M2-type microglial cell exosome and preparation method and application thereof

Abstract

This invention discloses an M2 type microglia exosome, its preparation method, and its application, belonging to the field of biotechnology, comprising: preparing microglia at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / mL. After cell adhesion, they were treated with IL-4 to induce polarization to the M2 phenotype. The culture medium was collected, and M2-type microglia exosomes (M2-EXO) were obtained by ultracentrifugation. Studies revealed that M2-EXO, through miR-1949-mediated microglia phenotype transformation, inhibited neuroinflammation and improved sepsis-related encephalopathy in mice. Further research showed that M2-EXO, through miR-127-5p, inhibited neuroinflammation and improved MK-801-induced schizophrenia in mice. Therefore, this invention also discloses the use of M2-EXO and its miRNAs in the treatment of sepsis-related encephalopathy and schizophrenia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to exosomes derived from M2 microglia, their preparation methods, and applications. Background Technology

[0002] Sepsis is a life-threatening systemic infectious disease caused by a dysregulation of the body's response to infection. Sepsis-associated encephalopathy (SAE) is a severe brain dysfunction caused by sepsis, characterized by high morbidity and mortality. Currently, there is a lack of targeted treatments for SAE, and molecular mechanism-based therapies remain urgently needed. When sepsis occurs, peripheral inflammatory factors can enter the central nervous system through multiple pathways, causing neuroinflammation and subsequently leading to brain damage. This suggests that neuroinflammation plays a crucial role in the pathogenesis of SAE. Schizophrenia is a major mental illness with a global prevalence of approximately 1%. Patients have a life expectancy that is 10-15 years shorter than the general population and have a higher suicide rate. Studies have shown a direct and close relationship between schizophrenia and neuroinflammation.

[0003] Neuroinflammation is often accompanied by abnormal activation of microglia. Microglia are the main immune cells of the central nervous system (CNS). When the CNS is damaged or an inflammatory response occurs, microglia transition from a resting state to an activated state. Activated microglia can be classified into two polarized phenotypes based on surface markers and functional characteristics: pro-inflammatory (M1) and anti-inflammatory (M2). Regulating excessive microglia activation or promoting their phenotypic transformation may be an effective strategy for improving neuroinflammation in schizophrenia (SAE), but the dynamic changes in microglia during the course of SAE remain unclear, and the molecular mechanisms of phenotypic transformation need further elucidation. Furthermore, given the potential role of neuroinflammation in the pathogenesis of schizophrenia, improving the intracranial inflammatory environment is likely to be an effective treatment for schizophrenia.

[0004] Exosomes are extracellular vesicles secreted by various cells, possessing a phospholipid bilayer structure and a diameter of 30-150 nm. They contain bioactive molecules such as miRNAs, messenger RNAs (mRNAs), proteins, and lipids. They interact with target cells through paracrine or humoral pathways, playing crucial roles in intercellular communication, biomolecular transport, gene expression regulation, and the development of various diseases. miRNAs are known to be a class of non-coding small RNAs, 18-24 bases in length, which regulate target gene expression by specifically binding to the 3'UTR of the target gene, inhibiting translation or degrading mRNA. Currently, the effects of M2 microglia exosomes (M2-EXO) and their miRNAs on SAE neuroinflammation are unknown, and their role in schizophrenia remains unclear. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides an M2-type microglia exosome (M2-EXO), its preparation method, and its application in treating neurological diseases (such as SAE and schizophrenia). Through in-depth research, the inventors discovered that M2-EXO can at least improve sepsis-related encephalopathy in mice by inhibiting neuroinflammation through miR-1949-mediated microglia phenotypic transformation. Furthermore, the research found that M2-EXO can at least improve MK-801-induced schizophrenia in mice by inhibiting neuroinflammation through miR-127-5p.

[0006] The specific solution of the present invention is as follows:

[0007] A method for preparing M2 type microglial cell exosomes includes the following steps:

[0008] Microglia were divided into 1×10 5 The cells were seeded at a density of cells / mL. After the cells adhered, they were treated with IL-4 to induce them to polarize to the M2 phenotype. The culture medium was collected and M2-EXO was obtained by ultracentrifugation.

[0009] M2-EXO was traced and labeled using the PKH26 fluorescent dye method, and it was found that M2-EXO could be taken up by M1 microglia.

[0010] Analysis of the miRNA expression profile in M2-EXO using Exosome UID miRNA-seq technology revealed a variety of differentially expressed miRNAs, among which four were highly expressed: miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p, with miR-1949 showing the most significant differential expression.

[0011] Further GO and KEGG enrichment analyses showed that miR-1949 target genes are closely related to the occurrence of neurological diseases and are involved in the regulation of the Wnt signaling pathway.

[0012] In a mouse model of SAE, M2-EXO and miR-1949 significantly improved behavioral and cognitive impairments, reduced cortical microglia activation, and regulated the expression of inflammatory factors such as IL-10, IL-6, iNOS, and TNF-α. This invention also verified the targeted binding relationship between miR-1949 and the 3'UTR of DKK1 using a dual-luciferase reporter gene system, indicating that DKK1 may be a target of miR-1949. Based on these findings, this invention provides the application of M2-EXO and miR-1949 in regulating neuroinflammatory diseases and in the treatment of SAE.

[0013] This invention also provides a method for inducing the transformation of M1 microglia to the M2 phenotype using M2-EXO. Through the action of M2-EXO, the polarization state of microglia can be regulated, thereby alleviating neuroinflammation.

[0014] A modified calcium chloride method was used to load exosomes with miR-127-5p or miR-1949 mimics or inhibitors. This method can enhance exosome function and provide new strategies for the treatment of specific diseases.

[0015] This invention tested the effects of M2-EXO and miR-127-5p on positive symptoms, anxiety-like behaviors, and cognitive impairment symptoms of schizophrenia in mice, as well as their effects on neuronal damage, microglia morphology, and inflammatory factor levels in the cerebral cortex of schizophrenic mice. The results indicate that M2-EXO and miR-127-5p have potential roles in improving schizophrenia-related symptoms and pathological mechanisms. Therefore, this invention also provides the application of M2-EXO and miR-127-5p in the preparation of drugs for treating schizophrenia.

[0016] This invention also tested the effects of M2-EXO and miR-127-5p on cell survival in neuronal inflammatory damage, as well as their effects on neuronal inflammation, microglial inflammation, and phenotype. The results showed that M2-EXO and miR-127-5p could alleviate neuronal inflammatory damage, reduce inflammatory cytokine levels, inhibit the inflammatory response of microglia, and promote the conversion of microglia to an anti-inflammatory (M2) phenotype. These findings indicate that M2-EXO and miR-127-5p play an important role in the regulation of neuroinflammation and neuroprotection, providing potential interventional targets for the treatment of neuroinflammation-related diseases. Therefore, this invention also provides the application of M2-EXO and miR-127-5p in the preparation of drugs for alleviating neuroinflammation, neuronal damage, and providing neuroprotection.

[0017] The application of M2-EXO in neurological diseases relies not only on the regulatory role of miRNAs, but also on the synergistic participation of various bioactive molecules such as proteins, lipids, mRNAs, and circRNAs.

[0018] The beneficial effects of this invention are as follows: In this invention, M2-EXO, through miR-1949-mediated microglial phenotypic transformation, inhibits neuroinflammation and improves sepsis-related encephalopathy in mice. Cellular experiments have confirmed that M2-EXO can induce the transformation of M1-type microglia into the M2 phenotype, and that miR-1949 in M2-EXO mediates the M1 / M2 phenotypic transformation by inhibiting DKK1 activation of the Wnt / β-catenin pathway. Animal experiments have confirmed that M2-EXO, at least through its miR-1949, can mediate M1 / M2 phenotypic transformation and improve neuroinflammation after SAE in mice by inhibiting DKK1 activation of the Wnt / β-catenin pathway. Furthermore, M2-EXO can at least improve MK-801-induced schizophrenia in mice by inhibiting neuroinflammation through miR-127-5p. This invention has demonstrated through cell experiments that miR-127-5p in M2-EXO plays a crucial role in the transformation of M1 microglia to the M2 phenotype; and that miR-127-5p in M2-EXO plays a significant role in the levels of inflammatory factors in M1 microglia and HT22 neurons. Animal experiments further demonstrate that M2-EXO can improve schizophrenia in mice, and that M2-EXO exerts its important role at least through miR-127-5p. Attached Figure Description

[0019] Figure 1Effects of M2-CM on iNOS and CD206 protein levels in M1 microglia (immunofluorescence staining; Scalebar = 50 μm). A represents iNOS protein level, B represents CD206 protein level, and C and D represent quantitative analyses of the relative fluorescence intensity of iNOS and CD206, respectively. *** p < 0.001 ### p < 0.001.

[0020] Figure 2 Extraction and identification of M2-EXO. A is the extraction flowchart, B is the transmission electron microscope image (scale bar = 500 nm), C is the particle size distribution map, and D is the identification of protein markers.

[0021] Figure 3 : Verify that M2-EXO is taken up by M1 type microglia.

[0022] Figure 4 Effects of M2-EXO on iNOS and CD206 protein levels in M1 microglia (immunofluorescence staining; scale bar = 50 μm). A and C represent iNOS and CD206 protein levels, respectively; B and D represent quantitative analysis of relative fluorescence intensity. *** p < 0.001 ### p < 0.001.

[0023] Figure 5 : Exosome UID miRNA-seq technology was used to analyze the differential expression of miRNAs in M2-EXO. A is a heatmap of miRNA expression, and B is a volcano plot of differential expression.

[0024] Figure 6 : Verify the expression levels of miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p in M2-EXO; * p < 0.05 ** p < 0.01, *** p < 0.001.

[0025] Figure 7 GO and KEGG enrichment analysis of miR-1949 target genes.

[0026] Figure 8 Validation of miR-1949 mimic and inhibitor via a modified calcium chloride method mediated by RT-qPCR. A represents the validation of miR-1949 mimic, and B represents the validation of miR-1949 inhibitor. *** p < 0.001 ### p < 0.001.

[0027] Figure 9 Effects of miR-1949 mimic and inhibitor on iNOS and CD206 protein levels in M1 microglia (immunofluorescence staining; scale bar = 50 μm). AH represents the quantitative analysis of CD206 and iNOS protein levels and their relative fluorescence intensity, respectively. ** p < 0.01, *** p < 0.001 ### p < 0.001.

[0028] Figure 10 : Validate miR-1949 targeting DKK1. A is a schematic diagram of the effect of DKK1 on the Wnt / β-catenin signaling pathway, B is the binding site and mutation site of DKK1 and miR-1949, and C is the effect of miR-1949 mimic on the relative luciferase activity of DKK1-WT; *** p < 0.001, no significant difference in ns.

[0029] Figure 11 Effects of miR-1949 mimic and inhibitor on the mRNA levels of DKK1, Wnt3A, and β-catenin in M1 microglia (RT-qPCR); * p < 0.05 ** p < 0.01, # p < 0.05 ## p < 0.01.

[0030] Figure 12-13 Effects of M2-EXO and miR-1949 on MSS and cortex in mice after SAE. A is the experimental treatment flowchart, B is the effect on MSS, and C is the effect on cortical brain tissue (HE staining; scale bar = 50 μm). *** p < 0.001 ### p < 0.001 $$$ p < 0.001.

[0031] Figure 14-15 Effects of M2-EXO and miR-1949 on the morphology of microglia after SAE in mice (immunofluorescence). A represents microglia morphology, B represents complexity, and C represents changes in total branch length, total number of branches, and number of terminal branches. *** p < 0.001 # p < 0.05 ## p < 0.01, ### p < 0.001 $$ p < 0.01, $$$p < 0.001, no significant difference in ns.

[0032] Figure 16-17 Effects of M2-EXO and miR-1949 on mRNA levels of inflammatory cytokines (Arg1, CD206, IL-10, IL-6, iNOS, TNF-α) in the cortical epithelium after SAE in mice (RT-qPCR); ** p < 0.01, *** p < 0.001 # p < 0.05 ## p < 0.01, ### p < 0.001 $ p < 0.05 $$ p < 0.01, $$$ p < 0.001, no significant difference in ns.

[0033] Figure 18-19 Effects of M2-EXO and miR-1949 on the levels of DKK1, Wnt3A, and β-catenin mRNA in the posterior cortex of mice after SAE (RT-qPCR); *** p < 0.001 # p < 0.05 ## p < 0.01, ### p < 0.001 $ p < 0.05 $$ p < 0.01, $$$ p < 0.001, no significant difference in ns.

[0034] Figure 20 Expression level of miR-127-5p in exosomes (RT-qPCR); *** p < 0.001.

[0035] Figure 21-23 M2-EXO and miR-127-5p showed positive symptoms in mice with schizophrenia. Figure 21 A is the total distance traveled. Figure 21 B represents movement speed), anxiety-like behavior ( Figure 22 A represents the trajectory of motion. Figure 22 B represents the quantitative analysis of movement trajectories and the impact on cognitive impairment symptoms. Figure 23 ); * p < 0.05 ** p < 0.01, *** p < 0.001, no significant difference in ns.

[0036] Figure 24-25Effects of M2-EXO and miR-127-5p on neuronal damage in schizophrenic mice (immunofluorescence; scale bar = 50 μm). A represents immunofluorescence staining, and B represents relative fluorescence intensity. * p < 0.05 ** p < 0.01, *** p < 0.001, no significant difference in ns.

[0037] Figure 26-27 Effects of M2-EXO and miR-127-5p on the morphology of microglia in schizophrenic mice (immunofluorescence). A represents microglia morphology, B represents microglia complexity, and C represents the total length of microglia branches, the total number of branches, and the number of terminal branches. *** p < 0.001.

[0038] Figures 28-29 Effects of M2-EXO and miR-127-5p on neuroinflammation in schizophrenic mice (RT-qPCR and Western blot). Group A shows the effect on the mRNA levels of neuroinflammatory factors, Group B shows the effect on the protein levels of TNF-α, β-actin, and IL-1β, and Group C shows the quantitative analysis. * p < 0.05 ** p < 0.01, *** p < 0.001, no significant difference in ns.

[0039] Figure 30 Effects of M2-EXO and miR-127-5p on the survival of neuronal cells damaged by inflammation; ** p < 0.01, *** p < 0.001, no significant difference in ns.

[0040] Figure 31 Effects of miR-127-5p on the mRNA levels of neuronal inflammatory factors (TNF-α, IL-6, IL-1β) (RT-qPCR); * p < 0.05 *** p < 0.001.

[0041] Figure 32 Effects of miR-127-5p on inflammation levels and phenotypic transformation in M1 microglia (RT-qPCR); ** p < 0.01, *** p < 0.001. Detailed Implementation

[0042] Example 1: Effect of M2-type microglia (M2-CM) on the phenotypic transformation of M1-type microglia

[0043] (1) Culture of N9 microglia

[0044] The complete culture medium was based on IMDM (Gibco, USA), supplemented with 5% fetal bovine serum (FBS, USA) and 1% penicillin antibiotics (Gibco, USA). N9 microglia (a gift from Dr. Jimin Wang of the National Cancer Institute (NCI)) were placed in the above culture medium and cultured in a 37°C, 5% CO2 incubator (HealForce, China). When the cell confluence reached 80%-90%, the cells were digested with 0.05% trypsin (Gibco, USA) and passaged at a 1:4 ratio.

[0045] (2) Phenotypic induction of N9 microglia

[0046] Lipopolysaccharide (LPS) (Sigma, USA) was prepared into a 1 mg / mL stock solution using IMDM basal medium and stored at -20°C protected from light. Interleukin-4 (IL-4) (Peprotech, USA) was prepared into a 10 μg / mL stock solution using trehalose-containing phosphate-buffered saline (DAKEWE, China) and stored at -20°C protected from light. During experimental procedures, the LPS and IL-4 stock solutions should be diluted to the required working concentrations using complete culture medium. Treating N9 microglia with 1 μg / mL LPS for 24 h induced M1 microglia, and treating N9 microglia with 20 ng / mL IL-4 for 24 h induced M2 microglia (M2-CM).

[0047] (3) Preparation of conditioned culture medium for M2 type microglia

[0048] N9 microglia were used at a rate of 1×10 5 Microglia were seeded at a density of cells / mL in culture dishes. After cell attachment, they were treated with IL-4 (20 ng / mL) for 24 h to induce M2 polarization. M2-CM cells were collected, centrifuged at 1300 rpm for 5 min to remove cell debris, and used immediately.

[0049] (4) Cell viability detection

[0050] Cell viability was determined using the methyl thiazolyl tetrazolium (MTT, Sigma, USA) method. First, the cells were incubated with 5 mg / mL MTT solution at 37°C for 4 h. Then, the culture supernatant was removed, and an equal volume of dimethyl sulfoxide (DMSO, Sigma, USA) was added to dissolve the formed formazan crystals. Finally, the absorbance of each well was measured at 492 nm using a microplate reader (Thermo Fisher Scientific, USA).

[0051] (5) Immunofluorescence

[0052] For cell samples, fixation was first performed with 4% paraformaldehyde (PFA, Macklin, China) at room temperature for 15 min. Then, permeabilization with 0.1% saponin (Amresco, USA) was performed for 10 min, followed by blocking with 3% BSA at room temperature for 30 min. After discarding the blocking solution, primary antibodies such as Anti-iNOS antibody (1:500, Abcam, UK) and Anti-CD206 antibody (1:500, Abcam, UK) were added and incubated overnight at 4°C. The next day, equilibration was performed at room temperature for 30 min. Secondary antibodies such as Alexa Fluor 555-labeled donkey anti-rabbit IgG (1:1000, Abcam, UK) were then added and incubated in the dark for 60 min, followed by DAPI (1:500, Sigma, USA) staining solution and incubation in the dark for 30 min. Finally, images were acquired and analyzed using a laser confocal microscope (Nikon, Japan).

[0053] For tissue samples, mice were perfused with PBS (pH 7.4), followed by perfusion with 4% PFA fixative. The brain was then post-fixed in 4% PFA, and 40 μm brain slices were prepared using a vibratory microtome (Leica, Germany). The slices were blocked for 1 h at room temperature in 5% porcine serum containing 0.3% Triton X-100 (Amresco, USA). After incubation overnight at 4°C with primary antibodies such as Anti-iNOS antibody (1:150, HUABIO, China), Anti-CD206 antibody (1:500, Abcam, UK), and Anti-IBA1 antibody (1:500, Millipore, USA), the samples were warmed to room temperature, washed with PBST, and incubated for 1 h with secondary antibodies such as Alexa Fluor 555-labeled donkey anti-rabbit IgG (1:1500, Abcam, UK) and Alexa Fluor 647-labeled donkey anti-mouse IgG (1:1000, Abcam, UK) under light-protected conditions. Nuclear staining with DAPI (1:500, Sigma, USA) for 30 min, followed by confocal microscopy to observe and acquire images.

[0054] The protein levels of CD206 and iNOS were examined using immunofluorescence assays. The results are as follows: Figure 1 As shown, compared with the M0 microglia conditioned medium (M0-CM) group, M2-CM treatment of M1 microglia for 24 h significantly increased CD206 expression and significantly decreased iNOS expression. Notably, the addition of the neutral sphingomyelinase inhibitor GW4869 (10 μM) to inhibit exosome secretion during M2-CM preparation significantly reversed the aforementioned effects of M2-CM on CD206 and iNOS. These results indicate that M2-CM can induce M1 microglia to transform into the M2 phenotype, and exosomes play an important role in this process.

[0055] Example 2: Preparation and Characterization of Exosomes

[0056] (1) Preparation of exosomes

[0057] Exosomes were extracted using an ultracentrifuge (Hitachi, Japan). Figure 2As shown in Figure A, firstly, the suspended cells in the culture medium (the M2 type microglia conditioned culture medium obtained in the preparation of M2 type microglia conditioned culture medium in Example 1(3)) were removed by centrifugation at 300g for 10 min at 4℃. Then, dead cells were removed by centrifugation at 2000g for 10 min, and cell debris was removed by centrifugation at 10000g for 30 min. Next, the exosomes were obtained by ultracentrifugation at 100000g for 70 min at 4℃, and the supernatant was discarded. Finally, the exosomes were resuspended in sterile PBS and washed again by centrifugation at 100000g for 70 min. After discarding the supernatant, the exosomes were dissolved in PBS and stored at -80℃ for later use.

[0058] (2) Preparation and characterization of exosomes, and immunoblotting experiments

[0059] Transmission electron microscopy (TEM, Hitachi, Japan) is used to identify the morphology of exosomes. Figure 2 B). Nanoparticle tracking analysis (Omec, China) is used to measure the particle size of exosomes. Figure 2 C). The protein content of exosomes was determined using the BCA (Beyotime Biotechnology, China) protein assay. The expression of exosome markers (CD63, CD9, TSG101) was detected by Western blot. Figure 2 D).

[0060] For cell samples, after washing with PBS, press 1×10 6Cells were treated with 50 μL of RIPA lysis buffer (Beyotime Biotechnology, China) containing a protease inhibitor (Solarbio, China); tissue samples were lysed at a ratio of 10 mg tissue to 100 μL of lysis buffer. Samples were sonicated (Scientz, China), incubated on ice for 30 min, and then centrifuged to collect the supernatant. Protein concentration was determined using the BCA method and diluted with loading buffer. After denaturation at 100°C for 10 min, the samples were stored at -80°C for later use. For Western blotting analysis, SDS-polyacrylamide gel electrophoresis was performed first, followed by wet transfer of proteins. After transfer, the membrane was blocked at room temperature for 60 min with blocking buffer, and then incubated at room temperature for 30 min with specific primary antibodies such as Anti-CD9 antibody (1:2000, Abclonal, China), Anti-CD63 antibody (1:1000, Abclonal, China), Anti-TSG101 antibody (1:1000, Abclonal, China), Anti-β-actin antibody (1:1000, Elabscience, China), Anti-β-Catenin antibody (1:1000, Cell Signaling, USA), and Anti-DKK1 antibody (1:2000, Proteintech, China) for 4°C overnight. After rinsing with TBST, it was incubated at room temperature for 60 min with horseradish enzyme-labeled goat anti-rabbit IgG (1:5000, ZSGB-BIO, China) for 50 min. After thorough rinsing, TBST was developed using ECL luminescent substrate (NCM, China), and finally analyzed using a gel imaging system (Bio-Rad, USA).

[0061] The results are as follows Figure 2 As shown in Figure B-2D, TEM revealed that the exosomes exhibited a "round cup-like" or "cup saucer" structure. Nanoparticle size analysis showed that the exosome particle size distribution ranged from 30 to 150 nm. Western blotting detected the expression of exosome markers CD9, CD63, and TSG101. These results indicate that the M2-EXO exosomes from N9 microglia were successfully extracted.

[0062] Example 3: Verification of M2-EXO uptake by M1 type microglia

[0063] M2-EXO was labeled using the PKH26 fluorescent dye (Sigma, USA). In short, M2-EXO was resuspended in PKH26 fluorescent dye staining solution and incubated at room temperature in the dark for 10 min. The labeled exosomes were then collected by ultracentrifugation at 100,000 g for 70 min at 4 °C. After washing with PBS, the precipitate was purified by centrifugation again. Finally, the labeled exosomes were resuspended in an appropriate amount of PBS and stored at 4 °C. Cellular uptake of exosomes was observed and analyzed using laser confocal microscopy. The results are as follows: Figure 3 As shown, red fluorescence represents PKH26-labeled exosomes, and blue fluorescence represents the nuclear dye DAPI. PKH26-labeled M2-EXO (5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 24 h) was localized around the nucleus of M1 microglia. These results indicate that M2-EXO can be taken up by M1 microglia.

[0064] Example 4: Effects of M2-EXO on iNOS and CD206 expression in M1 microglia

[0065] The expression levels of CD206 and iNOS were examined using immunofluorescence assays. The results are as follows: Figure 4 As shown, red fluorescence represents iNOS protein, green fluorescence represents CD206 protein, and blue fluorescence represents the nuclear dye DAPI. Compared with the CON group (blank control group), LPS (1 μg / mL) treatment of microglia for 24 h significantly increased the expression level of iNOS and significantly decreased the expression level of CD206. Compared with the LPS group, M2-EXO (20 μg / mL) treatment of M1 type microglia for 24 h significantly decreased the expression level of iNOS and significantly increased the expression level of CD206. These results indicate that M2-EXO can induce M1 type microglia to transform into the M2 phenotype.

[0066] Example 5: Analysis of differential expression of miRNAs in M2-EXO using Exosome UID miRNA-seq technology

[0067] This study used Exosome UID miRNA-seq technology (Wuhan Kangce Technology Co., Ltd.) to investigate differentially expressed miRNAs in M2-EXO. First, the total RNA samples underwent quality testing. After passing the quality testing, libraries were constructed. Then, the effective concentration of the libraries was accurately quantified using RT-qPCR (effective concentration > 2 nM) to ensure library quality. After passing library testing, different libraries were sequenced and analyzed according to their effective concentrations.

[0068] The results are as follows Figure 5As shown, M2-EXO contains a variety of differentially expressed miRNAs, including four highly expressed differentially expressed miRNAs: miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p.

[0069] Example 6: Expression verification of miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p in M2-EXO

[0070] Total RNA was extracted from cell and tissue samples using RNAiso Plus (Takara, Japan). PrimeScript was used. TM Reverse transcription was performed using the RT reagent kit (Takara, Japan) and the Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The transcription was then processed via TB. Premix Ex Taq TM mRNA quantification was performed using a transaminamide (Takara, Japan) kit. Primer sequences used in the amplification experiments are shown in Tables 1 and 2. The amplification program consisted of initial denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing extension at 60°C for 45 seconds.

[0071] Table 1 Primer sequences

[0072]

[0073] Table 2 Primer sequences

[0074]

[0075]

[0076] The mRNA levels of miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p in M2-EXO were examined using RT-qPCR. The results are as follows: Figure 6 As shown, compared with M0-EXO, the expression of miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p in M2-EXO were significantly increased, by 3791.42%, 85.85%, 143.08%, and 115.66%, respectively, with the most significant difference in miR-1949 expression. This suggests that miR-1949 may play a crucial role in the biological functions mediated by M2-EXO.

[0077] Example 7: Enrichment analysis of miR-1949 target genes GO and KEGG in M2-EXO

[0078] Downstream target genes of mmu-miR-1949 were predicted using the online websites miRTarBase, miRDB, and miranda. The obtained mouse-derived genes were homologously converted to human genes using the homologene package based on the NCBI HomoloGene database and R software (v4.3.3) to obtain the corresponding human target genes. Gene Ontology (GO) enrichment analysis was performed on the human-mouse homologous conversion human target genes using the DAVID online tool, including biological process (BP), cellular component (CC), and molecular function (MF). Simultaneously, signaling pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). KEGG database analysis was used to identify significantly enriched signaling pathways.

[0079] The results are as follows Figure 7 As shown, GO enrichment analysis revealed that in the BP module, target genes were mainly enriched in items such as "negative regulation of canonical Wnt signaling pathway," "cell morphogenesis," "negative regulation of neuron apoptotic process," and "neuron migration." In the CC module, target genes were mainly enriched in items such as "neuron projection" and "postsynaptic density." In the MF module, target genes were mainly enriched in items such as "protein binding," "ubiquitin-protein transferase activity," "transcription coactivator activity," and "protein kinase activity." KEGG enrichment analysis showed that the most significantly enriched target genes were those in the "Cytokine-cytokine receptor interaction" and "Wnt signaling pathway." These results suggest that miR-1949 target genes are closely related to the occurrence of neurological diseases and are associated with the regulation of the Wnt pathway.

[0080] Example 8: Validation of miR-1949 mimic and miR-1949 inhibitor loaded onto exosomes via a modified calcium chloride method

[0081] miR-1949 mimic (Ribobio, China) or miR-1949 inhibitor (Ribobio, China) were loaded into M2-EXO using a modified calcium chloride method. First, 0.2 nmol miR-1949 mimic or 0.2 nmol miR-1949 inhibitor and 20 μg M2-EXO were dissolved in 100 μL of sterile PBS and stored on ice. After preparing 100 μL of 0.3 M CaCl2 solution, the exosome suspension was gently mixed with the miRNA working solution, and pre-chilled CaCl2 solution was added to a final concentration of 0.1 M. The mixture was then incubated on ice for 30 min to promote complex formation. Subsequently, the mixture was heated in a 42°C metal bath (MIULAB, China) for 1 min, followed immediately by an ice bath for 5 min. 5 μg / mL RNase A (Thermo Fisher, USA) was added, and the mixture was incubated at 37°C for 30 min to remove free nucleic acids. Finally, the exosomes were collected by ultracentrifugation at 100,000g and 4℃ for 70 min, sterilized by filtration through a 0.22μm filter membrane, and stored at -80℃.

[0082] The levels of miR-1949 in exosomes after loading miR-1949 mimic and miR-1949 inhibitor using the calcium chloride method were investigated using RT-qPCR. The results are as follows: Figure 8 As shown, compared with the mimic NC group, the expression of miR-1949 in exosomes was significantly increased after loading miR-1949mimic; compared with the inhibitor NC group, the expression of miR-1949 in exosomes was significantly decreased after loading miR-1949inhibitor. This indicates that exosomes were successfully loaded with miR-1949mimic and miR-1949inhibitor.

[0083] Example 9: Effects of miR-1949 mimic and miR-1949 inhibitor on iNOS and CD206 expression in M1 microglia

[0084] The protein levels of CD206 and iNOS were examined using immunofluorescence assays. The results are as follows: Figure 9As shown, red fluorescence represents iNOS protein, green fluorescence represents CD206 protein, and blue fluorescence represents the nuclear dye DAPI. Compared with the mimic NC group, treatment of M1 microglia with miR-1949mimic (0.2 nmol / mL) for 24 h significantly increased CD206 expression and significantly decreased iNOS expression. Compared with the inhibitor NC group, treatment of M1 microglia with miR-1949inhibitor (0.2 nmol / mL) for 24 h significantly decreased CD206 expression and significantly increased iNOS expression. This suggests that miR-1949mimic can induce the transformation of M1 microglia into the M2 phenotype, while miR-1949inhibitor treatment enhances the expression of M1 polarization.

[0085] Example 10: Validation of miR-1949 targeting DKK1

[0086] 293T cell culture: The complete culture medium was based on DMEM (Gibco, USA), supplemented with 10% FBS and 1% penicillin antibiotics. 293T cells were placed in the above culture medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin (Gibco, USA) and then passaged.

[0087] Dual-luciferase reporter gene assay: The targeting relationship between miR-1949 and DKK1 3'UTR was verified using a dual-luciferase reporter gene system (Promega, USA). Wild-type and mutant plasmids of DKK1 3'UTR (GenePharma, China) were co-transfected with miR-1949 mimic or mimic NC (Ribobio, China) for 48 h after transfection using the Promega dual-luciferase reporter assay kit. All assays were performed under light-protected conditions to ensure data accuracy.

[0088] The dual-luciferase reporter system was used to investigate whether DKK1 is a target of miR-1949. For example... Figure 10 As shown in Figure A, DKK1 blocks the binding of the Wnt protein to the receptor complex (Frizzled-LRP5 / 6) by binding to the Wnt co-receptor LRP5 / 6 with high affinity, thereby inhibiting classical Wnt signaling. Therefore, wild-type and mutant plasmids of DKK1 were constructed. Figure 10 Figure B shows the binding site and mutation site of DKK1 with miR-1949. (The rest of the text appears to be unrelated and likely refers to a different topic.) Figure 10As shown in Figure C, compared with the mimic NC group, miR-1949mimic (100 nM, 48 h) significantly reduced the relative luciferase activity of DKK1-WT, but had no significant effect on the relative luciferase activity of DKK1-MUT. This suggests that miR-1949 can bind to the 3'UTR of DKK1, and DKK1 may be a target of miR-1949.

[0089] Example 11: Effects of miR-1949mimic and miR-1949inhibitor on the expression of DKK1, Wnt, and β-catenin in M1 microglia

[0090] The expression levels of DKK1, Wnt, and β-catenin in M1 microglia after 24 h of treatment with miR-1949mimic and miR-1949inhibitor were investigated using RT-qPCR. Results are as follows: Figure 11 As shown, compared with the mimic NC group, miR-1949mimic (0.2 nmol / mL) treatment of M1 microglia for 24 h significantly reduced the expression level of DKK1 and significantly increased the expression levels of Wnt and β-catenin; compared with the inhibitor NC group, miR-1949inhibitor (0.2 nmol / mL) treatment of M1 microglia for 24 h significantly increased the expression level of DKK1 and significantly reduced the expression levels of Wnt and β-catenin.

[0091] Example 12: Effects of M2-EXO on MSS and Cortex in Mice After SAE

[0092] (1) Animal experimental design

[0093] C57BL / 6 mice, weighing 18g-22g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Production License No. SCXK(Liaoning)2020-0001). All animal experiments were approved by the Experimental Animal Ethics Committee of Shenyang Pharmaceutical University (Registration No.: SYPU-IACUC-S2024-0930-103). Before stereotactic brain surgery, mice were anesthetized with 4% isoflurane (RWD, China), and the anesthesia concentration was maintained at 1.5% during the operation. The mice were then placed on a stereotactic frame for surgery (RWD, China). Exosome suspension (10μg / mouse) was injected into the lateral ventricle using a PBS-prewashed microsyringe. An SAE mouse model was established using intraperitoneal injection of LPS. LPS was dissolved in sterile PBS to prepare a 10mg / kg solution. After weighing the mice, the calculated dose was precisely injected intraperitoneally. 24 hours after injection, the mouse sepsis score (MSS) and sample collection were performed for subsequent experimental procedures.

[0094] (2) Mouse sepsis score

[0095] As shown in Table 3, MSS scored mice post-surgery based on seven aspects: appearance, level of consciousness, activity, response to stimuli, eyes, respiratory rate, and respiratory quality. The scores were given once a day from before modeling to seven days after modeling (starting from zero and increasing daily after modeling).

[0096] Table 3 Sepsis scores in mice

[0097]

[0098]

[0099] (3) Hematoxylin-eosin (HE) staining

[0100] Mouse brain tissue was fixed with PFA, dehydrated, and embedded in paraffin. The paraffin block was cut into 5 μm sections. These sections were then dewaxed and rehydrated. Subsequently, they were stained with hematoxylin and eosin (HE, Solarbio, China) and observed and imaged using a whole-slide scanning system (Olympus, Japan).

[0101] First, a mouse SAE model was established using intraperitoneal injection of LPS. Then, using lateral ventricle injection, the effects of M2-EXO on MSS and brain tissue damage after mouse SAE were investigated by MSS and HE staining. The procedure is as follows: Figure 12 As shown in A. (As indicated by...) Figure 12 As shown in Figure B, compared with the sham-operated group, the MSS of mice 24 hours after SAE was significantly increased; compared with the SAE group, M2-EXO (10 μg / mouse, 24h) and dexamethasone (DEX) (5 mg / kg, 24h) significantly decreased MSS after SAE. Figure 12 As shown in Figure C, the cortical neurons in the sham-operated group had normal morphology, uniform cytoplasmic staining, and no degenerated or necrotic cells. Compared with the sham-operated group, the cortical neurons in the SAE group had reduced volume, pyknosis, and deeply stained nuclei, with some cells showing vacuolization. Compared with the SAE group, M2-EXO (10 μg / needle, 24h) and DEX (5 mg / kg, 24h) reduced nuclear pyknosis in cortical neurons after SAE, and the cell morphology was close to normal. This suggests that M2-EXO and DEX can improve SAE.

[0102] Example 13: Effects of miR-1949 on MSS and cortex in mice after SAE

[0103] Next, we will examine the effects of miR-1949 on MSS and brain tissue damage in mice after SAE. The procedure is as follows: Figure 13 As shown in A. (As indicated by...) Figure 13 As shown in Figure B, compared with the sham-operated group, the MSS of mice 24 hours after SAE was significantly increased; compared with the SAE group, miR-1949mimic (0.1 nmol / mouse, 24h) significantly decreased MSS after SAE; compared with the miR-1949mimic group, miR-1949mimic + XAV939 (40 mg / kg, 24h) could reverse the above effects of miR-1949mimic. Figure 13 As shown in Figure C, the cortical neurons in the sham-operated group had normal morphology, uniform cytoplasmic staining, and no degenerated or necrotic cells. Compared with the sham-operated group, the cortical neurons in the SAE group showed reduced volume, pyknosis, and deep staining of the nuclei, with some cells exhibiting vacuolization. Compared with the SAE group, miR-1949mimic (0.1 nmol / needle, 24h) reduced nuclear pyknosis in cortical neurons after SAE, resulting in near-normal cell morphology. Compared with the miR-1949mimic group, miR-1949mimic + XAV939 (40 mg / kg, 24h) reversed the above effects of miR-1949mimic. This suggests that miR-1949 improves SAE by activating the Wnt / β-catenin pathway.

[0104] The results of Examples 12 and 13 suggest that M2-EXO and its miR-1949 can significantly improve SAE and are related to the activation of the Wnt / β-catenin pathway.

[0105] Example 14: Effect of M2-EXO on the morphology of microglia after mouse SAE

[0106] The effect of M2-EXO on the morphology of cortical microglia in mice 24 hours after SAE was investigated using immunofluorescence assays. Figure 14 As shown in Figure A, green fluorescence represents Iba1-labeled microglia, and blue fluorescence represents the nuclear dye DAPI. The results showed that, compared to the sham-operated group, microglia were activated after SAE, characterized by significantly enlarged cell bodies and shortened or absent processes. Figure 14 As shown in B and 14C, Sholl analysis revealed that microglial complexity, total branch length, total number of branches, and number of terminal branches were significantly reduced after SAE. Compared with the SAE group, M2-EXO (10 μg / mouse) significantly increased microglial complexity, total branch length, total number of branches, and number of terminal branches 24 h after SAE in mice. Compared with the SAE group, DEX (5 mg / kg) had no significant effect on microglial complexity, total branch length, total number of branches, and number of terminal branches. This suggests that M2-EXO can significantly reduce the activation of cortical microglia in mice 24 h after SAE.

[0107] Example 15: Effects of miR-1949 on the morphology of microglia after SAE in mice

[0108] The effect of miR-1949 on the activation of cortical microglia 24 h after mouse SAE was investigated using immunofluorescence assays. Results are as follows: Figure 15 As shown in Figure A, green fluorescence represents Iba1-labeled microglia, and blue fluorescence represents the nuclear dye DAPI. The results showed that, compared to the sham-operated group, microglia were activated after SAE, characterized by significantly enlarged cell bodies and shortened or absent processes. Figure 15 As shown in B and 15C, Shollen analysis revealed that microglial complexity, total branch length, total number of branches, and number of terminal branches were significantly reduced after SAE. Compared with the SAE group, miR-1949mimic (0.1 nmol / mouse) significantly increased microglial complexity, total branch length, total number of branches, and number of terminal branches 24 h after SAE in mice. Compared with the miR-1949mimic group, miR-1949mimic + XAV939 (40 mg / kg) treatment in mice 24 h after SAE reduced microglial complexity, total branch length, and total number of branches. This suggests that miR-1949 significantly reduces the activation of cortical microglia in mice 24 h after SAE by activating the Wnt / β-catenin pathway.

[0109] Example 16: Effects of M2-EXO on the mRNA levels of Arg1, CD206, IL-10, IL-6, iNOS, and TNF-α in the post-SAE cortex of mice.

[0110] The mRNA levels of Arg1, CD206, IL-10, IL-6, iNOS, and TNF-α in the cortex of M2-EXO-treated mice 24 hours after SAE were investigated using RT-qPCR. Figure 16 As shown, compared with the sham-operated group, the expression levels of Arg1, CD206, and IL-10 in the cortex of mice after SAE were significantly reduced, while the expression levels of IL-6, iNOS, and TNF-α were significantly increased. Compared with the SAE group, M2-EXO (10 μg / mouse) treatment for mice 24 h after SAE significantly increased the expression levels of Arg1, CD206, and IL-10 in the cortex of mice and significantly reduced the expression levels of IL-6, iNOS, and TNF-α. Compared with the SAE group, DEX (5 mg / kg) treatment for mice 24 h after SAE significantly increased the expression levels of CD206 and IL-10 in the cortex of mice and significantly reduced the expression levels of IL-6 and iNOS, but the efficacy of DEX was not as significant as that of M2-EXO.

[0111] Example 17: Effects of miR-1949 on the mRNA levels of Arg1, CD206, IL-10, IL-6, iNOS, and TNF-α in the post-SAE cortex of mice.

[0112] Next, RT-qPCR was used to examine the expression levels of Arg1, CD206, IL-10, IL-6, iNOS, and TNF-α in the cortex of mice treated with miR-1949 for SAE 24 hours. Figure 17 As shown, compared with the sham-operated group, the expression levels of Arg1, CD206, and IL-10 in the cortex of mice after SAE were significantly reduced, while the expression levels of IL-6, iNOS, and TNF-α were significantly increased. Compared with the SAE group, 24 h after treatment with miR-1949mimic (0.1 nmol / mouse) in mice with SAE, the expression levels of Arg1, CD206, and IL-10 in the cortex of mice were significantly increased, while the expression levels of IL-6, iNOS, and TNF-α were significantly decreased. Compared with the miR-1949mimic group, miR-1949mimic + XAV939 (40 mg / kg) could reverse the above effects of miR-1949mimic.

[0113] Example 18: Effects of M2-EXO on the expression of DKK1, Wnt, and β-catenin in the cortex of mice after SAE

[0114] The expression levels of DKK1, Wnt, and β-catenin in the cortex of M2-EXO-treated mice 24 hours after SAE were investigated using RT-qPCR. Results are as follows: Figure 18 As shown, compared with the sham-operated group, the expression level of DKK1 in the cortex of mice after SAE was significantly increased, while the expression levels of Wnt and β-catenin were significantly decreased. Compared with the SAE group, M2-EXO (10 μg / mouse) treatment for 24 h after SAE significantly decreased the expression level of DKK1 in the cortex of mice and significantly increased the expression levels of Wnt and β-catenin. Compared with the SAE group, DEX (5 mg / kg) treatment for 24 h after SAE significantly decreased the expression level of DKK1 in the cortex of mice, but had no significant effect on the expression levels of Wnt and β-catenin.

[0115] Example 19: Effects of miR-1949 on the expression of DKK1, Wnt, and β-catenin in the cortex of mice after SAE

[0116] The expression levels of DKK1, Wnt, and β-catenin in the cortex of mice treated with miR-1949 for SAE were investigated using RT-qPCR. Figure 19As shown, compared with the sham-operated group, the expression level of DKK1 in the cortex of mice after SAE was significantly increased, while the expression levels of Wnt and β-catenin were significantly decreased. Compared with the SAE group, treatment with miR-1949mimic (0.1 nmol / mouse) for 24 hours after SAE significantly decreased the expression level of DKK1 in the cortex of mice and significantly increased the expression levels of Wnt and β-catenin. Compared with the miR-1949mimic group, miR-1949mimic + XAV939 (40 mg / kg) could reverse the above effects of miR-1949mimic.

[0117] Example 20: Validation of miR-127-5p mimic loaded onto exosomes via a modified calcium chloride method

[0118] The content of miR-127-5p mimic in exosomes after loading miR-127-5p mimic via a modified calcium chloride method was investigated using RT-qPCR. The results are as follows: Figure 20 As shown, compared with the mimic NC group, the expression of miR-127-5p in exosomes was significantly increased after loading miR-127-5p mimic. This suggests that this method can effectively load miR-127-5p mimic into exosomes.

[0119] Example 21: Effects of M2-EXO and miRNA on Schizophrenia in Mice

[0120] (1) Animal experimental design

[0121] C57BL / 6 mice, weighing 18-22g, were anesthetized with 4% isoflurane before stereotactic brain surgery, maintaining a 1.5% anesthetic concentration during the procedure. The mice were then placed on a stereotactic frame for surgery. Exosome suspension (10μg / mouse) was injected into the lateral ventricle using a PBS-washed microsyringe. Twenty-four hours later, a mouse model of schizophrenia was established by intraperitoneal injection of MK-801 (0.2mg / kg). A positive control group received haloperidol (1mg / kg) 30 minutes before injection. All intraperitoneal drugs were dissolved in physiological saline to prepare a 10mg / kg solution. Mice were weighed and injected intraperitoneally according to the calculated dosage. Behavioral experiments were conducted 30 minutes after MK-801 injection.

[0122] (2) Effects of M2-EXO and miR-127-5p on positive symptoms of schizophrenia in mice

[0123] The open field test (OFT) was used to detect positive symptoms of schizophrenia in mice. The experimental setup consisted of a rectangular open box measuring 100cm long, 100cm wide, and 50cm high, equipped with a high-definition infrared camera on top. After 30 minutes of MK-801 (0.2mg / kg) induction, mice were gently placed in the open field with their heads facing a corner and allowed to explore freely for 5 minutes before recording was stopped. The total distance and speed of movement in the open field were recorded and analyzed. After each test, the odor was cleaned with 75% ethanol. The acquired videos were analyzed using the Etho Vision XT 8.0 video analysis system. Results are as follows: Figure 21 As shown, compared with the sham-operated group, the total distance and speed of movement in the open field were significantly increased in mice with schizophrenia. Compared with the schizophrenia group, M2-EXO (10 μg / mouse, 24 h), M2-EXO loaded with miRNA mimic NC (0.1 nmol / mouse, 24 h), and the positive control drug dopamine receptor antagonist haloperidol (1 mg / kg, 30 min) significantly reduced the total distance and speed of movement in mice with schizophrenia. Compared with the M2-EXO (0.1 nmol / mouse, 24 h) loaded with miRNA mimic NC, M2-EXO loaded with miR-127-5p (0.1 nmol / mouse, 24 h) further reduced the total distance and speed of movement in the open field in mice with schizophrenia.

[0124] Example 22: Effects of M2-EXO and miR-127-5p on anxiety-like behavior in schizophrenic mice

[0125] The effects of M2-EXO and miR-127-5p on anxiety behavior in schizophrenic mice were investigated by analyzing the movement trajectories of mice in an open field experiment. Figure 22 As shown, compared with the sham-operated group, the time spent in the central region of the open field was significantly increased in the schizophrenia group, indicating that schizophrenia mice may exhibit anxiety-like behavior. Compared with the schizophrenia group, M2-EXO (10 μg / mouse, 24 h) significantly reduced the time spent in the central region of the open field in mice, while haloperidol (1 mg / kg, 30 min) had no such effect. Compared with the miRNA mimic NC group, administration of M2-EXO loaded with miR-127-5p (0.1 nmol / mouse, 24 h) significantly reduced the time spent in the central region of the open field in schizophrenia mice.

[0126] The results suggest that M2-EXO can improve anxiety-like behavior in MK-801-induced schizophrenia in mice, and miR-127-5p may play an important role in the anti-anxiety effect of M2-EXO. Haloperidol had no significant effect on anxiety-like behavior in schizophrenia in mice.

[0127] Example 23: Effects of M2-EXO and miR-127-5p on cognitive impairment symptoms in mice with schizophrenia

[0128] The Y-maze test was used to assess cognitive impairment in mice. The Y-maze apparatus consists of three identical arms and a connecting area, with the three arms forming a 120° angle. In the experiment, a clean, odorless animal bedding layer was placed at the bottom of the apparatus, and the three arms were labeled A, B, and C for differentiation. After 30 minutes of modeling with MK-801 (0.2 mg / kg), the mice were gently placed in the center of the connecting area of ​​the Y-maze and allowed to explore freely for 8 minutes. The total number of arm entries, the number of correct alternation responses, and the percentage of alternation responses were recorded within 8 minutes. The number of correct alternation responses was the number of times the rat entered three different arms consecutively. The percentage of alternation responses was calculated using the formula: percentage of alternation (%) = number of alternations / (number - 2) × 100. Results are as follows: Figure 23 As shown, compared with the sham-operated group, the spontaneous alternation rate in the open field was significantly decreased in mice with schizophrenia. Compared with the schizophrenia group, M2-EXO (10 μg / mouse, 24 h) significantly improved the spontaneous alternation rate in the Y maze, while haloperidol (1 mg / kg, 30 min) had no such effect. Compared with the miRNA mimic NC group, administration of M2-EXO loaded with miR-127-5p (0.1 nmol / mouse, 24 h) significantly improved the spontaneous alternation rate in mice with schizophrenia.

[0129] Example 24: Effects of M2-EXO and miR-127-5p on neuronal damage in the cerebral cortex of schizophrenic mice

[0130] The effects of M2-EXO and miR-127-5p on neuronal damage in the cerebral cortex of schizophrenic mice were investigated using immunofluorescence assays. Figure 24 As shown, the neuronal marker protein NeuN is labeled with red fluorescence, and the microglia marker protein IBA1 is labeled with green fluorescence. Compared with the sham-operated group, the level of NeuN positivity in the cerebral cortex of mice in the schizophrenia group was significantly reduced. M2-EXO (10 μg / mouse) pretreatment for 24 hours did not affect NeuN positivity in mice; however, compared with the schizophrenia group, M2-EXO (10 μg / mouse) pretreatment for 24 hours significantly increased NeuN positivity. Figure 25As shown, compared with the mimic NC group, M2-EXO loaded with miR-127-5p mimic (0.1 nmol / mouse, 24h) significantly increased the NeuN positivity level in the cerebral cortex of mice. These results suggest that M2-EXO can improve neuronal damage in schizophrenic mice. miR-127-5p may play an important role in the effects of M2-EXO.

[0131] Example 25: Effects of M2-EXO and miR-127-5p on microglial morphology in schizophrenic mice

[0132] The morphology of microglia obtained from immunofluorescence experiments was analyzed using Sholl analysis and scaffold analysis. Results are as follows: Figure 26 As shown in A and 27A, compared with the sham-operated group, microglia were activated in the cerebral cortex of schizophrenic mice, specifically manifested as enlarged cell bodies, retracted processes, and a reduced number of processes. Figure 26 As shown in B and 26C, Sholl analysis revealed significantly reduced microglial complexity, total branch length, total number of branches, and number of terminal branches in schizophrenic mice. Compared to the schizophrenia group, M2-EXO (10 μg / mouse, 24h) significantly increased these morphologies. This suggests that M2-EXO can significantly alter microglial morphology, shifting them from a pro-inflammatory to an anti-inflammatory form. The next step is to investigate the effect of miR-127-5p on the morphology of cortical microglia in schizophrenic mice. Figure 27 As shown in B and 27C, compared with the mimic NC group, M2-EXO (0.1 nmol / mouse, 24h) with high expression of miR-127-5p significantly increased the complexity, total branch length, total number of branches, and number of terminal branches of microglia in schizophrenic mice. This suggests that miR-127-5p may play an important role in the effect of M2-EXO on improving microglia morphology.

[0133] Example 26: Effects of M2-EXO and miR-127-5p on the levels of inflammatory factors in microglia of schizophrenic mice

[0134] The effects of M2-EXO on the level of inflammation in the brain of schizophrenic mice were investigated using RT-qPCR and Western blotting experiments. Figure 28 As shown in Figure A, compared with the sham-operated group, the mRNA expression levels of TNF-α, IL-6, and IL-1β in the cerebral cortex of schizophrenic mice were significantly increased; compared with the schizophrenic group, pretreatment with M2-EXO (10 μg / mouse) for 24 h significantly reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β in the cerebral cortex of mice. Figure 28As shown in B and 28C, compared with the sham-operated group, the protein expression levels of TNF-α and IL-1β in the cerebral cortex of schizophrenic mice were significantly increased; compared with the schizophrenic group, pretreatment with M2-EXO (10 μg / mouse) for 24 h significantly reduced the protein expression levels of TNF-α and IL-1β in the cerebral cortex of mice.

[0135] Subsequently, the effect of miR-127-5p on the level of inflammation in the brain of schizophrenic mice was examined. Figure 29 As shown in Figure A, compared with the mimic NC group, M2-EXO (10 μg / mouse, 24h) with high expression of miR-127-5p significantly reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β in the mouse cerebral cortex. Figure 29 As shown in B and 29C, compared with the mimic NC group, M2-EXO (0.1 nmol / mouse, 24h) with high expression of miR-127-5p significantly reduced the protein expression levels of TNF-α and IL-1β in the mouse cerebral cortex.

[0136] The above results suggest that M2-EXO can improve neuroinflammation in schizophrenic mice, and miR-127-5p plays an important role in M2-EXO.

[0137] Example 27: Effects of M2-EXO and miR-127-5p on cell survival in neuronal inflammatory injury

[0138] 1. Culture of HT-22 neurons

[0139] The complete culture medium was based on IMDM, supplemented with 5% fetal bovine serum and 1% penicillin-dextrose antibodies. HT-22 neuronal cells were placed in the above culture medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were digested with 0.05% trypsin and passaged at a ratio of 1:4.

[0140] 2. Preparation of M1 type microglia conditioned culture medium (M1-CM)

[0141] N9 microglia were used at a rate of 1×10 5 Microglia were seeded at a density of cells / mL in culture dishes. After cell attachment, they were treated with LPS (1 μg / mL) for 24 h to induce M1 polarization. M1-CM cells were collected, centrifuged at 1300 rpm for 5 min to remove cell debris, and used immediately.

[0142] 3. Establishment of a neuronal injury model

[0143] HT-22 neurons were fed at a rate of 1×10 5The cells were seeded at a density of cells / mL in culture dishes. After the cells adhered, the medium was completely replaced with M1-CM and incubated for 24 hours.

[0144] 4. N9 microglia were induced with LPS (1 μg / mL, 24 h) to obtain M1-CM cells. These M1-CM cells were then used to treat HT-22 neurons for 24 h to establish a neuronal injury model. The effects of M2-EXO and miR-127-5p on the decreased HT-22 cell survival rate induced by M1-CM were investigated using the MTT assay. Figure 30 As shown, compared with the Con group, the cell viability in the Model group decreased significantly after 24 h of treatment with M1-CM. Compared with the Model group, simultaneous administration of M2-EXO (20 μg / mL, 24 h) during M1-CM treatment significantly inhibited the decrease in cell viability. Administration of M2-EXO loaded with mimic NC (0.2 nmol, 20 μg / mL, 24 h) also had a similar effect. Notably, administration of M2-EXO with high miR-127-5p expression further improved cell viability.

[0145] Example 28: Effects of miR-127-5p on neuronal inflammation

[0146] M2-EXO loaded with miR-127-5p mimic and mimic NC was prepared using a modified calcium chloride method. M1-type N9 microglia were treated with this solution at a dose of 0.2 nmol / mL for 24 h. The conditioned culture medium was then collected and used to treat HT-22 neurons for 24 h. The effect on the levels of inflammatory factors in HT-22 cells was investigated using RT-qPCR. Figure 31 As shown, compared with the Con group, the mRNA levels of TNF-α, IL-6, and IL-1β in HT-22 cells were significantly increased after M1-CM treatment; compared with the administration of M2-EXO loaded with mimic NC (0.2 nmol, 20 μg / mL, 24 h), the administration of M2-EXO (0.2 nmol, 20 μg / mL, 24 h) with high miR-127-5p expression during M1-CM treatment significantly reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β in HT-22 cells.

[0147] Example 29: Effects of miR-127-5p on microglial inflammation and phenotype

[0148] (1) Culture of N9 microglia

[0149] The complete culture medium was based on IMDM, supplemented with 5% fetal bovine serum and 1% penicillin-dextrose antibodies. N9 microglia were placed in the above culture medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were digested with 0.05% trypsin and passaged at a ratio of 1:4.

[0150] (2) Induction of M1 phenotype N9 microglia

[0151] LPS was prepared as a 1 mg / mL stock solution using IMDM basal medium and stored at -20°C protected from light. N9 microglia were cultured at a concentration of 1 × 10⁻⁶ mg / mL. 5 Microglia were seeded at a density of cells / mL in culture dishes. After the cells adhered, they were treated with LPS (1 μg / mL) for 24 h to induce M1 polarization.

[0152] (3) M2-EXO cells loaded with miR-127-5p mimic and control sequences (mimic NC) were prepared using a modified calcium chloride method and treated with M1-type N9 microglia at a dose of 0.2 nmol / mL for 24 h. The effect of miR-127-5p on the expression levels of inflammation- and phenotype-related mRNAs in M1-type microglia was investigated by RT-qPCR. Figure 32 As shown, in M1 microglia (LPS, 1 μg / mL, 24 h), administration of miR-127-5p-overexpressing M2-EXO (0.2 nmol, 20 μg / mL, 24 h) significantly reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β compared to administration of M2-EXO loaded with mimic NC (0.2 nmol, 20 μg / mL, 24 h). Figure 32 As shown, compared with the mimic NC group, high expression of M2-EXO by miR-127-5p significantly increased the mRNA levels of M2 microglia markers Arg-1 and CD206, and decreased the mRNA level of M1 microglia marker iNOS. The relative decrease in the mRNA expression levels of M1 phenotypic markers and the relative increase in the mRNA levels of M2 phenotypic markers suggest that the cell phenotype of microglia may be changing from M1 to M2.

[0153] The above results suggest that miR-127-5p may play an important role in both alleviating microglial inflammation and promoting the transformation of M1 microglia to M2 in M2-EXO.

Claims

1. An M2 type microglial cell exosome, characterized in that, The M2 microglia exosomes contain multiple differentially expressed miRNAs, including miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p. These miR-1949, miR-127-5p, miR-674-5p, and miR-322-5p are highly expressed in the M2 microglia exosomes, with miR-1949 showing the most significant differential expression. The preparation method of the M2 microglia exosomes includes the following steps: N9 microglia were used at a rate of 1×10 5 M2 microglia were seeded at a density of cells / mL and cultured. After cell adhesion, they were treated with 20 ng / mL IL-4 for 24 h to induce M2 polarization. M2 microglia were collected and centrifuged at 1300 rpm for 5 min to remove cell debris. Exosome extraction by ultracentrifugation: 1) Centrifuge at 300g for 10 min at 4℃ to remove suspended cells; 2) Centrifuge at 2000g for 10 min to remove dead cells; 3) Centrifuge at 10000g for 30 min to remove cell debris; 4) Centrifuge at 100000g for 70 min at 4℃ to obtain exosome precipitate; 5) Resuspend the precipitate in sterile PBS and wash again by centrifugation at 100000g for 70 min; 6) Dissolve the exosomes in PBS and store at -80℃ for later use.

2. The M2 type microglial cell exosome according to claim 1, characterized in that, The M2 microglia exosomes can be taken up by M1 microglia.

3. The M2 type microglial cell exosome according to claim 1, characterized in that, The M2 microglia exosomes can at least target and bind to the 3'UTR of DKK1 via miR-1949, inhibiting DKK1 expression and activating the Wnt / β-catenin signaling pathway, thereby inducing the transformation of M1 microglia into M2 microglia.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the M2 type microglia exosomes of claim 1 and a pharmaceutically acceptable carrier; the pharmaceutical composition is used to prepare a medicament for treating neurological disorders, including sepsis-associated encephalopathy and schizophrenia.

5. The use of the M2 type microglial exosomes according to any one of claims 1-3 in the preparation of drugs for treating sepsis-associated encephalopathy and drugs for treating schizophrenia.

6. The application according to claim 5, characterized in that, The M2 microglia exosomes can at least inhibit neuroinflammation through miR-127-5p, and improve positive symptoms, anxiety-like behavior, and cognitive impairment in mice with schizophrenia.

7. The application according to claim 5, characterized in that, The M2 microglia exosomes can at least suppress neuroinflammation and improve sepsis-associated encephalopathy in mice by mediating microglia phenotypic transformation through miR-1949.

8. The use of miR-1949 contained in the M2 type microglia exosomes of claim 1 in the preparation of a drug for treating sepsis-associated encephalopathy.

9. The use of miR-127-5p contained in the M2 type microglia exosomes of claim 1 in the preparation of a drug for treating schizophrenia.

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