Application of reagent for regulating and controlling expression of miRNA of extracellular vesicles in relieving cognitive impairment

Abstract

The invention provides application of a reagent for regulating and controlling miRNA expression of extracellular vesicles in relieving cognitive impairment, and researches find that the extracellular vesicles released by intestinal epithelial cells of inflammatory bowel disease and aging mice are rich in miRNA-16-5p, and the miRNA-16-5p can be transferred into hippocampal neural stem cells. The miRNA-16-5p is used for delaying hippocampal neurogenesis by targeting CDK1 protein, so that inflammatory bowel diseases and age-related cognitive impairment are induced. In addition, the invention also finds that the skatole generated by the bacteroides promotes cognitive impairment by up-regulating miRNA-16-5p in individuals suffering from inflammatory bowel diseases and aging. Compared with the prior art, another intestinal bacterial metabolite vitamin B12 can down-regulate miRNA-16-5p, so that the development of IBD and senescence individual cognitive impairment is relieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology technology, and particularly relates to the use of a reagent for regulating the expression of extracellular vesicle miRNA in alleviating cognitive impairment. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disease, including ulcerative colitis (UC) and Crohn's disease (CD), with characteristic relapse and remission periods. IBD has been shown to be a risk factor for cognitive impairment (CI). However, the mechanism of CI during IBD, especially the direct effect of IBD on hippocampal neurons, remains an unknown field. Aging is a normal physiological phenomenon characterized by systemic chronic inflammation. As people age, their cognitive abilities decline. In addition, how aging induces brain damage is largely unknown, and there is a lack of effective intervention strategies for IBD and age-related cognitive impairment.

[0003] Extracellular vesicles (EVs) are highly heterogeneous nanoscale particles released by almost all cells. Increasing evidence suggests that EVs play a key role in information exchange (proteins, miRNAs, metabolites, etc.) between cells. EVs have been shown to be involved in the formation of CI. EVs from the central nervous system are involved in the development of CI in Alzheimer's disease. In addition, peripheral adipocyte-derived EVs can cross the blood-brain barrier (BBB) and promote obesity-related CI by inducing synaptic damage. Therefore, EVs can mediate cross-organ communication between the gut and the brain, thereby participating in the occurrence of IBD and aging-related CI.

[0004] Germ-free (GF) mouse models have demonstrated that the commensal gut microbiota (GM) is crucial in shaping brain function and behavior. Acetate-producing bacteria in GM have a protective effect on CI in diabetic mice. GM also leads to obesity-related CI. During the development of IBD, the composition of GM has changed greatly. Therefore, GM may be involved in the pathogenesis of IBD-related CI. In addition, GM is closely related to aging. Fecal microbiota transplantation from aged rats to young rats impairs the cognitive behavior of young rats. Among the various products of GM, metabolites are crucial for gut-brain cross-talk and are therefore related to age-related CI. Isovalamine promotes age-related CI by driving microglial cell death. Oxidized trimethylamine mediates synaptic damage and promotes the development of age-related CI. However, whether GM mediates gut-brain cross-talk through gut EVs in IBD and age-related CI remains to be studied.

[0005] Therefore, it is of great significance to study the mechanism of IBD and aging-related CI and to provide new strategies and means for the treatment of related CI. SUMMARY

[0006] In view of the problems existing in the prior art, the purpose of the present application is to provide the use of a reagent for regulating the expression of extracellular vesicle miRNA in relieving cognitive impairment. Through research, it is found that the extracellular vesicles (IEC-sEVs) released by the intestinal epithelial cells of inflammatory bowel disease (IBD) and aging mice degrade CDK1 of hippocampal nerve cells through miRNA-16-5p, inhibit the proliferation of hippocampal nerve cells, thereby reducing the number of mature neurons, and thus induce the occurrence of cognitive impairment (CI). The research results of the present application provide strong evidence for supporting IBD and aging-mediated CI from the perspective of molecular mechanism, thereby providing a theoretical basis for preventing and treating IBD and aging-related CI.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] On the one hand, the present application provides the use of a reagent for regulating proteins or genes in extracellular vesicles for preparing a reagent for relieving cognitive impairment.

[0009] IBD has been considered as one of the high-risk factors of cognitive impairment, but there is still a lack of substantial evidence. Pain caused by the disease and long-term illness inevitably lead to anxiety, depression and other mental symptoms in patients, which also affects the patient's reaction to external things and slows down the action to a certain extent. However, it is not clear whether the neurons responsible for cognitive function in IBD patients are damaged and the corresponding molecular mechanism. In some embodiments of the present application, experimental research results show that compared with healthy mice, the hippocampal neural stem cells (NSCs) in the hippocampus of dextran sulfate sodium (DSS)-induced chronic colitis (IBD DSS ) mice decrease, the doublecortin (DCX) protein level, newborn neurons (NDNs) and DCX + cells also decrease, proving that chronic colitis damages the neurogenesis of the hippocampus.

[0010] In view of the importance of extracellular vesicles (EVs) in communication between different cells, in some embodiments, the present application verifies the important role of EVs in chronic colitis-induced CI through experiments. The results show that intestinal sEVs lead to CI in chronic colitis mice, and intestinal sEVs include A33 + IEC-sEVs and CD11c + DC-sEVs, so the present application finds in further experimental research that IEC-sEVs lead to CI in healthy mice, while DC-sEVs do not, and IEC-sEVs promote IBD-related CI by inhibiting hippocampal neurogenesis.

[0011] In some embodiments of the present application, it is also found that the intestinal sEVs of colitis can cross the blood-brain barrier and be absorbed by hippocampal cells, and compared with healthy mice, IBD DSS The permeability of the blood-brain barrier of the mouse is increased, and more IEC-sEVs enter the brain. Therefore, IEC-sEVs in the IBD state can also up-regulate effector molecules targeting the blood-brain barrier, increase the permeability of the blood-brain barrier, and allow more pathological sEVs to enter the central nervous system. In addition to inhibiting hippocampal neural stem cell proliferation, we found that IEC-sEVs from IBD mice also activated microglia, suggesting an increase in central inflammatory response, which can be one of the pathogenic mechanisms of CI. These results suggest that IEC-sEVs in the IBD state can exacerbate CI in multiple ways, and the specific mechanisms are worthy of further study.

[0012] Further, in order to study the specific pathway of IEC-sEVs damaging hippocampal neurogenesis, the present application performs bioinformatics analysis on the hippocampal tissue of IEC-sEVs-treated mice in some embodiments. Single-cell RNA sequencing results show that IEC-sEVs from IBD DSS The expression level of neural stem cell-related genes in the hippocampal neural stem cells of mice treated with IEC-sEVs (DSS-IEC-sEVs) of mice is significantly reduced; functional pathway enrichment analysis shows that the differentially expressed genes are mainly enriched in pathways related to nervous system diseases, cell signaling pathways, cell cycle and apoptosis, and cell structure and function, and GGPlot analysis shows that DSS-IEC-sEVs inhibit the cell cycle pathway the most significantly. Further experimental verification results show that DSS-IEC-sEVs cause neural stem cell cycle arrest at the G2 / M phase, and after DSS-IEC-sEVs treatment, the total CDK1 protein in hippocampal neural stem cells is significantly reduced, and the CDK1 inhibitor eliminates the DSS-IEC-sEVs-induced G2 / M phase arrest and CDK1 reduction, indicating that DSS-IEC-sEVs induce G2 / M phase arrest of hippocampal neural stem cells in a CDK1-dependent manner, thereby reducing hippocampal neurogenesis.

[0013] Further, the agent for regulating the extracellular vesicle protein or gene includes an agent for inhibiting the generation of the extracellular vesicle protein or an agent for inhibiting the expression of the extracellular vesicle gene.

[0014] To further explore how DSS-IEC-sEVs down-regulate CDK1 in neural stem cells, in some embodiments, the present application uses proteinase K and RNase I to digest the protein and RNA of electroporated DSS-IEC-sEVs, and it is found that miRNA plays an important role in exosome-mediated intercellular communication. Further verification results show that the level of miRNA-16-5p in the hippocampus of mice treated with DSS-IEC-sEVs is significantly up-regulated, CDK1 is a direct target of miRNA-16-5p, and specific overexpression of miRNA-16-5p and adeno-associated virus targeting neurons inhibit the proliferation of hippocampal neural stem cells and newborn neurons, while reducing CDK1 protein in cells, and intrarectal administration of an inhibitor specifically expressing miRNA-16-5p can increase the proliferation of hippocampal neural stem cells and newborn neurons and the content of CDK1 protein, and alleviate CI in mice. The above results show that under the condition of IBD, IEC-sEVs can degrade CDK1 in the hippocampus through miRNA-16-5p, inhibit the proliferation of hippocampal neural stem cells, thereby reducing the number of mature neurons, and leading to CI. Therefore, the research results of the present application provide strong evidence for supporting IBD-mediated CI from the perspective of molecular mechanism, thereby providing a theoretical basis for preventing and treating IBD-related CI.

[0015] Further, the gene is miRNA-16-5p, which has a nucleotide sequence as shown in Seq ID NO. 1.

[0016] Further, the cognitive dysfunction includes colitis and / or aging-induced cognitive dysfunction.

[0017] Since inflammation is a hallmark of aging, in some embodiments, the present application also reveals the corresponding mechanism of age-related CI through experiments, and finds similar changes in the intestinal microbiota of old mice and an increase in skatole. miRNA-16-5p in intestinal epithelial cells is also a potential target for treating age-related CI, and skatole induces the level of miRNA-16-5p in IEC-sEVs in aging mice, leading to age-related CI, and specific inhibition of miRNA-16-5p expression or blocking IEC-sEV release can effectively improve cognitive impairment caused by aging; VB12 alleviates the development of age-related CI by targeting miRNA-16-5p, so that the cognitive function of the elderly can be improved by supplementing VB12.

[0018] Further, the agent for inhibiting the expression of miRNA-16-5p includes vitamin B12.

[0019] The microbiota-gut-brain axis is widely involved in the pathological process of central nervous system diseases. In some embodiments, the present application finds by metagenomic sequencing that Bacteroides is enriched in the gut of IBD patients DSS The mouse is enriched in large quantities, and the library screening of intestinal microbial metabolites finds that the fecal odor produced by Bacteroides significantly up-regulates the level of miRNA-16-5p, and the colonization of Bacteroides in germ-free mice or the injection of fecal odor in healthy mice can cause the proliferation of hippocampal neural stem cells and newborn neurons and the inhibition of CDK1 protein, accelerating the development of CI. Therefore, the above research results provide ideas for the prevention and treatment of cognitive dysfunction promoted by microorganisms, for example, the balance of intestinal flora can be maintained by supplementing probiotics or prebiotics, or harmful microorganisms can be removed using antibiotics.

[0020] Although clinical studies have shown that VB12 can improve the cognitive function of the elderly, the related mechanism is not clear. Previous studies have shown that VB12 mainly improves cognitive function through its neurosupportive function, as VB12 is essential in the process of myelination. In addition, VB12 has an antioxidant function by reducing homocysteine in the body. However, in the research of the present application, the experimental results show that VB12 directly targets miRNA-16-5p, inhibits the inhibitory effect of IEC-sEVs on hippocampal neural stem cell proliferation, thereby improving CI in IBD and aging mice. In addition, we also detected that IBD patients and the elderly taking VB12 had lower levels of miRNA-16-5p in their feces, and the ability of EVs to inhibit the proliferation of human hippocampal neural stem cells was inhibited, indicating that VB12 may prevent and treat human IBD and age-related CI. In summary, our research reveals a mechanism that has not been recognized, that is, VB12 intervenes in IBD and age-related CI by promoting hippocampal neural stem cell proliferation. Therefore, VB12 should be used as a routine drug for the prevention and treatment of both types of CI.

[0021] Further, the vitamin B12 relieves induced cognitive dysfunction by promoting hippocampal neurogenesis.

[0022] Further, the vitamin B12 relieves cognitive dysfunction by promoting the production of CDK1 in the hippocampus.

[0023] In another aspect, the present application provides the use of miRNA-16-5p as a target for relieving colonitis and / or aging-induced cognitive dysfunction, characterized in that the miRNA-16-5p has a nucleotide sequence as shown in Seq ID NO. 1.

[0024] In another aspect, the present application provides the use of vitamin B12 for the preparation of a drug for relieving colonitis and aging-induced cognitive dysfunction.

[0025] In yet another aspect, the present application provides the use of extracellular vesicles as targets for alleviating colitis and aging-induced cognitive impairment.

[0026] The present application has the following beneficial effects:

[0027] 1、The research results of the present application prove that the extracellular vesicles (IEC-sEVs) released by the intestinal epithelial cells of IBD and aging mice degrade CDK1 in hippocampal neural stem cells through miRNA-16-5p, inhibit the proliferation of hippocampal neural stem cells, thereby reducing the number of mature neurons, and leading to CI; the research results of the present application provide strong evidence for supporting IBD-mediated CI from the perspective of molecular mechanism, thereby providing a theoretical basis for preventing and treating IBD and age-related CI.

[0028] 2、The research results of the present application also find that the fecal odor produced by the intestinal bacteria of IBD and aging mice up-regulates miRNA-16-5p in intestinal epithelial cells, causing IEC-sEVs to secrete more miRNA-16-5p, target CDK1 to inhibit its proliferation, and accelerate the development of CI.

[0029] 3、The present application also finds that another intestinal metabolite vitamin B12 (VB12) inhibits the inhibition of IEC-sEVs on the proliferation of hippocampal neural stem cells through miRNA-16-5p in IEC-sEVs of IBD and elderly patients, and the present application reveals the mechanism of VB12 in interfering with IBD and age-related CI by promoting the proliferation of hippocampal neural stem cells, and VB12 can be used as a conventional drug for preventing and treating the two types of CI. BRIEF DESCRIPTION OF DRAWINGS

[0030] Fig. 1A-L is IBD DSS The experimental results of CI induced in mice, wherein after DSS-induced mice, the body weight of the mice, the length of the colon and the morphological changes of the intestinal tissue were observed; A represents the fluctuation of the body weight of the mice with time after DSS induction; B represents the measurement of the length of the colon (n=5) after DSS induction (B); C represents the representative colon stained by H&E, the scale bar = 200 μm; D represents the DSS-induced IBD DSS The mice were subjected to the open field test, and the representative movement trajectory in the open field test was statistically analyzed, and the total movement amount (travel distance) and the time in the central area were statistically analyzed; E-F represents the IBD Il10- / - CI induced in mice, normal mice and Il10 - / - deficient mice (IBD Il10- / - ) were measured and statistically analyzed (E) and H&E staining showed the morphological changes of the intestinal tissue (F); G-I represents the IBD Il10- / -Time (G) to find the hidden platform in the Morris water maze, time spent in the target quadrant in the probe trial of the Morris water maze (H) and recognition index (RI) in the training and novel object recognition (NOR) phases in the novel object recognition test (I); J indicates IBD Il10- / - Representative motor trajectories in the open field test, statistical analysis of total movement (distance traveled) and time spent in the central area of the mice; K indicates detection of TUNEL in the hippocampus of these mice by immunostaining + Neuronal cell number (K); L indicates qPCR detection of mRNA content of IL-1 b, IL-6 and TNF-a, where ns indicates not significant, *** indicates P < 0.001 (unpaired t test).

[0031] Figures 2A-T are results of validation of IEC-sEVs inducing CI associated with IBD by impeding hippocampal neural signaling, where A indicates time (escape latency) to find the hidden platform in the Morris water maze of mice; B and C indicate statistical analysis of time in the target quadrant (B) in the probe trial of the Morris water maze and RI in the training and NOR phases in the novel object recognition test (C); D, F indicate detection of NeuN + Neurons, GFAP + Astrocytes, Olig2 + Oligodendrocytes and IBA + Microglia (D), Sox2 + Neural stem cells and DCX + Newborn neurons (F); E indicates detection of Sox2 and DCX proteins in the hippocampus of these mice by Western blotting; G indicates experimental protocol; H-J indicate time to find the hidden platform in the Morris water maze (H), time spent in the target quadrant in the probe trial of the Morris water maze (I) and statistical analysis of RI in the training and NOR phases of healthy mice treated with EVs (J); K-M indicate induction of chronic colitis in mice by drinking water containing DSS, time to find the hidden platform in the Morris water maze (K), time spent in the target quadrant in the probe trial of the Morris water maze (L) and RI in the training and NOR phases (M) of these mice; N-P indicate statistical analysis of mice treated with DC-sEVs or IEC-sEVs, respectively, time to find the hidden platform in the Morris water maze (N), time spent in the target quadrant in the probe trial of the Morris water maze (O) and RI in the training and NOR phases (P); Q-T indicate induction of chronic colitis in WT and IEC ΔRab27aMice with chronic colitis were treated with DC-sEVs or IEC-sEVs, and the time (Q) these mice took to find the hidden platform in the Morris water maze, the time (R) these mice spent in the target quadrant in the probe trial of the Morris water maze, and the RI (S) of these mice in the training and NOR phases were statistically analyzed, and the NeuN in the hippocampus of these mice was detected by immunostaining + Neurons, Sox2 + Hippocampal neural stem cells and DCX + Newborn neurons (T); the results shown are representative results of three repeated experiments, where ns indicates not significant, * indicates P < 0.05, *** indicates P < 0.01, *** indicates P < 0.001 (in K-M and Q-S, in addition to one-way ANOVA, independent sample t test uses Tukey test; mean ± SD; A-C: n = 10, D, F, T: n = 5).

[0032] Fig. 3A-M is the identification of DSS-sEVs, where A indicates the morphology of Ctrl-sEVs and DSS-sEVs detected by electron microscopy; B indicates the particle size distribution of Ctrl-sEVs and DSS-sEVs measured by nanoparticle tracking analysis (NTA); C indicates the detection of typical sEV protein markers in Ctrl-sEVs and DSS-sEVs by Western blotting; D-F indicates that after treating mice with Ctrl-sEVs and DSS-sEVs, the body weight (D), colon length (E) and histological morphological changes of the colon (F) of these mice were analyzed; G indicates that DSS-sEVs induced IBD DSS Representative movement trajectories of mice in the open field test and statistical analysis of total movement (distance traveled) and time in the center; H-J indicates that mice were treated with DC-sEVs and IEC-sEVs, and the time (H) these mice took to find the hidden platform in the Morris water maze, the time (I) these mice spent in the target quadrant in the probe trial of the Morris water maze, and the RI (J) of these mice in the training and NOR phases of the novel object recognition test were statistically analyzed; K indicates that DSS-induced mice were introduced to chronic colitis by rectal injection of GW4869 (2 mg / kg) 3 times a week, and then the sEV protein content in the colon tissue was evaluated by BCA method (K); L indicates the detection of specified protein components in IEC-sEVs and DC-sEVs by Western blotting; M indicates the analysis of WT and IEC ΔRab27a A33 + Percentage of sEVs.

[0033] Fig. 4A-J is the identification of DSS-sEVs, where A indicates the morphology of Ctrl-sEVs and DSS-sEVs detected by electron microscopy; B indicates the particle size distribution of Ctrl-sEVs and DSS-sEVs measured by nanoparticle tracking analysis (NTA); C indicates the detection of typical sEV protein markers in Ctrl-sEVs and DSS-sEVs by Western blotting; D-F indicates that after treating mice with Ctrl-sEVs and DSS-sEVs, the body weight (D), colon length (E) and histological morphological changes of the colon (F) of these mice were analyzed; G indicates that DSS-sEVs induced IBD DSSIn mice, IEC-sEVs cross the BBB more into the hippocampus, where A-B represent healthy and IBD DSS tdTomato mice were injected i.r. with 1 x 10 9 tdTomato mice were injected i.r. with 1 x 10 DSS Serum IFN-γ and TNF levels in mice; D represents healthy and IBD DSS Serum-treated bEnd3 cells were observed for tight junctions using ZO-1 staining; E represents healthy and IBD DSS Mice were injected i.v. with Evans Blue, and then the brains of these mice were photographed 1 h later, and the amount of Evans Blue in these mice was quantified; G represents healthy and IBD DSS Mice were injected i.v. with VivoTrack 680-labeled DSS-IEC-sEVs, and then the VivoTrack 680 in the brains of these mice was observed using an in vivo imaging system + Distribution levels of sEVs; F represents healthy and IBD DSS Hippocampal tissues of mice were stained to show Ig leakage from brain blood vessels; H represents healthy mice treated with Ctrl-IEC-sEVs or DSS-IEC-sEVs for 48 h, and then injected i.v. with Evans Blue, and the brains of these mice were photographed 1 h later, and the amount of Evans Blue in these mice was quantified; I represents healthy and IBD DSS Hippocampal tissues of mice were stained to show Ig leakage from brain blood vessels; J represents healthy mice treated with Ctrl-IEC-sEVs or DSS-IEC-sEVs for 48 h, and then injected i.v. with PKH26-labeled Ctrl-IEC-sEVs, and the PKH26 in hippocampal cells was observed using a confocal microscope 24 h later + sEVs; where ns represents not significant, ** represents P < 0.01, and *** represents P < 0.001 (unpaired t test).

[0034] FIGS. 5A-Y are scRNA-seq showing DSS-IEC-sEVs inhibit neural stem cell generation, wherein FIG. 5A is a uniform manifold approximation and projection (UMAP) plot of 30,713 cells colored by different cell clusters; FIG. 5B is a heatmap showing the expression pattern of marker genes in each cell cluster; FIG. 5C is a UMAP showing the expression level of cells marker in different clusters; FIGS. 5D-E are waterfall plots showing differentially expressed genes (DEGs) in NSCs in IBD-IEC-sEVs and Ctrl-IEC-sEVs treatment groups (D) and KEGG pathway enrichment analysis of these DEGs (E); FIG. 5F is a pathway enrichment analysis of differentially expressed genes in the hippocampus of mice treated with Ctrl-IEC-sEVs and DSS-IEC-sEVs; FIG. 5G is a UMAP plot showing the distribution of the module score of cell cycle-related genes in different cell types; FIG. 5H is a pathway enrichment analysis of neural stem cells in different treatment groups: a UMAP plot showing sub-clustering analysis of neural lineage cells, and a cell trajectory analysis plot showing the evolution of neural stem cells and neuronal cells over pseudo-time; FIG. 5I is a light microscope observation of mouse primary neural stem cell neurospheres (left panel) and identification by Sox2 staining (right panel); FIG. 5J is a flow cytometry analysis of neural stem cell apoptosis by PI and Annexin V staining after treating mouse primary NSCs with Ctrl-IEC-sEVs or DSS-IEC-sEVs for 48 h; FIG. 5K is a Western blot analysis of cyclin-dependent kinase 1 (CDK1) protein in mouse primary neural stem cells treated with DSS-IEC-sEVs in the presence of Ro-3306 for 48 h; FIGS. 5L-M are flow cytometry (A) and Western blot (B) analysis of DSS-IEC-sEVs surface proteins and total proteins after systematic digestion of proteins (L, M, and D) and RNase I (N and F) in DSS-IEC-sEVs treated by electroporation; FIG. 5N is a measurement of total RNA of sEVs using a NanoDrop Spectrophotometer; FIGS. 5O-P are RT-PCR measurement of Cdk1 mRNA levels in fetal mouse primary neural stem cells treated with DSS-IEC-sEVs without proteins (O) or without RNA (P) for 48 h; FIGS. 5Q-R are miRNA-seq analysis of miRNAs in Ctrl-IEC-sEVs and DSS-IEC-sEVs (Q) and in the hippocampus of mice treated with Ctrl-IEC-sEVs or DSS-IEC-sEVs (R), and heatmaps showing the enriched and upregulated miRNAs in each dataset;S is a Venn diagram showing the intersection of upregulated and enriched miRNAs in graph Q and graph R, wherein ns indicates not significant, *** indicates P < 0.001 (unpaired t test); T indicates RT-PCR overexpression detection results of miRNA-16-5p in mouse primary neural stem cells 24 h after transfection with miRNA-16-5p / M; U indicates that mouse primary hippocampal neural stem cells showed target sequences (U) of miRNA-16-5p in the 3'-untranslated region (UTR) of Cdk1 24 h after transfection with miR-16-5p / M; V indicates that the adeno-associated virus AAV-Nestin-miR-16-5p or AAV-Nestin-NC expressing green fluorescent protein (GFP) was injected into the IBD of mice; DSS The green fluorescent protein in the mouse brain was observed by confocal microscopy after 3 weeks in the hippocampus of the mice (V); W indicates that MCM2 + Sox2 + and CDK1 + Sox2 + Hippocampal neural stem cells and MCM2 + DCX + and CDK1 + DCX + Changes in newborn neurons (W); X-Y indicates that the mRNA level of CDK1 in these cells was analyzed by RT-PCR (X), and the level of miRNA-16-5p in the IEC-sEVs of these mice was detected by RT-PCR (Y).

[0035] FIGS. 6A-O are verification results of IEC-sEVs impairing hippocampal neurogenesis by inducing cell cycle arrest of neural stem cells, wherein A indicates a UAMP graph showing clusters identified in hippocampal cells of IEC-sEVs-treated mice; B-C indicate galaxy plots showing cell density (B) and frequency of subclusters (C) were statistically analyzed; D-E are heat maps showing expression of hippocampal neural stem cell-related genes in subgroups (D) and two groups (E); F indicates functional enrichment of IBD-IEC-sEVs relative to Ctrl-IEC-sEVs-treated mouse NSCs; G indicates GSEA analysis of cell cycle; H-I nuclear density curves of the graph show distribution of cell cycle scores in different subclusters (H) and groups (I); J indicates expression profiles of representative genes along cell trajectory analysis; K-M indicate that mouse primary hippocampal neural stem cells were treated with Ctrl-IEC-sEVs or DSS-IEC-sEVs, respectively, for 48 h, wherein Ki-67 +Neural stem cells (K), cell cycle of cells was analyzed by flow cytometry (L), and indicated proteins in these cells were detected by immunoblotting (M); N represents the cell cycle values of mouse primary hippocampal neural stem cells treated with DSS-IEC-sEVs for 48 h in the presence of Ro-3306 were analyzed by flow cytometry; O represents the observation of MCM2 in the hippocampus of mice treated with Ctrl-IEC-sEVs or DSS-IEC-sEVs, respectively, by immunofluorescence staining + Sox2 + and CDK1 + Sox2 + Neural stem cells and MCM2 + DCX + and CDK1 + DCX + New neurons, the results shown are representative results of three repeated experiments, where ns represents not significant, *** represents P<0.001 (independent sample t test; mean ± SD; O: n=5, others: n=3).

[0036] Figures 7A-P are the verification results of miRNA-16-5p in IEC-sEVs inducing cell cycle arrest of hippocampal neural stem cells by targeting CDK1, where A-B represent the detection of CDK1 mRNA and protein levels in mouse primary hippocampal neural stem cells transfected with miRNA-16s-5p / M for 24 h by real-time fluorescence quantitative PCR (A) and protein blotting (B); C represents the luciferase activity determination results of 3T3 cells co-transfected with WT or MUT fragments carrying Cdk1 3'-UTR, reninase plasmid and miRNA-16-5p / M for 24 h; D-G represent the injection of AAV-Nestin-miRNA-16-5p or AAV-Nestin-NC into the IBD DSS The levels of miRNA-16-5p in the hippocampus of mice (after drinking DSS for 7 days) were detected by real-time fluorescence quantitative PCR (D) after 3 weeks, the time to find the hidden platform in the Morris water maze (E), the time spent in the target quadrant in the Morris water maze probe test (F), and the RI of the training and NOR stages of these mice (G); H represents the detection of IBD DSS Mouse intestinal epithelial cells and circulating A33 of IBD patients + miRNA-16-5p levels in sEVs; I-L represent the IBD DSS After intrarectal injection of AAV-Villin-NC or AAV-Villin-miR-16-5p / I into mice for 3 weeks, the MCM2 in the hippocampus of mice was observed by immunofluorescence staining+ Sox2 + and CDK1 + Sox2 + hippocampal neural stem cells and MCM2 + DCX + and CDK1 1+ DCX + New neurons (I), statistical analysis of the time to find the hidden platform in the Morris water maze (J), the time in the target quadrant in the probe test of the Morris water maze (K), and the RI in the training and NOR phases (L); M-P represent mice treated with DSS-IEC-sEVs / NC or DSS-IEC-sEVs / miR-16-5p, respectively lo treatment, as shown in Figure 1G, where immunofluorescence staining was used to observe MCM2 + Sox2 + and CDK1 + Sox2 + hippocampal neural stem cells and MCM2 + DCX + and CDK1 + DCX + New neurons (M), statistical analysis of the time to find the hidden platform in the Morris water maze (N), the time in the target quadrant in the probe test of the Morris water maze (O), and the RI in the training and NOR phases (P), the results are shown graphically and are representative of three replicates, where ns means not significant, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001 (independent samples t test; mean ± SD; A, C, D, H: n = 5, E-G, J-L, N-P: n = 10).

[0037] Figures 8A-G show the analysis of the microbiota of healthy mice and IBD DSS Bacterial species in the feces of mice, where A represents the principal component analysis of the quantification differences; B is a heat map showing the different abundance of bacterial species; C represents the detection of the levels of miRNA-16-5p in MODE-K cells treated with the metabolites of the intestinal microbiota metabolome for 48 h by RT-PCR, the heat map shows the metabolites that upregulate miRNA-16-5p in MODE-K cells; D represents the detection of the levels of miRNA-16-5p in EVs from MODE-K cells stimulated with skatole for 48 h by RT-PCR; E-G represent the administration of fecal microbiota from IBD DSSMice treated with control MODE-K cell-derived extracellular vesicles (Ctrl-MK-sEVs) or with skatole-treated MODE-K cell-derived extracellular vesicles (Skl-MK-sEVs) were analyzed for the time (E) to find the hidden platform in the Morris water maze, the time spent in the target quadrant in the probe test of the Morris water maze (F), and the recognition index (RI) in the training and novel object recognition (NOR) phases (G), where ns indicates not significant, ** indicates P < 0.01, and *** indicates P < 0.001; H indicates germ-free (GF) mice colonized with B. Aci for 21 days, and the bacterial species in the feces of these mice were analyzed by metagenomic sequencing.

[0038] Figure 9A-S shows the validation of the effect of skatole extracted from Bacteroides on the upregulation of miRNA-16-5p in IEC-sEVs, where A indicates the upregulation of the expression level of miRNA-16-5p by metabolites in MODE-K cells; B-J indicate the detection of skatole in the feces of mice (B) and the detection of the level of miRNA-16-5p in intestinal epithelial cells and IEC-sEVs (C) in germ-free mice orally colonized with B. Aci for 10 days by ELISA (B) and real-time quantitative PCR (C), respectively; D-F indicate the flow cytometry analysis of Ki-67 + neural stem cells (D), the detection of CDK1 protein by immunoblotting (E), and the detection of the CCY of cells by flow cytometry (F); G-J indicate the IBD DSS Mice treated with Ctrl-IEC-sEVs or B.Aci-IEC-sEVs (G) were observed for MCM2 + Sox2 + and CDK1 + Sox2 + neural stem cells and MCM2 + DCX + and CDK1 + DCX + neurons (G), the time to find the hidden platform in the Morris water maze (H), the time spent in the target quadrant in the probe test of the Morris water maze (I), and the RI of these mice in the training and NOR phases (J); K indicates that mice were injected intraperitoneally with 30 mg kg -1SkI-IEC-sEVs for 48 h, then flow cytometry was used to detect the expression of Ki-67 (L-N) in hippocampal neural stem cells of mice + Hippocampal neural stem cells (L), Western blotting was used to detect the expression of CDK1 protein (M), and flow cytometry was used to detect the cell cycle (N) of the cells; O-R represents WT or IEC ΔRab27a IBD DSS Mice were injected intraperitoneally with 30 mg / kg of SkI every 2 days -1 The time taken to find the hidden platform in the Morris water maze (O) after SkI was administered for 21 days, the time spent in the target quadrant in the Morris water maze probe test (P), and the RI of the training and NOR stages (Q) of these mice, and the expression of MCM2 in the hippocampus of mice was observed by immunofluorescence staining + Sox2 + and CDK1 + Sox2 + Hippocampal neural stem cells and MCM2 + DCX + and CDK1 + DCX + Newborn neurons (R), wherein the results shown are representative results of three repeated experiments, ns represents not significant, ** represents P < 0.01, *** represents P < 0.001 (except for one-way ANOVA, independent sample t test uses Tukey test; mean ± SD; B-D, F, K, L, N: n = 3, G, S: n = 5; H-J, O-Q: n = 10); S represents the SkI in the feces and serum of IBD patients was detected by ELISA.

[0039] Figures 10A-G show the time taken to find the hidden platform in the Morris water maze (A) of young mice and old mice, the time spent in the target quadrant in the Morris water maze probe test (B), and the RI of the training stage and NOR stage (C), and the expression of NeuN positive neurons, Sox2 + Hippocampal neural stem cells and DCX +New neurons (D), E represents the injection of PKH26-labeled A-IEC sEVs into the tail vein of young mice and aged mice, and the distribution of PKH26-positive A-IEC sEVs in hippocampal cells was observed by confocal microscopy 24 h later; F represents the injection of PKH26-labeled A-IEC sEVs into the tail vein of young mice and aged mice, and the distribution of PKH26-positive A-IEC sEVs in hippocampal cells was observed by confocal microscopy 24 h later (F); G represents the rectal injection of adeno-associated virus AAV-Villin-miR-16-5p / NC or AAV-Villin-miR-16-5p / I into aged mice for 3 weeks, and the level of miR-16-5p in IECs of these mice was detected by RT-PCR; H represents the rectal injection of AAV-Villin-miR-16-5p / NC or AAV-Villin-miR-16-5p / I combined or not combined with GW4869 into aged mice, and the protein content in EVs derived from the colon of these mice was detected by BCA method.

[0040] Fig. 11A-Q are the verification results of IEC-sEVs participating in age-related CI through miRNA-16-5p, wherein A represents the detection of miRNA-16-5p level in IECs and IEC-sEVs of young and old mice by real-time fluorescent quantitative PCR method; B-D represent the flow cytometry analysis of Ki-67 + NSCs (B), immunoblotting method was used to detect the CDK1 protein in these cells (C), and flow cytometry was used to detect the CCY of the cells (D); E-H represent the observation of MCM2 + Sox2 + and CDK1 + Sox2 + Hippocampal neural stem cells and MCM2 + DCX + and CDK1 + DCX +New neurons (E), time to find hidden platform in Morris water maze (F), time spent in target quadrant in probe trial in Morris water maze (G) and RI of training and NOR phase of these mice (H); I-K represent young mice intrarectally injected with AAV-Villin-miR-16-5p / NC or AAV-Villin-miR-16-5p / I for 3 weeks and treated with A-IEC-sEVs, time to find hidden platform in Morris water maze (I), time spent in target quadrant in probe trial in Morris water maze (J) and RI of training and NOR phase of these mice (K); L-N represent aged mice intrarectally injected with AAV-Villin-miRNA-16-5p / NC or AAV-Villin-miRNA-16-5p / I for 3 weeks, time to find hidden platform in Morris water maze (L), time spent in target quadrant in probe trial in Morris water maze (M) and RI of training and NOR phase of these mice (N); O-Q represent aged mice intrarectally injected with AAV-Villin-miRNA-16-5p / NC or AAV-Villin-miRNA-16-5p / I with or without addition of GW4869 for 3 weeks, time to find hidden platform in Morris water maze (O), time spent in target quadrant in probe trial in Morris water maze (P) and RI of training and NOR phase of these mice (Q), wherein the results are representative of three replicates, ns represents not significant, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001 (except for one-way ANOVA, independent sample t-test with Tukey test; mean ± SD; A, B, D: n = 3, E: n = 5; F-Q: n = 10).

[0041] Figures 12A-N show the analysis of bacterial species in the feces of young and aged mice by metagenomic sequencing, wherein A shows principal component analysis of feces; B is a heatmap showing different abundance of bacterial species; C shows the detection of B.Aci abundance in the feces of young and aged mice by RT-PCR; D shows the detection of miRNA-16-5p levels in IECs of mice treated with or without OTA by RT-PCR after the gut microbiota (GM) of aged mice was cleared with OTA; E-G show the time taken by these aged mice to find the hidden platform in the Morris water maze (E); the time spent in the target quadrant in the Morris water maze probe test (F) and RI in the training phase and NOR phase (G); H-I show the analysis of bacterial species in the feces of young and aged mice by metagenomic sequencing, principal component analysis of quantified differences (H) and the presentation of different bacterial species abundance (I); J-K show the detection of miRNA-16-5p levels in miR-16-5p / I and miR-16-5p / M by RT-PCR (J) and (K); L shows the analysis of Ki-67 expression in humanized hippocampal neural stem cells transfected with human miRNA-16-5p mimic (miRNA-16-5p / M) for 48 h by flow cytometry + hippocampal neural stem cells; M-N show the detection of CDK1 protein in these cells by western blotting (M) and the analysis of cell cycle in these cells by flow cytometry (N).

[0042] Figures 13A-O show the verification results of fecal odor inducing miRNA-16-5p in IEC-sEVs of aged mice, wherein A shows the detection of fecal odor levels in the serum and feces of young and aged mice by ELISA; B-F show the detection of miRNA-16-5p levels in the intestinal epithelial cells and IEC-sEVs of young mice (B) after the young mice were co-fed with aged mice for 2 weeks, the time taken to find the hidden platform in the Morris water maze (C), the time spent in the target quadrant in the Morris water maze probe test (D) and RI in the training phase and NOR phase (E), the observation of MCM2 + Sox2 + and CDK1 + Sox2 + hippocampal neural stem cells and MCM2 + DCX + and CDK1 + DCX +Newborn neurons (F); G indicates the skatole levels in feces and serum of young and elderly people were measured by ELISA; H indicates the A33 expression in serum of young and elderly people was measured by real-time fluorescence quantitative PCR. + Absolute quantification of miRNA-16-5p levels in EVs; I represents relative and absolute quantification of miRNA-16-5p levels in HIEC-6 intestinal epithelial cells and their EVs after treatment with 1 μg of skatole for 48 h; JO represents treatment of humanized hippocampal neural stem cells with 2 μg of HIEC-6 EVs and Skl-HIEC-6-sEVs for 48 h (JL) or transfection with miRNA-16-5p / M for 24 h (MO), and flow cytometry detection of Ki-67 + Neural stem cells (J and M), CDK1 in cells was detected by immunoblotting (K and N), and cell cycle was detected by flow cytometry (L and O). The results shown are representative results of three repeated experiments, ns indicates not significant, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 (independent sample t test; mean ± SD; A, B, HJ, L, M, P: n = 3, CE: n = 10; G: n = 20).

[0043] Figure 14A-T shows the validation results of VB12 in preventing IBD and age-related CI by targeting miRNA-16-5p, where A represents the expression level of miRNA-16-5p in MODE-K cells after 48 h of VB12 treatment; BI represents IBD DSS and aging mice were intraperitoneally injected with 1.25 mg kg every 2 days -1 VB12 was administered for 3 weeks (BD), or AAV-Villin-miRNA-16-5p / NC or AAV-Villin-miRNA-16-5p / I was intrarectally injected into the mice before VB12 treatment for 3 weeks (FH), where B and F represent the time spent searching for the hidden platform in the Morris water maze, C and G represent the time spent in the target quadrant in the Morris water maze probe test, D and H represent the RI of the training and NOR phases of these mice; E represents the expression of Sox2 in the hippocampus of these mice observed by immunofluorescence staining + Hippocampal neural stem cells and DCX + Newborn neurons; I indicates qPCR detection of IBD after injection of VB12 DSS miRNA-16-5p in IEC-sEVs from senescent and aged mice; JL indicates miRNA-16-5p in IEC-sEVs from Ctrl- IBD ,VB12-IEC-sEVs IBD , Ctrl-IEC-sEVs Agingor VB12-IEC-sEVs Aging Mouse neural stem cells were treated for 48 h, and flow cytometry was used to detect Ki-67 + Hippocampal neurons (J), immunoblotting was used to detect CDK1 protein in cells (K), and flow cytometry was used to detect cell cycle of cells (L); M-O represent the use of Ctrl-IEC-sEVs IBD , VB12-IEC-sEVs IBD , Ctrl-IEC-sEVs Aging or VB12-IEC-sEVs Aging Time for mice to find the hidden platform in the Morris water maze (M), time spent in the target quadrant in the Morris water maze probe test (N), and RI of these mice in the training and NOR stages (O); P-T represent the morphology (P), size distribution (Q), and typical EVs protein markers (CD-Lyts, colon fragment lysate) (R) of EVs in the feces of IBD patients and the elderly before and after VB12 treatment; S represents qPCR detection of miRNA-16-5p in sEVs, and T represents the use of the CCK-8 method to detect the effect of EVs on the viability of hippocampal neural stem cells, wherein the results shown are representative results of three repeated experiments, ns represents not significant, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 (except for one-way ANOVA, independent sample t-test uses Tukey test; mean ± SD; A, I, J, L, M, P: n = 3, E: n = 5; B-D, F-H and M-O: n = 10). DETAILED DESCRIPTION

[0044] The application will be further described in conjunction with the following examples. It should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0045] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0046] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0047] Example 1: IBD impairs hippocampal neurogenesis

[0048] Dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) is similar to human ulcerative colitis (UC). To explain whether IBD can lead to secondary cognitive impairment, we performed behavioral tests in DSS-induced chronic colitis (IBD DSS ) mice (Figures 1A-1C) in the Morris water maze, novel object recognition test, and open field test.

[0049] I. Experimental Methods

[0050] The spatial memory ability of mice was evaluated by Morris water maze test, and the memory and discrimination ability of mice was evaluated by novel object recognition test, and the spontaneous activity behavior, exploration behavior and tension of mice in a new environment were evaluated by open field test.

[0051] Results found that compared with healthy mice (Ctrl), IBD DSS The escape latency of mice was prolonged in the hidden platform test, and the time in the target quadrant was shortened in the probe test (Figures 2A and 2B). In the target recognition test, there was no significant difference in the recognition index (RI) between healthy mice and IBD DSS mice. However, IBD DSS mice showed significantly lower recognition index (RI) in the novel object recognition (NOR) stage (Figure 2C). In addition, in the open field test, IBD DSS mice had similar walking distance, but reduced exploration in the central area (Figure 1D), indicating the presence of similar anxiety-like behavior.

[0052] IL-10 deficiency (Il10- / -) induces spontaneous colitis similar to human Crohn's disease. Like IBD DSS mice, CI and anxiety behavior were also observed in Il10- / - colitis (IBD Il10- / - ) mice (Figures 1E-1J). Considering the core role of hippocampus in cognitive function, we evaluated the effect of chronic colitis on hippocampal neural stem cell composition. Immunohistochemical staining (Figures 2D and 2F) detected a significant decrease in the number of NeuN DSS neurons in the hippocampus of IBD + mice, while the number of glial cells including GFAP + astrocytes and Olig 2+ oligodendrocytes was minimally affected by chronic colitis. In contrast, in IBD DSS mice, IBA + microglia and their activation were significantly increased. The above results preliminarily suggest that chronic colitis causes damage to hippocampal neurogenesis.

[0053] To investigate how chronic colitis reduces hippocampal neurons, we evaluated the apoptosis of neurons by TUNEL staining and found no significant difference (Fig. 2K). Consistent with the microglial phenotype, the mRNA levels of IL-1β, IL-6, and TNF-α were enhanced in the hippocampus (Fig. 2L), suggesting that chronic colitis can induce neuroinflammation, which damages hippocampal neurogenesis. Thus, we hypothesized that chronic colitis would suppress neurogenesis and verified this hypothesis by detecting the specific marker Sox2 of hippocampal neural stem cells by immunoblotting and immunofluorescent staining. As expected, Sox2 protein levels and Sox2 DSS Sox2 protein levels and Sox2 + cells were significantly lower in IBD mice than in healthy mice (Figs. 2E and 2F), indicating a decrease in hippocampal neural stem cells. The present disclosure also found that IBD mice had significantly lower levels of doublecortin (DCX) protein and DCX DSS Sox2 protein levels and Sox2 + cells were significantly lower in IBD mice than in healthy mice (Figs. 2E and 2F), indicating a decrease in hippocampal neural stem cells. The present disclosure also found that IBD mice had significantly lower levels of doublecortin (DCX) protein and DCX

[0054] Example 2: IEC-sEVs promote IBD-associated CI development by suppressing hippocampal neurogenesis

[0055] Extracellular vesicles (EVs) are essential for communication between different cells, and EVs can cross the blood-brain barrier. Therefore, the present disclosure further investigated whether EVs play a role in chronic colitis-induced CI. We isolated and identified sEVs (small extracellular vesicles) from the large intestine tissue of healthy mice or mice with chronic colitis induced by DSS (Ctrl-sEVs or DSS-sEVs) by density gradient centrifugation. The results showed that the morphology and size distribution of the two sEVs were similar (Figs. 3A and 3B). In addition, both sEVs contained comparable protein contents, including CD63, Tsg101, Alix, and IEC-A33-specific markers, but not endoplasmic reticulum resident protein GRP94 (Fig. 3C).

[0056] Subsequently, we injected the two sEVs into healthy mice (Fig. 2G), and the results showed that neither of the two sEVs could induce colitis phenotypes in mice (Figs. 3D-3F). In addition, all sEV-treated mice performed similarly in the open field analysis (Fig. 3G). However, the hidden platform test results showed (Figs. 2H-2J) that healthy mice injected with DSS-EVs had longer escape latencies, shorter times in the target quadrant, and significantly reduced novelty object preference, indicating that DSS-EVs induced more pronounced CI than Ctrl-sEVs.

[0057] The present application further uses sEVs of wild type (WT) mice (WT-sEVs) and sEVs from Il10 - / - sEVs of colitis mice (Il10 - / - sEVs) to treat healthy mice, and statistically analyzes the time for these mice to find the hidden platform in the Morris water maze, the time for these mice to stay in the target quadrant in the Morris water maze exploration test, and the RI of the training and NOR stages in the novel object recognition test. It is found that the use of Il10 - / - sEVs can cause CI of healthy mice, while WT-sEVs cannot (Fig. 3H-3J). Subsequently, this embodiment injects 2 mg / kg neutral sphingomyelinase inhibitor GW4869 into the rectum of healthy mice, and it is found that the inhibitor GW4869 inhibits the endogenous production of intestinal sEVs (Fig. 3K) and does not affect the cognitive ability of healthy mice, but significantly improves the CI of DSS-induced chronic colitis mice (Fig. 2K-2M). These results show that intestinal sEVs induce CI of chronic colitis mice.

[0058] The present application has found in previous studies that intestinal sEVs are composed of A33 + IEC-sEVs and CD11c + DC-sEVs. To explore which intestinal sEVs subpopulation is responsible for regulating CI, we first isolate small extracellular vesicles released by intestinal epithelial cells (IEC-sEVs) and dendritic cell-derived small extracellular vesicles (DC-sEVs) from DSS-induced chronic colitis mice (Fig. 3L), inject the isolated IEC-sEVs and DC-sEVs into healthy mice, and detect the cognitive function of these mice. It is found that IEC-EVs cause CI of healthy mice, while DC-EVs do not (Fig. 1N-P).

[0059] Further, the present application crosses Villin-Cre mice with Rab27afl / fl mice to generate IEC-specific Rab27a-deficient mice (IEC ΔRab27a mice), and the production of IEC-sEVs in the mice is specifically inhibited (Fig. 3M). By detecting the cognitive function of IEC ΔRab27a mice and WT mice, it is found that although Rab27a deficiency does not affect the cognitive ability of healthy mice, the CI of DSS-induced IEC ΔRab27a mice is significantly weakened (Fig. 2Q-2S), indicating that endogenous IEC-sEVs are involved in the induction of CI of chronic colitis mice. Similarly, the number of hippocampal neurons of IEC ΔRab27a colitis mice is significantly increased (Fig. 2T), and the number of hippocampal neurons of IEC ΔRab27aSox2 in hippocampus of colitis mice + Hippocampal neural stem cells and DCX + Neurons were significantly higher than Rab27afl / fl (wild type, WT) mice (Fig. 2T), the above results showed that IEC-sEVs played a key role in the pathological process of IBD-related CI by hindering the genesis of hippocampal neurons.

[0060] Example 3: IEC-sEVs enter the brain by destroying the integrity of the blood-brain barrier

[0061] To verify whether sEVs play a messenger role between the intestine and the brain, we first explored whether sEVs from the intestine would enter the brain. Based on the property that Cre recombinase can be transferred by sEVs and induce tdTomato expression in R26-LSL-tdTomato mice (referred to as tdTomato mice), we designed and carried out a series of systematic experiments. Intrarectal injection of Cre-expressing adenovirus (Cre-Ads) into healthy tdTomato mice can significantly induce red fluorescence in intestinal epithelial cells (Fig. 4A), and we detected a small amount of tdTomato + cells in the hippocampus of these mice, intrarectal injection of Cre-Ads induced more tdTomato DSS cells in the hippocampus of IBD + mice (Fig. 4B), indicating that colitis intestinal sEVs can cross the blood-brain barrier and be absorbed by hippocampal cells.

[0062] IFN-γ and TNF in the serum of IBD DSS mice were elevated (Fig. 4C), leading to the destruction of tight junctions between endothelial cells. Therefore, the present application hypothesized that the blood-brain barrier permeability of IBD DSS mice was elevated, and by constructing a bEnd3 mouse brain microvascular endothelial cell model, we found that the serum of IBD DSS mice could effectively destroy the tight junctions between endothelial cells (Fig. 4D), suggesting that there are active substances in the serum of IBD DSS mice that affect the integrity of the blood-brain barrier. After intravenous injection of Evans blue, we found that the accumulation of Evans blue in the brain of IBD DSS mice was significantly higher than that of healthy controls, directly suggesting an increase in blood-brain barrier permeability.

[0063] Immunoglobulin (Ig) leakage proved the destruction of the blood-brain barrier in IBD DSS mice (Fig. 4E), and more intravenous Evans blue accumulated in the brain of IBD DSS mice (Fig. 4F). In addition, compared with healthy mice, more intravenous Evans blue accumulated in the brain of IBD DSSAn increase in vivoTrack680-labeled IEC-sEVs was found in the brain of mice (Fig. 4G). The above results demonstrate that IBD DSS The permeability of the blood-brain barrier of mice was increased, and more IEC-sEVs entered the brain.

[0064] The Evans blue leakage test further demonstrated that IEC-sEVs from IBD DSS The blood-brain barrier permeability of IEC-sEVs of mice (DSS-IEC-sEVs) was higher than that of healthy mice (Ctrl-IEC-sEVs) (Fig. 4H). In addition, when the brain blood vessels were stained with lectin, obvious Ig leakage was observed in DSS-IEC-sEVs-treated mice (Fig. 4I), indicating that the blood-brain barrier was damaged. When DSS-IEC-sEVs or Ctrl-IEC-sEVs-treated mice were intravenously injected with PKH26-labeled Ctrl-IEC-sEVs, the former had more PKH26-labeled IEC-sEVs in the hippocampal cells (Fig. 4J). These results suggest that DSS-IEC-sEVs can damage the blood-brain barrier and promote their entry into the brain. + The blood-brain barrier permeability of IEC-sEVs of mice (DSS-IEC-sEVs) was higher than that of healthy mice (Ctrl-IEC-sEVs) (Fig. 4H). In addition, when the brain blood vessels were stained with lectin, obvious Ig leakage was observed in DSS-IEC-sEVs-treated mice (Fig. 4I), indicating that the blood-brain barrier was damaged. When DSS-IEC-sEVs or Ctrl-IEC-sEVs-treated mice were intravenously injected with PKH26-labeled Ctrl-IEC-sEVs, the former had more PKH26-labeled IEC-sEVs in the hippocampal cells (Fig. 4J). These results suggest that DSS-IEC-sEVs can damage the blood-brain barrier and promote their entry into the brain.

[0065] Example 4: IEC-sEVs impair hippocampal neurogenesis by inducing hippocampal neural stem cell cycle arrest

[0066] To further analyze how DSS-IEC-sEVs affect hippocampal neurogenesis, the present application performed single-cell RNA sequencing (scRNA-seq) on the hippocampal tissues of IEC-sEVs-treated mice. A total of 30714 cells were obtained from 6 samples and were divided into 32 major clusters (Fig. 5A). Eight cell types were identified according to known cell markers, and DSS-IEC-sEVs significantly reduced hippocampal neurons and hippocampal neural stem cells compared with Ctrl-IEC-sEVs (Figs. 6A-6C, Fig. 5B and Figure 5C ), which is consistent with their effect on hippocampal neurogenesis. Notably, the expression levels of neural stem cell-related genes were significantly reduced in hippocampal neural stem cells of DSS-IEC-sEVs-treated mice (Figs. 6D and 6E). These results suggest that DSS-IEC-sEVs can inhibit hippocampal neurogenesis by reducing hippocampal neural stem cells.

[0067] Subsequently, the present application performed functional pathway enrichment analysis on hippocampal neural stem cells of Ctrl-IEC-sEVs and DSS-IEC-sEVs-treated mice. The results consistently showed that the differentially expressed genes (DEGs) were mainly enriched in pathways related to nervous system diseases, cell signaling pathways, cell cycle and apoptosis, and cell structure and function (Figs. 5D and 5E). In particular, cell cycle ranked first in the cell cycle combined apoptosis pathway (Figure 5F ). In addition, GGPlot analysis showed that DSS-IEC-sEVs inhibited the cell cycle pathway most significantly (Figure 6F), and GSEA curve showed that the normalized enrichment score (NES) of cell cycle was -1.4636, highlighting the downregulation of DSS-IEC-sEVs on cell cycle process related genes (Figure 6G). AddModuleScore-UCell analysis can quantify the activity of a given marker gene set by summarizing the expression of the marker genes, and multiple gene sets related to cell cycle process also showed less enrichment in hippocampal neural stem cells of DSS-IEC-sEVs treated mice (Figure 6H, 6I and 5G).

[0068] Pseudo-time trajectory showed that hippocampal neural stem cells differentiated into neural precursor cells (Figure 5H), and the present embodiment found that some commonly used neural stem cell marker genes were initially highly expressed, including Apoe and Slclal, and then downregulated. In contrast, Dcx and Grin2b were initially turned off and upregulated by observing the temporal gene expression pattern. Hippocampal neural stem cells and neural marker expression of the DSS-IEC-sEVs treatment group were low throughout the differentiation process, indicating that they were inhibited during the differentiation process. At the same time, compared with Ctrl-IEC-sEVs, hippocampal neural stem cells began to express progenitor cell markers as DSS-IEC-sEVs entered S phase, significantly reducing proliferation and cycle markers (Figure 6J). Therefore, DSS-IEC-sEVs negatively affected the proliferation of neural stem cells by interfering with their cell cycle progression.

[0069] The present application further found that DSS-IEC-sEVs significantly reduced Ki-67 + proportion of neural stem cells (Figure 6K and 5I), and both sEVs had no effect on neural stem cell apoptosis (Figure 5J). Subsequently, the present embodiment found that DSS-IEC-sEVs caused cell cycle arrest of hippocampal neural stem cells at G2 / M phase (Figure 6L). Then, the present embodiment detected the content of Cyclin B, CDK1, CDC25 and Wee1 proteins that regulate G2 / M phase, and found that the total CDK1 protein in hippocampal neural stem cells was significantly reduced after DSS-IEC-sEVs treatment (Figure 6M). In addition, CDK1 inhibitor Ro-3306 eliminated DSS-IEC-sEV induced G2 / M phase arrest and CDK1 reduction (Figure 6N and 5K). In the in vivo experiment of the hippocampus of healthy mice, we also observed that DSS-IEC-sEV treatment inhibited the proliferation of hippocampal neural stem cells and newborn neurons (NDNs) and reduced CDK1 expression, manifested as MCM2 + Sox2+ and CDK1 + Sox2 + Neural stem cells and MCM2 + DCX + and CDK1 + DCX + Significant reduction in the number of newborn neurons Figure 6O ). Therefore, DSS-IEC-sEVs reduced hippocampal neurogenesis by inducing G2 / M phase arrest of hippocampal neural stem cells in a CDK1-dependent manner.

[0070] Example 5: miRNA-16-5p induces hippocampal neural stem cell cycle arrest by targeting CDK1

[0071] To investigate how DSS-IEC-sEVs downregulate CDK1 in hippocampal neural stem cells, in this embodiment, the proteins and RNAs of electroporated DSS-IEC-sEVs were digested by proteinase K (ProK) and RNase I, respectively (Figures 5L-5N), and it was found that the RNAs of DSS-IEC-sEVs inhibited the expression of CDK1 (Figures 5O and 5P), and the miRNAs enriched in sEVs are essential for exosome-mediated intercellular communication. To determine the miRNAs that affect the CDK1 downregulation induced by DSS-IEC-sEVs, in this embodiment, miRNAs in Ctrl-IEC-sEVs and DSS-IEC-sEVs were analyzed by miRNA array, and 30 enriched miRNAs were found in DSS-IEC-sEVs (Figure 5Q).

[0072] In addition, the miRNA array results showed that there were 14 upregulated miRNAs in the hippocampus of mice treated with DSS-IEC-sEVs, and after comparing the enriched and upregulated miRNAs, it was found that the expression level of miRNA-16-5p (miR-16-5p, sequence as shown in Seq ID NO. 1) was significantly upregulated (Figures 5R and 5S). Further, it was also found that the miRNA-16-5p mimic (miRNA-16-5p / M) reduced the mRNA and protein levels of CDK1 in neural stem cells (Figures 7A, 7B and Figure 5T). To detect whether CDK1 is a direct target of miRNA-16-5p, in this embodiment, alignment was performed in the Cdk1 3'-UTR and a predicted miRNA-16-5p target site was found (Figure 5U). Through luciferase reporter experiment, we confirmed that the miRNA-16-5p mimic significantly inhibited the luciferase activity of wild-type (WT) CDK1 3'-UTR, but had no significant effect on the mutant (MUT) sequence, which confirmed that miRNA-16-5p directly targets the 3'-UTR of CDK1.

[0073] (Figure 7C).

[0074] To test whether miRNA-16-5p inhibits hippocampal neurogenesis, this example further injected adeno-associated virus (AAV-Nestin-miR-16-5p) that specifically overexpresses miRNA-16-5p and targets neurons into the hippocampus of mice with DSS-induced chronic colitis (Figures 7D and 5V). As expected, AAV-Nestin-miR-16-5p treatment inhibited the proliferation of hippocampal neural stem cells and newborn neurons, while reducing CDK1 protein in these cells (Figure 5W), and the CI of mice was more severe (Figures 7E-7G), indicating that miRNA-16-5p plays a major role in hippocampal neurogenesis.

[0075] The present invention further found that miRNA-16-5p was expressed in the intestinal epithelial cells of chronic colitis mice and in the circulating A33 of IBD patients through enzyme-linked immunosorbent assay (ELISA) experiments. + sEVs increased (Figure 7H), and this finding suggests that miRNA-16-5p may mediate gut-brain axis signaling through sEVs, thereby participating in the occurrence of IBD-related CI. In order to study whether miRNA-16-5p is an effective target for the treatment of IBD-related CI, this example constructed an adeno-associated virus (AAV) that specifically expresses a miRNA-16-5p inhibitor (AAV-Villin-miR-16-5p / I) and its control (AAV-Villin-NC) in intestinal epithelial cells. Intrarectal administration of AAV-Villin-miR-16-5p / I increased the proliferation of hippocampal neural stem cells and newborn neurons and the content of CDK1 protein (Figures 7I and 5X), and the CI of mice was alleviated (Figures 7J-7L). In addition, this example isolated DSS-IEC-sEVs (DSS-IEC-sEVs / miR-16-5p from AAV-Villin-miR-16-5p / I-treated mice. lo ), a similar trend was obtained after AAV-Villin-NC treatment of healthy mice (Figures 7M-7P and 5Y). In summary, DSS-IEC-sEVs-dependent miRNA-16-5p inhibited hippocampal neurogenesis in chronic colitis mice, and miRNA-16-5p has the potential to treat IBD-related CI.

[0076] Example 6: Skatole produced by Bacteroides upregulates miRNA-16-5p in IEC-sEVs

[0077] In view of the key role of the symbiotic gut microbiota (GM) in the gut-brain axis, the present invention further explores whether GM is involved in the induction of pathogenic miRNA-16-5p in intestinal epithelial cells of mice with chronic colitis. First, this example analyzes the microbiota in the feces of healthy mice and IBD DSS mice, it is found that Bacteroides is enriched in the feces of IBD DSS mice (Figures 8A and 8B). Further, the present invention screens the metabolites in the gut microbial metabolome for regulating miRNA-16-5p and finds that 17 metabolites upregulate miRNA-16-5p in mouse small intestinal epithelial cells (MODE-K) (Figure 9A). Among these 17 metabolites, fecal odorant produced in large quantities by Bacteroides has the most obvious upregulation effect on miRNA-16-5p. Subsequently, the present invention further confirms by ELISA experiment that IBD DSS mice have significantly elevated fecal and serum levels of fecal odorant (Figure 8C). In addition, fecal odorant-stimulated MODE-K cells produce more miRNA-16-5p sEVs (Figure 8D). When transplanted into IBD DSS mice, miRNA-16-5p sEVs from mouse-treated MODE-K cells (Skl-MK-sEVs) induce more severe CI than mock-treated MODE-K cells (Ctrl-MK-sEVs) (Figures 8E-8G).

[0078] To confirm the role of Bacteroides in chronic colitis-related CI, this example orally colonizes the representative Bacteroides bacterium B. aci in germ-free mice and verifies successful colonization of B. aci in germ-free mice by metagenomic sequencing analysis (Figure 8H). The results show that B. aci colonization increases fecal odorant and induces miRNA-16-5p in the intestinal epithelial cells and extracellular vesicles released by intestinal epithelial cells of germ-free mice (Figures 9B and 9C). Accordingly, these mouse-derived IEC-sEVs (B. aci-IEC-sEVs) inhibit the proliferation and CDK1 protein expression of hippocampal neural stem cells in vitro and cause the hippocampal neural stem cells to be arrested in the G2 / M phase (Figures 9D-9F). In addition, B. aci-IEC-sEVs also inhibit the proliferation and CDK1 protein expression of hippocampal neural stem cells and newborn neurons (Figure 9G) and accelerate the development of CI in IBD DSS mice (Figures 9H-9J).

[0079] The present embodiment further injected healthy mice with skatole, and as a result, an increase in miRNA-16-5p was detected in the intestinal epithelial cells and EVs (FIG. 9K). The IEC-sEVs (Skl-IEC-sEVs) of these mice inhibited neural stem cell proliferation and CDK1, while causing G2 / M phase arrest of hippocampal neural stem cells (FIGS. 9L-9N). In addition, skatole accelerated the development of CI in mice DSS IBD mice (FIGS. 9O-9Q). Consistent with these results, skatole-treated IBD mice showed a decrease in the proliferation of hippocampal neural stem cells and newborn neurons and CDK1 protein (FIG. 9R). However, skatole did not induce IEC-sEVs in the intestinal epithelial cells of healthy mice (FIG. 9S). These results suggest that skatole can also cause the development of CI in IBD patients. In summary, B. Aci skatole caused CI in an IEC-sEV-dependent manner. DSS IBD mice (FIGS. 9O-9Q). Consistent with these results, skatole-treated IBD mice showed a decrease in the proliferation of hippocampal neural stem cells and newborn neurons and CDK1 protein (FIG. 9R). However, skatole did not induce IEC-sEVs in the intestinal epithelial cells of healthy mice (FIG. 9S). These results suggest that skatole can also cause the development of CI in IBD patients. In summary, B. Aci skatole caused CI in an IEC-sEV-dependent manner. ΔRab27a IBD mice (FIGS. 9O-9Q). Consistent with these results, skatole-treated IBD mice showed a decrease in the proliferation of hippocampal neural stem cells and newborn neurons and CDK1 protein (FIG. 9R). However, skatole did not induce IEC-sEVs in the intestinal epithelial cells of healthy mice (FIG. 9S). These results suggest that skatole can also cause the development of CI in IBD patients. In summary, B. Aci skatole caused CI in an IEC-sEV-dependent manner.

[0080] Example 7: IEC-sEVs involvement in age-related CI through miRNA-16-5p

[0081] Cognitive abilities weaken with age. The present invention confirmed that the cognitive abilities of aged mice were impaired relative to young mice by the time it took for the mice to find the hidden platform in the Morris water maze, the time spent in the target quadrant in the Morris water maze probe test, and the recognition index (RI) in the training phase and the novel object recognition (NOR) phase (FIGS. 10A-10C). Accordingly, the total number of neurons and Sox2 + Neural stem cells and DCX + Newborn neurons were significantly reduced (FIG. 10D). In addition, we found that the disruption and distribution of IEC-sEVs (A-IEC-sEVs) in the hippocampal cells of aged mice were significantly increased (FIGS. 10E and 10F), indicating that gut-brain signaling mediated by IEC-sEVs is enhanced with age. Therefore, the present invention further explored the role of IEC-sEVs in age-related CI.

[0082] First, we found that miRNA-16-5p was significantly increased in the intestinal epithelial cells and IEC-sEVs released from the intestinal epithelial cells of aged mice compared to young mice by RT-PCR detection (Figure 11A). A-IEC-sEVs were more potent than IEC-EC-EVs from young mice (Y-IEC-sEVs) in reducing neural stem cell proliferation and CDK1 protein and inducing G2 / M phase arrest of neural stem cells (Figures 11B-11D). Immunofluorescence staining results showed that A-IEC-sEVs significantly reduced the proliferation and CDK1 protein of hippocampal neural stem cells and newborn neurons in young mice (Figure 11E), and A-IEC-sEVs induced CI in young mice (Figures 11F-11H). According to the results of immunofluorescence experiments, intrarectal injection of AAV-Villin-miR-16-5p / I (A-IEC-sEVs / miR-16-5p / I) no longer caused CI (Figures 11I-11K and 10G), indicating that the ability of A-IEC-sEVs to impair cognitive ability is achieved in dependence on miRNA-16-5p. More importantly, AAV-Villin-miR16-5p / I alleviated CI in aged mice, which suggests that miRNA-16-5p in intestinal epithelial cells is also a potential target for treating age-related CI (Figures 11L-11N). To further explore the role of IEC-sEVs in age-related CI, we treated aged mice with GW4869, an inhibitor of sEVs secretion. The results showed that GW4869 treatment significantly alleviated CI in aged mice (Figure 10H). Notably, when GW4869 was combined with AAV-Villin-miR-16-5p / I treatment, no additional alleviation of CI was observed, suggesting that GW4869, by blocking IEC-sEVs secretion, has already prevented the delivery of miRNA-16-5p, and thus further inhibition of miRNA-16-5p failed to bring additional improvement (Figures 11O-11Q). In summary, our studies show that, consistent with the results of IBD, aging is accompanied by an increase in IEC-sEV-mediated long-distance delivery of miRNA-16-5p, which ultimately leads to CI by inhibiting hippocampal neurogenesis. Specific inhibition of miRNA-16-5p expression or blocking IEC-sEV release can effectively improve cognitive impairment caused by aging, suggesting that miRNA-16-5p and IEC-sEVs can be potential intervention targets for age-related CI.

[0083] Example 8: Skatole induces miRNA-16-5p in IEC-sEVs of aged mice

[0084] The present invention further analyzed the symbiotic intestinal microbiota of young and aging mice using metagenomic sequencing and found that B.Aci was highly enriched in the feces of aging mice (Figures 12A-12C). In addition, we found an increase in skatole in the feces and serum of aging mice (Figure 13A), indicating that the increase in miRNA-16-5p in intestinal epithelial cells may also be caused by the symbiotic intestinal microbiota of aging mice. To verify this speculation, the present invention removed the symbiotic intestinal microbiota of aging mice by oral antibiotics (OTA) and found that miRNA-16-5p in intestinal epithelial cells was significantly reduced (Figure 12D). Accordingly, antibiotics alleviated the CI phenotype of aging mice (Figures 12E-12G). In addition, the coexistence of young mice with old mice resulted in an increase in miRNA-16-5p in intestinal epithelial cells and IEC-sEVs of young mice (Figure 13B), while the CI symptoms of young mice increased significantly (Figures 13C-13E). Consistent with these results, decreased proliferation of hippocampal neural stem cells and newborn neurons and CDK1 protein were also observed in young mice ( FIG13F ).

[0085] To extend the findings of the present invention to humans, this example collected and analyzed feces and serum from young and elderly people. The results of large genome sequencing showed that Bacteroides were more abundant in the feces of the elderly (Figures 12H and 12I), and the number of Bacteroides in feces and serum increased (Figure 13G). In addition, in the serum of the elderly, A33 + The levels of miRNA-16-5p detected in EVs were higher than those in young people (Figure 13H). We then treated human HIEC-6 intestinal epithelial cells with skatole and detected a significant increase in miRNA-16-5p in HIEC-6 cells and their secreted sEVs (Skl-HIEC-6-sEVs) (Figure 13I). Like mouse IEC-sEVs, Skl-HIEC-6-sEVs inhibited the proliferation and CDK1 protein content of hippocampal neural stem cells and newborn neurons, and caused G2 / M phase arrest in human neural stem cells (Figures 13J-13L). However, when human neural stem cells were pre-transfected with a miRNA-16-5p inhibitor, the proliferation, G2 / M phase arrest, and CDK1 protein reduction of hippocampal neural stem cells were alleviated (Figures 13M-13O and 12J). Furthermore, similar results, including inhibition of neural stem cell proliferation and decreased CDK1 protein, were also observed in human neural stem cells transfected with miR-16-5p mimics (miR-16-5p / M) (Figures 12K-12N). Therefore, the commensal gut microbiota induces miRNA-16-5p levels in IEV-sEVs of aged mice, leading to age-related CI.

[0086] Example 9: VB12 prevents IBD and age-related CI by targeting miRNA-16-5p

[0087] In the process of screening the metabolites that modulate miRNA-16-5p, we also found that VB12 significantly downregulated miRNA-16-5p in MODE-K cells (Figure 14A). Since miRNA-16-5p in extracellular vesicles released by intestinal epithelial cells exacerbates IBD DSS and CI in aged mice, the present application further explored whether VB12 administration could improve IBD DSS and age-related CI by targeting miRNA-16-5p in IEC-sEVs. First, we performed VB12 intraperitoneal injection intervention on IBD DSS and aged mice. Subsequently, the cognitive function of mice was comprehensively evaluated by behavioral tests such as novel object recognition test and water maze test, and histological detection.

[0088] As expected, VB12 treatment significantly alleviated the development of CI in chronic colitis and aged mice (Figures 14B-14D). Accordingly, Sox2 + hippocampal neural stem cells and DCX + neurons were also reduced in these mice (Figure 14E). However, in the two types of mice pre-injected with AAV-Villin-miR-16-5p / I, VB12 did not affect the development of CI (Figures 14F-14H). These results indicate that VB12 can effectively alleviate IBD DSS and age-related CI by targeting miRNA-16-5p.

[0089] Subsequently, the present application further detected and found that miRNA-16-5p in intestinal epithelial cells and IEC-sEVs from VB12-treated IBD DSS and aged mice was reduced (Figure 14I). In addition, we extracted IEC-sEVs from VB12-treated IBD DSS mice and aged mice, which were labeled as VB12-IEC-sEVs IBD and VB12-IEC-sEVs Aging respectively. When the IEC-sEVs of the control group significantly inhibited CDK1 protein proliferation and led to G2 / M phase arrest of hippocampal neural stem cells, VB12-treated IBD DSS and aged mice (VB12-IEC-sEVs IBD and VB12-IEC-sEVs Aging ) hardly affected these phenotypes (Figures 14J-14L). Compared with Ctrl-IEC-sEVs IBD or Ctrl-IEC-sEVs Aging , VB12-IEC-sEVsIBD or VB12-IEC-sEVs Aging Mild CI was induced in healthy mice (Figures 14M-14O). Integrating the above results, it can be concluded that VB12 effectively alleviates IBD in a miRNA-16-5p-dependent manner DSS and age-related CI.

[0090] VB12 is an approved clinical drug, and VB12 levels decrease with age. In addition, VB12 supplementation can improve cognitive function in the elderly. These results prompted the present application to further investigate whether VB12 has therapeutic potential for human IBD and age-related CI by targeting miRNA-16-5p. Therefore, the present embodiment further collected feces from IBD patients and the elderly receiving VB12 treatment due to anemia or neuritis. Then, sEVs in the feces were isolated and identified (Figures 14P-14R). It was found that the level of miRNA-16-5p in these sEVs was significantly reduced after taking VB12 (Figure 14S). Consistent with these results, the ability of these sEVs to inhibit hippocampal neural stem cell proliferation was also significantly reduced (Figure 14T). In addition, the degree of reduction in miRNA-16-5p levels was positively correlated with the degree of reduction in the hippocampal neural stem cell proliferation inhibition index (Figure 14T). These results suggest that VB12 is also promising in the treatment of human IBD and age-related CI.

[0091] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

[0092] SEQUENCE LISTING:

[0093] Seq ID NO. 1:

[0094] miRNA-16-5p: 5'-UAGCAGCACGUAAAUAUUGGCG-3'.

Claims

1. Use of an agent modulating a protein or a gene in extracellular vesicles for the manufacture of an agent for alleviating cognitive dysfunction.

2. Use according to claim 1, characterized in that, The agent modulating a protein or a gene in extracellular vesicles comprises an agent inhibiting protein production in extracellular vesicles or an agent inhibiting gene expression in extracellular vesicles.

3. Use according to claim 1, characterized in that, The gene is miRNA-16-5p having a nucleotide sequence as shown in Seq ID NO.

1.

4. The use according to claim 1, characterized in that, The cognitive dysfunction comprises colitis and / or aging-induced cognitive dysfunction.

5. The use according to claim 3, characterized in that, The agent inhibiting miRNA-16-5p expression comprises vitamin B12.

6. Use according to claim 5, characterized in that, The vitamin B12 promotes hippocampal neurogenesis by targeting miRNA-16-5p to thereby alleviate induced cognitive dysfunction.

7. Use according to claim 6, characterized in that, The vitamin B12 alleviates cognitive dysfunction by promoting CDK1 production in hippocampal neurons.

8. Use of miRNA-16-5p as a target for the mitigation of colitis and / or age-induced cognitive dysfunction, characterized in that, The miRNA-16-5p has a nucleotide sequence as shown in Seq ID NO.

1.

9. Use of vitamin B12 for the manufacture of a medicament for alleviating colitis and / or aging-induced cognitive dysfunction.

10. Use of extracellular vesicles as a target for alleviating colitis and / or aging-induced cognitive dysfunction.

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