CN117298153B

CN117298153B - Application of extracellular vesicles derived from brain endothelial cells in neuroinflammation

Info

Publication number: CN117298153B
Application number: CN202311112402.6A
Authority: CN
Inventors: 江沛; 王长水; 崔昌萌; 党瑞丽; 王芳; 陈贝贝; 孙文学
Original assignee: JINING NO1 PEOPLE'S HOSPITAL
Current assignee: JINING NO1 PEOPLE'S HOSPITAL
Priority date: 2023-08-30
Filing date: 2023-08-30
Publication date: 2024-03-08
Anticipated expiration: 2043-08-30
Also published as: CN117298153A

Abstract

The invention relates to the use of extracellular vesicles derived from brain endothelial cells in neuroinflammation. Extracellular vesicles have sensitivity and specificity to specific diseases, are novel biomarkers, and the existing researches prove that the extracellular vesicles realize effective regulation of physiological functions by playing a transmitter role and can be used as novel biological therapeutic agents. The research of the invention proves that the vesicle from brain endothelial cells can inhibit the expression of inflammatory factors and promote autophagy by regulating various ways such as miRNA and the like, thereby realizing the treatment effect of neuroinflammation and having good medical prospect.

Description

Application of extracellular vesicles derived from brain endothelial cells in neuroinflammation

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of extracellular vesicles derived from brain endothelial cells in neuroinflammation.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Chronic subclinical inflammatory states are one of the most clear features shared by a variety of neuropsychiatric and neurodegenerative diseases. Sustained inflammatory activation affects neurotransmission, neuronal apoptosis and brain energy metabolism. Microglia are brain resident macrophages, the primary glial cells of the central nervous system. Similar to macrophages, microglia can differentiate into a pro-inflammatory M1 phenotype and an immunomodulatory M2 phenotype, responsible for the production of pro-inflammatory or anti-inflammatory cytokines, respectively. Previous studies have shown that neurovascular decoupling is associated with the pathogenesis of neuroinflammatory-related neurological diseases. Multicellular cross-talk between brain endothelial cells (CECs) and brain parenchymal cells (including neurons and glial cells) is critical for maintaining neurovascular homeostasis. Meanwhile, regulation of the microenvironment against the blood brain has been indicated as a novel therapeutic strategy for neurological dysfunction.

Extracellular vesicles (extracellular vesicles, EVs) are nanoscale (30-150 nm) lipid bilayer envelope vesicles secreted by various cells. Secreted EVs can be taken up by recipient cells and deliver their contents, including active proteins, RNA species, and small molecules, thereby coordinating the dynamic interactions between cells. Previous studies have shown that EVs secreted by brain endothelial cells have neuroprotective effects in animal models of cerebral ischemia, stroke and alzheimer's disease, preventing neuronal loss, oxidative damage and excessive neuroinflammatory responses. However, the molecular mechanism of these neural activities remains undefined.

Brain endothelial cell derived outer vesicles (CEC-EVs) are neuronal communication messengers, and prior studies have demonstrated that CEC-EVs play an important regulatory role in a variety of brain diseases, potentially as drug targets, monitoring tools and diagnostic tools.

Disclosure of Invention

In view of the current situation, the present invention has been made to investigate whether or not brain endothelial cell-derived outer vesicles (CEC-EVs) have a regulatory effect on chronic subclinical inflammation associated with neurological diseases or disorders. Based on the technical purpose, the invention provides the following technical scheme:

In a first aspect, the invention provides the use of brain endothelial cell-derived outer vesicles for inhibiting neuroinflammation.

The research of the invention shows that the brain endothelial cell-derived outer vesicle is an important neurotransmitter, can penetrate the blood brain barrier and is absorbed by neurons, astrocytes and microglia. In the inflammation model, CEC-EVs are effective in improving the inflammatory activation state of microglia. To further verify the anti-inflammatory effect of CEC-EVs, the present invention demonstrates the anti-inflammatory mechanism of CEC-EVs from the following aspects:

(1) CEC-EVs can deliver miR-672-5p so as to regulate a target related to inflammation of a downstream target, and realize an inflammation inhibition effect;

(2) CEC-EVs are capable of delivering miR-672-5p to promote autophagy and inhibit inflammatory small body activation.

In addition to the above anti-inflammatory activity, the present invention demonstrates that CEC-EVs are capable of improving neuroinflammation-related anxiety and depression-like behavior.

Further, the specific forms of the above application are as follows:

(1) Use of extracellular vesicles derived from brain endothelial cells or a composition comprising extracellular vesicles derived from brain endothelial cells for the preparation of a medicament for the treatment of anti-neuroinflammation;

(2) Use of extracellular vesicles derived from brain endothelial cells or a composition comprising extracellular vesicles derived from brain endothelial cells for the preparation of a medicament for promoting autophagy;

(3) Use of extracellular vesicles derived from brain endothelial cells or a composition comprising extracellular vesicles derived from brain endothelial cells for the preparation of an antidepressant or anxiolytic medicament.

The extracellular vesicles derived from the brain endothelial cells have the diameter ranging from 50 to 150nm, have double-layer phospholipid membranes and are in spherical forms, and one feasible extraction mode is as follows: cutting up a cerebral cortex part, adding 0.05-0.15% of type II collagenase for digestion, adding a gradient separation solution for separation to obtain endothelial cells, adding Dulbecco modified Eagle culture medium into the cells, supplementing fetal calf serum, penicillin/streptomycin and endothelial cell growth additives for culture, continuously culturing the cells after 75-85% of culture medium is fully paved, replacing the culture medium with DMEM (medium with exosome fetal calf serum) for continuous culture, and obtaining the culture supernatant for centrifugation to obtain the extracellular vesicles.

The extraction method of extracellular vesicles belongs to the technical content disclosed in the art, and the technical difficulty is not basically existed in the technical field for the person skilled in the art by digestion and separation of brain endothelial cells and separation of extracellular vesicles with the structural characteristics in cell culture supernatant. In one embodiment of the invention, the following specific technical parameters are provided:

The digestion temperature of the type II collagenase is 36-38 ℃, and the digestion time is 25-35 min.

The gradient separation liquid is Percoll, and the feasible concentration is 45-55%.

In the Dulbecco modified Eagle culture medium, the concentration of fetal bovine serum is 18-22%, the concentration of penicillin/streptomycin is 0.8-1.2%, and the concentration of endothelial cell growth additive is 0.8-1.2%.

The culture supernatant was centrifuged as follows: 300 g, centrifuging for 10 minutes to remove cell contamination; collecting supernatant, centrifuging for 20 minutes at 2000-g, and centrifuging for 13-17 minutes at 8000-12000 g to remove any possible dead cells and large cell debris; after centrifugation, the supernatant was collected and passed through a 0.22 μm sterile filter; transferring the obtained supernatant to a high-speed centrifuge tube, ultracentrifugating for 88-92 minutes at 80000-100,000 g, and adding PBS to resuspend the precipitate.

In the composition containing the extracellular vesicles derived from the brain endothelial cells, the extracellular vesicles derived from the brain endothelial cells are used as main active ingredients; in one embodiment, the composition further comprises an essential carrier, such as a buffer or a culture medium; in other embodiments, the composition further comprises other active ingredients, such as anti-inflammatory ingredients or nerve repair ingredients.

In a second aspect, the use of an extracellular vesicle agonist derived from brain endothelial cells in the manufacture of a medicament for the treatment of neuroinflammation.

The extracellular vesicle agonist derived from brain endothelial cells referred to in the second aspect mentioned above means a related substance capable of promoting secretion of extracellular vesicles from brain endothelial cells, and may be a small molecule compound, a polymer or a nucleic acid component, wherein the nucleic acid component comprises a polypeptide or a nucleotide.

In a third aspect, there is provided a method of treating neuroinflammation, the method comprising administering to an individual in need of treatment a therapeutic dose of an extracellular vesicle derived from brain endothelial cells or an extracellular vesicle agonist derived from brain endothelial cells as described above.

In the above-described methods of treatment, the external vesicles or agonists may be delivered to the focal site by a viable administration means such as injection, or by interventional means.

The subject is preferably a mammal, such as a mouse, rabbit, dog, monkey, human, more preferably a human.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a graph showing the effect of LPS treatment on CEC-EVs release and microglial uptake by CEC-EVs;

Wherein, (A) the morphology of CEC-EVs is observed by electron microscopy; (B) analyzing the concentration of CEC-EVs by NTA; (C) analyzing the CEC-EVs particle size by NTA; (D) analyzing the total protein concentration of CEC-EVs; (E) the amount of CEC-EVs released; (F) Detecting extracellular vesicle marker proteins syntenin, alix, CD, tsg101 and negative protein Calnexin by using Western blot; (G) Immunofluorescence analysis of uptake of microglia to CEC-EVs after LPS treatment in vivo; (H) An in vitro brain endothelial cell and microglial cell Transwell co-culture system; (I) Immunofluorescence analysis of the uptake of microglia to CEC-EVs after in vitro LPS treatment; all data are expressed as mean ± standard deviation (n=3); unpaired student t-test, ns = insignificant compared to control group.

FIG. 2 is a validation of CEC-EVs release of miR-672-5p to microglial cells;

wherein, (A) a schematic representation of sequencing analysis following treatment with microglial CEC-EVs; (B) Heat map analysis of up-and down-regulated mirnas following treatment with microglial CEC-EVs and PBS, respectively; (C) qRT-PCR validation of up-regulated mirnas; (D) After activated microglial cells are treated by CEC-EVs, qRT-PCR verifies the expression condition of miRNA; (E) Extracellular vesicle secretion inhibitor GW4869 inhibits miR-672-5p transfer from CEC to microglial cell; (F) qRT-PCR verifies the inhibition effect of GW4869 on miR-672-5p transfer; (G) qRT-PCR verifies that miR-672-5p knocks down in CEC; (H) After knocking down miR-672-5p of CEC, expressing miR-672-5p in CEC-EVs; (I) After knocking down miR-672-5p of CEC, miR-672-5p in microglial cells is expressed; all data represent mean ± standard deviation (n=3); ns represents insignificant, ×p <0.01 compared to the control group; # P<0.01 and miR-NC ^KD CEC or miR-NC ^KD -EVs group comparison.

FIG. 3 is a graph showing that miR-672-5p negatively regulates TAB2 expression, inhibiting TAB2 interaction with TAK 1;

wherein, (a) four different predictive software, targetScan, miRWalk, micro-T, PITA, are utilized to predict potential gene targets; (B) miR-672-5p targets the 3' UTR of TAB2 in a highly conserved manner; (C) construction of TAB2 3' UTR wild type and mutant plasmids; (D) Luciferase activity after co-transfection of miRNA and luciferase plasmid; (E) Expression of TAB2 protein after transfection of miR-672-5p micrometers; (F) TAB2 protein statistics; (G) Effect of transfection of miR-672-5p micrometers on TAB2 mRNA expression levels; (H) Effect of transfection miR-672-5p micrometers on TAK1 protein expression level and phosphorylation thereof; (I) Co-immunoprecipitation using anti-MYC antibodies in microglia expressing Flag-TAK1 and MYC-TAB 2; (J) Transfection of miR-672-5p micrometers or interference with TAB2 expression inhibits interaction between TAB2 and TAK 1; all data are expressed as mean ± SD (n=3); * P <0.01 compared to miR-672-5P-NC treatment group TAB2-WT, # P <0.01 compared to miR-672-5P MImic treatment group TAB 2-WT.

FIG. 4 is a CEC-EVs-derived miR-672-5p that modulates the interaction between TAB2 and TAK1, inhibiting NF- κB activation of microglia;

Wherein, (A) CEC-EVs-derived miR-672-5p affects TAB2, TAK1 and p-TAK1 protein levels; (B) CEC-EVs-derived miR-672-5p reduces the interaction between TAB2 and TAK 1; (C) schematic illustration of the ortho-ligation technique for TAB2 and TAK 1; (D) Ortho ligation experiments demonstrated that CEC-EVs-derived miR-672-5p inhibited TAB2 and TAK1 interactions; (E) CEC-EVs-derived miR-672-5p inhibits p-IKKK beta protein expression; (F) CEC-EVs-derived miR-672-5p has no effect on NFkB protein expression; (G) CEC-EVs-derived miR-672-5p inhibits NF- κB nuclear translocation; (H) representative images of nfkb immunofluorescence experiments in vitro; (I) CEC-EVs-derived miR-672-5p inhibits NF- κB activity; all data are expressed as mean ± SD (n=3); single factor analysis of variance; * P<0.01 compared to control group, #P<0.05，##P<0.01 in comparison with the LPS group,&&P<0.01 and LPS+miR-NC ^KD -EVs group comparison.

FIG. 5 is a CEC-EVs-derived miR-672-5p modulates the interaction between TAB2 and TAK1, inhibiting NF- κB activation in vivo;

wherein, (A) CEC-EVs are shown in the schematic diagram of in vivo experimental study; (B) Effects of CEC-EVs-derived miR-672-5p on TAB2, TAK1 and p-TAK1 protein levels; (C) Proximity Ligation Analysis (PLA) demonstrated that CEC-EVs-derived miR-672-5p inhibited TAB2 and TAK1 interactions; (D) Representative Western blot of p-IKKbeta, p-IkBα, ikBα and NF kB; (E) a p-ikkβ/ikkβ protein expression statistical map; (F) p-IκBα/IκBα protein expression statistics; (G) a statistical map of nfkb protein expression; (H) Effects of CEC-EVs-derived miR-672-5P on expression of NF- κB protein in cytoplasm; (I) CEC-EVs-derived miR-672-5p promotes nuclear translocation of NF-. Kappa.B; (J) representative images of nfkb in vivo immunofluorescence experiments; all data are expressed as mean ± SD (n=6); single factor analysis of variance, P <0.01 phase with control groupComparing; # P<0.01 compared to the LPS group;&&P<0.01 and LPS+miR-NC ^KD -EVs group comparison.

FIG. 6 is a graph showing that CEC-EVs-derived miR-672-5p promotes autophagy of microglial cells and inhibits activation of inflammatory corpuscles;

wherein (A) miR-672-5p from CEC-EVs promotes microglial cell LC3II and Beclin1 protein level, and inhibits p62 protein expression; (B) a statistical map of LC3II protein expression; (C) a statistical map of Beclin1 protein expression; (D) a statistical map of p62 protein expression; (E) Beclin1 and LC3II double-stained immunofluorescence images; (F) Immunofluorescence staining images after microglial cell transfection of mRFP-GFP-LC3 adenovirus; (G) Counting green fluorescence and red fluorescence of autophagy flow fluorescence images; (H) Number of autophagosomes and autophagosomes per cell; (I) NLRP3 and LC3II double-stained immunofluorescence images.

FIG. 7 is a CEC-EVs-derived miR-672-5p promotes autophagy in vivo and inhibits activation of NLRP3 inflammasome;

wherein (A) miR-672-5p from CEC-EVs promotes the in-vivo LC3II and Beclin1 protein level, and inhibits p62 protein expression; (B) a statistical map of LC3II protein expression; (C) a statistical map of Beclin1 protein expression; (D) a statistical map of p62 protein expression; (E) Autophagy inhibitor chloroquine assesses the effect of CEC-EVs treatment and LPS treatment on LC3II protein; (F) LC3II protein statistics; (G) qRT-PCR verifies the effect of CEC-EVs on inflammatory factors; (H) CEC-EVs inhibited LPS-induced activation of microglia.

FIG. 8 is the neuroprotective effect of CEC-EVs on LPS-induced behavioral deficits in mice;

wherein, (a) mice represent trajectory plots (right plot) in spatial exploration (left plot) and directional cruise test; (B) CEC-EVs treatment reduces the time for mice to find a platform; (C) CEC-EVs processing increases the number of passes across the platform; (D) CEC-EVs processing reduces the time spent in the target quadrant in which the platform is located; (E) elevated plus maze test mouse trajectory heatmaps; (F) CEC-EVs treatment increased the number of mice entering the open arms; (G) CEC-EVs treatment increases the time to open arms in mice; (H) CEC-EVs treatment increased sucrose preference in mice; (I) CEC-EVs treatment reduced immobility time in the forced swim test in mice.

FIG. 9 is a graph showing the effect of CEC-EVs on the uptake process by the verification of neurocyte uptake and neuroinflammatory activation;

wherein, (A) an in vivo assay schematic for verifying whether CEC-EVs are absorbable by nerve cells; (B) Immunofluorescence co-staining of PKH-67 with βIII tubulin (left) and data plots (right); (C) Immunofluorescence co-staining of PKH-67 with GFAP (left) and data plots (right); all data are expressed as mean ± standard deviation (n=3); unpaired student t-test; ns=is not significant compared to the control group.

FIG. 10 is a graph showing that CEC-EVs reduced LPS-induced inflammatory response and altered microglial polarization;

wherein (A) CEC-EVs reduce inflammatory factors released by LPS-induced microglia; (B) CEC-EVs reduce LPS-induced microglial activation; (C) Detecting the expression of microglial cell M1 markers (iNOS and CD 86) and M2 markers (CD 206, ARG-1) by Western blotting; (D) immunofluorescence images of iNOS and ARG-1; all data are expressed as mean ± standard deviation (n=3); single factor analysis of variance; ns=no significant difference compared to control group, P <0.05, P <0.01# # compared to LPS group P < 0.01.

FIG. 11 is a correlation verification that miR-672-5p promotes cell migration and reduces inflammatory responses;

wherein, (a) microglial cell migration inhibition results in each experimental group; (B) Expression of inflammatory factor IL-6 mRNA in each experimental group; (C) Expression of inflammatory factor IL-1 beta mRNA in each experimental group; (D) Expression of inflammatory factor TNF- α mRNA in each experimental group; (E) Expression of inflammatory factor IL-4 mRNA in each experimental group; all data are expressed as mean ± standard deviation (n=3); single factor analysis of variance P <0.01 compared to control group; ns=no significant &comparedto control group &p <0.01 compared to lps+mir-672-5P-NC group.

FIG. 12 is a validation of CEC-EVs-derived miR-672-5p in vitro inhibition of NF- κB activation and reduction of LPS-induced inflammation;

wherein, (a) p-iκbα, iκ B A, nfκb; (B) Statistical graphs of p-IκBα, Iκ B a, nfκB protein expression; (C) CEC-EVs derived miR-672-5p inhibits migration of migratory cells; (D) CEC-EVs derived miR-672-5p reduces the expression of inflammatory factor IL-6; (E) CEC-EVs derived miR-672-5p reduces the expression of inflammatory factor IL-1 beta; (F) CEC-EVs derived miR-672-5p reduces expression of inflammatory factor TNF-alpha; (G) CEC-EVs derived miR-672-5p increases expression of anti-inflammatory factor IL-4; all data are expressed as mean ± standard deviation (n=3); single factor analysis of variance P < 0.01# compared to control group and P < 0.01 compared to LPS group&&With LPS+miR NC ^KD EVs group was < 0.01 compared to P.

FIG. 13 is a correlation verification that CEC-EVs-derived miR-672-5p inhibits inflammatory small body activation in vitro;

wherein, (A) NLPR3, ASC, clear-Caspase-1 and mature IL-1β; (B) Statistical graphs of NLPR3, ASC, clear-Caspase-1 and mature IL-1 beta protein expression; (C) CEC-EVs derived miR-672-5p reduces expression of inflammatory factor TNF-alpha; (D) CEC-EVs derived miR-672-5p reduces the expression of inflammatory factor IL-1 beta; (E) CEC-EVs derived miR-672-5p reduces the expression of inflammatory factor IL-6; (F) CEC-EVs derived miR-672-5p increases expression of anti-inflammatory factor IL-10; all data are expressed as mean ± standard deviation (n=3); single factor analysis of variance; ns = no significance, P compared to control group <0.01; ns = no significant difference compared to LPS group (P<0.01 A) is provided; ns = insignificant, with lps+mir NC ^KD In contrast to the EVs group,&&P<0.01。

FIG. 14 is a CEC-EVs-derived miR-672-5p inhibiting activation of inflammatory bodies in vivo;

wherein, (A) NLPR3, ASC, clear-Caspase-1 and mature IL-1β; (B) Statistical graphs of NLPR3, ASC, clear-Caspase-1 and mature IL-1 beta protein expression; (C) Representative Western blots of M1 markers (iNOS and CD 86) and M2 markers (CD 206, ARG-1); (D) a statistical map of M1-tagged and M2-tagged protein expression; all data are averagedValue ± standard deviation (n=6); single factor analysis of variance; ns = no significance, P compared to control group<0.01; ns = no significant difference compared to LPS group (P<0.01 A) is provided; ns = insignificant, with lps+mir NC ^KD In contrast to the EVs group,&&P<0.01。

FIG. 15 is a correlation verification that CEC-EVs-derived miR-672-5p mitigates brain damage;

wherein, (a) HE staining, nissl staining and TUNEL detection; (B) quantification of normal cells; (C) percent of nikovia; (D) number of TUNEL positive cells; all data are expressed as mean ± standard deviation (n=6); single factor analysis of variance P < 0.01# compared to control group and P < 0.01 compared to LPS group &&With LPS+miR NC ^KD EVs group was < 0.01 compared to P.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, 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 belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

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

Example 1

1. Research method

1.1. Animal and treatment

Male C57BL/6J mice of 7-8 weeks old were purchased from Jinan Pengyue laboratory animal breeding Co., ltd (Jinan, china). The mice were placed under standard conditions (temperature: 22.+ -. 2 ℃ C.; 12:12 hours light/dark cycle; humidity: 50.+ -. 5%) and were free to access sterile food and water. Mice were acclimatized for 7 days prior to the experiment. Mice were randomly divided into control, LPS, LPS+NC-EVs and LPS+miR-672-5p EVs groups. In LPS+NC-EVs and LPS+miR-672-5pKD-EVs, 20 μg CEC-EVs was injected into mice 3 times per week via tail vein for 2 weeks, as described previously, while control and LPS groups were injected with the same volume of PBS via tail vein. Meanwhile, according to the previous study of this example, each mouse was injected by intraperitoneal injection of 500ug/kg, once every 2 days, for a total of 7 injections. The behavior study was performed 24 hours after the last injection, for 3 consecutive days. After the last behavioural test, mice were anesthetized deeply by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight), tissues were rapidly removed, and rapidly dissected on ice. All animal procedures were performed according to the guidelines for care and use of experimental animals and were approved by the ethical committee of the first human hospital in the economical Ning city (approval number JNMC-2022-DW-041).

1.2. Isolated culture of brain endothelial cells

Primary brain endothelial cells (CECs) were isolated from adult mice: the cortex was separated from the brain and minced with sterile scissors in a petri dish filled with cold D-Hank solution. The minced tissue was then digested with 0.1% collagenase type ii and digested at 37 ℃ for 30 minutes. Endothelial cell debris was isolated using a 50% Percoll gradient. Endothelial cells were cultured in complete Dulbecco modified Eagle medium supplemented with 20% fetal bovine serum (FBS, gibco), 1% penicillin/streptomycin (Gibco) and 1% endothelial cell growth additive (scientific). 5% CO of cells at 37 DEG C ₂ Culturing in an incubator. After the cells reached 80% confluence, the medium was changed to DMEM medium supplemented with exosome-free FBS. BV2 mouse microglial cells were purchased from Procell Life Science&amp, technology co., ltd (martial arts, china) and cultured as described above. BV2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, shanghai). Then, the cells were incubated at 37℃with 5% CO ₂ Culturing in incubator, and changing culture medium every two days.

1.3. Extracellular vesicle isolation and purification

Isolation and purification of extracellular vesicles from cell culture supernatants. The culture was centrifuged at 300 g for 10 minutes to remove cellular contamination, and then the supernatant was centrifuged at 10000 g for 15 minutes to remove any possible dead cells and large cell debris. After centrifugation, the supernatant was collected and passed through a 0.22 μm sterile filter. The resulting supernatant was transferred to a high speed centrifuge tube (3139-0050, thermo) and ultracentrifuged at 100,000 g for 90 minutes. The pellet was resuspended in PBS and the ultracentrifugation step was repeated. All centrifugation steps were performed at 4 ℃.

1.4. Extracellular vesicle characterization

Ultrastructural observation of extracellular vesicles was performed using a transmission electron microscope (HT 7700, hitachi). In addition, the concentration and size of extracellular vesicles were estimated using ZetaView and nanoparticle tracking analysis software. After determining the extracellular vesicle protein concentration, western blotting is performed to detect specific surface markers of extracellular vesicles.

1.5. In vivo extracellular vesicle tracking

Purified CEC-EVs were labeled with PKH67 green fluorescent membrane dye (Sigma, shanghai) according to the manufacturer's instructions. The labeled CEC-EVs pellet was washed with PBS and collected by ultracentrifugation and resuspended in PBS again. For in vivo tracking of extracellular vesicles, labeled extracellular vesicles (20 ug) were injected into 100 μl total PBS by tail vein. Brain tissue was collected 24 hours after injection for immunofluorescence analysis.

1.6. Transwell detection

To determine whether CEC-EVs can be efficiently taken up by activated microglia, a transwell co-culture system was established. CECs were inoculated in the upper layer of the transwell chamber and cultured in DMEM medium containing no exosome FBS. The lower layer was 600 ul DMEM medium (containing 10% exosome-free FBS) and was seeded with BV2 cells. transwell plates were placed at 37℃with 5% CO ₂ Is provided. After incubation of 24 h, the lower chamber BV2 cells were fixed with 4% paraformaldehyde and an inverted microscope (Olympus IX 73) was used to obtain images. For the purpose ofThe effect of miRNAs and CEC-EVs on inflammatory stimuli was studied and BV2 cells were seeded into the upper chamber of matrigel coated filters. The bottom chamber was filled with 0.5 ml DMEM medium as chemotactic agent. After incubation at 37 ℃ for 12 hours, the cells on the upper surface of the chamber were carefully cleaned with a cotton swab. Cells that invaded the lower chamber were then fixed with methanol, stained with 0.5% crystal violet, and counted under a microscope.

1.7. Transfection

miR-672-5p mimic, negative control (miR-672-5 p-NC), cy 3-labeled miR-672-5p and TAB 2-targeted small interfering RNA (siRNA) were synthesized by GenePharma (Shanghai, china) and transfected into BV2 cells. Cells were plated one day prior to transfection to achieve approximately 70% confluency on the day of transfection. The transfection procedure was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

1.8. Dual luciferase reporter detection

The 3' -UTR of TAB2 or the mutated miR-672-5p binding site was subcloned into the pmirGLO dual luciferase target expression vector (Promega, madison, wis., USA) to construct the reporter vectors pmirGLO-TAB2-WT and pmirGLO-TAB2-mut. miR-672-5p mimic or mimicNC was transfected into BV2 cells, followed by transfection with pmirGLO-TAB2-MT or pmirGLO-TAB2-Mut plasmids. Luciferase activity was determined by luciferase activity kit (promega) according to the manufacturer's protocol.

1.9. Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) assays were performed using the Co-IP kit (Absin Bioscience, china) according to the manufacturer's instructions. BV2 cells were transfected with Flag-TAK1 and/or MYC-TAB2 plasmids. Then, the cell lysates were incubated with anti-MYC antibodies (ab 32, abcam) overnight at 4 ℃. Samples were immunoprecipitated with anti-MYC antibodies overnight at 4 ℃. Proteins were eluted from the beads and separated by SDS-PAGE. For the label-free Co-IP assay, cell and tissue lysates were incubated with anti-TAB 2 antibodies (ab 264309, abcam) overnight at 4 ℃. Samples were immunoprecipitated overnight at 4℃with anti-TAB 2 antibodies. Proteins were eluted from the beads and separated by SDS-PAGE. The 3' -UTR of TAB2 or mutated miR-672-5p binding site was subcloned into the pmirGLO dual luciferase target expression vector (Promega, madison, wis., USA) to construct the reporter vectors pmirGLO-TAB2-WT and pmirGLO-TAB2-Mut. miR-672-5p mimic or mimic NC was transfected into BV2 cells, followed by transfection with pmirGLO-TAB2-MT or pmirGLO-TAB2-Mut plasmids. Luciferase activity was determined by luciferase activity kit (promega) according to the manufacturer's protocol.

1.10. Protein proximity ligation analysis technique

Protein Proximity Ligation Assay (PLA) was performed using the Duolink PLA kit (Sigma) according to the manufacturer's instructions. The cells were spun onto slides and fixed with 4% PFA. After permeabilization with 0.2% Triton X-100 for 5 min, cells were blocked with Duolink blocking buffer for 30 min. Subsequently, the cells were incubated with primary antibodies (anti-TAB 2 and anti-TAK 1) overnight at 4 ℃. PLA probes were diluted with Duolink's solid antibody diluent in the appropriate proportions, then ligated and amplified. Slides were washed with buffer and then mounted using the DAPI-containing Duolink in situ mounting agent. Slides were observed under a fluorescence microscope (Olympus). For tissues, sections were deparaffinized in xylene, hydrated by gradient ethanol and subjected to antigen retrieval. Following antigen retrieval, sections were incubated with primary antibodies overnight at 4 ℃. The remainder of the procedure is the same as for cells.

1.11. ELISA and NFkB Activity assay

TNF-alpha ELISA kit (MTA 00B), IL-1 beta ELISA kit (MLB 00C), IL-6 ELISA kit (M6000B) and IL-10 ELISA kit (M1000B) were purchased from R & D Systems. All operations were performed in accordance with the manufacturer protocol provided by the kit. Nfkb activity was detected using the Quanti-blue assay following the manufacturer's protocol (invitogen).

1.12. Real-time quantitative PCR analysis

Trizol reagent (Invitrogen, shanghai, china) was used to extract total RNA from tissues and cells according to the manufacturer's instructions. The final RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Total RNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen, shanghai, china). Quantitative real-time PCR was performed on a CFX96 Touch real-time PCR detection system (Bio-Rad). The experiment was repeated three times with three samples using the expression level of β -actin as an internal standard. Primers were ordered from origin and the sequences of the primers used are listed in Table 1.

TABLE 1 primer sequences for qPCR analysis

1.13. Western blot

For western blotting, total or nuclear proteins were extracted and the concentration was measured. Proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF). PVDF membranes were then blocked with 5% skim milk for 2 hours at room temperature, then incubated overnight at 4 ℃ with primary antibodies to syntenin (ab 19903, abcam), alix (ab 186429, abcam), CD63 (ab 217345, abcam), tsg101 (ab 125011, abcam), calnexin (ab 22595, abcam), TAB2 (DF 2368, affinity), TAK1 (AF 7616, affinity), p-TAK1 (AF 3019, affinity), ikkβ (ab 32135, abcam), p-ikkβ (AF 3010, affinity), ikbα (AF 5002, affinity), p-ikbα (AF 2002, affinity), nfκb (AF 6, affinity), LC3 (ab 192890, abcam), beclin 1 (ab 62557, abs), SQSTM1/p62 (ab 3562, ab67, and actin (ab 82, ab). The secondary antibody used was horseradish peroxidase (HRP) -conjugated goat anti-rabbit IgG (ab 6721, abcam). The gene expression level was normalized to the expression level of β -actin or PCNA and the signal was quantified using Image J software.

1.14. Immunofluorescent staining

Using Tyramine Signal Amplification (TSA) technology. Paraffin-embedded cortical tissue was cut into 4 μm thick sections. The sections were dewaxed in xylene and rehydrated in a gradient ethanol series. The sections were then immersed in EDTA antigen retrievalAntigen retrieval was performed in buffer. To block endogenous peroxidases, sections were exposed to 3% H ₂ O ₂ For 20 minutes. For immunocytochemistry, cultured cells were fixed on 4% formaldehyde and permeabilized with 0.5% Triton X-100 for 20 min. Cells were blocked with 3% BSA at room temperature. The sectioned or immobilized cells were then incubated with primary antibody overnight at 4℃and HRP-conjugated secondary antibody at room temperature for 30 min, followed by treatment with Cy3-TSA solution. The sections or fixed cells were immersed in EDTA buffer (pH 8.0) and microwaved to remove the primary and secondary antibodies. The sections or fixed cells were then incubated again with the secondary antibody, secondary antibody and FITC-TSA solution. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The antibodies used were as follows: IBA-1 (ab 178847, abcam), CD68 (ab 53444, abcam), nfkb (AF 5006, affinity), LC3 (ab 192890, abcam), beclin 1 (ab 62557, abcam), NLRP3 (DF 7438, affinity), iNOS (ab 178945, abcam), ARG-1 (DF 6657, affinity), HRP conjugated goat anti-rabbit IgG (ab 6751, abcam).

1.15. Behavioural detection

(1) Forced swimming experiments (FST).

The experiment was performed as described previously. The C57BL/6J mice were placed in a transparent plexiglas cylinder (height: 30 cm, diameter: 10 cm) filled with 15 cm of water. Mice were forced to swim for 6 minutes and the immobility time of the last 5 minutes was recorded. If the mouse floats in water without struggling, only the necessary action is taken to keep its head on the water surface, the mouse is considered to be in a resting state.

(2) Sucrose preference experiment (SPT).

Prior to the experiment, two bottles of 1% sucrose solution were placed on either side of the cage and the mice were trained to habituate to sucrose solution for 24 hours. One sucrose solution bottle was then replaced with water for 24 hours. After the adaptation phase, the mice were fasted for 24 hours. Each animal was free to feed two pre-weighed bottles at the same time, one containing 1% sucrose solution and the other containing equal weight tap water. To avoid spatial deviations, the positions of the two bottles are randomly placed on the left and right sides. After 12 hours, the weight of sucrose solution and water consumed was recorded. Sucrose preference is calculated using the following formula: sucrose preference (%) = (sucrose solution consumption/total liquid consumption) ×100%.

(3) Overhead plus maze test (EPM).

The experiment was performed as described previously. The EPM device consists of two opposite open arms (35 x 10 cm), perpendicular to the two opposite closed arms (35 x 10 cm), with a small central square (5 x 5 cm) sandwiched between the arms. The two closed arms are enclosed by vertical walls, while the open arms have edges that are not protected. The mice were gently placed in the center of the platform, facing the open arm on one side, and allowed to explore freely for 5 minutes. The total number of entries into the open and closed arms was recorded by the video camera and the time to stay in the open arm during the test.

(4) Morris water maze test (MWM).

MWM experiments were used to assess spatial memory and learning ability, for a total of 6 days. The MWM is a circular pool divided into four equal sized imaginary quadrants, with a circular platform immersed in the center one of the quadrants, 1 cm from the water surface. The navigation test was performed in the first 5 days. At each test, the mice were placed in a pool facing four different directional walls and allowed to find an escape platform for 60 seconds. If the mice reached the platform, the time was recorded as escape latency. If the mouse cannot find the plateau within a given time, it is guided to the plateau and left for 10 seconds, with a latency of 60 seconds recorded. After 24 hours, the platform was removed and the mice were released from the opposite quadrant for spatial detection testing. The time the mice remained in the target quadrant and the swimming speed were recorded.

1.16. Nissl staining

After waxing, the sections were immersed in a 1% Cresyl violet solution for 10 minutes at room temperature. The sections were washed with distilled water and differentiated in 95% ethanol. Finally, the sections were infiltrated in xylene, blocked with neutral resin and observed under an inverted optical microscope. For HE staining, brain tissue sections were first dewaxed with xylene for 20 min, twice, then rehydrated twice in absolute ethanol for 5 min, respectively, rehydrated in 75% ethanol for 5 min, and tap water for 5 min. Sections were stained with hematoxylin and eosin. Dehydrated with progressively coloured alcohol and cleared in xylene, then observed under an Olympus IX73 microscope. TUNEL staining was performed using TUNEL detection kit (Beyotime, china). Paraffin-embedded brain tissue sections were first de-waxed, treated with 20 μg/ml proteinase K for 20 min at 20 ℃, then rinsed three times with PBS, stained with 50 μl TUNEL reaction mix, incubated for 60 min at 37 ℃ and protected from light. The sections were blocked with an anti-fluorescent light-off flap and imaged using a fluorescent microscope (IX 73, olympus).

1.17. Statistical analysis

Data analysis was performed using SPSS v.13.0 software (SPPS inc., chicago, IL, USA) and results are expressed as mean ± standard deviation. Statistical analysis included unpaired student t-test and one-way analysis of variance. Data processing was performed using GraphPad Prism 7 software (GraphPad Software). P values less than 0.05 were considered significant.

2. Conclusion of the study

2.1. Inflammatory activation has no effect on the release of EVs in CECs, but increases microglial uptake of CEC-EVs

CECs were treated with LPS to extract EVs, and identified and analyzed by TEM, NTA and Western blotting. Spherical EVs with a double membrane structure in the diameter range of 50 to 150nm were observed in both the control group and the LPS treated group. No morphological differences in EVs were observed between the control and LPS treated groups in terms of shape, size or electron density (fig. 1A). NTA data also showed similar ion sizes (fig. 1B and 1C). LPS stimulation had no effect on total protein of EVs secreted by CECs (fig. 1D), while LPS stimulation had no effect on the number of EVs released per cell (fig. 1E). Considering that EVs are isolated from the same number of CECs, expression of EVs markers may reflect the ability of EVs to secrete. By protein analysis of Whole Cell Lysates (WCL) and specific markers Syntenin, alix, CD63 and Tsg101 for EVs in EVs, LPS exposure was found to have no effect on the expression of these proteins (fig. 1F). Calnexin is an endoplasmic reticulum protein that is difficult to detect in the EVs component, but is abundant in WCL, and the results show that Calnexin protein is not detected in CEC-EVs, indicating successful isolation of CEC-EVs (FIG. 1F).

Next, this example analyzes whether CEC-EVs can be successfully absorbed by nerve cells and whether neuroinflammatory activation alters the absorption process. After labeling CEC-EVs with PKH67 green fluorescent dye, this example injected PKH67 labeled EVs via the tail vein (fig. 9A). The results demonstrate that CEC-EVs can cross the blood brain barrier and are readily absorbed by neurons, astrocytes and microglia, as demonstrated by the co-localization of PKH67 green fluorescence with NEUN, GFAP and IBA-1, respectively (FIGS. 1G and 9B, 9C). Interestingly, treatment with LPS significantly enhanced the uptake of CEC-EVs by microglia, but did not alter the ability of neurons and astrocytes to uptake CEC-EVs (FIGS. 1G and 9B, 9C). Given that microglial cells are the primary immune responsive cells in the central nervous system, this example established a Transwell co-culture system to confirm in vivo findings (fig. 1H). Lipid membranes of CECs were stained with PKH26 red fluorescent dye and co-cultured with DIO-stained BV2 microglia in a Transwell (membrane well = 0.4 μm). Consistent with the in vivo results, this example found that red fluorescence of PKH26 in microglia increased following LPS treatment, indicating that the EVs secreted by CECs were transferred from the upper chamber to microglia seeded in the lower chamber, further indicating that activated microglia can take up more CEC-EVs (fig. 1I).

CEC-EVs with anti-inflammatory effect

Considering that CEC-EVs were found to have neuroprotective effects from previous studies, the next step in this example was to evaluate their neuroimmunomodulatory effects directly. The data of this example show that microglia release inflammatory cytokines and inhibit the expression of anti-inflammatory factors after LPS stimulation (fig. 10A). As expected, CEC-EVs could directly improve LPS-induced microglial overactivation (fig. 10B). This example found that CEC-EVs would promote the transition of microglial cells from the M1 phenotype to the M2 phenotype and increased the M2 biomarker and decreased the M1 biomarker (fig. 10C). Immunofluorescence results also confirmed this finding and indicated that CEC-EVs treatment significantly reduced LPS-exposure-induced iNOS increase and ARG-1 decrease, highlighting the powerful anti-inflammatory effect of CEC-EVs (fig. 10D).

Transfer of miR-672-5p from CECs to microglia through EVs-mediated delivery mechanism

mirnas are the major inclusions of EVs and play an important role in communication between cells. To detect potential immune modulatory mechanisms, RNA was extracted from CEC-EVs or PBS-treated microglia, respectively, and subjected to miRNA microarray analysis (fig. 2A). The results showed that 37 miRNAs were up-regulated after CEC-EVs treatment >1.5 times, P<0.05 12 miRNAs were down-regulated (FIG. 2B). Based on the results of the miRNA profiling, the first five upregulated mirnas, including miR-128-3p, miR-672-5p, miR-3058-5p, miR-574-3p, and miR-762, were selected and validated by q-PCR (fig. 2C). This example found that CEC-EVs significantly increased expression of these mirnas in LPS-induced microglia, with the most significant increase in miR-672-5p (fig. 2D). To further confirm the EVs-mediated miR-672-5p transport mechanism, CECs were transfected with a Cy 3-labeled miR-672-5p mimetic and co-cultured with microglial cells in a Transwell to follow the intercellular transport of miR-672-5 p. Cy 3-labeled miR672-5p (red fluorescence) was transferred from CECs in the upper compartment of the Transwell to microglia in the lower compartment, and this process was blocked by extracellular vesicle secretion inhibitor GW4869, indicating that miR-672-5p was transported between cells by extracellular vesicles (FIG. 2E). In addition, the results of miR-672-5p transport were further confirmed by qPCR (FIG. 2F). Migration is an important component of microglial response to inflammatory stimuli. Thus, this example examined whether miR-672-5p regulates microglial migration and inflammatory response. Through migration experiments, this example found that the miR-672-5p mimic inhibited microglial migration (fig. 11A) and enhanced anti-inflammatory effects (fig. 11B-E). Next, this example successfully knocked down miR-672-5p in CECs (FIG. 2G). Accordingly, miR-672-5p was also significantly inhibited in the secreted EVs from the CECs from which miR-672-5p was knocked out (FIG. 2H). EVs (miR-NC) without knocking down miR-672-5p by microglial cells are respectively given ^KD -EVs) and miR-672-5p knockdown EVs (miR-67)2-5p ^KD -EVs), found to be identical to miR-NC ^KD -miR-672-5 p compared to EVs treated group ^KD -significant reduction in EVs treated microglial miR-672-5p expression (figure 2I).

miR-672-5p inhibits TAB2 expression and inhibits interaction between TAB2 and TAK1

To identify the downstream target of miR-672-5p, the potential targets of miR-672-5p were predicted by four databases. (FIG. 3A). Among these potential target genes, the 3' -UTR of TAB2, which is closely related to inflammation, was found to bind to miR-672-5p with high affinity and high conservation (FIG. 3B). TAB2 is an indispensable coactivator for TAK1 signaling, and can activate TAK1-TAB complex to phosphorylate IKK by inflammatory stimulus, and further cause NF- κB activation by inducing IκB phosphorylation and degradation, thereby promoting inflammatory process. To further confirm that the 3'-UTR of TAB2 is a direct target of miR-672-5p, this example constructed a TAB2 3' -UTR wild-type (TAB 2-WT) or mutant (TAB 2-Mut) plasmid in a pmirGLO dual-luciferase reporter vector (Promega) and measured luciferase activity after co-transfection of the miRNA and luciferase plasmids (FIG. 3C). This example shows that the miR-672-5p mimetic significantly reduced the luciferase activity of TAB2-WT compared to that of mock NC, whereas the luciferase activity of TAB2-Mut was not significantly altered, confirming the targeted binding of miR-672-5p (FIG. 3D). Furthermore, the miR-672-5p mock transfection also inhibited TAB2 expression at the protein and mRNA levels (FIG. 3E-G). In addition, this example also found that the miR-672-5p mimetic had no effect on TAK1 protein levels, but inhibited TAK1 phosphorylation (FIG. 3H). When TAB2 forms a complex with TAK1 and TAB1, TAK1 is activated by autophosphorylation. Next, this example performed a co-immunoprecipitation (co-IP) experiment in microglia to evaluate the interaction of TAB2 and TAK 1. Co-IP analysis showed that TAB2 can interact with TAK1 protein (FIG. 3I). This co-immunoprecipitation decreased with TAB2 knockdown (fig. 3J).

CEC-EVs derived miR-672-5p inhibits activation of pro-inflammatory NF-. Kappa.B by targeting TAB2

Considering that miR-672-5p can negatively regulate TAB2 expression, this example studied TAB2 and TWhether AK1 expression is affected by CEC-EVs-derived miR-672-5 p. Data of this example shows that miR-NC ^KD EVs treatment significantly attenuated LPS-induced TAB2 and p-TAK1 protein expression, which trend was seen in miR-672-5p ^KD Significantly blocked in the EVs treated group (fig. 4A). However, TAK1 protein is found in miR-NC ^KD -EVs or miR-672-5p ^KD No change in EVs handling (fig. 4A). Co-IP data shows that miR-NC ^KD EVs inhibited the interaction between the TAB2 and TAK1 proteins (FIG. 4B). This result was further confirmed by PLA experiments (fig. 4C and 4D). NF-. Kappa.B is a family of transcription factors that play an important role in immune and inflammatory responses. The activated TAB2-TAK1 complex can phosphorylate the IκB kinase (IKK) complex and further activate NF- κB. Thus, this example investigated whether CEC-EVs affect NF-kB activation. miR-NC ^KD EVs improve LPS-induced IKKbeta phosphorylation, IκB degradation and NF κB activation, miR-672-5p ^KD EVs treatment showed the opposite trend ((FIG. 4E, FIG. 12A and FIG. 12B.) nuclear translocation of NF- κB showed miR-NC ^KD EVs reduced translocation of NF-. Kappa.B from the cytoplasm to the nucleus (FIG. 4F-H). Likewise, miR-NC ^KD EVs treatment also inhibited NFkB activity (FIG. 4I). The present example also found miR-NC ^KD -EVs inhibit microglial migration, whereas miR-672-5p ^KD EVs promote cell migration (FIG. 12C). In addition, miR-NC ^KD EVs inhibited inflammatory cytokines produced by activated microglia (FIGS. 12D-G).

To further explore the mechanism of CEC-EVs-derived miR-672-5p action in vivo, miR-NC was injected through the tail vein ^KD -EVs or miR-672-5p ^KD EVs were injected into mice (FIG. 5A). The present example shows miR-NC ^KD -EVs inhibit LPS-induced TAB2 expression and TAK1 activation, which is inhibited by miR-672-5p ^KD Treatment by EVs counteracts (fig. 5B). miR-NC ^KD EVs impair the interaction of TAB2 and TAK1 (FIG. 5C). Further study of miR-NC based on this example ^KD -effect of EVs on NFkB activation. Western blot shows that miR-NC ^KD EVs also inhibit LPS-induced phosphorylation of IKKbeta and IkBα and expression of NF- κB(FIGS. 5D-G). Next, this example investigated the effect of CEC-EVs on NF-. Kappa.B transfer between the cytoplasm and the nucleus. LPS-induced translocation of NF- κB to the nucleus by use of miR-NC ^KD EVs treatment is effectively weakened, and miR-672-5p ^K D-EVs did not significantly improve nuclear translocation of NF- κB (FIGS. 5H and I), as was also demonstrated by immunofluorescent staining (FIG. 5J). These data indicate that the immunomodulatory activity of CEC-EVs is mediated through delivery of miR-672-5 p.

CEC-EVs derived miR-672-5p promotes autophagy and inhibits inflammatory body activation by targeting TAB2

In addition to the nfkb pathway, TAB2 may interact with Beclin-1, thereby attenuating the autophagy process. Autophagy also plays a key role in inhibiting excessive inflammatory responses. Thus, this example further evaluates the effect of CEC-EVs on autophagy. LPS treatment inhibits autophagy and miR-NC is administered ^KD EVs treatment enhanced the expression of autophagy-related proteins LC3II and Beclin-1 and inhibited the expression of SQSTM1/p62 (FIGS. 6A-D). Immunofluorescence staining further confirmed miR-NC ^KD EVs increased the levels of LC3II and Beclin1 (FIG. 6E). Autophagy is often referred to as a measure of autophagy degradation. Once the autophagosome was fused to the lysosome, GFP signal was quenched by the acidic environment and only RFP signal was observed. The results of this example show that LPS significantly reduces yellow and red spots, while miR-NC ^KD EVs promote autophagy flow (FIG. 6F-H). However, inhibition of miR-672-5p in CEC-EVs disrupts the autophagy-enhancing process, suggesting that miR-672-5p plays an important role in the NF-. Kappa.B pathway and autophagy signaling. These findings all demonstrate that CEC-EVs may confer immunomodulation and neuroprotection through miR-672-5p-TAB2 signaling. Autophagy is reported to be closely related to inflammation. This example then investigated whether CEC-EVs regulate the autophagy process of microglia, involving activation of NLRP3 inflammatory bodies. Fluorescence staining results show that miR-NC ^KD EVs promoted expression of LC3 II and reduced expression of NLRP3 inflammatory corpuscles (FIG. 6I), indicating miR-NC ^KD EVs enhance autophagy, which in turn inhibits activation of NLRP3 inflammatory corpuscles (FIGS. 13A and 13B). Also, ELISA experiments further demonstrated miR-NC ^KD EVs significantly improved the expression of TNF- α, IL-1β, IL-6 and IL-10 (FIGS. 13C-F).

To further understand the relationship between CEC-EVs-derived miR-672-5p and autophagy in vivo, the present example examined the expression of autophagy-related proteins in vivo. CEC-EVs treatment improved down-regulation of autophagy markers by LPS exposure and was shown to occur at miR-672-5p ^KD No such phenomenon was observed in the EVs treated group (fig. 7A-D). This example further demonstrates the role of CEC-EVs in autophagy by using the autophagy inhibitor CQ. As shown in FIG. 7E, blocking autophagosome-lysosomal fusion using CQ induced an increase in LC3-II protein. Interestingly, miR-NC ^KD EVs treatment significantly improved LPS-induced reduction of LC3-II expression, CQ significantly increased LC3-II accumulation (FIGS. 7E and 7F). Together, these results indicate that LPS-induced autophagy dysfunction is mediated by miR-NC ^KD -EVs treatment is alleviated. Subsequently, this example further investigated NLRP3 inflammatory bodies. Protein expression of NLRP3, ASC, clear-caspase-1 and mature IL-1β was consistent with the in vitro experiments of this example (FIGS. 14A and 14B). Next, this example discusses miR-NC ^KD -immunomodulatory function of EVs in vivo. Accordingly, the study of this example shows that miR-NC ^KD EVs reduced LPS-induced neuroinflammation (FIGS. 7G and 7H), transitioning microglial cells from the M1 phenotype to the M2 phenotype (FIGS. 14C and 14D).

CEC-EVs to ameliorate LPS-induced behavioral deficits and neuroinflammation in mice

To demonstrate the neuroprotective effect of CEC-EVs in vivo, CEC-EVs were administered after repeated LPS stimulation. This example shows that CEC-EVs treatment significantly reduced escape latency compared to LPS-treated groups, and these results indicate that CEC-EVs treatment reduced LPS-induced cognitive decline. . CEC-EVs treatment also improved the performance of LPS-treated mice in spatial exploration, with greater numbers of platform crossings and longer time spent in target quadrants compared to LPS groups (fig. 8A-D). Furthermore, CEC-EVs improve LPS-induced anxiety-like and depression-like states. In the EPM test, LPS exposure reduced the time spent in and the number of open arm entries, which was restored by CEC-EVs administration (fig. 8E-G). Likewise, CEC-EVs also increase sucrose preference in SPT (fig. 8H) and decrease dead time in FST (fig. 8I). Meanwhile, CEC-EVs treatment alleviated LPS-induced neuronal apoptosis and neuronal loss, respectively, and inhibition of miR-672-5p expression attenuated neuroprotection of CEC-EVs in Nissl staining and Tunel staining (FIGS. 15A-D).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of extracellular vesicles derived from brain endothelial cells in preparing antidepressant or anxiety drugs is characterized in that the extracellular vesicles derived from brain endothelial cells have a diameter ranging from 50 to 150nm, have double-layer phospholipid membranes and are in a spherical form; the extraction mode of the outer vesicle is as follows: cutting up a cerebral cortex part, adding 0.05-0.15% of type II collagenase for digestion, then adding a gradient separation solution for separation to obtain endothelial cells, adding Dulbecco's modified Eagle culture medium into the cells, supplementing fetal calf serum, penicillin/streptomycin and endothelial cell growth additives for culture, continuously culturing the cells after 75-85% of culture medium is fully paved, replacing the culture medium with DMEM (medium supplemented with exosome fetal calf serum), and obtaining the extracellular vesicles by centrifugation of a culture supernatant;

the digestion temperature of the type II collagenase is 36-38 ℃ and the digestion time is 25-35 min;

The gradient separation liquid is Percoll, and the feasible concentration is 45-55%;

in the Dulbecco modified Eagle culture medium, the concentration of fetal bovine serum is 18-22%, the concentration of penicillin/streptomycin is 0.8-1.2%, and the concentration of endothelial cell growth additive is 0.8-1.2%;

the culture supernatant was centrifuged as follows: centrifuging for 13-17 minutes at 8000-12000 g to remove any possible dead cells and large cell debris; after centrifugation, the supernatant was collected and passed through a 0.22 μm sterile filter; transferring the obtained supernatant to a high-speed centrifuge tube, ultracentrifugating for 88-92 minutes at 80000-100,000 g, and adding PBS to resuspend the precipitate.