8% sucrose solution layer, and centrifuged at 120,000g - 150,000g for 90min - 120min. The two layers of bands between 8% - 30% and 30% - 45% are collected and transferred to a new ultracentrifuge tube, and then centrifuged at 120,000g - 180,000g for 90min - 120min to remove sucrose, and the platycodon grandiflorum exosome is obtained. The centrifugation temperature for all steps is 3 - 10°C.

[0013] Further preferably, the centrifugation temperature for all steps is 3 - 5°C.

[0014] Further preferably, the gradient sucrose solution refers to sucrose solutions with mass fractions of 8%, 30%, 45% and 60%.

[0015] The present invention provides the application of platycodon grandiflorum exosome in the preparation of drugs for treating lung diseases.

[0016] Preferably, the lung diseases include lung cancer, pneumonia, and acute lung injury.

[0017] Further preferably, the lung diseases include lung cancer acting on adenocarcinoma human alveolar basal epithelial cells (A549 cells), inflammation or lung injury acting on LPS-induced mouse mononuclear macrophage leukemia cells RAW264.7, and LPS-induced acute lung injury.

[0018] Even further preferably, the platycodon grandiflorum exosome is the platycodon grandiflorum exosome obtained by the above extraction method.

[0019] The present invention acts the platycodon grandiflorum exosome on adenocarcinoma human alveolar basal epithelial cells (A549 cells) to detect whether the exosome can be taken up by A549 cells and has the property of anti-tumor cell proliferation.

[0020] The present invention acts the platycodon grandiflorum exosome on an inflammation injury model of LPS-induced mouse mononuclear macrophage leukemia cells (RAW264.7) to detect whether the exosome can play roles such as inhibiting the expression of inflammatory cytokines and inhibiting cell damage.

[0021] The present invention applies platycodon grandiflorum exosomes to a mouse model of acute lung injury induced by LPS to detect whether they have a therapeutic effect against acute lung injury and biological activity.

[0022] Furthermore, the present invention studied the proteins of platycodon grandiflorum exosomes and obtained proteins that are correlated with lung diseases:

[0023] The present invention performed proteomic analysis on platycodon grandiflorum exosomes by Label Free protein quantification technology.

[0024] Preferably, a total of 112 proteins were obtained in the proteomic analysis, and KEGG enrichment analysis was performed on the obtained 112 proteins;

[0025] More preferably, a total of 50 metabolic signaling pathways were obtained in the KEGG enrichment analysis, and 3 signaling pathways related to inflammatory and cancerous diseases were screened out, namely: oxidative phosphorylation, glycolysis / gluconeogenesis, and MAPK signaling pathway.

[0026] Even more preferably, the proteins of the platycodon grandiflorum exosomes include one or more of the following proteins: nicotinamide adenine dinucleotide, nucleoside diphosphate kinase, calmodulin 2, phosphoglycerate kinase, fructose-bisphosphate aldolase, ATP synthase, and the proteins are directly related to lung diseases.

[0027] Compared with the prior art, the advantages of the present invention are:

[0028] 1. The inventors of the present invention found that by the method of the present invention, platycodon grandiflorum exosomes meeting the standards and having stable quality can be obtained, and the exosomes can be efficiently taken up by A549 cells and RAW264.7 cells in the lungs, and have biological activities of anti-proliferation of lung tumor cells and anti-lung inflammation. Therefore, the platycodon grandiflorum exosomes extracted by the method of the present invention can provide ideas for targeted therapy of clinical drugs and new drugs for the treatment of lung diseases such as anti-tumor therapy.

[0029] 2. The present invention obtained several proteins in platycodon grandiflorum exosomes that are directly related to lung diseases, which is beneficial for further subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments.

[0031] Figure 1 It is a diagrammatic illustration of the extraction steps of platycodon grandiflorum exosomes. In the figure: (A) A diagram of a fresh platycodon grandiflorum slice for standby, (B) A diagram of removing residues and small plant fragments by preliminary centrifugation after juicing, (C) A diagram after taking the supernatant and further centrifuging, (D) A diagram of centrifugation before purification, (E) A diagram of the band after purification by sucrose density gradient centrifugation, (F) A diagram of the precipitate of platycodon grandiflorum exosomes after resuspension with PBS;

[0032] Figure 2 These are the characterization results of Platycodon grandiflorum exosomes. In the figures: (A) Transmission electron microscope characterization of the morphology of Platycodon grandiflorum exosomes; (B) Determination of the protein content of Platycodon grandiflorum exosomes; (C) Agarose gel electrophoresis analysis of the RNA components of Platycodon grandiflorum exosomes.

[0033] Figure 3 These are the laser confocal results of A549 cells taking up Platycodon grandiflorum exosomes.

[0034] Figure 4 These are the CCK8 results of the inhibitory effect of Platycodon grandiflorum exosomes on the activity of A549 cells.

[0035] Figure 5 These are the results of the scratch assay of A549 cells intervened by Platycodon grandiflorum exosomes.

[0036] Figure 6 These are the results of the immunofluorescence assay for RAW264.7 cells taking up Platycodon grandiflorum exosomes.

[0037] Figure 7 These are the RT-qPCR results of Platycodon grandiflorum exosomes intervening in LPS-induced RAW264.7 cell injury. Among them, A is the comparison chart of OD values, B is the comparison chart of inflammatory factor IL-1β, and C is the comparison chart of inflammatory factor IL-6.

[0038] Figure 8 These are the graphs of the changes in body weight and lung index of mouse samples. Among them, A is the graph of body weight changes, and B is the graph of lung index changes.

[0039] Figure 9 These are the graphs of observing the pathological changes of the lung tissues of each group of mice by HE staining after collecting the lung tissues of each group of mice.

[0040] Figure 10 These are the result graphs of detecting the expression levels of inflammatory factors IL-6 and TNF-α in the bronchoalveolar lavage fluid of each group of mice by ELISA method. Among the inflammatory factors, A is the graph of the expression level of IL-6, and B is the graph of the expression level of TNF-α.

[0041] Figure 11 These are the result graphs of the total protein quantity in the proteomic analysis of Platycodon grandiflorum exosomes.

[0042] Figure 12 These are the KEGG enrichment graphs of 112 proteins of Platycodon grandiflorum exosomes.

[0043] Figure 13 These are the effective protein components in Platycodon grandiflorum exosomes. Specific implementation manners

[0044] Example 1 Preparation and characterization analysis of Platycodon grandiflorum exosomes

[0045] Example 1 of the present invention provides a method for isolating exosome-like vesicles from Platycodon grandiflorum, and its preparation and identification steps are as follows:

[0046] 1. Isolation of Platycodon grandiflorum exosomes

[0047] (1) Cut the washed fresh Platycodon grandiflorum into slices and add 8 times the amount of water to extract juice, and collect the juice by filtering through gauze;

[0048] (2) Centrifuge the Platycodon grandiflorum filtrate obtained in step (1). At 4°C, centrifuge at 2,000g for 20 min in sequence to remove large residues; centrifuge at 10,000g for 1 h to remove fine plant debris, and retain the supernatant;

[0049] (3) At 4°C, centrifuge the supernatant at 150,000g for 60 min, collect the precipitate, and resuspend it with PBS to obtain Platycodon grandiflorum exosomes (impure).

[0050] 2. Purification of Platycodon grandiflorum exosomes

[0051] (1) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60% using ultrapure water;

[0052] (2) Use a long needle to sequentially add sucrose solutions with mass fractions of 8%, 30%, 45%, and 60% from the bottom in a centrifuge tube to make a discontinuous sucrose density gradient solution. Transfer the resuspended crude extract of Platycodon grandiflorum exosomes to the top layer of the sucrose solution with a mass fraction of 8% in the sucrose density gradient solution;

[0053] (3) At 4°C, centrifuge at 150,000g for 120 min, combine and collect the 2 layers between 8% - 30% and 30% - 45% and transfer them to a new ultracentrifuge tube, then centrifuge at 150,000g for 60 min to remove sucrose, and resuspend the precipitate with PBS. The step diagram is as Figure 1 .

[0054] 3. Characterization analysis of Platycodon grandiflorum exosomes

[0055] a. Electron microscopy analysis

[0056] Fix the Platycodon grandiflorum vesicle particle precipitate and perform transmission electron microscopy using conventional procedures. The obtained transmission electron microscopy micrograph is as Figure 2 shown in A. The electron microscopy results show that there are round or oval vesicle structures, with uniform and complete sizes, and the diameters are mainly between 30 - 150 nm, indicating that Platycodon grandiflorum exosomes have been successfully extracted.

[0057] b. Protein analysis

[0058] Extract the PGEV protein using RIPA lysis buffer and collect the protein. After protein denaturation, perform electrophoresis using a 4×SDS polyacrylamide gel. Cut the 10% SDS-PAGE, stain with Coomassie Brilliant Blue for 2 hours, and then rinse 2 - 3 times, 1 - 2 hours each time (rinsing solution: 2250 mL of 95% ethanol + 250 mL of glacial acetic acid + 2500 mL of distilled water). Then use a gel imaging analysis system to analyze the proteins in the supernatant. The results are as Figure 2 shown in

[0059] c. RNA component analysis

[0060] Resuspend PGEV in PBS, and then extract exosomal RNA using an exosomal RNA purification kit (Hangzhou Xinjing Bioreagent Development Co., Ltd., Cat. No. 5202050, batch number: 20210410). Then perform RNA agarose electrophoresis on a 1.7% agarose gel. The results are as Figure 2 shown in

[0061] Example 2: Effects of Platycodon grandiflorum exosomes on adenocarcinoma human alveolar basal epithelial cells (A549 cells)

[0062] In Example 2 of the present invention, it was found that Platycodon grandiflorum exosomes have the property of inhibiting the proliferation of adenocarcinoma human alveolar basal epithelial cells. The verification process is as follows:

[0063] 1. Experiment on the uptake of Platycodon grandiflorum exosomes by A549 cells

[0064] a. Fluorescent labeling of PGEV

[0065] (1) Mix the purified exosomes evenly with 200 μL of Dilution C. Separately, mix 2 μL of PKH67 dye with 200 μL of Dilution C;

[0066] (2) Incubate the mixture at 25 °C for 1 - 5 minutes;

[0067] (3) Add an equal volume (400 μL) of serum (or 1% BSA) and incubate for 1 minute to terminate the staining;

[0068] (4) Add 800 μL of basal medium, transfer to an ultracentrifuge tube, and centrifuge at 4 °C, 100,000 × g for 70 minutes;

[0069] (5) Wash with PBS, centrifuge at 4 °C, 100,000 × g for 70 minutes to remove unbound dye;

[0070] (6) Repeat once.

[0071] b. Cell uptake experiment

[0072] (1) Extract exosomes according to the above method and perform fluorescent labeling;

[0073] (2) Mix the labeled exosomes with A549 cells, add them to a 12-well plate with coverslips, and incubate for 24 h;

[0074] (3) Fix, stain, and photograph with a confocal microscope. The results are as Figure 3 shown. Platycodon grandiflorum exosomes can be effectively taken up by A549 cells.

[0075] 2. Verification of the inhibitory effect of PGEV on the activity of A549 cells by CCK8 assay

[0076] (1) Seed A549 cells in a 96-well plate and incubate in a 5% CO2, 37 °C incubator until the cells adhere;

[0077] (2) Extract exosomes according to the above method;

[0078] (3) Treat the extracted exosomes with A549 cells, add CCK8 after 24 h, and detect the OD value with an enzyme-linked immunosorbent assay (ELISA) reader after 2 h. The results are as Figure 4 shown. The activity of A549 cells in the PGEV group decreased significantly compared with that in the normal group.

[0079] 3. Scratch assay

[0080] (1) Seed A549 cells in a 6-well plate and incubate in a 5% CO2, 37 °C incubator until the cells grow to confluence and adhere;

[0081] (2) The next day, use a pipette tip against a ruler to scratch as vertically as possible along the horizontal line on the back. The pipette tip should be perpendicular and not tilted;

[0082] (3) Extract exosomes according to the above method and treat them with A549 cells;

[0083] (4) Wash the cells 3 times with PBS to remove the scratched cells and add serum-free medium;

[0084] (5) Place in a 37 °C, 5% CO2 incubator and culture. Sample and photograph at 0, 6, 12, and 24 h. The results are as Figure 5 shown. PGEVs can significantly inhibit the migration ability of A549 cells. At different time points of 0, 6, 12, and 24 h, the cell spacing in the PGEVs group was significantly wider than that in the blank group.

[0085] The results show that Platycodon grandiflorum exosomes can be efficiently taken up by A549 cells and have the biological activity of inhibiting the proliferation of human alveolar basal epithelial cells with adenocarcinoma.

[0086] Example 3: The effect of Platycodon grandiflorum exosomes on the cell inflammation injury model of lipopolysaccharide (LPS)-induced murine monocytic macrophage leukemia cells (RAW264.7). In Example 3 of the present invention, it was found through cell experiments that Platycodon grandiflorum exosomes have the effect of resisting LPS-induced injury of lung macrophages (RAW264.7). The verification process is as follows:

[0087] 1. Cell uptake

[0088] a. Fluorescent labeling of PGEV

[0089] (1) Dilute and mix the purified exosomes with 200 μL of Dilution C. Separately, mix 2 μL of PKH67 dye with 200 μL of Dilution C;

[0090] (2) Mix the two and incubate at 25 °C for 1 - 5 min;

[0091] (3) Add an equal volume (400 μL) of serum (or 1% BSA) and incubate for 1 min to terminate the staining;

[0092] (4) Add 800 μL of basal medium, transfer to an ultracentrifuge tube, and centrifuge at 4 °C, 100,000 × g for 70 min;

[0093] (5) Wash with PBS, centrifuge at 4 °C, 100,000 × g for 70 min to remove the unbound dye;

[0094] (6) Repeat once.

[0095] b. Cell uptake experiment

[0096] (1) Extract exosomes and perform fluorescent labeling according to the above method;

[0097] (2) Mix the labeled exosomes with RAW264.7 cells, add them to a 12-well plate with coverslips, and incubate for 24 h;

[0098] (3) Fix, stain, and observe and photograph under a fluorescence microscope. The results are as Figure 6 shown, and Platycodon grandiflorum exosomes can be effectively taken up by RAW264.7 cells.

[0099] 2. RT-qPCR experiment

[0100] (1) Total RNA extraction: After collecting cells from each group, add 500 μL of TRIzol, pipette to mix, and lyse on ice for 5 min. Then add 100 μL of chloroform, mix well, let stand at room temperature for 5 min, and centrifuge at 10,000×g for 10 min. After taking out the upper - colorless aqueous layer of RNA, add 250 μL of isopropanol, mix well, and let stand at -20°C for 2 h. Centrifuge at 10,000×g for 10 min, discard the supernatant, add 500 μL of 75% cold ethanol, centrifuge at 5,000×g for 5 min, twice. After drying for 10 min, add 30 μL of RNase - free water and mix well;

[0101] (2) Measuring RNA concentration: Take 1 μL of RNA and measure its concentration using a spectrophotometer;

[0102] (3) Reverse transcription to synthesize cDNA: Reverse transcribe each RNA sample into cDNA according to the operating steps of the reverse transcription kit (NovoScript, E047 - 01B);

[0103] (4) Perform PCR on IL - 1β gene, IL - 6 gene and reference gene: Prepare the PCR system and perform amplification to obtain amplification curves, melting curves and experimental data, and conduct statistical analysis. The results are as Figure 7 shown. Compared with the model group, PGEV can down - regulate the expression of inflammatory factors IL - 1β and IL - 6.

[0104] The results show that platycodon grandiflorum exosomes can be efficiently taken up by RAW264.7 cells and have the effects of inhibiting the expression of inflammatory cytokines and inhibiting cell damage, etc.

[0105] Example 4: Effects of platycodon grandiflorum exosomes on LPS - induced mouse lung inflammation model

[0106] In Example 4 of the present invention, through animal experiments, it was found that platycodon grandiflorum exosomes have the effect of treating LPS - induced acute lung injury model in mice. The verification process is as follows:

[0107] 1. Animal preparation and model construction

[0108] (1) Animal preparation: C57 mice, weighing 16 - 18 g, half male and half female. Raise them under SPF - level for adaptation. Use ear tag method for animal identification, raise mice in cages, 6 mice per cage, change bedding every 2 d, temperature 22 ± 2°C, 12 - hour light - dark cycle, relative humidity 55 ± 15%.

[0109] (2) Model construction: After 3 days of adaptive feeding, the mice were randomly divided into four groups (n = 6 in each group): normal group (Control), model group (Model), and Platycodon grandiflorum exosome group (PGEV). PGEV was administered by gavage once a day for 7 consecutive days. After 24 hours of drug administration on the 7th day, LPS (5 mg / kg) was used to stimulate the mice through the nasal route to establish the model. All mice were sacrificed 24 hours later.

[0110] 2. Sample preparation: The same as before

[0111] 3. Evaluation of the effect on pneumonia / acute lung injury

[0112] a. Body weight and organ index

[0113] Collect the samples of mice in each group and weigh them. As Figure 8 shown, compared with the normal group, the body weight of the model group decreased significantly (P < 0.01), and the lung index increased significantly (P < 0.01). Compared with the model group, the body weight of the vesicle group increased significantly (P < 0.05), and the lung index decreased significantly (P < 0.01).

[0114] b. HE diagnostic pathological results

[0115] Collect the lung tissues of mice in each group and observe the pathological changes of the lung tissues of mice in each group by HE staining. The results are as Figure 9 shown. In the normal group, the alveolar pores of the lung tissues of mice were intact, and a clear capillary network, a small amount of red blood cells, fibroblasts and macrophages could be seen in the alveolar septum. Compared with the normal group, the sizes of the alveolar cavities in the lung tissues of the model group were different, a large number of inflammatory cells infiltrated in the alveolar cavities, the alveolar septum was significantly thickened, and there were obvious edema and bleeding in the lung interstitium. The vesicle group could improve the lung tissue injury induced by LPS, the pulmonary edema was reduced, and the infiltration of inflammatory cells was significantly reduced.

[0116] c. Detection of inflammatory factors by ELISA

[0117] Use ELISA to detect the bronchoalveolar lavage fluid of mice in each group. The results are as Figure 10 shown. Compared with the normal group, the expressions of IL-6 and TNF-α in the bronchoalveolar lavage fluid of the model group increased significantly (P < 0.01). Compared with the model group, the expressions of IL-6 and TNF-α in the bronchoalveolar lavage fluid of the vesicle group decreased significantly (P < 0.01).

[0118] The results show that Platycodon grandiflorum exosomes can inhibit the expression of inflammatory cytokines and have a certain therapeutic effect on LPS-induced acute lung injury.

[0119] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the technical field, within the technical scope disclosed by the present invention, according to the technical solution and concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the claims of the present invention.

[0120] Example 5 Proteomics Analysis and Enrichment Analysis of Platycodon grandiflorum Exosomes

[0121] In Example 2 of the present invention, it was found that Platycodon grandiflorum exosomes are rich in 112 proteins, and the proteins contained in the exosomes are enriched in the MAPK pathway related to lung diseases. The verification process is as follows:

[0122] 1. Proteomics of Platycodon grandiflorum Exosomes

[0123] a. Protein Extraction

[0124] (1) Take all exosome samples, add an appropriate amount of 2% sodium deoxycholate lysis buffer, heat at 95°C for 10 min, and use an ultrasonic disruptor for disruption treatment (total time 10 min, working for 2 s, stopping for 2 s).

[0125] (2) Centrifuge at 20,000g for 20 min, take the supernatant, take 5 μL for quantification, and freeze the rest at -80°C.

[0126] (3) Add DTT with a final concentration of 10 mM and incubate in a water bath at 37°C for 1 h;

[0127] (4) Add IAA with a final concentration of 20 mM and place in the dark for 30 min.

[0128] b. Protease Digestion

[0129] (1) Take 150 μg of protein from each sample;

[0130] (2) Add 3 μg of Trypsin enzyme according to the ratio of protein:enzyme = 50:1 and digest at 37°C for 14 - 16 h;

[0131] (3) Desalt the digested peptide segments using a Waters solid-phase extraction column and vacuum dry;

[0132] (4) Reconstitute the dried peptide segments with pure water and store at -20°C.

[0133] c. Mass Spectrometry Detection (Nano-LC-MS / MS)

[0134] (1) Reconstitute the dried peptide segment sample with 0.1% FA, centrifuge at 20,000g for 10 min, and take the supernatant for injection.

[0135] (2) Through the Thermo Scientific EASY-nLCTM The 1200 system was used for separation. The sample was introduced into a self-packed C18 column (inner diameter 100 μm, particle size of the column material 1.8 μm, column length approximately 35 cm) and separated at a flow rate of 300 nL / min using the following effective gradient: from 0 to 103 min, mobile phase B (98% ACN, 0.1% FA) linearly increased from 4% to 27%; from 103 to 111 min, mobile phase B increased from 27% to 40%; from 111 to 113 min, mobile phase B increased from 40% to 90%; from 113 to 120 min, 90% mobile phase B.

[0136] (3) The peptides separated by liquid chromatography were ionized by nanoESI source and then entered the mass spectrometer Orbitrap Exploris TM 480 (Thermo Fisher Scientific, San Jose, CA) for DDA (Data Dependent Acquisition) mode detection. The main parameter settings were as follows: the ion source voltage was 2.2 KV, the first-stage mass spectrometry scan range was 350 - 1,500 m / z; the resolution was set to 60,000; the Normalized AGC Target was set to 300%, and the maximum ion injection time (MIT) was 20 ms; the second-stage mass spectrometry fragmentation mode was HCD, and the fragmentation energy was set to 32%; the resolution was set to 15,000, and the dynamic exclusion time was set to 60 s. The starting m / z of the second-stage mass spectrometry was fixed at 110; the selection conditions for the precursor ions of the second-stage fragmentation were: charge 2+ to 6+; the Normalized AGC Target was set to standard, and the maximum ion injection time (MIT) was 22 ms.

[0137] (4) The results were as Figure 11 shown, and 112 proteins were rich in Platycodon grandiflorum exosomes.

[0138] 2. KEGG enrichment analysis of Platycodon grandiflorum exosome proteins

[0139] (1) The top 30 proteins of Platycodon grandiflorum exosomes were imported into the DAVID database for genome encyclopedia (KEGG) pathway enrichment analysis.

[0140] (2) With the condition of P < 0.05, the significant enrichment of the KEGG signal pathway annotation results was carried out to obtain the results of the protein KEGG pathway enrichment analysis.

[0141] The ImageGP tool was used to visualize the enrichment analysis results. The results were as Figure 12 、 Figure 13 shown, and a total of 9 important signal pathways related to Platycodon grandiflorum exosome proteins were obtained, including the MAPK signaling pathway related to lung diseases.

Claims

1. Application of platycodon grandiflorum exosomes in the preparation of drugs for treating pulmonary diseases, wherein the pulmonary diseases include lung cancer, pneumonia, and acute lung injury; the platycodon grandiflorum exosomes are obtained by the following extraction method, including the following specific steps: Fresh platycodon grandiflorum is sliced and juiced, and the juice is collected. It is centrifuged at 2,000 g - 3,000 g for 20 min - 30 min, and then centrifuged at 10,000 g - 15,000 g for 60 min - 90 min twice. The supernatant of the second time is collected and centrifuged at 150,000 g - 180,000 g for 60 min - 90 min. The precipitate is collected, resuspended with PBS and placed in a centrifuge tube to obtain a crude extract of platycodon grandiflorum exosomes after resuspension; Gradient concentration sucrose solutions with different concentrations are added successively from the bottom in the centrifuge tube. Finally, the crude extract of platycodon grandiflorum exosomes after resuspension is added to the resuspension solution at the 8% sucrose solution layer and centrifuged at 120,000 g - 150,000 g for 90 min - 120 min. The two bands between 8% - 30% and 30% - 45% are collected and transferred to a new ultracentrifuge tube, and then centrifuged at 120,000 g - 180,000 g for 90 min - 120 min to remove sucrose, thus obtaining platycodon grandiflorum exosomes. The centrifugation temperature for all steps is 3 - 10 °C; The gradient concentration sucrose solution refers to sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%.

2. The application according to claim 1, wherein The centrifugation temperature for all steps is 3 - 5 °C.

3. The application according to claim 1, characterized in that The pulmonary diseases include lung cancer acting on adenocarcinoma human alveolar basal epithelial cells A549 cells, inflammation or lung injury acting on LPS-induced mouse monocyte macrophage leukemia cells RAW264.7, and LPS-induced acute lung injury.

4. The application according to claim 1, wherein, The proteins of the platycodon grandiflorum exosomes include one or more of the following proteins: nicotinamide adenine dinucleotide, nucleoside diphosphate kinase, calmodulin 2, phosphoglycerate kinase, fructose bisphosphate aldolase, ATP synthase, and the proteins are directly related to pulmonary diseases.

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