Pericyte-derived mitochondria-rich extracellular vesicles and uses thereof in ischemic brain injury

Abstract

The application belongs to the technical field of medicine, and particularly relates to pericyte-derived mitochondria-rich extracellular vesicles and application thereof in ischemic brain injury. The extraction method of the extracellular vesicles comprises the following steps: (1) cutting a cerebral cortex part, adding collagenase type II and DNase I for digestion, and then adding a gradient separation liquid to separate pericytes; (2) spreading the pericytes on a culture dish coated with collagen type IV and culturing in a pericyte culture medium; (3) when the cell confluence degree reaches 80% to 90%, the cell culture medium is replaced with a fresh culture medium without exosomes FBS, the cells are subjected to OGD treatment and then reoxygenated for 24 hours, the culture supernatant is collected, and the culture supernatant is centrifuged to obtain the extracellular vesicles. The PC-EVs obtained after OGD stress are found to be capable of protecting the brain from ischemic stroke for the first time, and have important reference significance for researching and treating drugs related to ischemic brain injury.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a pericyte-derived extracellular vesicle rich in mitochondria and its application in ischemic brain injury. Background Technology

[0002] Ischemic stroke is a leading cause of morbidity and mortality worldwide, leading to severe and persistent neurological deficits. During ischemia, the interruption of blood flow rapidly depletes oxygen and glucose, thereby reducing ATP production and triggering cellular energy stress. This dysfunction can trigger a series of cellular events, including increased inflammation and oxidative stress, ultimately leading to neuronal apoptosis. Therefore, therapeutic strategies aimed at protecting mitochondrial integrity and function hold promise for improving neuroprotection and promoting recovery from ischemic brain injury.

[0003] Extracellular vesicles (EVs) are nanoscale, lipid-bilayered vesicles secreted by various cells. Secreted EVs can be taken up by recipient cells, delivering their contents, including active proteins, RNA species, and small molecules, thereby coordinating dynamic intercellular communication. Pericytic extracellular vesicles (PC-EVs) play multiple roles in vivo, and multiple pieces of evidence suggest that PC-EVs play important regulatory roles in various diseases, potentially serving 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 shortcomings of the prior art, the purpose of this invention is to provide a pericyte-derived extracellular vesicle rich in mitochondria and its application in ischemic brain injury.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides extracellular vesicles rich in mitochondria derived from pericytes, wherein the extraction method of the extracellular vesicles includes the following steps:

[0007] (1) The cerebral cortex was partially chopped, digested with type II collagenase and DNase I, and then separated by gradient separation solution to obtain pericytes;

[0008] (2) Place the pericytes on a culture dish coated with type IV collagen and culture them in pericyte medium;

[0009] (3) When the cell confluence reaches 80%~90%, the cell culture medium is replaced with fresh exosome-free FBS-free glucose-free culture medium. After OGD treatment, the cells are reoxygenated and cultured for 24 hours. The culture supernatant is collected and centrifuged to obtain the extracellular vesicles.

[0010] In some embodiments of the present invention, step (1) specifically involves: shredding the cerebral cortex in ice-cold PBS, then supplementing it with DMEM containing type II collagenase and DNase I, digesting for 1-2 hours to obtain a suspension, centrifuging the suspension, discarding the supernatant, resuspending the cell pellet in DMEM containing type II collagenase, dispersin and DNase I for 0.5-1.5 hours, centrifuging, discarding the supernatant, resuspending the cell pellet in DMEM, and then adding gradient separation solution to separate pericytes.

[0011] 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 DNase I is 10~20 μg / mL, the treatment concentration of the dispersant enzyme is 1~5 U / mL, and the digestion temperature of the type II collagenase and DNase I is 36℃~38℃.

[0012] In some embodiments of the present invention, the gradient separation liquid is Percoll, with a concentration of 30% to 35%.

[0013] In this invention, in DMEM containing type II collagenase and DNase I, the digestion time can be 1 h, 1.5 h, or 2 h, etc., and the digestion temperature can be 36 °C, 37 °C, or 38 °C, etc. In DMEM containing type II collagenase and dispersant enzyme, the digestion time can be 0.5 h, 1 h, or 1.5 h, etc., and the digestion temperature can be 36 °C, 37 °C, or 38 °C, etc. 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. The concentration of DNase I can be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, or 20 μg / mL. The concentration of the dispersant enzyme can be 1 U / mL, 2 U / mL, 3 U / mL, 4 U / mL, or 5 U / mL. The concentration of Percoll can be 30%, 32%, 33%, 34%, or 35%, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0014] In some embodiments of the present invention, the OGD treatment specifically involves placing pericytes in an anaerobic incubator containing 95% N2 and 5% CO2 and incubating at 37°C for 1-6 hours.

[0015] In some embodiments of the present invention, the OGD treatment specifically involves: when the cell confluence reaches 80% to 90%, the pericyte culture medium is replaced with exosome-free FBS and glucose-free culture medium, and incubated at 37°C for 1 to 6 hours in an oxygen-deficient incubator containing 95% N2 and 5% CO2; subsequently, it is reoxygenated at 37°C for 24 hours in a normal cell culture incubator containing 95% air and 5% CO2.

[0016] In some embodiments of the present invention, the exosome-free FBS glucose-free culture medium is glucose-free culture medium supplemented with 10% exosome-free fetal bovine serum.

[0017] In some embodiments of the present invention, the method for preparing the exosome-free fetal bovine serum is as follows: fresh fetal bovine serum is ultracentrifuged overnight at 100,000 x g at 4°C to remove exosomes, and the supernatant is collected.

[0018] In this invention, the incubation time can be 1h, 2h, 3h, 4h, 5h or 6h, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0019] In this invention, without causing significant pericyte death, OGD treatment for 1 hour increased the generation and secretion of PC-EVs. Compared with PC-EVs under normoxic conditions, PC-EVs pretreated with hypoxia had a better therapeutic effect on brain injury caused by ischemia-reperfusion.

[0020] In some embodiments of the present invention, the culture supernatant is centrifuged as follows: first, centrifuged at 200-400×g for 8-12 minutes at 4°C, then centrifuged at 1800-2200×g for 8-12 minutes to remove dead cells and large cell debris; after centrifugation, the supernatant is collected, filtered through a 0.22μm membrane, and then ultracentrifuged at 90000-110000×g for 88-92 minutes at 4°C, and the precipitate is collected to obtain total extracellular vesicles (PC-EVs).

[0021] Alternatively, centrifuge at 200-400×g for 8-12 minutes at 4°C, 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 9,000-11000×g for 68-72 minutes at 4°C, and collect the precipitate to obtain large extracellular vesicles (l-EVs).

[0022] Alternatively, centrifuge at 200-400×g for 8-12 minutes at 4°C, 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 for 68-72 minutes at 4°C to separate large extracellular vesicles; filter the supernatant after the large extracellular vesicle precipitation through a 0.22μm membrane, and then ultracentrifuge at 90,000-110,000×g for 88-92 minutes at 4°C to collect the precipitate to obtain small extracellular vesicles (s-EVs).

[0023] In some embodiments of the present invention, the culture supernatant is centrifuged as follows: first, centrifuged at 300×g for 10 minutes at 4°C, then centrifuged at 2000×g for 10 minutes to remove dead cells and large cell debris; after centrifugation, the supernatant is collected, filtered through a 0.22μm membrane, and then ultracentrifuged at 110,000×g for 90 minutes at 4°C to collect the precipitate and obtain total extracellular vesicles (PC-EVs).

[0024] Alternatively, centrifuge at 300×g for 10 minutes at 4°C, then centrifuge at 2000×g for 10 minutes to remove dead cells and large cell debris; after centrifugation, collect the supernatant and ultracentrifuge at 10000×g for 70 minutes at 4°C, and collect the precipitate to obtain large extracellular vesicles (l-EVs).

[0025] Alternatively, centrifuge at 300×g for 10 minutes at 4°C, then centrifuge at 2000×g for 10 minutes to remove dead cells and large cell debris; after centrifugation, collect the supernatant and ultracentrifuge at 10000×g for 70 minutes at 4°C to separate large extracellular vesicles; filter the supernatant after the large extracellular vesicle precipitation through a 0.22μm membrane, then ultracentrifuge at 100000×g for 90 minutes at 4°C, and collect the precipitate to obtain small extracellular vesicles (s-EVs).

[0026] In this invention, the size range of l-EVs is 50~400nm, and the size range of s-EVs is 50~200nm.

[0027] A second aspect of the invention provides a pharmaceutical composition comprising the pericyte-derived, mitochondrial-rich extracellular vesicles described in the first aspect and a pharmaceutically acceptable carrier or adjuvant.

[0028] In some embodiments of the present invention, the content of the excipients in the above-mentioned pharmaceutical composition may 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, and is usually about 80% by weight.

[0029] In some embodiments of the present invention, the pharmaceutical composition can be prepared by mixing the extracellular vesicles with a pharmaceutically acceptable carrier, for example, to obtain oral formulations 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 formulations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drops), topical formulations (e.g., skin formulations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory formulations (inhalers), eye drops, etc. In addition, these formulations can be used as controlled-release formulations (e.g., sustained-release microcapsules), such as immediate-release formulations, sustained-release formulations, etc. Such formulations can be obtained by preparation methods conventionally used in this art.

[0030] In some embodiments of the present invention, examples of the pharmaceutically acceptable carriers described above 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, gel, polyvinylpyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g., calcium carboxymethyl cellulose, talc, etc.), diluents (e.g., water for injection, saline, etc.), additives (e.g., stabilizers, preservatives, colorants, flavoring agents, solubilizers, emulsifiers, buffers, isotonic agents, etc.), and so on.

[0031] Drugs or pharmaceutical compositions containing the extracellular vesicles described in this application may be administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys). Administration may be by oral or parenteral route, such as intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, intravenous drip, intracerebral, rectal, vaginal, intraperitoneal, etc.

[0032] The dosage of extracellular vesicles described in this application for administration to subjects varies depending on the route of administration, symptoms, patient age, etc., and can be determined by clinicians in practice.

[0033] The drugs or drug compositions described in this application can also be used in conjunction with other existing known drugs for treating ischemic brain injury. When used together, there are no restrictions on the timing of administration of the respective drugs; two or more different drugs can be administered simultaneously, or at different times. The dosage of the known drugs can be determined according to clinically used dosages and appropriately selected based on the patient, route of administration, etc.

[0034] A third aspect of the invention provides the use of pericyte-derived, mitochondrial-rich extracellular vesicles as described in the first aspect in the preparation of products for the treatment or adjunctive treatment of ischemic brain injury.

[0035] In some embodiments of the present invention, the product reduces brain ischemia-reperfusion injury by reducing the activation of astrocytes.

[0036] In some embodiments of the present invention, the product is a drug.

[0037] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0038] (1) This invention establishes the protective effect of PC-EVs in cerebral ischemia-reperfusion injury. Pericytic extracellular vesicles (PC-EVs) are rich in mitochondria after oxygen-glucose deprivation (OGD), and mitochondrial components are packaged into EVs. These pericytic mitochondria are transferred and integrated into the mitochondrial network of astrocytes, thereby improving astrocyte activation.

[0039] (2) This invention demonstrates that PC-EVs obtained after OGD stress can protect the brain from ischemic stroke. Mechanistically, pericyte-derived EVs can reduce astrocyte activation, thereby alleviating ischemic brain injury. This invention reveals that pericytes can serve as a valuable source of mitochondrial transfer, and that ischemia-induced mitochondrial transfer may represent a compensatory mechanism for ischemic preconditioning. Attached Figure Description

[0040] Figure 1To illustrate how OGD increases the release of mitochondrial-containing extracellular vesicles from pericytes in this embodiment of the invention, a) is a schematic diagram of pericyte-derived EVs (PC-EVs) after OGD treatment; b) shows the concentration of PC-EVs (n=6) after 1, 3, and 6 hours of OGD treatment followed by reoxygenation for 24 hours; c) shows the Western blot analysis of EV marker proteins Alix, HSP70, and TSG101 in whole-cell lysates (WCLs) and PC-EVs; d) shows the concentrations of Alix and HSP70 in PC-EVsCon and PC-EVsOGD. Quantitative analysis of O and TSG101 protein expression (n=6); e is a representative TEM image of multivesicular bodies (MVBs) in OGD-treated and control pericytes, with red arrows indicating 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... The dashed line represents 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 plot of PC-EV proteome, mouse MitoCarta 3.0 list proteins, and mitochondrial-related GO terms; l is the 113 shared proteins filtered by mouse species and the Vesiclepedia database; m is the top 200 most significant proteins. Percentage of mitochondrial components in differentially expressed proteins; n represents double-labeled immunoelectron microscopy of PC-EVs, CD63 (large particles) and PDH (small particles), with red arrows marking examples of CD63-positive particles and green arrows marking examples of PDH-positive particles, scale bar = 200 nm; o represents PC-EVs co-immunostained with anti-CD63 and anti-mitochondrial protein PDH antibodies, scale bar = 500 nm; p represents representative TEM images of mitochondria in OGD-treated and control pericyte-derived PC-EVs, scale bar = 200 nm; q represents PC-EVs. Con and PC-EVs OGD Western blot analysis of mitochondrial oxidative phosphorylation (OXPHOS) complex protein levels; r represents real-time detection of PC-EVs by OCR. Con and PC-EVs OGD Oxygen consumption (n=6); s represents PC-EVs Con and PC-EVs OGDThe relative ATP content (n=6); data are expressed as mean ± SEM, and were analyzed by two-tailed unpaired t-tests (d,i,m,s), one-way ANOVA combined with Tukey post-hoc test (b) or two-way ANOVA combined with Tukey post-hoc test (r). *P<0.05, **P<0.01.

[0041] Figure 2 To illustrate how PC-EVs reduce the infarct area and improve long-term neurological function after cerebral ischemia-reperfusion injury in this embodiment of the invention, the following figures are presented: a) Schematic diagram of PC-EVs administration and behavioral analysis; b) Effect of PC-EVs administration 24 hours after MCAO on ipsilateral and contralateral brain water content (n=6); c) mNSS scores assessed at 1, 3, 7, and 14 days after MCAO (n=10); d) Effect of PC-EVs administration on body weight change (n=10); e) T2-weighted images of infarct volume in different groups; f) Quantitative analysis of infarct volume (n=6); g) Representative images of H&E and Nissl staining in the injured hemisphere, scale bar = 10 μm; h) Cortical area of ​​the injured hemisphere. Quantitative analysis of the percentage of damaged neurons in the brain region (n=6); i, j. are representative images (i) and statistical analysis (j) of cerebral blood flow in mice treated with PC-EVs 14 days after MCAO (n=6); kn are sensorimotor function assessed by grid walking test (k), cornering 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 ANOVA combined with Tukey post-hoc test (b, h, j) or two-way ANOVA combined with Tukey post-hoc test (c, d, kn). Con vs. MCAO or PC-EVs OGD vs.MCAO, *P<0.05, **P<0.01.

[0042] Figure 3 In this embodiment of the invention, PC-EVs are taken up by astrocytes and astrocyte activation is inhibited; wherein, a is PKH67-labeled PC-EVs. OGD Image (red circle); b is a representative fluorescence image. Statistical analysis shows that MCAO increases the response of astrocytes to PC-EVs. OGD The uptake of PC-EVs; c represents the differentially expressed gene analysis after PC-EVs administration, the upper figure is the Venn diagram of the intersection of the three groups of differentially expressed genes, and the lower figure is the heatmap of differentially expressed genes; d and e are the Venn diagram (d) and ternary diagram (e) of gene expression in reactive astrocytes, respectively; f represents the uptake of PC-EVs. OGDRepresentative fluorescence images of C3 and GFAP co-labeled astrocytes after drug administration; g is a Z-score heatmap of gene expression in primary astrocytes after treatment with OGD or PC-EVs; h is a Shollen analysis of GFAP-immunolabeled astrocytes in the cortex (shown in black), with concentric circles originating from the astrocyte cell body; i is a Z-score heatmap of PC-EVs inhibiting OGD-induced A1-responsive astrocytes in primary astrocytes; data are expressed as mean ± SEM, and a two-sided unpaired t-test (a, h) was used, *P<0.05, **P<0.01.

[0043] Figure 4 In this embodiment of the invention, mitochondria are transferred from pericytes to astrocytes via PC-EVs and integrated into the astrocyte mitochondrial network; wherein, a is the use of MitoDsRed⁺-PC-EVs OGD Representative fluorescence images of treated astrocytes show the fusion of transferred mitochondria 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 system for 24 hours; ce is a representative fluorescence image (c) and statistical analysis (d, e) of astrocytes after OGD or GW4869 treatment showing the fusion of transferred MitoDsRed 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 astrocytes internalizing APEX2+ mitochondria from pericytes via PC-EVs, and astrocytes that have not internalized APEX2+ mitochondria; data are expressed as mean ± SEM, and one-way ANOVA combined with Tukey post-hoc test (d, e), *P<0.05, **P<0.01.

[0044] Figure 5This invention illustrates how OGD increases the release of MVBs and PC-EVs. Specifically, a) is a schematic diagram of pericytes under OGD conditions; b) is a representative fluorescence image of TUNEL staining at different time points during OGD treatment (scale bar = 10 μm); c) is a statistical analysis of TUNEL-positive cells at different time points during OGD treatment (n = 6); d) is a representative TEM image of PC-EVs secreted by control and OGD-treated pericytes, with red triangles indicating PC-EVs already secreted extracellularly and green triangles indicating PC-EVs currently 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) and the size of each 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 (IEM) image showing LBPA-labeled MVBs in OGD-treated and control pericytes. Bs, scale bar = 200 nm; i is the dot blot analysis of CD63 and LBPA protein expression in OGD-treated and control pericytes; j is a schematic diagram of the isolation of large and small EVs from pericyte conditioned medium; k is the Western blot analysis of Alix, HSP70 and TSG101 protein expression in large and small EVs; l, m are the quantitative analysis of Alix, HSP70 and TSG101 protein expression in large EV (l) and small EV (m) respectively (n=6); n is the particle size distribution of large and small EVs secreted by control and OGD-treated pericytes, analyzed by nanoparticle tracking (NTA); o is the morphology of large and small EVs observed by negative staining TEM, scale bar = 200 nm; data are expressed as mean ± SEM, and one-way ANOVA combined with Tukey post-hoc test (c) or two-sided unpaired t test (e,f,l,m) was used, *P<0.05, **P<0.01; ns, no significant difference.

[0045] Figure 6In this embodiment of the invention, OGD-treated pericyte-derived PC-EVs are rich in mitochondrial components; a) is the top 10 cellular components in GO analysis of differentially expressed proteins (p<0.05); b) is the top 10 cellular components in GO analysis of upregulated proteins (p<0.05); c) is all upregulated proteins sorted by abundance level; d) is the cellular components in GO analysis of the top 200 upregulated proteins (p<0.05); e) is 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 and g are the quantitative analyses of PDH, SDHA, and CS protein expression in mitochondria (f) and PC-EVs (g), respectively (n=6); h) is the expression of PDH, SDHA, and CS proteins in large and small EVs. Western blot analysis of expression; i, j are quantitative analyses of PDH, SDH, and CS protein expression in large EVs (i) and small EVs (j), respectively (n=6); k is flow cytometry 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 representative TEM images of pericyte mitochondrial structure 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 two-sided unpaired t-tests (f, g, i, j, l) or one-way ANOVA combined with Tukey post-hoc test (n) were used. *P<0.05, **P<0.01; ns, no significant difference.

[0046] Figure 7 To illustrate how PC-EVs reduced the cerebral infarction area in MCAO mice in this embodiment of the invention; where a represents a representative image and statistical analysis of brain slices stained with TTC 24 hours after MCAO; b represents a representative image of brain slices stained with hematoxylin and eosin (H&E) 3 days after MCAO; data are expressed as mean ± SEM, and one-way ANOVA combined with Tukey post-hoc test (a) was used, **P<0.01.

[0047] Figure 8In this embodiment of the invention, OGD treatment enhances the uptake of PC-EVs by astrocytes; 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 co-culturing PKH67-labeled PC-EVs with OGD-treated astrocytes; d is a representative fluorescence image showing that the uptake of PC-EVs by astrocytes increases with the extension of OGD treatment time, scale bar = 10 μm; e is a schematic diagram of the construction of Cre-dependent color conversion vector; f is a representative fluorescence image of astrocytes uptake of Cre-containing PC-EVs, with fluorescence changing 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.

[0048] Figure 9 In this embodiment of the invention, PC-EVs inhibited astrocyte activation after MCAO; wherein, a and b are the PCA map (a) and differentially expressed gene volcano map (b) of the sample in the transcriptional analysis, respectively; c is the heatmap 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 injected with an equal volume of PBS; e and f are the representative binary image (e) and cytoskeleton image (f) converted from the astrocyte image, respectively; g is the Sholl analysis of astrocyte branches; h is the PC-EVs after MCAO. OGD Western blot analysis of GFAP and EAAT1 protein expression in the treated cortex; i represents the quantitative analysis of GFAP and EAAT1 protein expression (n=6); data are expressed as mean ± SEM, and two-way ANOVA combined with Tukey post-hoc test (g) was used. *P<0.05, **P<0.01.

[0049] Figure 10 In this embodiment of the invention, mitochondria are transferred from pericytes to astrocytes via PC-EVs and integrated into the astrocyte mitochondrial network; wherein, a) is flow cytometry analysis of pericytes infected with MitoDsRed-labeled cells expressing MitoDsRed adenovirus; b) is analysis of OGD-treated cells after OGD treatment with MitoDsRed... + PC-EVs OGD Schematic diagram of astrocyte treatment; c and d are OGD treatment followed by MitoDsRed, respectively. + PC-EVs OGDFlow cytometry (c) and immunofluorescence (d) analysis of MitoDsRed in treated astrocytes (n=6), scale bar=10μm; e shows the results of MitoDsRed treatment at different OGD times. + PC-EVs OGD Flow cytometry analysis of processed astrocytes (n=6); f is a schematic diagram of co-culturing MitoDsRed-labeled pericytes with GFP-labeled astrocytes; g is a representative fluorescence image of co-culturing GFP-labeled astrocytes with MitoDsRed-labeled pericytes, showing mitochondria obtained from MitoDsRed-labeled pericytes and fused with GFP-labeled mitochondria from astrocytes; h is a schematic diagram of TEM observation of APEX2+ mitochondria: after transfecting cells with plasmids expressing APEX2, the cells were fixed and treated with diaminobenzidine (DAB) and hydrogen peroxide. APEX2 catalyzes DAB polymerization, and treatment with osmium tetroxide 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-tests (c, d) or one-way ANOVA combined with Tukey's post-hoc test (e), **P<0.01. Detailed Implementation

[0050] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0052] To enable those skilled in the art to better 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.

[0053] Example

[0054] 1. Experimental Methods:

[0055] Primary pericyte culture

[0056] 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 separated and minced in ice-cold PBS with ophthalmic scissors. It was then digested in DMEM supplemented with type II collagenase (1 mg / mL) and DNase I (15 μg / mL) for 1.5 h, incubated at 37 °C, and centrifuged at 1000 × g for 5 min at 4 °C. The resulting pellet was resuspended in DMEM containing 20% ​​BSA and centrifuged at 1000 × g for 20 min. After discarding the supernatant, the pellet was resuspended in DMEM containing type II collagenase (1 mg / mL), dispersin (2 U / mL), and DNase I, and incubated with shaking at 37 °C for 60 min. The cell pellet was resuspended in DMEM and separated using a 33% discontinuous Percoll gradient, centrifuged at 1000 g for 10 min at 4 °C. Pericellular cells were plated onto culture dishes coated with type IV collagen and cultured in pericellular culture medium (ScienCell, 1201) for approximately 2 weeks.

[0057] Culture of primary astrocytes

[0058] Primary glial cell cultures were prepared from the cerebral cortex of C56BL / 6J mice (day 1 after birth). The collected cerebral cortex was cut into approximately 1 mm cubes in ice-cold PBS and digested with 0.25% trypsin at 37°C for 30 minutes. An equal volume of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added to inactivate the trypsin. The digestion solution was then mixed, repeatedly pipetted, 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 with 5% CO2. The medium was changed every 3 days. After 14 days of culture, microglia growing on the upper layer were removed from the mixed glial cell culture by shaking at 180 rpm for 30 minutes. The remaining adherent cells in the culture flasks were astrocytes.

[0059] Primary neuron culture

[0060] Obtained from the cerebral cortex of C56BL / 6J mice (day 15 of embryonic development). Cut into 1mm pieces. 3Cells of the cerebral cortex were digested with 0.25% trypsin at 37°C for 30 minutes. An equal volume of basal neural culture medium (containing 2% B27, 1% penicillin / streptomycin, and 1% glutamine enhancer) was added to terminate trypsin digestion. The tissue digestion solution was repeatedly mixed using a pipette and then filtered through a 40 μm cell sieve. The filtrate was centrifuged at 1500g for 10 minutes and resuspended in the same basal neural culture medium. The cell suspension was placed in a culture flask coated with poly-D-lysine and incubated at 37°C in a 5% CO2 incubator. In subsequent cultures, half of the culture medium was replaced every 3 days for a total of 10 days.

[0061] Oxygen deprivation / reoxygenation (OGD / R)

[0062] When the cell confluence reaches 80%–90%, the medium is replaced with exosome-free FBS and glucose-free medium. This medium is composed of glucose-free medium (Life Technology, 11966-025) with 10% exosome-free fetal bovine serum (FBS). Fresh FBS is ultracentrifuged overnight at 100,000 x g at 4°C to remove exosomes. The cells are then incubated at 37°C for 1–6 hours in an anoxic incubator containing 95% N2 and 5% CO2, followed by reoxygenation at 37°C for 24 hours in a normal cell culture incubator containing 95% air and 5% CO2.

[0063] Isolation and identification of extracellular vesicles (EVs)

[0064] Once the cell confluence reached 90%, the medium was replaced with exosome-free FBS and glucose-free medium. After a specific period of OGD treatment, the cells were reoxygenated for 24 hours. The conditioned medium (pericellular culture supernatant after reoxygenation) was collected. The supernatant was first centrifuged at 300×g for 10 minutes at 4°C, then at 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 separated 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 separate small EVs.

[0065] Transmission electron microscopy (TEM)

[0066] Standard electron microscopy analysis was performed on PC-EVs, pericytes, astrocytes, and brain tissue samples. PC-EVs or cells were fixed overnight at 4°C with 2.5% glutaraldehyde, washed three times (3 minutes each) with 0.1M phosphate-buffered saline (pH 7.4), and then embedded in 1% agarose. Mice were perfused transcranially with a 0.1MPB mixture of 2.5% glutaraldehyde and 2% paraformaldehyde. Freshly collected cortical tissue was minced to 1 mm in cold fixative. 3 PC-EVs, cells, and tissues were fixed with 1% osmium tetroxide at room temperature in the dark for 2 hours, dehydrated with a series of acetone solutions (30%, 50%, 70%, 80%, 95%, 100%), and embedded in Epon 812. Semi-thin sections were stained with toluidine blue, and ultrathin sections were stained with 2% uranium acetate and lead citrate. Imaging was performed using a Hitachi HT-7800 transmission electron microscope (80kV) equipped with a CMOS camera.

[0067] Immunogold electron microscopy (IEM)

[0068] For immunoelectron microscopy analysis of cells, cells were fixed with dedicated IEM fixative (Servicebio, G1124), washed with 0.1M PB solution, dehydrated with a series of acetones, and embedded into sections. Ultrathin sections were placed on Formvar / carbon-coated 100-mesh nickel grids, blocked with 1% BSA solution at room temperature for 30 minutes, incubated overnight at 4°C with anti-CD63 (ab217345, Abcam) or LBPA (MABT837, Sigma) primary antibody, and then incubated for 20 minutes with 12nm colloidal gold conjugated secondary antibody (111-205-114 or 115-205-146, Jackson Immuno Research), stained with 2% uranium 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 onto a Formvar / carbon-coated mesh for 20 min, quenched with PBS / 50 mM glycine, and blocked with 1% BSA. The EVs were then combined with mouse anti-PDH (dilution 1:40; sc-377092, Santa Cruz) alone or with rabbit anti-CD63 (dilution 1:20; ab217345, Abcam). Samples were incubated with the corresponding gold-labeled secondary antibody (12nm anti-mouse, 111-205-146 or 12nm anti-rabbit, 111-205-114 / 4nm anti-mouse, 115-185-146; Jackson Immuno Research) at a 1:100 dilution. The samples were negatively stained with 2% uranium acetate and embedded with methylcellulose / uranium acetate (2% / 0.5%). The images were then imaged using a Hitachi HT7800 transmission electron microscope (80kV).

[0069] Proteomics analysis

[0070] PC-EVs were purified from pericyte conditioned medium by ultracentrifugation, lysed with 1% SDS protein lysis buffer and a protease inhibitor cocktail, and protein concentration was determined using a BCA kit (Thermo Scientific). After protein quantification, SDS-PAGE electrophoresis was performed. The samples were reduced-alkylated with iodoacetamide and then digested overnight at 37°C with trypsin at a protein mass ratio of 1:50 to form a peptide mixture. The peptides were vacuum dried, acidified with 1% (v / v) trifluoroacetic acid, desalted on an HLBC18 column, and quantified using a peptide quantification kit (Thermo Science). Separation and identification were performed using a Vanquish Neo-Orbitrap Astral mass spectrometer (Thermo, USA). A uPAC high-throughput column (75 μm × 5.5 cm, Thermo) was used. Mobile phase A consisted of water containing 2% acetonitrile and 0.1% formic acid, and mobile phase B consisted of water containing 80% acetonitrile and 0.1% formic acid. The mass spectrometer operates 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.

[0071] Transcriptomics analysis

[0072] Total RNA was extracted from mouse cells and brain tissue using TRIzol reagents according to the manufacturer's protocol. Subsequent RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd. In short, poly(A) mRNA was purified and fragmented from total RNA using oligo(dT) magnetic beads. The fragmented RNA was reverse transcribed into first-strand cDNA, followed by the synthesis of double-stranded cDNA (dscDNA). The purified dscDNA underwent end repair and 3' adenylation before ligation with sequencing adapters. The resulting product was purified and amplified by PCR to generate a cDNA library. The library fragment size was analyzed using an Agilent 2100 bioanalyzer, and after passing the analysis, it was sequenced on the Illumina NovaSeq platform. Raw paired-end reads were trimmed and quality-controlled using FASTP. Clean reads were aligned to a reference genome using HISAT2 in directional mode. Aligned reads from each sample were assembled using a reference-based method using StringTie. Differentially expressed genes (DEGs) were identified using DEseq2; genes with |log2FC|>1 and FDR<0.05 were considered DEGs.

[0073] TUNEL staining

[0074] Pericytes were treated with OGD for 1, 3, or 6 hours and then stained with TUNEL. Cells were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, and then incubated with 0.3% Triton X-100 in PBS at room temperature for 5 minutes to increase cell membrane permeability. After washing twice with PBS, 50 μL of TUNEL detection solution was added to the sample, and the sample was incubated at 37ºC in the dark for 60 minutes. The sample was washed three more times with PBS, mounted with anti-fluorescence quenching mounting medium, and observed under a fluorescence microscope.

[0075] ATP measurement

[0076] 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 the relative luminescent units (RLUs) were measured using a luminometer.

[0077] Oxygen consumption rate (OCR)

[0078] For OCR analysis of PC-EVs, the 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 MEGTA, 0.2% BSA), added to an XFe24 microplate, and centrifuged at 2000 × g for 10 min at 4 °C. After centrifugation, glutamate (10 mM) and malic acid (2 mM) were added to the MAS to make a final volume of 500 μL per well. The microplate was incubated at 37 °C in a CO2-free environment for 10 min, then transferred to an XFe24 analyzer. 40 mM MADP was injected sequentially at 37 °C, and OCR was recorded.

[0079] Flow cytometry analysis

[0080] To detect mitochondria in PC-EVs, pericyte supernatant was collected, and PC-EVs were isolated after incubation at 37°C for 30 minutes with 100 nM Mito-Tracker Red CMXRos (Beyotime). For analysis of mitochondria in MitoDsRed-labeled PC-EVs, PC-EV isolation was performed after staining the pericyte supernatant. To detect MitoDsRed-labeled mitochondria in astrocytes, astrocytes were co-cultured with PC-EVs containing MitoDsRed, then incubated with 100 nM Mito Tracker Red CMXRos, centrifuged, and resuspended in fresh culture medium. All samples were analyzed by flow cytometry (BDBioscience, CA).

[0081] Adenovirus Construction and Infection

[0082] Recombinant adenovirus expressing MitoDsRed (Ad-MitoDsRed) and control adenovirus were constructed by Weizhen Biotechnology Co., Ltd. (Shandong Weizhen) using the pAdM-CMV vector system. In short, 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. For adenovirus infection, cells were planted at a density of 5 × 10⁶ cells per well. 4 Ad-MitoDsRed cells were seeded at a density of 100 in 24-well plates and infected with a multiplicity of infection (MOI) of 100. Fresh medium was added 24 hours after infection. Transfection efficiency was assessed under a fluorescence microscope 48 hours after transfection, and cells were screened with 1 μg / mL puromycin.

[0083] PC-EVs Tracking

[0084] 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 were dissolved in 100 μL PBS and injected into the lateral ventricle. Brain tissue was collected 24 hours after injection for immunofluorescence staining.

[0085] Constructing mitoAPEX2-labeled pericytes

[0086] Periperocytes were transfected with the pCMV-N-mito-Flag-APEX2 (Beyotime, D3047) plasmid to construct mitoAPEX2-labeled periperocytes. This plasmid was designed to express an N-terminal fused mitochondrial matrix-targeting peptide and APEX2. When cell confluence reached 80%, the plasmid was transfected into periperocytes using Lipo8000, and stable expression of the mitoAPEX2 reporter gene was achieved after selection with neomycin. Cells were fixed with 2% glutaraldehyde and stained with cold 3,3'-diaminobenzidine (0.5 mg / mL) and H2O2 (10 mM) for 15 minutes. APEX2 activity was observed by TEM. Samples were then stained with 2% OsO4, which reacted with DAB polymers to deposit electron-dense osmium for electron microscopy contrast. Cells were then stained with 2.5% uranium acetate, dehydrated in a gradient of ethanol, infiltrated, embedded, and observed under an electron microscope.

[0087] RNA extraction and quantitative real-time PCR

[0088] 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 CFX96Touch system using SuperRealPreMixPlus (Tiangen, Beijing). Amplification conditions were 95℃ for 2 minutes, followed by 40 cycles: 95℃ for 10 seconds, 60℃ for 30 seconds. β-actin was used as an internal reference gene, and gene expression was calculated using the 2^(-ΔΔCT) method. Primer sequences are shown in Table 1.

[0089] Mitochondrial DNA Extraction and Quantification

[0090] 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 using a SuperRealPreMixPlus (Tiangen, Beijing) Bio-Rad CFX96Touch system. 18S ribosomal RNA was used as an internal reference gene. Primer sequences are shown in Table 1.

[0091] Table 1 Primer Sequences

[0092]

[0093] animal

[0094] The animal research protocol was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical College and conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals". C57BL / 6 mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., and were housed under standard conditions (temperature: 22±2℃; 12:12 hour light / dark cycle; humidity: 50±5%), with free access to sterile food and water, and were allowed to acclimatize to the environment for 7 days before the experiment. All experimental mice were 7-week-old males and were randomly assigned to either the control or experimental groups.

[0095] Middle cerebral artery occlusion model (MCAO)

[0096] Focal cerebral ischemia was induced by a modified MCAO procedure as previously described. Mice were anesthetized and their skin exposed. A silicone-coated 6-0 nylon monofilament was inserted into the internal carotid artery via the external 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 heated pad with their body temperature maintained at 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).

[0097] brain water content

[0098] Mice were deeply anesthetized, and the entire brain was carefully extracted from the skull. The contralateral and ipsilateral hemispheres were then separated, and the samples were weighed using recoded values ​​to obtain the wet weight. The samples were then dried in an oven at 100°C for 48 hours and weighed again, with this value recorded as the dry weight. Brain water content was calculated as follows: Brain water content (%) = [(wet weight - dry weight) / wet weight] × 100%

[0099] Neurological deficit

[0100] Neurological deficits were assessed using the modified neurological deficit score (mNSS), which ranges from 0 to 14 points. Higher scores indicate more severe neurological deficits. mNSS testing was performed at 1, 3, 7, and 14 days after MCAO.

[0101] Behavioral analysis

[0102] Grid walking test: Mice were placed on a 32cm×20cm×50cm (length×width×height) elevated metal grid with 12cm×12cm mesh openings. They were trained for 4 days prior to 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, and the number of "foot slips" was recorded (if stride length did not provide support, the foot penetrated the mesh opening, or the mouse's wrist rested against the grid). The total number of steps was recorded at the end of each test, and the percentage of foot slips was calculated as: (number of foot slips / total steps) × 100%.

[0103] Corner test: Place the mouse in a device formed by two transparent plates at a 30-degree angle, with 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. Test each mouse 10 times and calculate: (number of turns to the ischemic side / total number of turns) × 100%.

[0104] Rotary bar test: Mice were pre-trained for 3 days before MCAO. During the test, the mice were placed on a rotating drum that was steadily accelerated from 5 rpm to 40 rpm within 5 minutes. The time it took for the mice to fall was recorded. The test was conducted 3 times a day with an interval of 15 minutes. The time spent on the bar 1 day before the surgery was recorded as the baseline value. The average of the 3 tests after MCAO was used as the fall latency.

[0105] Cylinder test: The mouse was placed in a plastic cylinder 15cm high and 10cm in diameter and recorded for 5 minutes. The exploration / pressing score was calculated as: (number of right paw contact - number of left paw contact) / (number of right paw contact + number of left paw contact + number of both paw contact).

[0106] TTC staining

[0107] Twenty-four hours after MCAO, mice were euthanized under deep anesthesia. 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 then fixed with 4% paraformaldehyde. Non-infarcted tissue was stained red, while infarcted areas remained white. After photographing the sections, the infarct area was quantified using ImageJ, and the percentage of infarct area was calculated as the ratio of the infarct area to the area of ​​the contralateral brain.

[0108] H&E and Nissl staining

[0109] The brain was fixed with 4% paraformaldehyde, embedded in paraffin, and serially sectioned. The paraffin sections were dewaxed, rehydrated, and routinely stained with hematoxylin-eosin or 0.1% cresol violet. The sections were then photographed under an Olympus IX73 microscope.

[0110] Magnetic resonance imaging (MRI)

[0111] On day 14 after MCAO, mice underwent MRI using a Bruker Biospect 7.0T 20cm MR imaging system. Mice were anesthetized with 2% isoflurane and maintained with 1.5% isoflurane via a small animal anesthesia machine. Throughout the test, mice were kept at a body temperature of 37°C on a heating pad. T2-weighted imaging parameters: 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, mean number of slices = 6. Infarct volume was analyzed using ImageJ.

[0112] Immunoblotting

[0113] EVs, cells, and brain tissues were lysed with RIPA buffer. Protein concentration was determined using a BCA protein assay kit. Lysates were mixed with loading buffer, boiled, and then separated by SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% skim milk in TBST, and incubated overnight at 4°C with a suitable primary antibody. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody, developed using the ChemiDoc+ system, and quantified using ImageJ software. For dot blot analysis, 2 μL of sample was spotted onto a nitrocellulose membrane, blocked, incubated with a suitable primary antibody, and imaged after secondary antibody treatment.

[0114] Immunofluorescence staining

[0115] For immunofluorescence staining of brain tissue, mouse brains were fixed with 4% PFA at 4°C, embedded in paraffin, and cut into 4μm thick sections. The sections were dewaxed, rehydrated, and subjected to antigen retrieval with EDTA buffer. Endogenous peroxidase activity was blocked with 3% H2O2, and non-specific binding was blocked with 3% BSA. The sections were incubated overnight with a suitable primary antibody, followed by incubation with a fluorescently labeled secondary antibody. For cell staining, cultured cells were fixed, permeabilized, blocked, and then incubated with a suitable primary antibody and a fluorescent secondary antibody. Immunofluorescence staining of PC-EVs was performed according to previous protocols. Pericytic cell-derived PC-EVs were treated with PEG10000, incubated overnight with a primary antibody mixture, washed, and then purified using a fluorescently conjugated secondary antibody. Unbound antibodies were removed by column purification. PC-EVs were placed on slides and imaged using a confocal microscope.

[0116] Immunohistochemistry

[0117] In brain immunohistochemistry, brain tissue was dewaxed with xylene, rehydrated with ethanol, and after antigen retrieval, endogenous peroxidase activity was blocked with 3% H2O2 for 20 minutes, and non-specific binding sites were blocked with 3% BSA. Sections were incubated overnight at 4°C with anti-GFAP (ab7260, Abcam) primary antibody, followed by incubation at room temperature with HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech). Freshly prepared DAB solution was added, and the colorimetric reaction was terminated with distilled water. Sections were counterstained with hematoxylin staining solution, differentiated with hematoxylin differentiation solution, treated with hematoxylin blue solution, and images were acquired under a conventional optical microscope (E100, Nikon).

[0118] Statistical analysis

[0119] All data are expressed as mean ± standard error of mean (SEM). Normality and homogeneity of variance were tested using the Shapiro-Wilk test and the Levene test, respectively. Two-tailed unpaired Student's test was used for comparisons between two groups; one-way ANOVA combined with Tukey's post-hoc test was used for comparisons among multiple groups; and two-way ANOVA combined with Tukey's post-hoc test was used for simultaneous analysis of two factors among multiple groups. Data were analyzed using GraphPad Prism 8.0 software, and p < 0.05 was considered statistically significant. All participants were blinded to the experimental conditions during data collection and analysis and received prior training.

[0120] 2. Results

[0121] OGD induces the release of PC-EVs containing mitochondria.

[0122] Pericytes possess stem cell-like properties and play a crucial role in the brain microenvironment. Previous studies have shown that stem cell-derived vesicles have neuroprotective effects, particularly under hypoxic conditions. Therefore, we explored the potential of PC-EVs in brain injury. We cultured pericytes for 1, 3, or 6 hours under OGD conditions (…). Figure 5 a), and TUNEL staining was used to assess apoptosis. The results showed that 1 hour of OGD did not induce apoptosis, while 3 hours of OGD significantly increased the number of apoptotic cells (a). Figure 5 b, c). We then isolated EVs from the pericyte conditioned medium by ultracentrifugation after 24 hours of reoxygenation following 0, 1, 3, or 6 hours of OGD treatment. Figure 1 a). Nanoparticle tracking analysis (NTA) showed that PC-EVs levels increased significantly after 1 hour of OGD treatment, but did not increase further at 3 and 6 hours, indicating that PC-EVs were rapidly released upon 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 also positive for untreated pericyte-derived EVs (PC-EVs). Con Compared to periodontal cell-derived EVs (PC-EVs) exposed to OGD for 1 hour, OGD The expression of these markers was significantly upregulated in ( ) Figure 1 c, d). EVs are typically packaged within intracellular MVBs and released into the extracellular space after the MVBs fuse with the plasma membrane. Transmission electron microscopy (TEM) confirmed that OGD leads to an increase in the number of EVs and MVBs. Figure 5 d). After OGD treatment, the number of intraluminal vesicles (ILVs) within MVBs in pericytes significantly increased ( Figure 1 e, Figure 5 e, f). Immunofluorescence staining showed that OGD induced the aggregation of CD63-positive and lysophosphatidylcholine (LBPA)-positive multivesicular bodies (MVBs) and increased the number of punctate structures. Figure 1 f, Figure 5 This result was further confirmed by immunoelectron microscopy (IEM). Figure 1 g, Figure 5 h). Dot blot analysis showed that the upregulation of CD63 and LBPA after OGD indicated that OGD affected the formation or dynamics of MVB (h). Figure 5 i). Measurements of the PC-EV diameter revealed two main subgroups, with sizes of approximately 100 nm and 350 nm, respectively. Figure 1 Next, we isolated large EVs (l-EVs) and small EVs (s-EVs) from the pericyte culture medium. Figure 5 Both expressed Alix, TSG101, and HSP70, and the expression of these proteins was significantly upregulated after OGD. Figure 5 NTA shows that the size range of l-EVs is 50-400 nm, and the size range of s-EVs is 50-200 nm. Figure 5 TEM further confirmed these dimensions (n). Figure 5 o).

[0123] For PC-EVs Con and PC-EVs OGD Proteomics analysis was performed, identifying 1,494 proteins, many of which are associated with mitochondria and other organelles. Figure 1 j, Figure 6 a). Comparison with mitochondrial proteins (GO:0005739 and MitoCarta3.0) revealed 137 overlapping proteins. Figure 1 k). Of these, 113 proteins were identified by the Vehiclepedia database as vesicle cargo proteins (k). Figure 1 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). Furthermore, 198 upregulated proteins (log2 FC>2) were enriched in exosome components ( Figure 6 c, d). Western blot analysis confirmed the presence of mitochondrial proteins in PC-EVs and their subsets (large EVs and small EVs), which were significantly upregulated after OGD (c, d). Figure 6 ej). IEM further demonstrated an increase in the number of mitochondrial protein-positive spots in PC-EVs after OGD ( Figure 1 n). Co-localization of pyruvate dehydrogenase E1α (PDH) and CD63 showed that most CD63-positive PC-EVs contained mitochondrial proteins after OGD, indicating an increase in mitochondrial components released through PC-EVs. Figure 1 o). Transmission electron microscopy (TEM) shows that PC-EVs OGD The number of mitochondria encapsulated in the middle layer is significantly greater than that in PC-EVs. Con ( Figure 1 These results indicate that OGD promotes mitochondrial conversion to PC-EVs. OGD The packaging of mitochondria. Mitochondrial dysfunction is usually caused by 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 6k,l). WB analysis further confirmed that PC-EVs OGD Contains the complete mitochondrial respiratory chain complex ( Figure 1 q). Importantly, these PC-EVs OGD It exhibits a higher oxygen consumption rate (OCR) and a higher ATP content. Figure 1 The r,s) indicates that PC-EVsOGD contains more structurally and functionally intact mitochondria. Notably, 1 hour of OGD did not affect the mitochondrial structure in pericytes (r,s). Figure 6 m,n).

[0124] PC-EVs can reduce cerebral infarction and improve neurological damage after cerebral ischemia.

[0125] Immediately after inducing cerebral ischemia in mice, 0.5 μg of PC-EVs were administered. 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, the mNSS score shows PC-EVs OGD Early recovery of neurological function was observed in the group, and this recovery became more pronounced on days 7 and 14. Figure 2 c), indicating that PC-EVs OGD It not only initiated early recovery but also enhanced recovery over time. Furthermore, PC-EVs treatment did not affect weight ( Figure 2 d). 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that PC-EVs OGD The infarct area was significantly reduced after 24 hours of ischemia. Figure 7 a). Hematoxylin and eosin (H&E) staining also significantly reduced the ischemic area on day 3 after ischemia ( Figure 7 b). T2-weighted magnetic resonance imaging (T2WI) further showed a continued reduction in infarct area ( Figure 2 e, f). H&E staining and Nissl staining also confirmed PC-EVs. OGD Protective effect against neuronal damage ( Figure 2 g, h). In addition, PC-EVs OGD The effect of enhancing cerebral blood flow is more significant. Figure 2 (i, j). Next, we investigated the effect of PC-EVs on motor function recovery. Mice treated with PC-EVs showed significant improvement in sensorimotor deficits and asymmetry for up to 14 days after ischemia, with PC-EVs... OGDIt has a greater recovery effect on performance in behavioral tests. Figure 2 These findings highlight PC-EVs (kn). OGD Its protective effect after cerebral ischemia.

[0126] PC-EVs are absorbed by astrocytes and inhibit the activation of astrocytes after ischemia.

[0127] PC-EVs were labeled with the film dye PKH67. OGD To track the uptake of PC-EVs by nerve cells after stroke ( Figure 3 a). In the case of PC-EVs OGD After injection into the lateral ventricle and allowing uptake for 24 hours, immunostaining showed that labeled PC-EVs were internalized in neurons, astrocytes, and microglia. OGD Among them, astrocytes showed a significant increase in uptake after cerebral ischemia ( Figure 3 b, Figure 8 a, b). Furthermore, primary astrocytes exhibited 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-transition reporter protein, which is activated upon uptake of Cre-containing EVs released by pericytes. Figure 8 e). PC-EVs expressing Cre OGD It is efficiently taken up by astrocytes, inducing a change in fluorescence from green (GFP+) to red (RFP+). Figure 8 f). These findings indicate that PC-EVs OGD It is efficiently taken up by astrocytes after OGD.

[0128] PC-EVs OGD Impact on brain gene expression. Principal component analysis showed significant clustering among the three groups, with the Sham group showing the most significant clustering with PC-EVs. OGD The groups partially overlapped, and both groups showed significant differences from the MCAO group. Figure 9 a, b). With Sham group and PC-EVs OGD Compared to the control group, a total of 1,567 genes were significantly upregulated and 862 genes were significantly downregulated in the MCAO group. Figure 3 c). Astrocyte activation plays a crucial role in the progression of MCAO. Genes related to astrocyte activation are significantly upregulated after MCAO, while those in PC-EVs are... OGD Significantly downregulated after administration ( Figure 3 d, Figure 9 c). The ternary plot shows that, with Sham groups or PC-EVs OGDCompared to the control group, these activation-related genes were significantly enriched in the MCAO group. Figure 3 e). Immunofluorescence and immunohistochemical staining confirmed that MCAO induced glial fibrillary acidic protein (GFAP) reactivity, while PC-EVs OGD This reaction was blocked. Figure 3 f, Figure 9 d). Further analysis showed that the number and length of astrocyte branches were reduced in the MCAO group, while PC-EVs... OGD The treatment significantly restored these indicators ( Figure 9 e, f). Sholl analysis confirmed that PC-EVs OGD It inhibited the reduction of astrocyte branching ( Figure 3 g, Figure 9 g). Genes associated with astrocyte activation were further validated in vivo by Western blotting. Figure 9 h, i), and further validated in vitro by qPCR ( Figure 3 h).

[0129] Pericytes transfer mitochondria to astrocytes via PC-EVs and integrate them into the astrocyte mitochondrial network.

[0130] Cells can enhance intercellular communication by transferring functional mitochondria to recipient cells via EVs. Further, this study investigated whether pericytes mediated mitochondrial transfer to astrocytes via PC-EVs. Adenovirus expressing MitoDsRed was used to label mitochondria in pericytes (…). Figure 10 a). PC-EVs isolated from labeled pericytes OGD (MitoDsRed) + -PC-EVs OGD Then it was co-incubated with astrocytes for 24 hours. Figure 10 b). Compared with untreated astrocytes, OGD-treated astrocytes showed increased MitoDsRed fluorescence (b). 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, followed by MitoDsRed+-PC-EVs OGD The cells were incubated for 24 hours to further investigate whether mitochondrial uptake in astrocytes increased with prolonged OGD exposure time. The proportion of MitoDsRed fluorescence in astrocytes increased with prolonged OGD treatment time. Figure 10 e).

[0131] Using BacMam technology, endogenous mitochondria from astrocytes were labeled with green fluorescent protein (GFP) and then compared with MitoDsRed+-PC-EVs. OGD Incubate for 24 hours to determine PC-EVs OGD The fusion of mitochondria with the host cell's endogenous mitochondrial network was observed. Following OGD treatment, MitoDsRed fluorescence was observed in astrocytes and showed fusion with the host mitochondrial network. Figure 4 a). Furthermore, MitoDsRed-labeled pericytes were co-cultured with GFP-labeled astrocytes ( Figure 10 f). 24 hours later, MitoDsRed was observed in OGD-treated astrocytes, co-localizing with the mitochondrial network ( Figure 10 g). Using a transwell co-culture system, it was demonstrated that MitoDsRed-labeled pericyte mitochondria can be transferred non-contactly to astrocytes, and this transfer is significantly increased under OGD conditions. Figure 4 be). As a negative control, treatment of pericytes with GW4869 significantly reduced the co-localization of RFP-labeled mitochondria and GFP in astrocytes (be). Figure 4 be). Mitochondria in pericytes were further labeled using engineered ascorbate peroxidase (APEX2) and observed under an electron microscope. Figure 10 These labeled mitochondria are transferred to astrocytes via the transwell system (h,i). Figure 4 f,g).

[0132] Pericytes, located at the center of neurovascular units (NVUs), mediate neurovascular coupling through their own contraction. This invention demonstrates that perivascular endothelial cells (PC-EVs) also possess regenerative properties and contribute to the brain repair process. Interestingly, the neuroprotective effect of PC-EVs is more pronounced after hypoxic pretreatment. One hour of oral glucose dispensing (OGD) increased PC-EV production and secretion without inducing significant perivascular cell death. Compared to PC-EVs under normoxic conditions, PC-EVs pretreated with hypoxia showed better 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 pretreatment enhanced angiogenesis, alleviated allergic airway inflammation and airway remodeling, and accelerated bone regeneration. This study is the first to demonstrate that perivascular pluripotent cells secrete more EVs, providing enhanced protection as a compensatory mechanism against ischemia-induced brain injury, suggesting that PC-EVs share common protective characteristics with other stem cell / progenitor cells.

[0133] The brain is one of the most energy-demanding organs and is highly susceptible to metabolic disorders. During ischemia, the lack of oxygen and glucose leads to a sharp decline in ATP production, resulting in energy depletion and impaired cellular function. Mitochondria play an indispensable role in the pathogenesis of cerebral ischemia-reperfusion injury because they play a crucial role in energy metabolism, oxidative stress, and cell death pathways. Mitochondria and specific signaling molecules can be transferred between cells via direct cell-to-cell contact or extracellular vehicles (EVs), thereby promoting intercellular communication. Transferred mitochondria provide essential support for energy and metabolic regulation in damaged cells, a mechanism particularly critical for maintaining the homeostasis of the neurovascular unit (NVU). Astrocytes are a vital component of the NVU, and their mitochondria maintain the integrity of the blood-brain barrier by improving 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 proteomics data showed that after short-term OGD, the levels of mitochondrial components (including the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM)) significantly increased and were integrated into PC-EVs. These PC-EVs' mitochondria were confirmed to be functional by OCR assays and ATP production detection. Subsequent integrated transcriptome analysis was performed to elucidate the potential protective mechanism. We found that PC-EVs... OGD It reduced astrocyte activation in the brain of the MCAO model.

[0134] In summary, this study establishes the protective role of pericytes (PC-EVs) in cerebral ischemia-reperfusion injury. By transferring mitochondria from pericytes to astrocytes, PC-EVs alleviated astrocyte reactivation. These data suggest that pericytes, in addition to regulating blood flow through their own contraction according to the metabolic microenvironment, can also share their mitochondria during ischemia to improve neurometabolism. This evidence provides new insights into the potential stem cell-like properties of pericytes.

[0135] Finally, it should be noted that the above description is merely 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A pericyte-derived extracellular vesicle rich in mitochondria, characterized in that, The extraction method of the extracellular vesicles includes the following steps: (1) The cerebral cortex was partially chopped, digested with type II collagenase and DNase I, and then separated by gradient separation solution to obtain pericytes; (2) Place the pericytes on a culture dish coated with type IV collagen and culture them in pericyte medium; (3) When the cell confluence reaches 80%~90%, the cell culture medium is replaced with fresh exosome-free FBS-free glucose-free culture medium. After OGD treatment, the cells are reoxygenated and cultured for 24 hours. The culture supernatant is collected and centrifuged to obtain the extracellular vesicles. The OGD treatment specifically involves placing pericytes in an oxygen-deficient incubator containing 95% N2 and 5% CO2 and incubating at 37°C for 1 hour; OGD treatment for 1 hour increases the generation and secretion of extracellular vesicles derived from pericytes.

2. The pericyte-derived, mitochondrial-rich extracellular vesicles as described in claim 1, characterized in that, Step (1) is as follows: The cerebral cortex is chopped in ice-cold PBS, then DMEM containing type II collagenase and DNase I is added and digested for 1-2 hours to obtain a suspension. The suspension is centrifuged, the supernatant is discarded, and the cell pellet is resuspended in DMEM containing type II collagenase and dispersin and digested for 0.5-1 hours. After centrifugation, the supernatant is discarded, and the cell pellet is resuspended in DMEM. Then, gradient separation solution is added to separate pericytes.

3. The pericyte-derived, mitochondrial-rich extracellular vesicles as described in claim 2, characterized in that, The treatment concentration of the type II collagenase is 0.8~1.2 mg / mL, the treatment concentration of DNase I is 10~20 μg / mL, the treatment concentration of the dispersant enzyme is 1~5 U / mL, and the digestion temperature of the type II collagenase and DNase I is 36℃~38℃.

4. The pericyte-derived, mitochondrial-rich extracellular vesicles as described in claim 1, characterized in that, The gradient separation solution is Percoll, with a concentration of 30% to 35%.

5. The pericyte-derived, mitochondrial-rich extracellular vesicles as described in claim 1, characterized in that, The culture supernatant was centrifuged as follows: first at 4°C at 200-400×g 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 was collected and ultracentrifuged at 90,000~110,000×g for 88~92 minutes at 4℃, 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, then centrifuge 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 9,000~11000×g for 68~72 minutes at 4℃. The precipitate was collected to obtain large extracellular vesicles. Alternatively, centrifuge at 200-400×g for 8-12 minutes at 4°C, then centrifuge 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℃ to separate large extracellular vesicles; the supernatant after the precipitation of large extracellular vesicles was filtered through a 0.22μm membrane and then ultracentrifuged at 90,000~110,000×g for 88~92 minutes at 4℃, and the precipitate was collected to obtain small extracellular vesicles.

6. A pharmaceutical composition, characterized in that, It comprises pericyte-derived mitochondrial-rich extracellular vesicles as described in any one of claims 1-5 and a pharmaceutically acceptable carrier or adjuvant.

7. The use of pericyte-derived mitochondrial-rich extracellular vesicles as described in any one of claims 1-5 in the preparation of products for the treatment or adjunctive treatment of ischemic brain injury.

8. The application as described in claim 7, characterized in that, The product reduces brain ischemia-reperfusion injury by decreasing the activation of astrocytes.

9. The application as described in claim 7, characterized in that, The product is a medicine.

Images