Preparation method of Brassica rapa L.-derived extracellular vesicle-like particles and application of Brassica rapa L.-derived extracellular vesicle-like particles in pulmonary fibrosis resistance
The use of extracellular vesicle-like particles (BREVLPs) prepared from coriander roots to treat pulmonary fibrosis has solved the problems of limited efficacy and complex and costly preparation of existing drugs, and has achieved a low-cost and effective treatment for pulmonary fibrosis.
Patent Information
- Application Number
- CN202512043157.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-02-10
AI Technical Summary
Existing drugs for treating pulmonary fibrosis have limited efficacy, significant side effects, and high costs. The preparation of existing animal-derived extracellular vesicle-like particles is complex and costly, limiting their application in the treatment of pulmonary fibrosis.
Using coriander root as raw material, extracellular vesicle-like particles (BREVLPs) were extracted through a simple preparation method and applied to a bleomycin-induced pulmonary fibrosis mouse model. The results significantly reduced the expression of related inflammatory factors and collagen, and inhibited the progression of pulmonary fibrosis.
BREVLPs significantly reduce the expression of inflammatory factors in mice with pulmonary fibrosis, reduce collagen deposition, and inhibit the progression of pulmonary fibrosis. They are low-cost and have a simple preparation process, making them suitable for large-scale production.
Smart Images
Figure CN121495826A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine and relates to a method for preparing extracellular vesicle-like particles derived from coriander root and its application in the treatment of pulmonary fibrosis. Background Technology
[0002] Pulmonary fibrosis, a chronic, progressive, and irreversible fibrotic lung disease, is the end-stage pathological manifestation of various interstitial lung diseases. Its core feature is abnormal remodeling of lung tissue, accompanied by continuous activation of fibroblasts and massive deposition of extracellular matrix, which leads to thickening of lung septa and impaired gas exchange, ultimately causing respiratory failure.
[0003] Many interstitial lung diseases can progress to pulmonary fibrosis, with idiopathic pulmonary fibrosis (IPF) being the most common subtype. IPF patients primarily present with progressive dyspnea, dry cough, and shortness of breath on exertion, and often die from respiratory failure in the terminal stage of the disease. With continuous advancements in diagnostic technology and the increasing aging of the population, the incidence and mortality rates of pulmonary fibrosis continue to rise.
[0004] The pathogenesis of interstitial pneumonia (IPF) is complex, involving interactions among various cells, including epithelial cells, macrophages, and fibroblasts. Fibroblast activation and their differentiation into myofibroblasts are key aspects of disease progression. IPF initiation is often associated with repeated epithelial cell damage; damaged epithelial cells release pro-inflammatory factors and pro-fibrotic mediators, which in turn activate fibroblasts and promote extracellular matrix deposition. Excessive extracellular matrix deposition further increases tissue stiffness, creating a vicious cycle that leads to the continued progression of IPF. Although considerable research has been conducted on the regulatory mechanisms of fibroblast activation and extracellular matrix deposition, the complex interactions involving multiple signaling pathways and cell types in IPF present significant challenges in developing drugs that can precisely intervene in key stages with minimal side effects.
[0005] Currently, only two drugs, pirfenidone (PFD) and nintedanib, are approved by the U.S. Food and Drug Administration (FDA) for the treatment of IPF. These two drugs primarily slow disease progression by inhibiting fibroblast proliferation and extracellular matrix deposition; however, their efficacy is limited, and they have significant side effects, such as gastrointestinal reactions, diarrhea, nausea, and hepatotoxicity, and are also costly. Furthermore, existing treatments can only slow disease progression and cannot reverse pulmonary fibrosis or significantly improve patients' quality of life. Therefore, developing novel treatment options that are highly effective, have fewer side effects, and are cost-effective is a pressing issue in the field of pulmonary fibrosis treatment.
[0006] Yuan root is a traditional Tibetan medicine of the plant genus Brassica in the family Brassicaceae, mainly growing in alpine regions above 3,800 meters above sea level. Yuan root is rich in bioactive components such as flavonoids, polysaccharides, glucosinolates, various mineral elements, alkaloids and organic acids. Previous studies have shown that yuan root has various biological functions such as anti-hypoxia and anti-oxidation. In addition, studies have confirmed that yuan root can alleviate intestinal injury induced by low pressure and hypoxia in mice and the protective effect on skeletal muscle homeostasis in mice induced by long-term exercise fatigue. At the same time, yuan root also has the functions of anti-fatigue and regulating immune homeostasis.
[0007] Extracellular vesicle-like particles are a type of vesicles released into the extracellular matrix by exocytosis after the multivesicular bodies formed by the invagination of the plant cell plasma membrane fuse with the cell membrane. Their diameter ranges from 40 to 160 nm and they have a typical bilayer lipid membrane structure. Extracellular vesicle-like particles are rich in bioactive substances such as DNA, RNA, lipids, and proteins, and can participate in various important physiological processes such as immune response, cell communication, and antigen presentation, and are the key medium for information exchange between cells.
[0008] Currently, studies have reported that plant extracellular vesicle-like particles show application potential in the treatment of various diseases, but the treatment research for lung diseases is relatively scarce, especially the related exploration in the field of IPF treatment is more limited. It is worth noting that in the existing studies, the sources of extracellular vesicle-like particles used for the treatment of lung diseases mostly focus on animal cells. The cultivation, production, and daily maintenance costs of such cells are relatively high, and the process methods for preparing exosomes are relatively complex. These factors have limited the further promotion and practical application of extracellular vesicle-like particles in the field of lung disease treatment to a certain extent. Based on the above research status, we speculate that extracellular vesicle-like particles derived from yuan root are expected to be a new treatment method for anti-pulmonary fibrosis. Summary of the Invention
[0009] The primary object of this application is to provide a method for preparing extracellular vesicle-like particles derived from yuan root.
[0010] The second object of this invention is to provide the application of the above-mentioned extracellular vesicle-like particles derived from yuan root in anti-pulmonary fibrosis.
[0011] This invention is achieved through the following technical solutions:
[0012] On the one hand, this invention provides a method for preparing extracellular vesicle-like particles derived from yuan root (Brassica rapa derived Extracellular Vesicle-like Particles, BREVLPs):
[0013] (1) Raw material pretreatment and homogenization preparation: After washing and grinding the roots, soak them in PBS equivalent to 1.5 times the weight of the medicinal material for 180 minutes, then put them into a homogenizer to make a homogenate, filter and let stand, and take the supernatant.
[0014] (2) Supergradient centrifugation purification: Perform gradient centrifugation with the following parameters: centrifuge at 300g for 30 minutes, centrifuge at 2000g for 30 minutes, and centrifuge at 12000g for 60 minutes. Take the supernatant and discard the precipitate.
[0015] (3) Ultra-high speed centrifugation collection: The supernatant was subjected to ultra-high speed centrifugation at 120000g for 120 minutes to collect the precipitate;
[0016] (4) Resuspension and preservation: The precipitate is obtained by re-vortexing with PBS and stored at ultra-low temperature for later use;
[0017] The morphology of the extract was observed by transmission electron microscopy, and the average particle size distribution of BREVLPs was analyzed by dynamic light scattering, which showed that the BREVLPs had a zeta potential of -5 to -15 mV.
[0018] A second aspect of this invention provides the application of *Coix lacryma-jobi*-derived extracellular vesicle-like granules (BREVLPs) in the treatment of pulmonary fibrosis. Specifically, BREVLPs demonstrate a significant therapeutic effect on bleomycin-induced pulmonary fibrosis in mice, as shown in the following ways:
[0019] (3) BREVLPs can significantly reduce the expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-β1) in the serum of mice with pulmonary fibrosis, as well as the lung coefficient and hydroxyproline expression levels, effectively slowing down the progression of pulmonary fibrosis and inhibiting lung tissue inflammation;
[0020] (4) Using muslin staining, it was shown that BREVLPs can significantly reduce collagen deposition in the lung tissue of mice with pulmonary fibrosis model;
[0021] (5) Immunohistochemical staining showed that BREVLPs could significantly reduce the expression levels of TGF-β1, fibronectin, and α-smooth muscle actin (α-SMA) in the lung tissue of mice with pulmonary fibrosis model.
[0022] The technical effects and advantages of this invention are as follows:
[0023] This invention demonstrates that BREVLPs, prepared in this invention, exhibit a significant therapeutic effect on bleomycin-induced pulmonary fibrosis in mice, specifically as follows:
[0024] (1) BREVLPs can significantly reduce the expression levels of IL-1β, IL-6, TNF-α and TGF-β1 in the serum of mice with pulmonary fibrosis model, and at the same time reduce the lung coefficient and lung hydroxyproline expression level, which can effectively slow down the process of pulmonary fibrosis and inhibit lung tissue inflammation.
[0025] (2) According to the observation of masson staining, BREVLPs can significantly reduce the collagen expression level in the lung tissue of mice with pulmonary fibrosis model;
[0026] (3) Immunohistochemical staining showed that BREVLPs could significantly reduce the expression levels of TGF-β1, Fibronectin, and α-SMA in the lung tissue of mice with pulmonary fibrosis.
[0027] (4) This invention is the first to prepare extracellular vesicle-like particles (BREVLPs) from coriander root. The preparation method is simple and convenient, and the raw material coriander root is widely available and inexpensive, making it suitable for large-scale production. It solves the problems of high cost and complex process of existing animal-derived extracellular vesicle-like particles culture. The extracellular vesicle-like particles prepared by this invention are derived from coriander root and are widely available from plants, and are inexpensive, providing a new possibility for the treatment of pulmonary fibrosis. Attached Figure Description
[0028] Figure 1 A schematic diagram of the extraction process for Brassica rapa-derived extracellular vesicle-like particles (BREVLPs). Figure 1 (A) Figure 1 The middle BD represents the characterization of BREVLPs. The morphology of BREVLPs was observed using transmission electron microscopy (TEM). Figure 1 B). Particle size distribution of BREVLPs obtained through dynamic light scattering analysis ( Figure 1 (C) and Zeta potential ( Figure 1 (D).
[0029] Figure 2 BREVLPs can alleviate the progression of pulmonary fibrosis and inflammation in a mouse model of bleomycin-induced idiopathic pulmonary fibrosis. The lung coefficient after BREVLPs treatment ( Figure 2 in A), pulmonary hydroxyplasine ( Figure 2 The expression level of IL-1β in serum and serum IL-1β (B) Figure 2C), IL-6 ( Figure 2 D), TNF-α ( Figure 2 (E), TGF-β1 ( Figure 2 (F) expression level;
[0030] Figure 3 Representative images of lung tissue collagen expression levels detected by Masson staining after BREVLPs treatment in mice with idiopathic pulmonary fibrosis (BPF) model. Figure 3 (A) and statistical charts ( Figure 3 (Middle B)
[0031] Figure 4 Immunohistochemical staining was performed on BREVLPs-treated mice with idiopathic pulmonary fibrosis to detect TGF-β1, Fibronectin, and α-SMA levels in lung tissue. Figure 4 (A) Representative images and corresponding statistical charts illustrating the level of expression ( Figure 4 (B) Detailed Implementation
[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0033] Example 1: Preparation of BREVLPs
[0034] Includes the following steps:
[0035] (1) After washing and grinding the root, soak it in PBS with 1.5 times the weight of the medicinal material for 180 minutes, then put it into a homogenizer to make a homogenate, filter and let it stand, and then take the supernatant.
[0036] (2) Centrifuge the supernatant from step (1) in a gradient: 300g for 30 minutes, 2000g for 30 minutes, and 12000g for 60 minutes. Take the supernatant and discard the precipitate.
[0037] (3) Centrifuge the supernatant from step (2) at 120000g for 120 minutes to obtain the precipitate.
[0038] (4) The precipitate from step (3) is BREVLPs by re-rotating with PBS and stored at ultra-low temperature for later use.
[0039] (5) The morphology of the extract of BREVLPs in step (4) was observed by transmission electron microscopy, and the particle size and zeta potential were analyzed by dynamic light scattering instrument.
[0040] Example 2: Application of BREVLPs in Anti-pulmonary fibrosis
[0041] Specific experimental steps:
[0042] Establishment of a mouse model of pulmonary fibrosis and experimental grouping: Six-week-old male C57BL / 6J mice were selected as experimental subjects and randomly divided into a normal control group (control), a pulmonary fibrosis model group (IPF-PBS), a BREVLPs treatment group (IPF-BREVLPs), and a pirfenidone (IPF-PFD) treatment group.
[0043] Mice in the normal control group were fed a standard diet. Mice in the pulmonary fibrosis model group were induced with bleomycin (1.5 U / kg) via endotracheal intubation. Starting from day 14 after bleomycin treatment, they were injected with saline via the tail vein daily for 7 consecutive days. The BREVLPs treatment group received 50 mg / kg BREVLPs via the tail vein daily for 7 consecutive days starting from day 14 after bleomycin treatment; the pirfenidone treatment group received 50 mg / kg pirfenidone via the tail vein daily for 7 consecutive days starting from day 14 after bleomycin treatment. All groups were euthanized on day 21. The left lung of each mouse was fixed and embedded, and lung tissue sections were prepared for Masson's staining and immunohistochemical staining. The histopathological staining steps are as follows:
[0044] 1. Tissue dehydration and embedding: Lung tissue was fixed in 4% paraformaldehyde for 24 hours and then dehydrated. After dehydration, it was embedded.
[0045] 2. Paraffin Sectioning and Drying: Preheat the tissue microtome to the appropriate temperature. Carefully install the microtome blade and group and label the slides. Freeze the paraffin at -20°C. After freezing, trim the paraffin block with a blade, leaving 1-2 mm around the lung area. Place the trimmed paraffin block on the microtome and trim it to a thickness of 20 μm until the lung outline is complete. Adjust the thickness to 5 μm for continuous sectioning. Gently place the slide into the microtome using tweezers and a brush, spreading the slide completely flat. Carefully transfer the slide to a glass slide. After sectioning, bake in an oven at 60°C for 1 hour for staining or for later use.
[0046] 3. Dewaxing and hydration: Soak the dried tissue sections in xylene I, II and III in a fume hood for 20 minutes each, then in anhydrous ethanol I and II for 15 minutes each, then in 95% anhydrous ethanol I and II for 5 minutes each, then in 80% anhydrous ethanol for 10 minutes, and finally rinse slowly with running water for 3-5 minutes.
[0047] 4. Masson staining
[0048] (1) Potassium dichromate mordant: Immerse the hydrated sections in 2.5% potassium dichromate mordant solution and incubate overnight at room temperature; the next day, transfer the sections to a 60 ℃ oven and incubate for 30 minutes. After taking them out, rinse them slowly with pure water for 60 seconds until the yellow color on the tissue surface completely fades.
[0049] (2) Cell nuclear staining: Immerse the slices rinsed in step (1) in Weigert iron hematoxylin staining solution and stain for 1 minute; after staining, rinse gently with pure water 2-3 times to remove residual staining solution on the surface of the slices.
[0050] (3) Hydrochloric acid alcohol differentiation: The sections are transferred into 1% hydrochloric acid alcohol for differentiation, and the time is controlled at 30-60 seconds. During the differentiation process, it is necessary to observe frequently under a microscope until the cell nuclei are gray-black and the tissue background is nearly colorless or light gray when observed with the naked eye. Then the differentiation is terminated immediately.
[0051] (4) Ponceau S Acid Fuchsin Staining: Gently rinse the differentiated sections with pure water, then gently blot the surface moisture along the edges of the sections with absorbent paper; then immerse the sections in the preheated Ponceau S Acid Fuchsin staining solution for 5 minutes. During the staining process, dynamic observation under a microscope is required. If the lung tissue has turned bright red, the staining can be stopped early; if the red color is light, the staining time can be extended appropriately according to the actual situation. After staining, gently rinse with pure water for about 30 seconds, observe the color of the rinsing solution, and stop rinsing when the outflowing water is colorless.
[0052] (5) Phosphomolybdic acid differentiation: Gently absorb the moisture from the surface of the slice with absorbent paper, immerse it in a preheated 1% phosphomolybdic acid solution for differentiation, and observe it under a microscope during the differentiation process until the collagen fibers change from red to light red.
[0053] (6) Aniline blue staining: Take the section out of the 1% phosphomolybdic acid solution, gently drain the surface liquid, and directly transfer it into the preheated 2.5% aniline blue solution for staining. The standard staining time is 30 seconds, and the time can be adjusted according to the staining effect observed under the microscope.
[0054] (7) Acetic acid rinsing and differentiation: The stained sections are sequentially transferred into three tanks of 1% acetic acid aqueous solution for rinsing and differentiation, each tank for about 7 seconds; when rinsing to the third tank, the staining of the sections should be observed under a microscope (to avoid over-differentiation that would cause the staining to become lighter).
[0055] (8) The sections from step (7) were placed in anhydrous ethanol for dehydration, with ethanol I for 3 seconds and ethanol II and III for 5 seconds each. After soaking in xylene for 5 minutes, the sections were cleared, then mounted with neutral resin, dried, and photographed. The results are shown below. Figure 3The results indicated that the lung tissue of mice in the pulmonary fibrosis model group (IPF+PBS group) was extensively damaged, with a large amount of collagen deposition forming dense fibrotic tissue, leading to the loss of normal lung structure. In contrast, the lung tissue damage in mice treated with BREVLPs was less severe than that in the pulmonary fibrosis model group, and the amount of collagen deposition was significantly reduced. The degree of pulmonary fibrosis in mice treated with pirfenidone was also significantly reduced compared to the pulmonary fibrosis model group.
[0056] 5. Immunohistochemical staining steps
[0057] (1) The paraffin sections were placed in xylene and ethanol in sequence to complete the dewaxing and gradient hydration. Finally, the sections were transferred to distilled water for later use.
[0058] (2) Place the slide in citrate buffer and heat to repair the antigen. After cooling to room temperature, rinse with PBS.
[0059] (3) Immerse the slides in 3% hydrogen peroxide solution to block endogenous peroxidase activity. Then rinse the slides with PBS to remove excess reagent.
[0060] (4) Add 10% goat serum to the surface of the slice and seal at room temperature for 30 minutes.
[0061] (5) Add primary antibody working solution, including anti-TGF-β1, anti-Fibronectin and anti-α-SMA antibodies, to the surface of the slide. Incubate overnight at 4°C to ensure adequate binding.
[0062] (6) The next day, the slides were brought to room temperature and washed with PBS, and then the secondary antibody working solution was added. Incubate at room temperature for 30 minutes to achieve specific binding.
[0063] (7) Add DAB colorimetric solution to the slide and observe it under a microscope. The brownish-yellow deposit is a positive signal. After the color reaches an appropriate depth, immediately rinse with distilled water to stop the reaction.
[0064] (8) Counterstain the cell nuclei with hematoxylin, and rinse with running water after staining for 30 seconds.
[0065] (9) The sections were dehydrated with ethanol and then cleared with xylene. Finally, they were mounted with neutral resin and dried for observation. The results are shown in […]. Figure 4 The results indicated that the expression of TGF-β1, Fibronectin, and α-SMA in the lung tissue of mice in the pulmonary fibrosis model group (IPF+PBS group) was significantly increased. Figure 4 (AB). The expression levels of TGF-β1, Fibronectin, and α-SMA in the lung tissue of mice treated with BREVLPs were significantly reduced, and the pulmonary fibrosis indicators in the pirfenidone-treated group were also significantly reduced compared with the pulmonary fibrosis model group.
[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing extracellular vesicle-like granules derived from *Corydalis yanhusuo* root, characterized in that, Includes the following steps: (1) Raw material pretreatment and homogenization preparation: After washing and grinding the roots, soak them in PBS equivalent to 1.5 times the weight of the medicinal material for 180 minutes, then put them into a homogenizer to make a homogenate, filter and let stand, and take the supernatant. (2) Supergradient centrifugation purification: Perform gradient centrifugation, collect the supernatant and discard the precipitate; (3) Ultra-high speed centrifugation collection: The supernatant was subjected to ultra-high speed centrifugation at 120000g for 120 minutes to collect the precipitate; (4) Resuspension and preservation: The precipitate is obtained by re-vortexing with PBS and stored at ultra-low temperature for later use.
2. The method for preparing extracellular vesicle-like granules derived from *Corydalis yanhusuo* root according to claim 1, characterized in that: In step (2), the specific parameters for gradient centrifugation are: 300g centrifugation for 30 minutes, 2000g centrifugation for 30 minutes, and 12000g centrifugation for 60 minutes.
3. The method for preparing extracellular vesicle-like granules derived from *Corydalis yanhusuo* root according to claim 1, characterized in that: The morphology of the extract was observed by transmission electron microscopy, and the average particle size distribution of BREVLPs was analyzed by dynamic light scattering, which showed that the BREVLPs had a zeta potential of -5 to -15 mV.
4. The application of the extracellular vesicle-like particles (BREVLPs) derived from coriander root obtained by the preparation method according to any one of claims 1-3 in the preparation of anti-pulmonary fibrosis drugs.
5. The application according to claim 4, characterized in that, BREVLPs can significantly reduce the expression levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-β1) in the serum of mice with pulmonary fibrosis, as well as the lung coefficient and hydroxyproline expression levels, effectively slowing down the progression of pulmonary fibrosis and inhibiting lung tissue inflammation.
6. The application according to claim 4, characterized in that, Masson staining showed that BREVLPs significantly reduced collagen deposition in the lung tissue of mice with pulmonary fibrosis.
7. The application according to claim 4, characterized in that, Immunohistochemical staining showed that BREVLPs could significantly reduce the expression levels of TGF-β1, fibronectin, and alpha-smooth muscle actin (α-SMA) in the lung tissue of mice with pulmonary fibrosis.