Therapeutic application of targeted exosome in improvement of blood-brain barrier destruction of acute ischemic stroke

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to treatment application of a targeted exosome in improving blood-brain barrier destruction of acute ischemic stroke, and the targeted exosome is an exosome from brain microvascular endothelial cells subjected to oxygen-glucose deprivation treatment, and is an exosome carrying CD151-siRNA (siCD151). According to the technical scheme, the screened CD151 is a key molecule for regulating and controlling the immune homeostasis of the endothelial cells. Meanwhile, the exosome from brain microvascular endothelial cells subjected to oxygen-glucose deprivation treatment is obtained to serve as a delivery carrier, and siCD151 is carried to construct an Exo-siCD151 system. The system can maintain the immune homeostasis of the endothelial cells by reducing the CD151 level of the ischemic-damaged endothelial cells in a targeted manner, so that the aims of repairing the blood-brain barrier and improving the cerebral ischemia prognosis are fulfilled.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the therapeutic application of targeted exosomes in improving blood-brain barrier disruption in acute ischemic stroke. Background Technology

[0002] Ischemic stroke is one of the leading causes of death and disability worldwide. Currently, effective treatments include reperfusion therapy such as intravenous thrombolysis and mechanical thrombectomy. However, due to the short time window and high risk of bleeding associated with these therapies, only a small number of patients benefit from them.

[0003] Therefore, new treatment methods for ischemic stroke urgently need to be developed, and revascularization is the cornerstone of ischemic stroke treatment. As a major component of blood vessels, the restoration of vascular endothelial cell immune homeostasis plays a crucial role in revascularization.

[0004] CN116334004A discloses the use of RVG-Exo-3059-5p in the treatment of stroke, disclosing a membrane structure containing miR-3059-5p. This membrane structure includes a tissue-targeting protein and miR-3059-5p, with the tissue-targeting protein linked to a transmembrane protein within the membrane structure. The membrane structure is disclosed to include exosomes, microvesicles, microparticles, endosome-derived vesicles, multivesicular bodies, or apoptotic bodies. This technical solution has applications in improving neurocognitive function during the recovery period of stroke patients. This technical solution requires the linkage between the targeting protein and the transmembrane protein. While transmembrane proteins facilitate entry into specific regions within tissues, the targeting protein may still be recognized as a foreign substance and rejected, affecting its performance. Furthermore, its exogenous nature also poses potential immune risks. Summary of the Invention

[0005] The purpose of this invention is to provide a therapeutic application of targeted exosomes in improving blood-brain barrier disruption in acute ischemic stroke. By utilizing the intrinsic targeting of ischemic exosomes to ischemic endothelial cells, precise delivery is achieved while avoiding complex chemical modifications, simplifying the production process, and reducing the immune risks caused by the introduction of exogenous proteins, thereby overcoming the shortcomings of traditional targeted protein therapies.

[0006] To achieve the above objectives, this application employs the following technical solution:

[0007] A therapeutic application of targeted exosomes in improving blood-brain barrier disruption in acute ischemic stroke, wherein the targeted exosomes are exosomes derived from oxygen-glucose deprived brain microvascular endothelial cells.

[0008] Furthermore, the targeted exosomes are exosomes carrying CD151-siRNA.

[0009] Furthermore, the targeted exosomes maintain the immune homeostasis of endothelial cells by downregulating the CD151 level in ischemic endothelial cells.

[0010] Furthermore, the application of the targeted exosomes in the preparation of drugs for the prevention or treatment of stroke.

[0011] Furthermore, the application of the targeted exosomes in drugs that improve the prognosis of stroke patients.

[0012] Furthermore, the drug is a pharmaceutical composition comprising targeted exosomes.

[0013] Furthermore, the pharmaceutical composition may be in any pharmaceutically acceptable form.

[0014] The beneficial effects of this invention are:

[0015] This technical approach, combining RNA-seq sequencing with previous work from our research group, identified CD151 as a key molecule regulating endothelial cell immune homeostasis. Simultaneously, exosomes derived from oxygen-glucose deprived brain microvascular endothelial cells were obtained as delivery vectors, carrying CD151-siRNA to construct the Exo-siCD151 system. By targeting and downregulating CD151 levels in ischemic endothelial cells, this system can maintain endothelial cell immune homeostasis, thereby achieving the goal of repairing the blood-brain barrier and improving the prognosis of cerebral ischemia. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating CD151 as a potential key molecule for regulating vascular endothelial cell immune homeostasis during the acute phase of cerebral ischemia.

[0017] Figure 2 Construction of an exosome-derived brain microvascular endothelial cell (BMVEC) system carrying siCD151 via OGD / R treatment (Exo-siCD151).

[0018] Figure 3 The diagram shows that Exo-siCD151 can target vascular endothelial cells in the ischemic area of ​​the brain and effectively downregulate CD151 protein levels.

[0019] Figure 4 A schematic diagram illustrating how Exo-siCD151 significantly reduces neurological functional impairment in rats after cerebral ischemia-reperfusion.

[0020] Figure 5 This is a diagram illustrating the analysis of differentially expressed genes based on transcriptome sequencing results.

[0021] Figure 6 A schematic diagram illustrating how Exo-siCD151 maintains immune homeostasis of vascular endothelial cells in the ischemic penumbra region 3 days after perfusion.

[0022] Figure 7 This is a schematic diagram showing how Exo-siCD151 maintains immune homeostasis in BMVECs 48 h after OGD / R.

[0023] Figure 8 This diagram illustrates how Exo-siCD151 exerts its neuroprotective effect by regulating the MAPK / ERK and PI3K / AKT signaling pathways.

[0024] Figure 9 Additional detailed diagrams illustrating the preparation and intervention process of Exo-siCD151.

[0025] Figure 10 This is a diagram showing the in vivo distribution and biosafety assessment of Exo-siCD151. Detailed Implementation

[0026] The technical solutions of the present invention will be described in detail below through embodiments. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solutions of the present invention, and should not be construed as limiting the technical solutions of the present invention.

[0027] This application proposes using exosomes derived from oxygen-glucose deprived brain microvascular endothelial cells (BMVECs) as delivery vectors, carrying CD151-siRNA, and injecting it via tail vein to target vascular endothelial cells in the ischemic brain region and inhibit CD151 expression, in order to further explore the role of CD151 in regulating vascular endothelial cell immune homeostasis, blood-brain barrier repair, and neutrophil infiltration.

[0028] Exosomes (Exo) were isolated from primary BMVECs subjected to oxygen-glucose deprivation treatment. An Exo-siCD151 system was established by loading the exosomes with siCD151 via electroporation. In in vitro experiments, Exo-siCD151 was co-cultured with primary BMVECs to investigate its cell targeting and role in regulating vascular endothelial cell immune homeostasis. In in vivo experiments, a rat model of transient middle cerebral artery occlusion (tMCAO) was constructed, and Exo-siCD151 was injected via the tail vein to assess its tissue targeting and its effect on vascular endothelial cell immune homeostasis. Furthermore, RNA sequencing and Western blot analysis were used to identify the signaling pathways related to the neuroprotective effects of Exo-siCD151.

[0029] Figure 1CD151 is a potential key molecule regulating vascular endothelial cell immune homeostasis during the acute phase of cerebral ischemia. (A) Volcano plot showing differentially expressed DEGs between the tMCAO and sham groups; (B) GO analysis of DEGs between the tMCAO and sham groups; (C) TPM levels of CD151 in the ischemic penumbra of the tMCAO group and the corresponding location in the sham group (based on RNA-seq); (D) Relative expression levels of CD151 mRNA (24 h post-reperfusion) and protein (48 h post-reperfusion) in BMVECs of the control and OGD / R groups; (E) Silent efficiency of different siCD151 sequences (50 nM) on CD151 in BMVECs of the OGD / R group (24 h and 48 h post-transfection). Figure 1 The data are expressed as mean ± standard deviation, n=3.

[0030] Figure 2 Construction of an exosome-derived brain microvascular endothelial cell (BMVEC) system using Exo and siCD151 (Exo-siCD151). (A) Transmission electron microscopy was used to analyze the morphology of exosomes (scale bar = 100 nm). (B) Nanoparticle tracking analysis was used to determine the size distribution and peak diameter of exosomes. (C) Western blot was used to detect exosome marker proteins, with BMVECs as the control group. Compared with the control group, the extracted exosomes expressed more CD81, CD9, and HSP70, but less Calnexin. (D) Immunofluorescence staining and scatter plot were used to detect the colocalization of exosomes and siCD151 after electroporation. Scale bar = 2 μm. (E) BMVECs were co-cultured with 20 μg / ml Exo-siNC, 20 μg / ml Exo-siCD151, or an equal volume of PBS and OGD / R for 48 h, and cell viability of each group was detected by CCK-8 assay (n = 6). (F) Stability of Exo-siRNA and free siRNA under nuclease conditions.

[0031] Figure 3Exo-siCD151 can target vascular endothelial cells in the ischemic brain region and effectively downregulate CD151 protein levels. (A) Representative fluorescence images and quantitative analysis of fluorescence intensity after co-culturing OGD / R-treated BMVECs with DiD-labeled 20 μg / mL naïve-Exo, OGD-Exo, or cRGD-blocked OGD-Exo for 24 h. Scale bar = 25 μm. (B) Representative immunoblot images and quantitative analysis of β3 protein in naïve-Exo and OGD-Exo. (C) Fluorescence imaging and quantitative analysis of brain tissue 24 h after tail vein injection of DiD-labeled 100 μg Exo-siCD151 in tMCAO rats. (D) Representative fluorescence images of the ischemic brain region 24 h after tail vein injection of DiD-labeled 100 μg Exo-siCD151 in tMCAO rats (scale bar = 25 μm). (E) Relative expression levels of CD151 mRNA in BMVECs 24 h after OGD / R and representative immunoblot and quantitative analysis of CD151 protein in BMVECs 48 h after OGD / R. (F) Relative expression levels of CD151 mRNA in the ischemic penumbra region of the brain 3 days after reperfusion and representative immunoblot and quantitative analysis of CD151 protein. Figure 3 The data are expressed as mean ± standard deviation, n=3.

[0032] Figure 4 Exo-siCD151 significantly reduced neurological function impairment in rats following cerebral ischemia-reperfusion. (A) Overall design of the in vivo experiment. Within 0.5 h after cerebral ischemia, rats were treated with 100 μg of Exo-siNC or Exo-siCD151 via tail vein injection. Three days after reperfusion, rats were sacrificed to collect brain tissue or perform neurobehavioral tests. (BE) Representative images and quantitative analysis of brain TTC staining, Evans blue staining, and brain water content measurement (n = 6). (FG) Representative images and infiltration count analysis of neutrophils, monocytes, and T lymphocytes. MPO, CD115, and CD3 represent neutrophils, monocytes, and T lymphocytes, respectively. Scale bar = 50 μm, black arrows indicate positive cells (n = 3). Figure 4 Data are expressed as mean ± standard deviation.

[0033] Figure 5This paper presents an analysis of differentially expressed genes (DEGs) based on transcriptome sequencing results. (A) A PCA plot shows the distribution of the four sample groups. (B) A Venn diagram shows the overlap of DEGs between the (tMCAO vs. sham) and (Exo-siNC vs. Exo-siCD151) groups. The overlapping portion includes 2684 DEGs. (C) A volcano plot depicts the DEGs between the tMCAO and sham groups, and between the Exo-siNC and Exo-siCD151 groups. (D) A visualization heatmap of DEG clusters among the four groups. (E) KEGG pathway enrichment analysis and GO enrichment analysis were performed on the 2684 overlapping DEGs. (F) The abundance of monocyte infiltration in the four groups was calculated using ImmucellAI. Figure 5 The data are expressed as mean ± standard deviation, n=3.

[0034] Figure 6 Three days after reperfusion, Exo-siCD151 maintained immune homeostasis of vascular endothelial cells in the ischemic penumbra region. (AB) Representative fluorescence images and positive cell count analysis of TUNEL / CD31. (CD) Representative fluorescence images and quantitative analysis of fluorescence intensity of ZO-1 / CD31. (EF) Representative immunoblot images and quantitative analysis of Bcl-2 and Bax. (GH) Representative immunoblot images and quantitative analysis of TNF-α and IL-1β. (IJ) Representative immunoblot images and quantitative analysis of VCAM-1 and ICAM-1. (KL) Representative immunoblot images and quantitative analysis of ZO-1 and Occludin. Figure 6 The data are expressed as mean ± standard deviation, n=3.

[0035] Figure 7 The experiment showed that Exo-siCD151 maintained immune homeostasis in BMVECs 48 h after OGD / R reperfusion. (A) Overall design of the in vitro experiment. BMVECs were subjected to OGD for 4 h and then reperfused with 20 μg / mL Exo-siNC or Exo-siCD151. Cells were harvested for protein detection 48 h after reperfusion. (BC) Representative immunoblot maps and quantitative analysis of Bcl-2, Bax, cleaved caspase 3, TNF-α, IL-1β and IL-6 in BMVECs. (DE) Representative immunoblot maps and quantitative analysis of VCAM-1 and ICAM-1 in BMVECs. (FG) Representative immunoblot maps and quantitative analysis of ZO-1 and Occludin in BMVECs. Figure 7 The data are expressed as mean ± standard deviation, n=3.

[0036] Figure 8Exo-siCD151 exerts its neuroprotective effect by regulating the MAPK / ERK and PI3K / AKT signaling pathways. (AB) Representative immunoblot images and quantitative analysis of p-ERK / ERK, p-PI3K / PI3K, and p-Akt / Akt in the ischemic penumbra. (CD) Representative immunoblot images and quantitative analysis of p-ERK / ERK, p-PI3K / PI3K, and p-Akt / Akt in BMVECs. (E) In vitro experimental design using inhibitor treatment. BMVECs were subjected to OGD for 4 h and reperfused with PBS or 20 μg / mL Exo-siCD151 during reperfusion. 24 h after reperfusion, PD98059 (ERK 1 / 2 inhibitor, 30 μM) or LY294002 (PI3K inhibitor, 20 μM) or no inhibitor was added. Cells were cultured for 48 h after reperfusion and then harvested for protein detection. Representative immunoblot and quantitative analysis of p-ERK / ERK in (FG)OGD / R BMVECs after administration of the ERK inhibitor PD98059. Representative immunoblot and quantitative analysis of p-PI3K / PI3K and p-AKT / AKT in (HI)OGD / R BMVECs after administration of the PI3K inhibitor LY294002. Representative immunoblot and quantitative analysis of TNF-α, IL-1β, IL-6, and Occludin in (JK)OGD / R BMVECs after administration of the ERK 1 / 2 inhibitor PD98059 or the PI3K inhibitor LY294002. Figure 8 The data are expressed as mean ± standard deviation, n=3.

[0037] Figure 9 Supplementary details of the preparation and intervention process of Exo-siCD151 are shown. (A) Light micrographs of BMVECs under normal culture conditions or after 12h of OGD reoxygenation for 36h. Scale bar = 100 μm (n = 3). (B) Representative immunoblots and quantitative analysis of CD151 protein in naïve-Exo and OGD-Exo (n = 3). (C) Body weight and neurobehavioral tests of tMCAO rats 3 days after reperfusion, including Longa score, Bederson score, limb placement test and crossbeam walking test (n = 8). (D) Intracellular ROS levels and neutrophil extracellular trap (NET) levels in cell supernatant after 3 days of co-culturing Exo-siCD151 with HL-60 cells (n = 3). (E) Intracellular ROS levels and sCD14 and sCD163 levels in cell supernatant after co-culturing Exo-siCD151 and THP-1 cells for 3 days (n = 3). Figure 9 Data are expressed as mean ± standard deviation.

[0038] Figure 10 This study assesses the in vivo distribution and biosafety of Exo-siCD151. (A) Levels of liver and kidney function markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), and blood urea nitrogen (BUN), in rats in the tMCAO or Exo-siCD151 groups (n = 4). (BC) Fluorescence imaging and quantitative analysis of fluorescence intensity in major organs of tMCAO rats 24 h after tail vein injection of 100 μg of DiD-labeled Exo-siCD151 (n = 3). (D) H&E-stained images of sections of major organs from rats in the tMCAO or Exo-siCD151 groups, including heart, liver, spleen, lung, and kidney (n = 3). Scale bar = 100 μm. Figure 10 Data are expressed as mean ± standard deviation.

[0039] Experimental steps:

[0040] In addition to the steps explicitly mentioned below, the experimental procedures of this application also include the preparation of siCD151, siCD151 transfection, real-time quantitative reverse transcription polymerase chain reaction, Western blotting, electroporation, electroporation encapsulation rate detection, cell proliferation and toxicity detection, agarose gel electrophoresis, immunofluorescence staining, in vivo experimental grouping and technical roadmap, neurobehavioral testing, cerebral infarction volume measurement, Evans blue assay, cerebral edema measurement, endothelial cell apoptosis assessment, immunohistochemistry, HL-60 cell and THP-1 cell culture, reactive oxygen species detection, human neutrophil extracellular trap network enzyme-linked immunosorbent assay, Scd14 and sCD163S enzyme-linked immunosorbent assay, blood biochemistry detection, paraffin sectioning and H&E staining, etc.

[0041] 1. Description of the animal and cell lines used in the experiments of this application:

[0042] The animal experimental protocol for this study was approved by the Experimental Animal Use and Management Committee of Tsinghua University (No.: 18-WJ1.G23-1). The animals used in this study were SPF-grade male SD rats, provided by Vital River Pharmaceuticals, Inc. The rats were approximately 8 weeks old and weighed 280-300g. All animals were housed in a barrier facility at the Experimental Animal Center of Tsinghua University.

[0043] The primary rat brain microvessel endothelial cells (BMVECs) used in this study were purchased from Wuhan Pronosai Co., Ltd., and were cultured in 2% gelatin-coated culture dishes.

[0044] 2. Establishment of the rat tMCAO model:

[0045] A rat tMCAO model was established using the suture occlusion method. The specific steps are as follows:

[0046] (1) Rats were induced to be anesthetized by inhalation of 5% isoflurane (Reward, China). After the rats were deeply anesthetized, anesthesia was maintained by 1.5-2.5% isoflurane through a face mask. During this period, the rats' respiratory status was monitored and the isoflurane flow rate was adjusted in a timely manner.

[0047] (2) The rats were fixed in a supine position. To ensure the stability of their physiological state during the operation, a rectal probe was inserted into the anus to dynamically monitor their body temperature, and a heating pad was used to maintain their body temperature at around 37°C.

[0048] (3) Shave the hair in the surgical area, disinfect the skin with iodine, and make a longitudinal incision about 2 cm long along the midline of the neck. Bluntly separate the subcutaneous tissue to clearly expose the surgical field.

[0049] (4) Carefully separate the right common carotid artery, external carotid artery, superior thyroid artery, occipital artery, internal carotid artery, and the anastomosing branches between the external and internal carotid arteries in sequence. Exercise caution throughout the procedure to avoid stimulating the vagus nerve or compressing the trachea.

[0050] (5) Using a surgical electrocoagulation knife (Jiuyang Medical, China), the superior thyroid artery, occipital artery, and the anastomosing branches between the external carotid artery and the internal carotid artery were electrocoagulated and severed in sequence.

[0051] (6) Perform temporary ligation on the right common carotid artery and internal carotid artery in sequence, and permanent ligation on the external carotid artery at a distance.

[0052] (7) Use the ligation suture to pull the external carotid artery and cut the blood vessel at the proximal end of the ligation.

[0053] (8) Gently insert a silicone-coated suture plug (Xinong, China) moistened with saline through the external carotid artery incision. Untie the internal carotid artery ligation suture and advance it along the internal carotid artery to the origin of the middle cerebral artery (marked by feeling slight resistance and by the black dot on the suture plug). Then, ligate the reserved suture of the external carotid artery to fix the suture plug.

[0054] (9) Loosen the ligature at the common carotid artery and complete the skin suturing.

[0055] (10) The suture thrombus is retained for 2 hours to achieve cerebral ischemia. After 2 hours, the skin suture is cut off, the suture thrombus is gently removed to achieve reperfusion, the suture is left at the incision site of the external carotid artery is ligated, and the skin is sutured.

[0056] (11) After the operation, the rats were placed on a 37°C warming pad until they woke up, and were kept in the same cage as healthy rats or sham-operated rats.

[0057] (12) After surgery, meloxicam (2mg / kg) was administered subcutaneously for pain relief.

[0058] (13) Rats in the sham-operated group underwent the same surgical procedure, but without the insertion of a suture plug.

[0059] (14) Preliminary behavioral criteria for successful tMCAO model: Rats whose bodies turned to the left and whose left upper limb movement was restricted immediately after awakening and 24 hours after awakening, when their tails were lifted, were considered to have successfully established the model. Rats that did not succeed or died were removed.

[0060] (15) Final evaluation of the success of the tMCAO model: at the experimental endpoint, the formation of cerebral infarction foci is confirmed by TTC staining or histological staining.

[0061] 3. Sample preparation for transcriptome sequencing:

[0062] (1) Three days after reperfusion, rats in the sham-operated group and the tMCAO group were anesthetized by intraperitoneal injection of Sutacetin 50 combined with xylazine.

[0063] (2) After deep anesthesia, fix the rat’s limbs, disinfect the abdomen with alcohol, and cut the skin from the middle of the abdomen upwards in a V-shape along both sides.

[0064] (3) Cut open the chest cavity to expose the heart and cut open the left atrial appendage.

[0065] (4) The heart is rapidly perfused with pre-cooled enzyme-free PBS buffer. The perfusion is stopped when the fluid flowing out of the left atrial appendage becomes clear.

[0066] (5) Decapitation, separation of brain tissue, removal of pia mater, and preservation of both cerebral hemispheres.

[0067] (6) Perform the procedure on ice. Collect brain tissue from the ischemic penumbra area of ​​rats, divide it into uniform small pieces of 50 mg each (about the size of a soybean), immediately place them in pre-cooled cryovials, quickly freeze them in liquid nitrogen for several minutes, and then transfer them to an ultra-low temperature freezer at -80℃ for storage.

[0068] 4. RNA extraction:

[0069] (1) Place the tissue in a grinding tube, add 1.5 ml of Trizol lysis buffer, grind with a tissue grinder for 30 seconds, remove and place on ice for 5 minutes to allow the tissue cells to fully lyse.

[0070] (2) Centrifuge at 4℃, 12,000×g for 5 minutes.

[0071] (3) Take the supernatant, add chloroform / isopropanol, and mix by inverting and shaking.

[0072] (4) Centrifuge at 4℃, 12,000×g for 8 minutes.

[0073] (5) Take the supernatant, add isopropanol, gently invert and mix, and let stand in a -20℃ refrigerator for more than 2 hours.

[0074] (6) Centrifuge at 4℃, 17,500×g for 25 minutes.

[0075] (7) Discard the supernatant, wash the precipitate with 75% alcohol, and mix by inverting.

[0076] (8) Centrifuge at 4℃, 17,500×g for 3 minutes.

[0077] (9) Discard the supernatant, remove the residual liquid, and let it air dry.

[0078] (10) Dissolve the precipitate in DEPC water or enzyme-free water.

[0079] 5. Sample quality assessment and analysis:

[0080] In this study, RNA-seq was performed by BGI Genomics Co., Ltd., using the DNBSEQ sequencing platform. To assess the reliability of biological replicates, the following analyses were conducted:

[0081] (1) Sample correlation heatmap: The Pearson correlation coefficient between samples is calculated based on the expression levels of all genes, reflecting the similarity of expression patterns between samples.

[0082] (2) Principal component analysis (PCA): The spatial distribution of samples is observed through the principal component dimensionality reduction algorithm. If the samples are closely clustered within groups and clearly separated between groups, it indicates that the experimental data are reliable and the treatment effect is significant.

[0083] 6. Screening for differentially expressed genes (DEGs):

[0084] DEGs between groups were screened using DESeq2 (v 1.42.1) software. The screening criteria were set as follows: adjusted P < 0.01, |log2 FC| ≥ 0.5. The analysis results were visualized as follows:

[0085] (1) Volcano plot: Visually shows the relationship between the difference multiple (x-axis) and statistical significance (y-axis), and highlights significant up- and down-regulated DEGs.

[0086] (2) Venn diagram: shows the number of DEGs that are common or unique between different comparison groups.

[0087] (3) Clustering heatmap: Perform hierarchical clustering analysis on DEGs to show the expression trend of DEGs among different samples.

[0088] 7. Functional enrichment analysis (KFGG and GO):

[0089] To further explore the pathways and biological functions involved by DEGs, the following analysis was conducted:

[0090] (1) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis: Analysis of the distribution of DEGs in signal transduction or metabolic pathways.

[0091] (2) Gene ontology (GO) analysis: DEGs were mapped to the GO database, and significantly enriched entries were screened from three dimensions: biological process (BP), cell component (CC), and molecular function (MF).

[0092] 8. Immune cell infiltration analysis:

[0093] To further analyze the changes in the abundance of immune cells in the brain parenchyma, the transcriptome data were analyzed using ImmuCellAI (Immune Cell Abundance Identifier), a computational tool based on gene expression characteristics. ImmuCellAI calculated the abundance score of 24 immune cell types (including 18 T cell subsets, B cells, NK cells, monocytes, etc.) in each sample, comparing the differences in immune cell composition among different groups.

[0094] 9. Culture of rat brain microvascular endothelial cells:

[0095] The complete culture medium for primary rat BMVECs consisted of DMEM-F12 medium, 10% fetal bovine serum, 0.5% insulin, 1% endothelial growth factor, heparin sodium, 1% penicillin-streptomycin, and 0.2% mycoplasma inhibitor. Cells were cultured in a normal incubator at 37°C, 95% O2, and 5% CO2. BMVECs within the first 10 passages were selected for the study.

[0096] 10. Establishment of a cellular oxygen-glucose deprivation model:

[0097] Select cells in good growth condition, and when their confluence reaches a certain level, perform 4 hours of oral glucose tolerance (OGD) treatment to simulate ischemia. The specific steps are as follows:

[0098] (1) Discard the old culture medium, add 2-3 ml of sterile PBS to each culture dish, gently shake to clean, discard the PBS, and repeat twice.

[0099] (2) Add 10 ml of DMEM sugar-free medium to each culture dish.

[0100] (3) Transfer the cells to an oxygen-deficient incubator, replace the original air in the incubator with a mixture of 95% N2 and 5% CO2, create an oxygen-deficient environment after 10 minutes, seal the incubator, and continue culturing at 37°C for 4 hours.

[0101] (4) After 4 hours, the cells were transferred from the hypoxic incubator (Billups-Rothenberg, USA) to a clean bench, the DMEM sugar-free medium was discarded, the cells were washed twice with PBS, and the medium was replaced with DMEM-F12 containing glucose to achieve reoxygenation and reglucose. The cells were then placed in a normal incubator (37°C, 95% O2 and 5% CO2). The usual time for OGD of BMVECs is 4 hours, during which cell viability may decrease and cell morphology may be damaged, such as cell shrinkage and rupture.

[0102] (5) Control group cells were cultured in a normal incubator and in DMEM-F12 complete medium for the same period of time.

[0103] 11. Exosome extraction:

[0104] This study yielded two types of exosomes: naïve-Exo, derived from BMVECs under normal culture conditions, and OGD-Exo, derived from BMVECs after OGD / R treatment. For naïve-Exo, BMVECs were inoculated, and when their confluence reached 60-70%, the culture medium was replaced with complete medium prepared from exosome-free serum (Enzekangtai, China) to avoid interference from serum exosomes. After 48 hours of further culture, the cell supernatant was collected, and exosomes were extracted by differential centrifugation at 4°C. For OGD-Exo, BMVECs were inoculated, and when their confluence reached 70-80%, they were treated with OGD for 12 hours and reoxygenated without glucose for 36 hours. Since the glucose-free medium does not contain serum, interference from serum exosomes was avoided. The cell supernatant was collected, and exosomes were extracted by differential centrifugation at 4°C. The specific steps were as follows:

[0105] (1) Centrifuge the supernatant at 300×g for 10 minutes to remove residual cells.

[0106] (2) Collect the supernatant and centrifuge at 2,000×g for 10 minutes to remove dead cells.

[0107] (3) Collect the supernatant and centrifuge at 10,000×g for 30 minutes to remove cell debris.

[0108] (4) The supernatant was filtered through a 0.22-μm filter (Nest, China) and centrifuged at 110,000×g for 90 minutes using an ultracentrifuge (Beckman Coulter, USA).

[0109] (5) Discard the supernatant, resuspend the precipitate with PBS (all PBS used in this part are pre-cooled and sterile), and centrifuge again at 110,000×g for 90 minutes.

[0110] (6) Discard the supernatant and resuspend the precipitate with PBS to obtain the prepared exosomes.

[0111] (7) The protein concentration of exosome suspension was determined using the BCA protein quantitative kit, and different batches of exosome samples were standardized to a uniform concentration.

[0112] (8) Exosome samples should be stored in an ultra-low temperature freezer at -80°C and used as soon as possible, with a maximum storage period of 2 weeks.

[0113] 12. Exosome fluorescent labeling:

[0114] If fluorescent labeling of exosomes is required, after the first centrifugation at 110,000×g, discard the supernatant, add an appropriate amount of DiO or DiD membrane dye (Beyotime, China) to resuspend the exosomes and incubate (select the dye type according to experimental needs). Then, after ultracentrifugation at 110,000×g for 90 minutes, discard the supernatant, collect the precipitate and resuspend it in PBS. The resulting product is the fluorescently labeled exosome, which should be stored in the dark.

[0115] 13. Exosome identification:

[0116] (1) Morphological identification using transmission electron microscopy (TEM), the specific steps are as follows:

[0117] (a) The exosome suspension was dropped onto a copper grid for electron microscopy.

[0118] (b) After allowing the liquid to stand at room temperature for several minutes to adsorb, use filter paper to remove excess droplets from the edge of the copper mesh.

[0119] (c) Negative stain with 1% uranium acetate solution for several minutes, then blot away excess stain with filter paper.

[0120] (d) Dry at room temperature, then take pictures via TEM (Tecnai Spirit, Czech Republic).

[0121] (2) Nanoparticle tracking analysis (NTA) is used to determine the particle size distribution. The specific steps are as follows:

[0122] (a) Instrument calibration: The Zetaview nanoparticle tracking analyzer (Particle Metrix, Germany) was used, and the instrument was rinsed with ultrapure water. 1 ml of standard solution was used for automatic instrument calibration. After successful calibration, the instrument was rinsed.

[0123] (b) Instrumental detection: Dilute the exosome sample to a suitable concentration with PBS solution, and inject an appropriate amount of the diluent into the instrument's sample cell. Enter the corresponding dilution factor in the accompanying software, and then start the detection program.

[0124] (c) Data analysis: After the test, the instrument generates data such as diameter distribution and particle concentration.

[0125] (3) Western blot detection of exosomal protein markers.

[0126] To detect exosome-specific and negative markers, proteins from exosomes were extracted and analyzed by Western blotting to determine the expression levels of CD9, CD81, HSP70, and Calnexin. Since CD9 and CD81 are also cell membrane proteins, to avoid false positives caused by residual cells in the exosome samples, proteins from BMVECs were also extracted as a control group.

[0127] 14. Cellular uptake of Exo-siCD151:

[0128] To evaluate the uptake efficiency of OGD-Exo and Native-Exo by BMVECs after OGD / R, this study conducted a cellular uptake experiment, the specific steps of which are as follows:

[0129] (1) BMVECs were seeded into confocal culture dishes (White Shark, China). When the cell confluence reached 60-70%, the cells were subjected to OGD for 4 hours. At the beginning of reoxygenation and reglucose administration, 20 μg / ml of DiD-stained OGD-Exo, Native-Exo, or cRGD-blocking OGD-Exo was added and incubated for 24 hours. For the cRGD blocking experiment, 15 μg / mL of cyclo-(RGDfK) (cRGD; Shanghai Apeptide) was pre-incubated with 20 μg / mL of OGD-Exo for 30 minutes to neutralize αvβ3 integrin on the surface of exosomes.

[0130] (2) After the exosomes and cells are co-incubated, the culture medium is discarded and the cells are washed with PBS three times for five minutes each time.

[0131] (3) Fix cells with 4% PFA for 10-30 minutes, remove the fixative, and wash with PBS 3 times for 5 minutes each time.

[0132] (4) Stain with DAPI for 5-10 minutes and image the cells under a confocal microscope (STELLARIS 8 Falcon, Leica, Germany).

[0133] 15. In vivo imaging:

[0134] (1) Within 0.5 h after cerebral ischemia, 100 μg of DiD-labeled Exo-siCD151 (based on exosomal protein quantification) was injected into tMCAO rats via the tail vein. Reperfusion was achieved by removing the thrombus 2 h after cerebral ischemia.

[0135] (2) After reperfusion for 24 hours, the rats were anesthetized by inhaling 5% isoflurane. After the rats were deeply anesthetized, their necks were quickly broken, and their brains, hearts, livers, spleens, lungs and kidneys were immediately collected and rinsed in pre-cooled PBS to remove surface blood.

[0136] (3) Use sterile filter paper to absorb the liquid on the surface of the organ and arrange them neatly on a black background tray.

[0137] (4) The organs were imaged using an in vivo imaging system (IVIS) (IVIS LUMINA III, PerkinElmer, USA).

[0138] (5) Turn on IVIS and pre-cool the CCD camera to -90℃.

[0139] (6) Place the organ tray in the imaging dark box and set the imaging parameters: excitation wavelength 620nm, emission wavelength 670nm; exposure time set to automatic adjustment; focus automatically; field of view covers all organs.

[0140] (7) Collect fluorescence images of each organ.

[0141] (8) Use Living Image 4 software (Caliper, USA) to delineate the regions of interest for each organ, measure and record the average fluorescence intensity, and perform quantitative analysis.

[0142] 16. Confocal imaging:

[0143] (1) 100 μg of DiD-labeled Exo-siCD151 was injected intravenously within 30 minutes of cerebral ischemia.

[0144] (2) After 24 hours of reperfusion, the rats were sacrificed, and 10 μm thick frozen brain sections were obtained for immunofluorescence staining, following the same procedure as in 29. The primary antibodies included anti-CD31 (R&D systems, AF3628, 10 µg / mL), anti-NeuN (HUABIO, ET1602-12, 1:1000), anti-GFAP (Cell Signaling Technology, 3670T, 1:400), and anti-Iba-1 (Abcam, ab283319, 1:100).

[0145] (3) The tissue sections were imaged using a confocal microscope (STELLARIS 8 Falcon, Leica, Germany). The images were processed using LAS X (version 7.0, Lecia, Germany) software.

[0146] 17. In vitro experimental grouping and technical roadmap:

[0147] BMVECs were randomly divided into four groups: control group, OGD / R+PBS group (referred to as OGD / R group), OGD / R+Exo-siNC group (referred to as Exo-siNC group), and OGD / R+Exo-siCD151 group (referred to as Exo-siCD151 group).

[0148] The Exo-siNC and Exo-siCD151 groups were reperfused 4 hours after OGD, with 20 μg / ml of Exo-siNC or Exo-siCD151 suspension added during reperfusion. The OGD / R groups were given an equal volume of PBS without exosomes. Cell samples were collected for Western blot (WB) 48 hours after reperfusion.

[0149] 18. In vitro experimental grouping and technical roadmap for using pathway inhibitors:

[0150] To further investigate the signaling pathways involved in the neuroprotective effects of Exo-siCD151, this study validated the effects in vitro using pathway-specific inhibitors. Primary BMVECs were used, subjected to OGD for 4 hours and then reperfused. At 24 hours after reperfusion, PD98059 (ERK1 / 2 inhibitor, 30 μM) or LY294002 (PI3K inhibitor, 20 μM) were added, respectively. Cells were cultured for another 48 hours after reperfusion and then harvested for protein analysis. The experimental groups are as follows: (1) OGD / R+PBS group: only PBS buffer was added during reperfusion; (2) OGD / R+Exo-siCD151 group: 20 μg / ml Exo-siCD151 was added during reperfusion; (3) OGD / R+PD98059 group: PD98059 was added 24 h after reperfusion; (4) OGD / R+Exo-siCD151+LY294002 group: 20 μg / ml Exo-siCD151 was added during reperfusion, and LY294002 was added 24 h after reperfusion.

[0151] 19. Statistical Analysis:

[0152] Each experiment was independently repeated at least three times. Statistical analysis of all data was performed using GraphPad Prism 9.1.0 (GraphPad Software, USA). Quantitative data are expressed as mean ± standard deviation (Mean ± SD). Results from RT-qPCR and WB experiments were normalized relative to the control group, i.e., data were divided by the corresponding control group value. The assessment of differences between groups followed these principles: when data conformed to a normal distribution, an unpaired two-tailed t-test was used for comparisons between two groups; when multiple group comparisons were involved, one-way analysis of variance (ANOVA) was used, followed by post-hoc analysis using Tukey's method.

[0153] The above are preferred embodiments of the present invention. The basic principles and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. All such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A therapeutic application of targeted exosomes in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The targeted exosomes are exosomes derived from brain microvascular endothelial cells subjected to oxygen-glucose deprivation treatment.

2. The therapeutic application of targeted exosomes according to claim 1 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The targeted exosomes are exosomes carrying CD151-siRNA.

3. The therapeutic application of targeted exosomes according to claim 2 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The targeted exosomes maintain endothelial cell immune homeostasis by downregulating CD151 levels in ischemic endothelial cells.

4. The therapeutic application of targeted exosomes according to claim 1 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The application of the targeted exosomes in the preparation of drugs for the prevention or treatment of stroke.

5. The therapeutic application of the targeted exosomes according to claim 1 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The application of the targeted exosomes in drugs that improve the prognosis of stroke patients.

6. The therapeutic application of targeted exosomes according to claim 4 or 5 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The drug is a pharmaceutical composition containing targeted exosomes.

7. The therapeutic application of the targeted exosomes according to claim 6 in improving blood-brain barrier disruption in acute ischemic stroke, characterized in that, The pharmaceutical composition may be in any pharmaceutically acceptable form.

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