New application of small extracellular vesicles with high NAMPT expression in medicine

Abstract

The invention relates to a novel application of small extracellular vesicles with high expression of NAMPT in preparation of drugs for relieving or treating traumatic brain injury. The invention finds that the NAMPT-sEV administration in the nose can obviously increase the expression of Sirtuin 1 of an rmTBI patient, so that tau is deacetylated. Besides, the NAMPT-sEV inhibits neuroinflammation, maintains the polarity of aquapoprotein-4, promotes the repair of a lymphatic system and a meningeal lymphatic system, and is beneficial to the removal of ac-tau protein in brain tissues, and the reduction of the ac-tau protein can prevent the degeneration of an initial axon segment and the wrong positioning of the tau protein. In conclusion, the invention provides that NAMPT-sEV reduces neuron loss of rmTBI and improves cognitive function through various mechanisms, and the small extracellular vesicles highly expressing NAMP can be well used for alleviating or treating traumatic brain injury and dementia including Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a new application of small extracellular vesicles highly expressing NAMPT in medicine. Background Art

[0002] The annual incidence of traumatic brain injury (TBI) is approximately 50 million cases, causing losses of approximately $400 billion. In recent years, the long-term chronic effects of repetitive mild traumatic brain injury (rmTBI) have received increasing attention, especially among military personnel and professional athletes. Clinically, rmTBI is associated with long-term memory deficits and significantly increases the risk of developing Alzheimer's disease (AD). Growing evidence indicates that there is a common pathological mechanism involving acetylation between these two diseases. In addition, studies have also reported an increase in acetylated tau (ac-tau) in patients with AD and chronic traumatic encephalopathy. Research has shown that tau acetylation can dissociate Tau from microtubules and promote Tau aggregation, leading to memory loss associated with the early stage of AD. Therefore, treatment targeting tau acetylation may be a promising therapeutic approach for early intervention in rmTBI and AD.

[0003] Alzheimer's disease (AD) is an irreversible neurodegenerative disease mainly characterized by cognitive impairment. As the disease progresses, the patient's ability to take care of themselves gradually deteriorates, bringing a heavy burden to society and families. The incidence of AD increases with age, approximately 5 - 8% in patients over 65 years old, and increasing to 25 - 50% in patients over 85 years old. Neuritic plaques formed by Aβ (amyloid-β) and neurofibrillary tangles formed by Tau protein are the most important pathological features. Since there is currently no cure for AD and most patients seek medical treatment at an advanced stage of the disease, which is difficult to control, early diagnosis, treatment, and intervention are particularly important. Neurodegenerative dementia, in addition to Alzheimer's disease (AD), also includes dementia with Lewy bodies (DLB), which is characterized by fluctuating cognitive impairment, visual hallucinations, and Parkinson-like motor symptoms. Frontotemporal dementia (FTD) is mainly characterized by personality changes, disinhibited behavior, and language disorders in the early stage, such as behavioral variant (bvFTD) and primary progressive aphasia (PPA). Most types of the above neurodegenerative dementia are irreversible, but early intervention can delay the progression.

[0004] Most of the recent drugs for AD are antibody drugs targeting Aβ, aiming to clear Aβ in the brain, but the curative effect is not significant. On the one hand, after binding to Aβ, antibody drugs need to be excreted through the brain clearance system. Therefore, the Glymphatic system in the brain may be an important factor affecting the drug efficacy. On the other hand, more and more evidence also shows that the pathological changes of Tau protein are more directly related to the cognitive impairment of AD patients, which also explains the poor curative effect on symptom relief. Tau protein, as a potential therapeutic target for AD, has attracted more and more attention.

[0005] Sirtuin 1 (SIRT1) is the most important member of the third deacetylase family and plays an important role in energy metabolism, inflammation and oxidative stress. SIRT1 is a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase. NAD + is the substrate for SIRT1 deacetylation. When SIRT1 deacetylates protein substrates, NAD + is broken down into nicotinamide (NAM) and adenosine diphosphate ribose (NR). Interestingly, NAM is converted back to NAD + through nicotinamide phosphoribosyltransferase (NAMPT). As the rate-limiting enzyme for intracellular NAD + synthesis, NAMPT is the key link between mammalian NAD + biosynthesis and SIRT1 activity. In addition, studies have shown that extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in plasma decreases with the increase of human age, and it is the only protein in young blood that can significantly delay aging and extend lifespan.

[0006] Exosomes are multivesicular vesicles (MVs) approximately 30-150 nm in diameter that are secreted extracellularly after budding from endosomes and fusing with the cell membrane. According to the guidelines of the International Society for Extracellular Vesicles (ISEV), vesicles smaller than 200 nm are collectively referred to as small extracellular vesicles (sEVs). Exosomes, primarily composed of proteins, nucleic acids, and lipids, mediate intercellular communication and the transfer of macromolecules between cells, promoting the transport of proteins, lipids, mRNA, miRNA, and DNA, and contributing to disease progression, playing important biological roles. Mesenchymal stem cell exosomes / sEVs contain miRNAs, lncRNAs, and at least 200 proteins with immunomodulatory properties. Due to their unique phospholipid bilayer structure, they are stable in body fluids and can freely cross the blood-brain barrier. Current research suggests that engineering sEVs, using them as carriers for beneficial proteins tailored to disease pathogenesis, can provide specific therapeutic effects. However, how to construct small extracellular vesicles with excellent biological activity for the treatment of traumatic brain injury and AD and achieve good therapeutic effects still needs to be explored and studied. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide small extracellular vesicles that highly express NAMPT and new applications thereof in medicine.

[0008] The technical solutions for achieving the above-mentioned purpose include the following.

[0009] Application of small extracellular vesicles (NAMPT-sEV) that highly express NAMPT in the preparation of products for alleviating or treating traumatic brain injury.

[0010] In some embodiments, the traumatic brain injury is mild traumatic brain injury.

[0011] In some embodiments, the traumatic brain injury is repetitive mild traumatic brain injury.

[0012] In some embodiments, the small extracellular vesicles highly expressing NAMPT can be derived from various cells, such as stem cells, HEK293T cells, HEK293F cells, CHO cells, Sf9 cells, Sf21 cells or Hi5 cells, preferably stem cells. The small extracellular vesicles highly expressing NAMPT in the present invention are preferably derived from mesenchymal stem cells.

[0013] In some preferred embodiments, the mesenchymal stem cells are derived from induced pluripotent stem cells, or are derived from materials such as umbilical cord, bone marrow, fat, urine, and uterine blood.

[0014] In some of these embodiments, the small extracellular vesicles highly expressing NAMPT are obtained by the following preparation method: constructing a recombinant lentiviral vector containing the NAMPT protein coding gene, transfecting mesenchymal stem cells to obtain mesenchymal stem cells stably expressing the NAMPT protein coding gene, culturing, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

[0015] In some of these embodiments, the promoter that initiates the expression of the NAMPT protein coding gene on the recombinant lentiviral vector is a dual promoter containing CMV and EF1α.

[0016] In some of these embodiments, the recombinant lentiviral vector is transfected into mesenchymal stem cells at a multiplicity of infection (MOI) of 50 - 150, preferably MOI is 80 - 120, and more preferably MOI is 90 - 110.

[0017] Preferably, the recombinant lentiviral vector is 293T cells. It can also be HEK293t cells, 293T / F cells, 293SG cells or cells developed based on 293T for specific purposes, or CHO cells, etc.

[0018] In some of these embodiments, the method of introducing the recombinant expression vector into stem cells can be achieved by various conventional infection methods, such as viral infection method, liposome transfection method or electroporation transfection method, etc.

[0019] In some of these embodiments, the product is a drug or a biological agent.

[0020] In some of these embodiments, the dosage form of the drug can be various dosage forms acceptable in pharmacy, including oral dosage form, aerosol, ointment, injection (including intravenous injection, intra-articular injection, corpora cavernosa injection, organ targeted injection, etc.), or dosage forms suitable for intravascular administration.

[0021] In some of these preferred embodiments, the dosage form of the drug is a dosage form suitable for intranasal administration.

[0022] In some of these embodiments, the application includes increasing the expression of Sirtuin 1 in the subject.

[0023] In some of these embodiments, the application includes inhibiting neuroinflammation in the subject, maintaining the polarity of aquaporin - 4, and promoting the repair of the lymphatic system and the meningeal lymphatic system.

[0024] In some of these embodiments, the application includes reducing neuronal loss in the subject.

[0025] In some of these embodiments, the application includes improving the cognitive function of the subject.

[0026] The second aspect of the present invention is to provide the use of small extracellular vesicles highly expressing NAMPT in the preparation of a medicament for treating Alzheimer's disease.

[0027] In some embodiments, the use includes promoting the clearance of acetylated tau protein (ac-tau) in a subject.

[0028] In some embodiments, the subject for the use is a mammal.

[0029] In some embodiments, the mammal is a human.

[0030] The third aspect of the present invention is to provide the use of small extracellular vesicles highly expressing NAMPT in the preparation of a product for preventing anti-aging.

[0031] The third aspect of the present invention is to provide a medicament for treating traumatic brain injury, and the active ingredient of the medicament includes small extracellular vesicles highly expressing NAMPT. Preferably, the small extracellular vesicles highly expressing NAMPT are obtained by the following preparation method: constructing a recombinant lentiviral vector containing the NAMPT protein-coding gene, transfecting mesenchymal stem cells, obtaining mesenchymal stem cells with stable expression of the NAMPT protein-coding gene, culturing, collecting the culture supernatant, and extracting small extracellular vesicles therefrom to obtain NAMPT-sEV (small extracellular vesicles highly expressing NAMP).

[0032] The research results of the present invention show that intranasal administration of NAMPT-sEV can significantly increase the expression of Sirtuin 1 in rmTBI mice, thereby deacetylating tau. In addition, NAMPT-sEV inhibits neuroinflammation, maintains the polarity of aquaporin-4, promotes the repair of the lymphatic system and the meningeal lymphatic system, is beneficial to the clearance of ac-tau in the brain tissue, and the reduction of ac-tau protein can prevent the degeneration of the initial segment of axons and the mislocalization of tau protein.

[0033] In summary, the present invention provides that NAMPT-sEV reduces neuronal loss in rmTBI and improves cognitive function through multiple mechanisms. The small extracellular vesicles highly expressing NAMPT can be well used to alleviate or treat traumatic brain injury, or for preventing and treating dementia, including treating Alzheimer's disease, and for anti-aging and other effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Preparation and detection of NAMPT-iPSC-MSCs and NAMPT-sEV; among which, A. Schematic diagram of the preparation of NAMPT-iPSC-MSCs and NAMPT-sEV, B. Detection of mCherry in purified NAMPT-MSCs under a fluorescence microscope, C. Nanoparticle tracking analysis of MSC-sEV / NAMPT-sEV, D. Transmission electron microscopy detection of MSC-sEV / NAMPT-sEV, E. Western blot showing the protein expression pattern of MSC-sEV / NAMPT-sEV, F, G. Schematic diagram of the intranasal treatment process of mccherry-sEV and its distribution in the cortex and hippocampus (CA1, CA3, DG).

[0035] Figure 2 Intranasal treatment with NAMPT-sEV protects against spatial and recognition memory deficits in mice with repetitive mild traumatic brain injury (rmTBI); among which, A. Schematic diagram of the experimental design, B. Comparison of mNSS in rmTBI mice in different treatment groups, C. Comparison of the fall latency in the rotarod test in rmTBI mice in each group 1 month after rmTBI, D. Comparison of object recognition indexes in four groups of subjects, E. Representative movement trajectories in the novel object test, F. Representative swimming paths in the spatial memory probe test, G. Comparison of the latency to the platform during the 5-day training in the Morris water maze, H. Comparison of the frequency of crossing the previous target platform, I. Comparison of the time in the target quadrant during the exploration test, n = 10, ns: no statistical significance, *p < 0.05, **p < 0.01, ****p < 0.0001.

[0036] Figure 3 Expression levels of Sirt1 and ac-tau in the hippocampal cortex of mice; among which, A, B Western blot shows the expression levels of Sirt1 and ac-tau in the cortex and hippocampus of mice, C, D Statistical chart of the comparison of Sirt1 in the cortex and hippocampus among different groups, E, F Statistical chart of the comparison of ac-tau / t-tau among different groups; n = 4, ns: no statistical difference, *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0037] Figure 4Intranasal treatment with NAMPT-sEV reduces acetylated tau, which can rescue axonal initial segment (AIS) degradation and pathological tau mislocalization to the somatodendritic compartment of neurons in rmTBI mice; among them, A. Representative images show that NAMPT-sEV protects mice from AIS degradation in the cerebral cortex after rmTBI, B. Comparison of normalized AnkG intensity in the cerebral cortex of each group, C. Representative images show that NAMPT-sEV protects mice from AIS degradation in the hippocampus after rmTBI, D. Comparison of normalized AnkG intensity in the hippocampus of each group, E, H. Representative images show that NAMPT-sEV protects mice from tau mislocalization in the cerebral cortex and hippocampus after rmTBI, F, I. Comparison of mislocalized tau indices between the cerebral cortex and hippocampus groups, G, J. Comparison of the degree of brain injury in different treatment groups; n = 6, ns: no statistical difference, ***p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0038] Figure 5 Small extracellular vesicles and NAMPT-sEV inhibit neuroinflammation in the cerebral cortex and hippocampus of rmTBI mice; among them, A. Immunofluorescence staining of microglia in the cerebral cortex and hippocampus, B, C. Comparison of the Iba1 area fraction shows that small extracellular vesicles and NAMPT-sEV in different groups both inhibit the activation of microglia in the cerebral cortex and hippocampus, D, E. Comparison of the GFAP area fraction shows that small extracellular vesicles and NAMPT-sEV in different groups both inhibit astrogliosis in the cerebral cortex and hippocampus, F. Representative images show that small extracellular vesicles and NAMPT-sEV inhibit astrogliosis and maintain AQP4 polarity, G, H. Comparison of AQP4 polarity among the Sham, rmTBI+Saline, rmTBI+sEV, and rmTBI+NAMPT-sEV groups; n = 6, ns: no statistical difference, ***p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0039] Figure 6 Small extracellular vesicles and NAMPT-sEV promote meningeal lymphatic reconstruction after rmTBI; among them, A, B. Representative images show buds near the transverse sinus and meningeal lymphatic vascular loops and lymphatic hotspots (injured sites), C. Representative images show dcln and OVA-555 drainage, D, E, F, G. Comparison of the percentage of the area coverage of whole-mount Lyve-1 antibody staining, the number of loops and buds in the meninges of different groups; n = 6, ns: no statistical difference, ***p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0040] Figure 7 Plasmid map of NAMPT. Detailed implementation methods

[0041] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0042] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions. For example, the fourth edition of "Molecular Cloning: A Laboratory Manual" edited by Green and Sambrook was published in 2013, or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0043] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0045] Definitions For the convenience of understanding the present technology, some terms and phrases are defined below.

[0046] rmTBI: Repetitive mild traumatic brain injury.

[0047] MSCs: Mesenchymal stem cells, mesenchymal stem cells.

[0048] iPSC-MSC-sEV: Small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells.

[0049] NAMPT: Nicotinamide phosphoribosyltransferase.

[0050] Small Extracellular vesicles: sEV: Small extracellular vesicles.

[0051] MSC-sEV: Small extracellular vesicles derived from mesenchymal stem cells.

[0052] MOI: Multiplicity of Infection, multiplicity of infection.

[0053] The terms "drug", "agent", "therapeutic agent", or "reagent capable of being used for treatment" are used interchangeably and refer to a molecule that confers some beneficial effect when administered to a subject, including nucleotides or protein polypeptides. The beneficial effect includes the achievement of a diagnostic determination; the amelioration of a disease, symptom, disorder, or pathological condition; the reduction or prevention of the onset of a disease, symptom, disorder, or pathological condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[0054] As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) has a disorder (e.g., a disorder caused by a disease-related gene defect).

[0055] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a beneficial or desired result. The therapeutically effective amount can vary depending on one or more of the subject being treated and the disease condition, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art.

[0056] As used herein, the process of "administering or acting on or inhibiting..." to an individual can occur in vitro, ex vivo, or in vivo.

[0057] Intranasal administration is a new route of administration to the central nervous system. It has the advantages of high efficiency, non-invasiveness, and repeatable administration, and is widely used in the research of treating central nervous system diseases.

[0058] In the present invention, small extracellular vesicles are used as a delivery system for carrying NAMPT (NAMPT-sEV), that is, small extracellular vesicles highly expressing NAMPT. Through intranasal administration, it is observed whether it can protect rmTBI mice from long-term memory defects. The research shows that NAMPT-sEV significantly increases the expression of SIRT1, maintains the polarity of AQP4, promotes lymphangiogenesis to reduce the ac-tau level, and inhibits neuroinflammation. These results also indicate that NAMPT-sEV is a promising treatment method for preventing long-term memory loss in rmTBI patients.

[0059] The present invention will be further described in detail below in conjunction with specific embodiments.

[0060] Example 1

[0061] The materials and methods involved in the experiments in this example are as follows.

[0062] Source of animals in the following experiments: Male C57 BL / 6 mice were purchased from GemPharmatech Co., Ltd. and raised at the Guangdong Laboratory Animal Monitoring Institute, China. These eight-week-old mice were raised in a 12:12 hour light-dark cycle under specific temperature and humidity conditions. Animals were randomly selected and assigned to different treatment groups.

[0063] Preparation and identification of NAMPT-iPSC-MSCs

[0064] The inventors found that the choice of lentiviral packaging system would affect the infection efficiency of the lentivirus obtained by packaging the recombinant expression vector containing the NAMPT protein coding gene of the present invention on recipient cells, especially on stem cells. The present invention preferably co-transfects the recombinant expression vector containing the NAMPT protein coding gene and the packaging plasmid into a suspension cell packaging system to prepare lentivirus, and a lentivirus supernatant with higher virus titer can be obtained. Further, after obtaining the virus supernatant, a suitable concentration method (secondary high-speed centrifugation) can further reduce impurities and increase the virus titer, and increase the activity of the recipient cells infected by the virus.

[0065] In the present invention, the recombinant expression vector is packaged into lentivirus and secreted into the packaging cell culture medium by using a suspension cell packaging system, and then the lentiviral vector is extracted and concentrated by combining with the ultracentrifugation method. The obtained lentiviral vector can more efficiently infect stem cells under the same MOI infection condition, so that the NAMPT protein is stably and abundantly expressed in stem cells.

[0066] The centrifugal force of the high-speed centrifugation method is 81000g - 83000g, and the centrifugation time is 1h - 2h; more preferably, the centrifugal force of the high-speed centrifugation is 81500g - 82500g, and the centrifugation time is 1.5h - 2h. In this example, it is preferred to perform two high-speed centrifugation concentrations, each centrifuging at 82000g for 1.5h.

[0067] Exploration and attempt were made to transfect 293T cells using recombinant lentiviral vectors constructed with plasmids containing different promoters and structures, including plasmids with CMV and EF1α dual promoters (LV-CMV-MCS-EF1a-coGFP-T2A-Puro-WPRE), Ubi promoter (LV-Ubi-H2B-EGFP-P2A-ENVA-WPRE), SFFV and CMV dual promoters (LV-SFFV-H2B-EGFP-6×His-CMV-mCherry-T2A-EGFP-WPRE), CBA and CMV dual promoters (LV-CBA-EGFP-6×His-CMV-mCherry-T2AEGFP-WPRE), and EF1α and CMV dual promoters (LV-EF1α-CMV-mCherry-P2A-EGFP-WPRE) as the backbone.

[0068] The results showed that the recombinant lentiviral vector packaged with the plasmid with LV-EF1α-CMV-mCherry-P2A-EGFP-WPRE as the backbone could efficiently infect 293T cells, while the lentiviral vectors obtained by packaging the above other plasmids had unsatisfactory infection effects and very low infection efficiency. It can be seen that the structure of the plasmid and the selection of the promoter have an important impact on the introduction and expression of the NAMPT protein-coding gene. Compressing some unnecessary components and tags such as Flag in the vector sequence, and using the recombinant lentiviral vector with CMV and EF1α dual promoters (LV-EF1α-CMV-mCherry-P2A-EGFP-WPRE) is most suitable for constructing a lentiviral vector for the NAMPT protein-coding gene and introducing the NAMPT gene into recipient cells. Therefore, it was selected to establish a stable transfection strain.

[0069] Using the three-plasmid method, in Opti-MEM medium, the NAMPT (Gene ID: 10135, see the map in Figure 7 ) plasmid (LV-EF1α-NAMPT-CMV-mCherry-P2A-EGFP-WPRE) and auxiliary plasmids (pspax2 plasmid and pMD2G plasmid) were used to transfect HEK293T cells to generate lentiviral vector particles. Dulbecco's modified Eagle's medium (DMEM medium; Cytiva, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; using Gibco, Grand Island, NY, USA) was used for culture. The supernatant was collected after 6 h, and the lentiviral vector was concentrated and extracted by performing two high-speed centrifugations (centrifuging at 82000g for 1.5 h each time) at 48 h.

[0070] The above NAMPT recombinant lentiviral vector was transfected into iPSC-MSCs at a specific concentration (50 - 150 MOI, preferably 100 MOI in this example), incubated for 6 h, and after 2 days of infection, the successfully infected cells were screened with purinomycin (InvivoGen, San Diego, CA, USA), the unsuccessfully infected cells were removed, and the efficiency of cell infection was confirmed using a fluorescence microscope.

[0071] Collect NAMPT-iPSC-MSCs supernatant: In tissue culture dishes, NAMPT-iPSC-MSCs were cultured using high-glucose DMEM medium (Cytiva, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), basic fibroblast growth factor (Thermo Fisher Scientific, Waltham, MA, USA), and epidermal growth factor (Thermo Fisher Scientific, Waltham, MA, USA) (NEST, Wuxi, Jiangsu, China). After NAMPT-iPSC-MSCs reached 80% density, the cells were washed and cultured with chemically defined protein-free (CDPF) medium (CD-CHO; Gibco, Grand Island, NY, USA), HT supplement (Gibco, Grand Island, NY, USA), l-glutamine (Gibco, Grand Island, NY, USA), D-(+)-glucose (Sigma-Aldrich, St. Louis, MO, USA), NEAA (Gibco, Grand Island, NY, USA), and vitamin solution (Gibco, Grand Island, NY, USA). After 6 h, the medium was discarded and fresh CDPF medium was replaced. After 42 h, the CDPF medium supernatant was harvested for small extracellular vesicle isolation.

[0072] Isolation and identification of NAMPT-sEV: NAMPT-sEV was isolated. In this example, an anion exchange resin (Cytiva, Pittsburgh, PA, USA) was loaded into an eco-pac chromatography column (Bio-Rad Laboratories, Hercules, CA, USA) and equilibrated with an equilibration buffer (containing 50 mM sodium chloride and 50 mM sodium phosphate buffer in sterile injection water). The supernatant was loaded onto the column, washed with a washing solution (100 mM sodium chloride and 50 mM sodium phosphate buffer, sterile injection water), and continuously eluted 8 times with an elution solution (containing 500 mM sodium chloride and 50 mM sodium phosphate buffer in sterile injection water). The concentration of sEV in 8 fractions was measured using a Bradford protein concentration assay kit (Dingguo, Beijing, China). The fraction with the highest sEV concentration was collected and dialyzed overnight in a phosphate buffer solution (PBS, 0.01 M) at 4°C. The sEV was further concentrated using an ultrafiltration centrifugal filtration device (50 kDa, Millipore, Billerica, MA, USA).

[0073] In addition to the above method in this example, a tangential flow (TFF) method can also be used. Using a Sartorius (Goettingen, Germany) 1-1000 kD pore size TFF filter cartridge (preferably, 100 kD is used), the CDPF culture medium supernatant was concentrated 10-100 times (preferably, 60 times), washed and filtered 5-10 times (preferably, 6 times) with PBS or other required buffer solutions, and the sEV was further concentrated with or without an ultrafiltration centrifugal filtration device (50 kDa, Millipore, Billerica, MA, USA) according to the use concentration.

[0074] Using a Bradford protein concentration assay kit and nanoparticle tracking analysis (NTA, NanoSight NS300; Malvern, UK); its structure was confirmed by transmission electron microscopy (Hitachi, Minato-ku, Tokyo, Japan), and the overexpression of NAMPT on sEV was detected by western blotting. NAMPT-sEV was obtained.

[0075] Controlled cortical impact Traumatic Brain Injury (Controlled cortical impact TBI): Male C57BL / 6 mice (n = 40) were repeatedly subjected to mild controlled cortical impact (CCI) injury or sham operation (n = 10). With 1.25% avertin (tribromoethanol, 1.25 g; tert-amyl alcohol, 2.5 mL dissolved in 97.5 mL of ddH2O, Sigma Aldrich), the mice were gently placed in an adapter (RWD, 68030) to fix the skull. Under sterile conditions, a small cranial window was drilled 2 mm posterior to the bregma, 2 mm lateral to the midline, with a diameter of approximately 1 mm. The skull cap was carefully removed without damaging the underlying dura mater. Before inducing injury, the impactor tip was tilted and perpendicular to the exposed cortical surface.

[0076] The CCI model was prepared by impacting with a precision impactor (RWD, 68099II) at a speed of 3 ms -1 and a depth of 2 mm, with a dwell time of 200 ms. The tapping was repeated 3 times every 48 hours. After hemostasis, the wound was sutured, and the mice were placed on a warm pad at 36 - 37 °C for recovery.

[0077] Intranasal treatment: One hour after the strike, 4% isoflurane (RWD, R510 - 22 - 10) was administered with a small animal anesthesia machine (RWD, R500IE) to achieve stable anesthesia. Before treatment, the mice were permeabilized with a 20 μL hyaluronidase (SigmaAldrich, CAS37326 - 33 - 3) solution to enhance the permeability of the nasal mucosa. After 30 minutes, the mice were gently held with their abdomens up and heads down, and 40 μL of NAMPT - sEV (rmTBI + NAMPT - sEV, n = 10), MSC - sEV (rmTBI + sEV, n = 10), and saline (rmTBI + saline, n = 10) were administered each time, for a total of three administrations.

[0078] Modified Neurological Severity Score (mNSS) to evaluate neurological function: The mNSS was used to evaluate the neurological function of the mice before injury, 1 day and 3 days after surgery. The mNSS evaluated the reflex, balance, sensory, and motor functions of the mice and scored the abnormal behaviors in each task. The mNSS score ranged from 0 to 18, and a higher score indicated more severe nerve injury.

[0079] Rotar - Rod test: Motor coordination was measured using the rotarod test 1 month after rmTBI. Briefly, each mouse was placed on an accelerating rotarod and rotated at a speed of 4 - 40 rpm for 5 minutes. The mice were tested three times a day for three days, with a 30 - minute interval between each test. The latency to fall was recorded for analysis. If the mouse remained on the rod throughout the test, a latency of 300 seconds was assigned. The average time for each mouse in the three tests was calculated.

[0080] Morris water maze: The Morris water maze (MWM) was conducted 1 month after rmTBI, as Figure 1 shown. Briefly, 40 mice were trained four times a day for the first five days and then underwent an exploratory test on the sixth day. The latency of the mice to reach the platform, the exploration time in each quadrant, and the number of times the platform was crossed were recorded.

[0081] Novel object recognition test: The novel object recognition (NOR) test was used to evaluate recognition memory. Mice were placed in the arena chamber (50×50×50 cm) and allowed to acclimate for 1 h. Two identical objects were placed at equal distances from the center of the arena, and the mice were allowed to freely explore the environment for 10 min. The object exploration time was defined as the nasal or oral contact time. One hour later, one of the objects was replaced with a novel object of similar size but different shape, and then the mice were returned to the open arena. The exposure time to the old and new objects was recorded for 10 min. The time difference between the exploration of the novel object and the familiar object was calculated as an index of novel object recognition.

[0082] Intracisternal injection: Mice were anesthetized with 1.25% avertin. The neck skin was shaved with iodine and 70% ethanol. Ophthalmic solution was used to prevent dry eyes, and the mouse head was fixed in a mouse adapter. After skin incision, the muscle layer contracted, and the cisterna magna was exposed. A Hamilton syringe was connected to a 33-gauge needle, and 10 μL of Alexa Fluor 555-conjugated ovalbumin (OVA-555; was injected into the cisterna magna over 10 min at a rate of 1 μL / min using an infusion pump (Harvard Apparatus, Holliston, MA). After injection, the syringe was left in place for 2 min to prevent cerebrospinal fluid reflux. Then the mice were perfused and fixed with 4% paraformaldehyde (PFA) for 30 min.

[0083] Immunofluorescence staining: Mice were perfused transcardially with 1× phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde in PBS (pH 7.4) under anesthesia. The deep cervical lymph nodes (dCLNs) and brains were carefully dissected and fixed in 4% PFA at 4 °C for 12 h. The dCLNs and brain tissues were dehydrated in 20% and 30% sucrose gradients and embedded in cryosections. The dCLNs and brains were coronally sectioned (30 and 15 μm). For whole-mount meningeal collection, the skin and muscle were stripped from the outer skull, the skull cap was removed with surgical scissors, and fixed in 2% PFA for 12 h at 4 °C. The meninges (dura mater and arachnoid mater) were carefully dissected from the skull using Dumont #5 forceps (Fine Science Tools). Then the whole meninges were transferred to PBS with 0.05% azide at 4 °C until further use. Brain sections were boiled in citrate buffer in a microwave for 5 min, treated with 0.3% Triton X-100 and 10% goat serum at room temperature for 1 h, and then incubated with primary antibodies overnight at 4 °C. The primary antibodies were [rabbit anti-Iba1 antibody (1:3000, Wako, Japan, Cat#019-19741), mouse anti-GFAP (1:3000, Cell signaling technology, Ca#3670), mouse anti-Tau (1:100, Abcam, USA, ab80579), rabbit anti-NeuN (1:5000, Millipore, ABN78), rabbit anti-AQP4 (1:2000, Alomone, Israel, AQP-014), mouse anti-AnkG (1:200, UC Davis / NIH NeuroMab Facility, Davis, CA, USA, N106 / 36, Ca#75-146)], and then incubated with secondary antibodies diluted in PBS containing 10% normal goat serum [1:3000 anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa 488 Conjugate), Cell signaling technology, USA; 1:30 anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa 555 Conjugate, Cell signaling technology, USA) at room temperature for 1 h. Images were acquired using a Nikon fluorescence microscope (Nikon, Japan) and a confocal microscope (Leica, Germany).

[0084] Immunoblotting: Four mice in each group were perfused with 50 mL of pre-cooled 0.1 m PBS. Cortical and hippocampal tissues were sonicated in RIPA buffer (Sigma-Aldrich, R0278) containing a protease and phosphatase inhibitor mixture (Thermo Scientific, #1861284), 1 mM phenylmethylsulfonyl fluoride (Sigma Aldrich, P7626), and histone deacetylase inhibitors such as 5 mM nicotinamide (Sigma-Aldrich, 72340) and 1 mM trichostatin a (Sigma-Aldrich, T8552). The lysate was centrifuged at 12,000 rpm for 25 min at 4 °C, and the protein concentration of the supernatant was detected using the Pierce TM Microplate BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (EpiZyme, PG112, CHN) at 200 V with 30 μg / gel lane, and the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) at 300 mA for 2 h. The membrane was blocked in 5% non-fat milk powder (R&D Systems, Minneapolis, MN, USA) for 1 h and incubated overnight at 4 °C with the following primary antibodies: mouse anti-Tau protein (1:2000, Abcam, USA, Ca#ab80579), rabbit anti-ac-tau protein (1:1000, USBiological, USA, Ca#542907), mouse anti-SIRT1 protein (1:1000, Abcam, USA, Ca#ab110304), and rabbit anti-β-actin protein (1:3000, Affinity, USA, Ca#AF7018). The membrane was rinsed with 1× tris-buffered saline containing Tween 20 (TBST). Then the membrane was incubated with a secondary antibody (anti-mouse IgG, horseradish peroxidase-conjugated antibody, or anti-rabbit IgG, horseradish peroxidase-conjugated antibody, both from Cell Signaling Technology, USA) in the darkroom for 1 h. The target protein bands were visualized and quantitatively analyzed using a chemiluminescence imaging system.

[0085] Immunohistochemical quantification: Analyze the polarity of AQP4. The AQP4 polarity was defined as the ratio of low-intensity to high-intensity regions based on the location of the overall immunoreactivity of AQP4. To analyze the mislocalization of tau protein, a 40x image was used. Briefly, as previously described, pathological tau was analyzed, which is characterized by its mislocalization to the somatodendritic compartment of neurons. In this study, a mislocalization tau index was introduced, defined as the ratio of the number of neurons surrounded by tau to the number of normal neurons, to analyze tau mislocalization.

[0086] Statistical analysis: The results were statistically compared using Prism software (La Jolla, CA, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze histological and western blotting results. One-way ANOVA and Tukey's post hoc test were used to analyze the differences between groups. A P < 0.05 was considered statistically significant. P values ≥ 0.05, 0.01, 0.001, 0.0001 were represented by *, **, ***, ****, respectively.

[0087] The above experimental results of this example are as follows:

[0088] I. Preparation and characterization of NAMPT-sEV

[0089] The preparation of NAMPT-iPSC-MSCs and NAMPT-sEV was as Figure 1 shown in A. NAMPT-iPSC-MSCs were successfully established, and almost 100% of NAMPT-MSCs were identified by mCherry positive staining ( Figure 1 B). Enriched MSC-sEV and sEV highly expressing NAMPT (NAMPT-sEV) were obtained from the supernatants of MSCs and NAMPT-iPSC-MSCs using anion exchange chromatography. NTA showed that the peak size of MSC-sEV was 87 nm and that of NAMPT-sEV was 98 nm, generally between 50 and 200 nm ( Figure 1 C). MSC-sEV and NAMPT-sEV were characterized by TEM as double lipid layers and nanoscale particles, respectively ( Figure 1 D). Western blotting showed an increased level of NAMPT protein in NAMPT-sEV cells. In addition, the expression of CD63, CD9, CD81, and ALIX was successfully detected in MSC-sEV and NAMPT-sEV ( Figure 1 E).

[0090] II. Distribution of NAMPT-sEV in the mouse brain

[0091] To determine whether intranasally administered small extracellular vesicles are effectively delivered to the brain, their distribution was visualized using mCherry-sEV. This process is shown in Figure 1 Figure F. After IN treatment, mCherry-sEV was visible in different regions of the cortex and hippocampus, including CA1, CA3, and DG ( Figure 1 Figure G).

[0092] III. NAMPT-sEV improves cognitive function in rmTBI mice

[0093] Neurobehavioral tests are shown in Figure 2 Figure A. Eight-week-old mice were treated with mNSS on the 1st and 3rd days after rmTBI. As shown in Figure 2 Figure B, the mNSS scores of the rmTBI+sEV and rmTBI+NAMPT-sEV groups were lower than those of the rmTBI+Saline group on the 1st day after rmTBI. This indicates that MSC-sEV and NAMPT-sEV have neuroprotective effects in promoting the recovery of neurological function in rmTBI mice. One month after rmTBI, there was no significant difference in the fall latency among the four groups in the rotarod test ( Figure 2 Figure C). This finding indicates that rmTBI does not cause long-term motor dysfunction. However, in the MWM and NOR tests, significant memory impairment was observed in rmTBI mice. After IN treatment with MSC-sEV and NAMPT-sEV, the cognitive function of the rmTBI+sEV and rmTBI+NAMPT-sEV groups in the MWM was improved. Specifically, in terms of the number of times the mice crossed the platform on the test day, the rmTBI+NAMPT-sEV group performed much better than the rmTBI+sEV group. This indicates that NAMPT further promotes the protection of long-term cognitive function after rmTBI ( Figure 2 Figures F-I). In the NOR test, the cognitive memory of the rmTBI+Saline group was impaired. The Sham and rmTBI+NAMPT-sEV groups spent more time exploring the new object than the old object. However, there was no significant difference between the rmTBI+Saline group and the rmTBI+sEV group ( Figure 2 Figure D and Figure 2 Figure E). These results indicate that although MSC-sEV improves the cognitive function of rmTBI mice, NAMPT-sEV significantly enhances the effects of mouse memory and recognition.

[0094] Figure 2 In Figure E, the representative movement trajectories in the novel object test show that the Sham and rmTBI+NAMPT-sEV groups spent more time exploring the new object, while the other group explored both objects on average.Figure 2 In F, representative swimming paths in the spatial memory probe test showed that mice in the rmTBI + sEV group and the rmTBI + NAMPT-sEV group crossed the former platform more times, and mice in the rmTBI + NAMPT-sEV group spent more time in the target quadrant.

[0095] IV. NAMPT-sEV increases SIRT1 expression to reduce tau acetylation

[0096] As a key enzyme in NAD+ recycling, NAMPT can recycle more NAD+ to provide substrates for SIRT1, which plays a role in deacetylation. Based on these hypotheses, the deacetylation ability of NAMPT-sEV was further tested. As expected, rmTBI led to a decrease in SIRT1 expression and an increase in tau acetylation in the cortex and hippocampus of mice ( Figure 3 ). Compared with the rmTBI + Saline and rmTBI + sEV groups, SIRT1 expression in the cortex and hippocampus of mice in the rmTBI + NAMPT-sEV group was significantly increased. This indicates that NAMPT-sEV activates SIRT1 and promotes its expression. As Figure 3 shown, the number of ac-tau in the cortex and hippocampus of the rmTBI + NAMPT-sEV group was significantly reduced. There was no significant statistical difference between the rmTBI + Saline group and the rmTBI + sEV group. Taken together, these results verified the ability of NAMPT-sEV to promote ac-tau deacetylation by increasing SIRT1 expression.

[0097] V. NAMPT-sEV can prevent axon initial segment degeneration (AIS) and pathological tau mislocalization, and reduce neuronal loss

[0098] It has been reported that tau acetylation is related to the number of ankyrin g (AnkG), a major component of AIS. As a guide to protect neurons, AIS maintains the main distribution of tau in the axon rather than mislocalizing to the somatodendritic compartment of neurons. The results of the study showed that rmTBI induced AIS degeneration because the intensity of normalized AnkG was significantly reduced compared with the sham-operated group. In the two intervention groups, the AnkG content in the cortex and hippocampus of mice in the rmTBI + NAMPT-sEV group was much higher than that in the rmTBI + sEV group ( Figure 3 A- Figure 3D), which is attributed to the reduction of ac-tau. In addition, the pathological tau mislocalization in mice after rmTBI was significantly increased. In the rmTBI+Saline group and the rmTBI+sEV group, most tau proteins were located in the somatodendritic compartment of neurons. In contrast, tau proteins in the Sham and rmTBI+NAMPT-sEV groups were mostly linearly normally distributed on the axons of the cortex and hippocampus( Figure 4 E, Figure 4 F, Figure 4 H and Figure 4 I). Based on the above findings, the protective effect of NAMPT-sEV on the survival of cortical and hippocampal neurons was analyzed. In the cortex and hippocampus, there was no difference in the number of neurons between the Saline group and the rmTBI+NAMPT-sEV group. In addition, the number of neurons in the rmTBI+NAMPT-sEV group was significantly higher than that in the rmTBI+sEV and rmTBI+Saline groups. The expression in the rmTBI+sEV group was also higher than that in the rmTBI group( Figure 4 G and Figure 4 J). Taken together, these results indicate that NAMPT-sEV has profound neuroprotective effects in AIS and regulates tau distribution by deacetylating tau.

[0099] VI. NAMPT-sEV alleviates neuroinflammation in rmTBI mice

[0100] It has been reported that MSC-sEV has anti-neuroinflammatory effects. In this study, the anti-neuroinflammatory effects of MSC-sEV were further verified. After rmTBI, the intensity of Iba1 in the cortex and hippocampus was significantly increased, suggesting the activation of microglia-related neuroinflammation. Similar to previous studies, the intensity of Iba1 in the rmTBI+sEV and rmTBI+NAMPT-sEV groups was significantly reduced. However, there was no significant difference between the two intervention groups( Figure 5 A - Figure C). Since astrocytes also play an important role in neuroinflammation, the astrogliosis among groups was also studied. As Figure 5 shown in D - Figure F, rmTBI caused a significant increase in the expression of GFAP throughout the brain, suggesting astrogliosis. Compared with the rmTBI+Saline group, the staining intensity of GFAP in the cortex and hippocampus of the two groups of mice was significantly reduced. This further demonstrated that MSC-sEV inhibited microglial activation and astrogliosis after rmTBI, but no additional anti-inflammatory effect of NAMPT was observed.

[0101] VII. NAMPT-sEV improves the lymphatic and meningeal lymphatic systems and promotes the clearance of acetylated tau

[0102] As a pathological protein, ac-tau is removed by the brain's lymphatic system and the meningeal lymphatic system. To explore the drainage function of the lymphatic system, the polarity of AQP4 was mainly studied. It plays a crucial role in the exchange of cerebrospinal fluid and interstitial fluid. Normally, AQP4 is located at the end feet of highly polarized astrocytes. Previously, the protective effects of small extracellular vesicles and NAMPT-sEV on astrocytes were demonstrated. The APQ4 polarity in the cortex and hippocampus regions of the rmTBI+sEV and rmTBI+NAMPT-sEV groups was superior to that of the rmTBI+Saline group, but there was no significant difference between the two groups. Therefore, MSC-sEV may contribute to more effectively draining and removing ac-tau from the brain parenchyma, while NAMPT has no further effect.

[0103] After the lymphatic system drains from the brain parenchyma, the meningeal lymphatic vessels further clear ac-tau. Traumatic brain injury damages the structure and function of the meningeal lymphatic vessels, leading to waste dysfunction and pathological protein clearance. The research results showed that rmTBI significantly damaged the meningeal lymphatic vessels. Compared with the sham, rmTBI+sEV, and rmTBI+NAMPT-sEV groups, the buds and clusters of the meningeal lymphatic system in the rmTBI+Saline group, indicating lymphangiogenesis and reconstruction, were much higher ( Figure 6 A, Figure 6 B, and Figure 6 D- Figure 6 F), which indicated that MSC-sEV and NAMPT-sEV promoted lymphangiogenesis and meningeal lymphatic vessel reconstruction. Correspondingly, after injecting OVA-555 into the cisterna magna, the fluorescence intensity of OVA-555 in the bilateral deep cervical lymph nodes increased significantly ( Figure 6 C, and Figure 6 G). This indicated that the function of the meningeal lymphatic system was well preserved.

[0104] The results of this study revealed that NAMPT-sEV plays a neuroprotective role in the rmTBI mouse model through multiple mechanisms, especially by activating SIRT1 to deacetylate tau. Although it has been reported that phosphorylated tau protein plays an important role in human TBI, previous studies have shown that tau acetylation dominates in the acute phase after TBI, and the ac-tau protein in cerebrospinal fluid increases significantly. In this study, persistent tau acetylation was observed in the rmTBI mouse model. By reducing ac-tau, IN treatment with NAMPT-sEV 1 hour after TBI injury improved cognitive function. Therefore, deacetylation of tau protein after traumatic brain injury may be crucial for preventing long-term memory deficits in the acute phase.

[0105] For neurological diseases, effectively delivering drugs to the central nervous system has always been a problem. Intrathecal injection is the most effective method. However, its invasiveness and inconvenient operation limit its application. Intranasal therapy is considered to have three routes to enter the central nervous system: the olfactory parenchyma route, the olfactory cerebrospinal fluid route, and the trigeminal cerebrospinal fluid route, making it a promising method for drug delivery to the central nervous system. Studies comparing different administration routes have found that IN therapy not only reaches the central nervous system fastest but also has the highest concentration in the central nervous system. In this study, as expected, after IN treatment, small extracellular vesicles were found to be effectively delivered to the mouse brain parenchyma, including the cortex and hippocampus.

[0106] Previous studies have reported that MSC-sEVs have neuroprotective effects. Due to their ability to cross the blood-brain barrier (BBB), the key enzyme NAMPT involved in deacetylation was loaded into MSC-sEVs. The research results also showed that MSC-sEVs contributed partially to the protection of cognitive function after rmTBI, but NAMPT-sEVs were superior to MSC-sEVs (see Figure 2 H, Figure 3 C- Figure 3 F, Figure 4 ).

[0107] NAMPT is a key enzyme in the NAD+ (the deacetylation substrate of SIRT1) recycling pathway. After rmTBI, tau acetylation in the mouse brain increased significantly. After treatment with NAMPT-sEVs by IN, a significant increase in SIRT1 expression was observed, followed by a decrease in ac-tau expression in the cortex and hippocampus. This indicates that NAMPT-sEVs exert a deacetylation effect by activating the SIRT1 pathway.

[0108] Based on the previous results, it can be expected that the maintenance of AIS and the increase in normalized tau are associated with a decrease in ac-tau levels. The amount of normal AnkG increased significantly in the rmTBI+NAMPT-sEV group. Correspondingly, in the rmTBI+NAMPT-sEV group, tau protein was limited and well located on the axons rather than entering the somatodendritic compartment of neurons. Overall, the number of neurons in the cortex and hippocampus was better preserved. As a result, the cognitive function of mice in the rmTBI+NAMPT-sEV group was significantly improved.

[0109] This study also demonstrated the anti-inflammatory effect of MSC-sEV by inhibiting microglial activation and astrocyte formation. The effect of NAMPT on neuroinflammation has been controversial. Some studies reported that microglia secrete NAMPT through small extracellular vesicles to promote neuroinflammation, and inhibitors targeting NAMPT can alleviate this inflammatory process. In contrast, other studies reported that NAMPT inhibits neuroinflammation by activating the SIRT1 signal. Notably, in this study, no additional effect of NAMPT on inflammation was found.

[0110] As a pathological protein, ac-tau was also evident in the lymphatic system and the meningeal lymphatic system. Previous studies have shown that both brain clearance systems are impaired after rmTBI. In this study, it was found that both MSC-sEV and NAMPT-sEV could inhibit astrocyte formation and maintain AQP4 polarity, which is beneficial to the function of the lymphatic system. TBI has also been reported to cause morphological changes and dysfunction, and the study also found that MSC-sEV promoted the reconstruction of damaged meningeal lymphatic vessels. The recovery of meningeal lymphatic function was also observed in the rmTBI + sEV and rmTBI + NAMPT-sEV groups after injecting fluorescent dye into the cisterna magna. Although MSC-sEV and NAMPT-sEV may promote the clearance of ac-tau by protecting the lymphatic system and the meningeal lymphatic system, the further mechanism remains to be explored.

[0111] In summary, through research, it was found that treatment with NAMPT-sEV reduced the ac-tau protein through multiple mechanisms, making it a promising treatment for preventing long-term cognitive dysfunction in patients with rmTBI.

[0112] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of small extracellular vesicles highly expressing NAMPT in the preparation of a product for treating traumatic brain injury.

2. The application according to claim 1, wherein, The traumatic brain injury is mild traumatic brain injury.

3. The application according to claim 1, wherein The traumatic brain injury is repetitive mild traumatic brain injury.

4. The application according to claim 1, wherein, The small extracellular vesicles highly expressing NAMPT are small extracellular vesicles derived from mesenchymal stem cells. Preferably, the mesenchymal stem cells are derived from induced pluripotent stem cells, or from umbilical cord, bone marrow, adipose tissue, urine, or menstrual blood.

5. The application according to any one of claims 1-4, wherein, The small extracellular vesicles highly expressing NAMPT are obtained by the following preparation method: constructing a recombinant lentiviral vector containing the NAMPT protein-coding gene, transfecting mesenchymal stem cells to obtain mesenchymal stem cells with stable expression of the NAMPT protein-coding gene, culturing, collecting the culture supernatant, and extracting small extracellular vesicles therefrom. Preferably, the promoter for initiating the expression of the NAMPT protein-coding gene on the recombinant lentiviral vector is a dual promoter containing CMV and EF1α; and / or Preferably, the recombinant lentiviral vector is transfected into mesenchymal stem cells at a multiplicity of infection of 50 - 150.

6. The application according to claim 1, wherein The product is a drug, and the dosage form of the drug is a dosage form suitable for intranasal administration, or the dosage form of the drug is a dosage form suitable for intravenous administration.

7. The application according to any one of claims 1-6, wherein, The application includes one or more of the following ways: Increasing the expression of Sirtuin 1 in a subject; Inhibiting neuroinflammation in a subject, maintaining the polarity of aquaporin-4, and promoting the repair of the lymphatic system and the meningeal lymphatic system; Reducing neuronal loss in a subject; Improving the cognitive function of a subject.

8. Use of small extracellular vesicles highly expressing NAMPT in the preparation of a product for treating Alzheimer's disease, or use of small extracellular vesicles highly expressing NAMPT in the preparation of an anti-aging product, or use of small extracellular vesicles highly expressing NAMPT in the preparation of a product for preventing and treating dementia.

9. The application according to claim 8, wherein, The application includes promoting the clearance of acetylated tau protein in a subject.

10. A drug for treating traumatic brain injury, wherein, The active ingredient of the drug includes small extracellular vesicles highly expressing NAMPT. Preferably, the small extracellular vesicles highly expressing NAMPT are obtained by the following preparation method: constructing a recombinant lentiviral vector containing the NAMPT protein-coding gene, transfecting mesenchymal stem cells to obtain mesenchymal stem cells with stable expression of the NAMPT protein-coding gene, culturing, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

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