Plant-derived hybrid exosome and application of plant-derived hybrid exosome in pulmonary fibrosis resistance

Abstract

The invention belongs to the technical field of application of plant exosomes, and discloses a plant-derived hybrid exosome and application of the plant-derived hybrid exosome in pulmonary fibrosis resistance. After screening a proper proportion, the obtained hybrid exosome has an obvious treatment effect on a mouse with pulmonary fibrosis induced by bleomycin, and the treatment effect is specifically shown as follows: (1) the hybrid exosome can obviously relieve lung inflammatory cell infiltration of the model mouse, protect a pulmonary alveolar structure and relieve the degree of pulmonary fibrosis; (2) the hybrid exosome can effectively reduce the expression levels of TNF-alpha, IL-1beta and IL-6, and can effectively slow down the pulmonary fibrosis process; the lung tissue inflammation is inhibited; and (3) the hybridized exosome inhibits the generation of ECM (extracellular matrix) and inhibits epithelial mesenchymal transition, so that the anti-fibrosis effect is achieved. The plant source hybrid exosome prepared by the invention is wide in plant source, simple in exosome preparation process and low in cost, and provides new possibility for treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of application of plant exosomes, and more specifically, relates to a hybrid exosome derived from a plant and its application in anti-pulmonary fibrosis. Background Art

[0002] Interstitial pneumonia is an inflammatory and fibrotic disease of the lung interstitium caused by various reasons. The lesions mainly invade the lung interstitium and alveolar cavity, including alveolar epithelial cells, capillary endothelial cells, basement membrane, and tissues around blood vessels and lymphatic vessels, eventually causing fibrosis of the lung interstitium and leading to loss of alveolar-capillary function.

[0003] The clinical manifestations of interstitial pneumonia are progressive lung function decline and diffusion dysfunction, which in turn lead to respiratory failure and death. Pulmonary fibrosis is the main pathological basis of lung dysfunction and respiratory failure. The root cause of pulmonary fibrosis is related to abnormal changes in the structure and function of epithelial cells, fibroblasts, macrophages, etc. in the lungs.

[0004] Existing treatments for pulmonary fibrosis include: (1) Western medicine, such as the well-known pirfenidone and nintedanib; however, these two drugs have been reported to have significant side effects, and imported drugs are expensive, making them unaffordable for most patients' families; (2) Traditional Chinese medicine. Many well-known active ingredients of traditional Chinese medicine, such as astragalus total flavonoids, saikosaponin D, galangin, angelica polysaccharides, andrographolide, quercetin, and salvianolic acid B, have been reported to have certain therapeutic effects on pulmonary fibrosis, but the extraction methods of active ingredients of traditional Chinese medicine are not standardized, and the therapeutic effects of traditional Chinese medicine are inconsistent. In addition, the research on the toxic and side effects caused by the cross-use of multiple active ingredients of traditional Chinese medicine is still unclear, which can easily cause other complications in patients; (3) Non-drug treatments, such as lung transplantation, are indeed a powerful means of treatment, but the lung supply is tight, the cost of surgery is high, and medical workers with lung transplant skills are scarce, making it difficult for most patients to achieve this goal.

[0005] Exosomes are multivesicular bodies formed by the invagination of the cytoplasmic membrane, which fuse with the cell membrane and are released into the extracellular matrix in the form of exocytosis. They have a diameter of 40 to 160 nm (average 100 nm) and a double-layer lipid membrane structure. Exosomes contain abundant DNA, RNA, lipids, proteins and other substances, and participate in physiological processes such as immune response, intercellular communication, and antigen presentation. They are an important tool for intercellular information exchange. Exosomes have been reported to be used to treat a variety of diseases, but there are few studies on exosomes for the treatment of lung diseases, and even fewer studies on the treatment of interstitial pneumonia. In addition, the known exosomes for the treatment of lung diseases are mostly derived from animal cells, such as macrophages and mesenchymal stem cells. The production and maintenance costs of these cells are high, and the methods for preparing exosomes are relatively complicated, which limits the promotion and application of exosomes in the treatment of lung diseases. Summary of the invention

[0006] In view of the deficiencies in the prior art, the primary purpose of the present application is to provide a plant-derived hybrid exosome.

[0007] The second object of the present invention is to provide applications of the above-mentioned plant-derived hybrid exosomes.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A plant-derived hybrid exosome is obtained by mixing Bacopa monnieri exosomes and cactus exosomes in a mass ratio of 3-8:10-16 and then performing water bath ultrasound.

[0010] Preferably, the preparation method of the Bacopa monnieri exosomes is:

[0011] (1) Grind Bacopa monnieri, dissolve in PBS, filter through gauze, centrifuge to remove impurities and debris, and collect the supernatant;

[0012] (2) The collected supernatant was subjected to ultracentrifugation at 9000 g for 60 min at 4°C, and the supernatant was discarded, while the precipitate was retained;

[0013] (3) Resuspend the precipitate retained in (2), and centrifuge again at 4°C and 9000 g for 60 min. After the centrifugation, collect the precipitate, resuspend it in PBS, and store it at ultra-low temperature for future use.

[0014] Preferably, the preparation method of the cactus exosomes is:

[0015] (1) Grind the cactus, dissolve it in PBS, filter it through gauze, centrifuge to remove impurities and debris, and collect the supernatant;

[0016] (2) The collected supernatant was subjected to ultracentrifugation at 8000 g for 70 min at 4°C, and the supernatant was discarded, while the precipitate was retained;

[0017] (3) Resuspend the precipitate retained in (2), and centrifuge again at 4°C and 8000 g for 70 min. After the centrifugation, collect the precipitate, resuspend it in PBS, and store it at ultra-low temperature for future use.

[0018] The present invention prepares Bacopa monnieri exosomes and cactus exosomes, combines them, and selects a suitable ratio to obtain hybrid exosomes, which have obvious therapeutic effects on mice with pulmonary fibrosis induced by bleomycin, specifically as follows: (1) hybrid exosomes can significantly reduce inflammatory cell infiltration in the lungs of model mice, protect alveolar structure, and reduce the degree of pulmonary fibrosis; (2) hybrid exosomes can effectively reduce the expression levels of TNF-α, IL-1β, and IL-6, and can effectively slow down the progression of pulmonary fibrosis; inhibit inflammation of lung tissue; (3) hybrid exosomes inhibit the production of ECM and inhibit epithelial-mesenchymal transformation, thereby playing an anti-fibrotic role.

[0019] Therefore, the present invention also provides the use of the hybrid exosomes in the preparation of a drug for reducing the expression of cellular inflammatory factors IL-1β, IL-6, and TNF-α and promoting the expression of anti-inflammatory factors IL-4 and IL-10.

[0020] Therefore, the present invention also provides the use of the hybrid exosomes in the preparation of a drug for reducing the cytokine TGF-β1.

[0021] Therefore, the present invention also provides the use of the hybrid exosomes in preparing a drug for inhibiting pulmonary fibrosis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention prepares Bacopa monnieri exosomes and cactus exosomes, combines them, and selects a suitable ratio to obtain hybrid exosomes, which have obvious therapeutic effects on mice with pulmonary fibrosis induced by bleomycin, specifically as follows: (1) hybrid exosomes can significantly reduce inflammatory cell infiltration in the lungs of model mice, protect alveolar structure, and reduce the degree of pulmonary fibrosis; (2) hybrid exosomes can effectively reduce the expression levels of TNF-α, IL-1β, and IL-6, and can effectively slow down the progression of pulmonary fibrosis; inhibit inflammation of lung tissue; (3) hybrid exosomes inhibit the production of ECM and inhibit epithelial-mesenchymal transformation, thereby playing an anti-fibrotic role.

[0024] The plant-derived hybrid exosomes prepared by the present invention have a wide range of plant sources, a simple exosome preparation process, and low cost, thus providing new possibilities for the treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a characterization diagram of exosome NTA according to an embodiment of the present invention; Figure 1 A is mixed exosomes, Figure 1 B is Bacopa monnieri exosomes, Figure 1 C is cactus exosomes, Figure 1 D is hybrid exosome;

[0026] Figure 2 It is an electron micrograph of hybrid exosomes;

[0027] Figure 3 The effect of hybrid exosomes on cell survival rate; the vertical axes 1, 2, 3, 4, D1, and D2 represent hybrid exosome 1, hybrid exosome 2, hybrid exosome 3, hybrid exosome 4, hybrid exosome D1, and hybrid exosome D2, respectively;

[0028] Figure 4 HE staining of lung tissue of mice with pulmonary fibrosis. Figure 4 A is the model group after bleomycin modeling, Figure 4 B is the drug control group, Figure 4 C is the hybrid exosome intervention group; Figure 4 D is the negative control group; the inset is magnified 20 times. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Example 1 Preparation and characterization of plant-derived hybrid exosomes

[0032] 1. Preparation of Bacopa monnieri exosomes

[0033] The method comprises the following steps: (1) removing the root of Bacopa monnieri, grinding the remaining tissues of Bacopa monnieri (including stem and leaf tissues), dissolving them with PBS at a mass ratio of 1:100, filtering them with gauze; centrifuging at 6000 rpm for 45 minutes to remove impurities and debris, and collecting the supernatant;

[0034] (2) The collected supernatant was transferred to an ultracentrifuge tube, accurately balanced under the condition that the error was less than 0.001 g, and then placed in a pre-cooled ultracentrifuge for ultracentrifugation at 4°C, 9000 g for 60 min, and the supernatant was discarded and the precipitate was retained;

[0035] (3) Resuspend the precipitate retained in (2) with PBS, balance it, and centrifuge it again at 4°C and 9000 g for 60 min. After the centrifugation, collect the precipitate, resuspend it with a small amount of PBS, and store it at ultra-low temperature for future use.

[0036] 2. Preparation of Cactus Exosomes

[0037] The method comprises the following steps: (1) after removing the thorns from the cactus, grinding the remaining cactus tissue, dissolving it with PBS at a mass ratio of 1:100, filtering it with gauze; centrifuging it at 8000 rpm for 60 minutes to remove impurities and debris, and collecting the supernatant;

[0038] (2) The collected supernatant was transferred to an ultracentrifuge tube, accurately balanced under the condition that the error was less than 0.001 g, and then placed in a precooled ultracentrifuge for ultracentrifugation at 4°C, 10,000 g for 70 min, and the supernatant was discarded, and the precipitate was retained;

[0039] (3) Resuspend the precipitate retained in (2) with PBS, balance it, and centrifuge it again at 4°C and 10,000 g for 70 min. After the centrifugation, collect the precipitate, resuspend it with a small amount of PBS, and store it at ultra-low temperature for later use.

[0040] 3. Preparation of Hybrid Exosomes

[0041] The exosomes of Bacopa monnieri and the exosomes of Cactus are mixed in a mass ratio of 5:12 to obtain mixed exosomes; the mixed exosomes are sonicated in a 100W water bath at 37°C for 30 minutes to obtain hybrid exosomes.

[0042] Exosomes were characterized by NTA. Figure 1 NTA results showed that the particle size of pure Bacopa monnieri exosomes was approximately 120 nm ( Figure 1 B), the particle size of pure cactus exosomes is about 150nm ( Figure 1 C), the particle size of the mixed exosomes after simple mixing is about 140nm ( Figure 1 A), the size of the exosomes increases after hybridization. In addition to the individual size peaks, there are also peaks of hybrid exosomes ( Figure 1 D), TEM results show that in addition to the particle size of pure exosomes, there are also larger particle sizes, namely the particle size of hybrid exosomes, indicating that the hybrid exosomes were successfully prepared.

[0043] The concentration of the prepared hybrid exosomes was determined by quantifying exosomal membrane proteins using a BCA kit. Figure 1 From the NTA results of hybrid exosomes, it can be seen that in the hybrid exosomes, the ratio of the number of particles of simple exosomes and hybrid exosomes is approximately 1:1.

[0044] Example 2 Optimization of hybrid exosomes

[0045] Four hybridization ratios of hybrid exosomes were set, and Bacopa monnieri exosomes and cactus exosomes were hybridized at mass ratios of 3:10, 5:12, 2:5, and 1:2, respectively, to obtain hybrid exosome 1, hybrid exosome 2, hybrid exosome 3, and hybrid exosome 4, respectively.

[0046] Using the same method, Bacopa monnieri exosomes and cactus exosomes were hybridized at a mass ratio of 2:15 and 1:1 as the control group, and hybrid exosomes D1 and hybrid exosomes D2 were obtained, respectively.

[0047] 1. Cytotoxicity Test

[0048] The MTT method was used to detect the toxicity of the hybrid exosomes prepared above (added at a concentration of 10 μg / μL) to human alveolar epithelial cells HPAEpiC, and the control group was added with an equal volume of PBS. The results showed that the survival rate of alveolar epithelial cells incubated with hybrid exosomes, hybrid exosomes 2, hybrid exosomes 3, and hybrid exosomes 4 reached more than 98%, and the survival rates of alveolar epithelial cells incubated with hybrid exosomes D1 and hybrid exosomes D2 were 99% and 78%, respectively. From the cytotoxicity experiment, it can be clearly seen that when the dosage of Bacopa monnieri exosomes is high, it will have a significant effect on the survival rate of alveolar epithelial cells, thereby damaging the alveolar epithelial cells, thereby significantly affecting the autonomous repair of damaged epithelium.

[0049] 2. Construction of mouse animal model and experimental grouping

[0050] C57BL / 6 male mice weighing 20-25 g were selected and randomly divided into a model group, a drug control group, a hybrid exosome intervention group, and a negative control group.

[0051] The model group was constructed by bleomycin endotracheal intubation injection, and the mice in the control group were raised normally. The specific method of using bleomycin to construct a mouse pulmonary fibrosis model is as follows: endotracheal intubation is performed on the mice in the model group, and 50 μL of bleomycin solution is taken with a pipette at a dose of 1.5 U / kg and injected once to establish a pulmonary fibrosis model.

[0052] The drug control group received 50 mg / kg of pirfenidone (PFD) instilled intratracheally every day for 7 consecutive days starting from the day after bleomycin treatment; the hybrid exosome intervention group received 50 mg / kg of hybrid exosomes instilled intratracheally every day for 7 consecutive days starting from the day after bleomycin treatment; the negative control group received normal saline instilled intratracheally every day for 7 consecutive days starting from the day after bleomycin treatment.

[0053] Mice were killed: All mice in the model group, drug control group, hybrid exosome intervention group and negative control group were killed after collecting lung tissues at week 7, i.e., 28 days later.

[0054] 1. Scoring of Pulmonary Fibrosis in Mice

[0055] HE and Masson staining were used to observe the degree of alveolitis and pulmonary fibrosis. The degree of alveolitis (grade 0 to +++) and pulmonary fibrosis (grade 0 to +++) were determined by scoring according to the Szapiel method. Five fields of view were randomly selected under a microscope to evaluate the degree of inflammation and fibrosis.

[0056] Table 1

[0057]

[0058]

[0059] The degree of lung fibrosis was scored according to the method of Szapiel SV. The results are shown in Table 2.

[0060] Table 2

[0061] Group Alveolar inflammation (points) Pulmonary fibrosis (points) Hybrid exosome 1+ model group 1.29±0.37 1.60±0.38 Hybrid exosome 2+ model group 1.05±0.27 1.57±0.61 Hybrid exosome 3+ model group 1.35±0.34 1.64±0.56 Hybrid exosome 4+ model group 1.40±0.24 1.61±0.43 Hybrid exosome D1+ model group 2.19±0.46 2.39±0.48 Hybrid exosome D2+ model group 2.48±0.63 2.79±0.45 Control group - - Model Group 3.36±0.13 3.73±0.51

[0062] Note: “-” in the table means not significant

[0063] Figure 4 HE staining of the lungs of mice in each group of animal models. Figure 4 A is the model group, in which obvious alveolar destruction, fibrosis and massive inflammatory cell infiltration were observed; Figure 4 B is the drug control group, from which it can be seen that the degree of fibrosis is alleviated, collagen deposition is reduced, and inflammatory cell infiltration is improved. Figure 4 C is hybrid exosome intervention (this figure is the HE image after hybrid exosome 2 intervention), which showed similar or better anti-fibrotic effects than the pirfenidone group, including less collagen deposition, more intact alveolar structure, and reduced inflammatory response.

[0064] From the data in Table 2, it can be seen that no obvious alveolar inflammation and pulmonary fibrosis were observed in the control group mice; a large number of inflammatory cells infiltrated the alveolar cavities of the model group mice. In the intervention model group mice, the alveolar inflammation of the model mice after hybrid exosome intervention was alleviated to a certain extent, and the lung tissue manifestations were alleviated; this shows that hybrid exosomes can significantly reduce the infiltration of inflammatory cells in the lungs of model mice, protect the alveolar structure, and reduce the degree of pulmonary fibrosis; among them, the relief effects of hybrid exosomes D1 and hybrid exosomes D2 on model mice were very limited. The reason for this was that the amount of Bacopa monnieri exosomes in hybrid exosomes D1 was small, and it could not cooperate with cactus exosomes to play the repair effect of hybrid exosomes; while the amount of Bacopa monnieri exosomes in hybrid exosomes D1 was large, which had a significant effect on the survival rate of alveolar epithelial cells, thereby affecting the reduction of alveolar inflammation and pulmonary fibrosis.

[0065] 2. Impact on inflammatory factors

[0066] The bronchoalveolar lavage fluid of mice was collected, and ELISA was used to detect the expression of inflammatory factors tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and transforming growth factor β1 (TGF-β1) in the bronchoalveolar lavage fluid of mice in each group.

[0067] The results are shown in Table 3. As can be seen from Table 3, compared with the control group, the expression levels of TNF-α, IL-1β, and IL-6 in the model group were increased. Compared with the model group, the expression levels of TNF-α, IL-1β, and IL-6 in the intervention model group were reduced to a certain extent. TNF-α and macrophages interact with each other in the development of pulmonary interstitial fibrosis, aggravate alveolar damage, and regulate and stimulate other cytokines in a complex cytokine network to jointly mediate pulmonary fibrosis. The significant reduction of TNF-α in the intervention model group indicates that the hybrid exosomes of the present invention can effectively slow down the process of pulmonary fibrosis; IL-6 and IL-1β are also considered to be important cytokines in bleomycin-induced lung injury. The significant reduction of IL-6 and IL-1β in the intervention model group indicates that the hybrid exosomes of the present invention can effectively inhibit lung tissue inflammation.

[0068] The lung tissue is induced to trigger an inflammatory response by bleomycin. In the fibrosis stage after the inflammatory response, pro-fibrotic mediators such as TGF-β1 can activate fibroblasts to transform into myofibroblasts, produce a large amount of collagen and ECM, and thus lead to the formation of pulmonary fibrosis. Therefore, TGF-β1 is a cytokine involved in the development of organ fibrosis. The significant reduction of TGF-β1 in the intervention model group indicates that the hybrid exosomes of the present invention can effectively inhibit the development of pulmonary fibrosis, thereby playing an anti-fibrotic role.

[0069] Table 3

[0070]

[0071]

[0072] From the data in Table 3, it can be seen that compared with the control group, the levels of various inflammatory factors in the model group mice were significantly increased. Compared with the model group mice, the levels of various inflammatory factors in the intervention model group mice were significantly reduced, and the difference was statistically significant. Specifically, the expression levels of various inflammatory factors in the model group mice intervened by hybrid exosomes 2 were the lowest. Among them, hybrid exosomes D1 and hybrid exosomes D2 had limited intervention effects on the levels of inflammatory factors in model mice.

[0073] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A hybrid exosome derived from a plant, characterized in that: The exosomes of Bacopa monnieri and cactus exosomes are mixed in a mass ratio of 3-8:10-16, and then the mixture is obtained by water bath ultrasound.

2. The plant-derived hybrid exosome according to claim 1, characterized in that The preparation method of the Bacopa monnieri exosomes is as follows: (1) Grind Bacopa monnieri, dissolve in PBS, filter through gauze, centrifuge to remove impurities and debris, and collect the supernatant; (2) The collected supernatant was subjected to ultracentrifugation at 9000 g for 60 min at 4°C, and the supernatant was discarded, while the precipitate was retained; (3) Resuspend the precipitate retained in (2), and centrifuge again at 4°C and 9000 g for 60 min. After the centrifugation, collect the precipitate, resuspend it in PBS, and store it at ultra-low temperature for future use.

3. The plant-derived hybrid exosome according to claim 2, characterized in that The preparation method of the cactus exosomes is: (1) Grind the cactus, dissolve it in PBS, filter it through gauze, centrifuge to remove impurities and debris, and collect the supernatant; (2) The collected supernatant was subjected to ultracentrifugation at 10,000 g for 70 min at 4°C, and the supernatant was discarded, while the precipitate was retained; (3) Resuspend the precipitate retained in (2) and centrifuge again at 4°C and 10,000 g for 70 min. After the centrifugation, collect the precipitate, resuspend it in PBS, and store it at ultra-low temperature for future use.

4. Use of the hybrid exosomes according to any one of claims 1 to 3 in the preparation of a drug for reducing the expression of cellular inflammatory factors IL-1β, IL-6, TNF-α and promoting the expression of anti-inflammatory factors IL-4 and IL-10.

5. Use of the hybrid exosomes according to any one of claims 1 to 3 in the preparation of a drug for reducing cytokine TGF-β1.

6. Use of the hybrid exosomes according to any one of claims 1 to 3 in the preparation of a drug for inhibiting pulmonary fibrosis.

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