Periocyte-derived extracellular vesicle rich in mitochondria and application of extracellular vesicle in ischemic brain injury

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a mitochondria-rich extracellular vesicle derived from periodontal cells and application of the extracellular vesicle in ischemic brain injury. The extraction method of the extracellular vesicles comprises the following steps: (1) chopping a cerebral cortex part, adding type II collagenase and DNaseI for digestion, and then adding a gradient separation medium for separation to obtain pericytes; (2) spreading the periodontal cells on a culture dish coated with IV-type collagen, and culturing in a periodontal cell culture medium; and (3) when the confluence degree of the cells reaches 80%-90%, replacing the cell culture medium with a fresh culture medium without exosome FBS, reoxygenating the cells for 24 hours after OGD treatment, collecting culture supernatant, and centrifuging the culture supernatant to obtain the extracellular vesicles. It is found for the first time that the PC-EVs obtained after OGD stress can protect the brain from ischemic stroke injury, and the PC-EVs have important reference significance for researching related drugs for treating ischemic brain injury.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to pericyte-derived mitochondria-rich extracellular vesicles and their application in ischemic brain injury. Background Art

[0002] Ischemic stroke is a leading cause of morbidity and mortality worldwide, resulting in severe and persistent neurological deficits. During ischemia, interrupted blood flow rapidly depletes oxygen and glucose, reducing ATP production and triggering cellular energy stress. This dysfunction triggers a cascade of cellular events, including increased inflammation and oxidative stress, ultimately leading to neuronal apoptosis. Therefore, therapeutic strategies aimed at preserving mitochondrial integrity and function have the potential to improve neuroprotection and promote recovery from ischemic brain injury.

[0003] Extracellular vesicles (EVs) are nanoscale, lipid-bilayer-coated vesicles secreted by various cells. Secreted EVs can be taken up by recipient cells and deliver their contents, including active proteins, RNA species, and small molecules, thereby coordinating dynamic intercellular communication. Pericyte-derived extracellular vesicles (PC-EVs) play multiple roles in vivo, and multiple lines of evidence suggest that PC-EVs play important regulatory roles in various diseases and may serve as drug targets, monitoring tools, and diagnostic tools. However, the mechanisms of PC-EV-mediated intercellular communication in the context of cerebral ischemia-reperfusion injury remain unclear. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a pericyte-derived mitochondria-rich extracellular vesicle and its application in ischemic brain injury.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, there is provided pericyte-derived mitochondria-rich extracellular vesicles, wherein the method for extracting the extracellular vesicles comprises the following steps: (1) Mince the cerebral cortex, add type II collagenase and DNase I for digestion, and then add gradient separation buffer to separate pericytes; (2) Plating pericytes on a culture dish coated with type IV collagen and culturing them in pericyte culture medium; (3) When the cell confluence reaches 80% to 90%, the cell culture medium is replaced with fresh exosome-free FBS and glucose-free culture medium. The cells are treated with OGD and then reoxygenated for 24 hours. The culture supernatant is collected and centrifuged to obtain the extracellular vesicles.

[0006] In some embodiments of the present invention, step (1) is specifically as follows: mincing the cerebral cortex in ice-cold PBS, then supplementing with DMEM containing type II collagenase and DNaseI, digesting for 1 to 2 hours to obtain a suspension, centrifuging the suspension, discarding the supernatant, resuspending the cell pellet in DMEM containing type II collagenase, dispase and DNaseI, digesting for 0.5 to 1.5 hours, centrifuging, discarding the supernatant, resuspending the cell pellet in DMEM, and then adding a gradient separation solution to separate and obtain pericytes.

[0007] In some embodiments of the present invention, the treatment concentration of the type II collagenase is 0.8-1.2 mg / mL, the treatment concentration of the DNaseI is 10-20 μg / mL, the treatment concentration of the dispase is 1-5 U / mL, and the digestion temperature of the type II collagenase and DNaseI is 36°C-38°C.

[0008] In some embodiments of the present invention, the gradient separation solution is Percoll, and its concentration is 30% to 35%.

[0009] In the present invention, in DMEM containing type II collagenase and DNaseI, the digestion time can be 1 h, 1.5 h or 2 h, and the digestion temperature can be 36 ° C, 37 ° C or 38 ° C. In DMEM containing type II collagenase and dispase, the digestion time can be 0.5 h, 1 h or 1.5 h, and the digestion temperature can be 36 ° C, 37 ° C or 38 ° C. The treatment concentration of the type II collagenase can be 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL or 1.2 mg / mL, etc., the treatment concentration of the DNaseI can be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 1,4 μg / mL, 15 μg / mL, 1,6 μg / mL, 17 μg / mL, 1,8 μg / mL, 19 μg / mL or 20 μg / mL, the treatment concentration of the dispase can be 1 U / mL, 2 U / mL, 3 U / mL, 4 U / mL or 5 U / mL, etc., and the Percoll concentration can be 30%, 32%, 33%, 34% or 35%, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally applicable.

[0010] In some embodiments of the present invention, the OGD treatment is specifically: placing the pericytes in an anoxic incubator containing 95% N2 and 5% CO2 at 37°C and incubating for 1 to 6 hours.

[0011] In some embodiments of the present invention, the OGD treatment is specifically as follows: when the cell confluence reaches 80% to 90%, the pericyte culture medium is replaced with an exosome-free FBS-free glucose-free medium, and the cells are placed in an anoxic incubator containing 95% N2 and 5% CO2 and incubated at 37°C for 1 to 6 hours; subsequently, the cells are reoxygenated and cultured in a normal cell culture incubator containing 95% air and 5% CO2 at 37°C for 24 hours.

[0012] In some embodiments of the present invention, the exosome-free FBS glucose-free culture medium is prepared by adding 10% exosome-free fetal bovine serum to glucose-free culture medium.

[0013] In some embodiments of the present invention, the exosome-free fetal bovine serum is prepared by ultracentrifuging fresh fetal bovine serum at 100,000 x g at 4° C. overnight to remove exosomes therein, and collecting the supernatant.

[0014] In the present invention, the incubation time can be 1 h, 2 h, 3 h, 4 h, 5 h or 6 h, but is not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0015] In the present invention, OGD treatment for 1 hour increased the generation and secretion of PC-EVs without causing significant pericyte death. Compared with PC-EVs under normoxic conditions, PC-EVs after hypoxia pretreatment had a better therapeutic effect on brain damage caused by ischemia-reperfusion.

[0016] In some embodiments of the present invention, the culture supernatant is centrifuged as follows: first, at 200-400 × g at 4°C for 8-12 minutes, then at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; after centrifugation, the supernatant is collected and filtered through a 0.22 μm membrane, and ultracentrifuged at 90,000-110,000 × g at 4°C for 88-92 minutes to collect the precipitate to obtain total extracellular vesicles (PC-EVs); Alternatively, first centrifuge at 200-400 × g for 8-12 minutes at 4°C, then at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; after centrifugation, collect the supernatant and ultracentrifuge at 9,000-11,000 × g for 68-72 minutes at 4°C to collect the precipitate to obtain large extracellular vesicles (l-EVs); Alternatively, first centrifuge at 200-400 × g at 4°C for 8-12 minutes, then centrifuge at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; after centrifugation, collect the supernatant and ultracentrifuge at 9,000-11,000 × g at 4°C for 68-72 minutes to separate large extracellular vesicles; after the large extracellular vesicles are precipitated, the supernatant is filtered through a 0.22 μm membrane and then ultracentrifuged at 90,000-110,000 × g at 4°C for 88-92 minutes to collect the precipitate to obtain small extracellular vesicles (s-EVs).

[0017] In some embodiments of the present invention, the culture supernatant is centrifuged as follows: first, at 300 × g for 10 minutes at 4°C, and then at 2000 × g for 10 minutes to remove dead cells and large cell debris; after centrifugation, the supernatant is collected and filtered through a 0.22 μm membrane, and then ultracentrifuged at 110,000 × g for 90 minutes at 4°C to collect the precipitate to obtain total extracellular vesicles (PC-EVs); Alternatively, the cells were centrifuged at 300 × g for 10 min at 4°C and then at 2000 × g for 10 min to remove dead cells and large cell debris. After centrifugation, the supernatant was collected and ultracentrifuged at 10,000 × g for 70 min at 4°C to obtain large extracellular vesicles (l-EVs). Alternatively, first centrifuge at 300 × g for 10 minutes at 4°C and then at 2000 × g for 10 minutes to remove dead cells and large cell debris; after centrifugation, collect the supernatant and ultracentrifuge at 10,000 × g for 70 minutes at 4°C to separate large extracellular vesicles; after the precipitation of large extracellular vesicles, the supernatant was filtered through a 0.22 μm membrane and then ultracentrifuged at 100,000 × g for 90 minutes at 4°C to collect the precipitate to obtain small extracellular vesicles (s-EVs).

[0018] In the present invention, the size range of l-EVs is 50~400nm, and the size range of s-EVs is 50~200nm.

[0019] The second aspect of the present invention provides a pharmaceutical composition comprising the pericyte-derived mitochondria-rich extracellular vesicles described in the first aspect and a pharmaceutically acceptable carrier or adjuvant.

[0020] In some embodiments of the present invention, the content of the excipients in the pharmaceutical composition can be 1% by weight to 98% by weight, including but not limited to 5% by weight, 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, 95% by weight, and 98% by weight, usually accounting for about 80% by weight.

[0021] In some embodiments of the present invention, the pharmaceutical composition can be prepared in the following form: the extracellular vesicles are mixed with a pharmaceutically acceptable carrier to obtain, for example, oral preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, instillations), external preparations (e.g., skin preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalers), eye drops, etc. In addition, these preparations can be used as controlled-release preparations (e.g., sustained-release microcapsules), such as immediate-release preparations, sustained-release preparations, etc. Such preparations can be obtained by conventional preparation methods in the art.

[0022] In some embodiments of the present invention, examples of the above-mentioned pharmaceutically acceptable carriers include excipients (e.g., starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gelatin, polyvinyl pyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g., carboxymethyl cellulose calcium, talc, etc.), diluents (e.g., water for injection, saline, etc.), additives (e.g., stabilizers, preservatives, colorants, flavorings, dissolution aids, emulsifiers, buffers, isotonic agents, etc.), and the like.

[0023] The drugs or pharmaceutical compositions containing the extracellular vesicles described herein can be administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, and monkeys). Administration can be oral or parenteral, for example, intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, rectal, vaginal, and intraperitoneal.

[0024] The amount of the extracellular vesicles described in the present application administered to a subject varies depending on the administration route, symptoms, patient age, etc., and can be determined by a clinician.

[0025] The medicine or pharmaceutical composition involved in this application can also be used together with other existing known medicines for treating cerebral ischemic injury. When used together, there is no restriction on the administration time of each medicine. Two or more different medicines can be administered simultaneously, and each medicine can be administered at different times. The dosage of known medicines can be determined according to the clinically used administration quantity, and can be appropriately selected according to the administration patient, administration route, etc.

[0026] The third aspect of the present invention provides the use of the pericyte-derived mitochondria-rich extracellular vesicles described in the first aspect in the preparation of a product for treating or assisting in the treatment of ischemic brain injury.

[0027] In some embodiments of the present invention, the product reduces cerebral ischemic injury by reducing the activation of astrocytes.

[0028] In some embodiments of the invention, the product is a pharmaceutical.

[0029] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) This study established the protective role of PC-EVs in cerebral ischemia-reperfusion injury. Pericyte-derived extracellular vesicles (PC-EVs) are enriched with mitochondria after oxygen-glucose deprivation (OGD), and mitochondrial components are packaged into EVs. These pericyte-derived mitochondria are transferred and integrated into the mitochondrial network of astrocytes, thereby improving astrocyte activation.

[0030] (2) This study demonstrates that PC-EVs obtained after OGD stress can protect the brain from ischemic stroke. Mechanistically, pericyte-derived EVs can mitigate astrocyte activation, thereby alleviating cerebral ischemic injury. This study reveals that pericytes can serve as a valuable source of mitochondrial transfer, and that ischemia-induced mitochondrial transfer may represent a compensatory mechanism of ischemic preconditioning. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Figure 2 shows that OGD increases the release of mitochondria-containing extracellular vesicles from pericytes in an embodiment of the present invention, wherein a is a schematic diagram of pericyte-derived EVs (PC-EVs) after OGD treatment; b is the concentration of PC-EVs after reoxygenation for 24 hours after 1, 3, and 6 hours of OGD treatment (n=6); c is a Western blot analysis of EV marker proteins Alix, HSP70, and TSG101 in whole cell lysates (WCLs) and PC-EVs; d is the concentration of Alix, HSP70, and TSG101 in PC-EVsCon and PC-EVsOGD Quantitative analysis of TSG101 and TSG0 protein expression (n = 6); e is a representative TEM image of multivesicular bodies (MVBs) in OGD-treated and control pericytes, red arrows indicate MVBs containing typical intraluminal vesicles (ILVs), scale bar = 1 μm; f is a representative fluorescence image of OGD-treated and control pericytes stained with anti-CD63 and anti-platelet-derived growth factor receptor β (PDGFRβ) antibodies, scale bar = 10 μm; g is an immunoelectron microscopy image showing CD63-labeled MVBs in OGD-treated and control pericytes, red arrows indicate MVBs containing typical intraluminal vesicles (ILVs), scale bar = 1 μm. The dotted line indicates the outline of PC-EVs, scale bar = 200 nm; h is the particle size distribution of PC-EVs determined by nanoparticle tracking analysis; i is the statistical analysis of particle size distribution; j is the volcano plot of differentially expressed proteins in PC-EVs secreted by OGD-treated and control pericytes (n = 3); k is the Venn diagram of the PC-EV proteome, mouse MitoCarta3.0 list proteins and mitochondria-related GO terms; l is the 113 shared proteins filtered by mouse species and Vesiclepedia database; m is the top 200 most significant The percentage of mitochondrial components in differentially expressed proteins; n is double-labeled immunoelectron microscopy of PC-EVs, CD63 (large particles) and PDH (small particles), red arrows mark examples of CD63-positive particles, green arrows mark examples of PDH-positive particles, scale bar = 200 nm; o is co-immunostained PC-EVs with anti-CD63 and mitochondrial protein PDH antibodies, scale bar = 500 nm; p is a representative TEM image of mitochondria in OGD-treated and control pericyte-derived PC-EVs, scale bar = 200 nm; q is PC-EVs Con and PC-EVs OGD Western blot analysis of mitochondrial oxidative phosphorylation (OXPHOS) complex protein levels in PC-EVs; r is real-time detection of PC-EVs by measuring OCR Con and PC-EVs OGD Oxygen consumption of PC-EVs (n=6); s is the oxygen consumption of PC-EVs Con and PC-EVs OGDRelative ATP content in the cells (n=6); Data are expressed as mean ± SEM and analyzed using two-sided unpaired t-test (d, i, m, s), one-way analysis of variance (ANOVA) with Tukey's post hoc test (b), or two-way analysis of variance (ANOVA) with Tukey's post hoc test (r); *P<0.05, **P<0.01.

[0032] Figure 2 Figure 3 shows that PC-EVs reduce the area of ​​cerebral infarction after cerebral ischemia and improve long-term neurological function in an embodiment of the present invention; wherein, a is a schematic diagram of PC-EVs administration and behavioral analysis; b is the effect of PC-EVs administration on ipsilateral and contralateral brain water content 24 hours after MCAO (n=6); c is the mNSS score evaluated 1, 3, 7 and 14 days after MCAO (n=10); d is the effect of PC-EVs administration on body weight change (n=10); e is a T2-weighted image of infarct volume in different groups; f is a quantitative analysis of cerebral infarction volume (n=6); g is a representative image of H&E and Nissl staining of the injured hemisphere, scale bar = 10μm; h is the cortical area of ​​the injured hemisphere Quantitative analysis of the percentage of damaged neurons in the cerebral cortex (n=6); i, j. Representative images (i) and statistical analysis (j) of cerebral blood flow in PC-EVs-treated mice 14 days after MCAO (n=6); kn. Sensorimotor function was assessed by grid walking test (k), corner turning test (l), rotarod test (m) and cylinder test (n) at 1, 3, 7 and 14 days after MCAO (n=10); Data are expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test (b,h,j) or two-way analysis of variance (ANOVA) with Tukey’s post hoc test (c,d,kn). PC-EVs Con vs. MCAO or PC-EVs OGD vs.MCAO, *P<0.05, **P<0.01.

[0033] Figure 3 In the embodiment of the present invention, PC-EVs are taken up by astrocytes and inhibit astrocyte activation; wherein a is PC-EVs labeled with PKH67 OGD Image (red circle); b is a representative fluorescence image and statistical analysis showing that MCAO increases astrocytes to PC-EVs OGD c is the analysis of differentially expressed genes after PC-EVs administration, the upper figure is the Venn diagram of the intersection of three groups of differentially expressed genes, and the lower figure is the heat map of differentially expressed genes; d, e are the Venn diagram (d) and ternary diagram (e) of reactive astrocyte gene expression, respectively; f is PC-EVs OGDRepresentative fluorescence images of co-labeling of C3 and GFAP in astrocytes after drug administration; g is a Z-score heat map of gene expression in primary astrocytes after treatment with OGD or PC-EVs; h is a Sholl analysis of GFAP immunolabeled astrocytes in the cortex (shown in black), with concentric circles originating from astrocyte cell bodies; i is a Z-score heat map of the inhibition of OGD-induced A1-reactive astrocytes by PC-EVs in primary astrocytes; data are expressed as mean ± SEM, and two-sided unpaired t-test was used (a, h), *P < 0.05, **P < 0.01.

[0034] Figure 4 In the embodiment of the present invention, mitochondria are transferred from pericytes to astrocytes through PC-EVs and integrated into the astrocyte mitochondrial network; wherein, a is the MitoDsRed⁺-PC-EVs OGD Representative fluorescence images of treated astrocytes, showing that the transferred mitochondria were fused with the host mitochondrial network in recipient astrocytes expressing GFP-labeled mitochondria (white arrows); b is a schematic diagram of MitoDsRed-expressing pericytes and GFP-labeled astrocytes co-cultured in a Transwell co-culture system for 24 hours; ce are representative fluorescence images (c) and statistical analysis (d, e) of MitoDsRed fluorescence uptake by astrocytes after OGD or GW4869 treatment, showing that the transferred MitoDsRed was fused with the astrocyte mitochondrial network (white arrows); f is a schematic diagram of the transfer of mitoAPEX2-labeled mitochondria from pericytes to astrocytes via PC-EVs; g is a representative TEM image showing that astrocytes internalized APEX2+ mitochondria from pericytes via PC-EVs, as well as astrocytes that did not internalize APEX2+ mitochondria; data are expressed as mean ± SEM, and analyzed by one-way analysis of variance (ANOVA) combined with Tukey's post hoc test (d, e), *P < 0.05, **P < 0.01.

[0035] Figure 5Figure 2 shows that OGD increases the release of MVBs and PC-EVs in an embodiment of the present invention; wherein, a is a schematic diagram of pericytes under OGD conditions; b is a representative fluorescence image of TUNEL staining at different time points of OGD, scale bar = 10 μm; c is a statistical analysis of TUNEL-positive cells at different time points of OGD (n = 6); d is a representative TEM image of PC-EVs secreted by control and OGD-treated pericytes, red triangles indicate PC-EVs that have been secreted outside the cells, and green triangles indicate PC-EVs being secreted, scale bar = 200 nm; e is the number of multivesicular bodies (MVBs) per cell (n = 18); f is the number of intraluminal vesicles (ILVs) of each MVB and the size of the MVB (n = 30); g is a representative fluorescence image of control and OGD-treated pericytes stained with anti-LBPA and anti-α-SMA antibodies, scale bar = 10 μm; h is an immunoelectron microscopy image (IEM) showing LBPA-labeled MVBs in OGD-treated and control pericytes Bs, scale bar = 200 nm; i, dot blot analysis of CD63 and LBPA protein expression in OGD-treated and control pericytes; j, schematic diagram of the isolation of large and small EVs from pericyte-conditioned medium; k, Western blot analysis of Alix, HSP70, and TSG101 protein expression in large and small EVs; l, m, quantitative analysis of Alix, HSP70, and TSG101 protein expression in large EVs (l) and small EVs (m), respectively (n = 6); n, size distribution of large and small EVs secreted by control and OGD-treated pericytes, as determined by nanoparticle tracking analysis (NTA); o, morphology of large and small EVs observed by negative-stained TEM, scale bar = 200 nm; data are expressed as mean ± SEM, and analyzed using one-way analysis of variance (ANOVA) combined with Tukey’s post hoc test (c) or two-sided unpaired t-test (e, f, l, m), *P < 0.05, **P < 0.01; ns, no significant difference.

[0036] Figure 6Figure 3 shows mitochondrial-rich components of pericyte-derived PC-EVs treated with OGD in an embodiment of the present invention; a shows the top 10 cellular components in the GO analysis of differentially expressed proteins (p<0.05); b shows the top 10 cellular components in the GO analysis of upregulated proteins (p<0.05); c shows the ranking of all upregulated proteins by abundance level; d shows the cellular components of the GO analysis of the top 200 upregulated proteins (p<0.05); e shows the Western blot analysis of pyruvate dehydrogenase E1α (PDH), succinate dehydrogenase complex flavoprotein subunit A (SDHA) and citrate synthase (CS) protein expression in whole cell lysate (WCL), mitochondria and PC-EVs; f, g show the quantitative analysis of PDH, SDHA and CS protein expression in mitochondria (f) and PC-EVs (g), respectively (n=6); h shows the PDH, SDHA and CS proteins in large EVs and small EVs Western blot analysis of expression; i, j are quantitative analyses of PDH, SDHA, and CS protein expression in large EVs (i) and small EVs (j), respectively (n=6); k is flow cytometric analysis of PC-EVs secreted by control and OGD-treated pericytes labeled with MitoTracker; l is statistical analysis of MitoTracker+PC-EVs secreted by control and OGD-treated pericytes (n=6); m is a representative TEM image of mitochondrial structure of pericytes under different OGD durations, scale bar = 200 nm; n is statistical analysis of mitochondrial cristae length (n=6); data are expressed as mean ± SEM, and analyzed by two-sided unpaired t-test (f,g,i,j,l) or one-way analysis of variance (ANOVA) combined with Tukey’s post hoc test (n), *P<0.05, **P<0.01; ns, no significant difference.

[0037] Figure 7 Figure 3 shows that PC-EVs reduce cerebral infarction area in MCAO mice in an embodiment of the present invention; wherein, a is a representative image and statistical analysis of TTC staining of brain sections 24 hours after MCAO; b is a representative image of hematoxylin-eosin (H&E) staining of brain sections 3 days after MCAO; data are expressed as mean ± SEM, and one-way analysis of variance (ANOVA) with Tukey's post hoc test was used (a), **P < 0.01.

[0038] Figure 8Figure 2 shows that OGD treatment enhances the uptake of PC-EVs by astrocytes in an embodiment of the present invention; wherein, a is a representative fluorescence image and statistical analysis showing that MCAO does not affect the uptake of PC-EVs by neurons, scale bar = 10 μm; b is a representative fluorescence image and statistical analysis showing that MCAO enhances the uptake of PC-EVs by microglia, scale bar = 10 μm; c is a schematic diagram of the co-culture of PKH67-labeled PC-EVs and OGD-treated astrocytes; d is a representative fluorescence image showing that the uptake of PC-EVs by astrocytes is enhanced with the extension of OGD treatment time, scale bar = 10 μm; e is a schematic diagram of the construction of Cre-dependent color conversion vectors; f is a representative fluorescence image of astrocytes uptake of PC-EVs containing Cre, with fluorescence converted from green to red, scale bar = 10 μm; data are expressed as mean ± SEM, and a two-sided unpaired t-test was used (a, b), *P < 0.05; ns, no significant difference.

[0039] Figure 9 Figure 2 shows that PC-EVs inhibit astrocyte activation after MCAO in an embodiment of the present invention; a and b are the PCA plot (a) and the volcano plot (b) of the samples in the transcriptional analysis; c is the heat map of differentially expressed genes in reactive astrocytes in the transcriptional analysis; d is a representative immunohistochemical image of GFAP+ astrocytes in the cortex, vector group: animals were injected with an equal amount of PBS; e and f are representative binary images (e) and cytoskeleton images (f) converted from astrocyte images; g is the Sholl analysis of astrocyte branches; h is PC-EVs after MCAO OGD Western blot analysis of GFAP and EAAT1 protein expression in the treated cortex; i: Quantitative analysis of GFAP and EAAT1 protein expression (n = 6); data are expressed as mean ± SEM, and analyzed by two-way analysis of variance (ANOVA) combined with Tukey's post hoc test (g), *P < 0.05, **P < 0.01.

[0040] Figure 10 The mitochondria in the present invention are transferred from pericytes to astrocytes through PC-EVs and integrated into the astrocyte mitochondrial network; wherein a is the flow cytometry analysis of MitoDsRed-labeled pericytes infected with adenovirus expressing MitoDsRed; b is the flow cytometry analysis of MitoDsRed-labeled pericytes after OGD treatment. + PC-EVs OGD Schematic diagram of astrocyte treatment; c, d are OGD treatment and MitoDsRed + PC-EVs OGDFlow cytometry (c) and immunofluorescence (d) analysis of MitoDsRed in treated astrocytes (n=6), scale bar=10μm; e is the expression of MitoDsRed in astrocytes treated with OGD at different time points. + PC-EVs OGD Flow cytometric analysis of treated astrocytes (n=6); f is a schematic diagram of co-culture of MitoDsRed-labeled pericytes and GFP-labeled astrocytes; g is a representative fluorescence image of co-culture of GFP-labeled astrocytes and MitoDsRed-labeled pericytes, showing that mitochondria were harvested from MitoDsRed-labeled pericytes and fused with GFP-labeled mitochondria from astrocytes; h is a schematic diagram of TEM observation of APEX2+ mitochondria: after transfection of the APEX2-expressing plasmid into the cells, the cells were fixed and treated with diaminobenzidine (DAB) and hydrogen peroxide. APEX2 catalyzes the polymerization of DAB, and after treatment with osmium tetroxide, it provides clear contrast for TEM; i is a representative TEM image of APEX2-labeled mitochondria in mitoAPEX2-transfected pericytes and unlabeled mitochondria in the control; data are expressed as mean ± SEM and analyzed by two-sided unpaired t-test (c, d) or one-way analysis of variance (ANOVA) with Tukey's post hoc test (e), **P < 0.01. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1 Experimental method: Culture of primary pericytes Primary pericytes were prepared from the cerebral cortex of C56BL / 6J mice. After brain dissection, the meninges and visible large blood vessels were carefully removed. The cortex was isolated and minced with ophthalmic scissors in ice-cold PBS. The cells were then digested in DMEM supplemented with type II collagenase (1 mg / mL) and DNase I (15 μg / mL) for 1.5 hours. After incubation at 37°C, the suspension was centrifuged at 1000 × g for 5 minutes at 4°C. The resulting pellet was resuspended in DMEM supplemented with 20% BSA and centrifuged at 1000 × g for 20 minutes. The supernatant was discarded, and the pellet was resuspended in DMEM supplemented with type II collagenase (1 mg / mL), dispase (2 U / mL), and DNase I and incubated at 37°C with shaking for 60 minutes. The cell pellet was resuspended in DMEM and separated on a 33% discontinuous Percoll gradient, centrifuged at 1000 × g for 10 minutes at 4°C. The pericyte pellet was plated on a culture dish coated with type IV collagen and cultured in pericyte medium (ScienCell, 1201) for approximately 2 weeks.

[0045] Culture of primary astrocytes Primary glial cell cultures were prepared from the cerebral cortex of C56BL / 6J mice (postnatal day 1). The collected cerebral cortex was cut into approximately 1 mm3 pieces in ice-cold PBS and digested with 0.25% trypsin at 37°C for 30 minutes. An equal volume of DMEM complete medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) was added to inactivate the trypsin. The tissue digest was then mixed, pipetted repeatedly, filtered through a 40 μm cell sieve, and centrifuged at 1000 rpm for 10 minutes. The cell pellet was resuspended in DMEM complete medium and seeded into poly-D-lysine-coated culture flasks and cultured at 37°C in 5% carbon dioxide. The medium was changed every 3 days. After 14 days of culture, microglia growing on the upper layer of the mixed glial culture were removed by shaking at 180 rpm for 30 minutes. The remaining adherent cells in the culture flask were astrocytes.

[0046] Primary neuronal culture The cerebral cortex was obtained from C56BL / 6J mice (embryonic day 15). The cerebral cortex was cut into 1 mm 3The cerebral cortex was digested with 0.25% trypsin at 37°C for 30 minutes. An equal volume of Neurobasal medium (containing 2% B27, 1% penicillin / streptomycin, and 1% glutamine enhancer) was added to terminate the trypsinization. The tissue digest was repeatedly mixed by pipetting and then filtered through a 40μm cell sieve. The filtrate was centrifuged at 1500g for 10 minutes and resuspended in the same Neurobasal medium as before. The cell suspension was placed in a culture flask coated with poly-D-lysine and incubated in a 37°C incubator with 5% carbon dioxide. During subsequent culture, half of the medium was replaced every 3 days for a total of 10 days.

[0047] Oxygen glucose deprivation / reoxygenation (OGD / R) When the cells reached 80%-90% confluence, the medium was replaced with exosome-free FBS and glucose-free medium (Life Technology, 11966-025) supplemented with 10% exosome-free fetal bovine serum (FBS) (exosomes were removed by ultracentrifugation at 100,000 x g overnight at 4°C) to glucose-free medium. The cells were incubated in an anoxic incubator containing 95% N₂ and 5% CO₂ at 37°C for 1-6 hours, followed by reoxygenation in a normal cell culture incubator at 95% air and 5% CO₂ for 24 hours.

[0048] Isolation and characterization of extracellular vesicles (EVs) After reaching 90% confluence, the cells were replaced with exosome-free FBS and glucose-free medium. After OGD treatment for a specified period of time, the cells were reoxygenated for 24 hours. The conditioned medium (pericyte culture supernatant after reoxygenation) was collected and centrifuged at 300 × g for 10 minutes at 4°C, followed by 2000 × g for 10 minutes to remove dead cells and large cell debris. The supernatant was then ultracentrifuged at 100,000 × g for 90 minutes at 4°C to collect total pericellular extracellular vesicles (PC-EVs), or large EVs were isolated by ultracentrifugation at 10,000 × g for 70 minutes at 4°C. For small EVs, the supernatant after large EV precipitation was filtered through a 0.22 μm membrane and then ultracentrifuged at 100,000 × g for 90 minutes at 4°C to isolate small EVs.

[0049] Transmission electron microscopy (TEM) Standard electron microscopy analysis was performed on PC-EVs, pericytes, astrocytes, and brain tissue samples. PC-EVs or cells were fixed with 2.5% glutaraldehyde overnight at 4°C, washed three times (3 minutes each) in 0.1 M phosphate buffer (pH 7.4), and then embedded in 1% agarose. Mice were perfused transcranially with a fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 MPBS. Freshly collected cortices were minced to 1 mm in cold fixative. 3PC-EVs, cells, and tissues were fixed with 1% osmium tetroxide for 2 hours at room temperature in the dark, dehydrated through a series of acetone solutions (30%, 50%, 70%, 80%, 95%, and 100%), and embedded in Epon 812. Semi-thin sections were stained with toluidine blue, and ultra-thin sections were stained with 2% uranyl acetate and lead citrate. Images were obtained using a Hitachi HT-7800 transmission electron microscope (80 kV) equipped with a CMOS camera.

[0050] Immunogold electron microscopy (IEM) For immunoelectron microscopy analysis, cells were fixed with a dedicated IEM fixative (Servicebio, G1124), washed with 0.1 M PB solution, serially dehydrated with acetone, and embedded in sections. Ultrathin sections were mounted on Formvar / carbon-coated 100-mesh nickel grids and blocked with 1% BSA solution at room temperature for 30 minutes. The sections were then incubated with primary antibodies against CD63 (ab217345, Abcam) or LBPA (MABT837, Sigma) at 4°C overnight. Subsequently, the sections were incubated with a diluted 12 nm colloidal gold-conjugated secondary antibody (111-205-114 or 115-205-146, Jackson ImmunoResearch) for 20 minutes, stained with 2% uranyl acetate, and imaged. For immunoelectron microscopy analysis of EVs, 100 μL of isolated EVs were fixed with an equal volume of 4% paraformaldehyde (PFA), adsorbed to Formvar / carbon-coated grids for 20 min, quenched with PBS / 50 mM glycine, and blocked with 1% BSA before incubation with mouse anti-PDH (dilution 1:40; sc-377092, Santa Cruz) alone or with rabbit anti-CD63 (dilution 1:20; ab217345, Abcam). After incubation, the samples were incubated with corresponding gold-conjugated secondary antibodies (12 nm anti-mouse, 111-205-146 or 12 nm anti-rabbit, 111-205-114 / 4 nm anti-mouse, 115-185-146; Jackson ImmunoResearch) at a dilution of 1:100. The samples were negatively stained with 2% uranyl acetate, embedded in methylcellulose / uranyl acetate (2% / 0.5%), and imaged using a Hitachi HT7800 transmission electron microscope (80 kV).

[0051] Proteomic analysis PC-EVs were purified from pericyte-conditioned medium by ultracentrifugation and lysed with 1% SDS protein lysis buffer and protease inhibitor cocktail. Protein concentration was determined using a BCA kit (Thermo Scientific). After protein quantification, SDS-PAGE electrophoresis was performed. Samples were reductively alkylated with iodoacetamide and digested overnight at 37°C with trypsin at a 1:50 protein ratio to generate a peptide mixture. Peptides were vacuum-dried, acidified with 1% (v / v) trifluoroacetic acid, and desalted on an HLBC18 column. Peptides were quantified using a peptide quantification kit (Thermo Scientific) and separated and identified using a Vanquish Neo coupled to an Orbitrap Astral mass spectrometer (Thermo, USA). uPAC high-throughput columns (75 μm × 5.5 cm, Thermo) were used. Mobile phase A consisted of 2% acetonitrile and 0.1% formic acid in water, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid in water. The mass spectrometer was operated in data-independent acquisition (DIA) mode with a mass-to-charge ratio range of 70–1050 m / z for MS1 and 150–2000 m / z for MS2.

[0052] Transcriptomic analysis Total RNA was extracted from mouse cells and brain tissue using TRIzol reagent according to the manufacturer's protocol. Subsequent RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. Briefly, poly(A) mRNA was purified from total RNA using oligo(dT) magnetic beads and fragmented. The fragmented RNA was reverse transcribed into first-strand cDNA, which was then synthesized into double-stranded cDNA (dscDNA). The purified dscDNA was end-repaired and 3'-end adenylated before ligation with sequencing adapters. The resulting product was purified and PCR amplified to generate a cDNA library. The library fragment size was analyzed using an Agilent 2100 Bioanalyzer and qualified for sequencing on an Illumina NovaSeq platform. Raw paired-end reads were trimmed and quality-controlled using Fastp. Clean reads were aligned to the reference genome using HISAT2 in directional mode. Aligned reads for each sample were assembled using a reference-based approach using StringTie. Differentially expressed genes (DEGs) were identified using DEseq2, and genes with |log2FC| > 1 and an FDR < 0.05 were considered DEGs.

[0053] TUNEL staining Pericytes were treated with OGD for 1, 3, or 6 hours before TUNEL staining. Cells were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, and then incubated with 0.3% Triton X-100 in PBS for 5 minutes at room temperature to permeabilize the cell membrane. After washing twice with PBS, 50 μL of TUNEL detection solution was added to the samples and incubated at 37°C in the dark for 60 minutes. The samples were then washed three times with PBS, mounted with anti-fade mounting solution, and observed under a fluorescence microscope.

[0054] ATP measurement ATP levels were determined using a luminescent ATP assay kit (S0026B, Beyotime). 20 μL of lysed PC-EVs or standards were added to the assay wells, and relative luminescence units (RLUs) were measured using a luminometer.

[0055] Oxygen consumption rate (OCR) For OCR analysis of PC-EVs, isolated PC-EVs were resuspended in MAS buffer (220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1.0 mM EGTA, 0.2% BSA), added to an XFe24 microplate, and centrifuged at 2000 × g for 10 minutes at 4°C. After centrifugation, glutamate (10 mM) and malate (2 mM) were added to the MAS buffer to a final volume of 500 μL per well. The microplate was incubated at 37°C in a CO2-free environment for 10 minutes before being transferred to the XFe24 analyzer. 40 mM ADP was injected sequentially at 37°C, and OCR was recorded.

[0056] Flow cytometric analysis To detect mitochondria in PC-EVs, pericyte supernatants were collected and incubated with 100 nM Mito-Tracker Red CMXRos (Beyotime) at 37°C for 30 minutes before PC-EV isolation. For analysis of mitochondria in PC-EVs labeled with MitoDsRed, pericyte supernatants were stained before the PC-EV isolation step. To detect MitoDsRed-labeled mitochondria in astrocytes, astrocytes were co-cultured with PC-EVs containing MitoDsRed and then incubated with 100 nM Mito Tracker Red CMXRos. After co-culture, astrocytes were centrifuged and resuspended in fresh culture medium. All samples were analyzed by flow cytometry (BD Bioscience, CA).

[0057] Adenovirus construction and infection Recombinant adenovirus expressing MitoDsRed (Ad-MitoDsRed) and control adenovirus were constructed by Weizhen Biotechnology Co., Ltd. (Shandong Weizhen) using the pAdM-CMV vector system. Briefly, the MitoDsRed sequence was subcloned and inserted into the shuttle vector pAdM-CMV digested with AsisI and MluI via homologous recombination. The recombinant plasmid was transfected into HEK293 cells, followed by amplification, purification, and concentration. During adenoviral infection, cells were plated at 5 × 10 cells per well. 4 The cells were seeded at a density of 100 in 24-well plates and infected with Ad-MitoDsRed at a multiplicity of infection (MOI) of 100. Fresh medium was replaced 24 h after infection. The transfection efficiency was assessed under a fluorescence microscope 48 h after transfection, and the cells were selected with 1 μg / mL puromycin.

[0058] PC-EVs tracing Isolated PC-EVs were labeled with PKH67 green fluorescent membrane dye (Sigma, Shanghai) according to the manufacturer's instructions to determine cellular uptake of PC-EVs. Labeled PC-EVs were washed with PBS, collected by ultracentrifugation, and resuspended in PBS. For in vivo tracking of PC-EVs, 20 μg of PC-EVs dissolved in 100 μL of PBS were injected into the lateral ventricle. Brain tissue was collected 24 hours after injection for immunofluorescence staining.

[0059] Construction of mitoAPEX2-labeled pericytes MitoAPEX2-labeled pericytes were constructed by transfecting pericytes with the pCMV-N-mito-Flag-APEX2 plasmid (Biyuntian, D3047). This plasmid expresses APEX2 fused to an N-terminal mitochondrial matrix-targeting peptide. When cell confluence reached 80%, the plasmid was transfected into pericytes using Lipo8000. Stable expression of the mitoAPEX2 reporter gene was achieved after neomycin selection. Cells were fixed with 2% glutaraldehyde and stained with cold 3,3'-diaminobenzidine (0.5 mg / mL) and 10 mM H2O2 for 15 minutes. APEX2 activity was visualized by TEM. Samples were then stained with 2% OsO4, which reacts with DAB polymer to deposit electron-dense osmium for electron microscopic contrast. Cells were then stained with 2.5% uranyl acetate, dehydrated through an ethanol gradient, permeabilized, and embedded for electron microscopic observation.

[0060] RNA extraction and quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Shanghai) and reverse-transcribed into cDNA using the SuperScript III Reverse Transcription Kit (Invitrogen). Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch system using SuperReal PreMix Plus (Tiangen, Beijing). Amplification conditions were 95°C for 2 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Gene expression was calculated using the 2-ΔΔCT method, using β-actin as an internal reference gene. Primer sequences are shown in Table 1.

[0061] Mitochondrial DNA extraction and quantification Total DNA was extracted from PC-EVs using a mitochondrial DNA extraction kit (ab65321, Abcam) according to the manufacturer's instructions. D-Loop mtDNA and cytochrome oxidase mtDNA were quantified on a Bio-Rad CFX96 Touch system using SuperReal PreMix Plus (Tiangen, Beijing). 18S ribosomal RNA was used as an internal reference gene. The primer sequences are shown in Table 1.

[0062] Table 1 Primer sequences

[0063] animal The animal study protocol was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical College and was performed in accordance with the Guide for the Care and Use of Laboratory Animals. C57BL / 6 mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. and housed under standard conditions (temperature: 22 ± 2°C; 12:12 hr light / dark cycle; humidity: 50 ± 5%) with free access to sterile food and water. Mice were acclimated for 7 days before the experiment. All mice used in the experiments were 7-week-old males and randomly assigned to either the control or experimental group.

[0064] Middle cerebral artery occlusion (MCAO) model Focal cerebral ischemia was induced by transient myocardial occlusion (MCAO) as previously described with modifications. After anesthesia, the mice were exposed to the skin. A silicone-coated 6-0 nylon monofilament was inserted through the external carotid artery into the internal carotid artery, occluding the origin of the middle cerebral artery. After 60 minutes of occlusion, the monofilament was removed to restore blood flow, the incision was sutured, and the mice recovered on a heating pad to maintain a body temperature of 37.0 ± 0.5°C. Cerebral blood flow (CBF) was monitored before and after surgery using a laser speckle contrast imaging system (RWD LifeSciences, RFLSI-ZW).

[0065] Brain water content The mice were deeply anesthetized and the whole brain was carefully extracted from the skull. The contralateral and ipsilateral hemispheres were then separated and weighed using the recoded value as the wet weight. The samples were then dried in an oven at 100°C for 48 hours and reweighed, and this value was recorded as the dry weight. Brain water content was calculated as follows: Brain water content (%) = [(wet weight - dry weight) / wet weight] × 100% Neurological deficits The modified neurological deficit score (mNSS) was used to detect neurological deficits. The mNSS range was 0-14, with higher scores indicating more severe neurological deficits. mNSS tests were performed 1, 3, 7, and 14 days after MCAO.

[0066] Behavioral analysis Grid walking test: Mice were placed on an elevated metal grid measuring 32 cm × 20 cm × 50 cm (length × width × height) with 12 cm × 12 cm mesh openings. They were trained for 4 days before surgery, and the average performance of each mouse at the end of training was used as the baseline. After MCAO, mice were allowed to walk freely on the grid for 5 minutes. The number of "foot slips" (i.e., strides without support, foot penetration through mesh openings, or resting of the mouse's wrist against the grid) was recorded. The total number of steps was recorded at the end of each test, and the foot slip percentage was calculated as: (number of foot slips / total number of steps) × 100%.

[0067] Corner turning test: Place the mouse in a device formed by two transparent plates at a 30-degree angle. Place the opening of the corner device as close as possible to the edge of the table to prevent the mouse from escaping. Allow the mouse to freely turn left or right to leave the corner. Each mouse is tested 10 times, and the calculation is: (number of turns toward the ischemic side / total number of turns) × 100%.

[0068] Rotarod test: Mice were pre-trained for 3 days before MCAO. During the test, the mice were placed on a rotating drum that steadily accelerated from 5 rpm to 40 rpm within 5 minutes. The time the mice fell was recorded. The test was conducted 3 times a day with an interval of 15 minutes. The time spent on the rod 1 day before surgery was recorded as the baseline value. The average of the 3 tests after MCAO was used as the latency to fall.

[0069] Cylinder test: The mouse was placed in a plastic cylinder 15 cm high and 10 cm in diameter and videotaped for 5 min. The exploration / pressing score was calculated as: (number of right paw contacts - number of left paw contacts) / (number of right paw contacts + number of left paw contacts + number of both paw contacts).

[0070] TTC staining Twenty-four hours after MCAO, mice were sacrificed under deep anesthesia, and the brains were dissected and cut into 2.0-mm-thick coronal sections. The sections were stained with 2% TTC solution for 20 minutes in the dark at 37°C and subsequently fixed with 4% paraformaldehyde. Non-infarcted tissue was stained red, while infarcted areas remained white. The sections were photographed and infarct area was quantified using ImageJ. The percentage of infarct area was calculated as the ratio of infarct area to contralateral brain area.

[0071] H&E and Nissl staining The brains were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin sections were serially sectioned, dewaxed, rehydrated, stained with conventional hematoxylin-eosin or 0.1% cresyl violet, and photographed under an Olympus IX73 microscope.

[0072] Magnetic resonance imaging (MRI) Fourteen days after MCAO, mice underwent MRI using a Bruker Biospect 7.0T 20cm MR imaging system. Mice were anesthetized with 2% isoflurane and maintained at 1.5% isoflurane using a small animal anesthesia machine. Body temperature was maintained at 37°C on a heating pad throughout the test. T2-weighted imaging parameters included: repetition time (TR) = 4000 ms, echo time (TE) = 48 ms, field of view (FOV) = 2.5 cm × 2.5 cm, slice thickness = 500 μm, number of slices = 20, image matrix size = 256 × 256, and averages = 6. Infarct volumes were analyzed using ImageJ.

[0073] Immunoblotting EVs, cells, and brain tissue were lysed with RIPA buffer. Protein concentration was determined using a BCA protein assay kit. The lysates were mixed with loading buffer, boiled, and separated by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked with 5% skim milk in TBST. The membranes were incubated with appropriate primary antibodies overnight at 4°C and subsequently with HRP-conjugated secondary antibodies. The staining was developed using the ChemiDoc+ system and quantified using ImageJ software. For dot blotting, 2 μL of sample was spotted onto nitrocellulose membranes, blocked, incubated with appropriate primary antibodies, and imaged after secondary antibody treatment.

[0074] Immunofluorescence staining For immunofluorescence staining of brain tissue, mouse brains were fixed with 4% PFA at 4°C, embedded in paraffin, and cut into 4-μm-thick sections. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval with EDTA buffer. Endogenous peroxidase activity was blocked with 3% H₂O₂, and nonspecific binding was blocked with 3% BSA. Sections were then incubated overnight with the appropriate primary antibody and subsequently with a fluorescent-conjugated secondary antibody. For cell staining, cultured cells were fixed, permeabilized, and blocked before incubation with the appropriate primary and fluorescent secondary antibodies. Immunofluorescence staining of PC-EVs was performed according to a previously described protocol. Pericyte-derived PC-EVs were treated with PEG10000 and incubated overnight with a mixture of primary antibodies. After washing, a fluorescent-conjugated secondary antibody was applied, and unbound antibodies were removed by column purification. PC-EVs were then mounted on slides and imaged using confocal microscopy.

[0075] Immunohistochemistry For brain immunohistochemistry, brain tissue was dewaxed with xylene and rehydrated with ethanol. After antigen retrieval, endogenous peroxidase activity was blocked with 3% H2O2 for 20 minutes, and nonspecific binding sites were blocked with 3% BSA. Sections were incubated with anti-GFAP (ab7260, Abcam) primary antibody overnight at 4°C and then with HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech) at room temperature. Freshly prepared DAB solution was added, and the color development reaction was terminated with distilled water. Sections were counterstained with hematoxylin staining solution, differentiated with hematoxylin differentiation solution, and treated with hematoxylin bluing solution. Images were acquired under a conventional light microscope (E100, Nikon).

[0076] Statistical analysis All data are presented as mean ± standard error of the mean (SEM). Data normality and homogeneity of variance were tested using the Shapiro-Wilk test and the Levene test, respectively. Two-group comparisons were performed using a two-sided unpaired Student's t-test. Multiple-group comparisons were performed using one-way analysis of variance (ANOVA) with Tukey's post hoc test. Simultaneous analysis of two factors between multiple groups was performed using two-way analysis of variance (ANOVA) with Tukey's post hoc test. Data were analyzed using GraphPad Prism 8.0 software. P < 0.05 was considered statistically significant. All participants were blinded to the experimental conditions during data collection and analysis and received prior training.

[0077] 2. Results OGD induces the release of mitochondria-containing PC-EVs Pericytes have stem cell-like properties and play a key role in the brain microenvironment. Previous studies have shown that vesicles derived from stem cells have neuroprotective effects, especially under hypoxic conditions. Therefore, we explored the potential of PC-EVs in brain injury. We cultured pericytes under OGD conditions for 1, 3, or 6 hours ( Figure 5a) and TUNEL staining was used to assess cell apoptosis. The results showed that 1 hour of OGD did not induce cell apoptosis, while 3 hours of OGD significantly increased the number of apoptotic cells ( Figure 5 b, c). We then isolated EVs from pericyte-conditioned medium by ultracentrifugation after 24 h of reoxygenation following 0, 1, 3, or 6 h of OGD treatment ( Figure 1 a). Nanoparticle tracking analysis (NTA) showed that PC-EVs levels increased significantly after 1 h of OGD treatment, but did not increase further at 3 and 6 h, indicating that PC-EVs were rapidly released under OGD stimulation ( Figure 1 b). Western blot (WB) analysis confirmed that PC-EVs were positive for multivesicular body (MVB) markers such as Alix, TSG101, and HSP70 and were significantly different from untreated pericyte-derived EVs (PC-EVs Con ) compared to pericyte-derived EVs (PC-EVs) exposed to OGD for 1 hour. OGD ) were significantly upregulated in these markers ( Figure 1 c, d). EVs are usually packaged in MVBs within cells and released into the extracellular space after the fusion of MVBs with the plasma membrane. Transmission electron microscopy (TEM) confirmed that OGD led to an increase in the number of EVs and MVBs ( Figure 5 d). After OGD treatment, the number of intraluminal vesicles (ILVs) in MVBs of pericytes increased significantly ( Figure 1 e, Figure 5 e,f). Immunofluorescence staining showed that OGD induced the aggregation of CD63-positive and lysophosphatidic acid (LBPA)-positive multivesicular bodies (MVBs) and increased the number of punctate structures ( Figure 1 f, Figure 5 g), this result was further confirmed by immunoelectron microscopy (IEM) ( Figure 1 g, Figure 5 h). Dot blot analysis showed that CD63 and LbpA were upregulated after OGD, indicating that OGD affects the formation or dynamics of MVBs ( Figure 5 i). Measurements of PC-EV diameters revealed two major subpopulations, approximately 100 nm and 350 nm in size ( Figure 1 h, i). Next, we isolated large EVs (l-EVs) and small EVs (s-EVs) from pericyte culture media ( Figure 5 j), both expressed Alix, TSG101, and HSP70, and the expression of these proteins was significantly upregulated after OGD ( Figure 5 km). NTA showed that the size range of l-EVs was 50-400 nm and the size range of s-EVs was 50-200 nm ( Figure 5 n), TEM further confirmed these sizes ( Figure 5 o).

[0078] PC-EVs Con and PC-EVs OGD performed a proteomic analysis and identified 1,494 proteins, many of which were associated with mitochondria and other organelles ( Figure 1 j, Figure 6 a). Comparison with mitochondrial proteins (GO:0005739 and MitoCarta3.0) showed 137 overlapping proteins ( Figure 1 k). Among them, 113 proteins were identified as vesicle cargo proteins by the Vesiclepedia database ( Figure 1 l). Analysis of the top 200 differentially expressed proteins showed that mitochondrial proteins were significantly enriched among the upregulated proteins ( Figure 1 m). Gene Ontology (GO) analysis of all upregulated proteins further confirmed this finding ( Figure 6 b). In addition, 198 upregulated proteins (log2 FC>2) were enriched in exosome fractions ( Figure 6 c, d). Western blot analysis confirmed the presence of mitochondrial proteins in PC-EVs and their subpopulations (large EVs and small EVs), which were significantly upregulated after OGD ( Figure 6 ej). IEM further demonstrated that the number of mitochondrial protein-positive spots increased in PC-EVs after OGD ( Figure 1 n). Colocalization of pyruvate dehydrogenase E1α (PDH) and CD63 showed that most CD63-positive PC-EVs contained mitochondrial proteins after OGD, indicating an increase in the release of mitochondrial components by PC-EVs ( Figure 1 o). Transmission electron microscopy (TEM) showed that PC-EVs OGD The number of mitochondria encapsulated in EVs was significantly higher than that in PC-EVs. Con ( Figure 1 These results suggest that OGD promotes the conversion of mitochondria to PC-EVs. OGD Mitochondrial dysfunction is often due to defects in mitochondrial structural integrity and loss of respiratory chain complexes. Therefore, we analyzed mitochondrial integrity and function in PC-EVs. PC-EVs were positive for MitoTracker staining ( Figure 6 k, l). WB analysis further confirmed that PC-EVs OGD Contains complete mitochondrial respiratory chain complexes ( Figure 1 q). Importantly, these PC-EVs OGD showed a higher oxygen consumption rate (OCR) and higher ATP content ( Figure 1 r,s), indicating that PC-EVsOGD contained more mitochondria with intact structure and function. It is worth noting that 1 hour of OGD did not affect the mitochondrial structure in pericytes ( Figure 6 m,n).

[0079] PC-EVs can reduce cerebral infarction and improve neurological damage after cerebral ischemia Immediately after inducing cerebral ischemia in mice, 0.5 μg of PC-EVs were injected into the Con and PC-EVs OGD Injected into the lateral ventricle to study the role of PC-EVs in stroke ( Figure 2 a). The results showed that PC-EVs significantly reduced brain water content, among which PC-EVs OGD The effect is more significant ( Figure 2 b). On day 3, mNSS scores showed PC-EVs OGD The group showed early neurological recovery, which became more significant on days 7 and 14 ( Figure 2 c), indicating that PC-EVs OGD Not only did it initiate early recovery, but it also enhanced the recovery effect over time. In addition, PC-EVs treatment did not affect body weight ( Figure 2 d). 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that PC-EVs OGD The infarct size was significantly reduced 24 hours after ischemia ( Figure 7 a). Hematoxylin and eosin (H&E) staining also significantly reduced the ischemic area on day 3 after ischemia ( Figure 7 b). Magnetic resonance T2-weighted imaging (T2WI) further showed that the infarct size continued to decrease ( Figure 2 e, f). H&E staining and Nissl staining also confirmed PC-EVs OGD Protective effect on neuronal damage ( Figure 2 g, h). In addition, PC-EVs OGD The enhancement effect on cerebral blood flow is more significant ( Figure 2 i, j). Next, we investigated the effects of PC-EVs on motor function recovery. Mice treated with PC-EVs showed significant improvements in sensorimotor deficits and asymmetry up to 14 days after ischemia. OGD There was a greater recovery effect on performance in behavioral tests ( Figure 2 kn). These findings highlight the role of PC-EVs OGD Protective effect after cerebral ischemia.

[0080] PC-EVs are taken up by astrocytes and inhibit astrocyte activation after ischemia Labeling of PC-EVs with the membrane dye PKH67 OGD , to track the uptake of PC-EVs by neurons after stroke ( Figure 3 a). When PC-EVs OGD After injection into the lateral ventricle and allowing 24 hours for uptake, immunostaining revealed that neurons, astrocytes, and microglia internalized labeled PC-EVs. OGD , in which astrocytes showed a significant increase in uptake after cerebral ischemia ( Figure 3 b, Figure 8 a, b). In addition, primary astrocytes showed time-dependent PC-EVs after OGD / R. OGD Increased intake ( Figure 8 Next, we used the Cre-loxP system to induce astrocytes to express a color-switching reporter protein, which was activated upon uptake of Cre-containing EVs released by pericytes ( Figure 8 e). PC-EVs expressing Cre OGD Efficiently taken up by astrocytes, inducing fluorescence conversion from green (GFP+) to red (RFP+) ( Figure 8 f). These findings suggest that PC-EVs OGD Efficiently taken up by astrocytes after OGD.

[0081] PC-EVs OGD Effects on brain gene expression. Principal component analysis showed that there were obvious clusters among the three groups, among which the Sham group and PC-EVs OGD The two groups partially overlapped, but both groups showed significant differences from the MCAO group ( Figure 9 a, b). Compared with the Sham group and PC-EVs OGD Compared with the control group, a total of 1,567 genes were significantly up-regulated and 862 genes were significantly down-regulated in the MCAO group ( Figure 3 c). Astrocyte activation plays a key role in the progression of MCAO. Astrocyte activation-related genes were significantly upregulated after MCAO, and in PC-EVs OGD After administration, the Figure 3 d, Figure 9 c). Ternary plot showed that compared with the Sham group or PC-EVs OGD Compared with the MCAO group, these activation-related genes were significantly enriched in the MCAO group ( Figure 3 e). Immunofluorescence and immunohistochemistry staining confirmed that MCAO induced glial fibrillary acidic protein (GFAP) reactivity, while PC-EVs OGD Blocking this reaction ( Figure 3 f, Figure 9d). Further analysis showed that the number and length of astrocyte branches were reduced in the MCAO group, while PC-EVs OGD Treatment significantly restored these indicators ( Figure 9 e, f). Sholl analysis confirmed that PC-EVs OGD Inhibited the reduction of astrocyte branches ( Figure 3 g, Figure 9 g). Genes associated with astrocyte activation were further verified in vivo by Western blotting ( Figure 9 h, i, and further validated in vitro by qPCR ( Figure 3 h).

[0082] Pericytes transfer mitochondria to astrocytes via PC-EVs and integrate them into the astrocyte mitochondrial network Cells can transfer functional mitochondria to recipient cells via EVs, thereby enhancing intercellular communication. Furthermore, we investigated whether pericytes mediate the transfer of mitochondria to astrocytes via PC-EVs. Mitochondria in pericytes were labeled using adenovirus expressing MitoDsRed. Figure 10 a). PC-EVs isolated from labeled pericytes OGD (MitoDsRed + -PC-EVs OGD ) were then incubated with astrocytes for 24 h ( Figure 10 b). OGD-treated astrocytes showed increased MitoDsRed fluorescence compared to untreated astrocytes ( Figure 10 c). Immunofluorescence analysis also confirmed the transfer of MitoDsRed to astrocytes, and a further increase in fluorescence intensity was observed after OGD ( Figure 10 d). Astrocytes were treated with OGD for 3 or 6 hours and then incubated with MitoDsRed+-PC-EVs. OGD Incubation was performed for 24 h to further investigate whether mitochondrial uptake by astrocytes increased with prolonged OGD exposure. The proportion of MitoDsRed fluorescence in astrocytes increased with prolonged OGD treatment ( Figure 10 e).

[0083] Using BacMam technology, endogenous mitochondria of astrocytes were labeled with green fluorescent protein (GFP) and compared with MitoDsRed+-PC-EVs. OGD Incubate for 24 h to identify PC-EVs OGDAfter OGD treatment, MitoDsRed fluorescence was expressed in astrocytes and fused with the host mitochondrial network ( Figure 4 In addition, MitoDsRed-labeled pericytes were co-cultured with GFP-labeled astrocytes ( Figure 10 f). After 24 h, MitoDsRed was observed in OGD-treated astrocytes, where it colocalized with the mitochondrial network ( Figure 10 g). Using a transwell co-culture system, we demonstrated that MitoDsRed-labeled pericyte mitochondria could be transferred to astrocytes in a non-contact manner, and this transfer was significantly increased under OGD conditions ( Figure 4 As a negative control, treatment of pericytes with GW4869 significantly reduced the colocalization of RFP-labeled mitochondria and GFP in astrocytes ( Figure 4 be). They further used engineered ascorbate peroxidase (APEX2) to label mitochondria in pericytes and visualize them under an electron microscope ( Figure 10 h,i). These labeled mitochondria were transferred into astrocytes using a transwell system ( Figure 4 f, g).

[0084] Pericytes are located at the center of the neurovascular unit (NVU) and mediate neurovascular coupling through their contraction. This study demonstrates that PC-EVs also possess regenerative properties and contribute to brain repair processes. Interestingly, the neuroprotective effects of PC-EVs were enhanced after hypoxic preconditioning. A 1-hour exposure to oxygenated globulin (OGD) increased PC-EV production and secretion without causing significant pericyte death. Compared with normoxic PC-EVs, PC-EVs preconditioned with hypoxia exhibited superior therapeutic effects against ischemia-reperfusion-induced brain injury. Consistent with our findings, short-term hypoxia has been shown to enhance the therapeutic properties of mesenchymal stem cells (MSCs). Furthermore, hypoxic preconditioning enhanced angiogenesis, attenuated allergic airway inflammation and airway remodeling, and accelerated bone regeneration. This study demonstrates for the first time that pericytes, multipotent cells surrounding blood vessels, secrete increased amounts of EVs, resulting in enhanced protection against ischemia-induced brain injury as a compensatory mechanism, suggesting that PC-EVs share protective features with other stem / progenitor cells.

[0085] The brain is one of the organs with the highest energy demands and is susceptible to metabolic disturbances. During ischemia, oxygen and glucose deprivation leads to a drastic decrease in ATP production, triggering energy failure and impaired cellular function. Mitochondria play an integral role in the pathogenesis of cerebral ischemia-reperfusion injury, as they play a crucial role in energy metabolism, oxidative stress, and cell death pathways. Mitochondria and specific signaling molecules can be transferred between cells through direct intercellular contact or via EVs, thereby facilitating intercellular communication. Transferred mitochondria provide essential support for energy and metabolic regulation in injured cells, a mechanism that is particularly critical for maintaining homeostasis in the neurovascular unit (NVU). Astrocytes are an essential component of the NVU, and their mitochondria maintain the integrity of the blood-brain barrier by ameliorating endothelial cell dysfunction. Astrocyte mitochondria also provide metabolic support to pericytes to maintain vascular stability. Furthermore, astrocyte mitochondria are transferred to neurons, enhancing energy metabolism and promoting neurological recovery after stroke. Our proteomic data showed that after short-term OGD, the levels of mitochondrial components, including the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM), were significantly increased and incorporated into PC-EVs. The mitochondria in these PC-EVs were functional as determined by OCR assays and ATP production. We subsequently performed integrated transcriptome analysis to elucidate the underlying protective mechanisms. We found that PC-EVs OGD Reduced astrocyte activation in the brain of the MCAO model.

[0086] In summary, this study establishes a protective role for PC-EVs in cerebral ischemia-reperfusion injury. By transferring mitochondria from pericytes to astrocytes, PC-EVs mitigate astrocyte reactivation. These data suggest that, in addition to regulating blood flow through contraction according to the metabolic microenvironment, pericytes can also share their mitochondria to improve neuronal metabolism during ischemia. This evidence provides new insights into the potential stem cell-like properties of pericytes.

[0087] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A pericyte-derived mitochondria-rich extracellular vesicle, characterized in that: The method for extracting extracellular vesicles comprises the following steps: (1) Mince the cerebral cortex, add type II collagenase and DNase I for digestion, and then add gradient separation buffer to separate pericytes; (2) Plating pericytes on a culture dish coated with type IV collagen and culturing them in pericyte culture medium; (3) When the cell confluence reaches 80% to 90%, the cell culture medium is replaced with fresh exosome-free FBS and glucose-free culture medium. The cells are treated with OGD and then reoxygenated for 24 hours. The culture supernatant is collected and centrifuged to obtain the extracellular vesicles.

2. The pericyte-derived mitochondria-rich extracellular vesicles according to claim 1, wherein Step (1) is as follows: mince the cerebral cortex in ice-cold PBS, then add DMEM supplemented with type II collagenase and DNaseI, digest for 1-2 hours, obtain a suspension, centrifuge the suspension, discard the supernatant, resuspend the cell pellet in DMEM containing type II collagenase and dispase, digest for 0.5-1 hour, centrifuge, discard the supernatant, resuspend the cell pellet in DMEM, and then add gradient separation solution to separate pericytes.

3. The pericyte-derived mitochondria-rich extracellular vesicles according to claim 2, wherein The treatment concentration of the type II collagenase is 0.8-1.2 mg / mL, the treatment concentration of the DNaseI is 10-20 μg / mL, the treatment concentration of the dispase is 1-5 U / mL, and the digestion temperature of the type II collagenase and DNaseI is 36° C.-38° C.

4. The pericyte-derived mitochondria-rich extracellular vesicles according to claim 1, wherein The gradient separation solution is Percoll, and its concentration is 30% to 35%.

5. The pericyte-derived mitochondria-rich extracellular vesicles according to claim 1, wherein The OGD treatment specifically includes placing the pericytes in an oxygen-deficient incubator containing 95% N2 and 5% CO2 and incubating them at 37°C for 1-6 hours.

6. The pericyte-derived mitochondria-enriched extracellular vesicles according to claim 1, wherein The culture supernatant was centrifuged as follows: first at 200-400 × g for 8-12 minutes at 4°C, then at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; After centrifugation, the supernatant was collected and ultracentrifuged at 90,000–110,000 × g for 88–92 min at 4°C, and the precipitate was collected to obtain total extracellular vesicles (PC-EVs); Alternatively, centrifuge at 200-400 × g for 8-12 minutes at 4°C and then at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; After centrifugation, the supernatant was collected and separated by ultracentrifugation at 9,000-11,000 × g for 68-72 min at 4°C, and the precipitate was collected to obtain large extracellular vesicles; Alternatively, centrifuge at 200-400 × g for 8-12 minutes at 4°C and then at 1800-2200 × g for 8-12 minutes to remove dead cells and large cell debris; After centrifugation, the supernatant was collected and ultracentrifuged at 9,000-11,000 × g for 68-72 minutes at 4°C to separate large extracellular vesicles; the supernatant after the large extracellular vesicles were precipitated was filtered through a 0.22 μm membrane and then ultracentrifuged at 90,000-110,000 × g for 88-92 minutes at 4°C to collect the precipitate to obtain small extracellular vesicles.

7. A pharmaceutical composition, characterized in that The invention comprises the pericyte-derived mitochondria-rich extracellular vesicles according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier or auxiliary agent.

8. Use of the pericyte-derived mitochondria-rich extracellular vesicles according to any one of claims 1 to 6 in the preparation of a product for treating or assisting in the treatment of ischemic brain injury.

9. The use according to claim 8, characterized in that The product reduces cerebral ischemic damage by reducing the activation of astrocytes.

10. The use according to claim 8, characterized in that The product described is a drug.

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