Extracellular vesicles derived from wine gastrodia elata and application of extracellular vesicles in cerebral ischemia reperfusion injury

Abstract

The invention provides wine gastrodia elata derived extracellular vesicles and application thereof in cerebral ischemia reperfusion injury, relates to the technical field of biological medicines, and provides application of the wine gastrodia elata derived extracellular vesicles in preparation of medicines for preventing and / or treating cerebral ischemia reperfusion injury. The traditional Chinese medicine composition can improve injury of nerve cells caused by oxygen deficit, inhibit phenotypic polarization of microglial cells to M1, promote phenotypic polarization of microglial cells to M2, reduce the area of cerebral infarction and improve neurological impairment after cerebral ischemia reperfusion. The invention reveals and verifies that the wine gastrodia elata-derived extracellular vesicle has an excellent nerve protection effect for the first time, and the vesicle can obviously relieve the injury of an anoxic environment to nerve cells, effectively maintain the cell activity and improve the survival ability of the cells in a stress state, so that the protection on brain tissue injury is realized from the cellular level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a cell extracellular vesicle from wine Gastrodia elata and application thereof in cerebral ischemia-reperfusion injury. BACKGROUND

[0002] Stroke is a common and extremely harmful cerebrovascular disease in clinical practice, which has complex causes and is mainly caused by damage to the brain blood vessels, leading to focal or whole brain tissue damage. Stroke has the typical characteristics of high morbidity, high disability rate, high recurrence rate and high mortality, which seriously threatens human life and health. Among all stroke cases, ischemic stroke accounts for about 75% to 90%, and hemorrhagic stroke accounts for about 10% to 25%. Among them, men, obese people and diabetic patients are considered as high-risk groups. Cerebral ischemia-reperfusion refers to the pathological phenomenon that the function and structure of damaged cells cannot be repaired after the blood supply to the brain tissue is restored, but the dysfunction of cells is further aggravated and the damage to the tissue structure is increased, which seriously limits the clinical treatment effect and is also a big problem in the treatment of ischemic stroke.

[0003] Extracellular vesicles (EVs) are nanoscale membrane vesicles actively released by various types of cells, and play a key role in information transmission and material exchange between cells and different species. According to its biological origin, size and biophysical characteristics, EVs can be divided into exosomes, microvesicles and apoptotic bodies. As early as the 1960s, Halperiun and other scholars first observed EVs of plant origin in carrots. Since then, the existence of EVs has been found in animals and microorganisms. EVs contain various bioactive molecules such as proteins, lipids, DNA, RNA and amino acids, and are therefore considered as an important medium for cell communication and play an irreplaceable role in signal transduction. With further research, people have gradually realized that EVs not only play a role in cell communication, but also can be used as an important tool for diagnosing and treating diseases. Its complex composition and unique role in diseases have attracted widespread attention in clinical medicine, and it can be used as a biomarker for disease diagnosis, as a drug carrier or directly as a treatment method, providing new possibilities for disease prevention and treatment.

[0004] In recent years, a wealth of research evidence has shown that plant cells can also release vesicle-like nanoparticles (EVs) containing a variety of bioactive components. Researchers have discovered vesicle-like nanoparticles in various fruits and plants, and these plant-derived EVs have exhibited certain therapeutic effects. For example, EVs extracted from edible plants such as grapes, grapefruits, ginger, and carrots have been shown to possess anti-inflammatory properties. These findings have greatly expanded the research scope and application prospects of EVs. Plant-derived extracellular vesicles, as natural sources of drugs, are considered to have great potential in disease treatment due to their abundant resources, wide availability, and ease of acquisition. Studies have shown that EVs from various plant sources not only possess anti-inflammatory effects but also exhibit antibacterial and antitumor activities. For example, EVs derived from turmeric and dandelion can effectively inhibit inflammatory responses, thereby alleviating related disease symptoms; EVs derived from kudzu root improve osteoporosis symptoms by enhancing autophagy; furthermore, EVs derived from bitter melon have been shown to cross the blood-brain barrier in animal models of cerebral ischemia-reperfusion injury, reducing the area of ​​cerebral infarction and significantly improving neurological deficits.

[0005] Gastrodia elata ( Rhizoma Gastrodiae ) is the orchid family plant Gastrodia elata ( Gastrodia elata The tuber of Gastrodia elata (Bl.) is a traditional Chinese medicine with a long history and wide application. It is neutral in nature, slightly aromatic, and sweet in taste, entering the liver meridian. It has the effects of calming wind and stopping spasms, suppressing liver yang, and dispelling wind and unblocking the meridians. Clinically, Gastrodia elata is often used to treat headaches, dizziness, numbness of limbs, infantile convulsions, epilepsy, tetanus, hemiplegia, and rheumatic pain. Modern pharmacological studies have shown that Gastrodia elata has various pharmacological effects, including neuroprotection, analgesia, antitumor, antioxidant, antihypertensive, hepatoprotective, and immune-enhancing effects. Numerous clinical and experimental studies have demonstrated that Gastrodia elata is significantly effective in treating neurological diseases such as headaches, hemiplegia, and stroke.

[0006] Wine-processed Gastrodia elata is a common processed form of Gastrodia elata. It is made by removing impurities, washing, steaming until soft, slicing thinly, drying, and then stir-frying using the wine-processing method. Wine-processed Gastrodia elata is sweet in taste, neutral in nature, and enters the liver meridian. It has the functions of calming wind and stopping spasms, soothing the liver and suppressing yang, and dispelling wind and unblocking the meridians. It is mainly used for infantile convulsions, epilepsy, tetanus, headache and dizziness, hemiplegia, numbness of limbs, and rheumatic pain. Through wine-processing, the meridian-unblocking function of Gastrodia elata can be further enhanced. However, although wine-processed Gastrodia elata has been widely used in traditional medicine, whether it can exert a protective effect on the brain or treat cerebral ischemia-related diseases through extracellular vesicles has not yet been clearly reported in research. Those skilled in the art cannot infer from the existing technology the extracellular vesicles (wine-processed Gastrodia elata) derived from it. Gastrodia Elata Does WGE-EV (derived extracellular vesicles) have the potential to treat diseases?

[0007] Based on this, the inventors conducted the first systematic study and confirmed that extracellular vesicles derived from *Gastrodia elata* can effectively alleviate neuronal damage caused by hypoxia. This discovery not only enriches the pharmacological content of *Gastrodia elata*, but also provides a new research direction and treatment strategy for the prevention and treatment of cerebral ischemia-reperfusion injury. Summary of the Invention

[0008] In view of this, the present invention proposes an extracellular vesicle derived from Gastrodia elata and its application in cerebral ischemia-reperfusion injury, in order to solve the problems existing in the prior art. The extracellular vesicles derived from Gastrodia elata extracted by the present invention can significantly protect against cell damage caused by hypoxia in nerve cells, thereby achieving the effect of protecting cells and providing a new treatment approach for the prevention and treatment of hypoxia-induced nerve cell damage diseases.

[0009] To achieve the above objectives, this invention proposes the application of extracellular vesicles derived from Gastrodia elata in the preparation of medicaments for the prevention and / or treatment of cerebral ischemia-reperfusion injury.

[0010] Furthermore, the application aims to improve nerve cell damage caused by hypoxia.

[0011] Furthermore, the application is to inhibit microglia polarization toward the M1 phenotype.

[0012] Furthermore, the application is to promote microglia polarization toward the M2 phenotype.

[0013] Furthermore, the application is to reduce the area of ​​cerebral infarction.

[0014] Furthermore, the application is to improve neurological deficits after cerebral ischemia-reperfusion.

[0015] This invention also provides a method for preparing extracellular vesicles derived from *Gastrodia elata*, comprising the following steps: (a) Take the sliced ​​Gastrodia elata, add water for reflux extraction, filter, and obtain the extract; (b) Concentrate the extract obtained in step (a); (c) Centrifuge the concentrate and collect the precipitate to obtain the extracellular vesicles derived from the wine-processed Gastrodia elata.

[0016] The present invention also provides an extracellular vesicle derived from Gastrodia elata prepared by the above preparation method.

[0017] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of extracellular vesicles such as those derived from Gastrodia elata, and a pharmaceutically acceptable carrier or excipient, for the prevention and / or treatment of cerebral ischemia-reperfusion injury.

[0018] Furthermore, the effective dose range of the drug is 20-160 μg / mL, which can significantly improve cell damage caused by nerve cell hypoxia and exhibits good blood compatibility and biocompatibility in vivo.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to reveal and demonstrate that extracellular vesicles derived from *Gastrodia elata* (a type of orchid) possess excellent neuroprotective effects. These vesicles can significantly reduce the damage to nerve cells caused by hypoxic environments, effectively maintain cell activity, and improve the survival ability of cells under stress, thereby achieving protection against brain tissue damage at the cellular level.

[0020] This invention reveals that the mechanism of action of this extracellular vesicle is closely related to the regulation of microglial polarization. It can effectively inhibit the activation of pro-inflammatory M1 microglia while promoting the polarization of anti-inflammatory and reparative M2 microglia. By regulating the neuroimmune microenvironment, it fundamentally alleviates the inflammatory damage process after cerebral ischemia-reperfusion and promotes the recovery of neurological function.

[0021] Furthermore, the extracellular vesicles described in this invention exhibit excellent biocompatibility and safety. Experiments show that these vesicles are essentially non-cytotoxic, do not cause hemolysis, and do not cause pathological damage to major organs and tissues, demonstrating extremely high in vitro and in vivo safety.

[0022] In summary, this invention not only provides a novel, highly efficient, and safe active substance for the treatment of cerebral ischemia-reperfusion injury, but also expands the applications of processed traditional Chinese medicine products and their active ingredients in the neurological field, possessing significant development value and broad clinical application prospects. Attached Figure Description

[0023] Figure 1 Transmission electron microscopy images of extracellular vesicles derived from Gastrodia elata; where A is scaled at 1000 nm and B is scaled at 200 nm. Figure 2 The particle size distribution of extracellular vesicles derived from Gastrodia elata. Figure 3 Zeta potential map of extracellular vesicles derived from Gastrodia elata using dynamic light scattering analysis; Figure 4 A protein quantification curve of extracellular vesicles derived from Gastrodia elata; Figure 5 The images show the cytotoxicity results of extracellular vesicles derived from *Gastrodia elata* (wine-processed gastrodia elata); A. Cytotoxicity results of WGE-EVs on BV2 cells after 24 h of treatment; B. Cytotoxicity results of WGE-EVs on HT22 cells after 24 h of treatment. p<0.0001; ##: p <0.01; #: p <0.05; Figure 6 The image shows the fluorescence signal detection results of microglia (BV2) after internalization and uptake of PKH67-labeled Gastrodia elata-derived extracellular vesicles within 12 hours, as studied by flow cytometry. Figure 7 The figure shows the results of BV2 cells uptake of extracellular vesicles derived from intact Gastrodia elata. Figure 8 Image showing the damage results of BV2 cells induced by the oxygen-glucose deprivation / reoxygenation (OGD / R) model of extracellular vesicles derived from Gastrodia elata; ###: p <0.0001; ##: p <0.01; #: p <0.05; Figure 9 The effect of extracellular vesicles derived from Gastrodia elata on the expression of CD86 and CD206 in BV2 cells is shown in the figure. Figure 10 The effect of extracellular vesicles derived from Gastrodia elata on in vitro hemolysis is shown in the figure. Figure 11 H&E staining results for different organs to assess the toxicity of extracellular vesicles derived from Gastrodia elata.

[0024] Figure 12 The result of using extracellular vesicles derived from Gastrodia elata to reduce the area of ​​cerebral infarction. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0027] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0028] Example 1: Preparation of extracellular vesicles (WGE-EVs) derived from Gastrodia elata. The WGE-EVs of the present invention are prepared by the following method: (1) Take 100 g of Gastrodia elata slices, add 12 times the amount of distilled water and reflux for 90 min, then filter; add 12 times the amount of distilled water to the residue and reflux for 90 min again, then filter; combine the two filtrates and concentrate them to 200 mL under reduced pressure in a rotary evaporator.

[0029] (2) The concentrated Gastrodia elata decoction was subjected to gradient centrifugation. Specifically, the Gastrodia elata decoction was centrifuged at 2000×g for 10 min, 7500×g for 20 min, and 10000×g for 30 min at 4℃. The supernatant was collected and filtered through a 0.22 μm filter membrane. The supernatant was then centrifuged at 100000×g for 90 min. The precipitate was collected after two centrifugations. The obtained sample was stored at -80℃ or resuspended in an appropriate amount of pure water before storage.

[0030] Example 2 Characterization and protein quantification of WGE-EVs The WGE-EVs samples prepared in Example 1 were characterized as follows: (1) Transmission electron microscopy (TEM) characterization Accurately pipette 10 μL of sample onto a copper grid using a 10 μL pipette, repeating 5-8 times. Absorb excess liquid with filter paper. Negatively stain with 3% phosphotungstic acid solution at room temperature for 5 min, wash once with double-distilled water, dry at room temperature for 2 min, and then image under a transmission electron microscope.

[0031] See results Figure 1 The WGE-EVs stock solution has a high particle density, with most particles having a diameter of about 126.3 nm and exhibiting a typical teacup-shaped vesicle structure.

[0032] (2) Particle size determination Testing instrument: Nanosight nanoparticle tracking analyzer.

[0033] First, rinse the sample cell three times with pure water. After calibrating the instrument, dilute the sample with PBS and load it onto the sample. Observe the particle dynamics and collect data through the instrument. Detect each sample three times.

[0034] See results Figure 2 The average particle size of WGE-EVs was 158.3 nm, with a standard deviation (SD) of 62.1 nm; the particle size range of 126.3 nm had the largest number of particles.

[0035] (3) Dynamic light scattering analysis and zeta potential measurement The zeta potential of the sample was detected using a Zetasizer Nano ZS instrument via dynamic light scattering (DLS) technology.

[0036] See results Figure 3 The zeta potential of WGE-EVs detected by DLS was -13.53 ± 0.31 mV.

[0037] (4) Protein quantification Samples were prepared and their absorbance was measured using the BCA protein quantification kit according to the instructions.

[0038] Establish a BCA standard curve and substitute the values ​​into the BCA standard curve to calculate the protein content. Perform a linear regression plot of absorbance value y against mass concentration x.

[0039] See results Figure 4 Within a concentration range of 0–2.0 μg / μL, protein concentration and absorbance showed a good linear relationship. The protein content in WGE-EVs samples determined by the BCA method can be used to characterize the subsequent dosage of WGE-EVs.

[0040] Example 3 Cytotoxic effects of WGE-EVs The cytotoxic effects of WGE-EVs on microglia (BV2) and mouse hippocampal neurons (HT22) were evaluated using the CCK-8 assay.

[0041] BV2 and HT22 cells were divided into three groups: a normal group (Sham), a blank group (Blank), and drug-treated groups (5 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL). The toxicity of WGE-EVs to BV2 and HT22 cells was investigated and cells were screened using the CCK-8 assay.

[0042] BV2 and HT22 cells at 5×10 4 The cells were seeded at a density of 10 cells / mL in 96-well plates and cultured in DMEM at 37°C and 5% CO2 for 24 h. After incubation, the cells were further cultured for 24 h after being injected with 5 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL and 320 μg / mL.

[0043] After incubation, add 10 μL of CCK-8 reagent to each well and incubate at 37℃ and 5% CO2 for 1-2 h. Measure the absorbance at 450 nm using a microplate reader. A)value.

[0044] Cell proliferation activity was assessed according to the CCK-8 kit instructions. Each group was tested in triplicate, and cell viability was calculated using the following formula.

[0045] Cell survival rate % = ( A 样品 - A 空白 ) / ( A 对照 - A 空白 )×100% The results are as follows Figure 5 As shown, BV2 and HT22 cells did not exhibit cytotoxicity after treatment with different concentrations of WGE-EVs, and the cell viability of BV2 cells increased with increasing WGE-EVs concentration after treatment.

[0046] Example 4: Flow cytometry detection of cellular internalization and uptake of WGE-EVs The endocytosis of WGE-EVs by BV2 cells was studied by flow cytometry, and the mechanism of WGE-EV uptake was preliminarily revealed.

[0047] BV2 cells were used at a rate of 1×10 6 BV2 cells were seeded at a density of 1 cell / mL in six-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The old culture medium was discarded, and BV2 cells were co-cultured with WGE-EVs (160 μg / mL) labeled with PKH67 green fluorescent dye at 37°C for 12 h.

[0048] After incubation, cells were collected, resuspended in PBS, and analyzed using flow cytometry to detect intracellular fluorescence signals.

[0049] The results are shown in Figure 6. Flow cytometry analysis revealed that, compared with the control group without WGE-EVs, the proportion of cells containing PKH67 fluorescent signals significantly increased with the addition of WGE-EVs. Quantitative analysis showed that approximately 82.0% of BV2 cells successfully internalized PKH67-labeled WGE-EVs, indicating that WGE-EVs can be effectively taken up by cells.

[0050] Example 5: Detection of WGE-EVs uptake by BV2 cells using a fluorescence imaging system To further observe the uptake of intact vesicle structures by cells more directly, a fluorescence imaging experiment was conducted: BV2 cells at 1×10 6The cells were seeded at a density of 10 cells / mL in six-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then discarded, and a 10 μM DiI red fluorescent dye for cell membranes was added to the six-well plates. After incubation in the dark for 30 min, the cells were aspirated and washed three times with PBS.

[0051] Hoechst nuclear blue fluorescent dye was added, and the cells were incubated in the dark for 10 min before being aspirated. PKH67 fluorescently labeled WGE-EVs at a concentration of 160 μg / mL were then co-incubated with BV2 cells at 37℃. Photos were taken at 0 h and 24 h. The uptake of WGE-EVs by BV2 cells was observed by comparing the drug-treated group with the control group.

[0052] The results are shown in Figure 7: the blue and red fluorescence patterns represent the cell number and distribution, respectively. The cell nuclei did not overlap, and the cell membranes were clearly distinguishable, indicating that there was no overlap after 24 hours of cell culture, and the cells grew compactly, forming a dense monolayer under these growth conditions. Compared with the control group without WGE-EVs, green fluorescent nanovesicles were visible near the cell nuclei in the treated group, indicating that WGE-EVs could be taken up and internalized into the cells with an intact structure.

[0053] Example 6: Protective effect of WGE-EVs against Oxygen-Glucose Deprivation / Reoxygenation (OGD / R)-induced BV2 cell damage. The protective effect of WGE-EVs against OGD / R-induced BV2 cell damage was determined using the CCK-8 assay.

[0054] BV2 cells at 5×10 4 Cells were seeded at a density of 10 cells / mL in 96-well plates and incubated in DMEM medium at 37°C and 5% CO2 for 12 h. After incubation, the medium was discarded, and the cells were incubated in glucose-containing DMEM treated with WGE-EVs at 37°C and 5% CO2 for 12 h. Then, the medium was replaced with glucose-free DMEM, and the cells were placed in an anaerobic incubator at 37°C (5% CO2, 1% O2, 94% N2) for 9 h to simulate oxygen-glucose deprivation (OGD). The medium was then replaced with glucose-containing DMEM, and the cells were incubated under normal oxygen conditions (37°C, 5% CO2) for 6 h to complete reoxygenation (R).

[0055] After OGD / R treatment, 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated at 37℃ and 5% CO2 for 1 hour. The absorbance was then measured at 450 nm using a microplate reader. A )value.

[0056] Cell viability is calculated as follows: Cell viability % = A con / test / A OGD / R The results are shown in Figure 8: After OGD / R treatment, the survival rate of BV2 cells in the model group was significantly reduced. p <0.05 indicates successful model establishment. Within the range of 20-160 μg / mL, the survival rate of hypoxic-damaged BV2 cells increased with increasing WGE-EVs dosage, and within the dosage range, the cell survival rate in each treatment group was significantly higher than that in the model group (…). p The result showed a significant dose-dependent effect (<0.05), indicating that WGE-EVs have a protective effect against OGD / R-induced cell damage, and that WGE-EVs have a significant protective effect against cerebral ischemia-reperfusion injury.

[0057] Example 7: Effect of immunofluorescence WGE-EVs on phenotypic polarization of microglia M1 / M2 The effect of WGE-EVs on the polarization phenotype of BV2 cells was detected by immunofluorescence.

[0058] BV2 cells at 3×10 5 Inoculate at a density of 160 μg / mL into 24-well plates, administer 160 μg / mL of drug for 12 h, and after OGD / R treatment, aspirate the DMEM medium, add 500 μL of paraformaldehyde (4%) to each well for fixation for 15 min, aspirate, and wash 3 times with PBS for 2 min each time.

[0059] Add 0.1% Triton X-100 (150 μL per well), incubate at room temperature for 15 min, then aspirate. Wash three times with PBS for 2 min each time. Add 5% BSA (1 mL per well) to block the incubator, then aspirate after 1 h.

[0060] Add 150 μL of primary antibody CD86 rabbit monoclonal antibody (M1 marker, 1:100 dilution) and CD206 polyclonal antibody (M2 marker, 1:200 dilution) to each well, seal with sealing film, and incubate in an incubator for 1.5 h. Then wash three times with 500 μL of 1×PBS for 5 min each time.

[0061] Add 150 μL of goat anti-rabbit fluorescent secondary antibody (1:200 dilution) to each well, incubate at 37°C in the dark for 45 min, then aspirate and wash each well with 500 μL of PBS for 5 min each time. Add 200 μL of 10 μM DAPI reagent to each well, incubate in the dark for 10 min, and then take pictures on a high content analysis system.

[0062] The results are shown in Figure 9: Compared with the normal group, the expression of CD86, a marker of M1 microglia, in BV2 cells of the model group was significantly enhanced. Compared with the model group, the expression of CD86 in BV2 cells of the WGE-EVs (160 μg / mL) group was significantly weakened. These results indicate that WGE-EVs can reduce the expression of M1 microglia. Furthermore, compared with the normal group, the expression of CD206, a marker of M2 cell type, in BV2 cells of the model group did not change significantly. Compared with the model group, the expression of CD206 in BV2 cells of the WGE-EVs (160 μg / mL) group was significantly enhanced. These results indicate that WGE-EVs can inhibit pro-inflammatory M1 polarization and promote anti-inflammatory M2 polarization.

[0063] Example 8 In vitro biocompatibility Assess the in vitro blood compatibility of WGE-EVs.

[0064] Transfer anticoagulated whole blood to a 15 mL centrifuge tube, centrifuge at 1000 g at 4℃ for 10 min, remove the supernatant serum, add 10 mL PBS solution to resuspend the precipitate, mix well, centrifuge at 1000 g for 10 min, remove the supernatant, repeat 3 times, and resuspend the red blood cells in physiological saline at a ratio of 1:50 to prepare a 2% red blood cell physiological saline suspension.

[0065] The experimental group was divided into 6 groups, with 3 parallel tubes in each group: negative control group (2.5 mL physiological saline + 2.5 mL red blood cell suspension), positive control group (2.5 mL distilled water + 2.5 mL red blood cell suspension), solvent control group (2.5 mL PBS + 2.5 mL red blood cell suspension), and WGE-EVs experimental group (WGE-EVs were added to a final concentration of 200, 500, and 1000 μg / mL, respectively, then PBS was added to bring the volume to 2.5 mL, and then 2.5 mL red blood cell suspension was added).

[0066] After gently mixing, place it in a 37 °C water bath. After 3 h, observe the color of the liquid in the centrifuge tube to judge the hemolysis situation. If the upper layer liquid is transparent or yellow and the lower layer is red and opaque, it is non-hemolytic; if the upper layer liquid is red and turbid, it is partially hemolytic; if the upper layer liquid is red and transparent, it is completely hemolytic. Then, centrifuge each group of sample tubes at 800 g for 5 min. Aspirate the supernatant in the centrifuge tube and add it to the enzyme-labeled plate. Use an enzyme-labeled instrument to detect the absorbance value at a wavelength of 545 nm in each well, and calculate the hemolysis rate of the sample. The formula is hemolysis rate (%) = ( A 样品 - A 阴性 ) / ( A 阳性 - A 阴性 )×100%. When the hemolysis rate < 5%, the hemolysis test is qualified.

[0067] The results are as Figure 10 shown: The normal saline group is the negative control group (NC), the upper layer liquid is colorless and clear, and no hemolysis occurs; the distilled water group is the positive control group (PC), the upper layer liquid is red and transparent, and obvious hemolysis occurs; then comes the PBS group with WGE-EVs as the solvent, and the groups with different concentrations added. The upper layer liquids are all clear and transparent, and no obvious hemolysis phenomenon is seen. After centrifuging each sample tube, take the upper layer liquid and add it to a 96-well enzyme-labeled plate. Use an enzyme-labeled instrument to detect the absorbance value of the sample at a wavelength of 545 nm to calculate the degree of hemolysis. Taking the degree of hemolysis of the positive control group as 100%, statistical calculations are carried out. Different concentration groups of WGE-EVs did not cause red blood cell rupture and hemolysis phenomenon. The data of each group were compared with the positive control group by t-test, P the values were all < 0.005, which was statistically significant. At the same time, the hemolysis degree of each group of samples was lower than 5%, meeting the international standard, indicating that WGE-EVs has no in vitro hemolytic property.

[0068] Example 9 Evaluation of the in vivo biocompatibility of WGE-EVs Evaluate the in vivo safety of WGE-EVs through a rat gavage administration experiment.

[0069] Take several rats. The experimental group was continuously gavaged with WGE-EVs for 7 days, and the blank control group was given an equal volume of pure water. All rats had normal diet during the experiment and were fasted for 12 hours before the last administration. After the last administration, all rats were euthanized under pentobarbital sodium anesthesia.

[0070] Major organs such as the heart, liver, spleen, lungs, and kidneys were dissected, cleaned with physiological saline, fixed with chloral hydrate, and stained with H&E. Finally, the pathological changes of each tissue and organ were observed under a microscope.

[0071] The results are shown in Figure 11. Microscopic analysis of H&E stained sections of various tissues and organs revealed that, compared with the blank control group, rats administered WGE-EVs by gavage for 7 consecutive days showed no obvious pathological changes or signs of damage in the heart, liver, spleen, lungs, and kidneys. This indicates that WGE-EVs have good in vivo biocompatibility and safety at the experimental dose.

[0072] Example 10: Evaluation of the performance of WGE-EVs in reducing cerebral infarction area in mice The therapeutic performance of WGE-EVs in cerebral ischemia-reperfusion injury was evaluated through a mouse gavage administration experiment.

[0073] Healthy C57BL / 6 mice were selected and anesthetized with isoflurane. The mice were fixed in a supine position, and the bifurcation of the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) was located. The ICA was clamped, and the CCA and ECA were ligated. A suture was inserted through an incision between the ligated CCA and the bifurcation. The arterial clamp at the ICA was opened, and the suture was inserted into the ICA, continuing to penetrate into the cranium until it reached the thinner anterior cerebral artery. At this point, blood flow occlusion of the right middle cerebral artery was achieved. The ICA was ligated, and the sutures were sutured layer by layer. After 30 minutes of embolization, the suture was removed and reperfusion was performed. The mice were placed on a heated pad after surgery and returned to their cages after recovery. Mice in the treatment group received a tail vein injection of WGE-EVs 80 mg / kg. After anesthesia, the brains of the mice were harvested and rapidly frozen at -20°C for 20 minutes. Brain tissue was cut into 2 mm thick sections, fixed with 4% paraformaldehyde for 30 min, stained with 1% toluidine blue solution for 4 min, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0074] The results are as follows Figure 12 As shown, analysis of TTC staining results of brain tissue sections from each group revealed that, compared with the model control group, 80 mg / kg WGE-EVs administered via tail vein injection 12 h significantly reduced the infarct volume and cerebral edema induced by the MCAO model. This indicates that WGE-EVs have the potential to treat cerebral ischemia-reperfusion injury at experimental doses.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The wine Gastrodia elata-derived extracellular vesicles for use in the preparation of a medicament for preventing and / or treating cerebral ischemia-reperfusion injury.

2. Use according to claim 1, characterized in that, The use is to improve the damage of nerve cells caused by hypoxia.

3. The use according to claim 1, characterized in that, The use is to inhibit the polarization of microglia to M1 phenotype.

4. The use according to claim 1, characterized in that, The use is to promote the polarization of microglia to M2 phenotype.

5. The use according to claim 1, characterized in that, The use is to reduce the area of cerebral infarction.

6. The use according to claim 1, characterized in that, The use is to improve the neurological deficit after cerebral ischemia-reperfusion.

7. A method of preparing wine-derived extracellular vesicles, characterized by, The method comprises the following steps: (a) taking wine Gastrodia elata decoction pieces, adding water to reflux extraction, filtering to obtain the extract; (b) concentrating the extract obtained in step (a); (c) centrifuging the concentrated solution, collecting the precipitate, to obtain the wine Gastrodia elata-derived extracellular vesicles.

8. The wine Gastrodia elata-derived extracellular vesicles prepared by the preparation method of claim 7.

9. A pharmaceutical composition, characterized by, The medicament comprises a therapeutically effective amount of the wine Gastrodia elata-derived extracellular vesicles of claim 8, and a pharmaceutically acceptable carrier or excipient, for preventing and / or treating cerebral ischemia-reperfusion injury.

10. The pharmaceutical composition of claim 9, wherein, The effective dose range of the medicament is 20-160 μg / mL, which can significantly improve the cell damage of nerve cells caused by hypoxia, and show good blood compatibility and biological safety in vivo.

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