Application of hypoxia pretreatment extracellular vesicles / miR-27b-3p in ischemic stroke treatment

Abstract

The invention discloses an application of a hypoxia pretreated extracellular vesicle / miR-27b-3p in the treatment of ischemic stroke. The research proves that both the neuron-derived EV and H-EV can promote in-vivo and in-vitro MCAO repair. The two vesicles secreted by neuronal cells can significantly influence phenotypic change and apoptosis of astrocytes, wherein the effect of H-EV is more obvious than that of EV. Besides, the research result shows that H-EV secreted by neurons can regulate a PI3K / AKT signal channel by transmitting miR-27b-3p, so that astrocytes are promoted to be converted from the A1 phenotype to the A2 phenotype. The research of the invention provides a new therapeutic target and strategy for treating ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of hypoxia preconditioned extracellular vesicles / miR-27b-3p in the treatment of ischemic stroke. Background Art

[0002] Stroke is the leading cause of long-term disability and death worldwide, among which ischemic stroke (IS) is the most common. Middle cerebral artery occlusion (MCAO) is an important type of ischemic cerebrovascular disease, which is usually related to intracranial atherosclerosis. Currently, thrombolytic therapy is the main treatment for patients with ischemic stroke. However, reperfusion after ischemia may cause secondary damage to brain tissue and neurological dysfunction, a condition known as cerebral ischemia-reperfusion injury (CIRI). Given that CIRI has a significant impact on the prognosis of cerebrovascular disease, effective strategies must be developed to alleviate CIRI to improve the prevention and treatment of cerebral ischemia.

[0003] Neurons are particularly vulnerable to ischemic stroke. However, the recovery of neurological function depends on the survival of neurons and glial cells. Neurons and glial cells interact closely during brain tissue remodeling, laying the foundation for the effective recovery of neurological function. Astrocytes are the most numerous cells in the central nervous system and play a variety of roles, such as maintaining the blood-brain barrier, regulating environmental balance, and transmitting immune and synaptic signals. Recent studies have shown that astrocytes have two phenotypes, namely A1 and A2, which are similar to macrophages. When a stroke occurs, astrocytes are rapidly activated. Classically activated astrocytes (A1 subtype) exhibit neurotoxic effects by releasing proinflammatory mediators (including specific complement component C3 and inflammatory cytokines), leading to the death of neurons and glial cells. In contrast, alternatively activated astrocytes (A2 subtype) express the specific protein S100A10 and secrete anti-inflammatory cytokines and neurotrophic factors, thereby exerting a neuroprotective effect. Notably, studies have shown that fibroblast growth factor 2 (FGF2) from A2 astrocytes plays a key role in stimulating cell proliferation and differentiation through the PI3K / AKT signaling pathway. Targeting astrocytes for phenotypic switching (A1 / A2 astrocyte polarization) is a promising strategy for the treatment of ischemic stroke.

[0004] Extracellular vesicles (EVs) are small phospholipid bilayer vesicles secreted by cells, containing various biomacromolecules such as nucleic acids, proteins and metabolites, which are essential for intercellular communication and signal transduction. Increasing evidence shows that EVs have the potential to become biomarkers for a variety of pathological conditions. They play an important role in intercellular communication, including neuron-glia crosstalk, tissue development and maintenance, apoptosis, cell homeostasis and synaptic plasticity. Exosomes (EXOs) and microvesicles (MVs) are subtypes of EVs, which vary in biogenesis, vesicle size and composition. Studies have shown that microvesicles are released earlier than exosomes, so that they can quickly transmit information under external stimuli and promote cells to respond promptly to internal changes. In a recent study, Souza et al. used differential centrifugation to isolate extracellular vesicles from cerebral vascular endothelial cells and found that after acute cerebral ischemia, it was microvesicles rather than exosomes that promoted the transfer of mitochondria from endothelial cells to cerebral cortex and hippocampal neurons. These studies suggest that microvesicles may play a more important role after brain injury. In addition, extracellular vesicles have been shown to have the ability to regulate apoptosis pathways, promote cell survival, and reduce neuronal loss. Neuronal cells are very sensitive to hypoxia and nutrient deficiency and play a key role in regulating brain function. However, the specific mechanism of action of neuronal cell-derived extracellular vesicles on cerebral ischemic injury is still unclear and needs further study.

[0005] miRNA (microRNA) is an important component of extracellular vesicles and contributes to local and systemic intercellular communication. Studies have shown that miR-22-3p from extracellular vesicles of mesenchymal stem cells (MSCs) can significantly reduce cerebral ischemia-reperfusion injury and reduce cell apoptosis in vivo. In addition, compared with normoxic conditions, the extracellular vesicles of MSCs pretreated with hypoxia have a stronger inhibitory effect on neuronal apoptosis and a stronger effect on promoting angiogenesis. At present, most researchers believe that pretreating cells with hypoxia can better simulate the in vivo injury environment, thereby improving the efficacy of the extracellular vesicles they secrete. Astrocytes are the most important non-neuronal cell population in the central nervous system and play a vital role in providing structural and functional support to neurons. They release glial transmitters to regulate synaptic activity and participate in the formation and remodeling of synapses. At the same time, signal exchange between neurons and glial cells is essential for maintaining synaptic transmission in the central nervous system (CNS). Abnormalities in this communication can lead to imbalances in a variety of physiological functions, including learning and memory. However, it is still unclear whether hypoxia-preconditioned EVs derived from neurons can exert better effects than normal EVs, especially in terms of the effect on astrocyte phenotype changes. If so, whether it can affect astrocyte phenotype by delivering specific miRNAs, thereby promoting MCAO recovery, has not been reported in the prior art. Summary of the invention

[0006] The purpose of the present invention is to address the above problems and provide an application of hypoxia preconditioned extracellular vesicles / miR-27b-3p in the treatment of ischemic stroke.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of hypoxia pre-treated extracellular vesicles in any of the following:

[0009] (a1) Use in the preparation of a medicament for treating ischemic stroke;

[0010] (a2) Use in the preparation of a preparation for promoting the transformation of astrocyte phenotype from type A1 to type A2;

[0011] The extracellular vesicles are extracellular vesicles derived from neuronal cells.

[0012] The application described above, wherein the hypoxia pre-treated extracellular vesicles reduce the infarct volume of brain tissue after ischemic stroke and improve neurological deficits, inhibit brain cell apoptosis, and promote recovery of motor function in subjects.

[0013] The method for preparing hypoxia pretreated extracellular vesicles comprises the following steps:

[0014] When the neuronal cells reached 60%-80% confluence, the culture medium was replaced with a complete medium prepared with FBS, and after culturing in a hypoxic cell culture incubator for 3.5-4.5 hours, the culture medium was replaced with normoxic conditions for 22-26 hours;

[0015] The conditioned medium is collected, cell debris is removed, the collected conditioned medium is subjected to differential centrifugation, the supernatant is completely discarded, the precipitate is resuspended in PBS, washed in PBS and then centrifuged, the supernatant is discarded, and the hypoxia-pretreated extracellular vesicles are obtained.

[0016] The culture conditions in the hypoxic cell culture incubator were 37°C and 5% CO 2 and 0% O 2 ;

[0017] The normoxic conditions refer to 37°C and 5% CO 2 and 21% O 2 ;

[0018] The differential centrifugation means that the collected conditioned medium is centrifuged at 300×g and 4°C for 10 minutes, then at 2000×g and 4°C for 10 minutes, and then at 10,000×g and 4°C for 30 minutes.

[0019] The miR-27b-3p in the hypoxic preconditioning extracellular vesicles promoted the transformation of astrocytes from the A1 phenotype to the A2 phenotype, thereby enhancing recovery after ischemic stroke.

[0020] The hypoxic preconditioned extracellular vesicles exert neuroprotective effects through the miR-27b-3p / PI3K / AKT signaling pathway, promote the transformation of type A1 astrocytes to type A2 astrocytes, and ultimately help recovery after ischemic stroke; hypoxic preconditioned extracellular vesicles regulate the PI3K / AKT signaling pathway by transmitting miR-27b-3p.

[0021] The second aspect of the present invention provides the use of miR-27b-3p in any of the following:

[0022] (b1) Use in the preparation of a medicament for treating ischemic stroke;

[0023] (b2) Use in the preparation of a preparation for promoting the transformation of astrocyte phenotype from type A1 to type A2.

[0024] miR-27b-3p inhibits the PI3K / AKT signaling pathway activated by OGD / R, reduces cerebral infarction area and neurological deficits after ischemic stroke, and inhibits cell apoptosis.

[0025] The nucleotide sequence of the miR-27b-3p is 5'-UUCACAGUGGCUAAGUUCUGC-3'.

[0026] The beneficial effects of the present invention are:

[0027] The present study confirmed that both neuron-derived EVs and H-EVs can promote the repair of MCAO in vivo and in vitro. Both vesicles secreted by neuronal cells can significantly affect the phenotypic changes and apoptosis of astrocytes, among which the effect of H-EVs is more obvious than that of EVs. In addition, our results show that H-EVs secreted by neurons can regulate the PI3K / AKT signaling pathway by transmitting miR-27b-3p, thereby promoting the transformation of astrocytes from A1 phenotype to A2 phenotype. The present study provides new therapeutic targets and strategies for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The extraction and characterization of EVs and H-EVs in neuronal cells PC12 are shown: (A) Particle size analysis of EVs and H-EVs by NTA; (B) Western blot analysis of three protein markers of EVs and H-EVs (Calnexin, TSG101 and CD63); (C) Morphological images of EVs and H-EVs under TEM.

[0029] Figure 2 Figure 4 shows increased apoptosis and activation of A1 / A2 subtype astrocytes after focal cerebral ischemia: (AB) Cerebral blood flow was monitored by cerebral blood flow laser spot before MCAO and after reperfusion; * indicates significant difference between Sham group and MCAO group (n=3); (CE) Representative images and quantitative analysis of protein immunoblotting of two astrocyte subtypes after MCAO (C3: A1 astrocytes; S100A10: A2 astrocytes) (n=3); (FI) Representative images and quantitative analysis of protein immunoblotting of cell apoptosis markers (Caspase-3, Bcl-2 and Bax) after MCAO (n=3); (*p<0.05; **p<0.01; ***P<0.001; ns, not statistically significant).

[0030] Figure 3Figure 3: Injection of EV and H-EV improves ischemic brain injury in rats; (A) Fluorescence images of brain slices were taken using a live imager after injection of EV and H-EV; (BC) TTC staining and quantification of brain infarct volume; EV and H-EV significantly reduced the brain infarct volume in MCAO rats (n=3, *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant); (DG) Delivery of EV and H-EV prevents ischemia-induced motor coordination disorders; The results of the modified neurological severity score, corner turning test, balance beam test, and paw slip were measured 1 day before stroke and 2, 5, 7, and 14 days after stroke (n=4-9, *p<0.05; **P<0.01, SHAM vs PBS; #p<0.05; ##P<0.01, PBS vs EV; &p<0.05; &&P<0.01, PBS vs H-EV; ns, not statistically significant).

[0031] Figure 4 It was shown that in vivo, EVs and H-EVs can promote the phenotype transition of astrocytes from type A1 to type A2;

[0032] (AC) Representative images and quantitative analysis of western blot of two astrocyte subtypes (C3: A1 astrocytes, S100A10: A2 astrocytes) in the SHAM, PBS, EV and H-EV groups (n=3); (DG) Representative images and quantitative analysis of immunofluorescence of two astrocyte subtypes in the SHAM, PBS, EV and H-EV groups (n=3 / group, C3 / S100A10: red, GFAP: green, DAPI: blue); (*P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant).

[0033] Figure 5 The apoptosis of astrocytes after MCAO was detected by immunoblotting; (AD) Representative images and quantitative analysis of apoptosis markers (Caspase-3, Bcl-2 and Bax) in the SHAM, PBS, EV and H-EV groups (n=3); (*P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant).

[0034] Figure 6Figure 3 showed that miR-27b-3p was upregulated in H-EV and transported to astrocytes; (A) Volcano plot of the difference in miRNA expression in the H-EV group compared with the EV group; the horizontal axis represents multiple miRNAs in the H-EV group compared with the EV group, and the vertical axis represents the Log10 of the P value; (B) qRT-PCR was performed to verify the difference in miR-27b-3p expression levels between EV and H-EV (n=3); (C) qRT-PCR verified the difference in miR-27b-3p expression levels in CTX-TNA2 astrocytes in the PBS group, EV group and H-EV group (n=3); (D) Immunofluorescence showed that PKH26-labeled H-EV was transferred to astrocytes; *p<0.05; **P<0.01; P<0.001; ns, not statistically significant).

[0035] Figure 7 It was shown that miR-27b-3p promotes the phenotypic switch of astrocytes from A1 to A2 in vitro;

[0036] (AC) Representative images and quantitative western blot analysis of two astrocyte subtypes (C3: A1 astrocytes, S100A10: A2 astrocytes) in CON+NC, OGD+NC and OGD+27b mimic groups (n=3); (DG) Representative images and quantitative immunofluorescence analysis of two astrocyte subtypes in CON+NC, OGD+NC and OGD+27b mimic groups (n=3 / group, C3 / S100A10: red, GFAP: green, DAPI: blue); (HK) Representative images and quantitative western blot analysis of apoptosis markers (Caspase-3, Bcl-2 and Bax) in CON+NC, OGD+NC and OGD+27b mimic groups (n=3); (*P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant).

[0037] Figure 8 The effect of miR-27b-3p on the activation of the PI3K / AKT pathway in CTX-TNA2 astrocytes detected by immunoblotting is shown. (A-C) Representative images and quantitative analysis of PI3K, P-PI3K, AKT, and P-AKT protein immunoblotting in CON+NC, OGD+NC, and OGD+27b mimic groups (*P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant).

[0038] Fig. 9Figure 3 miR-27b-3p reduced brain injury area and neurological deficits after MCAO in mice. (A) After miR-27b-3p treatment, qRT-PCR verified that miR-27b-3p expression in rat brain tissue increased (n=3). (BC) TTC staining and infarct volume quantification. miR-27b-3p significantly reduced the infarct volume in MCAO rats (n=3, *p<0.05; **p<0.01; **p<0.001; ns, not statistically significant). (DG) miR-27b-3p treatment improved ischemia-induced motor coordination deficits. The results of modified neurological severity score, angle test, balance beam test, and paw slip were measured 1 day before stroke and 2, 5, and 7 days after stroke (n=6). (*p<0.05; **p<0.01, SHAM vs MCAO+NC; #p<0.05; ##P<0.01, MCAO+NC vs MCAO+27b simulation; ns, not statistically significant).

[0039] Fig.10 It was shown that miR-27b-3p promoted the transformation of rat astrocytes from A1 to A2 after MCAO and had an anti-apoptotic effect; (AC) Representative images of protein immunoblotting and quantitative analysis of two astrocyte subtypes (C3: A1 astrocytes, S100A10: A2 astrocytes) in the SHAM, MCAO+NC and MCAO+27b mimic groups (n=3); (DG) Representative images and Western blot quantitative analysis of apoptosis markers Caspase-3, Bcl-2 and Bax proteins in the SHAM, MCAO+NC and MCAO+27b mimic groups (n=3); (*p<0.05; **P<0.01, SHAM vs MCAO+NC; #p<0.05; ##P<0.01, MCAO+NC vs MCAO+27b mimic; ns, not statistically significant). DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0042] Example 1

[0043] 1. Main experimental reagents

[0044]

[0045]

[0046] 2. Methods

[0047] 2.1 Experimental animals

[0048] 2.1.1 Source of experimental animals

[0049] This study used male Sprague Dawley (SD) rats aged 8-10 weeks, weighing 200-220 g. The experimental animals used in this experiment were from the Animal Experiment Center of Chongqing Medical University. All rats were kept in ventilated cages at a temperature of 20-25°C, a relative humidity of 30-50%, a light / dark cycle of 12 hours, and free access to food and water. In accordance with the Animal Management Regulations and the Animal Management Regulations of Chongqing Medical University, all operations were carried out after obtaining approval from the Animal Ethics Committee (No.: IACUC-CQMU-2023-09067).

[0050] 2.1.2 Experimental Animal Grouping

[0051] To evaluate the therapeutic effects of EVs (extracellular vesicles) and H-EVs (hypoxia-pretreated extracellular vesicles) on rats after MCAO (middle cerebral artery occlusion), the animals were randomly divided into four groups: (1) Sham group; (2) MCAO control group (PBS group), in which 6 μl PBS was injected intracerebroventricularly after MCAO as a control; (3) EV group and (4) H-EV group, in which an equal volume (6 μl) of EVs or H-EVs was injected intracerebroventricularly 30 minutes after MCAO model establishment (a total of approximately 800 ng EVs or H-EVs were injected into each rat).

[0052] To verify whether H-EVs exert beneficial effects in vivo through miR-27b-3p delivery, rats were randomly divided into three groups: (1) Sham surgery group; (2) MCAO control group, in which 6 μl of negative control mimic (mimic NC) was administered ICV after MCAO as a control; (3) MCAO+27b mimic group, in which an equal volume (6 μl) of miR-27b-3p mimic (mimic) was administered ICV 30 minutes after MCAO.

[0053] The mice were kept in separate cages and fasted for 12 hours before surgery, with free movement and drinking water.

[0054] 2.1.3 Animal model establishment

[0055] The abdominal skin of SD rats was carefully disinfected with iodine, and then 2% sodium pentobarbital (30 mg / kg) was injected with a 5 ml syringe 1 cm to the left of the midline of the lower abdomen. A heating pad was used during the operation to maintain the body temperature at 37.0 ± 0.5 degrees Celsius. Subsequently, the rats were fixed in a supine position and their limbs and teeth were gently tied. After disinfection with iodine, the hair of the neck was shaved and the middle of the neck was cut with fine scissors. The muscle tissue was carefully separated with fine forceps, and the left common carotid artery (LCCA), left external carotid artery (LECA) and left internal carotid artery (LICA) were exposed and carefully separated in turn, paying attention to separating the vagus nerve attached to the LCCA and avoiding excessive traction. The LCCA and LECA were then ligated, a slipknot was placed at the distal end of the LCCA, and the LICA was temporarily occluded with an artery clamp. A small incision was made on the LCCA, and a silicone thread plug was inserted through the incision and advanced to the middle cerebral artery until the correct position was confirmed and fixed. The head of the silicone plug was pushed forward about 1.8 cm until resistance was felt, indicating that the target position had been reached. After ischemia lasted for 1 hour, the plug was withdrawn, the blood vessels were ligated at the incision, and reperfusion was performed. The muscles and skin were immediately sutured with interrupted sutures, disinfected with iodine, and placed on an electric blanket. After careful observation, the rats that woke up and were in good condition were placed in a clean cage and fed with normal water and food; rats in poor condition continued to be observed. In the sham operation group, rats only received neck incisions and structural exposure, without any occlusion or plugging procedures. After modeling, the neurological function of the rats was scored according to the Zea-Longa scoring criteria. If the rat's Zea-Longa score was between 1 and 3, it was included in the study. If the rat's Zea-Longa score was 0 or 4, or the rat died prematurely, it was not included in the study.

[0056] The specific scoring criteria are as follows:

[0057]

[0058] 2.1.4 Animal tissue collection and processing

[0059] (1) Inject 2% sodium pentobarbital intraperitoneally to anesthetize the rat. Lift the ribs and quickly open the chest with surgical scissors. To gain a wider field of vision, clamp the sternum with a vascular clamp and fold it over to expose the heart.

[0060] (2) Insert the perfusion needle into the left ventricle (at the apex) and perfuse pre-cooled saline at a constant rate.

[0061] (3) Use a small pair of surgical scissors to make a small incision in the right atrium to allow blood and perfusion fluid to flow out.

[0062] (4) Use a 50 ml syringe to evenly inject 50 ml of normal saline. If the color of the liquid discharged from the right atrium becomes colorless and the color of the liver turns white, it means that the blood is fully perfused.

[0063] (5) Use a new 50 ml syringe to draw 40 ml of pre-cooled 4% paraformaldehyde (4% PFA, pH 7.4), and then perfuse at a constant speed.

[0064] The performance of successful perfusion is as follows: the mouse has neck bending and stiffness, limb twitching, tail upturned, etc. This means that the fixation is complete and the sample can be taken. Use scissors to cut the perfused rat along the head and neck, and then use forceps to peel off the rat skull and completely remove the rat brain. If the tissue is used for protein immunoblotting experiments, put the tissue block into liquid nitrogen for quick freezing (10-30 seconds), and then transfer it to a -80℃ refrigerator for storage. If the tissue is used for immunofluorescence experiments, place it in a freshly prepared 4% paraformaldehyde in a 4℃ refrigerator and fix it overnight. On this basis, dehydrate it with 10%, 20%, and 30% sucrose solutions in sequence until the brain sinks naturally. Then store it in a -80℃ freezer. Use Leica cryostat for frozen sectioning. When slicing, the blade fixes the tissue in the coronal position, cut the target tissue into 10μm thick tissue slices and stick them on adhesive slides. After drying the slices at room temperature for 3-4 days, put them in a slice box and store them at -20℃.

[0065] 2.1.5 Modified Neurological Deficit Score (mNSS)

[0066] The modified neurological deficit score (mNSS) was used to evaluate the nervous system function of the rats in each group 1 day before surgery and 2, 5, 7, and 14 days after surgery.

[0067] The specific scoring criteria are as follows:

[0068]

[0069]

[0070] The higher the score, the more severe the neurological impairment.

[0071] 2.1.6 Behavioral testing

[0072] The corner turning test was performed according to the method of Li et al. The rat was placed in a 30° corner. When the rat approached the corner, the whiskers on both sides would first touch the plate. At this time, the rat would move forward or tilt upward, then turn 180°, facing one end of the opening, and finally turn to the left or right. Only when the rat stood up completely on its hind legs along the angle and then turned to one side was it recorded as a trial. The test was repeated 10 times with at least 30 seconds between each test, and then the percentage of right turns was calculated. Corner turning test score = [(R) / (R+L)]×100; R: right turn; L: left turn.

[0073] Motor coordination and balance in rats were assessed by the balance beam test. The test requires the rat to remain upright and cross an elevated narrow beam to a safe platform. Motor coordination was quantified by measuring the time required for the rat to cross the balance beam and the number of paw slips that occurred during the measurement. The test was considered successful if the rat did not stall while crossing the balance beam. The experimenter was required to record the time for three tests and calculate the average as the result.

[0074] 2.1.7 Detection of mouse brain blood flow using laser speckle blood flow imaging system

[0075] At 24 hours after ischemia, cortical blood flow in each rat was assessed by skull constant laser speckle imaging. The rat skull was fully exposed under anesthesia, and ipsilateral and contralateral cortical blood flow were assessed. Each analysis was performed at the same region of interest, and the average measurements were analyzed simultaneously.

[0076] 2.1.8 TTC staining

[0077] TTC is a lipid-soluble photosensitive complex and a proton receptor for the pyridine-nucleoside structural enzyme system in the respiratory chain. It reacts with dehydrogenase in normal tissues to present a red color. It reacts with dehydrogenase in normal tissues to present a red color, while the dehydrogenase activity in ischemic tissues is reduced and cannot react with TTC, so no changes occur and it presents a pale white color. TTC staining was performed 7 days after MCAO according to the method described previously. Briefly, rats were anesthetized and killed, and brain tissues were quickly dissected and frozen at minus 20°C for 20 minutes, and then cut into 5 consecutive coronal sections of approximately 2 mm. The sections were stained with 2% TTC solution at 37°C in the dark for 30 minutes, then fixed in 4% paraformaldehyde solution for 2 hours and photographed. The infarct area was quantified by Image-J analysis software. The infarct volume was obtained by multiplying the total infarct area by the thickness of the brain slice. The percentage of infarct volume was calculated as follows: ipsilateral infarct volume / contralateral hemisphere volume × 100%.

[0078] 2.1.9 Intracerebroventricular Microinjection

[0079] For intraventricular injection, anesthetized rats were placed in a stereotaxic apparatus and a burr hole was made 0.9 mm posterior to bregma, 1.5 mm lateral to the sagittal suture, and 3.6 mm deep. Thirty minutes after the suture plug was removed, intraventricular injection was performed at a rate of 0.8 μl / min with 6 μl EV, 6 μl H-EV, 6 μl miR-27b-3p mimic NC, 6 μl miR-27b-3p mimic, or the same volume of PBS, and the needle was left in place for 10 minutes. The amount of EV administered in each rat was equal, with a total protein amount in the range of 800 ng. The amount of mimic administered in each rat was 100 pmol.

[0080] 2.2 In vitro experiments

[0081] 2.2.1 Cell culture

[0082] Immortalized rat astrocyte cell line CTX-TNA2 was purchased from Shanghai Saibaikang Biotechnology Co., Ltd. In order to simulate neuronal cells, this experiment used the highly differentiated rat adrenal medullary pheochromocytoma cell line PC12, which was purchased from the American Cell Bank. CTX-TNA2 and PC12 cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator at 37°C containing 5% CO2, and cells in the logarithmic growth phase were used for subsequent experiments.

[0083] 2.2.2 Cell passaging and cryopreservation

[0084] After 2 days, when 80% to 90% of the cells have attached to the wall, the cells can be digested. Use a pipette to aspirate the culture medium in the T25 bottle, rinse twice with 2ml 0.01mol / LPBS, remove the residual culture medium, add 1ml 0.25% trypsin and digest at 37℃ for 1 minute, observe under a microscope, and when the cells become round, tap the culture bottle, add 2ml DMEM complete medium to stop digestion, gently blow and mix with the pipette tip 10-15 times, and then subculture them into a new T25 culture bottle at a ratio of 1:2.

[0085] When the cells cover the bottom of the bottle, digest the cells according to the cell passage method, add DMEM complete medium to stop digestion, centrifuge at 1000rpm for 5 minutes, remove the medium and trypsin in the supernatant, add serum-free cell freezing solution, and mix gently with a pipette. Store at 4℃ for 30 minutes, -20℃ for 2 hours, -80℃ for 24 hours, and finally store in a liquid nitrogen tank.

[0086] 2.2.3 Cell experiment grouping

[0087] To verify whether H-EV affects the phenotypic changes of CTX-TNA2 astrocytes through the delivery of miR-27b-3p, the cells were divided into three groups: (1) CON+NC group, 6 μl mimicNC was added to the normally cultured CTX-TNA2 astrocytes; (2) OGD+NC group, 6 μl mimic NC was added after 4 h of oxygen-glucose deprivation, and then cultured under normal conditions for 24 h; (3) OGD+27b mimic group: an equal volume (6 μl) of miR-27b-3p mimic was added after 4 h of oxygen-glucose deprivation, and then cultured under normal conditions for 24 h.

[0088] 2.2.4 Establishment of cellular oxygen-glucose deprivation model

[0089] To establish the in vitro ischemia-reperfusion model, the

[27] The oxygen-glucose deprivation / reperfusion model (OGD / R) was induced. When the cells reached 80-90% confluence, they were washed twice with PBS. At the beginning of OGD / R, the standard culture medium was replaced with glucose-free DMEM and the cells were kept under hypoxic conditions (95% N) at 37°C. 2 and 5% CO 2 ) After 4 h, the cells were cultured in high-glucose DMEM to terminate OGD, and then reoxygenated under normoxic conditions for 24 h.

[0090] EV uptake by CTX-TNA2 astrocytes

[0091] According to the instructions of the reagent manufacturer, EVs were fluorescently labeled with PKH26. In brief, the prepared PKH26 dye working solution (freshly prepared and used) was added to the extracellular vesicles, vortexed for 1 minute and incubated for 10 minutes, and an appropriate amount of 0.01 mol / L PBS was added to the incubated extracellular vesicle dye complex and mixed evenly. The extracellular vesicles were extracted again according to the extracellular vesicle extraction method to remove the free dye, and finally an appropriate amount of 0.01 mol / L PBS was used to resuspend the precipitate, which was the stained extracellular vesicle. These PKH26-labeled EVs were added to CTX-TNA2 astrocytes and cultured for 24 hours, and then the cells were washed with PBS and fixed in 4% paraformaldehyde. The absorption of PKH26-labeled EVs by CTX-TNA2 astrocytes was then observed using an inverted fluorescence microscope. 2.2.6 Cell transfection of miR-27b-3p mimics

[0092] miR-27b-3p mimic and mimic NC were purchased from Beijing Qingke Biotechnology Co., Ltd. They were dissolved and diluted according to the instructions provided by the manufacturer. After OGD, cell transfection was performed. According to the manufacturer's protocol, 100 nM aliquots of miR-27b-3p mimic or mimic NC were transfected into CTX-TNA2 astrocytes using TSnanofect V2 transfection reagent.

[0093] 2.3 Isolation and identification of EVs and H-EVs

[0094] Neuronal PC12 cells were cultured under normal conditions: in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, at 5% CO. 2 Cultured in a constant temperature 37°C incubator.

[0095] When the PC12 neurons reached 80% confluence, the culture medium was replaced with complete culture medium containing 10% FBS without exosomes and the cells were cultured in normoxia (37°C, 5% CO 2 and 21% O 2 ) for 24 hours in a cell culture incubator or in hypoxia (37°C, 5% CO 2 and 0% O 2 ) After 4 hours in the cell culture incubator, the cells were cultured under normal conditions for 24 hours. The conditioned medium was collected and the cell debris was removed. The collected conditioned medium was centrifuged at 300×g and 4°C for 10 minutes, then at 2000×g and 4°C for 10 minutes, and then at 10,000×g and 4°C for 30 minutes. The supernatant was completely discarded, and the pellet was resuspended in PBS, washed once in PBS, and centrifuged again at 10,000×g and 4°C for 30 minutes. Finally, the supernatant after centrifugation was discarded and resuspended in PBS to obtain EV and H-EV suspensions. EV and H-EV were either stored at -80°C or used immediately in downstream experiments.

[0096] To characterize EVs and H-EVs, the size and concentration of EVs were assessed by nanoparticle tracking analysis (NTA) equipment, and the morphological characteristics of EVs were observed by transmission electron microscopy (TEM). EV markers such as Calnexin, TSG101, and CD63 were detected by immunoblotting.

[0097] 2.4 Immunofluorescence

[0098] 2.4.1 Tissue immunofluorescence

[0099] 2.4.2 Cell Immunofluorescence

[0100] 2.5 Immunoblotting

[0101] 2.5.1 Preparation of protein samples

[0102] 2.5.2 BCA method for protein concentration measurement

[0103] 2.5.3 Preparation of SDS-PAGE gel

[0104] 2.5.4SDS-PAGE electrophoresis

[0105] 2.5.5 Transfer and blocking

[0106] 2.5.6 Antibody incubation

[0107] 2.5.7 ECL Development

[0108] 2.6 Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR)

[0109] 2.6.1 Extraction of small RNA from cells

[0110] 2.6.2 miRNA quality and concentration detection

[0111] 2.6.3 RNA Reverse Transcription to cDNA

[0112] 2.6.4 Real Time-PCR

[0113] (1) Prepare the following mixture in a qPCR tube:

[0114]

[0115] (2) Perform qPCR reaction according to the following conditions

[0116]

[0117] 2.6.5 Primer design and synthesis

[0118] The nucleotide sequence of miR-27b-3p mimic (SEQ ID NO.1):

[0119] 5'-UUCACAGUGGCUAAGUUCUGC-3'.

[0120]

[0121] 2.7 Statistical analysis

[0122] All experimental data were statistically analyzed to evaluate the significance of differences between groups. Data are expressed as mean ± standard deviation (Mean ± SD). In this study, we used ImageJ and Graph PadPrism8.0 for statistics, and used unpaired t-test to analyze the differences between the two groups, with a significance level set at p < 0.05.

[0123] 3. Results

[0124] 3.1 Extraction and identification of EVs and H-EVs from PC12 neurons

[0125] EVs and H-EVs were isolated by high-speed centrifugation from exosome-free culture media under normal and OGD / R conditions. They were then analyzed using NTA, WB, and TEM and used in subsequent experiments after characterization to prevent contamination by cellular components or other vesicles. NTA showed similar size distributions in normoxic and hypoxic groups (median 219.2 nm vs.

[0126] 223.5nm)( Figure 1 A). WB showed the same surface markers in both groups, with typical EV markers (TSG101 and CD63) enriched, while calnexin (a typical negative marker) was not detected ( Figure 1 B). TEM shows that the vesicles exhibit obvious biconcave morphology ( Figure 1 C). No morphological differences in size, shape, or electron density were observed between the two groups.

[0127] 3.2 Increased polarization and apoptosis of A1 / A2 subtype astrocytes after focal cerebral ischemia

[0128] To test the success of the model preparation, we used a laser speckle imaging system to detect changes in cerebral blood flow. Before the establishment of MCAO (baseline), the cerebral blood flow of rats was the highest, and it decreased to about 39% of the baseline value 7 days after ischemia ( Figure 2 AB), indicating that the MCAO model was successfully established.

[0129] Recent studies have shown that ischemic stroke can also induce two different polarization states of reactive astrocytes, namely the neurotoxic A1 type and the neuroprotective A2 type. In addition, C3 and S100A10 are considered to be markers of A1 and A2 astrocytes, respectively.

[0130] In this study, Western blotting showed that ( Figure 2 CI) After cerebral ischemia, the expressions of C3 and S100A10 increased, the expressions of pro-apoptotic proteins (Caspase-3, Bax) increased, and the expression of anti-apoptotic protein Bcl-2 decreased.

[0131] In summary, ischemic stroke can induce astrocytes to transform into two different phenotypes and promote apoptosis.

[0132] EV and H-EV administration reduces infarct volume and improves neurological deficits after MCAO

[0133] To determine the effects of neuronal cell-derived extracellular vesicles on focal cerebral ischemia, extracellular vesicles isolated by high-speed centrifugation were injected into the lateral ventricle of the injured side of rats. We labeled EV / H-EV with DIR fluorescent dye and evaluated its uptake into brain tissue by in vivo imaging ( Figure 3 A) The results show that extracellular vesicles circulate in the cerebrospinal fluid and are widely expressed in brain tissue.

[0134] In this study, to investigate whether H-EV has a more beneficial effect on motor function after MCAO than EV, we first evaluated the functional recovery of rats in the SHAM, PBS, EV, and H-EV groups using multiple behavioral methods. The results showed that both EV and H-EV groups showed better functional improvement compared with PBS rats. However, in this study, we noticed that the TTC improvement in the H-EV group was more significant than that in the EV group ( Figure 3 BC).

[0135] Neurobehavioral assessments included mNSS scores, corner turning test, balance beam test, and paw slip counts. EV delivery resulted in better test performance in all behavioral tests compared with the PBS group. In the mNSS test, the scores of rats in the PBS group were significantly higher than those in the sham group at 2, 5, 7, and 14 days after reperfusion ( Figure 3 D). After reperfusion, the scores of both the EV and H-EV groups decreased, but the score of the H-EV group was significantly lower. Figure 3 E), rats in the PBS group showed obvious bias, severe spatial orientation and cognitive function defects. The EV group and H-EV group showed some improvement, and the H-EV group performed better. In the balance beam test and the claw slip test ( Figure 3 FG), the PBS group showed obvious instability, frequent claw slipping, indicating a certain degree of nervous system dysfunction or decreased motor coordination ability. The EV and H-EV groups showed better balance ability, and the improvement in the H-EV group was more obvious.

[0136] However, these beneficial effects did not appear immediately on the second day after stroke induction, and significant improvements were not seen until the 7th and 14th days. This suggests that EV improvement of neurological dysfunction is a time-dependent process. These behavioral results are also consistent with the results of TTC staining. The infarct area in the PBS group showed obvious paleness or colorlessness, and the infarct area was larger. The color of the infarct area in the EV and H-EV groups was partially restored, showing red or light red, indicating that brain tissue ischemic damage was improved, and drug intervention may have played a positive role by reducing the infarct area or promoting neuroprotection. Similarly, the H-EV group showed better therapeutic effects than the EV group. In summary, these results show that both EV and H-EV administration can promote functional behavioral recovery after MCAO in rats, and these beneficial effects are more obvious in the H-EV group than in the EV group.

[0137] EV and H-EV promote the transformation of astrocyte phenotype from A1 to A2 in vivo

[0138] Given that astrocytes can exhibit two distinct phenotypes, we sought to determine whether EV and H-EV administration could induce a phenotype shift in astrocytes from A1 to A2 after MCAO. Seven days after MCAO, we performed western blot analysis using representative A1-associated C3 and A2-associated S100A10 markers to examine protein expression levels ( Figure 4 AC). The results showed that the expression levels of C3 and S100A10 in the PBS group were increased compared with the sham group. The expression of C3 in the EV group and the H-EV group was significantly lower than that in the PBS group, while the expression of S100A10 was significantly higher than that in the PBS group. It is worth noting that compared with the EV group, the expression of C3 in the H-EV group showed a downward trend, while the expression of S100A10 in the H-EV group was higher than that in the EV group. To further confirm our findings, we performed immunofluorescence on brain sections ( Figure 4 DG), and the results were consistent with those of western blot. Our results showed that both EV and H-EV promoted the transformation of astrocytes from A1 phenotype to A2 phenotype. However, the promoting effect of H-EV was more obvious than that of EV.

[0139] 3.5 EV and H-EV administration inhibits apoptosis after focal cerebral ischemia

[0140] Also 7 days after MCAO, we used WB to detect the expression levels of pro-apoptotic proteins (Bax and Caspase-3) and anti-apoptotic proteins (Bcl-2) in brain tissue. The results showed that compared with the SHAM group, the pro-apoptotic proteins (Bax and Caspase-3) in the PBS group were significantly increased, while the anti-apoptotic protein (Bcl-2) was significantly decreased. Compared with the PBS group, both EV and H-EV treatments significantly reduced the expression levels of pro-apoptotic proteins (Bax and Caspase-3) and increased the expression level of anti-apoptotic protein (Bcl-2). However, compared with EV treatment, H-EV treatment had a more obvious effect on promoting the expression of anti-apoptotic proteins (Bcl-2) and inhibiting the expression of pro-apoptotic proteins (Bax and Caspase-3) ( Figure 5 AD). Taken together, these results indicate that both EV and H-EV administration can promote anti-apoptotic effects after MCAO in rats, and these beneficial effects in the H-EV group are more pronounced than those in the EV group.

[0141] 3.6 miR-27b-3p is upregulated in H-EVs and can be transported to astrocytes

[0142] Comparison of the in vivo efficacy of EV and H-EV revealed that H-EV was superior to EV in terms of motor function, astrocyte polarization, and apoptosis. Since many studies have found that miRNA plays an important role in its efficacy, we performed miRNA sequencing on EV and H-EV and found that two miRNAs were downregulated (miR-451-5p and miR-486) ​​and one miRNA was upregulated (miR-27b-3p) in H-EV compared with EV ( Figure 6 A). We then selected the only upregulated miR-27b-3p to verify whether it could alter CTX-TNA2 astrocyte polarization. First, we confirmed the upregulation of miR-27b-3p in H-EVs by qRT-PCR ( Figure 6 B). Then PBS, EV and H-EV were added to CTX-TNA2 astrocytes to extract their RNA, and the expression level of miR-27b-3p in astrocytes was verified by qRT-PCR ( Figure 6 C). The results showed that the expression level of miR-27b-3p in the H-EV group was significantly increased compared with that in the PBS group and the EV group.

[0143] To determine whether CTX-TNA2 astrocytes took up EVs, EVs were labeled with PKH26 dye and co-cultured with CTX-TNA2 cells for 24 h. Fluorescence microscopy was used to analyze the uptake of EVs by CTX-TNA2 astrocytes ( Figure 6D). The results showed that CTX-TNA2 astrocytes could successfully take up EVs, and that EVs were mainly distributed inside the cells rather than on the cell surface.

[0144] 3.7H-EVs promote the transformation of astrocytes from A1 to A2 by transporting miR-27b-3p and have an anti-apoptotic effect

[0145] To determine whether miR-27b-3p has a therapeutic effect similar to the changes in astrocyte phenotype observed in vivo by H-EV, we provided a hypoxic and low-glucose environment. The cells were placed in a hypoxic environment for 4 hours to induce the CTX-TNA2 astrocyte OGD model to simulate MCAO in vivo, and then co-cultured with mimic NC or miR-27b-3p mimic for 24 hours at room temperature and normoxia. The cells were collected and the cell proteins were extracted. The expression levels of C3 and S100A10 in CTX-TNA2 astrocytes were detected by western blotting. The results showed that compared with the OGD+NC group, the expression level of C3 in the OGD+27b mimic group was significantly decreased, and the expression level of S100A10 was significantly increased ( Figure 7 AC). Immunofluorescence ( Figure 7 DG) also confirmed the results of western blot. Taken together, these results indicate that H-EV can convert the phenotype of CTX-TNA2 astrocytes from A1 to A2 in vitro via miR-27b-3p, which is consistent with the results observed in vivo.

[0146] Similarly, in vitro, to determine whether the anti-apoptotic effect of miR-27b-3p is similar to the results of H-EV in vivo, we collected cells after OGD / R and extracted their proteins to evaluate the therapeutic effect. The expression levels of pro-apoptotic proteins (Bax and Caspase-3) and anti-apoptotic proteins (Bcl-2) in CTX-TNA2 astrocytes were detected by immunoblotting ( Figure 7 HK). The results showed that compared with the OGD+NC group, the pro-apoptotic proteins (Bax and Caspase-3) in the OGD+27b mimic group were significantly reduced. At the same time, the expression level of the anti-apoptotic protein (Bcl-2) was significantly increased. These results are consistent with those observed in vivo, indicating that H-EV can inhibit cell apoptosis in vitro through miR-27b-3p.

[0147] Effect of miR-27b-3p on PI3K / AKT pathway activation in CTX-TNA2 astrocytes

[0148] Studies have shown that the PI3K / AKT signaling pathway has a neuroprotective effect on cerebral ischemia and is an important signaling pathway that regulates cell growth, differentiation and migration, and plays a key role in regulating the phenotypic changes of astrocytes. Based on this finding, we continued to detect the expression levels of P-PI3K, PI3K, P-AK and AKT in the CON+NC, OGD+NC and OGD+27b mimic groups by immunoblotting ( Figure 8 AC). The results showed that OGD / R significantly activated the PI3K / AKT pathway, including P-PI3K and P-AKT. Compared with the CON+NC group, the expression levels of P-PI3K and P-AKT in the OGD+NC group were significantly increased, while after taking miR-27b-3pmimic, the expression levels of P-PI3K and P-AKT were significantly inhibited. These findings suggest that miR-27b-3p has an inhibitory effect on the PI3K / AKT signaling pathway activated by OGD / R.

[0149] 3.9miR-27b-3p reduces cerebral infarction area and neurological deficits in rats after MCAO

[0150] To determine the effect of miR-27b-3p on focal cerebral ischemia-reperfusion, the MCAO model was induced in rats, and then miR-27b-3p was injected into the lateral ventricle. The expression of miR-27b-3p in brain tissue was detected by qRT-PCR 7 days after MCAO. The results showed that miR-27b-3p treatment significantly increased the expression of miR-27b-3p in brain tissue ( Fig. 9 A). TTC staining results showed that the infarct area in the MCAO+27b mimic group was about 33% smaller than that in the MCAO+NC group. This result proves that miR-27b-3pmimics can reduce the infarct area in rats with cerebral ischemia and has a protective effect against cerebral ischemia ( Fig. 9 BC). We also conducted a series of neurobehavioral assessments on the three groups of rats one day before modeling and 2, 5, and 7 days after modeling, including mNSS scores, corner turning test, balance beam test, and paw slip count ( Fig. 9 DG). The results showed that the delivery of miR-27b-3p resulted in improved performance in all behavioral tests analyzed, and these results were consistent with the results of H-EV administration in vivo, providing strong evidence for the key role of miR-27b-3p in the recovery of MCAO rats. In conclusion, treatment with miR-27b-3p has a positive effect on the recovery of neurological function and motor coordination after ischemic stroke.

[0151] 3.10miR-27b-3p promotes the transformation of rat astrocytes from A1 to A2 after MCAO and has an anti-apoptotic effect

[0152] In addition, we used WB analysis to evaluate the changes in astrocyte phenotype in brain tissue of MCAO rats. The results showed that ( Fig.10 AC), compared with the MCAO+NC group, the expression of C3 in the MCAO+miR-27b-3p mimic group was significantly decreased, and the expression of S100A10 was significantly increased. The results confirmed that miR-27b-3p indeed promoted the transformation of astrocytes from A1 phenotype to A2 phenotype in MCAO rats. At the same time, WB was used to detect the expression changes of apoptosis-related markers ( Fig.10 DG), the results showed that compared with the MCAO+NC group, the expression levels of pro-apoptotic proteins (Bax and Caspase-3) in the MCAO+miR-27b-3p mimic group were significantly reduced, and the expression level of anti-apoptotic protein Bcl-2 was increased. These results are consistent with those observed in cells, indicating that miR-27b-3p can inhibit cell apoptosis in MCAO rats.

[0153] 4 Analysis and summary

[0154] The present study showed that both intracerebroventricular injection of EV and H-EV can promote the recovery of motor function in MCAO rats, among which H-EV has a better therapeutic effect than EV. In addition, compared with EV, H-EV showed a stronger anti-apoptotic effect after MCAO. This study focused on regulating the phenotype of astrocytes after ischemic stroke. Both EV and H-EV promoted the transformation of astrocytes from A1 phenotype to A2 phenotype. It is worth noting that the effect of H-EV on phenotypic change was more obvious than that of EV. Through sequencing analysis, we further found that miR-27b-3p in H-EV promoted the transformation of astrocytes from A1 phenotype to A2 phenotype, thereby enhancing recovery after MCAO. Mechanistic studies have shown that H-EV may play a neuroprotective role through the miR-27b-3p / PI3K / AKT signaling pathway, promote the transformation of A1 astrocytes to A2 astrocytes, and ultimately help recovery after MCAO.

[0155] Recent studies have shown that astrocytes are abundant cells in the central nervous system and play a key role in the regulation of various neurological diseases. For example, in the mouse ischemia-hypoxia model induced by middle cerebral artery occlusion, astrocytes were observed to transform into the A2 phenotype, secrete neurotrophic factors and inhibit inflammatory responses, thereby exerting a neuroprotective effect.

[35] Homer1 is known for its neuroprotective properties, and studies have shown that Homer1 can enhance functional recovery after intracerebral hemorrhage in mice by promoting the transformation of A1 astrocytes to A2 astrocytes.

[36] . This study found that after MCAO, the expression of type A1 astrocytes was reduced and the expression of type A2 astrocytes was increased through EV administration, while cell apoptosis was improved, brain tissue infarction was reduced, and motor function was restored. Therefore, promoting the transformation of astrocytes from type A1 to type A2 may provide new insights and great potential for the repair of neural injury. However, in terms of central nervous system injury, research has mainly focused on the mechanisms and treatments of neurons and astrocytes, with limited attention paid to intercellular communication.

[0156] EVs are widely studied intercellular communication carriers that contain a wide variety of lipids, proteins, RNAs, and DNAs. In recent years, researchers have become increasingly interested in using EVs as a means to study neurological recovery after stroke. For example, neural progenitor cell-derived EVs have demonstrated the ability to rescue cultured neurons from cell death-inducing stimuli associated with the ischemic cascade. In addition, EVs derived from human neural and glial cells have neuroprotective properties against oxygen-glucose deprivation-induced neuronal injury. Consistent with these findings, the present study demonstrated that EVs extracted from neural cells by intracerebroventricular injection after MCAO can act directly at the site of injury and effectively improve neural damage caused by MCAO.

[0157] It is well known that the microenvironment at the time of injury is very different from that under normal cell culture conditions. Experimental studies conducted in stroke models have emphasized that EVs pre-treated with mesenchymal stem cells are more adaptable to the in vivo post-injury microenvironment than EVs cultured under normoxic conditions, can more realistically simulate the in vivo injury state, and improve the therapeutic effect to a greater extent. Similarly, studies have shown that EVs released by hypoxic microglia play a vital role in neuroprotection and neurorecovery after stroke. Therefore, in this study, we cultured neural cells (PC12) under normoxic and hypoxic conditions and extracted their secreted extracellular vesicles, and then used them to treat MCAO rats. The results showed that EVs and H-EVs played a key role in the assessment of motor function behavior and histological and cytological evaluation of lesion size and cell apoptosis. Notably, H-EVs showed superior efficacy in functional motor recovery and reduced cell apoptosis after MCAO compared with EVs. In addition, H-EVs can more effectively convert A1 astrocytes into A2 astrocytes compared with EVs. Neurobehavioral assessments of rats, including mNSS scores, turning tests, balance beam tests, and paw slip counts, showed that rats treated with H-EV had reduced neurological dysfunction, improved spatial orientation, and enhanced motor coordination compared with rats treated with EV. In conclusion, these findings suggest that H-EV has the potential to promote functional recovery in the later stages of MCAO, with efficacy exceeding that of EV, and may provide a new idea for how to improve the prognosis of MCAO in the future.

[0158] Recently, an increasing number of studies have shown that the potential role of miRNAs in extracellular vesicles is gaining more and more attention. One study showed that EV-derived miR-23a-5p derived from M2 microglia promoted white matter repair and functional recovery after cerebral ischemia in mice. Similarly, another study showed that overexpression of miR-22-3p can lead to reduced infarct volume, inhibit apoptosis, and improve neuronal viability. To further understand the specific mechanisms of the difference in the therapeutic effects of EV and H-EV, we performed miRNA sequencing analysis on EV and H-EV. We hypothesized that the enrichment of specific miRNAs in H-EVs has potential rejuvenating effects; therefore, upregulated miRNAs were the focus of the study. miR-27b-3p was identified as the only upregulated miRNA in H-EVs compared with EVs. To investigate the role of miR-27b-3p in astrocyte transformation, miR-27b-3pmimic was incubated with astrocytes. The results showed that miR-27b-3p plays an irreplaceable role in promoting the transformation of astrocytes from type A1 to type A2. In addition, cell fluorescence results showed that H-EVs can be transported into astrocytes instead of staying on the cell surface. It can be concluded that H-EVs promote MCAO repair by transforming type A1 astrocytes into type A2 astrocytes by delivering miR-27b-3p. This finding provides convincing evidence to support the observed differences in the efficacy of EVs and H-EVs, especially in terms of the effects of changing astrocyte phenotype. To further investigate the potential role of miR-27b-3p in MCAO rats, we injected miR-27b-3p mimic and mimic NC into the lateral ventricle. The results of mNSS score, turning test, and balance beam test indicated that miR-27b-3p may contribute to the recovery of motor function in rats with ischemic stroke.

[0159] Studies have shown that the PI3K / AKT signaling pathway plays a key role in the phenotypic changes of astrocytes. An article confirmed that the PI3K / AKT signaling pathway is a key downstream mechanism regulating the transformation of reactive astrocytes during chronic postoperative pain. In addition, studies have shown that the PI3K / AKT pathway is activated after ischemic stroke. Conversely, inhibiting its activity through drug intervention can alleviate ischemic brain damage. Similarly, this study analyzed the expression levels of PI3K / AKT in cells of the mimic NC group and miR-27b-3p mimic group after OGD / R by protein immunoblotting. The results showed that after oxygen-glucose deprivation, the expression levels of P-PI3K and P-AKT increased, and the transformation of astrocytes to the A1 type also increased. After the addition of miR-27b-3pmimic, the expression of P-PI3K and P-AKT was significantly reduced, and the number of A1 astrocytes decreased, while the number of A2 astrocytes continued to increase. These findings provide compelling evidence for the regulatory relationship between miR-27b-3p and PI3K / AKT. After inhibition of this pathway, changes in astrocyte polarization and suppression of apoptosis may create a favorable microenvironment for the recovery of neurological function, which deserves further investigation. In conclusion, the observed differences in the efficacy of EVs and H-EVs in vivo may not be entirely attributed to this single mechanism; the specific mechanisms of action are complex, and the connections between various miRNAs or proteins are constantly evolving. Our results further reveal the mechanism of action of EVs, suggest directions for future research, and provide new possibilities for improving the prognosis of ischemic stroke.

[0160] The present study illustrates that both neuron-derived EVs and H-EVs can promote the repair of MCAO in vivo and in vitro. Both vesicles secreted by neuronal cells can significantly affect the phenotypic changes and apoptosis of astrocytes, among which the effect of H-EVs is more obvious than that of EVs. In addition, our results show that H-EVs secreted by neurons can regulate the PI3K / AKT signaling pathway by transmitting miR-27b-3p, thereby promoting the transformation of astrocytes from A1 phenotype to A2 phenotype. The present study provides new therapeutic targets and strategies for the treatment of MCAO.

Claims

1. Application of hypoxic preconditioned extracellular vesicles in any of the following: (a1) Use in the preparation of a medicament for treating ischemic stroke; (a2) Use in the preparation of a preparation for promoting the transformation of astrocyte phenotype from type A1 to type A2; The extracellular vesicles are extracellular vesicles derived from neuronal cells.

2. The use according to claim 1, characterized in that: The hypoxia pretreated extracellular vesicles reduce the infarct volume of brain tissue after ischemic stroke and improve neurological function deficits, inhibit brain cell apoptosis, and promote the recovery of motor function of subjects.

3. The use according to claim 1 or 2, characterized in that: The method for preparing hypoxia pretreated extracellular vesicles comprises the following steps: When the neuronal cells reached 60%-80% confluence, the culture medium was replaced with a complete medium prepared with FBS, and after culturing in a hypoxic cell culture incubator for 3.5-4.5 hours, the culture medium was replaced with normoxic conditions for 22-26 hours; The conditioned medium is collected, cell debris is removed, the collected conditioned medium is subjected to differential centrifugation, the supernatant is completely discarded, the precipitate is resuspended in PBS, washed in PBS and then centrifuged, the supernatant is discarded, and the hypoxia-pretreated extracellular vesicles are obtained.

4. The use according to claim 3, characterized in that: The culture conditions in the hypoxic cell culture incubator are 37° C., 5% CO 2 and 0% O 2 ; The normoxic conditions refer to 37°C, 5% CO2 and 21% O2; The differential centrifugation means that the collected conditioned medium is centrifuged at 300×g and 4°C for 10 minutes, then at 2000×g and 4°C for 10 minutes, and then at 10,000×g and 4°C for 30 minutes.

5. The use according to claim 1, characterized in that: The miR-27b-3p in the hypoxic preconditioning extracellular vesicles promoted the transformation of astrocytes from the A1 phenotype to the A2 phenotype, thereby enhancing recovery after ischemic stroke.

6. The use according to claim 5, characterized in that: The hypoxic preconditioned extracellular vesicles exert neuroprotective effects through the miR-27b-3p / PI3K / AKT signaling pathway, promote the transformation of type A1 astrocytes to type A2 astrocytes, and ultimately help recovery after ischemic stroke; hypoxic preconditioned extracellular vesicles regulate the PI3K / AKT signaling pathway by transmitting miR-27b-3p.

7. Application of miR-27b-3p in any of the following: (b1) Use in the preparation of a medicament for treating ischemic stroke; (b2) Use in the preparation of a preparation for promoting the transformation of astrocyte phenotype from type A1 to type A2.

8. The use according to claim 7, characterized in that: miR-27b-3p inhibits the PI3K / AKT signaling pathway activated by OGD / R, reduces cerebral infarction area and neurological deficits after ischemic stroke, and inhibits cell apoptosis.

9. The use according to claim 7, characterized in that: The nucleotide sequence of the miR-27b-3p is 5'-UUCACAGUGGCUAAGUUCUGC-3'.

Images