Application of lycium barbarum-derived extracellular vesicles in preparation of medicine for treating acute kidney injury

Abstract

The invention relates to the technical field of medicines, in particular to application of lycium barbarum-derived extracellular vesicles in preparation of a medicine for treating acute kidney injury. The used extracellular vesicles derived from Chinese wolfberry fruits have a lipid bilayer membrane structure, are good in stability, free of remarkable toxicity, good in blood compatibility and high in-vivo safety after long-term intravenous injection, are used for preparing the medicine for treating the acute kidney injury, and can reduce apoptosis, inhibit rising of blood urea nitrogen and creatinine levels and relieve injury of kidney tissues; the invention further provides application of the extracellular vesicles derived from the Chinese wolfberry fruits in preparation of the medicine for treating the lung injury disease, the weight loss is relieved, lung tissue is protected, meanwhile, the pulmonary alveolar-capillary barrier can be repaired, and in addition, the medicine can be used for treating the lung injury disease. The invention also provides application of the extracellular vesicles derived from Chinese wolfberry fruits in preparation of nuclear transcription factor kappa B inhibitors and / or inhibition of apoptosis and / or repair of pulmonary alveolar capillary barriers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the application of extracellular vesicles from Lycium barbarum in the preparation of a medicament for treating acute kidney injury. BACKGROUND

[0002] Lycium barbarum is a traditional and precious Chinese medicinal material and is listed as a "medicinal and edible" variety. It is recorded in Compendium of Materia Medica that it has the effects of "tonifying liver and kidney, moistening lung and generating essence, and benefiting qi". Modern research has confirmed that Lycium barbarum contains rich active ingredients such as Lycium barbarum polysaccharide, amino acids, vitamins, flavonoids, and terpenes, and has the effects of antioxidant, anti-inflammatory, liver protection, immune enhancement, and promotion of tissue repair. Current researches are mostly focused on Lycium barbarum polysaccharide and decoction extract, but they have problems such as low bioavailability and unclear mechanism of action.

[0003] Extracellular vesicles are membrane structures secreted by cells and wrapped by lipid bilayers, with a diameter of about 30-1000 nm, containing active substances such as nucleic acids (DNA, RNA, miRNA), proteins, lipids, and secondary metabolites, and can mediate cell-to-cell communication and participate in various physiological and pathological processes. In recent years, plant-derived extracellular vesicles have attracted widespread attention. Compared with animal-derived extracellular vesicles, plant-derived extracellular vesicles have the characteristics of wide source, easy separation, high yield, and good biocompatibility. Due to the rich active ingredients unique to plants, plant-derived extracellular vesicles can exhibit biological activities comparable to or even better than plants. However, the application of extracellular vesicles from Lycium barbarum in the treatment of acute kidney injury, lung injury, and nuclear transcription factor kappa B inhibitors has not been reported. SUMMARY

[0004] In view of this, the technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide the application of extracellular vesicles from Lycium barbarum in the preparation of a medicament for treating acute kidney injury.

[0005] Another technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide the application of extracellular vesicles from Lycium barbarum in the preparation of a medicament for treating lung injury.

[0006] A third technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide the application of extracellular vesicles from Lycium barbarum in the preparation of a nuclear transcription factor kappa B inhibitor.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides the application of extracellular vesicles from Lycium barbarum in the preparation of a medicament for treating acute kidney injury.

[0009] Further, the acute kidney injury is acute kidney injury induced by rhabdomyolysis.

[0010] Further, the drug can reduce blood urea nitrogen and / or reduce creatinine levels.

[0011] In a second aspect, the application provides a use of the extracellular vesicle derived from wolfberry in the preparation of a drug for treating a lung injury disease.

[0012] Further, the lung injury is at least one of acute respiratory distress syndrome and chronic obstructive pulmonary disease.

[0013] Further, the drug can break the vicious cycle of inflammation-oxidative stress-alveolar capillary barrier destruction, and play the functions of anti-inflammation, anti-oxidation and repairing alveolar capillary barrier.

[0014] In a third aspect, the application provides a use of the extracellular vesicle derived from wolfberry in the preparation of a drug as a nuclear transcription factor kappa B inhibitor and / or for repairing alveolar capillary barrier and / or for inhibiting cell apoptosis.

[0015] Further, the preparation method of the extracellular vesicle derived from wolfberry comprises the following steps:

[0016] S1 step: taking wolfberry, soaking, breaking the wall and homogenizing to obtain a homogenate;

[0017] S2 step: centrifuging and filtering the homogenate;

[0018] S3 step: taking the liquid and centrifuging again, collecting the precipitate, resuspending the precipitate in a solvent, and obtaining the extracellular vesicle derived from wolfberry after centrifuging, resuspending again and filtering.

[0019] Further, the S1-S3 steps are all carried out at 2-4℃.

[0020] Further, the wolfberry in the S1 step includes dry wolfberry and fresh wolfberry.

[0021] In some optional embodiments, the S1 step is cleaned before soaking, and the cleaning liquid is first ultrapure water, and the last cleaning is replaced with PBS buffer.

[0022] In some optional embodiments, the breaking wall operation in the S1 step is short-term multiple breaking by a breaking wall machine, such as breaking for 3 minutes for 5 times, with an interval of 5 minutes, so as to reduce the possibility of degradation caused by instrument heating during breaking.

[0023] Further, the centrifugation in the S2 step comprises a first centrifugation, a second centrifugation and a third centrifugation, the centrifugal force of the first centrifugation is 500-2000xg, preferably 1000xg, the time of the first centrifugation is 8-12min, preferably 10min; the centrifugal force of the second centrifugation is 2000-5000xg, preferably 3000xg, the time of the second centrifugation is 20-40min, preferably 30min; the centrifugal force of the third centrifugation is 7000-12000xg, preferably 10000xg; the time of the third centrifugation is 0.5h-1.5h, preferably 1h.

[0024] Further, the filtering operation in the S2 step is gauze filtering or microporous sterilization filter membrane filtering.

[0025] In some optional embodiments, after the first centrifugation, the residues are filtered out with gauze, and after the third centrifugation, the first filtering is performed with a water-based microporous sterilization filter membrane with a pore size of 0.45μm, and the second filtering is performed with a water-based microporous sterilization filter membrane with a pore size of 0.22μm.

[0026] Further, the centrifugation in the S3 step has a centrifugal force of 100000-160000xg, preferably 150000xg, and a time of 1-3h, preferably 1.5h.

[0027] Further, the solvent used for resuspension and re-resuspension in the S3 step is a buffer solution or a liquid medium.

[0028] In some optional embodiments, the buffer solution is a PBS buffer solution.

[0029] In some optional embodiments, the PBS buffer solution has a pH value of 6.5-7.5, preferably 7.

[0030] In some optional embodiments, the liquid medium is at least one of DMEM liquid medium, MEM liquid medium, RPMI 1640 liquid medium, F12 liquid medium or IMDM liquid medium.

[0031] In some optional embodiments, the re-resuspension operation in the S3 step can be directly performed after centrifugation, or the precipitate after centrifugation can be added to the solvent and refrigerated at 4℃ overnight, and the next day the precipitate is resuspended by blowing.

[0032] In some optional embodiments, after the extraction of the cell extracellular vesicles from the wolfberry is successful, the cell extracellular vesicles can be subpackaged and stored at -80℃ for a long time.

[0033] Further, the particle size of the cell extracellular vesicles from the wolfberry is 100-230nm.

[0034] The protein concentration of the wolfberry-derived extracellular vesicle is 0.01-1000 μg / mL.

[0035] Further, the dosage form is a tablet, a capsule, a granule, an oral solution, an injection solution, a sustained-release preparation, a controlled-release preparation or a nano-preparation.

[0036] The technical scheme of the present application has the following advantages:

[0037] The wolfberry-derived extracellular vesicle provided by the present application has a lipid bilayer membrane structure, good stability, no significant toxicity to cells, good blood compatibility, high safety in long-term intravenous injection in vivo. In addition, it can reduce cell apoptosis and play a protective role. In acute kidney injury mice caused by rhabdomyolysis, it can inhibit the increase of blood urea nitrogen and creatinine levels caused by kidney injury, and reduce the damage to the kidney tissue.

[0038] The wolfberry-derived extracellular vesicle provided by the present application is used for preparing a drug for treating lung injury diseases. Studies have shown that in mice with acute respiratory distress syndrome induced by lipopolysaccharide (LPS), the wolfberry-derived extracellular vesicle can reduce body weight loss, protect lung tissue, improve pathological characteristics such as alveolar collapse and inflammatory infiltration, reduce lung injury score and pulmonary edema, and also effectively repair alveolar-capillary barrier, reduce lung oxidative stress and inhibit cell apoptosis, thereby playing a therapeutic role. At the same time, in a mouse model of chronic obstructive pulmonary disease, the wolfberry-derived extracellular vesicle not only can improve the overall health status of the mouse, but also can significantly improve its respiratory function, indicating that the wolfberry-derived extracellular vesicle has a therapeutic effect on chronic obstructive pulmonary disease.

[0039] The wolfberry-derived extracellular vesicle provided by the present application is used for preparing a nuclear transcription factor kappa B inhibitor. Studies have shown that LPS activates the nuclear transcription factor kappa B (NF-κB) pathway, and the use of wolfberry-derived extracellular vesicle treatment can effectively inhibit the activation of the LPS-induced NF-κB pathway, and is used for the preparation of NF-κB inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0041] Figure 1 FIG. 1 is a transmission electron microscope image of the wolfberry-derived extracellular vesicle in Experimental Example 1.

[0042] Figure 2 is a dynamic light scattering result chart of the extracellular vesicles from wolfberry in experimental example 1, the vertical coordinate Intensity is translated as intensity, and the horizontal coordinate Size is translated as particle size;

[0043] Figure 3 is a 5-day stability of the extracellular vesicles from wolfberry in experimental example 1 in phosphate buffer and lung lavage fluid, the vertical coordinate Size is translated as particle size, and the horizontal coordinate Time is translated as time;

[0044] Figure 4 is a result chart of MTT detection of the cell compatibility of the extracellular vesicles from wolfberry in experimental example 2, the vertical coordinate Cell viability is translated as cell viability;

[0045] Figure 5 is a live / dead cell staining chart of the extracellular vesicles from wolfberry after being co-cultured with human umbilical vein endothelial cells (HUVEC) for 24 hours in experimental example 2, Calcein AM is calcium calcein acetoxy methyl ester, PI is propidium iodide, and Merge means merging layers;

[0046] Figure 6 is a result chart of the blood compatibility of the extracellular vesicles from wolfberry in experimental example 3, the vertical coordinate Hemolysis ratio is translated as hemolysis rate;

[0047] Figure 7 is a chart of the apoptosis of human umbilical vein endothelial cells (HUVEC) after different treatments in experimental example 4, Merge means merging layers, AO is acridine orange, and EB is ethidium bromide;

[0048] Figure 8 is a chart of the apoptosis of mouse alveolar epithelial cells (MLE-12) after different treatments in experimental example 4, 7-AAD is 7-aminoactinomycin D, Annexin V is a calcium-dependent phospholipid binding protein, and Annexin V-APC is a fluorescent probe complex formed by the combination of Annexin V protein and APC fluorescent dye;

[0049] Figure 9 is a fluorescence image of tight junction protein (ZO-1) in mouse alveolar epithelial cells (MLE-12) after different treatments in experimental example 5;

[0050] Figure 10 is a Western blot chart of the expression of tight junction protein (ZO-1) in mouse alveolar epithelial cells (MLE-12) after different treatments in experimental example 5, Fold Change is translated as fold change;

[0051] Figure 11These are Western blot images of different proteins expressed in primary bone marrow macrophages (BMDM) of mice after different treatments in Experiment Example 6.

[0052] Figure 12 This is a quantitative heatmap of different protein phosphorylation ratios in primary bone marrow macrophages (BMDM) of mice after different treatments in Experiment Example 6;

[0053] Figure 13 This refers to the weight changes of mice in each group in Experiment Example 7. "Change of the mouse weight" is translated as "change of mouse weight," and "Day" is translated as "number of days."

[0054] Figure 14 These are HE-stained images of the lung tissues of mice in each group in Experiment Example 7;

[0055] Figure 15 These are the pathological scores (Score) based on HE staining for each group in Experiment Example 7;

[0056] Figure 16 These are the lung indices for each group in Experiment Example 7;

[0057] Figure 17 This is the principal component analysis (PCA) diagram of mRNA expression in lung tissue of mice with acute respiratory distress syndrome treated with extracellular vesicles of wolfberry without wolfberry in Experiment Example 7.

[0058] Figure 18 This is an immunofluorescence image showing the distribution of tight junction protein (ZO-1) and E-cadherin in lung tissue in each group of Experiment Example 7;

[0059] Figure 19 These are the immunoblot images of tight junction protein (ZO-1) in lung tissue from each group in Experiment Example 7. FoldChange is translated as fold change.

[0060] Figure 20 This is a graph showing the protein concentration results in the bronchoalveolar lavage fluid of each group in Experiment Example 7.

[0061] Figure 21 This is a graph showing the cell count results in the bronchoalveolar lavage fluid of each group in Experiment Example 7;

[0062] Figure 22 These are immunofluorescence images of apoptosis detection in the lung tissues of mice in each group of Experiment Example 7;

[0063] Figure 23Figure 7 is a graph showing the results of the immunofluorescence semi-quantitative analysis of the apoptosis detection of the lung tissue of the mice in each group in Example 7, Integrated density is the cumulative fluorescence intensity, and Cell number is the cell count;

[0064] Figure 24 Figure 8 is a graph showing the results of the blood biochemical indicators urea nitrogen (Bun) and (Cre) of the mice in each group in Example 8;

[0065] Figure 25 Figure 9 is a graph showing the HE staining images of the kidney tissue of the mice in each group in Example 8;

[0066] Figure 26 Figure 10 is a graph showing the weight (Weight) changes of the mice in each group in Example 9;

[0067] Figure 27 Figure 11 is a graph showing the low-density lipoprotein cholesterol (LDL-C) of the blood of the mice in each group in Example 9;

[0068] Figure 28 Figure 12 is a graph showing the semi-quantitative results of the cross-sectional area (Cross-sectional Area) of the gastrocnemius muscle of the mice in each group in Example 9;

[0069] Figure 29 Figure 13 is a graph showing the HE staining images of the humerus, gastrocnemius muscle, liver, and spleen of the mice in each group in Example 9;

[0070] Figure 30 Figure 14 is a graph showing the analysis of the trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), bone surface tissue volume ratio (BV / TV), and trabecular bone separation degree (Tb.Sp) of the humerus of the mice in each group in Example 9;

[0071] Figure 31 Figure 15 is a graph showing the weight (Weight) changes of the mice in each group in Example 10;

[0072] Figure 32 Figure 16 is a graph showing the results of the lung function determination indicators of the mice in each group in Example 10, PEF is the peak expiratory flow rate, and Penh is the airway resistance value. DETAILED DESCRIPTION

[0073] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application or the content of the protection, and do not constitute a limitation on the content and scope of protection of the application, and any person who obtains any product identical or similar to the application under the inspiration of the application or combines the application with other prior art features falls within the protection scope of the application.

[0074] The specific experimental procedures or conditions are not specified in the examples, which can be carried out according to the conventional experimental procedures described in the literature or the operating conditions. The reagents or instruments used are not specified by the manufacturer, which are conventional reagent products that can be obtained by purchase.

[0075] Example 1

[0076] This example provides the preparation of extracellular vesicles (LBNVs) from wolfberry. The preparation process is as follows:

[0077] (1) Washing and soaking step: after washing the dry wolfberry surface of the dust, the wolfberry is soaked in PBS buffer (pH 7.4) for 10 minutes;

[0078] (2) Breaking wall homogenate step: using a breaking wall machine to break 5 times, 3 minutes each time, and 5 minutes interval for the next time;

[0079] (3) Impurity removal step: the homogenate liquid is centrifuged at 1000xg for 10 minutes, then the supernatant is collected by filtering with gauze; the supernatant is centrifuged at 3000xg for 30 min; the supernatant is centrifuged at 10000xg for 1h; the supernatant is filtered with 0.45μm and 0.22μm filter membranes;

[0080] (4) Extracellular vesicle preparation step: then the obtained supernatant is ultracentrifuged at 150000xg for 1.5h, the precipitate is collected, 13mL PBS buffer (pH 7.4) is added to resuspend the precipitate; it is ultracentrifuged at 150000xg for 1.5h again, the solvent is removed, the precipitate is obtained, 1mL PBS buffer (pH 7.4) is added and left overnight; the next day, the precipitate is resuspended by blowing, then the liquid filtered with 0.22μm filter membrane is obtained as the wolfberry-derived extracellular vesicle solution.

[0081] The protein concentration of the wolfberry-derived extracellular vesicles was detected using the BCA protein concentration determination kit (purchased from Biyun Tian Biotechnology Co., Ltd., product number P0011) according to the instructions. The initial protein concentration of the wolfberry-derived extracellular vesicles prepared in Example 1 was 187μg / mL.

[0082] Experimental Example 1 Characterization of Wolfberry-Derived Extracellular Vesicles

[0083] 1. Transmission electron microscopy (TEM) observation of the morphology of wolfberry-derived extracellular vesicles

[0084] (1) Experimental method

[0085] The extracellular vesicle solution of Example 1 was diluted with PBS buffer to a protein concentration of 5 pg / mL (determined by BCA method), mixed well; the TEM copper mesh was taken out with tweezers; 10 pL of the diluted extracellular vesicles was taken up with the gun head, 10 pL of the extracellular vesicles was dropped on the copper mesh, and stood for 15 minutes; the liquid beads on the copper mesh were gently absorbed with filter paper, and then the copper mesh was placed in a 37°C oven to dry overnight; the copper mesh was placed in a 120 kV transmission electron microscope to observe the extracellular vesicles from wolfberry.

[0086] (2) Experimental results

[0087] The morphology of the extracellular vesicles from wolfberry observed by TEM is shown in Figure 1 , which shows that the extracellular vesicles from wolfberry have a cup-shaped lipid bilayer membrane structure.

[0088] 2. Particle size of extracellular vesicles from wolfberry detected by dynamic light scattering (DLS)

[0089] (1) Experimental method

[0090] The extracellular vesicle solution of Example 1 was diluted with PBS buffer to a protein concentration of 2 ng / mL (determined by BCA method), mixed well; the diluted solution was loaded into a cuvette; the particle size distribution of the extracellular vesicles from wolfberry was detected by dynamic light scattering (DLS).

[0091] (2) Experimental results

[0092] The particle size distribution of the extracellular vesicles from wolfberry observed by DLS is shown in Figure 2 , which shows that the size of the extracellular vesicles from wolfberry is 100-230 nm.

[0093] 3. Stability evaluation of extracellular vesicles from wolfberry

[0094] (1) Experimental method

[0095] The prerequisite for the therapeutic effect of extracellular vesicles from wolfberry is their stability in the storage solvent and the target microenvironment. In order to evaluate its stability, the extracellular vesicle solution of Example 1 was diluted with bronchoalveolar lavage fluid and PBS buffer to a protein concentration of 2 ng / mL (determined by BCA method) respectively, mixed well, and placed at 37°C for 5 days. Starting from the first day, the particle size of the extracellular vesicles from wolfberry in the bronchoalveolar lavage fluid and the PBS buffer was tested daily.

[0096] (2) Experimental results

[0097] The extracellular vesicles from wolfberry were incubated in phosphate buffered saline (PBS) and bronchoalveolar lavage fluid (BALF) at 37°C for 5 days to simulate the in vivo alveolar environment, as shown inFigure 3 As shown, the sustained DLS monitoring confirmed their sustained stability in both physiological and pathological lung environments.

[0098] Experimental Example 2: Detection of cell compatibility of Gouqi-derived extracellular vesicles

[0099] 1. Experimental method

[0100] MTT method: 1) Human umbilical vein endothelial cells (HUVEC) were seeded in a 96-well plate at a concentration of 2000 cells / well, 100 μL of DMEM medium containing 10% serum was added to each well, and randomly divided into experimental groups (5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL) and control groups (Blank); 2) After overnight culture, the Gouqi-derived extracellular vesicle solution of Example 1 was diluted with DMEM medium containing 10% (v / v) serum to a protein concentration of 5 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL, respectively, and mixed well; 3) Remove the old culture medium in all wells, add 100 μL of DMEM medium containing 10% (v / v) serum to the control group, and add 100 μL of 5 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL Gouqi-derived extracellular vesicle solution to the experimental groups, respectively; 4) After 24 hours of incubation, remove the culture medium, rinse once with 100 μL of PBS buffer, and then add 100 μL of 10% MTT-containing culture solution; 5) After 3 hours of incubation, remove the MTT culture solution, add 100 μL of DMSO to each well, and shake well; 6) Detect the 490 nm absorbance value with a microplate reader.

[0101] MTT experimental method for MLE-12 cells is the same as above.

[0102] Live / dead cell staining experiment: 1) Human umbilical vein endothelial cells (HUVEC) were seeded in 6-well plates at a concentration of 100,000 cells per well in 2 mL of DMEM medium containing 10% serum, and randomly divided into experimental groups (5 pg / mL, 10 pg / mL, 25 pg / mL, 50 pg / mL) and a control group (CON); 2) After overnight culture, the Gynostemma pentaphyllum-derived extracellular vesicle solution of Example 1 was diluted to a protein concentration of 5 pg / mL, 10 pg / mL, 25 pg / mL, and 50 pg / mL, respectively, with DMEM medium containing 10% (v / v) serum, and mixed well; 3) The old culture medium in all wells was removed, and 2 mL of DMEM medium containing 10% (v / v) serum was added to the control group, and 100 pL of Gynostemma pentaphyllum-derived extracellular vesicle solution at a concentration of 5 pg / mL, 10 pg / mL, 25 pg / mL, and 50 pg / mL was added to the experimental groups, respectively; 4) After incubation for 24 hours, the culture medium was removed, and the cells were rinsed once with 2 mL of PBS buffer, and then 2 mL of culture solution containing 0.1% (v / v) Calcein AM and 0.1% (v / v) PI was added; 5) After incubation at 37°C in the dark for 15 minutes, the staining solution was removed, and 2 mL of PBS was added to each well; 6) The cells were observed using a fluorescence microscope, and live cells emitted green fluorescence, while dead cells emitted red fluorescence.

[0103] 2. Experimental results

[0104] The experimental results of MTT and live / dead cell staining are shown in Figure 4 , 5 It can be seen that the Gynostemma pentaphyllum-derived extracellular vesicles have good biocompatibility. Good biocompatibility is a prerequisite for extracellular particle-based therapeutic methods to be used in clinical practice, which can avoid secondary damage to damaged organs. The alveolar capillary barrier includes alveolar epithelium and capillary endothelium, and the present application selected mouse capillary alveolar epithelial cells (MLE-12 cells) and human umbilical vein endothelial cells (HUVEC) to evaluate the cell compatibility of Gynostemma pentaphyllum-derived extracellular vesicles. After HUVEC and MLE-12 cells were exposed to different concentrations of Gynostemma pentaphyllum-derived extracellular vesicles for 24 hours, MTT assay showed that HUVEC had high cell survival ability, and MLE-12 cells had a certain proliferative effect. Live / dead cell staining further confirmed these findings, with live cells (green fluorescence) predominating, indicating that Gynostemma pentaphyllum-derived extracellular vesicles have good biocompatibility.

[0105] Example 3: Hemolysis experiment to detect the blood compatibility of Gynostemma pentaphyllum-derived extracellular vesicles

[0106] 1. Experimental method

[0107] Take 2 mL of C57BL / 6J mouse blood, add 10 mL of PBS buffer containing 1 x 104 units of heparin sodium, centrifuge at 1000 rpm for 10 min, and separate the red blood cells. After washing twice with 2 mL of PBS buffer, a diluted 4% blood cell solution is obtained.

[0108] The Gynostemma pentaphyllum-derived extracellular vesicle solution of Example 1 was diluted with PBS buffer to a protein concentration of 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The diluted Gynostemma pentaphyllum-derived extracellular vesicle solution and the diluted blood cells were added to a test tube in a volume ratio of 1:1, so that the final solution had a Gynostemma pentaphyllum-derived extracellular vesicle protein concentration of 5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. The test tube was incubated in a 37°C constant temperature incubator for 3 h. Water was used as a positive control group (H2O) instead of the diluted Gynostemma pentaphyllum-derived extracellular vesicle solution and the diluted blood cells, and the same operation was performed. PBS buffer was used as a negative control group (CON) instead of the diluted Gynostemma pentaphyllum-derived extracellular vesicle solution and the diluted blood cells, and the same operation was performed. Finally, all sample tubes were centrifuged at 1000 rpm for 15 min, and 200 μL of supernatant was added to a 96-well plate. The absorbance was measured at 540 nm using a microplate reader.

[0109] 2、Experimental results

[0110] To test the blood compatibility, Gynostemma pentaphyllum-derived extracellular vesicles were incubated with mouse red blood cells. As shown in Figure 6 there was no significant difference in the color of the supernatant absorbance value in the Gynostemma pentaphyllum-derived extracellular vesicle-treated sample and the PBS control group. Given the recognized biocompatibility of PBS buffer, this indicates that Gynostemma pentaphyllum-derived extracellular vesicles are unlikely to cause significant damage to red blood cells during systemic circulation, and therefore Gynostemma pentaphyllum-derived extracellular vesicles have excellent blood compatibility in vitro.

[0111] Example 4: Detection of the protective effect of Gynostemma pentaphyllum-derived extracellular vesicles on apoptosis

[0112] 1、Experimental method

[0113] Human umbilical vein endothelial cells (HUVEC) and mouse alveolar epithelial cells (MLE-12) were used for the apoptosis experiment.

[0114] Preparation method of Infla-Medium: RAW cells were inoculated in 6-well plates at a concentration of 3 million per well; after overnight incubation, the old culture medium was removed, 2 mL of DMEM culture medium containing 1 μg / mL LPS was added for continued culture at 37°C for 2 hours, then the DMEM culture medium was removed and 2 mL of DMEM culture medium was added, and the culture medium was collected after 24 hours of culture, centrifuged at 2000 rpm, and filtered with a 0.22 μm filter to obtain the inflammatory culture medium.

[0115] Cell culture: 1) The Gynostemma pentaphyllum-derived extracellular vesicles of Example 1 were diluted to 1 μg / mL with DMEM culture medium containing 10% (v / v) serum, and mixed well; 2) The cells (HUVEC and MLE-12) were each inoculated in 6-well plates at a concentration of 100,000 per well; and randomly divided into an experimental group (Infla-Medium + LBNVs), a control group (CON), and a model group (Infla-Medium + PBS); 3) After overnight culture, the culture medium of the control group was replaced with 2 mL of DMEM containing 10% (v / v) serum, the culture medium of the model group was replaced with 2 mL of inflammatory culture medium containing 10% (v / v) serum, and the culture medium of the experimental group was replaced with 2 mL of inflammatory culture medium containing 10% serum and 1 μg / mL of Gynostemma pentaphyllum-derived extracellular vesicles, and the cells were collected after 24 hours of incubation.

[0116] Detection method: The MLE-12 cells were stained according to the apoptosis kit (purchased from Kaygen Biotech Co., Ltd., product code KGA1025), and detected using a flow cytometer; the HUVEC cells were stained using AO / EB (acridine orange / ethidium bromide) reagent (purchased from Shanghai McLean Biochemical Technology Co., Ltd.), and the cultured cells of each group were each replaced with 2 mL of DMEM containing 0.1 mg / mL of AO and EB for incubation for 10 min, then washed twice with DMEM, and then added with 2 mL of DMEM, and detected using an inverted fluorescence microscope.

[0117] 2、Experimental results

[0118] The protective effect of Gynostemma pentaphyllum-derived extracellular vesicles on endothelial cell apoptosis caused by LPS is shown in Table 1, and it can be seen that the inflammatory culture medium can cause cell apoptosis, and the Gynostemma pentaphyllum-derived extracellular vesicles can reduce cell apoptosis, thereby playing a protective role on cells. Figure 7 、 8

[0119] Experimental Example 5: Repairing effect of Gynostemma pentaphyllum-derived extracellular vesicles on alveolar-capillary barrier

[0120] 1、Experimental method

[0121] ​Cell culture: 1) Dilute the extracellular vesicles derived from Lycium barbarum in Example 1 to 1 μg / mL and 5 μg / mL respectively with MLE-12 special medium, and mix well; 2) Seed MLE-12 cells at a concentration of 200,000 / well in 6-well plates, and randomly divide them into control group ( Figure 9 For CON, Figure 10 PBS), model group (LPS), low-dose experimental group ( Figure 9 For LBNVs+LPS, Figure 10 The experimental groups were divided into two groups: a low-dose group (LPS + 1 μg / mL) and a high-dose group (LPS + 5 μg / mL); 3) Remove the old culture medium, add 2 mL of MLE-12 special culture medium to both the model group and the control group, add 2 mL of extracellular vesicle solution of Lycium barbarum with a protein concentration of 1 μg / mL to the low-dose experimental group, and add 2 mL of extracellular vesicle solution of Lycium barbarum with a protein concentration of 5 μg / mL to the high-dose experimental group; 4) After 12 hours, the culture medium of the control group was replaced with DMEM inflammatory culture medium containing 10% (v / v) serum, the culture medium of the model group was replaced with 2 mL of inflammatory culture medium, the culture medium of the low-dose experimental group was replaced with 2 mL of extracellular vesicle inflammatory culture medium of Lycium barbarum with a protein concentration of 1 μg / mL, and the culture medium of the high-dose experimental group was replaced with 2 mL of extracellular vesicle inflammatory culture medium of Lycium barbarum with a protein concentration of 1 μg / mL. After incubation for 24 hours, the cells were collected.

[0122] DAPI staining: 1) After cell treatment, rinse twice with PBS buffer to remove residual culture medium; 2) Add 4% paraformaldehyde and incubate at room temperature for 15 min, followed by rinsing three times with PBS buffer for 5 min each time; 3) Dilute with PBS buffer to obtain 0.5% Titon X-100, incubate at room temperature for 15 min, followed by rinsing three times with PBS buffer for 5 min each time; 4) Use PBST (containing Tween) to stain the cells. 1) Prepare 3% BSA (bovine serum albumin) with 20% phosphate buffer, block at room temperature for 2 hours, then wash three times with PBST for 5 minutes each time; 2) Prepare 3% BSA with PBST, add ZO-1 primary antibody (purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number P39447) at a ratio of 1:1000, incubate overnight at 4°C, then wash three times with PBST for 5 minutes each time; 3) Prepare 1% BSA with PBST, add fluorescently labeled secondary antibody at a ratio of 1:200, incubate at room temperature in the dark for 90 minutes, then wash three times with PBST for 5 minutes each time; 4) Dilute DAPI dye 1000 times with PBS buffer, incubate for 10 minutes to stain nuclei, then wash with PBS buffer, add 1 mL of PBS buffer, and take pictures using a confocal microscope.

[0123] Western blot (WB):

[0124] 1) Extraction of protein: In accordance with the above cell culture method, the cells were taken out of the incubator, washed with PBS for 3 times, 100 μL cell lysis solution, 1 μL protease inhibitor and 1 μL protein phosphatase inhibitor were added to each well, lysed on ice for 30 min, scraped down with a cell scraper and transferred to a 1.5 mL EP tube and stored in a -80°C refrigerator;

[0125] 2) Protein denaturation: The protein was taken out of the -80°C refrigerator, dissolved on ice, centrifuged at 12000 rpm for 30 min at 4°C in a high-speed refrigerated centrifuge, the precipitate was discarded, and the supernatant was carefully transferred to a new EP tube, 5x SDS loading buffer was added at a ratio of 4:1, mixed well, sealed with sealing film, and placed in a 98°C metal bath for 5 min;

[0126] 3) Gel preparation, loading and electrophoresis: a glass plate was prepared, washed and dried, installed after removing impurities, prepared the separation gel and added to the glass plate, added anhydrous ethanol to flatten, after complete solidification, the pure water was poured out, 8% concentrated gel was added, immediately inserted into a 10-hole comb, after complete solidification and molding, it was placed into an electrophoresis tank, and the electrophoresis solution was prepared in advance; 20 μL of protein was loaded into each well, the voltage was adjusted to 90 V, after the marker band was pulled apart, the voltage was changed to 120 V, and the electrophoresis was stopped after the marker band was fully separated;

[0127] 4) Transfer: according to the size of the gel, a PVDF membrane of the corresponding size was cut, after the electrophoresis was almost completed, the PVDF membrane was soaked in methanol for 1 min, then transferred to the transfer buffer; after the protein was fully separated, the electrophoresis was completed, the transfer device was installed in the transfer buffer, the transfer clamp was firmly clamped, placed into the electric transfer tank, connected the positive and negative electrodes, then the transfer device was placed in ice water, surrounded by multiple ice bags to ensure constant temperature during the transfer process, the voltage was 250 mA, and the transfer time was 90 min;

[0128] 5) Blocking: the device was opened, the side of the PVDF membrane in contact with the electrophoresis gel was observed as the positive side, marked with a ballpoint pen, and the membrane was carefully taken out with tweezers and placed in the prepared blocking solution for room temperature blocking;

[0129] 6) Immune response: the positions of the target protein and the internal reference were determined according to the molecular weight of the marker, the required PVDF membrane was cut out at the corresponding position, marked, and placed in the required primary antibody (purchased from Wuhan Sivier Biological Technology Co., Ltd., product number P39447) for 4°C overnight; the next day, the membrane was washed with 1x TBST for 3 times, each for 10 min. According to the source of the primary antibody, the anti-rabbit secondary antibody diluted with blocking solution at a ratio of 1:3000 was added, and incubated at room temperature for about 1 hour, and the PVDF membrane was washed for 3 times, each for 10 min;

[0130] 7) Chemiluminescence test: After the PVDF membrane is slightly absorbed with filter paper, it is placed in a developing instrument, an appropriate amount of chemiluminescence reagent is uniformly added and completely covers the membrane, and an image is collected for analysis.

[0131] 2. Experimental results

[0132] The alveolar capillary barrier is composed of epithelial tight junctions and endothelial adhesion complexes, which are susceptible to oxidative and inflammatory damage. Therefore, the ability of Gynostemma pentaphyllum extracellular vesicles to maintain barrier integrity was evaluated by examining the tight junction protein ZO-1 (Zonula occludens-1), which is a peripheral membrane protein that anchors tight junction transmembrane proteins to the actin cytoskeleton and regulates cell permeability. Figure 9 Immunofluorescence confirmed LPS-induced ZO-1 fragmentation and degradation, while Gynostemma pentaphyllum extracellular vesicles maintained continuous junctional localization. Western blot experiments, as shown in Figure 10 Figure 6, showed that LPS significantly reduced ZO-1 expression to 78% of the control level in MLE-12 cells, and ZO-1 expression could be completely restored by Gynostemma pentaphyllum extracellular vesicle treatment, thereby maintaining the integrity of the alveolar-capillary barrier.

[0133] Example 6 Inhibitory effect of Gynostemma pentaphyllum-derived extracellular vesicles on nuclear transcription factor κB (NF-κB)

[0134] 1. Experimental method

[0135] Cell culture: 1) Dilute the Gynostemma pentaphyllum-derived extracellular vesicle solution of Example 1 to a protein concentration of 1 μg / mL and 5 μg / mL with DMEM medium, and mix well; 2) Seed mouse primary bone marrow macrophages (BMDM) in a 6-well plate at a concentration of 3 million cells per well, and randomly divide them into a control group (CON), a model group (LPS), a low-dose experimental group (LPS + 1 μg / mL), and a high-dose experimental group (LPS + 5 μg / mL); 3) After overnight incubation, remove the old culture medium, add 2 mL of DMEM medium containing 1 μg / mL LPS and 10% (v / v) serum to the model group, add 2 mL of DMEM medium containing 10% (v / v) serum to the control group, and add 2 mL of DMEM medium containing 1 μg / mL LPS and 1 μg / mL and 5 μg / mL Gynostemma pentaphyllum-derived extracellular vesicles to the low-dose and high-dose experimental groups, respectively; 4) After 12 hours of incubation, collect the cells and extract the proteins in the cells with radioimmunoprecipitation method lysis buffer (RIPA lysis buffer) containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors; 5) Use the obtained proteins for WB experiments.

[0136] Western Blot (WB):

[0137] 1) Protein denaturation: Take the protein from the -80℃ refrigerator, dissolve on ice, high-speed refrigerated centrifuge at 4℃, 12000 rpm, centrifuge for 30 min, discard the precipitate, carefully transfer the supernatant to a new EP tube, add protein: 5x SDS loading buffer = 4:1, mix well, seal with sealing film, 98℃ water bath for 5 min, store at -20℃;

[0138] 2) Gel preparation, loading and electrophoresis: Prepare the glass plate, wash and dry, install after removing impurities, prepare the separation gel and add it to the glass plate, press flat with pure water, after complete solidification, pour off the pure water, add the prepared 12% concentrated gel, immediately insert the 10-hole comb, after complete solidification and molding, place it in the electrophoresis tank, add the prepared electrophoresis solution; load 20 μL of protein per well, adjust to 90V constant voltage current, after the marker band is pulled apart, change to constant voltage 120V, stop electrophoresis after the marker band is fully separated;

[0139] 3) Transfer: According to the size of the gel, cut the corresponding size of PVDF membrane, after the electrophoresis is almost completed, soak in methanol for 1 min, then transfer the PVDF membrane to the transfer buffer; after the protein is fully separated, complete the electrophoresis, install the electrotransfer device in the transfer buffer, firmly clamp the transfer clamp, place it in the electrotransfer tank, connect the positive and negative electrodes, then place the transfer device in ice water, surround it with several ice bags to ensure constant temperature during the transfer process, the transfer time is 90 min at 250mA voltage;

[0140] 4) Blocking: Open the device, observe the side of the PVDF membrane in contact with the electrophoresis gel as the positive side, cut a small corner and mark it, carefully take out the membrane with tweezers and place it in the prepared blocking solution at room temperature;

[0141] 5) Immunoreaction: According to the molecular weight of the marker, determine the position of the target protein and the internal reference, cut out the required PVDF membrane at the corresponding position, mark it, and place it in the required primary antibody (the primary antibody is from the NF-κB signaling pathway antibody sample kit of Cell Signaling TECHNOLOGY company, item number #9936) at 4℃ overnight; the next day, wash the membrane with 1x TBST 3 times for 10 min each time. According to the source of the primary antibody, add the corresponding species secondary antibody diluted with blocking solution at 1:3000, incubate at room temperature for about 1 hour, wash the PVDF membrane 3 times for 10 min each time;

[0142] 6) Chemiluminescence detection: After the PVDF membrane is slightly absorbed with filter paper, place it in the developer, evenly add an appropriate amount of chemiluminescence reagent to completely cover the membrane, expose to collect images for analysis.

[0143] 2、Experimental results

[0144] The WB experiment and phosphorylation quantification results are as followsFigure 11 、 12 As shown in FIG. 6, LPS significantly increased the ratio of p-IKK-a / IKK-a (about 1.70 times compared with the control group) and p-p65 / p65 (about 1.86 times compared with the control group), thereby activating the NF-κB pathway, which is consistent with previous studies. Treatment with Lycium barbarum-derived extracellular vesicles significantly reduced this ratio to a level almost equivalent to that of the control group, indicating that extracellular vesicles effectively inhibited LPS-induced activation of the NF-κB pathway.

[0145] Example 7 Treatment effect of Lycium barbarum-derived extracellular vesicles on a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mouse model

[0146] 1. Experimental animals: C57BL / 6 mice, male, 6-8 weeks old, weighing 20±3 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0147] 2. Experimental animal grouping: Randomly divided into 12 groups, divided into control group (CON), lipopolysaccharide group (LPS), Lycium barbarum-derived extracellular vesicle experimental group (LPS+LBNVs), dexamethasone experimental group (LPS+DEX).

[0148] 3. Animal modeling and drug administration method:

[0149] Control group: 40 μL / 20 g of mouse body weight of PBS buffer was given, and the lung was atomized and administered through the trachea by an atomizing needle;

[0150] Lipopolysaccharide group: Mix PBS buffer and LPS solution to obtain a mixed liquid, so that the final administration concentration of LPS is 5 mg / kg, and the mixed liquid is administered by lung atomization through the trachea by an atomizing needle at a dose of 40 μL / 20 g of mouse body weight;

[0151] Lycium barbarum-derived extracellular vesicle experimental group: Mix Lycium barbarum-derived extracellular vesicle PBS solution and LPS solution to obtain a mixed liquid, so that the final Lycium barbarum-derived extracellular vesicle protein concentration is 2 μg / mL, and the final administration concentration of LPS is 5 mg / kg, and the mixed liquid is administered by lung atomization through the trachea by an atomizing needle at a dose of 40 μL / 20 g of mouse body weight;

[0152] Dexamethasone experimental group: Mix dexamethasone PBS solution and LPS solution to obtain a mixed liquid, so that the final dexamethasone concentration is 2 μg / mL, and the final administration concentration of LPS is 5 mg / kg, and the mixed liquid is administered by lung atomization through the trachea by an atomizing needle at a dose of 40 μL / 20 g of mouse body weight.

[0153] 4. Sampling method: sacrifice the mice 24 hours after administration, remove the lungs and fix them in paraformaldehyde, embed the fixed lung tissue, section and perform HE staining.

[0154] Lavage fluid acquisition method: 1) After the mouse is sacrificed, fix it on the operating table; 2) use a 1 mL syringe to suck pre-cooled PBS buffer, then replace the needle of the syringe with a 10 mL syringe needle with the tip cut off; 3) after the above operation, use scissors and tweezers to cut open the skin tissue on the neck of the mouse, use tweezers to pull the tissue and muscle at the trachea to both sides, taking care not to tear the trachea; 4) use tweezers to carefully remove the mucosa tissue wrapped around the trachea, ensuring that the trachea remains completely exposed; 5) use the left hand to hold the trachea with tweezers, and use the right hand to hold the 1 mL syringe needle at the top of the trachea and repeatedly pass through the needle hole, making it visible to the naked eye; 6) after the needle hole in the trachea is visible to the naked eye, the left hand holds the tweezers and the right hand holds the needle, then holds the suture line with the tweezers and delivers it to the left hand, and then pulls the suture line to the bottom of the trachea (equivalent to pre- placing the suture line under the trachea to facilitate subsequent direct ligation of the trachea); 7) then, aim the 10 mL syringe needle with the tip cut off at the previous needle hole, and deliver it horizontally without lifting it up or down to avoid damaging the trachea; 8) start ligation, the suture line is located at a certain distance from the needle hole to avoid displacement of the suture line during ligation (ligation must be done by tightly fixing the needle body and the trachea together); 9) after ligation, remove the left and right tweezers, hold the 1 mL syringe containing pre-cooled PBS buffer, and start injecting the lavage fluid; 10) aspirate the lavage fluid and recover as much as possible; 11) after recovery, fix the needle to prevent displacement, separate the syringe needle and needle tube, and repeat the operation with 1 mL of PBS buffer. (equivalent to recovering 2 mL of lavage fluid per mouse).

[0155] 5. Detection index and method

[0156] (1) Body weight change observation

[0157] Weigh the mice once before modeling and administration, and once before sacrificing the mice 24 hours after administration, and record the body weight change of the mice.

[0158] (2) Hematoxylin-eosin (HE) staining

[0159] Put the slices into the environment-friendly dewaxing liquid I for 20 min - environment-friendly dewaxing liquid II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% alcohol for 5 min, and then wash with tap water; put the slices into the high-definition constant dyeing pretreatment liquid for 1 min; hematoxylin staining: put the slices into the hematoxylin dyeing liquid for 3-5 min, wash with tap water, differentiate with the differentiation liquid, wash with tap water, return to blue with the return blue liquid, and then wash with running water; eosin staining: put the slices into 95% alcohol for 1 min, and then put into the eosin dyeing liquid for 15 s; dehydration and mounting: put the slices into anhydrous ethanol I for 2 min - anhydrous ethanol II for 2 min - anhydrous ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min, and then transparentize, and mount with neutral balsam; microscope examination, image collection and analysis.

[0160] Semi-quantitative lung injury scoring criteria: three fields are taken for scoring for each slice, and the average value is taken as the final score of the slice, and the specific scoring content is shown in the following table:

[0161]

[0162] Total score = [(20 x A) + (14 x B) + (7 x C) + (7 x D) + (2 x E)]

[0163] (3) Lung index

[0164] After the whole lung is taken out, wash with normal saline, absorb the water with clean filter paper, weigh the lung, and calculate the lung index, the formula being lung index = lung weight / body weight x 100%.

[0165] (4) RNA sequencing

[0166] After the total RNA of the lung tissue is extracted, rRNA is removed through a conventional kit, eukaryotic mRNA with a polyA tail is enriched through magnetic beads with Oligo(dT), the mRNA is broken by ultrasonic waves, the fragmented mRNA is used as a template, random oligonucleotides are used as primers, the first strand of cDNA is synthesized in an M-MuLV reverse transcriptase system, then the RNA strand is degraded by RNaseH, and the second strand of cDNA is synthesized in a DNA polymerase system with dNTPs as raw materials. The purified double-stranded cDNA is subjected to end repair, A tailing and sequencing adapter ligation, screened with AMPure XP beads for cDNA of about 200 bp, subjected to PCR amplification and purified again using AMPure XP beads, and finally a library is obtained. Data analysis is performed on the Genedio bioinformatics cloud platform.

[0167] (5) Immunofluorescence

[0168] 1) After the end of cell processing, rinse with PBS buffer for 2 times to wash away residual medium; 2) add 4% paraformaldehyde, incubate at room temperature for 15 min, then rinse with PBS buffer for 3 times, each for 5 min; 3) dilute with PBS buffer to obtain 0.5% titon X-100, incubate at room temperature for 15 min, then rinse with PBS buffer for 3 times, each for 5 min; 4) prepare 3% BSA (bovine serum albumin) with PBST (phosphate buffer containing Tween 20), block at room temperature for 2 h, then rinse with PBST for 3 times, each for 5 min; 5) prepare 3% BSA with PBST, add ZO-1 primary antibody (purchased from Abeam Company, item number ab221547) or E-cadherin primary antibody (purchased from Cell Signaling TECHNOLOGY Company, item number #3195) at 1:1000, incubate at 4°C overnight, then rinse with PBST for 3 times, each for 5 min; 6) prepare 1% BSA with PBST, add fluorescently labeled secondary antibody at 1:200, incubate at room temperature in the dark for 90 min, then rinse with PBST for 3 times, each for 5 min; 7) dilute DAPI dye 1000 times with PBS buffer, incubate for 10 min to stain the nucleus, then wash away with PBS buffer, add 1 mL of PBS buffer, and take pictures using a confocal microscope.

[0169] (6) Western blot (WB)

[0170] 1) Protein extraction: after washing the lung tissue with PBS buffer, freeze it quickly with liquid nitrogen, then divide each lung tissue into 5 equal parts, take one part and add 300 μL cell lysis solution, 1 μL protease inhibitor and 1 μL protein phosphatase inhibitor, grind at 4°C, then centrifuge at high speed in a refrigerated centrifuge at 12000 rpm for 30 min, discard the precipitate, carefully transfer the supernatant to a new EP tube, add protein: 5x SDS loading buffer = 4:1, mix well, seal with sealing film, 98°C water bath for 5 min, store at -80°C;

[0171] 2) Gel preparation, loading and electrophoresis: prepare a glass plate, wash and dry, install after removing impurities, prepare the separation gel and add it to the glass plate, press flat with pure water, after complete solidification, discard the pure water, add the prepared 8% concentrated gel, immediately insert the 10-hole comb, after complete solidification and molding, place it in the electrophoresis tank, add the prepared electrophoresis solution; load 10 μL of protein per well, adjust to a constant voltage of 90 V, after the marker band is separated, change to a constant voltage of 120 V, stop electrophoresis after the marker band is fully separated;

[0172] 3) Transfer: according to the size of the gel cut the corresponding size of the PVDF membrane, when the electrophoresis is almost finished, soak in methanol for 1 min, then transfer the PVDF membrane to the transfer buffer; after the protein is fully separated, complete the electrophoresis, install the electrotransfer device in the transfer buffer, firmly clamp the transfer clamp, put it into the electrotransfer tank, connect the positive and negative electrodes, then put the transfer device into ice water, surround it with several ice bags to ensure the constant temperature during the transfer process, the transfer time is 90 min at 250 mA voltage;

[0173] 4) Blocking: open the device, observe the side of the PVDF membrane that is in contact with the electrophoresis gel as the front side, cut a small corner and mark it, carefully take out the membrane with tweezers and place it in the prepared blocking solution, block at room temperature;

[0174] 5) Immunoreaction: according to the molecular weight of the marker, determine the position of the target protein and the internal reference, cut out the required PVDF membrane at the corresponding position, mark it, and place it in the required primary antibody (purchased from Wuhan Sivier Biotechnology Co., Ltd., item number P39447) at 4°C overnight; the next day, wash the membrane with 1x TBST for 3 times, each for 10 min. According to the source of the primary antibody, add the anti-rabbit secondary antibody diluted with blocking solution at 1:3000, incubate at room temperature for about 1 hour, and wash the PVDF membrane for 3 times, each for 10 min;

[0175] 6) Chemiluminescence detection: after the PVDF membrane is slightly absorbed with filter paper, place it in the developing instrument, add an appropriate amount of chemiluminescence reagent evenly and completely cover the membrane, expose to collect images for analysis.

[0176] (7) Protein concentration determination

[0177] After collecting the perfusate, centrifuge at 2000xg, then take the supernatant and use the BCA protein concentration determination kit (purchased from Biyun Tian Biotechnology Co., Ltd., item number P0011) to detect the protein concentration.

[0178] (8) Cell counting

[0179] After collecting the perfusate, centrifuge at 2000xg, remove the supernatant, add the red cell lysing solution and blow it apart, then centrifuge at 2000xg for 5 minutes, repeat 2 times, finally add 200μL PBS buffer and count with a cell counter.

[0180] (9) Dihydroethidium (DHE) staining

[0181] Put the slices into xylene I 15 min-xylene II 15 min-absolute ethanol I 5 min-absolute ethanol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water; the group of the organization around the circle to prevent the liquid flow, the addition of PBS dilution of ROS working fluid in the dark incubated at 37℃ for 1h, throw off containing ROS dye, PBS washing 3 times; the slide is placed in PBS buffer (PH7.4) in the decolorization shaking bed under the light shaking wash 3 times, 5 min each time. Slightly dry after the slice drop DAPI dye room temperature light dye nucleus 10 min; the slide is placed in PBS (buffer PH7.4) in the decolorization shaking bed under the light shaking wash 3 times, 5 min each time. Slightly dry after the slice with anti-fluorescence quenching mounting agent mounting; microscopy: the slice under the fluorescence microscope observation and image acquisition.

[0182] (10) TUNEL staining

[0183] Paraffin section dewaxing to water, environmental protection transparent agent (three cylinders, 10 min each cylinder), gradient alcohol (absolute, 95%, 75%) 5 min each cylinder, tap water rinse clean, rinse with distilled water once and soak in distilled water; 0.1% Triton X-100, 0.1% sodium citrate dropwise penetration at room temperature for 8 min; TBS wash 3 times, 5 min each time; each slice drop 3% BSA and 20% normal calf serum mixed solution to block non-specific reaction, room temperature 30 min, no washing; shake off the serum, each slice drop TUNEL reaction mixture, 50uL per slice, prepared before use (A liquid: B liquid = 1:9) mixed, avoid light wet box incubation for 60 min, 37℃, negative light with TBS instead of working solution; after completion, TBS wash 3 times, 5 min each time; DAPI dye nucleus, light room temperature 10 min; anti-fluorescence attenuation mounting agent mounting, avoid light 4℃ preservation, ready for examination and photography.

[0184] 6. Statistical method: measurement data using one-way ANOVA, the data is shown as mean ± SD; Western blot results will be normalized to the protein expression level of β-tubulin (β-tubulin), and expressed as the difference multiple relative to the control group, *P<0.05, **P<0.01, ***P<0.001.

[0185] 7. Experimental results

[0186] (1) Body weight change

[0187] Body weight can reflect the overall health of mice, with the progress of ARDS, such as Figure 13As shown in Fig. 2A, compared with the CON group, the body weight of the LPS group decreased by about 15.0%, while both treatment interventions alleviated the weight loss, and the LPS+LBNVs group showed a more superior protective effect, reducing the weight loss to about 3.0%, and the LPS+DEX group had a weight loss rate of 5.0%.

[0188] (2) Protective effect of LBNVs on lung tissue

[0189] HE staining of lung tissue Figure 14 As shown in Fig. 3A, the alveoli of the ARDS mice in the LPS group were collapsed, with inflammatory cell infiltration and interstitial thickening, while both the LPS+LBNVs group and the LPS+DEX group attenuated these pathological features, and the LPS+LBNVs group almost completely restored the alveolar structure. The semi-quantitative lung injury score based on interstitial / alveolar neutrophils, hyaline membrane, alveolar septum thickening and protein debris is shown in Fig. 3B. Figure 15 As shown in Fig. 3B, the lung injury score of the LPS group was 3.95 times higher than that of the CON group, while both the LPS+LBNVs group and the LPS+DEX group showed significant improvement, and the LPS+LBNVs group (13.33±1.67) showed superior therapeutic effect compared with the LPS+DEX group (23.89±3.33).

[0190] The lung index results are shown in Fig. 4A. Figure 16 As shown in Fig. 4A, compared with the LPS group (0.94±0.07), the LPS+LBNVs group (0.648±0.08) significantly reduced lung edema.

[0191] Principal component analysis of lung tissue RNA sequencing data of LPS+LBNVs and LPS groups of mice is shown in Fig. 5A. Figure 17 As shown in Fig. 5A, there was a clear separation between the two groups, reflecting the excellent therapeutic effect of LBNVs derived from wolfberry on acute respiratory distress syndrome mice.

[0192] (3) Repair effect of LBNVs on alveolar-capillary barrier

[0193] Immunofluorescence analysis Figure 18 As shown in Fig. 6A, compared with the LPS group, both the LPS+DEX group and the LPS+LBNVs group can improve the reduction of ZO-1 and E-cadherin fluorescence signals caused by LPS, and the fluorescence intensity of LBNVs derived from wolfberry is stronger than that of DEX.

[0194] WB experiment Figure 19As shown, further indicated that compared with the CON group, LPS significantly reduced the levels of ZO-1 and E-cadherin, and compared with the LPS group, the LPS+DEX group did not significantly improve, while the LPS+LBNVs group significantly restored the expression levels of ZO-1 (p=0.0135) and E-cadherin (p=0.0022).

[0195] The destruction of the lung epithelial-endothelial barrier can lead to protein leakage and excessive inflammatory cell invasion, therefore, the protein concentration and cell number in the bronchoalveolar lavage fluid were measured. The results are shown in Figure 20 、 21 As shown, compared with the LPS group of mice, the LPS+LBNVs group effectively reduced the protein concentration and cell number in the lung to a level similar to that of healthy mice.

[0196] (4) The effect of LBNVs on oxidative stress and apoptosis in the treatment of ARDS

[0197] Since oxidative stress is a key component of the pathological network in the progression of ARDS, dihydroethidium (DHE) was used to detect oxidative activity in living cells. DHE can penetrate into the cell membrane and be oxidized to form red fluorescent ethylene glycol, and the signal intensity is positively correlated with the level of ROS. Fluorescence imaging and semi-quantitative analysis are shown in Figure 22 and 23 Compared with the CON group, the DHE signal in the lungs of the LPS group of ARDS mice increased. Compared with the LPS group, the DHE level of the LPS+LBNVs group decreased significantly, and its effect was superior to that of the LPS+DEX group. Given the close relationship between oxidative stress and cell death, the effect of LBNVs on apoptosis in ARDS was evaluated next. TUNEL staining and semi-quantitative analysis are shown in Figure 22 and 23 As shown, there was extensive apoptosis in the LPS group, and the number of apoptotic cells (red) increased significantly compared with the CON group. Compared with the LPS group, the LPS+LBNVs group significantly inhibited apoptosis, and the therapeutic effect was significantly superior to that of the LPS+DEX group.

[0198] In summary, LBNVs derived from wolfberry can repair the alveolar-capillary barrier, reduce oxidative stress and inhibit apoptosis, better protect lung tissue, and thus achieve the effect of treating ARDS.

[0199] Experimental Example 8 Treatment effect of LBNVs derived from wolfberry on glycerol-induced acute renal injury in a mouse model induced by striated muscle

[0200] 1. Experimental animals: C57BL / 6 mice, male, 6-8 weeks old, weighing 20±3 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0201] 2、Experimental animal grouping: Randomly grouped, 6 in each group, divided into control group (CON), model group (Glycerine), experimental group (LBNVs).

[0202] 3、Animal modeling method: After 16 hours of water deprivation, glycerol and PBS buffer were mixed at a volume ratio of 1:1, and then injected into the posterior lateral muscle of the thigh of mice at a dose of 5 mL / kg to model.

[0203] 4、Group administration method:

[0204] Control group: No modeling, water after all other groups are modeled, and 100 μL / 20 g of PBS buffer is injected through the tail vein, and 100 μL / 20 g of PBS buffer is injected again 24 hours after all other groups are modeled.

[0205] Model group: Water after modeling, and 100 μL / 20 g of PBS buffer is injected through the tail vein, and 100 μL / 20 g of PBS buffer is injected again 24 hours after modeling.

[0206] Experimental group: Water after modeling, and 40 μg / 20 g of extracellular vesicles solution from wolfberry is injected through the tail vein, and 40 μg / 20 g of extracellular vesicles solution from wolfberry is injected again 24 hours after modeling.

[0207] 5、Sampling method: Sacrifice the mice 24 hours after the second injection of extracellular vesicles from wolfberry, collect the right kidney and blood of the mice. Remove the right kidney of the mice and fix it in paraformaldehyde, embed the fixed kidney tissue, section and perform HE staining. After 4 hours of standing, the supernatant is collected and used to detect the blood biochemical indicators blood urea nitrogen (BUN) and creatinine (Cre) levels.

[0208] 6、Detection index and method

[0209] (1) Blood biochemical index detection method

[0210] The serum samples were sent to the Central Laboratory for Experimental Animals of Hong Kong University of Science and Technology (Guangzhou) for testing according to the instructions of the Kehua Biological Creatinine Test Kit (Sarcosine Oxidase Method) and Urea Determination Kit.

[0211] (2) HE staining

[0212] Put the slices into the environment-friendly dewaxing liquid I for 20 min - environment-friendly dewaxing liquid II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% alcohol for 5 min, and then wash with tap water; put the slices into the high-definition constant dyeing pretreatment liquid for 1 min; hematoxylin staining: put the slices into the hematoxylin dyeing liquid for 3-5 min, wash with tap water, differentiate with the differentiation liquid, wash with tap water, return to blue with the return blue liquid, and then wash with running water; eosin staining: put the slices into 95% alcohol for 1 min, and then put into the eosin dyeing liquid for 15 s; dehydration and mounting: put the slices into anhydrous ethanol I for 2 min - anhydrous ethanol II for 2 min - anhydrous ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min, and then transparentize and mount with neutral balsam; microscope examination, image collection and analysis.

[0213] 7. Experimental results

[0214] The blood biochemical results are shown in Table 2. Compared with the CON group, the kidney injury indicators in the Glycerine group increased significantly, while the LBNVs group recovered. Figure 24 The HE staining results are shown in Figure 2. In the CON group, the kidney tissue structure was complete, the renal tubules were closely arranged, and the glomerulus was not congested or swollen, indicating normal kidney function. In the Glycerine group, the renal tubules were significantly dilated, and transparent cast was observed. In addition, there was obvious necrosis of cells and infiltration of inflammatory cells. In contrast, the kidney tissue pathology of the LBNVs group was significantly improved. These results show that LBNVs effectively alleviate the kidney injury induced by rhabdomyolysis. Figure 25

[0215] In summary, the cell extracellular vesicles derived from wolfberry may play a role by resisting oxidation and protecting cells, thereby achieving the effect of treating acute kidney injury.

[0216] Example 9: Long-term safety evaluation of cell extracellular vesicles derived from wolfberry

[0217] 1. Experimental animals: C57BL / 6 mice, male, 6-8 weeks old, weighing 23±3 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0218] 2. Experimental animal grouping: randomly divided into 5 groups, including a control group (Healthy), a dexamethasone group (DEX), and a cell extracellular vesicle group derived from wolfberry (LBNVs).

[0219] 3. Animal modeling and drug administration method:

[0220] Control group: no treatment;

[0221] ​DEX group: 1.5 mg / kg of dexamethasone (powder dissolved in PBS buffer) was injected into the tail vein of mice every day for the first 6 days of each week for 3 consecutive weeks.

[0222] LBNVs group: 1.5 mg / kg of Lycium barbarum-derived extracellular vesicles solution was injected into the tail vein of mice every day for the first 6 days of each week for 3 consecutive weeks.

[0223] 4. Sampling method: After 3 weeks of administration, the mice were sacrificed, and the liver, gastrocnemius muscle, femur, and spleen of the mice were collected and fixed in paraformaldehyde. The fixed tissues were embedded, sectioned, and stained with HE. The supernatant was collected after the blood was allowed to stand for 4 hours, and was used to detect low-density lipoprotein cholesterol.

[0224] 5. Detection index and method

[0225] (1) Body weight change observation

[0226] The mice were weighed once a day for 3 weeks after administration, and the body weight changes of the mice were recorded.

[0227] (2) Measurement of low-density lipoprotein cholesterol (LDL-C) in blood

[0228] The serum samples were sent to the Central Laboratory for Experimental Animals of Hong Kong University of Science and Technology (Guangzhou) for testing according to the instructions of the Kewei Biological Small and Dense Low-Density Lipoprotein Cholesterol Assay Kit (Peroxidase Method).

[0229] (3) Semi-quantitative method of gastrocnemius muscle cross-sectional area

[0230] Semi-quantitative measurement of gastrocnemius muscle cross-sectional area was performed by ImageJ. Three regions were selected on each gastrocnemius muscle cross-sectional HE section for cross-sectional area measurement, and the final cross-sectional area of the section was the average of the three regions.

[0231] (4) Micro-CT bone analysis method

[0232] Micro-CT analysis of bone tissue was performed using Analyze 15.0 software. Starting from the 30th layer above the growth plate, 100 consecutive layers of bone tissue were selected for analysis, and the bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) parameters were extracted for quantitative evaluation of bone structure.

[0233] (5) HE staining

[0234] The sections were sequentially immersed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinsed with tap water. The sections were then treated with high-resolution constant staining pretreatment solution for 1 min. For hematoxylin staining, the sections were stained with hematoxylin solution for 3-5 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, and then stained with blue solution and rinsed with running water. For eosin staining, the sections were dehydrated in 95% ethanol for 1 min, then stained with eosin solution for 15 s. For dehydration and mounting, the sections were sequentially immersed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min for clearing, then mounted with neutral resin. Microscopic examination and image acquisition and analysis were then performed.

[0235] 6. Experimental Results

[0236] (1) Weight changes

[0237] Weight monitoring such as Figure 26 As shown, compared with the Healthy group, mice treated with DEX showed a significant decrease in body weight, approximately 20%. In contrast, mice treated with LBNVs exhibited a similar pattern of weight gain as the Healthy group mice (p>0.05).

[0238] (2) Low-density lipoprotein cholesterol level

[0239] The results are as follows Figure 27 As shown, DEX treatment led to a significant increase in serum low-density lipoprotein cholesterol (LDL-C) levels, approximately 2.0 times higher than the Healthy group, while the LDL-C concentration in the LBNVs group was similar to that in the Healthy group. This suggests that LBNVs may circumvent the metabolic disorders associated with long-term DEX treatment.

[0240] (3) Histological analysis of the gastrocnemius muscle

[0241] The results are as follows Figure 28 and 29 As shown, compared with the Healthy group, mice in the DEX group exhibited significant muscle atrophy, with a reduction of approximately 41.2% in the cross-sectional area of ​​the gastrocnemius muscle. In contrast, mice in the LBNVs group maintained normal muscle structure and fiber size distribution.

[0242] (4) Histological analysis of the femur

[0243] Long-term use of DEX treatment can induce osteoporosis. Micro-CT analysis results are as follows... Figure 30The results showed that compared with the Healthy group, the bone trabecular structure of the DEX group mice was impaired, the bone surface tissue volume ratio (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were reduced, and the trabecular separation (Tb.Sp) was increased. HE staining of femur sections Figure 29 Further confirmed these results, showing that DEX-treated mice thinning of trabecular bone and expansion of bone marrow space, while LBNVs group of mice remain similar to the Healthy group of physiological bone parameters.

[0244] (5) Histological analysis of liver, spleen

[0245] In order to evaluate the extent of organ damage during long-term administration, histological analysis of liver, spleen was also performed. The results of HE staining are shown in Figure 29 Compared with the Healthy group, long-term treatment with DEX induced significant hepatocyte edema and disruption of spleen structure, characterized by blurred medullary boundary and pale central area, which indicated that DEX might induce potential liver dysfunction and immune suppression. In contrast, LBNVs-treated mice maintained normal liver and spleen tissue structure.

[0246] In summary, long-term injection of dexamethasone caused abnormal metabolism and immunity, osteoporosis, muscle atrophy and liver edema in mice, while long-term injection of Lycium barbarum-derived extracellular vesicles did not cause such adverse effects.

[0247] Experimental Example 10 Treatment effect of Lycium barbarum-derived extracellular vesicles on chronic obstructive pulmonary disease mouse model

[0248] 1. Experimental animals: C57BL / 6 mice, male, 6-8 weeks old, body weight 20±3g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0249] 2. Experimental animal grouping: Randomly divided into 6 groups, each group of 6, divided into control group (CON, Healthy), model group (COPD, LPS), experimental group (LBNVs, LPS+LBNVs). Figure 31 Figure 32 Figure 31 Figure 32 Figure 31 Figure 32

[0250] 3. Animal modeling method:

[0251] ​​​​​​On the 1st day and the 15th day of the modeling, LPS was dissolved in PBS buffer to obtain a PBS+LPS mixed liquid, so that the final administration concentration of LPS was 2 mg / kg, and the mixed liquid was administered by pulmonary aerosolization through a tracheal aerosol needle at 40 μL / 20 g of mouse body weight; other days were smoked by a smoking machine (Tawang) (10 cigarettes per day), and samples were collected on the 30th day.

[0252] 4. Group administration method:

[0253] Control group: no treatment;

[0254] Model group: On the 1st day and the 15th day of the modeling, LPS was dissolved in PBS buffer to obtain a PBS+LPS mixed liquid, so that the final administration concentration of LPS was 2 mg / kg, and the mixed liquid was administered by pulmonary aerosolization through a tracheal aerosol needle at 40 μL / 20 g of mouse body weight; other days were smoked by a smoking machine (Tawang) (10 cigarettes per day), and samples were collected on the 30th day.

[0255] Experimental group: On the 1st day and the 15th day of the modeling, LPS was dissolved in PBS buffer, and then Gynostemma pentaphyllum-derived extracellular vesicles were added to obtain a PBS+LPS mixed liquid, so that the final administration concentration of LPS was 2 mg / kg, and the final administration concentration of Gynostemma pentaphyllum-derived extracellular vesicles was 2 μg / kg, and the mixed liquid was administered by pulmonary aerosolization through a tracheal aerosol needle at 40 μL / 20 g of mouse body weight; other days were administered by aerosolization (2 μg / kg, 10 mL per day) and smoked by a smoking machine (Tawang) (10 cigarettes per day), and samples were collected on the 30th day.

[0256] 5. Detection index and method:

[0257] (1) Body weight change observation

[0258] The mice were weighed on the day of modeling and on the 2nd, 5th, 8th, 11th, 14th, 16th, 20th, 24th, 28th, and 30th days after modeling, and the body weight changes of the mice were recorded.

[0259] (2) Lung function determination method

[0260] Before the sample collection on the 30th day of modeling, a small animal whole body plethysmography system (Tawang Technology, WBP-8MII) was used to evaluate the lung function. First, the mice were adapted in the chamber for 30 minutes, and then a 5-minute monitoring period was performed under unrestricted conditions. Subsequently, the recorded lung function parameters were analyzed to evaluate the lung function.

[0261] 6. Experimental results

[0262] During the experiment, there were significant differences in the body weight changes of experimental animals in different groups, such as Figure 31As shown, the body weight of the Healthy group animals continued to grow, the body weight of the COPD group animals grew significantly slower and decreased significantly after LPS administration, while the intervention of LBNVs from Gynostemma pentaphyllum could slow down this body weight decrease. Statistical analysis showed that the body weight of the Healthy group was significantly different from that of the COPD group on day 30 (p < 0.05), while the body weight of the LBNVs group was significantly higher than that of the COPD group (p < 0.05), indicating that LBNVs treatment had a certain effect on the overall health recovery of COPD animals.

[0263] The results of lung function determination are shown in FIG. 6. Figure 32 As shown, the airway resistance Penh value of the LPS group increased significantly, which was significantly different from that of the CON group (p < 0.001); while the Penh value of the LPS+LBNVs group decreased significantly, which was significantly different from that of the LPS group (p < 0.001), and was close to the normal level of the CON group. In addition, the peak expiratory flow PEF value of the LPS group decreased significantly (p < 0.001), while the PEF value of the LPS+LBNVs group increased significantly, which was statistically significant compared with the LPS group (p < 0.001).

[0264] The above results show that LBNVs from Gynostemma pentaphyllum treatment not only can improve the overall health status of mice with chronic obstructive pulmonary disease, but also can significantly improve their respiratory function. These data further verify the potential efficacy of LBNVs from Gynostemma pentaphyllum treatment for chronic obstructive pulmonary disease, suggesting that it may play a role by improving lung function.

[0265] Obviously, the above examples are only examples for clarity, and are not limitations of the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or modifications. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

1. Application of extracellular vesicles derived from wolfberry in the preparation of drugs for treating acute kidney injury.

2. The application according to claim 1, characterized in that, The acute kidney injury mentioned is acute kidney injury caused by rhabdomyolysis; And / or, the drug can reduce blood urea nitrogen and / or reduce creatinine levels.

3. Application of extracellular vesicles derived from wolfberry in the preparation of drugs for treating lung injury.

4. The application according to claim 3, characterized in that, The lung injury is at least one of acute respiratory distress syndrome and chronic obstructive pulmonary disease; and / or, the drug can break the vicious cycle of inflammation-oxidative stress-alveolar capillary barrier damage and exert anti-inflammatory, antioxidant and alveolar capillary barrier repair functions.

5. Application of extracellular vesicles derived from Lycium barbarum in the preparation of drugs as inhibitors of nuclear transcription factor κB and / or inhibitors of apoptosis and / or repair of the alveolar-capillary barrier.

6. The application according to any one of claims 1-5, characterized in that, The method for preparing extracellular vesicles derived from wolfberry includes the following steps: Step S1: Soak goji berries, break down the cell walls, and homogenize them to obtain a homogenate solution; Step S2: Centrifuge and filter the above homogenate; Step S3: Take the liquid and centrifuge it again to collect the precipitate. Resuspend the precipitate in a solvent, centrifuge, resuspend it again, and filter to obtain extracellular vesicles derived from wolfberry.

7. The application according to claim 6, characterized in that, All steps S1-S3 are performed at 2-4℃; And / or, the goji berries in step S1 include dried goji berries and fresh goji berries; And / or, the centrifugation in step S2 includes a first centrifugation, a second centrifugation, and a third centrifugation. The centrifugal force of the first centrifugation is 500-2000×g, preferably 1000×g, and the centrifugation time is 8-12 min, preferably 10 min. The centrifugal force of the second centrifugation is 2000-5000×g, preferably 3000×g, and the centrifugation time is 20-40 min, preferably 30 min. The centrifugal force of the third centrifugation is 7000-12000×g, preferably 10000×g, and the centrifugation time is 0.5 h-1.5 h, preferably 1 h. And / or, the filtration operation in step S2 is filtration with gauze or microporous sterile filter membrane; Preferably, after the first centrifugation, the residue is filtered off with gauze, and after the third centrifugation, the residue is first filtered with a 0.45μm aqueous microporous sterilization filter membrane, and then filtered a second time with a 0.22μm aqueous microporous sterilization filter membrane. And / or, in step S3, the centrifugal force for the second centrifugation and centrifugation is 100,000-160,000×g, preferably 150,000×g, and the time is 1-3h, preferably 1.5h.

8. The application according to claim 6, characterized in that, The solvent used for resuspension and re-resuspension in step S3 is a buffer solution or liquid culture medium. Optionally, the buffer solution is a PBS buffer. Optionally, the pH of the PBS buffer is 6.5-7.5, preferably 7; Optionally, the liquid culture medium is at least one of DMEM liquid culture medium, MEM liquid culture medium, RPMI 1640 liquid culture medium, F12 liquid culture medium or IMDM liquid culture medium.

9. The application according to any one of claims 1-5, characterized in that, The extracellular vesicles derived from wolfberry have a particle size of 100-230 nm; And / or, the protein concentration of the extracellular vesicles derived from wolfberry is 0.01-1000 μg / mL.

10. The application according to any one of claims 1-5, characterized in that, The drug is available in either injectable or oral formulation.

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