Application of stem cell exosome in treatment of depression-like behavior caused by high-altitude hypoxia

Abstract

The invention discloses application of a stem cell exosome in treating depression-like behaviors caused by high-altitude hypoxia, and belongs to the technical field of biomedicine. The stem cell exosome is an exosome derived from human umbilical cord mesenchymal stem cells, and is used for drugs for preventing and / or treating depression-like behaviors caused by high-altitude hypoxia. The exosome can effectively improve depression-like core behavior symptoms caused by high-altitude hypoxia by regulating a brain-intestinal axis; the method starts from remodeling intestinal flora so as to influence the intracerebral metabolite spectrum, and finally plays a role in neuroprotection by regulating and controlling a 5-HT6 / cAMP-PKA key signal channel. In addition, the combination of the hUC-MSC-exo and probiotics shows synergistic interaction potential, and a potential treatment strategy which is novel in mechanism, remarkable in effect and high in safety is provided for the field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to the application of stem cell exosomes in treating depression-like behavior caused by high-altitude hypoxia. BACKGROUND

[0002] Depression is a common mental disorder that affects millions of people worldwide. Epidemiological surveys and experimental studies have shown that high-altitude environments may induce or exacerbate such conditions, and residents in high-altitude areas are more likely to develop such psychological problems compared to those in low-altitude areas. It is currently believed that the onset of this condition is related to the imbalance of neurotransmitter systems (such as serotonin, GABA, norepinephrine, and dopamine), as well as the overactivation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to abnormal elevation of stress hormones such as cortisol, and functional and structural changes in specific brain regions such as the prefrontal cortex and hippocampus. The gut microbiota is one of the key regulators of the brain-gut axis, and it participates in physiological and pathological changes in the brain and intestinal tissues through multiple pathways such as neural, immune inflammation, and endocrine. The gut microbiota communicates with the brain by producing neuroactive substances and regulating the neural network of the intestinal immune system and the enteric nervous system (ENS). Numerous studies have shown that regulating the gut microbiota based on the "brain-gut axis" theory has become a new therapeutic target for the prevention and treatment of depression.

[0003] Exosomes are extracellular vesicles with lipid bilayer membranes, with a diameter ranging from 30 to 200 nanometers, and can carry biological information from the parent cells. Various cell types, including neurons, immune cells, epithelial cells, and mesenchymal stem cells, can secrete exosomes under normal and pathological conditions. Mesenchymal stem cell exosomes have low immunogenicity, small size, can penetrate the blood-brain barrier, have enhanced stability, and are easy to store. Numerous studies have shown that exosomes have significant therapeutic effects in treating brain injury and nervous system diseases.

[0004] Many studies have shown that the use of exosomes derived from mesenchymal stem cells can significantly improve depression-like behavior and inhibit inflammatory response. These exosomes not only regulate key signaling pathways in the brain, but also alleviate depressive symptoms by regulating the expression of inflammatory cytokines, but the mechanism of improving depression-like behavior caused by high-altitude hypoxia is not clear enough. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an application of stem cell exosomes in treating depression-like behavior caused by high-altitude hypoxia, which solves the problem of lack of safe, effective, and mechanism-specific treatment methods for high-altitude hypoxia-induced depression-like behavior in the prior art.

[0006] The technical problem to be solved by the present application is solved by the following technical solution:

[0007] The use of stem cell exosomes in the preparation of drugs for the prevention and / or treatment of depressive-like behaviors induced by high-altitude hypoxia.

[0008] Preferably, in the above technical solution, the drug is used to improve one or more of the following pathological changes caused by high-altitude hypoxia:

[0009] (a) Elevated serum oxidative stress markers and decreased antioxidant markers;

[0010] (b) Elevated serum inflammatory factor levels;

[0011] (c) Impaired intestinal barrier function;

[0012] (d) Cytotoxic edema of brain tissue and ultrastructural damage to neurons;

[0013] (e) Intestinal flora imbalance.

[0014] Preferably, in the above technical solution, the stem cell exosomes are exosomes derived from human umbilical cord mesenchymal stem cells, which are administered via intraperitoneal injection.

[0015] Preferably, in the above technical solution, the preventive and / or therapeutic effects of the drug are achieved by regulating the brain-gut axis.

[0016] Preferably, in the above technical solution, the regulation of the brain-gut axis includes:

[0017] (a) Improves gut microbiota composition and increases the abundance of short-chain fatty acid-producing bacteria;

[0018] (b) Repairing the intestinal barrier and upregulating the expression of intestinal tight junction proteins Occludin and ZO-1;

[0019] (c) Regulate the levels of metabolites in the brain that are related to energy metabolism and neural activity.

[0020] Preferably, in the above technical solution, the regulation of the brain-gut axis ultimately involves activating the 5-HT6 / cAMP-PKA signaling pathway in the brain.

[0021] A pharmaceutical composition comprising:

[0022] (a) Therapeutic doses of exosomes derived from human umbilical cord mesenchymal stem cells;

[0023] (b) Live Bifidobacterium longum or its fermentation supernatant; and

[0024] (c) A pharmaceutically acceptable carrier;

[0025] The pharmaceutical composition is used to prevent and / or treat depressive-like behaviors caused by high-altitude hypoxia.

[0026] The use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of depressive-like behaviors caused by high-altitude hypoxia.

[0027] The application of stem cell exosomes in the preparation of one or more of the following drugs, all of which are used for depressive-like behavior induced by high-altitude hypoxia and have the following corresponding functions:

[0028] (a) Drugs that lower serum oxidative stress markers and increase antioxidant markers;

[0029] (b) Medications that lower serum inflammatory factor levels;

[0030] (c) Drugs that repair damage to the intestinal barrier function;

[0031] (d) Drugs that reduce cytotoxic edema of brain tissue and damage to the ultrastructure of neurons;

[0032] (e) Medications that correct gut microbiota dysbiosis.

[0033] The above-described technical solution of the present invention has the following beneficial effects:

[0034] This application provides a novel application of human umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSC-exo) in the prevention and treatment of depressive-like behaviors induced by high-altitude hypoxia. hUC-MSC-exo can effectively improve core depressive-like behavioral symptoms caused by high-altitude hypoxia by regulating the "brain-gut axis," and significantly repair intestinal barrier damage, reduce systemic inflammation and oxidative stress, and protect brain tissue ultrastructure. Its mechanism of action is clear, beginning with the remodeling of the gut microbiota, then influencing the brain metabolite profile, and ultimately exerting neuroprotective effects by regulating the key 5-HT6 / cAMP-PKA signaling pathway. Furthermore, the combination of hUC-MSC-exo and probiotics shows synergistic potential, providing a novel, effective, and safe potential therapeutic strategy for this field. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0036] Figure 1Characterization of human umbilical cord mesenchymal stem cells and exosomes. (A) Morphological characteristics and multi-lineage differentiation potential of human umbilical cord mesenchymal stem cells (hUC-MSCs); (B) Detection of surface markers of human umbilical cord mesenchymal stem cells; (C) Transmission electron microscopy (TEM) images showing that the isolated vesicles have a typical cup-shaped morphology and an intact lipid bilayer membrane structure; (D) Nanoparticle tracking analysis (NTA) maps showing the particle size distribution of the isolated particles, with most particle sizes falling within the expected range (50-150 nm) for exosomes; (E) Western blot analysis confirming the presence of exosome-positive protein markers.

[0037] Figure 2 Human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-exo) can improve depressive-like behavior, pathological changes, and serum biomarkers induced by high-altitude hypoxia in rats. (A) Representative movement trajectories of each experimental group in the open field test (OFT); (B) Quantitative analysis of OFT parameters: number of central region entries, stationary time, and central region dwell time; (C) Immobility time recorded in the 5-minute forced swimming test (FST); (D) Representative images of H&E staining and immunohistochemical staining; (E) Serum T-SOD activity; (F) Serum malondialdehyde (MDA) level; (G) Serum IL-6 concentration measurement results; (H) Serum TNF-α concentration measurement results.

[0038] Figure 3 hUC-MSC-exo can alleviate hypoxia-induced brain injury and improve the ultrastructural pathological changes in brain and intestinal tissues after hypoxia injury. (A) Representative SWI-weighted magnetic resonance imaging (MRI) images of rat brains. Arrows indicate microhemorrhagic foci present in the hypoxia model (DY) group, which were not present in the normoxic control group (CY) and the hUC-MSC-exo treatment group; (BE) Quantitative analysis of apparent diffusion coefficient (ADC) in specific brain regions. Compared with the CY group, hypoxia exposure (DY group) led to a significant increase in ADC values ​​in all measured regions, indicating cytotoxic edema, which was improved by hUC-MSC-exo treatment; (F) Transmission electron microscopy (TEM) images of neuronal cell bodies and mitochondria; (G) TEM images of ileal villi and mitochondria.

[0039] Figure 4Results of multi-omics analysis. 16S results: (A) Shannon index; (B) Simpson index; (C) Principal component analysis (PCA); (D) Genus-level species composition; (E) Species-level species composition. Metabolomics: (F) Principal component analysis (PCA); (G) Volcano plot of differentially expressed metabolites; (H) Differentially expressed metabolites in the H_vs_DY group. Transcriptomics analysis: (I) Principal component analysis (PCA); (J) Volcano plot of differentially expressed genes; (K) Heatmap of differentially expressed genes.

[0040] Figure 5 For pathway selection and validation, as well as in vitro bacterial growth curve plotting and cell viability assays. (A) DY vs CYKEGG enrichment of upregulated pathways; (B) H vs DYKEGG enrichment of downregulated pathways. (CF): Representative protein blot images and grayscale statistical analysis of 5-HT6, cAMP, and PKA in rat cerebral cortex tissue; (GL): RT-qPCR validation of related genes; (M) Bacterial growth curves; (N) Imaging of PKH67-labeled exosomes in CTX-TNA2; (OP) Cell viability assays.

[0041] Figure 6 Results were validated in vitro. (AD) Representative Western blot images and grayscale statistical analysis of 5-HT6, cAMP, and PKA in CTX-TNA2. (EJ) RT-qPCR validation of related genes.

[0042] Figure 7 A schematic diagram illustrating the mechanism by which hUC-MSCs-exo alleviates depressive-like behavior induced by high-altitude hypoxia. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0045] Example 1: Preparation of human umbilical cord stem cell exosomes (hUC-MSC-exo)

[0046] Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were provided by Shandong Qilu Stem Cell Engineering Co., Ltd. (Approval No.: QLSC2024-001). Human umbilical cord-derived mesenchymal stem cell exosomes (hUC-MSC-exo) were extracted by Beijing Enzekangtai Biotechnology Co., Ltd. (Product No.: CTE-05) and diluted before use. Nanoparticle tracking analysis (NTA) was used to analyze the particle size distribution and concentration of the separated particles, and transmission electron microscopy (TEM) was used to observe their morphology. Western blot analysis confirmed the presence of specific exosome marker proteins.

[0047] Results analysis:

[0048] Characterization of human umbilical cord mesenchymal stem cells and their exosomes: Under a microscope, human umbilical cord mesenchymal stem cells (hUC-MSCs) appear as slender, spindle-shaped, fibroblast-like cells with multi-lineage differentiation potential. Figure 1 Figure A). Flow cytometry detected positive markers CD81, CD63, and TSG101 on the surface of human umbilical cord mesenchymal stem cells (hUC-MSCs), while CD45, CD34, and HLA-DR were negative. Figure 1 Figure B). Transmission electron microscopy confirmed the presence of the characteristic bilayer membrane structure of exosomes, and nanoparticle tracking analysis (NTA) showed that the exosome size distribution was mainly between 50 and 150 nanometers. Figure 1 The CD image), Western blot analysis confirmed the presence of positive exosome protein markers ( Figure 1 (E diagram).

[0049] Example 2: The effect of hUC-MSC-exo on improving depressive-like behavior induced by high-altitude hypoxia in rats (in vivo pharmacodynamic evaluation)

[0050] 1. Laboratory animals

[0051] SD rats aged 6-8 weeks (weighing 200-250 grams) were selected and provided by Henan Scripps Biotechnology Co., Ltd. (License No.: SCXK; Yu,2020–0005). This study protocol followed the National Research Council of the United States’ Guidelines for the Care and Use of Laboratory Animals and was approved by the Laboratory Animal Ethics Committee of Qinghai University (Approval No.: PJ202501-162).

[0052] 2. Animal Experiment Design and Sample Collection

[0053] Thirty rats were randomly divided into five groups: normoxic group (CY), hypoxic group (DY), low-dose group (L, 2×10⁻⁶), and low-dose group (L, 2×10⁻⁶). 5 particles / mL, 0.5mL), medium dose group (M, 2×10) 6particles / mL, 0.5mL) and high-dose group (H, 2×10) 6 Six animals per group (particles / mL, 1 mL). Except for the normoxic group, all animals were kept in a hypobaric chamber at an altitude of 6000 meters for 7 days. During this period, the L, M, and H groups were injected intraperitoneally with the corresponding dose of hUC-MSC-exo daily, while the CY and DY groups were injected with the same volume of physiological saline daily and allowed free access to food. After modeling, the animals were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital solution (1 ml / 100 g). After blood collection from the abdominal aorta, the animals were euthanized by blood loss. Subsequently, samples of prefrontal cortex tissue, ileum tissue, and feces were collected.

[0054] 3. Forced swimming test (FST)

[0055] After 7 days of modeling, the rats were placed in transparent cylindrical tanks (40 cm high, 20 cm in diameter) filled with 15-18 cm of warm water (23-25°C). All animals had received 15 minutes of pre-swimming training beforehand, followed by a 5-minute swimming test. The time the rats remained still within 5 minutes was recorded to assess their stamina and fatigue levels.

[0056] 4. Open Field Experiment (OFT)

[0057] Motor activity was measured using an open-field apparatus (100 cm × 100 cm × 40 cm). Rats were placed in the center of the apparatus, and their behavior was recorded for 5 minutes using a camera. Images were acquired on an IBM computer equipped with ANY-maze software. Total distance traveled and average speed were considered indicators of motor activity.

[0058] Results analysis:

[0059] The results of the open field test in rats showed that the number of times rats entered the central area, the immobility time, and the time spent in the central area were significantly improved in group H compared with those in groups L and M. Figure 2 (AB diagram). Forced swimming test (FST) showed that the immobility time of rats in group DY was significantly longer than that in group CY within 5 minutes. After drug administration, the immobility time of rats gradually decreased with increasing dose, with group H showing the most significant effect. Therefore, behavioral experiments indicate that group H had the best effect. Figure 2 (Figure C).

[0060] Example 3: The effect of hUC-MSC-exo on improving tissue damage, inflammation, and oxidative stress caused by high-altitude hypoxia.

[0061] 1. HE staining

[0062] The ileum segments of SD rats were placed on ice and fixed with 4% paraformaldehyde solution for 48-72 hours. The tissue was cut into small pieces, dehydrated overnight, embedded in paraffin, and then dewaxed with xylene. The tissue sections were rehydrated with alcohols of varying concentrations and rinsed with tap water. After rehydration, the tissue sections were stained with hematoxylin for 3 minutes, rinsed with tap water for 20 seconds, differentiated with 1% hydrochloric acid solution in ethanol for 30 seconds, and rinsed with water for 10 seconds. They were then transferred to eosin staining solution for 60 seconds, washed with alcohols of varying concentrations, and then washed again with xylene. After mounting, randomly selected areas were photographed under an optical microscope.

[0063] 2. Immunohistochemical staining

[0064] Ileal tissue was fixed in 4% formaldehyde and stored at 4°C for 24 hours, followed by paraffin embedding. After dewaxing and rehydration, the sections were placed in sodium citrate antigen retrieval solution (EDTA) and subjected to antigen retrieval in a microwave oven for 15 minutes. After washing with 3% hydrogen peroxide solution, bovine serum albumin (BSA) blocking solution was added. ZO-1 antibody (Servicebio, GB115686) (1:200) and Occlulin (Servicebio, GB111401) (1:150) were added and incubated overnight. After washing, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:200) was added and incubated at room temperature for 50 minutes. Finally, the antibodies and cell nuclei were stained using an immunohistochemical DAB staining kit (ELabscicence, E-IR-R217) and hematoxylin, respectively.

[0065] 3. Measurement of serum oxidative stress and inflammatory markers

[0066] Rat serum samples were collected, and the levels of IL-6 (Elabscience, EL-R0015C), TNF-α (Elabscience, KE20018), T-SOD (Elabscience, E-BC-K020-M), and MDA activity (Elabscience, E-BC-K025-M) in the serum were measured according to the kit instructions. Blood routine indicators were also detected using a blood routine analyzer.

[0067] Results analysis:

[0068] HE staining results showed that the small intestinal villi in group DY exhibited severe structural damage, shortening, and deformation, accompanied by significant inflammatory cell infiltration. Group L showed a relatively small improvement effect, followed by group M, while group H showed the most significant improvement effect. Immunohistochemical staining results showed that no significant positive expression of the intestinal barrier proteins Occludin and ZO-1 was observed in group DY, while the positive expression was most significant in group H. Figure 2(Figure D). ELISA results showed that the T-SOD value in the DY group was significantly reduced, while the levels of MDA, IL-6, and TNF-α were significantly increased. Among them, the treatment effect of human umbilical cord mesenchymal stem cells (HUMSCs) in the H group was the most significant. Figure 2 (EH diagram).

[0069] Based on the combined results of behavioral experiments, HE staining, immunohistochemistry, serum oxidative stress, and inflammatory marker analysis, the H group showed the most significant treatment effect.

[0070] Example 4: Imaging and ultrastructural evidence of hUC-MSC-exo's improvement of brain-gut axis structure and function induced by high-altitude hypoxia.

[0071] 1. MRI

[0072] Adult male Sprague-Dawley rats (weighing 250-300 g) were anesthetized with isoflurane (induction concentration: 5%, maintenance concentration: 1.5-2%) via a mixture of medical air and oxygen. The rats were placed in a specialized rodent receiver coil, and their heads were secured with a rack and earring to minimize motion artifacts. Respiratory rate was monitored throughout the MRI examination using a physiological monitoring system (Model 1025, SA Instruments), and body temperature was maintained at 37.0 ± 0.5 °C. All MRI experiments were performed on a PharmaScan 70 / 16 US imaging system. The following high-resolution sequences were acquired after rapid localization scans for quantitative analysis:

[0073] Susceptibility-weighted imaging (SWI): Flow-compensated three-dimensional gradient echo sequence was used, with the following parameters set: repetition time (TR) = 30 ms, echo time (TE) = 15 ms, flip angle (FOV) = 15°, and field of view (FOV) = 35 × 35 × 16 mm. 3 The matrix size (M) = 256 × 192 × 32, resulting in an isotropic resolution of 0.137 × 0.182 × 0.5 mm. Diffusion-weighted images were acquired using a spin-echo plane echo imaging (SE-EPI) sequence with b-values ​​of 0 and 800 s / mm. 2 The apparent diffusion coefficient (ADC) is then calculated using the formula ADC = ln(S0 / S1) / (b1 - b0), where S0 and S1 represent the signal strength when b is 0 and 800 s / mm², respectively.

[0074] 2. Transmission electron microscopy (TEM)

[0075] Brain tissue samples were fixed with 2% paraformaldehyde, followed by fixation with 5% glutaraldehyde and 1% osmium tetroxide. They were then dehydrated with ethanol and a series of acetone solutions, embedded, stained with uranyl acetate and lead nitrate, and finally cut into ultrathin sections with a thickness of 60-70 nm. The sections were then observed using a JEM1200EX transmission electron microscope.

[0076] Results analysis:

[0077] MRI results showed hemorrhages in the DY group, but they were not obvious. Subsequently, the ADC values ​​of white matter, gray matter, corpus callosum, and hippocampus were measured. The ADC values ​​showed an increase after exposure to high-altitude hypoxia, suggesting cytotoxic edema, and high-dose hUC-MSC-exo administration significantly improved the condition. Figure 3 (AE diagram).

[0078] TEM observation revealed that hypoxia caused deformation of neuronal cell bodies, uneven cell body density, blurred cell body boundaries, mitochondrial deformation, and damage to cristae structures. hUC-MSC-exo treatment showed a significant improvement effect. Figure 3 (Figure F). Transmission electron microscopy (TEM) results of the ileum tissue showed that the DY group exhibited dissolution and destruction of the intestinal villi structure, mitochondrial swelling, and disappearance of cristae. In contrast, the H group showed significant improvement in villi remodeling and mitochondrial structure after treatment. Figure 3 (G diagram).

[0079] Example 5: Study on the Mechanism of Action of hUC-MSC-exo Based on Multi-omics—Regulation of Gut Microbiota and Brain Metabolism / Transcription

[0080] 1. Fecal 16S rRNA sequencing and analysis

[0081] First, genomic DNA was extracted and analyzed by 1% agarose gel electrophoresis. After PCR amplification, the DNA was purified using the Agencourt AMPure XP nucleic acid purification kit. Libraries were constructed for next-generation high-throughput sequencing and subsequently analyzed. After obtaining the raw data, quality control was performed. Optimized sequences were obtained through sequence assembly, filtering, and dechimerization. Alpha diversity analysis, beta diversity analysis, and correlation analysis between sample composition and differences in inter-sample community structure were performed on these sequences.

[0082] 2. Non-targeted metabolomics analysis of the frontal cortex

[0083] Weigh approximately 100 mg of tissue sample, add 200 μl of pre-cooled water, and vortex thoroughly. Add 0.8 mL of pre-cooled methanol-acetonitrile mixture and vortex for 60 seconds. Perform cryogenic sonication extraction for 30 minutes. Incubate at -20°C for 1 hour to precipitate proteins, then centrifuge at 12,000 rpm for 10 minutes at 4°C. Remove the supernatant, vacuum dry, redissolve in 200 μl of 30% acetonitrile solution, vortex, and centrifuge at 14,000 rpm for 15 minutes at 4°C. Collect the supernatant for instrumental analysis. Raw data were first preprocessed using Progenesis QI software (Waters Corporation, Milford, USA), followed by data quality control and analysis.

[0084] 3. Transcriptomic analysis of the frontal cortex

[0085] First, total RNA was extracted from tissue samples. The concentration and purity of the extracted RNA were assessed using a Nanodrop 2000 analyzer. RNA integrity was verified by agarose gel electrophoresis, followed by library preparation. mRNA was enriched from the total RNA using magnetic beads coated with oligodeoxythymidine (Oligo(dT)). The mRNA was then fragmented, followed by reverse transcription and PCR amplification, and finally sequenced on a second-generation high-throughput sequencing platform.

[0086] Results analysis:

[0087] 16S sequencing results showed that, at the phylum level, Bacteroidetes were significantly reduced in the DY group, while Firmicutes were increased, and this was improved after drug treatment; at the species level, the abundance of major genera producing short-chain fatty acids (such as Lactobacillus and Clostridium) increased after drug treatment. Figure 4 (AE diagram). Therefore, it can be inferred that changes in the gut microbiota led to alterations in short-chain fatty acids. Short-chain fatty acids can cross the blood-brain barrier and enter the brain, affecting the host's emotional state and neurotransmitter synthesis, thereby causing changes in metabolites.

[0088] The role of hUC-MSC-exo in metabolomics and transcriptomics:

[0089] (1) Metabolomics

[0090] Based on metabolomics results, the DY group and the H group were significantly separated in principal component analysis (PCA). Key differentially expressed metabolites included FAD, ADP-ribose, adenine, NAD+, 3-AMP, GMP, palmitoylcarnitine, 2-deoxyguanosine-5-monophosphate, adenosine, pantothenic acid, and succinate. These metabolites involve metabolic pathways including the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, oxidative phosphorylation, and metabolic pathways involving short-chain fatty acids (such as acetic acid, butyric acid, and propionic acid). This further confirms that alterations in the gut microbiota induce changes in short-chain fatty acids, leading to changes in metabolites. Figure 4 (FH diagram).

[0091] (2) Transcriptomics

[0092] Based on transcriptomics results, principal component analysis (PCA) showed a significant separation between the DY and H groups. Volcano plot and heatmap analysis indicated specific differences between the two groups. Figure 4 IK diagram). KEGG enriched pathways include "neuroactive ligands and receptors" and "cAMP signaling pathway," etc. Figure 5 (A / B diagram). After screening for differentially expressed genes in the 'neuroactive ligand and receptor pathway', we found that the differentially expressed gene htr6 encodes a 5-HT6 receptor that can mediate the 'cAMP signaling pathway'. Therefore, we hypothesize that the improvement of depressive-like behavior in high-altitude exposed SD rats by mesenchymal stem cell exosomes is related to the regulation of the 5-HT6 / cAMP-PKA signaling pathway.

[0093] Example 6: Validation of the core signaling pathway of hUC-MSC-exo and its synergistic effect with probiotics (in vitro validation)

[0094] 1. In vitro experiments

[0095] 1.1 Bacterial growth curve

[0096] 150 μL of frozen Bifidobacterium longum culture was mixed with 150 μL of human umbilical cord mesenchymal stem cell exosomes, and 15 mL of MRS medium was added. The medium was divided into a control group and a human umbilical cord mesenchymal stem cell exosome group, with three replicates in each group. The cultures were placed in an anaerobic incubator at 37℃, and the absorbance (OD) at 600 nm was measured every 4 hours. Growth curves were plotted by calculating the average OD values ​​of each group at different time points.

[0097] 1.2 Cell viability

[0098] Cell culture: Rat type I astrocytes (CTX-TNA2) were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells in the CY group were cultured at 37°C under 5% CO2 gas, while cells in the DY group and other treatment groups were cultured at 37°C under 1% O2. The culture medium was changed every 48-72 hours, and cells were passaged when the confluence reached 80-90%.

[0099] Cell viability assay: Cell viability was determined using the CCK-8 cell viability assay kit. CTX-TNA2 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates. Cell viability was assessed at six time points (6 h, 12 h, 18 h, 24 h, 30 h, and 36 h) to determine the time point at which cell death reached 50%. Subsequently, 5 μL, 10 μL, 15 μL, 20 μL, and 30 μL of hUC-MSC-exo (2 × 10³ cells per well) were added to each well, respectively. 6 (particles / mL) to determine the optimal concentration.

[0100] 2. Western Blot

[0101] Proteins were extracted from rat brain tissue and rat type I astrocytes (CTX-TNA2). After ultracentrifugation, denaturation, and electrophoresis, quantification was performed using the bis(N-octanoyl-L-cysteine) (BCA) method. Following electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After brief blocking, the membrane was incubated overnight at 4°C with a primary antibody targeting 5-HT6 (Bioss, bs-12058R), cAMP (Proteintech, 820940-1-RR), and PKA (Proteintech, 28205-1-AP). After washing, the membrane was incubated with secondary antibody for 1 hour, followed by chemiluminescence detection, and the results were analyzed using ImageJ software.

[0102] 3. RT-qPCR

[0103] Total RNA was extracted from tissues using the procedures provided in the RNA extraction kit. Subsequently, the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit according to the manufacturer's instructions. The resulting cDNA was used as a template for qRT-PCR amplification, and the transcriptional level of the target gene mRNA in the tissue samples was detected using real-time quantitative qRT-PCR.

[0104] Results analysis:

[0105] (1) hUC-MSC-exo exerts its function through the 5-HT6-mediated cAMP signaling pathway.

[0106] To further verify whether hUC-MSC-exo improves hypoxia-induced depressive-like behavior in rats through the 5-HT6 / cAMP-PKA signaling pathway, we verified the expression levels of 5-HT6, cAMP, and PKA proteins using Western blotting analysis; qRT-PCR verified the 5-HT6 / cAMP-PKA signaling pathway; and simultaneously detected genes (htr6, cAMP, PKA, Drd2, End1, htr1b) that showed differential expression in both the neuroactive ligand and receptor pathway and the cAMP signaling pathway. The results indicate that human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-exo) ameliorated the hypoxia-induced changes in the expression of these related proteins and genes. Figure 5 (CL diagram).

[0107] (2) hUC-MSC-exo promotes the growth of lactic acid bacteria in vitro.

[0108] Bacterial growth curves showed that hUC-MSC-exo promoted the growth of Bifidobacterium longum and shortened the time required for it to reach the stationary phase. Figure 5 (M diagram).

[0109] (3) Human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-exo) and lactic acid bacteria supernatant exerted effects on neurons after hypoxia treatment.

[0110] Results and Discussion:

[0111] First, hUC-MSC-exo was labeled with PKH67, and its intracellular localization and uptake were observed. Figure 5 (N diagram). Cell viability assays at different time points in the CY and DY groups showed that the cell viability at 18 hours was approximately 50%. Figure 5 (O diagram). The optimal dose of hUC-MSC-exo was then determined, and the results showed that adding 15 μL of hUC-MSC-exo (at which point the hUC-MSC-exo concentration was 2.6 × 10⁻⁶) yielded the desired result. 5 The highest cell viability recovery rate was obtained at a concentration of particles / mL, thus establishing it as the optimal dose. Figure 5 (Image edited by Photoshop).

[0112] To verify the function of this pathway in vitro and determine its association with the gut microbiota, the following treatment groups were used: human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-exo), Bifidobacterium longum supernatant (stationary phase), MRS medium, hUC-MSC-exo, and Bifidobacterium longum supernatant. Cells were divided into CY group, DY group, DY+hUC-MSC-exo group, DY+BL group, DY+MRS group, and DY+hUC-MSC-exo+BL group. Results showed that the expression of all proteins increased in the DY treatment group. Treatment with hUC-MSC-exo or Bifidobacterium longum alone resulted in a slight decrease in expression levels; combined treatment significantly reduced expression levels, while MRS medium treatment did not cause significant changes and even exacerbated the above effects. Figure 6 ).

[0113] In summary, this study indicates that human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-exo) can improve depressive-like behaviors induced by high-altitude hypoxia exposure through the brain-gut axis pathway. hUC-MSC-exo can also improve gut microbiota, thereby regulating brain metabolite levels. Furthermore, transcriptomic analysis suggests that the regulatory role of hUC-MSC-exo may be achieved through the 5-HT6 / cAMP-PKA signaling pathway. Figure 7 ).

[0114] While specific embodiments of the invention have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. The use of stem cell exosomes in the preparation of a drug for the prevention and / or treatment of depressive-like behaviors induced by high-altitude hypoxia.

2. The application according to claim 1, characterized in that, The drug is used to improve one or more of the following pathological changes caused by high-altitude hypoxia: (a) Elevated serum oxidative stress markers and decreased antioxidant markers; (b) Elevated serum inflammatory factor levels; (c) Impaired intestinal barrier function; (d) Cytotoxic edema of brain tissue and ultrastructural damage to neurons; (e) Intestinal flora imbalance.

3. The application according to claim 1 or 2, characterized in that, The stem cell exosomes are derived from human umbilical cord mesenchymal stem cells and are administered via intraperitoneal injection.

4. The application according to claim 3, characterized in that, The preventive and / or therapeutic effects of the drug are achieved by modulating the brain-gut axis.

5. The application according to claim 4, characterized in that, The regulation of the brain-gut axis includes: (a) Improves gut microbiota composition and increases the abundance of short-chain fatty acid-producing bacteria; (b) Repairing the intestinal barrier and upregulating the expression of intestinal tight junction proteins Occludin and ZO-1; (c) Regulate the levels of metabolites in the brain that are related to energy metabolism and neural activity.

6. The application according to claim 4, characterized in that, The regulation of the brain-gut axis ultimately involves activating the 5-HT6 / cAMP-PKA signaling pathway in the brain.

7. A pharmaceutical composition, characterized in that, Include: (a) Therapeutic doses of exosomes derived from human umbilical cord mesenchymal stem cells; (b) Bifidobacterium longum ( Bifidobacterium longum Live bacteria or their fermentation supernatant; and (c) A pharmaceutically acceptable carrier; The pharmaceutical composition is used to prevent and / or treat depressive-like behaviors caused by high-altitude hypoxia.

8. The use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for the prevention and / or treatment of depressive-like behavior caused by high-altitude hypoxia.

9. The use of a stem cell exosome in the preparation of one or more of the following drugs, all of which are used for depressive-like behavior induced by high-altitude hypoxia and each has the following corresponding functions: (a) Drugs that lower serum oxidative stress markers and increase antioxidant markers; (b) Medications that lower serum inflammatory factor levels; (c) Drugs that repair damage to the intestinal barrier function; (d) Drugs that reduce cytotoxic edema of brain tissue and damage to the ultrastructure of neurons; (e) Medications that correct gut microbiota dysbiosis.

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