Targeted intestine-brain axis combined medicine and application thereof

Abstract

The invention discloses a targeted intestine-brain axis combined medicine which contains mesenchymal stem cells and exosomes derived from the mesenchymal stem cells, and through a stem cell and exosome combined double-targeted treatment strategy, neural immune dysfunction and intestinal flora imbalance are synchronously improved. The combined medicine disclosed by the invention can prevent and / or treat nervous system diseases through various ways of inhibiting prefrontal cortex neuroinflammation, enhancing antioxidation defense of the prefrontal cortex, relieving oxidative damage, repairing a synaptic ultrastructure and adjusting intestinal flora.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a combined drug targeting the gut-brain axis and applications thereof. Background Art

[0002] Autism Spectrum Disorder (ASD), a representative neurodevelopmental disorder, has a global incidence that has climbed from 1 / 88 in 2010 to 1 / 54 in 2024, showing a continuous growth trend. Current clinical treatment mainly relies on behavioral intervention and symptomatic drug treatment, but the existing technology system has significant defects. In terms of drug treatment, although drugs such as risperidone and aripiprazole are approved for improving aggressive behavior and anxiety symptoms in ASD patients, these drugs are almost ineffective for core social disorders and stereotyped behaviors, and are often accompanied by adverse reactions such as drowsiness and weight gain.

[0003] Although the biotherapeutic technologies that have emerged in recent years have shown potential, they still face major technical bottlenecks. Although intravenous stem cell therapy can work through immune regulation, clinical data show that its incidence of pulmonary embolism is as high as 3-7%, which seriously limits its clinical application. Exosomes, as a new type of nanocarrier, can penetrate the blood-brain barrier, but the existing nasal delivery technology has two major defects: first, the half-life of exosomes in a physiological environment is less than 4 hours, and second, only less than 0.1% of the administered dose can ultimately reach the target area of ​​the brain. More importantly, a 2024 study in the Cell journal pointed out that the use of exosomes alone cannot achieve bidirectional regulation of the gut-brain axis, which is the core link in the pathological mechanism of ASD.

[0004] The gut-brain axis theory offers new insights into the treatment of ASD, but existing solutions have significant limitations. Commercially available probiotics can only regulate specific gut microbiota, failing to inhibit neuroinflammation and lacking systemic immune regulation. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a combined drug targeting the gut-brain axis and its application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a combination drug targeting the gut-brain axis, which contains a first active ingredient and a second active ingredient, wherein the first active ingredient is mesenchymal stem cells and the second active ingredient is exosomes derived from mesenchymal stem cells.

[0008] In some embodiments, the mesenchymal stem cells are selected from mesenchymal stem cells differentiated from induced pluripotent stem cells or mesenchymal stem cells isolated from human tissues.

[0009] In some embodiments, the human tissue is selected from bone marrow, adipose tissue, umbilical cord, umbilical cord blood, skeletal muscle, peripheral blood, lubricating membrane, or amniotic fluid.

[0010] In some embodiments, the first active ingredient is umbilical cord mesenchymal stem cells, and the second active ingredient is umbilical cord mesenchymal stem cell-derived exosomes.

[0011] In some embodiments, the umbilical cord mesenchymal stem cell-derived exosomes are prepared by the following method: umbilical cord mesenchymal stem cells are cultured in a conditioned medium, and the cell culture supernatant is centrifuged using differential ultracentrifugation.

[0012] In some embodiments, the differential ultracentrifugation method comprises the following steps: centrifugation at 300-350 × g for 8-12 minutes to remove residual cells; then centrifugation at 2,000-2,500 × g for 8-12 minutes to remove cell debris; and then centrifugation at 10,000-15,000 × g for 20-35 minutes to pellet microvesicles. The clarified supernatant is filtered through a polyethersulfone (PES) membrane and finally ultracentrifuged at 100,000-150,000 × g for 1-3 hours.

[0013] In some embodiments, the first active ingredient and the second active ingredient are in the same dosage unit.

[0014] In some embodiments, the first active ingredient and the second active ingredient are in different formulation units.

[0015] In some embodiments, the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially.

[0016] In some embodiments, the dosage form of the combination drug is a parenteral dosage form or a parenteral dosage form.

[0017] In some embodiments, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0018] In some embodiments, the non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0019] In some embodiments, the dosage form of the combination drug is any one of powder, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral solutions, ointments, emulsions, and irrigation solutions.

[0020] In some embodiments, the combination drug further comprises a pharmaceutically acceptable excipient.

[0021] In some embodiments, the excipients further include at least one of a filler, a diluent, a disintegrant, a binder, a lubricant, a glidant, a surfactant, a solvent, a flavoring agent, and a preservative.

[0022] In some embodiments, the filler or diluent includes sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0023] In some embodiments, the lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silicon dioxide; micropowder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0024] In some embodiments, the binder includes distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, ethyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, and compounds similar to the above excipients.

[0025] In some embodiments, the disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0026] In some embodiments, the surfactant includes Tween, sodium lauryl sulfate, sodium stearate sulfonate, and the like.

[0027] In some embodiments, the antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid, and the like.

[0028] In some embodiments, the preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0029] In some embodiments, the flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, mint oil, menthol, mint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0030] In some embodiments, the first active ingredient and the second active ingredient are in the same preparation unit, the combination drug is a lyophilized powder inhaler, and the lyophilized powder inhaler further contains trehalose and mannitol.

[0031] In some embodiments, the aerodynamic particle size of the lyophilized powder inhaler is 1-5 μm.

[0032] In some embodiments, the mass ratio of umbilical cord mesenchymal stem cells to umbilical cord mesenchymal stem cell-derived exosomes in the lyophilized powder inhaler is 1:(30-50).

[0033] In some embodiments, the preparation method of the lyophilized powder inhaler comprises: mixing umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 1:(30-50) in a lyophilization protective solution containing trehalose and mannitol, pre-freezing, freeze-drying to form a porous solid, and micronizing to obtain the lyophilized powder inhaler.

[0034] In some embodiments, the preparation method of the lyophilized powder inhaler comprises: mixing umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 1:(30-50) in a lyophilization protective solution containing 10% trehalose (w / v) and 5% mannitol (w / v), pre-freezing to -80°C and maintaining for 2 hours, freeze-drying at -40°C and 0.1 mbar vacuum conditions for 48 hours to form a porous solid, forming a porous solid, and obtaining the lyophilized powder inhaler after micronization treatment.

[0035] In some embodiments, the lyophilized powder inhaler is administered via the oral and nasal route.

[0036] In some embodiments, the first active ingredient and the second active ingredient are in different formulation units, the first active ingredient is in an intravenous injection, and the second active ingredient is in nasal drops.

[0037] The second aspect of the present invention provides the use of the combined drug of the first aspect in any of the following aspects:

[0038] preparing drugs that modulate the gut-brain axis;

[0039] preparing drugs for inhibiting neuroinflammation;

[0040] Preparation of drugs for preventing and / or treating diseases related to the nervous system.

[0041] In some embodiments, the nervous system-related disease is a gut-brain axis-related nervous system-related disease.

[0042] In some embodiments, the nervous system-related disease comprises at least one of Alzheimer's disease, autism spectrum disorder, vascular dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autoimmune encephalitis, meningitis, epilepsy, migraine, fibromyalgia, spinal muscular atrophy, mitochondrial encephalomyopathy, depression, anxiety, schizophrenia, attention deficit hyperactivity disorder, and Cushing's syndrome.

[0043] In some embodiments, the nervous system-related disease is autism spectrum disorder.

[0044] In some embodiments, inhibiting neuroinflammation comprises inhibiting microglial activation, inhibiting the expression of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α in the prefrontal cortex, and upregulating the expression of anti-inflammatory factors such as IL-10 in the prefrontal cortex.

[0045] In some embodiments, the combination drug prevents and / or treats neurological diseases by at least one of the following pathways: inhibiting prefrontal cortex neuroinflammation, enhancing prefrontal cortex antioxidant defense and reducing oxidative damage, repairing synaptic ultrastructure, and regulating intestinal flora.

[0046] In some embodiments, the inhibition of prefrontal cortex neuroinflammation includes inhibiting microglial activation, inhibiting the expression of pro-inflammatory factors such as IL-1β, IL-6 and TNF-α in the prefrontal cortex, and upregulating the expression of anti-inflammatory factors such as IL-10 in the prefrontal cortex.

[0047] In some embodiments, the inhibition of microglial activation reduces Iba1 protein levels in the CA1 region and prefrontal cortex, and reduces the density of Iba1+ microglial cells in the hippocampus.

[0048] In some embodiments, said enhancing the antioxidant defense of the prefrontal cortex and reducing oxidative damage comprises increasing the activities of GSH and SOD and reducing the levels of MDA and NO in the prefrontal cortex.

[0049] In some embodiments, the repairing of synaptic ultrastructure comprises increasing synaptic vesicle density in the hippocampal CA1 region.

[0050] In some embodiments, regulating the intestinal flora includes reducing the Firmicutes / Bacteroidetes ratio, increasing the abundance of Ruminococcaceae, and reducing the abundance of Enterobacteriaceae.

[0051] The present invention provides a combination drug targeting the gut-brain axis, pioneering a dual-targeted therapeutic strategy combining mesenchymal stem cells and exosomes to simultaneously improve neuroimmune dysfunction and intestinal microbial imbalance. This combination drug can prevent and / or treat neurological diseases by inhibiting prefrontal cortical neuroinflammation, enhancing prefrontal cortical antioxidant defenses and mitigating oxidative damage, repairing synaptic ultrastructure, and regulating intestinal microbiota. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Characterization and tumorigenicity of exosomes and human umbilical cord mesenchymal stem cells (hUCMSCs). (A-F) Flow cytometric analysis of hUCMSC surface markers. The cells highly express CD105, CD90, and CD73 (positive), but lack HLA-DR, CD34, and CD45 (negative). Blue: control cell population; red: sample cell population. (G) Transmission electron microscopy (TEM) images of hUCMSC-derived exosomes. Purified hUCMSC-Exo exhibit a typical cup-shaped morphology. (H) Karyotype analysis of hUCMSCs (G-banding). Analysis demonstrated a normal karyotype (46, XY). (I) Photographs of hUCMSC morphology. Scale bar = 100 μm. (J-L) Photographs demonstrating the multidirectional differentiation potential of hUCMSCs. (J) Osteogenic differentiation (Alizarin Red S staining); (K) Chondrogenic differentiation (Toluidine Blue staining); (L) Adipogenic differentiation (Oil Red O staining). Scale bar = 200 μm or 100 μm. (MV) Histological analysis of various organs (hematoxylin and eosin staining, H&E). From left to right, top to bottom: myocardium, liver, spleen, lung, kidney, brain, stomach, small intestine, pancreas, muscle tissue. Scale bar = 50 μm.

[0053] Figure 2 .Open field test and repetitive stereotypic behavior analysis of SD rats. Among them, (A) the number of crossing the central area; (B) the distance of movement in the central area; (C) the time spent in the central area; (D) the time of self-grooming; (E) the number of buried glass beads; (F) the number of upright times; (G) the representative open field movement trajectory of the control group; (H) the representative open field movement trajectory of the ASD model group; (I) the representative open field movement trajectory of the hUCMSC+Exo treatment group. Data are expressed as mean ± standard deviation ( Statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the ASD model group exposed to VPA.

[0054] Figure 3 Sprague-Dawley rat three-chamber social behavior analysis. (A) Indicators for the sociability phase: time spent in the stranger rat 1 compartment, time spent in the empty cage compartment, time spent interacting with stranger rat 1, and time spent interacting with objects. (B) Indicators for the social novelty preference phase: time spent in the stranger rat 1 compartment, time spent in the stranger rat 2 compartment, time spent interacting with stranger rat 1, and time spent interacting with stranger rat 2. (C) Representative movement heatmap of the ASD model group during the sociability phase (0-10 minutes). (D) Representative movement heatmap of the control group during the sociability phase (0-10 minutes). (E) Representative movement heatmap of the hUCMSC+Exo treatment group during the sociability phase (0-10 minutes). (F) Representative movement heatmap of the ASD model group during the social novelty phase (10-20 minutes). (G) Representative movement heatmap of the control group during the social novelty phase (10-20 minutes). (H) Representative movement heatmap of the hUCMSC+Exo treatment group during the social novelty phase (10-20 minutes). Data are expressed as mean ± standard deviation (n = 12). Compared with the VPA-exposed ASD model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Index definitions: Stranger 1 cage: time spent in the cage containing Stranger 1; Empty cage: time spent in the empty cage; Stranger 1 cage: time spent interacting with Stranger 1; Object: time spent interacting with inanimate objects; Stranger 2 cage: time spent in the cage containing Stranger 2; Stranger 2 cage: time spent interacting with Stranger 2.

[0055] Figure 4Morris water maze behavioral analysis. (A) Time spent in the target quadrant during the exploration test. (B) Total swimming distance (motor function control). (C) Escape latency during the acquisition phase. (D) Number of platform crossings during the exploration test. (E) Distribution of time spent in each quadrant during the exploration test. (F) Representative acquisition phase movement path: VPA-exposed group. (G) Representative acquisition phase movement path: control group. (H) Representative acquisition phase movement path: hUCMSC+Exo-treated group. (I) Representative exploration test movement path: VPA-exposed group. (J) Representative exploration test movement path: control group. (K) Representative exploration test movement path: hUCMSC+Exo-treated group. Data are expressed as mean ± standard deviation (n = 12). Compared with the VPA-exposed ASD model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0056] Figure 5 Analysis of neuroinflammation and oxidative stress levels in the prefrontal cortex. (A) IL-1β level; (B) IL-6 level; (C) TNF-α level; (D) IL-10 level; (E) GSH-Px (glutathione peroxidase) activity; (F) GSH (glutathione) content; (G) SOD (superoxide dismutase) activity; (H) MDA (malondialdehyde) content; (I) T-NOS (total nitric oxide synthase) activity; (J) NO (nitric oxide) content; (K) CAT (catalase) activity. Data are presented as mean ± SD ( Statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the ASD model group exposed to VPA.

[0057] Figure 6Analysis of microglial activation in the prefrontal cortex and hippocampal CA1 region of rats in the control, VPA-exposed, and treatment groups. (AJ) Hippocampal CA1 region: (AC) Control group: Shows resting microglia with a ramified morphology and low Iba1 expression. (DF) VPA-exposed group: Shows activated microglia, characterized by hypertrophy of the cell body, thickened processes, and strong Iba1 immunoreactivity. (HJ) hUCMSC+Exo treatment group: Shows reduced microglial activation. (G) Quantification of Iba1 fluorescence intensity (arbitrary units, AU): Confirms that microglial activation in the VPA group is significantly greater than in the control group (P < 0.0001), and activation returns to normal levels after treatment (P < 0.0001 compared to the VPA group). (KT) Prefrontal cortex: (KM) Control group: Shows resting microglia with weak Iba1 signal. (OQ) VPA-exposed group: Shows strong Iba1 expression, indicating proinflammatory activation. (RT) Treatment group: Shows reduced Iba1 immunoreactivity. (N) Quantification of cortical Iba1 fluorescence: Shows significantly enhanced activation in the VPA group (P < 0.0001 compared with the control group), and attenuated activation after intervention (P < 0.0001 compared with the VPA group). Methodological details: Data are presented as mean ± standard deviation (n = 6 biological replicates); intensity analysis was performed using ImageJ software; Immunofluorescence: Iba1 (green), DAPI nuclear counterstain (blue); Scale bar: 50 μm (consistent across all panels).

[0058] Figure 7Ultrastructural pathological changes and repair of neurons in the PFC and hippocampal CA1 regions. (A) Mitochondrial ultrastructure in the control group: regular cristae and intact membranes. (B) Mitochondrial pathological changes in the VPA-exposed ASD model group: disorganized cristae and ruptured membranes. (C) Mitochondrial repair in the hUCMSC+Exo-treated group: ultrastructural restoration to normal. (D) Synaptic ultrastructure in the control group: narrow synaptic cleft, dense postsynaptic density (PSD), and aggregated synaptic vesicles. (E) Synaptic pathological changes in the ASD model group: widened synaptic cleft, blurred membrane structure, and decreased vesicles. (F) Synaptic repair in the treated group: reorganized cleft structure and reaggregated vesicles. (G) Synaptic vesicle density in the hippocampal CA1 region at PND60: significantly decreased after VPA exposure (**P < 0.001 compared with the control group); partially recovered after treatment (P < 0.05 compared with the VPA group). (H) Synaptic vesicle density in the cerebral cortex at PND60: VPA exposure resulted in a decrease in density (**P<0.001 compared to the control group); treatment-mediated restoration of density (P<0.05 compared to the VPA group). Representative images are from n=3 independent experiments; synaptic vesicles from 30 nerve terminals were quantified per animal; data are presented as mean ± standard error of the mean (SEM); statistical comparisons: P<0.05, *P<0.01, **P<0.001, ***P<0.0001 (VPA group vs. control group in G / H; significant markers indicate treatment group vs. VPA group)

[0059] Figure 8 Analysis of gut microbiota composition and diversity in different experimental groups. (A-E) Stacked bar charts show the species composition distribution at the class (A), family (B), genus (C), order (D), and phylum (E) levels in the VPA-induced ASD rat model group (VPA), normal control group (Control), and hUCMSC+Exo treatment group (hCiPSCs). Taxa with a relative abundance below 1% were classified as "Others." (F) Venn diagrams show shared and unique operational taxonomic units (OTUs) among the groups. Data analysis was performed using the WekemoBioincloud platform.

[0060] Figure 9 Heatmap analysis of intestinal microbiota composition across experimental groups. (A-D) Hierarchical cluster heatmaps show the relative abundance patterns at the phylum (D), order (C), family (A), and genus (B) levels in the VPA-induced ASD rat model group (VPA), normal control group (Control), and hUCMSC+Exo treatment group (MSC+Exo). Darker colors represent higher abundance.

[0061] Figure 10Hierarchical analysis of changes in the gut microbiota between autism model rats and stem cell-treated rats. (A) Family-level phylogenetic cladogram: Red / green branches indicate taxa enriched / depleted in the VPA_RAT group (autism model group) relative to the control group (Control). (B) Family-level LDA score: Bar lengths represent the effect size (log10-transformed value) for significantly different bacterial families. (C) Genus-level phylogenetic cladogram: Phylogenetic distribution of enriched (red) and depleted (green) bacterial genera. (D) Genus-level LDA score: Key genera driving differences between groups. (E) Phylum-level phylogenetic cladogram: Extensive taxonomic changes between groups. (F) Phylum-level LDA score: Major phylum-level changes quantified by effect size. Groups: Control (healthy control group), VPA_RAT (autism model group), MSC+Exo (hUCMSC+Exo treatment group).

[0062] Figure 11 Volcano plot analysis of intestinal dysbiosis. Comparative analysis (DESeq2): (A) Genus-level differences (hUCMSC+Exo treatment group vs. control group). (B) Phylum-level differences (hUCMSC+Exo treatment group vs. control group). (C) Genus-level differences (hUCMSC+Exo treatment group vs. VPA-induced ASD model group). (D) Phylum-level differences (hUCMSC+Exo treatment group vs. ASD model group). (E) Genus-level differences (control group vs. ASD model group). (F) Phylum-level differences (control group vs. ASD model group).

[0063] Figure 12Alpha diversity indicators of the gut microbiota in the experimental groups. (A) Boxplots (AE) show the following for the control group, hUCMSC+Exo treatment group (MSC+Exo group), and VPA-induced ASD model group (VPA_RAT group): (A) Chao1 index (species richness); (B) FaithPD index (phylogenetic diversity); (C) ObservedFeatures (number of observed species, richness); (D) Shannon index (evenness); and (E) Simpson index (dominance). Higher Chao1, FaithPD, ObservedFeatures, and Shannon values ​​indicate greater diversity; lower Simpson values ​​indicate decreased dominance. (F) Rarefaction curves (based on the Shannon index) show how species richness changes with sequencing depth. The control group reaches a plateau at higher values, indicating consistently high species richness.

[0064] Figure 13 Analysis of gut microbiota β-diversity between experimental groups. (A) Bray-Curtis dissimilarity analysis between the control group and hUCMSC+Exo (MSC+Exo). (B) Bray-Curtis dissimilarity between the VPA-induced ASD model group (VPA_RAT) and the hUCMSC+Exo treatment group (MSC+Exo). (CD) Unweighted UniFrac analysis, showing differences based on species presence / absence. (EF) Weighted UniFrac analysis, including species abundance information. Boxplots show the interquartile range (boxes) and median (line) of differences between groups; statistical evaluation was performed using ANOSIM (analysis of similarities).

[0065] Figure 14 Analysis of intestinal microbiota composition in different experimental groups. (A) Phylogenetic tree heatmap with species identification (phylogenetic_tree_heatmap_ID). (B) Phylogenetic tree heatmap (phylogenetic_tree_heatmap).

[0066] Figure 15 Network correlation analysis. (A) Family-level network correlation analysis. (B) Genus-level network correlation analysis. (C) Order-level network correlation analysis. (D) Phylum-level network correlation analysis.

[0067] Figure 16 Functional and taxonomic profile analysis of the intestinal microbiota in the experimental group. (A) KEGG pathway level 1 classification (L1) histogram. (B) KEGG pathway level 2 classification (L2) histogram. (C) KEGG pathway level 3 classification (L3) histogram. (D) MetaCyc pathway histogram. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below through specific examples.

[0069] All operations involved in the embodiments of the present invention complied with the guidelines of the Institutional Animal Care Committee of the Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, and were performed under specific pathogen-free (SPF) conditions certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

[0070] All experimental protocols involved in the embodiments of the present invention were approved by the Animal Ethics Committee (IACUC) of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (approval number: IACUC-2022-0063; CAS ethics number: IACUC: 2023081). The experiments were carried out in an AAALAC-certified SPF-level facility and strictly followed the ARRIVE guidelines (Percie du Sert et al., 2020) and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (8th edition, 2011). The euthanasia procedure complies with the 2020 guidelines of the American Veterinary Medical Association and no banned drugs (such as chloral hydrate) are used. Analgesia and humane endpoint criteria were implemented during the experiment to minimize animal suffering.

[0071] Data in the Examples of the present invention were analyzed using GraphPad Prism software, and the results are expressed as mean ± standard deviation (SD) or standard error (SEM). Intergroup comparisons were performed using one-way ANOVA with Tukey's post hoc test. Statistical significance was defined as *p* < 0.05.

[0072] Example 1 Isolation and culture of hUCMSCs

[0073] 1. Isolation and Culture of hUCMSCs

[0074] a) Obtain the umbilical cord of a healthy fetus delivered by cesarean section at term and soak it in sterile saline containing 1% penicillin and streptomycin (HyClone) and store on ice. b) Remove the umbilical cord from the sterile saline containing 1% double-antibody and rinse it several times with saline until all blood stains are removed. Discard any deformed blood vessels and open ends. c) Cut the umbilical cord into 3-4 cm segments. Use forceps to remove the cord skin and blood vessels (for two arteries and one vein, cut along the vein and carefully remove the venous intima; for the artery, pull it directly out with forceps). Retain the intima, which is Whalton's jelly, and wash it 2-3 times in saline. d) Transfer the umbilical cord to a 1.5 mL centrifuge tube and add a 1:1 mixture of 0.1% collagenase type 4 (Sigma-Aldrich) and trypsin, mince the cord, and digest at 37°C for 1-2 hours, depending on the application. e) Add 3-5 volumes of complete medium (DMEM / F12 medium) supplemented with 10% serum to terminate the trypsinization. f) Filter the digested cell suspension through a 100 μm cell strainer (Corning) and centrifuge at 300 × g for 5 minutes. Collect the cell pellet, resuspend it in DMEM / F12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin / streptomycin, and then plate it into a 10 cm culture dish. After 72 hours, discard any unattached cells. Change the medium every 3 days until the cells are fully confluent and passaged. At this point, the cells are designated P0.

[0075] 2. Recovery of hUCMSCs

[0076] Before thawing cells, preheat a 37°C water bath and preheat DMEM / F12 medium. Remove the cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking them to quickly melt until only a small ice crystal remains. Remove from the water bath, spray with alcohol, wipe dry, and transfer to a biosafety cabinet. Mix the cell suspension and transfer it to a 15mL centrifuge tube containing DMEM / F12 medium. Add dropwise and centrifuge for 5 minutes. Remove the supernatant and resuspend in complete medium (DMEM / F12 medium) for counting. Inoculate the cells into a cell culture dish of appropriate size, shake well, and place in a cell culture incubator.

[0077] 3. Passaging of hUCMSCs

[0078] When the cells grow to 80-90% fusion rate, discard the culture medium, wash twice with PBS, remove excess culture medium, add appropriate amount of 0.05% trypsin and digest at 37℃ for 2min. Add twice the volume of complete culture medium (DMEM / F12 culture medium) containing serum to terminate digestion, blow the cells several times to accelerate the cells from the culture dish surface, collect them in a centrifuge tube for cell counting. Centrifuge the cell suspension at 300g for 5min and remove the supernatant.2 The cells were subcultured and expanded at a certain density.

[0079] 4. Cryopreservation of hUCMSCs

[0080] When the cells grow to 80-90% fusion rate, freeze the cells for seed storage: discard the culture medium, wash twice with PBS, add appropriate amount of trypsin to digest for 2 minutes, incubate at 37°C, stop digestion with twice the volume of complete culture medium (DMEM / F12 culture medium), collect the cells in a 15mL centrifuge tube, centrifuge, and remove the supernatant. Resuspend the cell pellet with complete culture medium (DMEM / F12 culture medium), count the cells, and adjust to 2×10 6 Cells / mL density was calculated. An equal volume of 2× freezing buffer (Nalgene) was added to the cell suspension in the centrifuge tube, mixing thoroughly. The suspension was then transferred to cryovials. Label the cryovials with information such as cell type, passage number, operator, time, and cell quantity. The cryovials were placed in a cryovial box overnight and then transferred to liquid nitrogen for long-term storage.

[0081] Example 2 Identification and tumorigenicity detection of hUCMSCs

[0082] Experimental methods

[0083] 1. Identification of hUCMSCs Surface Markers

[0084] After digestion and collection of cells, wash them again twice with 5 mL PBS and then add 1 × 10 6 Add 100 μL PBS to each cell to resuspend the cell pellet at the bottom of the tube, distribute it into multiple EP tubes, and add the corresponding antibodies (CD105-PE / Cy7 (1:100, BioLegend 344016), CD90-FITC (1:200, BioLegend 328108), CD73-APC (1:150, BioLegend323208) CD34-PerCP (1:100, BioLegend 343608), CD45-PE (1:200, BioLegend 202208), HLA-DR-APC / Cy7 (1:150, BioLegend 307616)), and leave one tube without antibody as a control. After incubation in the dark for 30 min with 4 mL of culture medium, the cells were washed twice with PBS. Finally, each tube was resuspended with 500 μL of PBS and analyzed and plotted on a BD FACSCalibur flow cytometer using FlowJo_V10 to identify MSC surface markers.

[0085] 2. Karyotype Analysis

[0086] Cells at 80-90% confluence were treated with 20 μg / ml colcemid for 2 hours to arrest in metaphase. After trypsinization and centrifugation, the cell pellet was resuspended in hypotonic solution (37°C, 30 minutes), fixed with methanol:glacial acetic acid (3:1), and dropped onto pre-chilled slides. Air-dried slides were stained with 1% Giemsa stain for 10 minutes. Karyotypes were analyzed and images were recorded under a microscope.

[0087] 3. Identification of Adipogenic Differentiation of hUCMSCs

[0088] hUCMSCs were cultured at 2×10 4 / cm 2 Cells were seeded at a density of 100 μg / mL in a 24-well plate, and 0.5 mL of low-glucose DMEM culture medium containing 15% FBS and 1 ng / mL bFGF was added to each well. When the cells reached 80% confluence, the induction medium was changed to high-glucose DMEM, 10% FBS, hydrocortisone, 0.1% isobutylmethylxanthine, indomethacin, and the medium was changed every 3 to 4 days. Oil red O staining was performed after 2 weeks of induction for identification.

[0089] 4. Identification of osteogenic differentiation of hUCMSCs

[0090] hUCMSCs were cultured at 2×10 4 / cm 2 The cells were seeded at a density of 100 μg / mL in a 24-well plate and cross-mixed. After 24 h of cell attachment, each well was replaced with 0.5 mL of osteogenic induction medium (high-glucose DMEM, 10% FBS, sodium β-glycerophosphate, dexamethasone, ascorbic acid). The medium was changed every 2-3 days, and the induction status was observed under a microscope. On the 21st day, alizarin red staining was performed, the culture medium was aspirated, the cells were washed three times with 1×PBS, fixed with 95% ethanol for 10 min, rinsed three times with double-distilled water, 200 μL of 2% alizarin red dye (pH = 4.2) was added, and the cells were stained at room temperature for 30 min. The excess dye was washed with double-distilled water and observed under a microscope.

[0091] 5. Identification of Chondrogenic Differentiation of hUCMSCs

[0092] Induction medium: high glucose DMEM + 1% penicillin-streptomycin solution 0.01% dexamethasone, 1% insulin iron selenium transfer protein, 0.1% sodium pyruvate, 0.1% proline, 1% transforming growth factor-β. Cell culture: take 2×10 5The cell suspension was dispensed into 15 mL centrifuge tubes, centrifuged again and the supernatant was carefully removed. The induction medium was gently added along the tube wall and placed in a CO2 incubator. The tube cap was first tightened and then loosened half a turn. The medium was changed every 3 days and the induction situation was observed. Alcian blue staining was performed on the 21st day. The culture medium was discarded and the tubes were washed 3 times with 1×PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 3 times with 1×PBS, stained with Alcian blue dye overnight (200 μL / tube), washed 3 times with 1×PBS until it did not fade, and pictures were taken under a stereomicroscope.

[0093] 6. Evaluation of hUCMSCs tumorigenicity

[0094] To comprehensively evaluate the safety of cell therapy, 1×10 7 hUCMSCs were injected into the left quadriceps region and abdominal subcutaneous tissue of 8-week-old NOD-SCID immunodeficient mice. Three months after cell transplantation, mice were euthanized, and muscle, skin, liver, kidney, myocardium, small intestine, brain, spleen, and lung samples were systematically collected. Paraffin-embedded sections were then sectioned and histopathologically examined using hematoxylin and eosin (H&E) staining. All animal experiments were conducted in strict compliance with the guidelines of the institutional animal ethics committee.

[0095] Experimental results: Flow cytometry was used to identify the expression of typical surface markers of mesenchymal stem cells. Flow cytometric analysis showed that cells isolated from the umbilical cord expressed almost no hematopoietic progenitor cell markers, such as CD45 (0.097%, Figure 1 Middle D), CD34 (0.024%, Figure 1 E) and HLA-DR (0.058%, Figure 1 Middle F); at the same time, the mesenchymal stem cell marker CD73 was highly expressed (99.8%, Figure 1 A), CD90 (99.9%, Figure 1 Middle B) and CD105 (99.9%, Figure 1 These results indicate that contamination by hematopoietic cells is negligible. Chromosome analysis by G-banding showed that hUCMSCs had a normal karyotype ( Figure 1 (H). Figure 1 As shown in Figure 1, hUCMSCs showed typical morphological characteristics. The results of hUCMSC multidirectional differentiation potential identification were shown in Figure 1. Figure 1 As shown in Figure 1, hUCMSCs have the potential to become osteoblasts ( Figure 1 Middle J), ​​adipocytes ( Figure 1 Middle L) and chondrocytes ( Figure 1 hUCMSC tumorigenicity test results are shown in Figure 2. Figure 1 As shown in the middle MV, no tumor formation was found in the myocardium, liver, spleen, lung, kidney, brain, stomach, small intestine, pancreas, and muscle tissues by histological analysis.

[0096] Example 3 Exosome isolation and characterization

[0097] Exosomes were isolated from hUCMSC-conditioned medium (serum-free DMENF12 medium) using differential ultracentrifugation according to the MISEV 2018 guidelines. Cell culture supernatants were centrifuged sequentially: first at 300 × g for 10 minutes (4°C) to remove residual cells; then at 2,000 × g for 10 minutes (4°C) to remove cellular debris; and finally at 10,000 × g for 30 minutes (4°C) to pellet microvesicles. The clarified supernatant was filtered through a 0.22 μm polyethersulfone (PES) membrane (Millipore) and finally ultracentrifuged at 100,000 × g for 2 hours (4°C; Optima XPN-100, Beckman Coulter). The purified exosome pellet was resuspended in PBS for subsequent use.

[0098] After obtaining sufficient amounts of exosomes, they were characterized and analyzed. Specifically, exosomes were extracted from hUCMSCs using ultracentrifugation. Transmission electron microscopy (TEM) observations showed that exosomes derived from hUCMSCs had a typical saucer-shaped (cup-shaped) structure with a diameter of approximately 30-150 nm ( Figure 1 Middle G).

[0099] Example 4 Combination of human umbilical cord mesenchymal stem cells (hUCMSCs) and exosomes for the treatment of autism spectrum disorders

[0100] The animal experiment method involved in this embodiment is as follows:

[0101] 1. Animal Husbandry and Reproduction: Adult Sprague-Dawley rats (24 females and 12 males; initial weight 200-250 g) were housed in a constant temperature (22 ± 1°C) and humidity (55 ± 5%) environment with a 12-h light / 12-h dark photoperiod (light period 07:00-19:00). Animals had free access to sterile drinking water and a standard chow diet. For timed mating experiments, male and female rats were housed together at 17:00 daily. Vaginal plugs were examined at 09:00 the following day, and the day of conception was designated as gestational day 0.5 (GD 0.5).

[0102] 2. Prenatal Sodium Valproate (VPA) Exposure Experiment: Twenty-four pregnant rats were randomly divided into two groups using a stratified randomization method. During the peak period of neocortical neurogenesis (GD11.5-GD13.5), rats in the VPA group (n=16) received daily intraperitoneal injections of sodium valproate solution prepared in endotoxin-free saline (400 mg / kg on GD11.5, 450 mg / kg on GD12.5, and 400 mg / kg on GD13.5). Rats in the control group (n=8) received an equal volume of 0.9% sodium chloride solution.

[0103] 3. Postnatal grouping: Male pups were placed in nulliparous dams for nursing and randomly divided into the following three groups on postnatal day 21 (P21): (1) Control group (n=12): pups born from control dams were treated with normal saline; (2) VPA-exposed group (n=12): pups exposed to sodium valproate prenatally; (3) MSC+exosome intervention group (n=12): VPA-exposed pups received combined intervention with human umbilical cord mesenchymal stem cells and exosomes.

[0104] All behavioral assessments were performed by researchers unaware of the group assignments, and subsequent histological analyses were performed. Data collection and processing were performed blindly to group assignments.

[0105] 1. Human umbilical cord mesenchymal stem cells (hUCMSCs) and exosomes combined transplantation

[0106] The MSC+Exo group (MSC+exosome intervention group) received combined treatment via two routes of administration: (1) Systemic administration (intravenous injection): once a week for 4 consecutive weeks, via tail vein injection for systemic circulation administration. Each injection dose contained 5×10 6 hUCMSCs (cells from passages 3 to 5 were used, and the cell viability was >95% as determined by trypan blue exclusion) were mixed with 200 μL of PBS solution. (2) Local administration (nasal administration): Local nasal administration was performed once a week for 4 consecutive weeks. 50 μL of PBS solution containing 100 μg of exosomes was instilled using a micropipette tip (the tip was about 2 mm deep into the nasal cavity). (3) Intraventricular booster injection: Four weeks after the last systemic administration (intravenous injection), the animals were subjected to stereotaxic surgery for intraventricular administration. The surgery was performed under anesthesia with 3% isoflurane (purchased from Shenzhen Ruiwode Life Science Technology Co., Ltd., China). The hUCMSCs cell suspension (concentration of 2×10 5Cells were injected into the bilateral lateral ventricles at a rate of 5 μL per injection site (cells / μL). The injection rate was -0.4 μL / min (slow injection). The stereotaxic coordinates of the injection site (relative to bregma) were as follows: anteroposterior: -0.8 mm; mediolateral: ±1.6 mm; dorsoventral: -3.8 mm. The injection needle was slowly withdrawn 15 minutes after implantation. To prevent potential immune reactions, animals were treated with 0.3% gentamicin for 3 days before and after transplantation.

[0107] Behavioral experiments were started one week after the last treatment (intracerebroventricular injection).

[0108] 2. Behavioral Experiment

[0109] Sprague-Dawley rats were acclimated to the experimental room for 1 hour before testing.

[0110] (1) Open Field Test

[0111] Experimental Methods: A square open field (100 × 100 × 40 cm) was used, with a defined central zone (60 × 60 cm). After a 10-minute acclimation period (free exploration), a 10-minute test was conducted. An overhead camera system recorded movement trajectories, and the number of entries into the central zone and the duration of stay in the central zone were quantified using Noldus EthoVision XT software. Recorded parameters included the number of central / peripheral crossings (all limbs crossed the zone boundary) and rearing activity (forelimb lifting ≥ 2 seconds).

[0112] Results: The open field test assessed autonomous exploratory behavior and anxiety-like responses in a novel environment. Valproic acid (VPA) exposure significantly inhibited SD rats' exploratory activity and increased anxiety-like behavior ( Figure 2 Subsequent hUCMSC+Exo treatment improved these behavioral deficits as follows: Exploratory behavior: VPA-exposed rats showed significantly fewer central zone crossings compared with the control group (P<0.001), and the hUCMSC+Exo-treated group increased the crossing frequency compared with the VPA group (P<0.001) ( Figure 2 Middle A); The VPA group showed a reduced movement distance in the central zone compared with the control group (P<0.0001), while the hUCMSC+Exo treated animals showed partial recovery, with a movement distance significantly greater than that of the VPA group (P<0.0001) ( Figure 2Anxiety-like behavior: The VPA-exposed rats spent less time in the central area than the control group (P<0.0001). The hUCMSC+Exo-treated group spent more time in the central area than the VPA group (P<0.0001), indicating that anxiety was reduced ( Figure 2 Middle C); the vertical activity score of the VPA group was lower than that of the control group (P<0.0001), and hUCMSC+Exo-treated animals showed improved standing behavior compared with VPA-exposed rats (P<0.001) ( Figure 2 Middle F); VPA-exposed rats showed a restricted movement pattern with minimal exploration of the central area ( Figure 2 Middle H), hUCMSC+Exo treated animals showed a similar trajectory to the control group ( Figure 2 Middle G), characterized by increased exploration and decreased haptotaxis in the central region ( Figure 2 Middle I).

[0113] (2) Analysis of repetitive stereotyped behaviors

[0114] 1. Marble-Burying Test

[0115] Experimental methods: Rats were individually placed in polycarbonate test cages filled with 5 cm deep fresh corn cob bedding. After 3 minutes of acclimation, the animals were temporarily transferred to a holding cage. Sixteen black glass beads (1.6 mm diameter) were arranged in a standardized 4×4 grid (3 cm spacing). The test rats were returned to the cage for a 10-minute test (50 lux illumination), and the test process was video recorded from an orthogonal angle. Three blinded observers independently scored based on the burial criterion of ≥75% coverage of the glass bead surface area. Consensus was required by ≥2 / 3 of the observers; ambiguous cases were resolved by frame-by-frame analysis.

[0116] Experimental results: The glass bead burying test (a validated paradigm for assessing repetitive behaviors in rodents) showed that valproic acid (VPA) exposure significantly increased the stereotyped burying behavior of SD rats ( Figure 2 Middle E). Compared with the control group, the number of glass beads buried by rats in the VPA-exposed group was significantly increased (P<0.0001). This repetitive behavior was significantly reduced in the hUCMSC+Exo-treated group compared with the VPA-exposed group (P<0.0001), indicating that the exploratory behavior pattern was partially restored ( Figure 2 Middle E).

[0117] 2. Self-Grooming Test

[0118] Experimental Methods: Rats were individually placed in clean polycarbonate cages (30 × 20 × 15 cm) under 50 lux illumination. After acclimation for 10 minutes, spontaneous grooming behaviors (face cleaning, body licking, and genital / tail grooming) were recorded for 10 minutes using a Logitech C920 HD Pro camera. Grooming count (discrete grooming events) and cumulative duration (total time engaged in stereotyped movements) were analyzed by three blinded observers.

[0119] Results: Mice exposed to VPA exhibited excessive self-grooming, a hallmark of repetitive, stereotypic behavior ( Figure 2 D). The frequency of grooming in the VPA group was significantly higher than in the control group (P<0.01). hUCMSC+Exo treatment significantly attenuated this behavior, with animals in the hUCMSC+Exo-treated group showing a reduced frequency of grooming compared to the VPA-exposed group (P<0.05). Compared to the control group, VPA-exposed SD rats exhibited significantly increased repetitive stereotyped behaviors (glass bead burying and grooming behaviors), suggesting neurodevelopmental abnormalities that may be associated with basal ganglia-cortical circuit dysfunction. hUCMSC+Exo treatment effectively alleviated these behaviors, supporting its therapeutic potential for modulating circuit-specific abnormalities in ASD-like phenotypes.

[0120] (3) Three-Chamber Social Interaction Test

[0121] Experimental Methods: Acclimation Procedure: Days 1-7: Animals were acclimated to the environment (23°C, 50 lux) for 2 hours daily. Days 8-10: Progressive exposure training, including 3 minutes of exploration of the central compartment daily, followed by 3 minutes of free movement in the entire area daily.

[0122] The experimental sequence included: Baseline period (5 min): an empty wire cage was placed in each side compartment. Sociability test (10 min): an age / sex-matched stranger rat (Stranger 1) was placed in one compartment, and an empty control cage was placed in the contralateral compartment. Social novelty preference test (10 min): the control cage was replaced with a new stranger rat (Stranger 2), while the original social stimulus (Stranger 1) remained unchanged. Critical control: The stranger rats were individually housed 48 hours before testing to standardize olfactory cues. Interaction time (nose tip <2 cm from the cage) was quantified using EthoVision XT 15.0 software (Noldus IT, Wageningen).

[0123] Experimental results: According to Figure 3hUCMSC+Exo treatment reversed VPA-induced social deficits (social avoidance / nonsocial preference; P < 0.0001). The VPA group also exhibited novelty preference deficits (fixation to stranger mouse 1 / avoidance to stranger mouse 2), while hUCMSC+Exo treatment enhanced novelty-seeking behavior by restoring cognitive flexibility (P < 0.05).

[0124] (IV) Assessment of learning and memory abilities

[0125] Morris water maze (MWM) was used to assess spatial learning and memory abilities through hidden platform training (acquisition test) and memory retention test (probe test).

[0126] Experimental Methods: The apparatus consisted of a black circular pool (150 cm diameter) filled with opaque water (23.0 ± 0.5°C; non-toxic white tempera paint) and a hidden platform (14 cm diameter) located 1.5 cm below the water surface in the target quadrant. Spatial cues consisted of four high-contrast geometric patterns with uniform illumination (50 ± 5 lux). Acquisition phase (5 days): Four trials were conducted daily (maximum 90 seconds, with a 25-minute inter-trial interval) with a randomized starting point (north-south-east-west); failed trials in which the platform was not found included a 10-second platform-guided localization. Probe trial: 24 hours after training, the rats swam freely for 60 seconds (without the platform). All test sessions were digitally acquired using EthoVision XT v15.0 software (Noldus Information Technology), and positional error was verified to be <2% using checkerboard calibration.

[0127] Experimental results:

[0128] according to Figure 4 VPA-exposed rats exhibited significant spatial memory deficits (prolonged latency, randomized search strategy, and impaired quadrant preference) independent of motor dysfunction. hUCMSC+Exo treatment ameliorated these deficits, enhancing spatial learning efficiency (P<0.01) and memory retention (P<0.05).

[0129] 3. Analysis of neuroinflammation and oxidative stress levels in the prefrontal cortex

[0130] Experimental methods:

[0131] 1. Tissue Collection and Processing: Animals were euthanized after behavioral testing. Anesthesia was induced by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). Sprague-Dawley rats were transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde (PFA; Merck, Germany). The brains were postfixed in 4% PFA for 24 hours, after which the hippocampus and prefrontal cortex tissue were dissected and isolated. Tissues were dehydrated using a graded ethanol series, embedded in paraffin (Merck, Germany), and cut into 5 μm thick sections using a rotary microtome (Microm HM335E, Walldorf, Germany).

[0132] 2. Biochemical Assays: Glutathione (GSH), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), catalase (CAT), total nitric oxide synthase (T-NOS), and nitric oxide (NO) levels were quantitatively measured using commercially available kits (Nanjing Jiancheng Bioengineering Institute). Interleukin-6 (IL-6), IL-10, IL-1β, and tumor necrosis factor-α (TNF-α) concentrations were determined using rat-specific ELISA kits (Fankel, Shanghai, China).

[0133] Experimental results: According to Figure 5 VPA exposure resulted in a shift in the proinflammatory state of the prefrontal cortex, as evidenced by elevated levels of IL-1β, IL-6, and TNF-α (P < 0.0001), accompanied by suppression of IL-10 (P < 0.0001). hUCMSC+Exo treatment reversed this imbalance by suppressing proinflammatory cytokines (IL-1β: P < 0.0001; IL-6: P < 0.0001; TNF-α: P < 0.0001) and restoring the anti-inflammatory cytokine IL-10 (P < 0.0001), suggesting its potential to ameliorate neuroinflammation in an ASD-like phenotype. VPA exposure induced systemic oxidative stress, as evidenced by depletion of antioxidants (CAT, GSH-Px, GSH, and SOD, all P < 0.0001) and elevated levels of oxidative damage markers (MDA, T-NOS, and NO, all P < 0.0001). hUCMSC+Exo treatment reversed the above effects by enhancing antioxidant defense and alleviating oxidative damage, suggesting that it may exert its therapeutic potential by activating the Nrf2 / ARE pathway and scavenging reactive oxygen species (ROS).

[0134] 4. Microglial Activation and Neuroinflammation in Autism Spectrum Disorder

[0135] The neuroinflammatory pathological changes in valproic acid (VPA)-exposed Sprague-Dawley (SD) rats were assessed by immunofluorescence staining of ionized calcium-binding adaptor 1 (Iba1) in the hippocampal CA1 region and prefrontal cortex.

[0136] Experimental Methods: Brain sections were deparaffinized and antigen retrieved using standard protocols and washed three times (5 minutes each) in phosphate-buffered saline (PBS, pH 7.4). Nonspecific binding was blocked with 4% (w / v) bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 for 1 hour at room temperature. Rabbit primary antibodies (diluted 1:200 in blocking buffer) were incubated in a humidified atmosphere at 4°C for 16 hours. After three 10-minute washes in PBS containing 0.1% Tween 20 (PBST), sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibodies (diluted 1:500) for 1 hour at room temperature in the dark. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1 μg / mL) for 10 minutes in the dark. Confocal imaging was performed using a TCS SP8 STED 3X system (Leica Microsystems) with standardized parameters: 488 nm laser (12% power), detector gain 650–750 V, and z-slice interval 0.5 μm. Integrated fluorescence density (IntDen) was quantified within threshold-optimized regions of interest (ROIs) using ImageJ software (v1.53k, NIH).

[0137] Experimental results: According to Figure 6 VPA exposure induced microglial hyperactivation (increased Iba1 expression in the CA1 region and prefrontal cortex; P < 0.0001), exhibiting a proinflammatory phenotype. hUCMSC+Exo treatment inhibited microglial activation (hippocampus: P < 0.0001; cortex: P < 0.0001), suggesting a therapeutic modulatory effect on ASD neuroinflammation by regulating microglial phenotype.

[0138] 5. Ultrastructural pathological changes and repair of neurons in the prefrontal cortex and hippocampal CA1 region Experimental methods: 1. Tissue preparation (PND60 rats, i.e., rats on the 60th day after birth): (1) Terminal anesthesia was performed by intraperitoneal injection of ketamine / xylazine (75:5 mg / kg). (2) Transcardial perfusion was performed via the ascending aorta: Vascular flushing: 200 mL ice-cold PBS (0.01 M sodium / potassium phosphate, pH 7.4); Primary fixation: 300 mL mixed aldehyde fixative (containing 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4; Sigma-Aldrich #G5882); (3) Microdissection: 1 mm3 2. Post-processing: Samples were subjected to the following steps: secondary fixation (fixation in primary fixative at 4°C for 20 hours → fixation in 1% osmium tetroxide (OsO4) / 0.8% potassium ferricyanide (K4[Fe(CN)6]) for 2 hours), ethanol gradient dehydration (50–100%), propylene oxide transition, EPON TM Embedded in 812 epoxy resin (polymerized at 60°C for 48 hours). 3. Imaging: Semi-thin sections (1 μm) were stained with toluidine blue for localization. Ultrathin sections (60 nm) were mounted on 200-mesh copper grids and stained for double electron microscopy with uranyl acetate (15 minutes) and then Reynolds' lead citrate (5 minutes). Images were acquired using a JEOL JEM-1200EX transmission electron microscope (80 kV) equipped with a MORADA CCD camera (Olympus iTEM v5.2 software; 3280 × 2464 pixels, 8-bit depth).

[0139] Experimental results: According to Figure 7 hUCMSC+Exo treatment ameliorates synaptic and mitochondrial pathological changes in VPA-exposed rats, restoring their ultrastructural features to levels close to those of the control group: Synaptic repair: narrowing of the synaptic cleft, clear boundaries of the postsynaptic density (PSDs), and reaggregation of synaptic vesicles (SVs) near the presynaptic membrane; mitochondrial restoration: orderly arrangement of cristae and intact membrane structure. hUCMSC+Exo treatment rescues synaptic ultrastructure (increased SV density and restoration of normal cleft morphology) and mitochondrial integrity in VPA-exposed rats, with features similar to those of the control group. These findings highlight its therapeutic potential for reversing ASD-related neuropathology.

[0140] 6. Intestinal flora analysis

[0141] Experimental methods: 1. Fecal sample collection: To minimize the influence of circadian rhythm, fresh fecal samples were collected aseptically between 09:00 and 11:00. The rats were briefly fixed and defecation was induced by gently lifting the tail and pressing the abdomen aseptically. Use disposable forceps to transfer the freshly excreted fecal pellets (3–5 pellets ≈ 100 mg) into a sterile 2 mL centrifuge tube (Axygen, USA), quickly freeze them with liquid nitrogen within 5 minutes, and store them at -80°C. 2. DNA extraction and amplification: Genomic DNA was extracted using the CTAB method combined with liquid nitrogen homogenization (0.1 mm zirconium oxide beads; FastPrep-24 homogenizer). The DNA purity (NanoDrop 2000 test: A260 / A280 = 1.8–1.9, A260 / A230> 2.0) was normalized to 1 ng / μL. Barcoded primers 341F / 806R and The V3-V4 region was amplified using a high-fidelity master mix under the following cycling conditions: 98°C / 1 minute; 30 cycles of 98°C / 10 seconds, 50°C / 30 seconds, and 72°C / 30 seconds; and 72°C / 5 minutes. 3. Library Construction and Sequencing: Amplicons (≈550 bp) were verified by agarose gel electrophoresis and purified using the TIANgel Midi Kit. Libraries were constructed using the NEBNext Ultra II Kit and verified for size distribution (Agilent 5400 Bioanalyzer: 450–650 bp) and concentration (Qubit 4.0: ≥20 nM). Paired-end sequencing (PE250) was performed on the Illumina NovaSeq 6000 platform (50,000 reads per sample). 4. Bioinformatics analysis: The original sequences were denoised and chimeras removed using DDA2 in QIIME2 (v2023.2), and an amplicon sequence variant (ASV) table was generated, and species classification annotation was performed based on the SILVA 138.1 database. Alpha diversity analysis: Chao1 index, Shannon index, Faith's PD index; beta diversity analysis: Bray-Curtis distance / UniFrac distance (principal coordinate analysis PCoA / non-metric multidimensional scaling analysis NMDS); differential species analysis: LEfSe (linear discriminant analysis LDA score > 2.0), ANCOM (W value > 0.7), DESeq2 (false discovery rate FDR < 0.05); function prediction: KEGG pathway prediction using PICRUSt2 (v2.4.1) (STAMP software: Welch t-test, FDR correction); network analysis: Gephi (v0.10.1; Spearman correlation coefficient |ρ| > 0.6, P < 0.01); multivariate modeling: redundancy analysis RDA (using the vegan package) and partial least squares discriminant analysis PLS-DA (using the mixOmics package).

[0142] Experimental results:

[0143] 1. Analysis of microbial composition (class, family, genus, order, phylum level) Figure 8As shown, ASD rats exhibited hierarchical taxonomic alterations at the class (Lactobacillales↑, Clostridia↓), family (Enterobacteriaceae↑, Ruminococcaceae_UCG_014↓), genus (Lactobacillus↑, Turicibacter↓), order (Enterobacteriales↑, Ruminococcaceae↓), and phylum (Firmicutes↑ / Bacteroidota↓) levels, reflecting a pro-inflammatory dysbiosis. hUCMSC+Exo treatment promoted the stabilization of mucosal repair-associated microbiota (Ruminococcaceae_UCG_014), anti-inflammatory metabolite producers (butyrate-producing bacteria), and diversity, supporting their regulatory role in the gut-brain axis in ASD.

[0144] 2. The distribution characteristics of intestinal flora at various taxonomic levels are as follows: Figure 9 As shown, the microbial remodeling characteristics of the ASD model (increased Firmicutes, decreased Prevotellaceae, and overgrowth of Lactobacillus) are consistent with the dysbiosis pattern in clinical ASD. The normalization of Bacteroidetes and Prevotella after treatment indicates that hUCMSC+Exo modulates the microbial network related to short-chain fatty acid (SCFA) metabolism. These changes may affect neurodevelopment through gut-brain axis pathways such as SCFAs and immune regulatory signaling.

[0145] 3. LEfSe analysis results of multi-level classification of intestinal flora Figure 10 As shown, ASD-associated dysbiosis manifests as multi-level taxonomic changes: Family-level changes: Christensenellaceae (anti-inflammatory bacteria) decreased, Butyricoccaceae (dysbiosis-associated bacteria) increased. Genus-level changes: Akkermansia (mucosal health-associated bacteria) decreased, Negativibacillus (pro-inflammatory bacteria) increased. Phylum-level changes: Bacilliota (pro-inflammatory bacteria) expanded, Verrucomicrobiota (metabolic health-associated bacteria) decreased. hUCMSC+Exo treatment promoted the proliferation of the following key functional bacterial communities: barrier integrity-associated bacteria (Akkermansia), immune regulation-associated bacteria (Verrucomicrobiota), and metabolic stability-associated bacteria (Christensenellaceae), suggesting that it can be regulated through multi-level microbial networks and may serve as a therapeutic strategy for ASD-associated dysbiosis.

[0146] 4. Comparative analysis of intestinal flora changes Figure 11As shown, the VPA-induced ASD model has characteristic intestinal flora imbalance, manifested by the proliferation of pro-inflammatory flora (Tyzzerella, Pseudomonadota) and the depletion of flora with fiber degradation (Bacteroidota, Ruminococcus) and immunomodulatory functions (Actinomycetota). hUCMSC+Exo treatment can partially reverse the above-mentioned flora disorders and specifically promote the recovery of bacteria related to immune homeostasis (Turicibacter) and metabolic health. These results suggest that hUCMSC+Exo may exert its therapeutic effect on ASD by regulating the composition of the intestinal flora and then affecting the signal transduction of the gut-brain axis, providing an experimental basis for the development of microbiome-based intervention strategies.

[0147] 5. Results of α-diversity analysis of intestinal flora Figure 12 As shown in the results, the partial recovery of α-diversity indicators in the treated rats suggested that hUCMSC+Exo therapy may regulate intestinal flora homeostasis through mechanisms such as immune regulation or barrier repair. These findings indicate that the gut-brain axis can be used as a therapeutic target for ASD-related flora imbalance.

[0148] 6. The results of the analysis of the beta diversity pattern of the intestinal flora are as follows Figure 13 As shown, the increased Bray-Curtis distance in the hUCMSC+Exo treatment group suggests that stem cell therapy may drive microbial remodeling by altering the intestinal microenvironment. Increased unweighted UniFrac distance indicates the restoration of phylogenetically diverse taxa, enhancing the functional redundancy of the ecosystem to maintain robustness. The weighted UniFrac pattern reflects selective regulation of metabolically active microbiota, potentially modulating host-microbe interactions. These changes highlight that microbiome-targeted intervention strategies can be an effective approach to influence the dynamic balance of the gut-brain axis in neurodevelopmental disorders.

[0149] 7. Analysis of hierarchical clustering patterns of intestinal flora, such as Figure 14 As shown, at the phylum level, the persistent presence of sulfate-reducing bacteria Thermodesulfobacteriota and inflammation-associated Pseudomonadota is consistent with the phenotype of gut redox imbalance in neurodevelopmental disorders. The widespread absence of Verrucomicrobiota (such as Akkermansia muciniphila) across all groups suggests impaired mucosal barrier integrity, a key factor in gut-brain axis dysregulation. At a finer taxonomic scale, the persistent reduction of short-chain fatty acid-producing bacteria (Roseburia, Blautia) and mucin-degrading bacteria (Bacteroides) reflects persistent metabolic dysfunction, which may exacerbate neuroinflammation by reducing the production of neuroprotective metabolites.

[0150] 8. Network correlation analysis results are as follows Figure 15 As shown, the hierarchical dysbiosis seen in the VPA-induced ASD model is consistent with the intestinal microecological imbalance seen in clinical ASD patients. The enrichment of pro-inflammatory taxa (such as Enterobacteriaceae) and the loss of immunomodulatory genera (such as Lactobacillus) may exacerbate intestinal barrier dysfunction and neuroimmune dysregulation. hUCMSC+Exo treatment restored microbial balance at multiple taxonomic levels, potentially attenuating intestinal inflammatory signals and improving ASD-related behavioral manifestations.

[0151] 9. What are the results of functional changes in intestinal flora-related metabolic pathways? Figure 16 As shown, carbohydrate-driven energy metabolism (glycolysis / gluconeogenesis) was enhanced in the control and hUCMSC+Exo treatment groups, which may reflect the adjustment of the microbiota to the host's energy needs. The glutamate / amino acid metabolism deviation associated with ASD is consistent with the neurotransmitter imbalance in neurodevelopmental disorders, suggesting that microbiota-derived glutamatergic signaling is involved in the pathogenesis of ASD. Treatment-induced inhibition of drug metabolic pathways suggests that stem cell intervention can regulate the microbiota's ability to handle exogenous compounds, thereby affecting drug dosing strategies. The persistently low levels of phenylalanine in ASD further support the role of microbiota-mediated aromatic amino acid imbalance in gut-brain axis dysfunction.

[0152] In summary, the combined use of human umbilical cord mesenchymal stem cells (hUCMSCs) and their exosomes can significantly alleviate the autism-like phenotype in rats induced by prenatal valproic acid (VPA) by regulating the gut-brain axis and inhibiting neuroinflammation, mainly including: (1) significant behavioral improvement - reduced social deficits, reduced repetitive behaviors, and restored spatial memory; (2) inhibition of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in the prefrontal cortex, and restoration of redox homeostasis (increased GSH and SOD; decreased MDA and NO); (3) transmission electron microscopy confirmed synaptic ultrastructure repair (increased vesicle density); (4) rebalancing of the intestinal flora - decreased Firmicutes / Bacteroidetes ratio, increased Ruminococcaceae, and decreased Enterobacteriaceae, which was associated with behavioral improvement.

[0153] Example 5 Lyophilized powder inhalation containing human umbilical cord mesenchymal stem cells (hUCMSCs) and exosomes

[0154] Composition and preparation of freeze-dried powder inhalation

[0155] Active ingredient:

[0156] Human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated and cultured from the umbilical cord Wharton's jelly (flow cytometry confirmed that the positive rates of CD73, CD90, and CD105 were >99%, and the negative rates of CD34, CD45, and HLA-DR were <0.1%). After trypsinization, 5×10 6 cells / mL concentration for standby use, refer to Example 1 for details;

[0157] Exosomes (Exos): Exosomes were isolated from hUCMSC-conditioned medium (serum-free DMENF12 medium) by differential ultracentrifugation. Cell culture supernatants were centrifuged sequentially: first at 300 × g for 10 minutes (4°C) to remove residual cells; then at 2,000 × g for 10 minutes (4°C) to remove cellular debris; and finally at 10,000 × g for 30 minutes (4°C) to pellet microvesicles. The clarified supernatant was filtered through a 0.22 μm polyethersulfone (PES) filter (Millipore) and finally ultracentrifuged at 100,000 × g for 2 hours (4°C; Optima XPN-100, Beckman Coulter). Transmission electron microscopy confirmed the presence of cup-shaped vesicles with diameters of 30–150 nm.

[0158] Lyophilization process: hUCMSCs and exosomes (mass ratio 1:40) were mixed in a lyophilization protection solution containing 10% trehalose (w / v) and 5% mannitol (w / v). After pre-freezing to -80°C for 2 hours, the mixture was freeze-dried at -40°C and 0.1 mbar vacuum for 48 hours to form a porous solid.

[0159] Micronization: The freeze-dried block is crushed and sieved using a ball mill to obtain micropowder with an aerodynamic particle size of 1-5 μm (this particle size range can optimize alveolar deposition and olfactory nerve uptake efficiency).

[0160] Testing has shown that the trehalose-mannitol protection system enables the freeze-dried powder to retain >90% of cell activity and >95% of exosome membrane integrity after storage at 4°C for 6 months, far exceeding the 15-day shelf life of the liquid preparation.

[0161] The lyophilized powder inhaler is administered orally and nasally via a dry powder inhaler device, once a week for 4 consecutive weeks;

[0162] A single dose contains 5×10 hUCMSCs 6 cells and exosomes 100 μg;

[0163] Delivery mechanism: After inhalation, exosome microparticles enter the brain directly through the olfactory nerve pathway, while stem cell microparticles enter the systemic circulation after exchange in the alveolar capillaries and migrate to the gut-associated lymphoid tissue.

[0164] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A combination drug targeting the gut-brain axis, comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is mesenchymal stem cells and the second active ingredient is exosomes derived from mesenchymal stem cells.

2. The combined drug according to claim 1, characterized in that The mesenchymal stem cells are selected from mesenchymal stem cells differentiated from induced pluripotent stem cells or mesenchymal stem cells isolated from human tissues.

3. The combined drug according to claim 2, characterized in that The human tissue is selected from bone marrow, adipose tissue, umbilical cord, umbilical cord blood, skeletal muscle, peripheral blood, lubricating membrane or amniotic fluid.

4. The combined drug according to claim 1, characterized in that The first active ingredient is umbilical cord mesenchymal stem cells, and the second active ingredient is exosomes derived from umbilical cord mesenchymal stem cells.

5. The combined drug according to claim 1, characterized in that The first active ingredient and the second active ingredient are in the same dosage unit.

6. The combined drug according to claim 1, characterized in that The first active ingredient and the second active ingredient are in different formulation units.

7. The combined drug according to claim 1, characterized in that The first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially.

8. The combined drug according to claim 1, characterized in that The dosage form of the combined drug is a gastrointestinal dosage form or a parenteral dosage form.

9. The combined drug according to claim 1, characterized in that The combined drug further includes pharmaceutically acceptable excipients.

10. Use of the combined drug according to any one of claims 1 to 9 in any of the following aspects: preparing drugs that modulate the gut-brain axis; preparing drugs for inhibiting neuroinflammation; Preparation of drugs for preventing and / or treating diseases related to the nervous system.

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