Application of extracellular vesicle particles derived from shenshisan in preparation of drugs for preventing or treating pulmonary fibrosis

Abstract

The application claims the extracellular vesicle particles of Shenshi Sanjie formula source and a preparation method thereof and application in preparing a medicine for preventing and treating pulmonary fibrosis. The extracellular vesicle particles of Shenshi Sanjie formula source provided by the application have good therapeutic effect on pulmonary fibrosis by inhibiting the release of early persistent inflammatory factors and reducing the degree of late fibrosis. The extracellular vesicle particles of Shenshi Sanjie formula source have definite anti-pulmonary fibrosis effect, low side effects and wide medical application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to the medical use of exosome particles derived from Shenshidanjie formula, in particular to the medical use of exosome particles derived from Shenshidanjie formula in the preparation of a drug for preventing or treating pulmonary fibrosis. BACKGROUND

[0002] Pulmonary fibrosis is the final pathological change of various interstitial lung diseases, characterized by long-term diffuse alveolitis, excessive proliferation of fibroblasts, and then excessive deposition of extracellular matrix, gradually replacing normal lung tissue, eventually causing lung dysfunction, and patients may die of respiratory failure. Pulmonary fibrosis can be broadly divided into two categories: known causes (such as radiation, drugs, silicosis, etc.) and unknown causes (such as idiopathic pulmonary fibrosis). Research suggests that pathogenic microorganisms, dust, drugs, and chemical agents can all induce the occurrence of pulmonary fibrosis, but the detailed mechanism is not yet clear. Pulmonary fibrosis can occur at any age, with a global incidence of 7-10 / 100,000, and the average survival time of patients is 3-4 years. As age increases, the incidence gradually increases and the average survival time gradually decreases. Therefore, pulmonary fibrosis seriously endangers human health and survival, and the research and development of drugs for treating pulmonary fibrosis is very important.

[0003] According to the classification of unknown causes, pulmonary fibrosis can be subdivided into primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis, and interstitial pneumonia; among them, pulmonary interstitial fibrosis has many different types, including secondary pulmonary interstitial fibrosis (secondary to immune diseases such as rheumatoid, dry syndrome, etc.), drug-induced pulmonary interstitial fibrosis, and idiopathic pulmonary interstitial fibrosis. Most pulmonary interstitial fibrosis is caused by viruses, mainly adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus, and measles virus. Among them, adenovirus and influenza virus are more common and more serious, often forming necrotizing bronchitis and bronchopneumonia, and are prone to evolve into chronic pneumonia.

[0004] The accurate concept of pulmonary interstitial fibrosis is interstitial lung disease (ILD), a group of diffuse lung diseases that mainly involve the lung interstitium and alveolar space, leading to loss of alveolar and capillary function, with high morbidity and mortality. Currently, glucocorticoids combined with immunosuppressive agents are considered the main method for treating interstitial lung disease. However, this combination therapy increases the risk of Yeerowei lung cyst pneumonia, which poses a great challenge to clinicians. Therefore, there is an urgent need to find safe and effective drugs to treat ILD.

[0005] Traditional Chinese medicine is more and more valued due to its outstanding curative effect and small adverse reactions. However, the complex components and processing technology limit its further application. Shenshisanjie formula (SJ) is composed of three traditional Chinese medicines, namely, Rhodiola, Trionyx sinensis and Coicis Semen, and is a famous formula widely used in the treatment of pulmonary diseases / pulmonary fibrosis in clinic. However, the complex processing technology and strict storage conditions limit its further application in clinic. Extracellular vesicles (EVs) contain various proteins, lipids and nucleic acids, and can play an important physiological function by mediating cell-to-cell communication. Almost all types of eukaryotic and prokaryotic cells secrete EVs. Plant EVs are similar to mammalian EVs in morphology and have cross-species regulation function, which can not only regulate the physiological function of mammalian cells, but also intervene and prevent the disease process, and play a therapeutic role in diseases. These research conclusions prove that plant EVs as a new type of natural product have the potential to become a good candidate source for new drug development. However, there is no literature report on the extracellular vesicles derived from Shenshisanjie formula. Therefore, it is unknown whether the extraction of extracellular vesicles from Shenshisanjie formula is feasible, and whether the extracted extracellular vesicles have biological activity or the related function of Shenshisanjie formula.

[0006] Therefore, it is significant to provide a method for extracting extracellular vesicles from Shenshisanjie formula and to further study the function of the extracted extracellular vesicles in order to explore the application potential of the extracellular vesicles in the treatment of pulmonary fibrosis. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a pharmaceutical use of extracellular vesicle nanoparticles derived from Shenshisanjie formula, in particular to the application of the extracellular vesicle nanoparticles derived from Shenshisanjie formula in the preparation of a drug for preventing or treating pulmonary fibrosis.

[0008] The present application provides extracellular vesicle nanoparticles derived from Shenshisanjie formula and a preparation method thereof and the application of the extracellular vesicle nanoparticles in the preparation of a drug for preventing or treating pulmonary fibrosis. The extracellular vesicle nanoparticles derived from Shenshisanjie formula provided by the present application have a good therapeutic effect on pulmonary fibrosis by inhibiting the release of early persistent inflammatory factors and reducing the degree of late fibrosis, and are safe and non-toxic, and therefore can be used for preparing a drug for preventing or treating pulmonary fibrosis.

[0009] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0010] In a first aspect, the present application provides the application of extracellular vesicle nanoparticles derived from Shenshisanjie formula in the preparation of a drug for preventing or treating pulmonary fibrosis, wherein the active ingredient of the drug comprises the extracellular vesicle nanoparticles derived from Shenshisanjie formula.

[0011] The application obtains a wetness penetrating and knot resolving formula derived extracellular vesicle-like particle, and through experiments, it is found that the wetness penetrating and knot resolving formula derived extracellular vesicle particle has a significant treatment effect on pulmonary fibrosis, is safe and non-toxic, and can be used to prepare a drug for preventing and treating pulmonary fibrosis.

[0012] The Chinese herbal medicine formula wetness penetrating and knot resolving formula is composed of three kinds of herbs: Rhodiola (dried Rhodiola), Trionyx sinensis Wiegmann carapace (dried Trionyx sinensis Wiegmann carapace) and Coix seed (dried Coix seed), wherein, Rhodiola 15 g, Trionyx sinensis Wiegmann carapace 15 g and Coix seed 30 g.

[0013] The wetness penetrating and knot resolving formula derived extracellular vesicle-like particle is obtained by extraction and separation of the wetness penetrating and knot resolving formula.

[0014] The pulmonary fibrosis is primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis and interstitial pneumonia.

[0015] Further, the pulmonary interstitial fibrosis is secondary pulmonary interstitial fibrosis, drug-induced pulmonary interstitial fibrosis or idiopathic pulmonary interstitial fibrosis.

[0016] Further, the secondary pulmonary interstitial fibrosis is pulmonary interstitial fibrosis secondary to immune diseases such as rheumatoid, Sjogren's syndrome and the like.

[0017] Further, the pulmonary interstitial fibrosis is virus-induced pulmonary interstitial fibrosis.

[0018] Further, the virus is adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus and / or measles virus.

[0019] Further, the virus is adenovirus or influenza virus.

[0020] In combination with the first aspect, the dosage form of the drug is an oral preparation.

[0021] Preferably, the drug for preventing and treating pulmonary fibrosis further comprises a pharmaceutically acceptable injection adjuvant or oral preparation adjuvant. The specific adjuvant and the corresponding preparation method can be conventionally selected according to the specific dosage form of the injection or oral preparation, and the pharmacological activity and physicochemical stability of the wetness penetrating and knot resolving formula derived extracellular vesicle particle are not adversely affected and meet the clinical drug use requirements, which are not limited in the application.

[0022] In the second aspect, the application provides a preparation method of the wetness penetrating and knot resolving formula derived extracellular vesicle particle.

[0023] The preparation method of the wetness penetrating and knot resolving formula derived extracellular vesicle particle comprises the following steps:

[0024] Step S1: preparation of wetness penetrating and knot resolving formula decoction

[0025] The dried Rhodiola, Trionyx and Coicis Semen are mixed and cut into pieces, extracted with distilled water, and a crude extraction system is used. The supernatant of the two extractions is homogenized, and the Shenshi Sanjie Decoction extract is obtained.

[0026] Step S2: Separation of Shenshi Sanjie Decoction-derived extracellular vesicle particles

[0027] The Shenshi Sanjie Decoction-derived extracellular vesicles are separated from the Shenshi Sanjie Decoction extract by differential centrifugation.

[0028] Further, the preparation method of the Shenshi Sanjie Decoction-derived extracellular vesicle particles comprises the following steps:

[0029] Step S1: Preparation of Shenshi Sanjie Decoction extract

[0030] The dried Rhodiola, Trionyx and Coicis Semen are mixed and cut into pieces, extracted with distilled water, and a crude extraction system is used. The supernatant of the two extractions is homogenized, and the Shenshi Sanjie Decoction extract is obtained.

[0031] Step S2: Separation of Shenshi Sanjie Decoction-derived extracellular vesicle particles

[0032] The collected Shenshi Sanjie Decoction extract is sequentially centrifuged at 1000xg for 5-15 minutes, 2000xg for 15-25 minutes, 3000xg for 25-35 minutes, and 10000xg for 50-70 minutes to remove large particles and cell debris. Then, the final supernatant is centrifuged at 150000xg for 1.5-2.5 hours in a centrifuge, and the obtained particles are gently shaken in PBS and resuspended at 4°C for at least 1 hour to obtain Shenshi Sanjie Decoction-derived extracellular vesicle particles.

[0033] Further, in the step S1, the weight ratio of Rhodiola, Trionyx and Coicis Semen is 1:1:2.

[0034] Further, in the step S1, the crude extraction system is 95±5°C, and the first extraction is performed for 2-5 hours; the second extraction is performed at 95±5°C for 0.5-2 hours.

[0035] Further, in the step S1, the crude extraction system is 95°C, and the first extraction is performed for 3 hours; the second extraction is performed at 95°C for 1 hour.

[0036] Further, in the step S2, 60g of Shenshi Sanjie Decoction uses about 500-700μL of culture medium.

[0037] Further, in the step S2, 60g of Shenshi Sanjie Decoction uses 600μL of culture medium.

[0038] Further, in step S2, 1000xg centrifugation for 10 minutes.

[0039] Further, in step S2, 2000xg centrifugation for 20 minutes.

[0040] Further, in step S2, 3000xg centrifugation for 30 minutes.

[0041] Further, in step S2, 10000xg centrifugation for 60 minutes.

[0042] Further, in step S2, the centrifuge is Himac ultracenfuge CP100NX.

[0043] Further, in step S2, 150000xg, centrifugation for 2h.

[0044] Further, in step S2, the obtained particles are gently shaken in 600μL PBS.

[0045] Further, in step S2, the obtained particles are resuspended at 4℃ for 1-5h.

[0046] The preparation method of the extracellular vesicle particles derived from the Shenshi Sanjie Decoction, specifically includes the following steps:

[0047] S1: Preparation of the Shenshi Sanjie Decoction extract

[0048] The dried Rhodiola, Trionyx and Coicis Semen are cut and mixed, extracted with distilled water, and a crude extraction system is used, 95±5℃, first extraction for 3h; 95±5℃, second extraction for 1h. The supernatants of the two extractions are homogenized, which is the Shenshi Sanjie Decoction extract, and stored in a sealed container.

[0049] S2: Isolation of the extracellular vesicle particles derived from the Shenshi Sanjie Decoction

[0050] The differential centrifugation method is used to separate the extracellular vesicles from the Shenshi Sanjie Decoction extract, 60g of the Shenshi Sanjie Decoction uses 600μL of medium. 23mL of the collected Shenshi Sanjie Decoction extract is sequentially centrifuged at 1000xg for 10 minutes, 2000xg for 20 minutes, 3000xg for 30 minutes, and 10000xg for 60 minutes to remove large particles and cell debris. Then, the sequential ultracentrifugation method is further used, including centrifuging the final supernatant at 150000xg for 2h in a Himac ultracenfuge CP100NX, Ti45 rotor. The obtained particles are gently shaken in 600μL PBS and resuspended at 4℃ for at least 1h, thereby obtaining the extracellular vesicle particles derived from the Shenshi Sanjie Decoction. The final sample of the extracellular vesicles derived from the Shenshi Sanjie Decoction is stored at -80℃.

[0051] In a third aspect, the present application provides a Shenshi Sanjie formula-derived extracellular vesicle particle, which is prepared by the above method.

[0052] In a fourth aspect, the present application provides a drug for preventing and treating pulmonary fibrosis, wherein the active ingredient of the drug comprises the Shenshi Sanjie formula-derived extracellular vesicle particle.

[0053] In combination with the fourth aspect, the pulmonary fibrosis is primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis, and interstitial pneumonia.

[0054] Further, the pulmonary interstitial fibrosis is secondary pulmonary interstitial fibrosis, drug-induced pulmonary interstitial fibrosis, or idiopathic pulmonary interstitial fibrosis.

[0055] Further, the secondary pulmonary interstitial fibrosis is secondary to immune diseases such as rheumatoid arthritis, Sjogren's syndrome, etc.

[0056] Further, the pulmonary interstitial fibrosis is viral-induced pulmonary interstitial fibrosis.

[0057] Further, the virus is adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus, and / or measles virus.

[0058] Further, the virus is adenovirus or influenza virus.

[0059] In combination with the fourth aspect, the drug for preventing and treating pulmonary fibrosis is in the form of an injection or an oral preparation.

[0060] Preferably, the drug for preventing and treating pulmonary fibrosis further comprises a pharmaceutically acceptable injection adjuvant or oral preparation adjuvant. The specific adjuvant and the corresponding preparation method can be routinely selected according to the specific dosage form of the injection or oral preparation, and the selection should not adversely affect the pharmacological activity and physicochemical stability of the Shenshi Sanjie formula-derived extracellular vesicle particle and meet the clinical drug requirements. The present application does not limit this.

[0061] The pharmacodynamic examples of the present application adopt intratracheal injection of bleomycin (BLM) to induce a mouse pulmonary fibrosis model to study the treatment effect of the extracellular vesicle particles derived from Shenshisan on pulmonary fibrosis, especially pulmonary interstitial fibrosis. After the experiment, the rat body weight, lung tissue pathological changes, pulmonary fibrosis degree and pro-fibrotic cytokines are detected by Masson trichrome staining, Sirius red staining and ELISA kit on the 21st day. HE staining and biochemical reagents are used on the 3rd day to detect the local area infiltration of mononuclear cells, inflammatory factors and total cell number in bronchoalveolar lavage fluid (BALF). In the in vitro experiment, the epithelial-mesenchymal transition (EMT) model of A549 cells induced by TGF-β is used to evaluate the effect of the extracellular vesicle particles derived from Shenshisan on alveolar epithelial cells. A series of indexes are detected by western blot, wound healing test and immunofluorescence. In addition, flow cytometry and Seahorse Assay kit are used to detect the anti-fibrosis effect of the extracellular vesicle particles derived from Shenshisan on the IL-4 activated M2 macrophage model. The in vivo and in vitro experiments show that the extracellular vesicle particles derived from Shenshisan have a significant treatment effect on pulmonary fibrosis, especially pulmonary interstitial fibrosis.

[0062] The present application has the following beneficial effects: the present application takes Shenshisan as raw material, uses a series of high-speed and ultra-centrifugal separation of extracellular vesicle particles derived from Shenshisan, and uses electron microscopy, particle size analyzer and agarose gel electrophoresis for characterization. In the in vivo study, the extracellular vesicle particles derived from Shenshisan have a good treatment effect on pulmonary fibrosis by inhibiting the release of early persistent inflammatory factors and reducing the degree of late fibrosis. In the in vitro experiment, the extracellular vesicle particles derived from Shenshisan can improve the EMT of A549 cells mediated by ROS-NLRP3 pathway. At the same time, the extracellular vesicle particles derived from Shenshisan improve the OXPHOS inhibition of M2 polarization. The extracellular vesicle particles derived from Shenshisan improve pulmonary fibrosis by reducing ROS / NLRP3 mediated EMT of ATII cells and inhibiting energy metabolism mediated polarization of M2 cells. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 Separation and characterization of EVs-SJ.

[0064] (A) Steps for extracting EVs from SJ decoction;

[0065] (B) RNA was separated by electrophoresis on 3% agarose gel and visualized with UVP imaging system;

[0066] (C) Macroscopic appearance observed by electron microscopy;

[0067] (D) zeta potential of EVs-SJ;

[0068] (E) particle size analysis by a particle size analyzer.

[0069] Figure 2 EVs-SJ ameliorated bleomycin (BLM)-induced pulmonary fibrosis.

[0070] After a single aerosol administration of BLM (5 mg / kg), EVs-SJ was administered by gavage daily for 21 days, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected on day 21.

[0071] (A) Masson staining for collagen fibers;

[0072] (B) Sirius red staining for the distribution and type of newly formed collagen during fibrogenesis;

[0073] (C) ELISA for TGF-β levels in BALF;

[0074] (D) Biochemical kit for hydroxyproline content in lung tissue;

[0075] (E) Mice weight change curve.

[0076] EVs-1-SJ: EVs isolated from SJ 200 mg / kg; EVs-2-SJ: EVs isolated from SJ 300 mg / kg; SJ: 200 mg / kg.

[0077] Figure 3 EVs SJ significantly anti-inflammatory capacity.

[0078] After a single aerosol administration of BLM (5 mg / kg), EVs-SJ and SJ were administered by gavage daily for 3 days, and BALF and lung tissue were collected.

[0079] (A) HE staining for inflammatory cell infiltration in damaged alveoli;

[0080] (B-E) ELISA for total cell number, IL-1β, TNF-α, IL-6 levels in BALF.

[0081] EVs-1-SJ: EVs isolated from SJ 200 mg / kg; EVs-2-SJ: EVs isolated from SJ 300 mg / kg; SJ: 200 mg / kg.

[0082] Figure 4 EVs-SJ modulates multiple key cells through cell-cell communication.

[0083] (A) DiI-stained EVs-SJ were administered to BLM mice. 3 hours later, mouse lung tissue was isolated. Under fluorescence microscopy, Dil-stained EVs were observed to express in SFTPC-labeled ATII cells and CD163-labeled macrophages in the lung tissue.

[0084] (B) Vimentin, a marker of fibroblasts, was observed by fluorescence microscopy;

[0085] (C) CD163, a marker of M2 macrophages, was observed by fluorescence microscopy.

[0086] Figure 5 EVs-SJ improved epithelial-mesenchymal transition (EMT) in A549 cells.

[0087] (A-B) Cell viability after 24 and 48 hours of A549 cells treated with different concentrations (0, 1, 3, 5, 10 pg / mL) of EVs-SJ;

[0088] (C) E-cadherin and Vimentin, a marker of mesenchyme, were observed in A549 cells by Western blotting, with GAPDH as an endogenous control;

[0089] (D) Quantitative analysis of the results of C;

[0090] (E) Wound healing results.

[0091] Figure 6 EVs-SJ modulated the ROS / NLRP3 pathway-mediated EMT.

[0092] A549 cells were pre-treated with EVs-SJ (1, 3, 5 pg / mL) for 1 hour, then stimulated with TGF-β for 24 hours, and then placed in LPS (500 ng / mL) and ATP (5 mM) for 4 h.

[0093] (A) ROS levels were determined using immunofluorescence techniques;

[0094] (B) The expression levels of IL-1β, Caspase 1, and NLRP3 in A549 cells were detected by Western blotting, with GAPDH as an endogenous control;

[0095] (C) Immunofluorescence staining of Vimentin, a marker of mesenchyme in A549;

[0096] (D) Wound healing: ROS scavenger NAC, 5 mM, and NLRP3 inhibitor MCC950, 10 mM, both significantly reduced the expression of EMT marker protein Vimentin in the wound area and inhibited wound healing.

[0097] Figure 7 EVs-SJ suppresses M2 repolarization by improving OXPHOS.

[0098] (A) RAW264.7 cells were cultured with EVs-SJ for 1 hour, then stimulated with IL-4 for 4 hours, and M2 polarization was detected by flow cytometry.

[0099] (BC) RAW264.7 cells were cultured with EVs-SJ for 1 hour, and then incubated in IL-4 for 24 hours with the addition of inhibitors OM (ATP synthase inhibitor), 2-DG, FCCP, ROT / AA, GLU, or without the addition of inhibitors. (BC) shows the results of OCR response to OM (ATP synthase inhibitor), FCCP (ex vivo mitochondrial uncoupling agent), or ROT / AA (electron transport inhibitor).

[0100] (DE) Extracellular acidification rate (ECAR) of glucose, OM and 2-DG (synthetic analogue of glucose). Detailed Implementation

[0101] To enable those skilled in the art to fully understand the present invention, the present invention is further illustrated below through specific embodiments. However, those skilled in the art should understand that the embodiments of the present invention do not limit the present invention in any way.

[0102] Example 1

[0103] S1: Preparation of decoction of the dampness-dispersing and nodule-resolving formula (SJ)

[0104] Chop and mix Rhodiola rosea (dried), turtle shell (Carapace of Trionyx sinensis Wiegmann), and coix seed (dried), using 15g of Rhodiola rosea, 15g of turtle shell, and 30g of coix seed. Extract with distilled water using a crude extraction system at 95℃ for the first extraction (3 hours) and the second extraction (1 hour) at 95℃. Homogenize the supernatants from both extractions; this is the extract of the decoction for dispersing dampness and stagnation, and store it in a sealed container.

[0105] S2: Separation of extracellular vesicle granules from the permeation and dissipation mechanism

[0106] Extracellular vesicles were isolated from the decoction of Shenshi Sanjie Fang (a traditional Chinese medicine formula) using differential centrifugation. 60g of Shenshi Sanjie Fang was used with 600μL of culture medium. The collected 23mL extract of Shenshi Sanjie Fang decoction was centrifuged sequentially at 1000×g for 10 min, 2000×g for 20 min, 3000×g for 30 min, and 10000×g for 60 min to remove large particles and cell debris. Further sequential ultracentrifugation was then performed, including centrifuging the final supernatant at 150000×g for 2 h on a Himac Ultracenfuge CP100NX Ti45 rotor. The resulting particles were then gently resuspended in 600μL PBS at 4℃ for 2 h to obtain the extracellular vesicle particles derived from Shenshi Sanjie Fang. The final sample of extracellular vesicles derived from Shenshi Sanjie Fang was stored at -80℃ until analysis.

[0107] Example 2

[0108] S1: Preparation of the decoction of the dampness-removing and nodule-dispersing formula

[0109] Chop and mix Rhodiola rosea, turtle shell, and coix seed, using 15g of Rhodiola rosea, 15g of turtle shell, and 30g of coix seed. Extract with distilled water using a coarse extraction system at 90℃ for 2 hours and 98℃ for 1.5 hours. Homogenize the supernatants from both extractions to obtain the extract of the decoction for dispersing dampness and stagnation, and store it in a sealed container.

[0110] S2: Separation of extracellular vesicle granules from the permeation and dissipation mechanism

[0111] Extracellular vesicles were isolated from the decoction of Shenshi Sanjie Fang (a traditional Chinese medicine formula) using differential centrifugation. 60g of Shenshi Sanjie Fang was used with 600μL of culture medium. The collected 23mL extract of Shenshi Sanjie Fang decoction was centrifuged sequentially at 1000×g for 15 minutes, 2000×g for 15 minutes, 3000×g for 35 minutes, and 10000×g for 65 minutes to remove large particles and cell debris. Further sequential ultracentrifugation was then performed, including centrifuging the final supernatant at 150000×g for 1.5 hours using a Himac Ultracenfuge CP100NX with a Ti45 rotor. The resulting particles were then gently resuspended in 600μL of PBS at 4°C for 1 hour to obtain the extracellular vesicle particles derived from Shenshi Sanjie Fang. The final sample of Shenshi Sanjie Fang-derived extracellular vesicles was stored at -80°C.

[0112] Example 3

[0113] S1: Preparation of the decoction of the dampness-removing and nodule-dispersing formula

[0114] Chop and mix Rhodiola rosea, turtle shell, and coix seed, using 15g of Rhodiola rosea, 15g of turtle shell, and 30g of coix seed. Extract with distilled water using a coarse extraction system at 98℃ for 3.5 hours for the first extraction and 95℃ for 1.5 hours for the second extraction. Homogenize the supernatants from both extractions to obtain the extract of the decoction for dispersing dampness and stagnation, and store it in a sealed container.

[0115] S2: Separation of extracellular vesicle granules from the permeation and dissipation mechanism

[0116] Extracellular vesicles were isolated from the decoction of Shenshi Sanjie Fang (a traditional Chinese medicine formula) using differential centrifugation. 60g of Shenshi Sanjie Fang was used with 500μL of culture medium. The collected 23mL extract of the decoction was centrifuged sequentially at 1000×g for 12 min, 2000×g for 25 min, 3000×g for 33 min, and 10000×g for 55 min to remove large particles and cell debris. Further sequential ultracentrifugation was then performed, including centrifuging the final supernatant at 150000×g for 2 h on a Himac Ultracenfuge CP100NX Ti45 rotor. The resulting particles were then gently resuspended in 600μL PBS at 4℃ for 3 h to obtain the extracellular vesicle particles derived from Shenshi Sanjie Fang. The final sample of Shenshi Sanjie Fang-derived extracellular vesicles was stored at -80℃.

[0117] Example 4

[0118] S1: Preparation of the decoction of the dampness-removing and nodule-dispersing formula

[0119] Chop and mix Rhodiola rosea, turtle shell, and coix seed, using 15g of Rhodiola rosea, 15g of turtle shell, and 30g of coix seed. Extract with distilled water using a coarse extraction system at 95℃ for the first extraction (3 hours) and 98℃ for the second extraction (1.5 hours). Homogenize the supernatants from both extractions to obtain the extract of the decoction for dispersing dampness and stagnation, and store it in a sealed container.

[0120] S2: Separation of extracellular vesicle granules from the permeation and dissipation mechanism

[0121] Extracellular vesicles were isolated from the decoction of Shenshi Sanjie Fang (a traditional Chinese medicine formula) using differential centrifugation. 60g of Shenshi Sanjie Fang was used with 650μL of culture medium. The collected 23mL extract of Shenshi Sanjie Fang decoction was centrifuged sequentially at 1000×g for 10 min, 2000×g for 20 min, 3000×g for 35 min, and 10000×g for 65 min to remove large particles and cell debris. Further sequential ultracentrifugation was then performed, including centrifuging the final supernatant at 150000×g for 2 h on a Himac Ultracenfuge CP100NX Ti45 rotor. The resulting particles were then gently resuspended in 600μL PBS at 4℃ for 1.5 h to obtain the extracellular vesicle particles derived from Shenshi Sanjie Fang. The final sample of extracellular vesicles derived from Shenshi Sanjie Fang was stored at -80℃.

[0122] Example 5: Characterization of extracellular vesicles (EVs-SJ) from the infiltration and dissipation mechanism

[0123] The content of EVs-SJ protein extracted in Example 1 was determined using a BCA kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0124] The morphology, particle size, zeta potential, purity, and chemical composition of the EVs-SJ extracted in Example 1 were characterized.

[0125] (1) Observe the morphology of EVs-SJ under a transmission electron microscope, such as Figure 1 As shown: the vesicles are cup-shaped with clearly visible cell membranes, sharp membrane boundaries, clean staining background, and obvious contrast.

[0126] (2) The particle size distribution and zeta potential of EVs-SJ were analyzed using a Zetasizer Nano ZSE nanoparticle size potentiometer, such as... Figure 2 As shown in (a) and (b), the particle size distribution is between 250 and 300 nm, with an average diameter of 286.5 nm. The zeta potential of EVs-SJ is -7.74 mV.

[0127] (3) Agarose gel electrophoresis, silver staining, and thin-layer chromatography were used to detect DNA, RNA, protein, and lipids in EVs-SJ, respectively. The results are as follows: Figure 3 As shown, RNA and protein are the main components of extracellular vesicles. BCA protein analysis confirmed that the EVs-SJ protein concentration was 60 mg / mL.

[0128] Example 1: Efficacy Study of Extracellular Vesicles (EVs-SJ) from the Dampness-Inducing and Nodule-Dissolving Formula on Bleomycin-Induced Pulmonary Fibrosis in Mice

[0129] 1. Materials

[0130] 1.1 Laboratory Animals

[0131] Thirty male C57BL / 6 mice, aged 6-8 weeks and with an average weight of 18-22g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Changzhou, Jiangsu Province). Procedures were strictly followed in accordance with NIH guidelines on the care and use of laboratory animals (NIH Publication 8023, revised in 1978). All experiments were conducted in accordance with the National Institute of Animal Health's "Regulations on the Care and Use of Laboratory Animals" and the guidance of the Qingdao University Ethics Committee (Animal Welfare Guarantee No.: 14-0027).

[0132] 1.2 Chemicals and Reagents:

[0133] Bleomycin sulfate (BLM) and N-acetylcysteine ​​were purchased from MedChemExpress (MCE, Princeton, NJ, USA). LPS, MCC950, TGF-β, and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). E-cadherin (24E10), NLRP3 (D4D8T), Vimentin (D21H3), CD163 (68922S), Cleaved Caspase-1 (Asp297), CD206 (24595S), and GAPDH (D16H11) antibodies were purchased from Cell Signaling (Danvers, MA, USA). SFTPC (PA5-71680) was purchased from Invitrogen (Carlsbad, California, USA). IL-1β (AF5103) antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA). The Seahorse XF Cell Mitochondrial Stress Assay Kit (103015-100) was purchased from Seahorse Bioscience (Hongkou District, Shanghai, China).

[0134] EVs-SJ dissolved in 0.9% NaCl.

[0135] 2 methods

[0136] 2.1 Experimental grouping, drug administration, and model establishment

[0137] All animals were randomly divided into 5 groups (n=6 / group): 1) control group; 2) BLM (5mg / kg) group; 3) BLM+EVs-1-SJ group (EVs isolated from SJ 200mg / kg); 4) BLM+EVs-2-SJ group (EVs isolated from SJ 300mg / kg); 5) BLM+SJ group (200mg / kg).

[0138] Mice were anesthetized by intraperitoneal injection of chloral hydrate 350 mg / kg. The normal control group was given physiological saline via tracheal instillation, while the other four groups were given bleomycin (5 mg / kg) via tracheal instillation. Immediately after instillation, the mice were rotated upright for 2 minutes to ensure even distribution of the drug in the lungs, and then returned to their cages for normal feeding.

[0139] 2.2 Detection Indicators and Methods

[0140] Mice were anesthetized with 4% chloral hydrate on days 3 and 21 and euthanized with an overdose of the anesthetic. Bronchoalveolar lavage fluid, blood, and lung tissue were then collected and fixed in 4% PFA.

[0141] 2.2.1 Detection of cytokines in bronchoalveolar lavage fluid

[0142] Mice were sacrificed on days 3 and 21. After exposing the trachea, a 20G intravenous cannula was inserted into the left bronchus of the mice, and the bronchoalveolar lavage fluid was collected after three irrigations with PBS (0.5 mL, 2 mM EDTA). The bronchoalveolar lavage fluid was then centrifuged at 500 g / min for 10 minutes at 4 °C. The supernatant was used to measure cytokine and total protein levels. The cells in the pellet were resuspended in 0.5 mL of PBS and counted using a hemocytometer.

[0143] According to the manufacturer's instructions, the levels of IL-1β, IL-6, TNF-α and TGF-β in bronchoalveolar lavage fluid were measured using a commercial mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA).

[0144] 2.2.2 Histopathology and Immunofluorescence Assay

[0145] Tissue samples from the right lung were fixed with 4% PFA for 48 hours, then embedded in paraffin and sectioned into 4-micron sections. HE staining was performed on the tissue sections according to the manufacturer's instructions to assess histopathological changes. Fibrotic areas in the lung sections were evaluated using Masson's trichrome staining and Sirius red staining.

[0146] Immunofluorescence staining was used to detect the expression of SFTPC, CD163, and Vimentin in cells. Cells were fixed with 4% PFA for 20 minutes and infiltrated with 0.1% Triton X100 for 15 minutes. The samples were then blocked with 5% BSA (Solarbio, China) for 30 minutes. Cells were then incubated overnight at 4°C with a fluorescent primary antibody. After additional washing with PBS, cell nuclei were labeled with DAPI (Beyotime, China). Fluorescent images of stained cells were then acquired using a laser scanning confocal microscope (Leica TCS SP8, Leica, Germany).

[0147] 2.2.3 Determination of Hydroxyproline Content

[0148] Lung tissue was homogenized on ice and centrifuged at 1000g for 10 min at 4°C. The lung homogenate was then centrifuged at 1000g for 10 min at 4°C. The hydroxyproline content in the lung tissue was determined using an alkaline hydrolysis kit (Jiancheng Biotechnology Institute, Nanjing, China) according to the manufacturer's instructions.

[0149] 2.2.4 Scratch Test

[0150] A549 cells (3×10) 5 Cells / well were cultured in 12-well plates at 37°C and 5% CO2 using 10% FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin until they reached 90-100% monolayer confluence. The monolayer was scratched in the center of each culture dish using the tip of a 200 μL sterile pipette. After scratching, the cells were gently washed three times with serum-free DMEM medium to remove the isolated cells. Then, different concentrations of EVs-SJ were added to the culture dishes. The control group was treated with an equal volume of PBS. Photographs of the same wound sites were taken at 0 h, 24 h, and 48 h using a Leica Qwin System (Leica, Germany) inverted microscope. Each experiment was repeated three times.

[0151] 2.2.5 Protein Blotting

[0152] Total protein collected from A549 cells was extracted using RIPA lysis buffer containing protease inhibitors. Approximately 50 μg of protein sample was loaded into each well of a 10% or 12% SDS-PAGE gel for electrophoresis. After SDS-PAGE, the protein was transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 1 hour, the PVDF membrane was incubated overnight at 4°C with primary antibodies. The primary antibodies used were: NLRP3 (1:1000), Vimentin (1:1000), E-cadherin (1:1000), IL-1β (1:1000), cleaved Caspase 1 (1:1000), and GAPDH (1:1000). The membrane was then incubated with an HRP-conjugated secondary antibody (1:5000) at room temperature for 1 hour. TBST washing was performed three times for 5 minutes each time. Protein bands were detected using an Amersham ImageQuant 800 (Cytiva, Cambridge, UK) with 200 μL of HRP substrate (Millipore, MA, USA), and quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0153] 2.2.6 Flow cytometry analysis

[0154] Approximately 1.0x10 6 Cells were seeded in 6-well plates overnight. Cells were pretreated for 1 hour with or without EVs-SJ and various inhibitors, followed by IL-4 stimulation. Cells were then incubated with fluorescent CD206 at 37°C for 30 minutes. After washing twice with PBS and separating with trypsin / EDTA, fluorescence signals were acquired using a FACScan™ flow cytometer (BD Biosciences) via the FITC channel. At least 1 x 10⁶ cells were collected from each sample. 4 Each cell.

[0155] 2.2.7 Statistical Analysis

[0156] All experimental data are expressed as mean ± standard error (SEM) and analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Statistical significance between groups was defined as p < 0.05. Data visualization was performed using GraphPad Prism (version 9.0; GraphPad Prism Software, La Jolla, CA, USA).

[0157] 3 Results

[0158] 3.1 Histopathological observation and hydroxyproline content determination of EVs-SJ and SJ in BLM-induced pulmonary fibrosis in mice

[0159] Mice were sacrificed on day 21 after treatment with drugs and BLM. Body weight, lung tissue pathological changes, degree of pulmonary fibrosis, and pro-fibrotic cytokines were measured. Figure 2 As shown in Figure A, BLM mice exhibit a large number of tightly packed type I collagen fibers (stained red or blue). Consistently, excessive collagen fibers with large blue fibrous streaks are observed in BLM mice. Figure 2 B). Furthermore, serum levels of collagen-derived hydroxyproline and the collagen inducer TGF-β were significantly elevated in BLM mice. Figure 2 C-2D). All the above changes were alleviated after administration of EVs-SJ and SJ. Notably, EVs-SJ isolated from 300 mg / kg SJ had similar effects to 200 mg / kg SJ, indicating that EVs-SJ is only the main active ingredient of SJ and its effects are not entirely equivalent to SJ. Furthermore, mice in the BLM group were observed to have lower body weight than the control group, and both SJ and EVs-SJ could restore body weight (C-2D). Figure 2 E). All data indicate that EVs-SJ improve the pathological process of pulmonary fibrosis in a dose-dependent manner.

[0160] 3.2EVs-SJ exhibits significant anti-inflammatory capabilities.

[0161] On day 3, the extent of focal inflammatory cell infiltration and the total cell count in the bronchoalveolar lavage fluid were assessed. Numerous inflammatory macrophages accumulated around the damaged alveoli in BLM mice, such as... Figure 3 As shown in Figure A. Furthermore, in the bronchoalveolar lavage fluid of BLM mice, a series of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, as well as the total cell count, were elevated, while EVs-SJ and SJ significantly reduced their production, such as... Figure 3 As shown in B-3E, EVs-SJ and SJ have significant anti-inflammatory advantages.

[0162] 3.3 EVs-SJ regulate multiple key cells through intercellular communication

[0163] Type II alveolar cells (ATII) and macrophages play important roles in pulmonary fibrosis. For example... Figure 4 As shown in Figure A, Dil-stained EVs (red) were observed entering SFTPC-localized ATII cells (green) and CD163-localized macrophages (green), indicating that EVs have an interventional effect on both ATII cells and macrophages. ATII cells receiving EMT are thought to promote a pro-fibrotic microenvironment. Simultaneously, metabolic reprogramming-mediated macrophage M2 polarization has been shown to promote fibroblast differentiation into myofibroblasts. Therefore, we investigated the effects of EVs-SJ on these key cell types. Figure 4 In B-4C, the key EMT biomarker Vimentin and the M2 macrophage biomarker CD163 were observed to be highly expressed in the damaged lung tissue of BLM mice, while EVs-SJ and SJ significantly reduced their overexpression. This suggests that EVs-SJ exerts a protective effect by inhibiting ATII'EMT and M2 polarization.

[0164] Example 2: In vitro cellular study of the bioactivity of extracellular vesicles (EVs-SJ) derived from the formula for penetrating dampness and dispersing nodules.

[0165] 1.1 Effects of EVs-SJ on in vitro EMT

[0166] First, the optimal concentration of EVs-SJ on A549 cells was screened using a cell viability assay. For example... Figure 5 As shown in Figure A, EVs-SJ concentrations of 1, 3, and 5 μg / mL had no effect on cell viability. However, 10 μg / L EVs-SJ significantly promoted A549 cell proliferation. Therefore, 5 μg / mL EVs-SJ was used in the following experiments.

[0167] To investigate the effects of EVs-SJ on in vitro EMT, an EMT model of lung epithelial cells was established by stimulating A549 cells with TGF-β. Under TGF-β stimulation, the expression of the epithelial marker E-cadherin was significantly decreased, while the expression of the mesenchymal marker Vimentin was significantly increased. Notably, EVs-SJ pretreatment reversed these phenomena in a dose-dependent manner. Figure 5 C-5D) leads to a reduction in the EMT process. Furthermore, scratch healing assays directly demonstrate that EVs-SJ inhibit EMT-induced cell migration (C-5D). Figure 5 E). All these results indicate that EVs-SJ alleviates EMT processes in alveolar epithelium in vitro.

[0168] Mounting evidence suggests that NLRP3 is activated in alveolar epithelial cells that lead to myofibroblast development, and reactive oxygen species (ROS) are a major mediator of NLRP3 inflammasome activation. Therefore, we investigated intercellular ROS generation and inflammasome activation. Figure 6 As shown in A-6B, LPS / ATP stimulation significantly increased ROS production in A549 cells, activating the NLRP3 / Caspase-1 / IL-1β axis. EVs-SJ pretreatment significantly inhibited intracellular ROS production and suppressed the NLRP3 / Caspase-1 / IL-1β pathway. Furthermore, both the ROS scavenger NAC and the NLRP3 inhibitor MCC950 significantly reduced the expression of the EMT marker protein Vimentin. Figure 6 C) and inhibits scratch healing ( Figure 6 D). The above evidence suggests that EVs-SJ improves ROS / NLRP3 pathway-mediated alveolar epithelial EMT.

[0169] 1.2 Effect of Evs-SJ on M2 polarization

[0170] In this study, macrophages were converted to the M2 phenotype using IL-4. The effect of EVs-SJ on M2 polarization was then investigated. Figure 7 As shown in Figure A, EVs-SJ significantly blocked M2 polarization in a dose-dependent manner. The changes in extracellular acidification rate (ECAR) in response to glucose, oligomycin (OM), and 2-deoxyglucose (2-DG) injections were then examined to calculate all glycolytic parameters, including glycolysis, glycolytic capacity, and glycolytic reserve. Simultaneously, OXPHOS characteristics were calculated based on changes in oxygen consumption rate (OCR) in response to OM, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone / antimycin A (Rot / AA) injections. Figure 7As shown, IL-4 significantly improved mitochondrial function in M2 macrophages, significantly increasing basal respiration, reserve respiratory capacity, and maximal respiration, while inhibiting OXPHOS in a dose-dependent manner. EVs-SJ reversed these changes to inhibit M2 polarization (…). Figure 7 B-7C). Furthermore, EVs-SJ exhibit a significant ability to enhance glycolysis when mitochondrial ATP production is blocked by OM (B-7C). Figure 7 (D-7E). The above data indicate that EVs-SJ regulate energy metabolism to improve M2 repolarization.

[0171] In summary, through in vivo and in vitro experiments, our study demonstrates that EVs-SJ improves pulmonary fibrosis by reducing ROS / NALRP3-mediated alveolar epithelial EMT and inhibiting energy metabolism-mediated M2 cell polarization. Our research supports the potential therapeutic role of EVs-SJ in pulmonary fibrosis.

Claims

1. The use of moisture-dispersing and nodule-resolving extracellular vesicle granules in the preparation of drugs for the prevention or treatment of pulmonary fibrosis, characterized in that, The extracellular vesicle particles derived from the Shenshi Sanjie formula are obtained by extraction and separation from the Shenshi Sanjie formula, which is composed of Rhodiola rosea, turtle shell, and coix seed, with a weight ratio of 1:1:

2. The method for preparing the aforementioned moisture-absorbing and nodule-dispersing extracellular vesicle particles includes the following steps: Step S1: Preparation of the decoction of the dampness-removing and nodule-dispersing formula Rhodiola rosea, turtle shell and coix seed were chopped and mixed, and extracted with distilled water using a crude extraction system. The supernatant from the two extractions was homogenized, which is the extract of the decoction of the dampness-dispersing and nodule-reducing formula. The crude extraction system was set at 95±5℃ for the first extraction for 2-5 hours and 95±5℃ for the second extraction for 0.5-2 hours. Step S2: Isolation of extracellular vesicle granules from the permeation and dissipation mechanism The collected extract of the decoction of the Shishi Sanjie formula was centrifuged sequentially at 1000 ×g for 5-15 minutes, 2000 ×g for 15-25 minutes, 3000 ×g for 25-35 minutes, and 10000 ×g for 50-70 minutes to remove large particles and cell debris. Then, the final supernatant was centrifuged at 150000 ×g for 1.5-2.5 hours using a sequential ultracentrifugation method. The resulting particles were then gently shaken in PBS and resuspended at 4°C for at least 1 hour to obtain the extracellular vesicle particles derived from the Shishi Sanjie formula. The particle size of the infiltration and dissipation type extracellular vesicle particles is 250–300 nm.

2. The use as described in claim 1, characterized in that, In step S1, the crude extraction system is set at 95°C for the first extraction for 3 hours; the second extraction is also set at 95°C for 1 hour. In step S2, centrifuge at 1000 ×g for 10 minutes; In step S2, centrifuge at 2000 ×g for 20 minutes; In step S2, centrifuge at 3000 ×g for 30 minutes; In step S2, centrifuge at 10000 ×g for 60 minutes; In step S2, the centrifuge is a Himac Ultracenfuge CP100NX; In step S2, centrifuge at 150,000 × g for 2 h. In step S2, the obtained particles are gently shaken in 600 μL PBS; In step S2, the obtained particles are resuspended at 4°C for 1-5 hours.

3. The use as described in claim 1, characterized in that, The method for preparing the aforementioned moisture-absorbing and nodule-dispersing extracellular vesicle particles includes the following steps: S1: Preparation of the decoction of the dampness-removing and nodule-dispersing formula Rhodiola rosea, turtle shell and coix seed were chopped and mixed, and extracted with distilled water. A coarse extraction system was used, and the first extraction was carried out at 95±5℃ for 3 hours; the second extraction was carried out at 95±5℃ for 1 hour. The supernatant from the two extractions was homogenized, which is the extract of the decoction of the dampness-dispersing and nodule-reducing formula, and stored in a sealed container. S2: Separation of extracellular vesicle granules from the permeation and dissipation mechanism Extracellular vesicles were isolated from the decoction of Shenshi Sanjie Fang using differential centrifugation. The collected 23 mL extract of Shenshi Sanjie Fang decoction was centrifuged sequentially at 1000 ×g for 10 minutes, 2000 ×g for 20 minutes, 3000 ×g for 30 minutes, and 10000 ×g for 60 minutes to remove large particles and cell debris. Then, a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000 ×g for 2 h on a Himac Ultracenfuge CP100NX Ti45 rotor, and resuspending the resulting particles in 600 μL PBS at 4°C for at least 1 h to obtain the extracellular vesicle particles derived from Shenshi Sanjie Fang.

4. The use as described in claim 1, characterized in that, The drugs for preventing and treating pulmonary fibrosis are available in injection or oral formulations.

5. The use as described in claim 4, characterized in that, The drugs for preventing and treating pulmonary fibrosis also include pharmaceutically acceptable injectable or oral excipients.

Images