Application of umbilical cord blood plasma-derived extracellular vesicles and related bioactive molecules in treatment of alzheimer's disease

Abstract

The application belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of umbilical cord blood plasma-derived extracellular vesicles and related bioactive molecules thereof in treatment of Alzheimer's disease. Specifically, the application isolates extracellular vesicles (UCBP-sEVs) from umbilical cord blood plasma, which has the effect of improving Alzheimer's disease. Further research finds that high enrichment of miR-16-2-3p therein is a key molecule for mediating treatment of Alzheimer's disease. miR-16-2-3p targets and inhibits expression of ROCK2, reduces phosphorylation level of TFEB and promotes nuclear translocation of the same, activates microglial autophagy-lysosome pathway, and thus promotes phagocytosis and degradation of intracerebral beta amyloid, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of extracellular vesicles derived from umbilical cord blood plasma and their related bioactive molecules in the treatment of Alzheimer's disease. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Alzheimer's disease (AD) is a central nervous system degenerative disease characterized by progressive cognitive impairment and behavioral disturbances. Its core pathological features include β-amyloid (Aβ) plaque deposition in the brain, neurofibrillary tangles, neuroinflammation, oxidative stress, synaptic degeneration, and neuronal death. In the neuroinflammation associated with AD and other neurodegenerative diseases, microglia, as resident immune cells of the central nervous system (CNS), play a crucial role in maintaining homeostasis. Under pathological conditions, microglia clear cellular debris and toxic protein aggregates through phagocytosis and autophagy; however, defects in lysosomal acidification lead to impaired phagocytic and autophagy functions, which not only perpetuates neuroinflammation but also accelerates the progression of neurodegenerative diseases. In recent years, numerous genes identified by genome-wide association studies (GWAS) have also highlighted the central role of microglia-specific functions in the pathophysiology of AD.

[0004] Exosomes are a class of natural nanovesicles with a diameter of 30-150 nm. They possess the characteristics of easily penetrating the blood-brain barrier and efficiently targeting lesion sites. They can release active substances to target cells through membrane fusion, regulating the phenotype and function of target cells, and have shown significant therapeutic potential in various disease models. As a class of natural nanovesicles rich in various stem cell-derived bioactive substances, human umbilical cord blood plasma-derived exosomes (UCBP-sEVs) have attracted widespread attention in recent years due to their unique therapeutic potential. Existing studies have confirmed the efficacy of UCBP-sEVs in the treatment of diseases such as wound healing, reversal of liver fibrosis, and repair of acute lung injury, but their role and molecular mechanism in the treatment of Alzheimer's disease (AD) remain unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of extracellular vesicles derived from umbilical cord blood plasma and their related bioactive molecules in the treatment of Alzheimer's disease. This invention involves isolating extracellular vesicles (UCBP-sEVs) from umbilical cord blood plasma, which have been shown to improve Alzheimer's disease. Further research revealed that highly enriched miR-16-2-3p is a key molecule mediating its therapeutic effect on Alzheimer's disease. miR-16-2-3p targets and inhibits ROCK2 expression, reduces TFEB phosphorylation levels and promotes its nuclear translocation, activates the microglial autophagy-lysosomal pathway, and thereby promotes the phagocytosis and degradation of β-amyloid protein in the brain. Based on the above research findings, this invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] A first aspect of the present invention provides the use of extracellular vesicles and related bioactive molecules in the preparation of medicaments for the treatment of Alzheimer's disease.

[0008] Specifically, the extracellular vesicles are extracellular vesicles derived from umbilical cord plasma, and more specifically, extracellular vesicles derived from human umbilical cord plasma; the relevant bioactive molecules include miRNA, and more specifically, miR-16-2-3p. In particular, in addition to miR-16-2-3p, the bioactive molecules may also include miR-16-2-3p expression promoters. These promoters can be exogenous delivery agents, i.e., directly supplementing miR-16-2-3p exogenously, such as miRNA mimics (mimic), umbilical cord plasma-derived exosomes, lentiviruses carrying the miR-16-2-3p encoding gene, adeno-associated virus (AAV) vectors, and endogenous promoters, i.e., those inducing cells to synthesize miR-16-2-3p themselves, such as transcription factor activators of the miR-16-2-3p encoding gene, etc., which are not limited here.

[0009] The specific manifestations of treatment for Alzheimer's disease are as follows: a1) Regulates the ROCK2 / TFEB axis and activates the microglia autophagy-lysosome pathway; a2) Improve cognitive function in AD; a3) Inhibits neuronal apoptosis in the brain; a4) Reduces intracranial inflammation and oxidative stress damage; a5) Enhances synaptic plasticity; a6) Relieve AD pathological damage.

[0010] Furthermore, a1) specifically manifests as: inhibiting the gene expression of Rho-associated protein kinase 2 (ROCK2), reducing the phosphorylation level of transcription factor EB (TFEB) and promoting its nuclear translocation, thereby activating the microglia autophagy pathway; the activation of this pathway can further upregulate the expression of lysosomal-associated functional proteins LAMP1, CTSD, and CTSB, enhance autophagy, and ultimately promote the phagocytosis and degradation of Aβ in the brain by microglia.

[0011] The extracellular vesicles and their associated bioactive molecules can serve as active ingredients and / or therapeutic carriers for Alzheimer's disease treatments, thereby exerting a dual effect of (targeted) therapy and loading other active ingredients.

[0012] Therefore, in a second aspect, the present invention provides a pharmaceutical preparation comprising at least extracellular vesicles and their associated bioactive molecules.

[0013] Specifically, the extracellular vesicles are extracellular vesicles derived from umbilical cord plasma, and the related bioactive molecules include miRNA, more specifically miR-16-2-3p. In particular, in addition to miR-16-2-3p, the bioactive molecules may also include promoters of miR-16-2-3p expression.

[0014] The pharmaceutical formulation is used for the treatment of Alzheimer's disease. Furthermore, as previously stated, the extracellular vesicles and their associated bioactive molecules (especially extracellular vesicles) exert at least a dual effect of (targeted) therapy and loading other pharmaceutically active ingredients.

[0015] Therefore, the pharmaceutical preparation may further include other active ingredients for treating Alzheimer's disease; and at least one non-pharmaceutical active ingredient.

[0016] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0017] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0018] Preferably, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0019] Preferably, the drug of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. It will be understood by those skilled in the art that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0020] Preferably, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees.

[0021] A third aspect of the present invention provides a method for improving Alzheimer's disease, the method comprising administering the above-described extracellular vesicles and their associated bioactive molecules or pharmaceutical preparations to a subject.

[0022] The beneficial technical effects of one or more of the above technical solutions are as follows: The UCBP-sEVs prepared using the above-mentioned technical solution are widely available, non-invasive to obtain, and have low immunogenicity. Compared with exosomes derived from umbilical cord mesenchymal stem cells (UCMSC-sEVs), they show more significant improvement in AD pathological and functional indicators. Furthermore, this technical solution elucidates for the first time the molecular mechanism of UCBP-sEVs in treating AD, namely, regulating the ROCK2 / TFEB axis through miR-16-2-3p, activating the microglial autophagy-lysosomal pathway, and promoting Aβ phagocytosis and degradation, providing a new target for targeted therapy of AD.

[0023] The above-mentioned technical solutions provide key experimental evidence for the development of AD treatment strategies. UCBP-sEVs are expected to become a new targeted therapy carrier for the treatment of AD and have broad clinical application prospects. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1To illustrate the characterization of exosomes and their uptake in the brain in this embodiment of the invention, (a) a schematic diagram of the separation process of small extracellular vesicles (sEVs) derived from human umbilical cord blood plasma (UCBP). (b) Representative transmission electron microscopy (TEM) images showing the morphology of UCBP-sEVs and UCMSC-sEVs. Scale bar = 200 nm. (c) Particle size distribution and concentration of UCBP-sEVs and UCMSC-sEVs as determined by nanoparticle tracking analysis (NTA). (d) Western blot analysis of purified sEVs to detect classic sEV markers (TSG101 and CD63) and the negative control endoplasmic reticulum marker (Calnexin). (e) Representative in vivo fluorescence images of 5XFAD mice at 4, 12, and 24 hours after intravenous injection (iv) of PKH26-labeled sEVs. (fg) Quantitative analysis of the fluorescence intensity of UCBP-sEVs and UCMSC-sEVs in the brain over time to indicate biodistribution (n = 3 per group). (h) Representative immunofluorescence images of the hippocampus of 5XFAD mice after treatment with PKH26-labeled UCBP-sEVs (red). Sections were immunostained (green) for astrocytes (GFAP), neurons (NeuN), or microglia (Iba-1). Nuclei were counterstained with DAPI (blue). (i) Colocalization analysis showing the spatial overlap between PKH26-labeled sEVs and GFAP+, NeuN+, or Iba-1+ cells. Data are expressed as mean ± standard error (mean ± sem).

[0026] Figure 2This invention illustrates how human umbilical cord blood plasma exosomes improve learning and cognitive function in mice. (a) A schematic timeline of intravenous injection (iv) of sEVs and behavioral tests (MWM, NOR, and OLM) in 5XFAD mice. (b) Representative swimming trajectories of each group of mice in the water maze (MWM) spatial exploration experiment. (ce) Quantitative analysis of spatial learning and memory performance in the MWM test. (c) The number of times the original platform was crossed in the exploration experiment, (d) the percentage of time spent in the target quadrant, and (e) the escape latency during the 5-day learning phase. (f) A learning curve representing the average daily escape latency during the 5-day training period. (g) Representative examples of mouse exploration trajectories in the New Object Recognition (NOR) and Object Location Memory (OLM) task testing phases. (hi) Quantitative analysis of recognition memory, expressed as the percentage of exploration time (or discrimination index) interacting with new objects in the NOR test (h) and the percentage of exploration time interacting with new locations in the OLM test (i). Data are expressed as mean ± standard error (mean ± sem) (n = 8 per group). Statistical significance was determined using one-way ANOVA and Bonferroni post-hoc test. P<0.05, P<0.01, P<0.001; ns indicates no significant difference.

[0027] Figure 3 In this embodiment of the invention, umbilical cord blood plasma exosomes can alleviate the pathological features of 5XFAD mice. Specifically, (a) brain sections of the cortex or parahippocampal gyrus of 5XFAD mice were stained with Aβ (6E10, red). Scale bar = 200 µm. (b) Quantitative analysis of the area and number of 6E10-labeled Aβ staining cores. (c) Mean ± standard error; n = 4; P<0.01, P < 0.001, Bonferroni-corrected univariate ANOVA was used. (d) Thioflavin S staining of cortical or parahippocampal gyrus brain sections of 5XFAD mice, scale bar = 200 µm. (e) Quantitative analysis of thioflavin S-labeled Aβ area and core number in brain tissue of 5XFAD, 5XFAD+hUCBP-sEVs, and 5XFAD+hUCBP-sEVs mice. (f) Mean ± standard error; n = 4; P<0.01, P<0.001, one-way ANOVA combined with Bonferroni post-hoc test. (g) Western blot analysis of APP, p-Tau, Tau, and Aβ1-42 in the hippocampus of WT, 5XFAD, and 5XFAD+hUCBP-sEVs and 5XFAD+hUCBP-sEVs mice. (hj) Quantification of the expression of APP, p-Tau, Tau, and Aβ1-42 relative to β-actin. Mean ± standard error; n=4; P<0.05, P<0.01, P < 0.001, one-way ANOVA and Bonferroni post-hoc test were performed. (k) Representative immunohistochemical images of P-Tau expression in hippocampal tissue. Scale bar = 50 μm. (l) Quantitative analysis of P-Tau staining positive signal. Mean ± standard error (mean ± sem); statistical significance was determined by one-way ANOVA and Bonferroni post-hoc test n = 4; P<0.05, P<0.01, P<0.001.

[0028] Figure 4 In this embodiment of the invention, umbilical cord blood plasma exosomes can reduce apoptosis in the brain of 5XFAD mice and increase dendritic spine density. Specifically, (a) representative immunofluorescence images of NeuN (red) in the hippocampus of 5XFAD mice treated with vectors, hUCMSC-sEVs, or hUCBP-sEVs. Scale bar: 50 µm. (b) Quantitative analysis of NeuN fluorescence intensity (n=4 per group). (c) Quantitative analysis of TUNEL positive signal. (d) Representative images of TUNEL staining (red) showing apoptotic cells in the hippocampus. Scale bar: 50 µm. (e) Western blot analysis of NeuN levels in hippocampal tissue lysate. β-actin was used as a control. (f) Quantitative analysis of NeuN expression relative to β-actin optical density (n=4). (g) Representative images of hippocampal neuronal reconstruction. (h) Quantitative analysis showing the number of branch crossings. Scale bar: 20 µm. (i) Representative images of Golgi staining. (j) Quantitative analysis of dendritic spine density (number of dendritic spines per 10 µm) (n=4). Data are expressed as mean ± standard error (mean ± sem) (n=4). One-way ANOVA and Bonferroni 1 test were used to determine statistical significance.

[0029] Figure 5 In this embodiment of the invention, hUCBP-sEVs alleviated oxidative stress and neuroinflammation in the brains of 5XFAD mice. (af) hUCBP-sEVs activated the Nrf2 antioxidant signaling pathway: the relative mRNA expression levels of antioxidant-related genes CAT(a), HO-1 (b), SOD2 (c), Nrf2 (d), NQO1 (e), and GSH-Px (f) in the hippocampus were detected by RT-PCR (n=4). (g) Representative immunofluorescence images of the expression of the pro-inflammatory factor iNOS (red) in the hippocampus of each group of mice. Scale bar: 50 μm. (h) Quantitative analysis of the immunofluorescence intensity of iNOS in the hippocampus and its (i) RT-PCR analysis of the relative mRNA expression level (n=4). (jm) hUCBP-sEVs reduced excessive activation of glial cells. (j) Representative immunofluorescence images showing the activation status of astrocytes (GFAP, red) in the hippocampus. Scale bar: 100 μm. (k) Quantitative analysis of GFAP immunofluorescence intensity in the hippocampus of mice in each group (n=4). (l) Representative immunofluorescence images of microglia (Iba-1, green) expression in the hippocampus of mice in each group. Scale bar: 100 micrometers. (m) Quantitative analysis of Iba-1 immunofluorescence intensity in the hippocampus of mice in each group (n=4). (nq) hUCBP-sEVs downregulate glial proliferation markers and pro-inflammatory cytokines: The relative mRNA expression levels of glial markers Iba-1 (n), GFAP (o), and pro-inflammatory factors IL-6 (p) and TNF-α (q) in the hippocampus were detected by RT-PCR (n=4). Data are presented as mean ± standard error (mean ± sem); statistical significance was determined by one-way ANOVA and Bonferroni post-hoc test (n=4). P<0.05, P<0.01, P<0.001, ns indicates no significant difference.

[0030] Figure 6This invention demonstrates how umbilical cord blood plasma exosomes enhance the autophagy-lysosome pathway in the brains of 5XFAD mice. Specifically, (a) a hierarchical clustering heatmap of differentially expressed genes (DEGs) in the hippocampus of wild-type (WT), 5XFAD, and hUCBP-sEVs-treated 5XFAD mice; (bc) relative expression levels of RNA sequencing of key genes involved in neural differentiation and autophagy (b) and RT-qPCR validation results (c); (d) KEGG pathway enrichment analysis of hUCBP-sEVs-treated DEGs (compared to hUCMSC-sEVs), highlighting the unique enrichment of FoxO and the lysosome-autophagy pathway; (e) a gene-pathway interaction network diagram showing the connections between hUCBP-sEVs-treated DEGs and their associated KEGG pathways; and (f) representative protein blots and quantitative analysis (gi) of autophagy-related proteins p62, Beclin-1, and LC3B in hippocampal tissue lysates. (j) Representative protein blots and quantitative analysis (km) of lysosomal markers LAMP1, CTSB, and CTSD. Data are expressed as mean ± standard error (n = 4). P<0.05, P<0.01, P<0.001.

[0031] Figure 7 This invention demonstrates how umbilical cord plasma exosomes enhance lysosomal function of microglia in the brains of AD mice and promote microglia phagocytosis of Aβ. Specifically, (a) representative immunofluorescence images of Iba1 (microglia) co-stained with Lamp1 in hippocampal sections from 5XFAD mice treated with the vector or hUCBP-sEVs. Scale bar: 10 μm. (b) Quantitative analysis of the Lamp1 signal area co-localized with Iba1-positive microglia. Data are expressed as mean ± standard error (n=4 mice per group). P<0.01 (two-tailed unpaired Student's t-test). (c) Representative images of Iba1-positive microglia co-localized with 6E10-positive Aβ plaques in hippocampal sections from 5XFAD and 5XFAD+hUCBP-sEVs groups. Scale bar: 20 μm. (d) Quantitative analysis of Aβ plaque-associated microglia. Data are expressed as mean ± standard error (n=4). P < 0.01 (two-tailed unpaired Student's t-test). (e) Representative images of GFAP (astrocytes) and 6E10 (Aβ) co-stained brain slices from each group of mice. Scale bar: 20 micrometers. (f) Quantitative analysis of Aβ plaque-associated astrocytes. Data are expressed as mean ± standard error (n=4). ns: no significant difference (two-tailed unpaired Student's t-test).

[0032] Figure 8 This is a microRNA sequencing analysis of umbilical cord blood plasma exosomes in this embodiment of the invention. (a) KEGG pathway enrichment analysis of miRNA target genes in hUCBP-sEVs (b) GO enrichment analysis of miRNA target genes in hUCBP-sEVs (c) 17 most highly expressed miRNAs specifically expressed in hUCBP-sEVs.

[0033] Figure 9 This invention demonstrates how miR-16-2-3p from hUCBP-sEVs enhances Aβ clearance by increasing autophagy-lysosome function in microglia. (a) Schematic diagram of the experimental procedure for assessing the effect of miR-16-2-3p on lysosome and autophagy function in HMC3 cells. (be) Effect of miR-16-2-3p on lysosomal function: (b) Representative Western blot maps of key lysosomal markers (Lamp1, CTSB, CTSD) in HMC3 cells. Quantitative analysis of CTSB (c), CTSD (d), and Lamp1 (e) protein levels normalized with beta-actin as an internal control (n=4). (fi) Effect of miR-16-2-3p on autophagic flux: (f) Representative Western blot maps of autophagy-related proteins (p62, Beclin-1, LC3B) in HMC3 cells. Quantitative analysis of Beclin-1 (g), LC3II / I ratio (h), and p62 (i) protein levels (n=4) relative to beta-actin normalization. (jk) Effect of miR-16-2-3p on autolysosome formation: (j) Representative fluorescence images of intracellular autophagosomes (yellow spots) and autolysosomes (red spots) after transfection with mCherry-GFP-LC3 adenovirus. (k) Quantitative statistics of the number of intracellular autophagosomes and autolysosomes in each group (n=4). (lm) Effect of miR-16-2-3p on Aβ uptake and degradation: (l) Representative immunofluorescence images showing the colocalization of FITC-labeled Aβ1-42 (green) and microglial marker Iba-1 (red), used to assess lysosomal uptake of Aβ. Scale bar: 20 micrometers. (m) Quantitative analysis of intracellular FITC-Aβ1-42 fluorescence intensity in each treatment group (n=4). (no) Detection of lysosomal acidic environment: (n) Representative fluorescence images showing the distribution of acidic organelles (lysosomes) using LysoTracker Red staining. Scale bar: 20 micrometers. (o) Quantitative statistics of corresponding LysoTracker fluorescence intensity (n=4). Data are expressed as mean ± standard error (n=4). In all figures, P<0.05, P<0.01, P<0.001 (one-way ANOVA and Bonferroni post-hoc test were used).

[0034] Figure 10 In this embodiment of the invention, miR-16-2-3p regulates the TFEB signaling axis by directly targeting ROCK2. (a) Bioinformatics interaction network diagram predicting potential downstream targets of miR-16-2-3p, with the core target gene ROCK2 highlighted in yellow. (b) Schematic diagram of sequence alignment between the miR-16-2-3p seed sequence and the conserved binding site of ROCK2 3'-UTR. (c) Results of dual-luciferase reporter gene assay in HMC3 cells, confirming the direct targeting and inhibition of ROCK2 3'-UTR wild-type (WT) but not mutant (Mut) (n=4). (d) Molecular docking simulation model of the interaction between ROCK2 and TFEB protein, showing that the two have a high binding affinity (-9.7 kcal / mol). (ef) Immunofluorescence staining analysis of the effect of miR-16-2-3p on ROCK2 expression: (e) Representative fluorescence images showing the expression and distribution of ROCK2 (red) in HMC3 cells of each treatment group. Scale bar: 20 μm. (f) Quantitative analysis of ROCK2 fluorescence intensity in each group of cells (n=4). (gi) Effect of miR-16-2-3p on ROCK2 / TFEB pathway protein levels: (g) Representative Western blot maps to detect the expression levels of ROCK2, phosphorylated TFEB (p-TFEB), and total TFEB after Aβ stimulation and the addition of hUCBP-sEVs, miR-16-2-3p mimics or inhibitors. Quantitative statistics based on the p-TFEB / TFEB ratio (h) and the relative protein level of ROCK2 (i) based on gray values ​​(n=4). Data are expressed as mean ± standard error (n=4). P<0.05, P<0.01, ns: no significant difference (one-way ANOVA combined with Bonferroni post-hoc test). Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0038] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0039] Example I. Materials and Methods 1. Mice We purchased 5XFAD mice from Nanjing Nanmo Biotechnology Co., Ltd. The mice were housed at the New Animal Model Center of Shandong University under a 12-hour light / 12-hour dark cycle, with free access to food and water. The animal use and experimental procedures have been approved by the Animal Care and Use Committee of Shandong University. 2. Acquisition and characterization of umbilical cord blood plasma exosomes and umbilical cord mesenchymal stem cell exosomes Exosome purification was performed using a fully automated exosome purification system (Shenzhen Huixin Biotechnology, EXODUS T-2800) based on ultrasonic nanofiltration. This system consists of an automated workstation and disposable, fully enclosed consumables (compliant with GMP requirements). 7 L of cell supernatant was pumped into a disposable sample bag, and 5 L of physiological saline was pumped into a buffer bag, connected to the corresponding tubing; the purification program was set to A20J15K5T2, with a recovery volume of 75 mL. 4 L of umbilical cord plasma was pumped into the sample bag, and 4 L of physiological saline was pumped into the buffer bag, connected to the corresponding tubing; the purification program was set to A23J20K3T8, with a recovery volume of 45 mL. After extraction, the exosome recovery station was sealed, the tubing was cut using a heat sealer, and the exosome solution was collected from the recovery station after being removed from the cooling chamber.

[0040] Nanoparticle tracking analysis (NTA) Before sample analysis, the sample chamber was rinsed with deionized water, and the ZetaView instrument was calibrated using polystyrene microspheres (110 nm). The sample cell was rinsed with 1×PBS buffer, diluted 200-fold with 1×PBS buffer, and then injected and analyzed. NTA software was used to assess size distribution and vesicle concentration. Transmission electron microscopy (TEM) Glutaraldehyde was added to the purified exosomes for fixation, and the solution was incubated overnight at 4°C. The suspension was dropped onto a copper grid on a carbon-supported membrane and left to stand for 3-5 minutes, then negatively stained with 2% uranium acetate. Excess liquid was blotted dry with filter paper, and images were collected and analyzed using a transmission electron microscope.

[0041] 3. Cell culture, transfection and treatment HMC3 cells were cultured in a constant temperature and humidity incubator at 37°C and 5% CO2. The culture medium used was a mixed solution containing 90% MEM, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin antibiotics, and the culture medium was changed every two days.

[0042] For cell transfection experiments, once cells reached 60%-70% confluence, miR-16-2-3p mimics or miR-16-2-3p inhibitors were transfected into the cells according to the Lipofectamine 2000 (Invitrogen) instructions. Twenty-four hours after transfection, cells were treated with Aβ and cultured for another 48 hours. Total RNA and total protein were then extracted for subsequent analysis.

[0043] 4. Alzheimer's disease mouse model and treatment This study used three-month-old 5XFAD mice. The vector-treated mice received saline solution. The 5XFAD mice were treated with hUC-MSC-sEVs and hUCBP-sEVs at a dose of 1:1 in 100 μL PBS. 10 9 The number of granules is determined by intravenous injection every other day for 45 days.

[0044] 5. Exosome markers Exosomes were stained using PKH26. Following the instructions for the PKH26 staining reagent, sEVs suspended in PBS were mixed with PKH26 dye and incubated at 37°C for 30 min. The reaction was terminated using FBS. After centrifugation at 120,000 g for 70 min, the supernatant containing unbound dye was discarded, and the precipitate was resuspended in PBS.

[0045] 6. In vivo and exosome tracking PKH-26-labeled exosomes were injected into mice via the tail vein, and the fluorescence signal in the mouse brain was measured using an IVIS spectral system at different time points (4h, 12h, 24h).

[0046] 7. Luciferase experiment To determine the common binding site between miR-16-2-3p and ROCK2, the 3′-UTR of ROCK2 or the mutated miR-16-2-3p binding site was subcloned into the pmirGLO dual-luciferase target expression vector to construct a reporter vector. Cells were seeded into 24-well plates and transfected using Lipofectamine 2000 with a mixture containing the reporter plasmid and miR-16-2-3p mimic. Luciferase activity was quantified using a dual-luciferase reporter gene assay kit.

[0047] 8.Aβ 1-42 Preparation of oligomers FITC-labeled Aβ1-42 peptide (MedChemExpress, HY-P3908) was dissolved to a concentration of 1 mM in ice-cold hexafluoroisopropanol (HFIP; Aladdin, H107503). The solution was incubated at room temperature for at least 1 hour to promote monomerization and remove pre-existing aggregates. HFIP was then evaporated to remove the HFIP, and the resulting peptide membrane was stored at -80 °C. Before use, the peptide membrane was resuspended in anhydrous dimethyl sulfoxide (DMSO) to a concentration of 5 mM, vortexed thoroughly, and then diluted to the desired working concentration with pre-cold PBS or serum-free, phenol red-free DMEM medium. To induce oligomerization, the diluted peptide solution was incubated at 4 °C for 24 hours before being added to the cell culture system.

[0048] 9. Golgi body staining The morphology and structure of dendritic spines in the hippocampus were stained and imaged using a Golgi apparatus staining kit (Cat# PK401, FD Neurotechnologies Inc., USA). Briefly, mouse brain tissue was extracted and immersed in Golgi apparatus staining solution for two weeks under light-protected conditions. The brain tissue was then transferred to a 30% sucrose solution and treated for another 72 hours. Afterward, the samples were cut into 200 μm thick brain slices, stained, and then dehydrated using a gradient of ethanol and xylene. Finally, the dendritic spine structure was imaged under a microscope, and spinal density was analyzed using ImageJ software.

[0049] 10. Lysosomal labeling Remove the cell culture medium, add 50 nM Lyso-Tracker Red staining working solution, incubate with the cells at 37ºC for 30 minutes, then remove the Lyso-Tracker Red staining working solution, add fresh cell culture medium, and observe under a fluorescence microscope.

[0050] 11. Thioflavin S staining Brain sections were stained for 8 minutes at room temperature in the dark with filtered 1% thioflavone S solution (Sigma, T1892-25G, prepared with 50% ethanol). Differentiation was initiated by rinsing with 50% ethanol (twice, 2 minutes each), followed by rehydration with PBS (three times, 5 minutes each). Sections were mounted with anti-fluorescence quenching mounting medium and images were acquired immediately using a fluorescence microscope (Olympus).

[0051] 12. TUNEL testing Apoptosis detection was performed strictly according to the manufacturer's instructions (Vazyme, A113-03). The simplified procedure is as follows: Brain slices were permeabilized with proteinase K at room temperature for 15 minutes, washed with PBS, and then incubated in a light-protected humidified chamber at 37°C for 1 hour with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP. Cell nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope (Olympus), and TUNEL-positive cells were quantitatively counted using ImageJ software.

[0052] 13. Immunofluorescence and Immunohistochemistry After anesthetizing and cardiac perfusion of mice, brain tissue was harvested and fixed in 4% paraformaldehyde (PFA). Following dehydration and embedding, brain slices with a thickness of 10-20 μm were prepared using a cryostat and stored at -80°C for later use. For immunofluorescence assays, the brain slices were equilibrated at room temperature for 30 minutes, washed three times with PBST for 5 minutes each time, and then treated with 2 mg / mL glycine solution for 15 minutes for sodium citrate antigen retrieval. Next, the slices were blocked with 10% serum at room temperature for 3 hours and incubated with primary antibody overnight at 4°C.

[0053] In the immunofluorescence assay, cell slides were first washed three times with PBS for 5 minutes each time, then fixed with 4% PFA at room temperature for 30 minutes, and treated with glycine for 15 minutes. Subsequently, blocking buffer containing 10% serum was prepared using 0.05% PBST, and the slides were blocked at room temperature for 3 hours, followed by overnight incubation with primary antibody at 4°C.

[0054] Paraffin-embedded brain tissue was serially sectioned to a thickness of 4 μm. After dewaxing with xylene and hydration with graded ethanol, the sections were placed in antigen retrieval buffer for antigen retrieval. To block endogenous peroxidase activity, the sections were treated with 3% H₂O₂ solution for 20 minutes. Subsequently, they were blocked at room temperature for 3 hours and incubated overnight with primary antibody at 4°C.

[0055] The primary antibody was recovered the next day and washed three times with PBST for 15 minutes each time. Then, the secondary antibody was incubated at room temperature in the dark for 1 hour, followed by three more washes with PBST for 15 minutes each time. The nuclei were stained with DAPI in the dark for 30 minutes. Finally, the slides were mounted with anti-fluorescence quenching mounting medium, and images were acquired and observed under a fluorescence microscope.

[0056] 14. Protein extraction and Western blotting experiments Brain tissue or cells were added to a lysis system containing RIPA lysis buffer, a mixture of protease inhibitors, and a mixture of phosphatase inhibitors. The sample was homogenized using a grinder for 60 seconds, followed by lysis at 4°C for 30 minutes. The cell lysate or tissue homogenate was centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal volumes of protein samples were separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. For Western blot analysis, the membrane was first blocked in TBST containing 5% skim milk at room temperature for 2 hours, then incubated overnight with primary antibody at 4°C with shaking. The next day, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour. The membrane was treated with ECL chemiluminescent substrate (Thermo Fisher Scientific) according to the manufacturer's instructions, and protein signals were imaged and analyzed using a Tanon 5200 multiplexer system (Tanon). The bands were quantified using ImageJ software, and the relative expression levels of each protein were normalized using β-actin as an internal reference.

[0057] 15. Water Maze Experiment The Morris Water Maze (MWM) experiment was used to assess the spatial learning and memory abilities of mice over a six-day period. The maze consisted of a circular pool virtually divided into four equal quadrants, with a hidden platform approximately 1 cm underwater in the center of one quadrant. For the first five days, mice were placed in the water facing the pool wall from one of four different entry points, given 60 seconds to find the platform. The time required to successfully find the platform was recorded as the escape latency. If a mouse failed to reach the platform within the time limit, it was guided to the platform and remained there for 30 seconds; this escape latency was recorded as 60 seconds. On day six, a spatial exploration experiment was conducted: the hidden platform was removed, and the mouse was placed in the water from the quadrant opposite the original platform location. The time spent in the original target quadrant and the number of times the mouse crossed the original platform location were recorded.

[0058] 16. New Object Recognition Experiment Before behavioral testing, mice were allowed at least 30 minutes to acclimatize to the testing environment. Testing was conducted in a white acrylic open field (35×35×25 cm). On Day 1, mice were allowed to freely explore the empty field for 10 minutes. On Day 2 (training phase), two identical objects were placed in the field, and each mouse was placed in a compartment facing away from the object, allowing for 10 minutes of free exploration. On Day 3 (testing phase), one of the familiar objects was replaced with a new object (for the new object recognition test, NOR), or a familiar object was moved to a new location (for the object localization memory test, OLM). Mice explored for 5 minutes. The discrimination index was calculated as: the time spent exploring the new or moved object divided by the total time spent exploring both objects. During the test intervals, the experimental field and objects were cleaned with a 5% ethanol solution to eliminate odor cues. All tests were recorded video using the Targetscan system.

[0059] 17. RNA extraction and real-time quantitative PCR analysis Total RNA was extracted from tissues and cells using Trizol reagent. The final RNA concentration was measured using a NanoDrop 2000 spectrophotometer. Subsequently, the total RNA was reverse transcribed into cDNA using reverse transcriptase. Quantitative real-time PCR was performed on a CFX96 Touch real-time PCR detection system (Bio-Rad). After amplification, the CT value was determined using a fixed threshold setting, and the average CT value was calculated through three replicates of PCR experiments. The relative expression of RNA was determined by the ΔΔCT method, with β-actin mRNA serving as an internal reference for mRNA quantification.

[0060] 18. Small RNA deep sequencing Small RNA libraries were constructed using the TruSeq Small RNA Library Preparation Kit (Illumina) and strictly followed the manufacturer's instructions. Library quality was assessed using the Agilent 2100 Bioanalyzer, and quantification was performed using the Quant-iT PicoGreends DNA assay kit. High-throughput sequencing was performed on the Illumina platform. After removing adapter sequences and low-quality reads, clean reads were aligned to the miRBase database (v22) to identify known miRNAs, and differential expression analysis was performed using DESeq2. TargetScan was then used to predict target genes for differentially expressed miRNAs, and functional enrichment analysis was conducted.

[0061] 19. RNA sequencing Total RNA from mouse brain tissues of different treatment groups was sequenced using transcriptome sequencing. RNA sequencing libraries were prepared by BGI Genomics Co., Ltd. (BGI Genomics, Wuhan, China). RNA sequencing was performed using the Illumina HiSeq™ 2000 platform.

[0062] 20. Statistical Analysis All quantitative data are expressed as mean ± standard deviation (SD) or standard error (SEM) and are from at least three independent experiments. The sample size (n) for each experimental group is clearly indicated in the corresponding legend. Statistical analysis was performed using GraphPad Prism 9.3.1. Unpaired two-tailed Student's t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons of three or more groups, combined with Bonferroni or Dunnett multiple comparison correction. P<0.05, P<0.01, P<0.001 and P < 0.0001 is considered statistically significant.

[0063] II. Experimental Results 1. Characterization of exosomes and their uptake in the brain To investigate the therapeutic potential of human umbilical cord plasma (UCBP)-derived factors, small extracellular vesicles (sEVs) were isolated using a fully automated exosome purification system based on ultrasonic nanofiltration (Figure 1a). For comparison, we also purified sEVs from human umbilical cord mesenchymal stem cells (UCMSC-sEVs). Transmission electron microscopy (TEM) confirmed that both UCBP-sEVs and UCMSC-sEVs exhibited typical goblet morphology and intact lipid bilayer structure, which are typical characteristics of sEVs. Figure 1 b). Nanoparticle tracking analysis (NTA) showed that both groups had similar particle size distributions, with peak values ​​around 150 nm. Western blot analysis confirmed the identity of these vesicles, showing significant enrichment of the classic sEV markers TSG101 and CD63, while the endoplasmic reticulum marker Calnexin was not detected, confirming the high purity of the isolates (Figure 1d).

[0064] A prerequisite for treating neurodegenerative diseases is the ability of drugs to cross the blood-brain barrier. To assess this, we intravenously injected PKH-26-labeled sEVs into 5XFAD mice. Long-term monitoring using the IVIS in vivo imaging system showed that both types of sEVs exhibited significant time-dependent accumulation of fluorescence signals in the brain, confirming their ability to cross the BBB (Figure 1e).

[0065] To further identify cellular targets within the central nervous system (CNS), we injected PKH26-labeled sEVs and performed immunofluorescence analysis on brain slices. Confocal microscopy revealed extensive uptake of sEVs in the brain parenchyma, with fluorescence signals primarily colocalizing with neurons (NeuN+) and microglia (Iba1+). Figure 1 (hi). These data indicate that peripherally administered UCBP-sEVs can target the brain and be internalized by key effector cells involved in the pathological process of AD.

[0066] 2. Human umbilical cord blood plasma exosomes improve learning and cognitive function in 5xFAD mice. To investigate the regulatory effects of small extracellular vesicles (sEVs) from different sources on cognitive function in Alzheimer's disease model mice, this study subjected 3-month-old 5XFAD mice to a 45-day alternating intervention via the tail vein of hUCBP-sEVs and hUCMSC-sEVs. Cognitive levels were then assessed using the Morris water maze (MWM) and a novel object / position recognition test system. Figure 2 a).

[0067] In the MWM water maze experiment, the escape latency of mice in all groups decreased with the increase of training days, but the learning curve of the hUCBP-sEVs treatment group decreased most significantly, showing superior learning ability compared to the AD+Saline group from day 2. Figure 2 f). In subsequent space exploration experiments, representative motion trajectories showed that the control group (AD+Saline) exhibited aimless random searching, while the hUCBP-sEVs treatment group showed a clear plateau-oriented target search pattern (f). Figure 2 b). Quantitative analysis confirmed that, compared with the hUCMSC-sEVs group, the hUCBP-sEVs group had a shorter escape latency and a significantly increased number of platform crossings (b). Figure 2 c) and extending the time spent in the target quadrant ( Figure 2 In terms of d), it showed a more significant statistical advantage (P<0.05).

[0068] In addition, to assess the mice's recognition memory and spatial location memory abilities, we conducted novel object recognition (NOR) and novel location recognition experiments. Figure 2 g). The results showed that the AD model group showed a significant decrease in its preference for exploring novel and displaced objects, while the hUCBP-sEVs treatment significantly reversed this trend, showing a higher proportion of preference for exploring new objects (g). Figure 2 i) and time spent exploring new locations ( Figure 2 (h), and the improvement effect was better than that of the hUCMSC-sEVs group.

[0069] In conclusion, hUCBP-sEVs, compared with hUCMSC-sEVs, can more effectively improve spatial learning and memory impairment and recognition memory impairment in 5XFAD mice.

[0070] 3. Umbilical cord blood plasma exosomes can alleviate pathological features in 5XFAD mice. To evaluate the therapeutic potential of extracellular vesicles (sEVs) targeting the two core neuropathological features of Alzheimer's disease—β-amyloid deposition and tau protein hyperphosphorylation—we systematically analyzed the brain tissue of 5XFAD transgenic mice. Quantitative immunohistochemical analysis using 6E10 anti-Aβ antibody and thioflavin S fluorescence staining revealed that, compared with the vector control group, both human umbilical cord blood plasma-derived sEVs (hUCBP-sEVs) and human umbilical cord mesenchymal stem cell-derived sEVs (hUCMSC-sEVs) significantly reduced Aβ plaque burden in the hippocampus. Figure 3 (af). Notably, hUCBP-sEVs exhibited superior efficiency compared to hUCMSC-sEVs in reducing Aβ deposition.

[0071] Western blot analysis results corroborated histological findings: hUCBP-sEVs administration significantly downregulated the expression levels of amyloid precursor protein (APP), insoluble Aβ1-42, and phosphorylated tau protein (p-Tau) in the brains of 5XFAD mice. Figure 3 g). hUCBP-sEVs had a significantly stronger regulatory effect on these pathological markers than hUCMSC-sEVs. Furthermore, immunohistochemical quantification showed that although both sEV interventions alleviated tau protein pathology, hUCBP-sEVs were more effective in reducing p-Tau immunoreactivity. Figure 3 In summary, these results indicate that although both types of sEVs can improve AD-related pathology, hUCBP-sEVs exhibit a stronger neuroprotective advantage in targeting the Aβ and tau protein pathological cascade.

[0072] 4. Umbilical cord blood plasma exosomes can reduce apoptosis in the brains of 5XFAD mice and increase dendritic spine density. To evaluate the neuroprotective efficacy of extracellular vesicles (sEVs), we used NeuN (neuronal nuclear antigen) staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) staining to detect neuronal activity and apoptotic cell death in the hippocampus. Compared with the untreated 5XFAD control group, both hUCBP-sEVs and hUCMSC-sEVs treatments significantly restored neuronal density. Figure 4 a, b), and simultaneously reduce apoptosis activity in brain cells ( Figure 4 (c, d). It is worth noting that hUCBP-sEVs exhibit superior neuroprotective efficacy compared to hUCMSC-sEVs.

[0073] Consistent with histological data, Western blot analysis showed that sEV administration modulated the balance of apoptosis regulators. Specifically, sEVs upregulated the expression of the neuronal marker NeuN (…). Figure 4 Similarly, the regulatory effects observed in the hUCBP-sEVs group were significantly more pronounced than those in the hUCMSC-sEVs group.

[0074] Synaptic loss is a structural marker of cognitive decline. To assess synaptic integrity, we used Golgi staining to examine dendritic morphology. Our study found that sEVs administration significantly restored dendritic spine density and branching complexity in the hippocampus. Figure 4(g,i). Notably, hUCBP-sEVs demonstrated superior efficacy compared to hUCMSC-sEVs in restoring synaptic plasticity. In summary, these data suggest that while both umbilical cord-derived sEV subtypes can alleviate neuronal apoptosis and synaptic dysfunction, hUCBP-sEVs possess greater therapeutic potential.

[0075] 5. Umbilical cord blood plasma exosomes can reduce oxidative stress and neuroinflammation in the brains of 5XFAD mice. To investigate the effects of extracellular vesicles (sEVs) on Alzheimer's disease-related oxidative stress and neuroinflammation, we first assessed the body's antioxidant defense levels. Results showed that sEV treatment significantly activated the nuclear factor E2-related factor 2 (Nrf2) signaling pathway in 5XFAD mice; qPCR analysis confirmed that the expression of Nrf2 and its downstream antioxidant molecules, including superoxide dismutase 2 (SOD2), catalase (CAT), heme oxygenase-1 (HO-1), glutathione peroxidase (GSH-Px), and nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase 1 (NQO1), was significantly upregulated. Figure 5 (a–f). Subsequently, we systematically evaluated the immunomodulatory effects of sEVs on chronic neuroinflammation. In the hippocampus of AD group mice, the protein and mRNA levels of the pro-inflammatory cytokine iNOS were significantly increased, while sEV intervention effectively inhibited the expression of this pro-inflammatory mediator (a–f). Figure 5 Furthermore, immunofluorescence and qPCR analysis showed that both sEVs significantly reduced abnormal glial cell activation, as evidenced by decreased expression levels of GFAP (an astrocyte marker) and Iba1 (a microglia marker) in the hippocampus. Figure 5 j–o), and simultaneously downregulated the levels of other key pro-inflammatory cytokines (IL-6 and TNF-α). Figure 5 (p, q). Notably, hUCBP-sEVs exhibited significantly stronger anti-inflammatory effects than hUCMSC-sEVs in inhibiting the expression of pro-inflammatory factors such as iNOS and improving glial cell overactivation. These data suggest that hUCBP-sEVs have superior efficacy in remodeling the neuroinflammatory microenvironment and inhibiting pro-inflammatory cascades.

[0076] 6. Umbilical cord blood plasma exosomes enhance the autophagy-lysosomal pathway in the brains of 5XFAD mice. To further explore the unique advantages of umbilical cord blood plasma exosomes (hUCBP-sEVs) in treating 5XFAD mice, we conducted an in-depth analysis of overlapping differentially expressed genes (DEGs) after treatment. Cluster heatmaps showed that after hUCBP-sEVs treatment, genes related to neural development (…) Ovol2, Foxp2, Otx2 ), neuropeptide receptor gene ( Npbwr1, Npsr1 ) and autophagy-related genes ( Dapk2 The expression of ) was significantly increased ( Figure 6 ab). The mRNA levels of these genes in the hippocampus of each group of mice were detected by reverse transcription PCR (RT-PCR), and the results were consistent with RNA-seq data. Figure 6 c).

[0077] Further KEGG enrichment analysis of genes whose expression changed after hUCBP-sEVs treatment but not after hUCMSC-sEVs (umbilical cord mesenchymal stem cell exosomes) treatment revealed that these genes were significantly enriched in the FoxO signaling pathway and the lysosomal-autophagy-related signaling pathway. Figure 6 d).

[0078] To elucidate the molecular regulatory framework of hUCBP-sEVs in Alzheimer's disease (AD), we constructed a gene-KEGG pathway interaction network based on differentially expressed genes (DEGs) and their enriched KEGG pathways. Analysis showed that the neuroactive ligand-receptor interaction pathway is the core hub with the highest connectivity in the network, and is closely linked to key genes. Npbwr1 (Neuropeptide B / W receptor 1) Nts (Neurotensin) Rxfp1 It is closely linked to receptors such as relaxin family peptide receptor 1. This pathway regulates fundamental neurophysiological processes such as central neurotransmitter transmission and synaptic plasticity—processes that are commonly disrupted in AD-related cognitive impairments (such as synaptic loss and impaired neural communication). The central role of this module suggests that hUCBP-sEVs may restore impaired neurophysiological functions in AD by repairing this pathway.

[0079] Network analysis also identified a submodule centered on autophagy and lysosomal functions, comprising three interconnected pathways: Animal autophagy is associated with the autophagy initiation regulatory gene Dapk2, which may regulate the initiation of autophagic flux. The FoxO signaling pathway is associated with the gene Irs4, which regulates metabolic stress and cell survival, and may mediate the balance between cell survival and stress response. The lysosome signaling pathway is associated with the lysosomal protein transport and degradation gene Ap1m2, and may be responsible for protein degradation (such as Aβ clearance). The co-activation of this submodule by hUCBP-sEVs suggests that it may promote the clearance of pathological protein aggregates in AD by enhancing the function of the autophagy-lysosome pathway. Figure 6 e).

[0080] Our experiments confirmed that in 5XFAD mice, the expression of autophagy-related proteins (LC3B, Beclin-1) in the hippocampus was decreased, while the expression of p62 protein was increased, and the expression of lysosomal production-related proteins (CTSD, CTSB, Lamp1) was reduced. Treatment with hUCBP-sEVs effectively rescued these abnormally expressed proteins. Figure 6 Therefore, our data indicate that umbilical cord blood plasma exosomes can significantly enhance autophagy-lysosomal capacity in the brains of 5XFAD mice.

[0081] 7. Umbilical cord blood plasma exosomes enhance lysosomal function of microglia in the brain of AD mice and promote phagocytosis of Aβ by microglia. Microglia, as resident innate immune cells in the brain, play a crucial role in coordinating immune responses. Simultaneously, their powerful phagocytic capacity forms the first line of defense in the central nervous system. As phagocytes, microglia are responsible for clearing cellular waste, debris, and toxic protein aggregates; this process is highly dependent on optimal lysosomal acidification and function. Impaired lysosomal acidification in microglia leads to dysfunction in phagocytosis and autophagy, thereby promoting persistent neuroinflammation and driving the progression of neurodegeneration. By analyzing the co-localization of IBA-1 and the lysosomal marker protein Lamp1, we found that umbilical cord blood plasma exosome treatment significantly increased the number of lysosomes in microglia in the brains of 5XFAD mice. Figure 7 a). To investigate whether enhanced microglial recruitment around plaques promoted Aβ phagocytosis and clearance, we further analyzed the co-localization of microglia with Aβ plaques. The results showed that umbilical cord blood plasma exosome treatment significantly enhanced the co-localization of microglia (but not astrocytes) with Aβ plaques. Figure 7 (cf). In summary, these findings indicate that umbilical cord blood plasma exosome therapy effectively enhances the phagocytic clearance capacity of microglia for Aβ.

[0082] 8. MicroRNA sequencing analysis in umbilical cord blood plasma exosomes Umbilical cord blood plasma exosomes contain a variety of bioactive molecules. Comparative analysis of the microRNA expression profiles of umbilical cord mesenchymal stem cell exosomes and umbilical cord blood plasma exosomes revealed a significantly richer variety of microRNAs in umbilical cord blood plasma exosomes. Small RNA sequencing identified conserved microRNAs in both types of exosomes. Notably, among these shared microRNAs, miR-548am-5p, miR-20b-5p, and miR-30b-5p have been shown to possess neuroprotective functions: miR-548am-5p maintains cellular homeostasis by inhibiting mitochondrial apoptosis pathways and reducing reactive oxygen species (ROS) levels; miR-20b-5p promotes neurite growth and axonal elongation; and miR-30b-5p has a significant protective effect against ischemic brain injury. KEGG pathway enrichment analysis of microRNAs specific to umbilical cord blood plasma exosomes showed significant enrichment in inflammation regulation, metabolism, and lysosomal-related signaling pathways. Figure 8 a) Gene Ontology (GO) analysis further revealed its enrichment in key biological processes, including: protein transport, cell cycle regulation, DNA repair, endocytosis, nervous system development, autophagic flux, neuronal projection development, autophagy regulation, negative regulation of apoptosis, and positive regulation of stem cell population maintenance. At the cellular component level, significantly associated key structures include: focal adhesion (cell adhesion), glutamatergic synapses, neuronal membrane and postsynaptic density (neural signal transmission), lysosomes, endosomes, mitochondrial membranes, autophagosomes, and proteasome complexes. Molecular functions are mainly enriched in kinase activity, ubiquitination modification, and transcriptional regulation. Figure 8 b).

[0083] We sorted the exosome-specific microRNAs in umbilical cord blood plasma according to their expression abundance. Figure 8 c) Notably, several of the highly abundant microRNAs exhibit clear anti-inflammatory functions: miR-150-3p alleviates the inflammatory response by inhibiting the TRAF6 / NF-κB signaling axis; miR-106a-5p can reverse LPS-induced macrophage glycolysis abnormalities and pyroptosis; miR-18a-3p alleviates inflammatory diseases by regulating the GSDMD / NLRP3 pathway; and miR-1283 protects cardiomyocytes from hypoxia-reoxygenation damage through the JNK / p38 MAPK pathway. Particularly noteworthy is that miR-16-2-3p, the most abundant microRNA, can improve diabetic microvascular complications by regulating ACADM-mediated fatty acid metabolism, and its potential function in neurological diseases remains to be explored.

[0084] 9 miR-16-2-3p derived from hUCBP-sEVs enhances β-amyloid clearance by activating the autophagy-lysosomal pathway in microglia. We further investigated the effect of miR-16-2-3p on the lysosomal autophagy mechanism in Aβ-stimulated HMC3 cells. Overexpression of miR-16-2-3p significantly upregulated key lysosomal markers (LAMP1, CTSD, and CTSB) in transfection with miR-16-2-3p mimics or negative controls. Figure 9 be) and autophagy-related proteins (Beclin-1 and LC3BII / I ratio); Figure 9 fh). This molecular amplification of lysosomal compartments is enhanced by LysoTracker fluorescence intensity (fh). Figure 9 n) was confirmed. To verify whether this enhanced lysosomal biosynthesis could translate into improved amyloid clearance, we used FITC-labeled Aβ 1-42 Oligomers were incubated in cells. Notably, compared to the control group, cells overexpressing miR-16-2-3p showed a higher FITC-Aβ content. 1-42 Colocalization with lysosomes increased significantly ( Figure 9 Furthermore, detection using the dual-fluorescent autophagy indicator system (mCherry-GFP-LC3) showed that miR-16-2-3p significantly increased the number of intracellular autolysosomes (red spots). Figure 9 Importantly, the addition of the miR-16-2-3p inhibitor significantly reversed the aforementioned lysosomal enhancement and Aβ degradation effects induced by hUCBP-sEVs. These results indicate that miR-16-2-3p improves Aβ pathology by enhancing microglial phagocytosis and subsequent lysosomal degradation pathways.

[0085] 10 miR-16-2-3p directly targets ROCK2 to modulate the TFEB signal axis. To identify downstream effector molecules mediating miR-16-2-3p function, we performed computer simulations to predict target sites using the TargetScan database. Figure 10 a). Analysis revealed ROCK2 (Rho-associated coiled-coil protein kinase 2) as a potential target, with a highly conserved miR-16-2-3p binding site within its 3′ untranslated region (3′-UTR). Figure 10 b). ROCK2 was chosen for further validation based on its proven negative regulatory role in mitophagy and its association in the pathology of neurodegenerative diseases. Molecular docking simulations further revealed a strong binding affinity (-9.7 kcal / mol) between ROCK2 and TFEB, suggesting a possible direct interaction between the two. Figure 10d). To confirm the direct interaction between miR-16-2-3p and ROCK2, we performed a dual-luciferase reporter gene assay. Co-transfection with the miR-16-2-3p mimic significantly inhibited luciferase activity in the wild-type ROCK2 3′-UTR (ROCK2-WT) reporter vector, while this inhibition was eliminated in the mutant 3′-UTR (ROCK2-Mut) group. Figure 10 c). Furthermore, at the protein level, overexpression of miR-16-2-3p not only downregulated ROCK2 expression but also reduced the level of phosphorylated TFEB (p-TFEB). Figure 10 d). Because ROCK2-mediated phosphorylation retains TFEB in the cytoplasm, and furthermore, immunofluorescence staining results showed that hUCBP-sEVs or miR-16-2-3p mimics significantly reduced the red fluorescence intensity of ROCK2 in HMC3 cells. Figure 10 e, f).

[0086] At the protein level, Western blot analysis confirmed that overexpression of miR-16-2-3p significantly downregulated ROCK2 expression, accompanied by a decrease in phosphorylated TFEB (p-TFEB) levels. Figure 10 (gi). Since ROCK2-mediated phosphorylation retains TFEB in the cytoplasm, the addition of miR-16-2-3p inhibitors can effectively reverse the inhibitory effect of hUCBP-sEVs on the ROCK2 / p-TFEB pathway. These data indicate that miR-16-2-3p promotes autophagic flux by relieving ROCK2-dependent inhibition of TFEB.

[0087] Matters not covered in this invention are common knowledge.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of extracellular vesicles and related bioactive molecules in the preparation of drugs for the treatment of Alzheimer's disease; among which, The extracellular vesicles are extracellular vesicles derived from umbilical cord plasma; the related bioactive molecules include miRNA, specifically miR-16-2-3p.

2. The application as described in claim 1, characterized in that, The extracellular vesicles are extracellular vesicles derived from human umbilical cord plasma.

3. The application as described in claim 1, characterized in that, The bioactive molecules also include promoters of miR-16-2-3p expression.

4. The application as described in claim 3, characterized in that, The promoter is an exogenous delivery agent and an endogenous promoter; The exogenous delivery reagents include miRNA mimics, umbilical cord plasma-derived exosomes, lentiviruses carrying the miR-16-2-3p encoding gene, and adeno-associated virus vectors. The endogenous promoters include transcription factor activators of the miR-16-2-3p encoded gene.

5. The application as described in claim 1, characterized in that, The specific manifestations of treatment for Alzheimer's disease are as follows: a1) Regulates the ROCK2 / TFEB axis and activates the microglia autophagy-lysosome pathway; a2) Improve cognitive function in AD; a3) Inhibits neuronal apoptosis in the brain; a4) Reduces intracranial inflammation and oxidative stress damage; a5) Enhances synaptic plasticity; a6) Relieve AD pathological damage.

6. The application as described in claim 5, characterized in that, Specifically, a1) is manifested as: inhibiting the gene expression of ROCK2, reducing the phosphorylation level of transcription factor TFEB and promoting its nuclear translocation, thereby activating the microglia autophagy pathway; this pathway activation further upregulates the expression of lysosomal-related functional proteins LAMP1, CTSD, and CTSB, enhances autophagy, and ultimately promotes the phagocytosis and degradation of Aβ in the brain by microglia.

7. The application as described in claim 1, characterized in that, The extracellular vesicles and their related bioactive molecules serve as active ingredients and / or therapeutic carriers for Alzheimer's disease treatment drugs.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains at least extracellular vesicles and their related bioactive molecules; The extracellular vesicles are extracellular vesicles derived from umbilical cord plasma; the related bioactive molecules include miRNA, specifically miR-16-2-3p.

9. The pharmaceutical preparation according to claim 8, characterized in that, The bioactive molecules also include promoters of miR-16-2-3p expression.

10. The pharmaceutical formulation as described in claim 8, characterized in that, The pharmaceutical preparation also contains other active ingredients for treating Alzheimer's disease; and at least one non-pharmaceutical active ingredient.

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