Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Neurocognitive Disorders in Chronic Obstructive Pulmonary Disease by Suppressing Neuroinflammation

Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Neurocognitive Disorders in Chronic Obstructive Pulmonary Disease by Suppressing Neuroinflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Neurocognitive Disorders in Chronic Obstructive Pulmonary Disease by Suppressing Neuroinflammation Hui Xiao, Xiao Yu, Yiding Dong, Shilong Bao, Jiayue Cui, Zhiyong Dong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9465519/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Patients with chronic obstructive pulmonary disease (COPD) frequently suffer from COPD-related neurocognitive disorders (COPD-NCDs), which severely impair their quality of life. Neuroinflammation is a key pathological mechanism, but effective therapies are still lacking. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) have anti-inflammatory and neuroprotective effects in other neurological disorders. However, their efficacy and underlying mechanisms in COPD-NCDs remain unclear. Methods Female BALB/c mice were divided into normal control (NC), COPD-NCDs (model), COPD-NCDs + PBS (vehicle), and COPD-NCDs + Exos (treatment) groups. COPD-NCDs was induced by 24 weeks of cigarette smoke exposure, and hUCMSC-Exos were administered via tail vein during weeks 20–23. Cognitive function, pulmonary function, lung and hippocampal pathology, microglial activation, astrocytic A1/A2 phenotype, inflammatory cytokines, and hippocampal RNA-seq were assessed. Results hUCMSC-Exos significantly improved lung function, reduced pulmonary inflammation, emphysema, collagen deposition, and systemic inflammation. Importantly, hUCMSC-Exos also improved cognitive function, attenuated hippocampal neuronal damage (increased Nissl-positive neurons and NeuN expression), inhibited microglial activation, and reduced inflammatory cytokine levels in brain tissue. Furthermore, hUCMSC-Exos downregulated the A1 astrocyte marker C3 and upregulated the A2 marker S100A10. RNA-seq suggested modulation of MAPK, Wnt, cAMP and PI3K-Akt signaling pathways. Conclusions hUCMSC-Exos improved cognitive function in COPD-NCDs mice, which was associated with inhibition of microglial activation and a shift from an A1-like to an A2-like astrocytic phenotype. These findings highlight the potential of hUCMSC-Exos as a therapeutic strategy for COPD-NCDs, although the causal relationship between phenotype shift and cognitive improvement requires further mechanistic validation. Chronic obstructive pulmonary disease Neurocognitive disorders Human umbilical cord mesenchymal stem cell-derived exosomes Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder characterized primarily by chronic airway inflammation and progressive airflow limitation, exhibiting marked heterogeneity [ 1 ]. COPD has become the third leading cause of death globally, resulting in approximately 3.23 million deaths annually and imposing a substantial economic and public health burden on healthcare systems worldwide [ 2 ]. Smoking is the primary risk factor, accounting for over 70% of cases in high-income countries and 30–40% in low- and middle-income countries [ 3 ]. Beyond impaired lung function, COPD frequently presents with multiple extrapulmonary complications, including cardiovascular diseases, anxiety, depression, osteoporosis, and metabolic disorders [ 4 – 6 ]. Among these, cognitive impairment has increasingly drawn attention. Research indicates that up to 61% of COPD patients exhibit cognitive impairment, primarily manifesting as memory decline, impaired executive function, and reduced attention [ 2 , 7 ]. Furthermore, the degree of cognitive decline correlates with disease severity, with patients with severe COPD demonstrating poorer overall cognitive function compared to those with mild or moderate COPD [ 8 ]. COPD-related neurocognitive disorders (COPD-NCDs) not only diminish treatment adherence and pulmonary rehabilitation outcomes but also significantly impair quality of life and prognosis, increasing mortality and disability rates [ 7 , 9 , 10 ]. However, effective interventions for COPD-NCDs remain scarce. Recent research suggests that persistent pulmonary inflammation and oxidative stress induced by cigarette smoke (CS) exposure may promote the spillover of inflammatory mediators into the systemic circulation. This disrupts the integrity of the blood-brain barrier (BBB), activates microglia and astrocytes within the central nervous system (CNS), and triggers neuroinflammation and neuronal damage, thereby contributing to cognitive impairment [ 2 , 11 – 13 ]. During this process, microglia undergo a transition from a homeostatic surveillance phenotype to an activated phenotype, characterized by cell body hypertrophy and shortened processes [ 11 , 13 ]. Previous research has shown that activated microglia release TNF-α, IL-1α and C1q, which further induce astrocytes to adopt an A1-like neurotoxic phenotype and exacerbate neuronal damage through the release of molecules such as complement C3 [ 14 , 15 ]. Consequently, inhibiting the neuroinflammatory response mediated by the abnormal activation of microglia and astrocytes may represent an important therapeutic strategy for managing COPD-NCDs. Mesenchymal stem cells (MSCs), owing to their potent immunomodulatory properties, have emerged as a promising candidate for anti-inflammatory therapy. MSCs possess self-renewal capacity, multipotent differentiation potential, and immunomodulatory properties, demonstrating therapeutic promise in various inflammatory and neurological disorders [ 16 , 17 ]. The biological effects of MSCs are primarily mediated by their paracrine actions, with exosomes (Exos) serving as key paracrine mediators [ 18 ]. Exos carry bioactive molecules, including proteins, lipids, and nucleic acids, and participate in intercellular communication [ 19 ]. MSC-derived exosomes (MSC-Exos) exhibit multiple biological functions, including immune modulation, angiogenesis induction, promotion of cell proliferation and migration, and acceleration of damaged neuron repair and regeneration [ 20 , 21 ]. Moreover, they attenuate allogeneic transplant rejection, do not exhibit tumorigenic potential, and have a low embolism risk, representing a promising cell-free therapeutic approach [ 22 , 23 ]. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos), in particular, have demonstrated significant neuroprotective effects in various CNS disorders [ 24 – 28 ]. However, their therapeutic efficacy and molecular mechanisms in COPD-NCDs remain understudied. Against this background, this study aims to establish a COPD-NCDs mouse model to evaluate the therapeutic effects of hUCMSC-Exos on cognitive impairment. We hypothesize that hUCMSC-Exos exert neuroprotective effects by modulating microglial activation and astrocytic A1/A2 phenotype conversion, thereby alleviating neuroinflammation. This study may provide novel experimental evidence and a theoretical basis for the treatment of COPD-NCDs. 2. Materials and Methods 2.1 Laboratory Animals Female BALB/c mice (6 weeks old, weighing 18–20 grams, Specific Pathogen Free (SPF) grade) were procured from Changchun Yisi Laboratory Animal Technology Co., Ltd. (Changchun, China; Licence No. SYXK(Ji)2023-0010). They were housed at the SPF-grade animal facility of the School of Basic Medical Sciences, Jilin University, maintained at 20 ± 5°C with 50% ± 10% humidity, and provided with ad libitum access to food and water. All animal experimentation protocols in this study were approved by the Animal Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.2025–773). 2.2 Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) Culture hUCMSCs (Sciencell, #7530) were cultured in MSC basal medium (Dakewe, #6114011) supplemented with 10% fetal bovine serum (XP BioMed, #C04400-500) (MSC complete medium). The cells were cultured at 37°C, 5% CO₂ incubator, passaged when confluence reached 80–90%, and cells from passages 3–7 were selected for subsequent experiments. 2.3 Phenotypic Characterization of hUCMSCs Cell phenotypes were validated by flow cytometry. The cells (1 × 10⁶/mL in PBS) were incubated with anti-CD34, anti-CD45, anti-CD73, anti-CD90, and anti-CD105 antibodies for 30 minutes at 4°C in the dark (Details of the antibodies are shown in Table 1 ). After washing, the cells were resuspended in 100 µL PBS and analyzed by flow cytometry. Table 1 List of antibodies for flow cytometry. Antibody Species Manufacturers/Cat.no. Conjugate CD73 Mouse BD Biosciences, #561254 FITC CD90 Mouse BD Biosciences, #555595 FITC CD105 Mouse BD Biosciences, #560839 PE CD34 Mouse BD Biosciences, #560942 FITC CD45 Mouse BD Biosciences, #561865 FITC 2.4 Multilineage Differentiation of hUCMSCs To evaluate the multipotent differentiation potential of hUCMSCs, 1 × 10⁴ cells were seeded into poly-L-lysine-coated 6-well plates and cultured in MSC complete medium. When cell confluence reached 80%–90%, the medium was replaced with either adipogenic differentiation induction medium (Dakewe, #6114531) or osteogenic differentiation induction medium (Dakewe, #6114541), and induction was continued for 18–21 days, with medium changes carried out regularly according to the manufacturer’s instructions. Upon completion of induction, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O staining solution (Dakewe, #4060711) and Alizarin Red staining solution (Dakewe, #4060611), respectively, followed by observation and photography under an inverted microscope. 2.5 Isolation of hUCMSC-Exos hUCMSCs at passages 3 to 7 were seeded at a density of 5 × 10⁵ cells per 10 cm culture dish and cultured in MSC complete medium. When cell confluence reached 60%–70%, the original medium was discarded and the cells were washed 2–3 times with PBS. Then, 10 mL of exosome-depleted medium, comprising MSC basal medium (Dakewe, #6114011) supplemented with 10% exosome-depleted fetal bovine serum (Umibio, #UR50202), was added. The cells were then cultured for a further 48 h to collect conditioned medium. After the culture supernatant was collected, differential centrifugation was performed at 4°C in the following order: 3,000 × g for 20 min, 10,000 × g for 45 min, filtration through a 0.22 µm filter membrane, and finally 120,000 × g for 120 min. The supernatant was discarded, the pellet was resuspended in an appropriate volume of PBS, the protein concentration was determined using the BCA method, and the samples were stored at − 80°C for future use. 2.6 Transmission Electron Microscope (TEM) A total of 20–30 µL of the hUCMSC-Exos suspension was dispensed onto a 200-mesh copper grid coated with formvar and allowed to adsorb at room temperature for 3–5 minutes. After excess liquid was removed, the grid was counterstained with 2% phosphotungstic acid (pH 7.0) filtered through a 0.22 µm membrane for 3–5 minutes, and the excess staining solution was then removed and the grid was allowed to dry. Once the sample was completely dry, the morphology of the hUCMSC-Exos was observed and photographed using a TEM at 80 kV. 2.7 Western Blot Total proteins were extracted from the samples using RIPA lysis buffer containing phenylmethylsulphonyl fluoride (PMSF). The protein samples were mixed with 5× protein loading buffer and denatured. Conventional proteins were heated at 100°C for 5 minutes, whereas membrane proteins were incubated at 37°C for 30 minutes. Twenty micrograms of total protein from each sample were loaded, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with a protein-free rapid blocking solution (Servicebio, #G2052-500ml) and then incubated with the primary antibodies overnight at 4°C. The primary antibodies used were anti-CD9 antibody (1:1000, Abcam, #ab263019), anti-HSP70 antibody (1:1000, Abcam, #ab181606), anti-TSG101 antibody (1:5000, Abcam, #ab125011), anti-Calnexin antibody (1:2500, Abcam, #ab133615), anti-C3 antibody (1:2500, Invitrogen, #PA521349), anti-S100A10 antibody (1:2500, Invitrogen, #PA595505), and anti-β-actin antibody (1:2500, Proteintech Group, #20536-1-AP). After washing the membrane, HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:5000, Proteintech Group, #RGAR001) was added and incubated at room temperature for 2 hours. Finally, the bands were visualized using enhanced ECL chemiluminescent substrate (Thermo Scientific, #34580), followed by image acquisition and analysis. 2.8 Establishment of COPD-NCDs Model and hUCMSC-Exos Therapy Mice were acclimatized for one week prior to the start of the experiment and were subsequently randomized into a normal control (NC) group and a CS-exposed group. Mice in the NC group were exposed to ambient indoor air; the CS-exposed group was used to establish a COPD-NCDs model using commercially available filtered CS (specifications: 10 mg tar/cigarette; 0.9 mg nicotine/cigarette; 11 mg carbon monoxide/cigarette). Mice were exposed for 5 days per week, at a rate of 9 cigarettes per day, for a total of 24 weeks. Body weight changes were recorded weekly throughout the experiment. After 20 weeks of CS exposure, the model mice were further randomly divided into three groups: the COPD-NCDs group, the COPD-NCDs + PBS group, and the COPD-NCDs + Exos group. Together with the NC group, four experimental groups were finally included: NC, COPD-NCDs, COPD-NCDs + PBS, and COPD-NCDs + Exos (n = 18 per group). The COPD-NCDs + Exos group received intravenous tail injections of hUCMSC-Exos from weeks 20–23, once weekly at a dose of 100 µg per mouse, for 4 consecutive weeks (The dosing frequency in this study was optimised by drawing on previous studies of systematic administration of hUCMSC-Exos in neurological disorders, while taking into account the cycle of the model used in this study and the intervention window [ 29 , 30 ]). As determined by the BCA assay, the protein concentration of hUCMSC-Exos was 0.5 mg/mL, with a single-dose volume of 200 µL. The COPD-NCDs + PBS group received an equal volume of sterile PBS during the same period. Neurocognitive and pulmonary function tests were conducted in week 24. Following completion of all functional assessments, samples were collected. Mice were anaesthetized via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg), and blood was collected via the retroorbital venous plexus. The mice were subsequently euthanized under deep anesthesia, and lung and brain tissues were rapidly isolated for subsequent analysis. 2.9 Invasive Pulmonary Function Testing After deep anesthesia with sodium pentobarbital, the mice were positioned supine and immobilized. Following routine disinfection of the neck skin, a longitudinal incision was made, the muscles were dissected to fully expose the trachea, and a small incision was made in the upper segment of the trachea to insert and secure a tracheal tube. The tube was then connected to the Buxco pulmonary function testing system and a small-animal ventilator. Following system calibration and once respiration had stabilized. The following parameters were measured: forced expiratory volume in 0.1 s/forced vital capacity (FEV0.1/FVC), airway resistance (RI) and dynamic compliance (Cdyn). 2.10 Non-invasive Pulmonary Function Testing Non-invasive pulmonary function testing was performed on awake, freely moving mice using a whole-body volumetric recording system. After the mice had acclimatized to the environment and the instrument had been calibrated, each mouse was placed individually in a sealed recording chamber for 5–10 minutes. Data were collected once breathing had stabilized. The following parameters were analyzed: inspiratory time (TI), expiratory time (TE), peak inspiratory flow (PIF), peak expiratory flow (PEF), enhanced pause (Penh), expiratory flow at 50% expired volume (EF50), the ratio of time to peak expiratory flow to expiratory time (Rpef), minute ventilation (MV) and tidal volume (VT), among other respiratory parameters. 2.11 Open Field Test (OFT) The OFT apparatus consisted of an open-field chamber (60 cm × 60 cm × 60 cm) in the form of an opaque black cube, with the floor divided into a central area and a peripheral area. Mice were gently placed in the central area of the open field and allowed to explore freely for 10 minutes. Their behavior was recorded using a video tracking system. After each experiment, the mice’s urine and feces were promptly removed, and the apparatus was wiped with 75% ethanol to eliminate odor interference. The following parameters were recorded and analyzed: total distance travelled, distance travelled within the central zone, and time spent in the central zone. A reduction in time spent in the central zone indicated an increase in anxiety-like behavior. 2.12 Novel Object Recognition (NOR) The NOR and OFT tests were conducted in the same apparatus. The experiment consisted of three phases: habituation, training and testing. (1) During the habituation phase, on Day 1, the mice were placed in an empty box and allowed to explore freely for 5 minutes. (2) During the training phase, on Day 2, two identical objects were placed diagonally opposite each other within the arena, and the mice were allowed to explore freely for 10 minutes. (3) Test phase: 24 hours after training, one of the familiar objects was replaced with a novel object, and the mice were again allowed to explore freely for 10 minutes. A video tracking system was used to record the time the mice spent exploring the familiar and novel objects. After each experiment, the mice’s urine and feces were removed, and the apparatus and object surfaces were wiped with 75% ethanol to eliminate any residual odors that might cause interference. 2.13 Morris Water Maze (MWM) The MWM was conducted in a quiet environment with stable lighting. The apparatus consisted of a circular tank with a diameter of 120 cm, a water depth of approximately 30 cm, and a water temperature maintained at 23 ± 1°C. A non-toxic white dye was added to the water to render it opaque, and a hidden platform was fixed at the center of the target quadrant, situated 1–2 cm below the water surface. The tank was divided into four quadrants, with different visual markers on the tank walls serving as spatial reference cues. The experiment comprised five days of orientation navigation training followed by a spatial exploration test on the sixth day. During the training period, the mice were placed into the water from one of the four different quadrants each day, and their escape latency and swimming trajectories were recorded while they searched for the platform within 60 seconds. If they failed to locate the platform, they were guided to it and held there for 10 seconds. Twenty-four hours after the final training session, the platform was removed. The mice were placed in the water from the side opposite the target quadrant and allowed to swim freely for 60 seconds. The time spent in the target quadrant, the number of platform crossings, and the swimming trajectories were recorded to assess spatial learning and memory abilities. 2.14 Histological Examination Following the collection of lung and brain tissue, the specimens were fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. After dewaxing and rehydration, the lung tissue sections were stained with hematoxylin and eosin (H&E) (Beyotime, #C0105S) and Masson’s stain (Solarbio, #G1340). Brain tissue sections were dewaxed and rehydrated, followed by staining with H&E (Beyotime, #C0105S) and Nissl staining (Solarbio, #G1430). 2.15 Enzyme-Linked Immunosorbent Assay (ELISA) Tissue samples were homogenized in pre-chilled PBS at a mass-to-volume ratio of 1:10 (w/v). After centrifugation at 4°C and 3000 × g for 5 minutes, the supernatant was collected, aliquoted, and stored at − 80°C for later use. Mouse whole blood was centrifuged at 4°C and 2000 rpm for 20 minutes, and the serum was then collected and stored at − 80°C for later use. The concentrations of TNF-α, IL-1β, and IL-6 in lung tissue homogenate supernatants and serum, as well as TNF-α, IL-1α, C1q, IL-1β, and IL-6 in brain tissue homogenate supernatants, were determined according to the instructions provided with the ELISA kits (ColorfulGene Biotech). Prior to the experiment, all reagents were allowed to reach room temperature. Standards or samples were added to each well in duplicate, followed sequentially by the enzyme-conjugated antibody and color development substrate, and the plates were incubated at 37°C. After the reaction was terminated, the absorbance at 450 nm was measured using a microplate reader. 2.16 Immunofluorescence (IF) Sections were permeabilized with 0.1% Triton X-100. Following blocking with 10% normal goat serum at room temperature for 1 hour, the sections were incubated overnight at 4°C with the following primary antibodies: anti-NeuN antibody (1:200, Abcam, #ab279297), anti-IBA1 antibody (1:500, Synaptic Systems, #234009), anti-GFAP antibody (1:300, Sigma, #MAB3402), anti-C3 antibody (1:500, Invitrogen, #PA521349), and anti-S100A10 antibody (1:500, Invitrogen, #PA595505). The following day, after the sections were equilibrated at room temperature for 30 minutes, they were incubated with Alexa Fluor 488-labelled goat anti-rabbit IgG (1:500, Invitrogen, #A11008), Alexa Fluor 555-labelled goat anti-chicken IgG (1:500, Invitrogen), Alexa Fluor 546-labelled goat anti-rabbit IgG (1:500, Invitrogen, #A11010), and Alexa Fluor 488-labelled goat anti-rat IgG secondary antibodies (1:500, Invitrogen, #A11006) at room temperature for 2 hours. Cell nuclei were counterstained with Hoechst dye. Images were acquired using a fluorescence microscope (Olympus BX53, Japan) and processed using ImageJ (Version 1.8.0) software. 2.17 Mouse RNA Sequencing Analysis Brain tissue samples were collected from mice in the NC group, the COPD-NCDs group and the hUCMSC-Exos group, with six samples per group. Total RNA was extracted using the TRIzol method, and its concentration, purity and integrity were assessed. Samples with a RIN ≥ 7.0 were selected for library preparation. From each sample, 1 µg of total RNA was taken. Following mRNA enrichment using oligo(dT) magnetic beads, sequencing libraries were constructed and subjected to 150-base paired-end sequencing on the Illumina platform. After quality control, clean reads were obtained from the raw data. These were aligned to the mouse reference genome using Hisat2, and transcript assembly and expression quantification were performed using StringTie. Differential expression analysis was performed using DESeq2, with differentially expressed genes identified based on |log₂Fold Change| ≥ 1 and FDR < 0.05. Subsequently, GO and KEGG enrichment analyses were conducted using ClusterProfiler. Bioinformatics analysis was performed using BMKCloud ( www.biocloud.net ). 2.18 Statistical Analysis GraphPad Prism 8.0 was used for statistical analysis and figure generation. Mean fluorescence intensity was measured using ImageJ (Version 1.8.0) software. Data were presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was assessed using Levene’s test. Comparisons between two groups were performed using the unpaired two-tailed Student’s t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. In all statistical analyses, P < 0.05 was considered statistically significant (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P 0.05). 3 Results 3.1 Characterization of hUCMSCs and hUCMSC-Exos The cultured hUCMSCs exhibited typical adherent growth, with uniform cell morphology characterized by a spindle-shaped appearance and a swirling arrangement under phase-contrast microscopy (Fig. 1 A). Following adipogenic and osteogenic induction, distinct lipid droplet and calcium nodule formation were observed, respectively, suggesting multipotent differentiation potential (Figs. 1 B–C). Flow cytometry results (Fig. 1 D) showed that the cells highly expressed MSC-specific markers CD73 (99.54%), CD90 (99.83%) and CD105 (99.94%), while expression levels of the hematopoietic markers CD34 (1.22%) and CD45 (1.08%) were extremely low. NTA results (Fig. 1 E) showed that hUCMSC-Exos had an average particle size of 126.2 nm, a peak particle size of 115.3 nm, and a particle concentration of 2.3 × 10¹⁰ particles/mL. TEM revealed a typical round or cup-shaped vesicular structure (Fig. 1 F). Western blot analysis confirmed the expression of CD9, HSP70 and TSG101, while Calnexin was undetectable (Fig. 1 G), indicating successful isolation of hUCMSC-Exos. 3.2 hUCMSC-Exos Improved Pulmonary Ventilation Function in Mice with COPD-NCDs Following 20 weeks of exposure to CS, mice received tail-vein injections of hUCMSC-Exos for four consecutive weeks between weeks 20 and 23. The experimental protocol is shown in Fig. 2 A. Longitudinal body weight monitoring (Fig. 2 B) showed that body weight gain in the COPD-NCDs group was significantly slowed compared with the NC group. Following treatment with hUCMSC-Exos, the trend in body weight gain improved markedly, while the PBS group exhibited a trend largely consistent with that of the COPD-NCDs group. Analysis of final body weight further indicated (Fig. 2 C) that at week 24, the body weight of mice in the COPD-NCDs group was significantly lower than that of the NC group (COPD-NCDs group: 20.98 ± 2.36 vs. NC group: 26.92 ± 1.29, P < 0.0001), whereas following treatment with hUCMSC-Exos, body weight was significantly higher than that of the COPD-NCDs group (23.01 ± 1.11, P 0.05). Mouse pulmonary ventilation function was further assessed via invasive and non-invasive pulmonary function testing. Results of invasive pulmonary function testing (Fig. 2 D–F) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited reduced FEV0.1/FVC (COPD-NCDs group: 0.49 ± 0.05 vs. NC group: 0.91 ± 0.01, P < 0.0001), increased RI (COPD-NCDs group: 7.98 ± 1.58 vs. NC group: 4.31 ± 0.54, P < 0.01) and reduced Cdyn (COPD-NCDs group: 0.006 ± 0.001 vs. NC group: 0.01 ± 0.001, P < 0.001). Following treatment with hUCMSC-Exos, the COPD-NCDs + Exos group showed increased FEV0.1/FVC (0.72 ± 0.07, P < 0.0001), decreased RI (5.24 ± 0.76, P < 0.05) and increased Cdyn (0.008 ± 0.001, P 0.05). Results from non-invasive pulmonary function testing (Fig. 2 G–O) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited prolonged TI (COPD-NCDs group: 0.069 ± 0.007 vs. NC group: 0.05 ± 0.006, P < 0.001) and TE (COPD-NCDs group: 0.09 ± 0.006 vs. NC group: 0.075 ± 0.005, P < 0.001), as well as increased Penh (COPD-NCDs group: 0.67 ± 0.04 vs. NC group: 0.53 ± 0.07, P < 0.01). In addition, PIF (COPD-NCDs group: 5.48 ± 0.32 vs. NC group: 7.44 ± 0.48, P < 0.0001), PEF (COPD-NCDs group: 4.83 ± 0.6 vs. NC group: 6.31 ± 0.31, P < 0.001), EF50 (COPD-NCDs group: 2.52 ± 0.29 vs. NC group: 3.32 ± 0.35, P < 0.001), Rpef (COPD-NCDs group: 0.39 ± 0.03 vs. NC group: 0.51 ± 0.05, P < 0.01), MV (COPD-NCDs group: 40.86 ± 3 vs. NC group: 55.5 ± 6.7, P < 0.001) and VT (COPD-NCDs group: 0.1 ± 0.01 vs. NC group: 0.16 ± 0.03, P < 0.001) were all significantly reduced, indicating impaired pulmonary ventilation in the COPD-NCDs group. Following treatment with hUCMSC-Exos, TI (0.055 ± 0.002, P < 0.05), TE (0.081 ± 0.003, P < 0.05) and Penh (0.55 ± 0.03, P < 0.05) were significantly lower than in the COPD-NCDs group, while PIF (6.67 ± 0.46, P < 0.01), PEF (5.71 ± 0.21, P < 0.05), EF50 (3.1 ± 0.14, P < 0.05), Rpef (0.47 ± 0.03, P < 0.05), MV (49.31 ± 2.83, P < 0.05) and VT (0.14 ± 0.01, P < 0.05) were significantly increased. These results indicated that hUCMSC-Exos improved pulmonary ventilation function in COPD-NCDs mice. No statistically significant difference was observed between the PBS group and the COPD-NCDs group ( P > 0.05). 3.3 hUCMSC-Exos Alleviated Pulmonary Histopathological Injury, Pulmonary Inflammation, and Systemic Inflammation in Mice with COPD-NCDs To evaluate the effects of hUCMSC-Exos on pathological damage in the lung tissue of COPD-NCDs mice, H&E staining was used to observe changes in lung tissue structure, while Masson’s staining was employed to assess collagen fiber deposition. The HE staining results (Fig. 3 A) showed that the lung tissue structure in the NC group was intact, with regular alveolar arrangement and no obvious inflammatory cell infiltration. In contrast, the COPD-NCDs group exhibited disorganized alveolar structures, thinning or rupture of alveolar septa, enlargement and fusion of alveolar spaces, and marked inflammatory cell infiltration. Following treatment with hUCMSC-Exos, inflammatory cell infiltration was reduced, and alveolar dilatation and tissue damage were significantly improved, whereas no significant differences were observed between the PBS group and the COPD-NCDs group. Masson’s staining results (Fig. 3 B) showed that the COPD-NCDs group exhibited a significant increase in collagen fiber deposition in the airway walls, indicating aggravated fibrosis. Following treatment with hUCMSC-Exos, the area of collagen deposition decreased, and the thickening of the airway walls and the degree of fibrosis improved markedly. In contrast, no significant improvement was observed in the PBS group. ELISA was further employed to detect the levels of TNF-α, IL-1β and IL-6 in the lung tissue and serum of mice in each group. The results showed (Fig. 3 C–E) that, compared with the NC group, the levels of the inflammatory factors TNF-α (COPD-NCDs group: 290.97 ± 26.33 vs. NC group: 233.69 ± 20.03, P < 0.01), IL-1β (COPD-NCDs group: 55.62 ± 7.27 vs. NC group: 42.24 ± 3.94, P < 0.05) and IL-6 (COPD-NCDs group: 69 ± 3.11 vs. NC group: 57.48 ± 1.54, P < 0.01) were significantly elevated in the lung tissue of the COPD-NCDs group, suggesting an enhanced inflammatory response. Treatment with hUCMSC-Exos significantly reduced the levels of TNF-α (248.13 ± 19.21, P < 0.05), IL-1β (44.2 ± 7.44, P < 0.05) and IL-6 (60.93 ± 1.25, P 0.05). Compared with the NC group, the serum levels of TNF-α (COPD-NCDs group: 370.64 ± 17.67 vs. NC group: 307.4 ± 7.4, P < 0.0001), IL-1β (COPD-NCDs group: 63.37 ± 3.02 vs. NC group: 51.1 ± 1.03, P < 0.0001) and IL-6 (COPD-NCDs group: 86.6 ± 6.15 vs. NC group: 63.23 ± 2.95, P < 0.0001) in the COPD-NCDs group were significantly elevated, indicating an enhanced systemic inflammatory response. There was no statistically significant difference between the PBS group and the COPD-NCDs group ( P > 0.05). Following treatment with hUCMSC-Exos, the levels of TNF-α (325.59 ± 7.06, P < 0.001), IL-1β (55.71 ± 1.66, P < 0.0001) and IL-6 (72.17 ± 2.23, P < 0.001) were significantly reduced compared with the COPD-NCDs group (Fig. 3 F–H), indicating that hUCMSC-Exos ameliorated the systemic inflammatory response in COPD-NCDs mice. Given the observed improvement in pulmonary inflammation and systemic inflammation, we next examined whether hUCMSC-Exos could ameliorate cognitive dysfunction in COPD-NCDs mice. 3.4 hUCMSC-Exos Alleviated Neurocognitive Impairment in Mice with COPD-NCDs To evaluate the neuroprotective effects of hUCMSC-Exos on neurocognitive function in mice with COPD-NCDs, behavioral tests including the OFT, NOR, and MWM were conducted after completion of treatment. Schematic diagrams of the experimental apparatus are shown in Figs. 4 A–B. OFT results (Fig. 4 C–F) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited a significantly reduced total distance travelled (COPD-NCDs group: 604.69 ± 70.08 vs. NC group: 2093.4 ± 462.21, P < 0.0001) and a greater tendency to remain in the peripheral area, suggesting decreased locomotor activity and increased anxiety-like behavior. Both the distance travelled in the central area (COPD-NCDs group: 130.12 ± 42.25 vs. NC group: 321.92 ± 90.79, P < 0.0001) and the time spent there (COPD-NCDs group: 15.32 ± 2.58 vs. NC group: 52.81 ± 6.82, P < 0.0001) were markedly reduced, suggesting decreased spontaneous activity and anxiety-like behavioral changes. Following hUCMSC-Exos treatment, the total distance travelled increased significantly (1568.31 ± 242.52, P < 0.0001), and both the distance travelled in the central area (238.17 ± 55.6, P < 0.01) and the time spent there (43.59 ± 7.87, P < 0.0001) increased significantly, whereas the tendency to remain in the peripheral area was reduced. No statistically significant differences were observed between the PBS group and the COPD-NCDs group ( P > 0.05). NOR results (Fig. 4 G) showed that the discrimination index in the COPD-NCDs group was significantly lower than that in the NC group (COPD-NCDs group: 0.23 ± 0.08 vs. NC group: 0.57 ± 0.08, P < 0.0001), indicating impaired recognition memory. In the COPD-NCDs + Exos group, the discrimination index increased significantly (0.4 ± 0.09, P 0.05). The MWM was further used to evaluate spatial learning and memory. During the training phase (Fig. 4 I), the escape latency on day 5 was significantly prolonged in the COPD-NCDs group compared with the NC group, indicating impaired spatial learning ability. hUCMSC-Exos treatment significantly shortened the escape latency. In the probe trial, trajectory plots (Fig. 4 H) showed that mice in the NC group mainly swam within the target quadrant, whereas mice in the COPD-NCDs group showed reduced swimming activity in the target quadrant. Statistical analysis further showed (Figs. 4 J–K) that the time spent in the target quadrant (COPD-NCDs group: 25.35 ± 8.76 vs. NC group: 58.27 ± 9.69, P < 0.0001) and the number of platform crossings (COPD-NCDs group: 1 ± 1 vs. NC group: 4 ± 1, P < 0.0001) were both significantly reduced in the COPD-NCDs group compared with the NC group. In contrast, both indicators were significantly increased in the COPD-NCDs + Exos group ( P 0.05). Having established that hUCMSC-Exos improved cognitive function, we next investigated whether these effects were associated with the alleviation of neuroinflammation and neuronal damage in the hippocampus. 3.5 hUCMSC-Exos Alleviated Hippocampal Neuronal Damage in Mice with COPD-NCDs The hippocampus is a critical brain region involved in learning, memory, and emotional regulation. To determine whether cognitive impairment in mice with COPD-NCDs was accompanied by hippocampal structural damage and neuronal loss, H&E staining, Nissl staining, and NeuN IF were performed to evaluate hippocampal histopathology and neuronal survival. H&E staining results (Fig. 5 A) showed that neurons in the NC group exhibited intact morphology and were arranged in a dense and orderly manner. In contrast, the COPD-NCDs group showed a reduced number of neurons, disordered arrangement, and obvious pathological changes, including nuclear shrinkage, hyperchromatic nuclei, blurred nuclear membranes, and cytoplasmic vacuolization. Nissl staining results (Figs. 5 B and 5 D–F) showed that the hippocampus of the NC group was rich in Nissl bodies, with clear boundaries, uniform staining, and regular arrangement. Compared with the NC group, the numbers of Nissl-positive neurons in the DG, CA3, and CA1 regions were significantly reduced in the COPD-NCDs group (DG: COPD-NCDs group: 38 ± 4 vs. NC group: 81 ± 7, P < 0.0001; CA3: COPD-NCDs group: 12 ± 5 vs. NC group: 52 ± 7, P < 0.0001; CA1: COPD-NCDs group: 35 ± 7 vs. NC group: 83 ± 6, P < 0.0001), accompanied by sparse distribution, partial dissolution of Nissl bodies, and abnormal neuronal morphology, indicating marked neuronal damage. No statistically significant difference was observed between the PBS group and the COPD-NCDs group ( P > 0.05). In contrast, in the COPD-NCDs + Exos group, treatment with hUCMSC-Exos significantly increased the numbers of Nissl-positive neurons in all hippocampal regions (DG: 61 ± 7, P < 0.001, CA3: 26 ± 4, P < 0.05, CA1: 59 ± 8, P < 0.01), and their arrangement tended to return to normal, suggesting that hUCMSC-Exos alleviated hippocampal neuronal damage. NeuN IF was further used to assess neuronal survival. The results (Figs. 5 C and 5 G–I) showed that, compared with the NC group, the fluorescence intensity of NeuN-positive neurons in the DG, CA3, and CA1 regions was significantly decreased in the COPD-NCDs group (DG: COPD-NCDs group: 19.84 ± 4.27 vs. NC group: 49.3 ± 3.88, P < 0.0001; CA3: COPD-NCDs group: 18.7 ± 3.2 vs. NC group: 42.05 ± 4.94, P < 0.001; CA1: COPD-NCDs group: 21.24 ± 7.1 vs. NC group: 47 ± 4.86, P 0.05). However, in the COPD-NCDs + Exos group, treatment with hUCMSC-Exos significantly increased NeuN fluorescence intensity in all hippocampal regions (DG: 33.76 ± 3, P < 0.01, CA3: 30.39 ± 1.99, P < 0.05, CA1: 36.11 ± 2.92, P < 0.05). Collectively, these results indicated that mice with COPD-NCDs exhibited significant hippocampal neuronal damage and loss, whereas hUCMSC-Exos markedly improved hippocampal histopathological changes and neuronal survival, thereby exerting a protective effect on hippocampal neurons. Having established that hUCMSC-Exos protected hippocampal neurons, we next investigated whether these effects were mediated by the modulation of neuroinflammation, including microglial activation and astrocytic A1/A2 phenotype conversion. 3.6 hUCMSC-Exos Alleviated Neuroinflammation by Suppressing Microglial Activation in Mice with COPD-NCDs To investigate the role of microglia-mediated neuroinflammation in COPD-NCDs, IBA1 IF staining combined with morphological observation was used to evaluate the activation status of microglia in the hippocampus. The results showed (Figs. 6 A–D) that microglia in the NC group displayed a typical resting-state morphology, characterized by small cell bodies, slender processes, and abundant branching. In contrast, an increased number of hypertrophic microglia was observed in the hippocampus of the COPD-NCDs group, characterized by markedly enlarged cell bodies, shortened and reduced processes. The mean IBA1 fluorescence intensity was significantly increased in the DG (COPD-NCDs group: 84.16 ± 5.79 vs. NC group: 51.54 ± 5.7, P < 0.01), CA3 (COPD-NCDs group: 86.1 ± 5.87 vs. NC group: 45.04 ± 4.2, P < 0.001), and CA1 (COPD-NCDs group: 87.34 ± 2.8 vs. NC group: 42.28 ± 6.01, P < 0.001) regions, indicating microglial activation in the hippocampus. The morphology of microglia in the PBS group was similar to that in the COPD-NCDs group. However, in the COPD-NCDs + Exos group, microglial morphology in all hippocampal regions was markedly improved, as evidenced by reduced cell body size, more numerous and elongated processes, and decreased mean IBA1 fluorescence intensity (DG: 62.06 ± 4.16, P < 0.05, CA3: 55.75 ± 5.26, P < 0.01, CA1: 64.28 ± 8.85, P < 0.05). The overall morphology was similar to that in the NC group, suggesting that hUCMSC-Exos suppressed microglial activation in the hippocampus of mice with COPD-NCDs. To further evaluate the regulatory effect of hUCMSC-Exos on neuroinflammation in brain tissue, ELISA was performed to detect the levels of inflammatory and complement-related factors, including TNF-α, IL-1α, C1q, IL-1β, and IL-6, in the brain tissue of mice from each group. The results showed (Figs. 6 E–I) that, compared with the NC group, the levels of TNF-α (COPD-NCDs group: 282.54 ± 20.38 vs. NC group: 230.91 ± 31.89, P < 0.05), IL-1α (COPD-NCDs group: 51.03 ± 5.7 vs. NC group: 30.68 ± 3.11, P < 0.0001), C1q (COPD-NCDs group: 5.79 ± 0.17 vs. NC group: 3.69 ± 0.13, P < 0.0001), IL-1β (COPD-NCDs group: 48.77 ± 3.64 vs. NC group: 35.68 ± 3.59, P < 0.001), and IL-6 (COPD-NCDs group: 145.88 ± 11.67 vs. NC group: 110.52 ± 6.61, P 0.05). However, in the COPD-NCDs + Exos group, the levels of TNF-α (236.92 ± 22.76, P < 0.05), IL-1α (40.67 ± 1.06, P < 0.05), C1q (4.45 ± 0.1, P < 0.0001), IL-1β (41.82 ± 2.36, P < 0.05), and IL-6 (120.25 ± 4.69, P < 0.01) in brain tissue were significantly reduced compared with the COPD-NCDs group, indicating that hUCMSC-Exos alleviated neuroinflammatory responses in mice with COPD-NCDs. Collectively, these results indicated that hUCMSC-Exos effectively inhibited excessive microglial activation and reduced the levels of inflammatory and complement-related factors in brain tissue, thereby attenuating neuroinflammation. Activated microglia can release IL-1α, TNF-α, and C1q, which are known to induce neurotoxic A1 astrocytes. We therefore next examined whether hUCMSC-Exos modulated astrocytic A1/A2 phenotype in the hippocampus. 3.7 hUCMSC-Exos Inhibited A1 Neurotoxic Astrocyte Activation in Mice with COPD-NCDs To further investigate the regulatory effects of hUCMSC-Exos on astrocyte reactivity and the A1 neurotoxic phenotype, IF staining was performed to detect the co-expression of the astrocyte marker GFAP and the A1 reactive astrocyte-associated marker C3. Western blot analysis was used to assess C3 protein expression. IF results (Figs. 7 A–D) showed that, compared with the NC group, the number of GFAP/C3 double-positive cells in the DG, CA3, and CA1 regions of the hippocampus was significantly increased in the COPD-NCDs group (DG: COPD-NCDs group: 32 ± 2.65 vs. NC group: 17.67 ± 2.52, P < 0.001; CA3: COPD-NCDs group: 31 ± 2.65 vs. NC group: 19.33 ± 1.15, P < 0.01; CA1: COPD-NCDs group: 40 ± 4 vs. NC group: 19 ± 3.61, P 0.05). In contrast, in the COPD-NCDs + Exos group, the number of GFAP/C3 double-positive cells in all hippocampal regions was significantly reduced (DG: 23 ± 2.65, P < 0.05, CA3: 22.67 ± 2.52, P < 0.01, CA1: 26.33 ± 1.53, P < 0.05), suggesting that A1 astrocyte activation was suppressed. Western blot results further confirmed (Figs. 7 E–F) that, compared with the NC group, C3 protein expression was significantly elevated in the COPD-NCDs group ( P < 0.01). In contrast, C3 protein expression was significantly reduced in the COPD-NCDs + Exos group compared with the COPD-NCDs group ( P 0.05). 3.8 hUCMSC-Exos Promoted A2 Neuroprotective Astrocyte Activation in Mice with COPD-NCDs To further evaluate the regulatory effect of hUCMSC-Exos on the protective A2 astrocyte phenotype, IF staining was performed to detect the co-expression of the astrocyte marker GFAP and the A2 astrocyte-associated marker S100A10. Western blot analysis was used to assess S100A10 protein expression. IF results (Figs. 8 A–D) showed that, compared with the NC group, the number of GFAP/S100A10 double-positive cells in the DG, CA3, and CA1 regions of the hippocampus was significantly reduced in the COPD-NCDs group (DG: COPD-NCDs group: 14.67 ± 2.52 vs. NC group: 32.67 ± 5.86, P < 0.01; CA3: COPD-NCDs group: 15 ± 3 vs. NC group: 27.33 ± 2.52, P < 0.01; CA1: COPD-NCDs group: 12 ± 3.61 vs. NC group: 31.33 ± 2.08, P 0.05). In contrast, in the COPD-NCDs + Exos group, the number of GFAP/S100A10 double-positive cells in all hippocampal regions increased significantly (DG: 25.67 ± 2.08, P < 0.05, CA3: 22.33 ± 1.53, P < 0.05, CA1: 22.67 ± 2.52, P < 0.05), indicating enhanced A2 astrocyte activation. Western blot results further confirmed (Figs. 8 E–F) that, compared with the NC group, S100A10 protein expression was significantly reduced in the COPD-NCDs group ( P < 0.01). In contrast, S100A10 protein expression was significantly increased in the the COPD-NCDs + Exos group compared with the COPD-NCDs group ( P 0.05). 3.9 Transcriptome Analysis Revealed the Molecular Mechanisms of hUCMSC-Exos Treatment in Mice with COPD-NCDs To investigate the potential molecular mechanisms underlying the therapeutic effects of hUCMSC-Exos in mice with COPD-NCDs, RNA-seq analysis was performed on brain tissue from the NC, COPD-NCDs, and COPD-NCDs + Exos groups. Differential expression analysis was conducted using DESeq2, with fold change ≥ 1.5 and P < 0.05 as the screening criteria. Venn diagram analysis (Fig. 9 A) showed the distribution and overlap of differentially expressed genes (DEGs) among the groups. Compared with the NC group, a total of 494 DEGs were identified in the COPD-NCDs group, including 207 up-regulated and 287 down-regulated genes (Fig. 9 B), indicating marked alterations in the gene expression profile of brain tissue in the COPD-NCDs group. Further comparison between the COPD-NCDs group and the COPD-NCDs + Exos group identified 671 DEGs, of which 450 were up-regulated and 221 were down-regulated (Fig. 9 C). These results suggested that COPD-NCDs markedly altered the transcriptional profile of brain tissue, whereas hUCMSC-Exos intervention further reshaped the expression patterns of relevant genes. GO enrichment analysis of the DEGs further showed that, in the comparison between the NC and COPD-NCDs groups, DEGs were primarily enriched in Biological Process (BP) categories such as cellular processes, biological regulation, response to stimulus, signaling, and metabolic processes. In Cellular Component (CC) categories, they were mainly enriched in intracellular components and protein complexes. In Molecular Function (MF) categories, they were primarily associated with binding and catalytic activity (Fig. 9 D). In the comparison between the COPD-NCDs and COPD-NCDs + Exos groups, DEGs were similarly enriched in BP categories including cellular processes, biological regulation, response to stimulus, metabolic processes, signaling, and immune system processes. CC categories were again dominated by intracellular components and protein complexes. MF categories mainly involved binding, catalytic activity, transport, and transcriptional regulation (Fig. 9 E). Based on the functional changes suggested by GO analysis, KEGG pathway enrichment analysis was further performed to identify the key signaling pathways involved. The results showed that, compared with the NC group, DEGs in the COPD-NCDs group were mainly enriched in the Wnt, cAMP, PI3K-Akt, TNF, and MAPK signaling pathways (Fig. 9 F). Compared with the COPD-NCDs group, DEGs in the COPD-NCDs + Exos group were mainly enriched in pathways including MAPK, PI3K-Akt, cAMP, and Wnt (Fig. 9 G). These results suggested that the Wnt, cAMP, PI3K-Akt, and MAPK pathways might be involved in the regulatory effects of hUCMSC-Exos on COPD-NCDs-related pathological processes. 4. Discussion Neurocognitive dysfunction is one of the common extrapulmonary complications of COPD, significantly affecting patients’ quality of life and disease prognosis. Currently, there is a lack of effective treatment strategies for COPD-NCDs. Previous studies have shown that exposure to CS can induce pulmonary inflammation and exacerbate oxidative stress, facilitating the entry of inflammatory mediators from the lungs into the systemic circulation. This further compromises the integrity of the BBB, activates microglia and astrocytes, and consequently leads to neuroinflammation, neuronal damage and cognitive decline [ 11 – 13 , 31 , 32 ]. Therefore, targeting inflammation- and oxidative stress-related pathways may represent a promising therapeutic strategy for COPD-NCDs. Existing research indicates that certain anti-inflammatory or antioxidant drugs hold potential value in this field. For example, ebselen, an organoselenium compound with glutathione peroxidase-like activity, can alleviate CS-induced cognitive impairment to some extent by reducing pulmonary inflammation and maintaining synaptophysin expression in the hippocampus [ 32 ]. However, its poor water solubility may limit bioavailability in vivo and increase the risk of potential toxicity, thereby affecting further clinical application [ 28 ]. Given the limitations of existing pharmacological treatments, the development of novel intervention strategies that combine biosafety with neuroprotective potential is of great significance. MSC-Exos can partially mimic the therapeutic effects of stem cells while avoiding the safety and technical constraints associated with cell transplantation. In comparison, hUCMSC-Exos are considered to have greater clinical translational potential due to their stable source, lower ethical burden and superior biosafety. Previous studies have demonstrated that hUCMSC-Exos can carry various cytokines, growth factors and functional RNA molecules, and are capable of crossing the BBB to exert neuroprotective and immunomodulatory effects in a range of CNS disorders. Furthermore, their low immunogenicity and absence of oncogenic risk further enhance their application prospects. However, there is currently a lack of direct evidence regarding the role of hUCMSC-Exos in COPD-NCDs. Therefore, this study employed hUCMSC-Exos to treat COPD-NCDs mice and systematically evaluated the therapeutic effects. A COPD-NCDs mouse model was successfully established using a 24-week CS exposure protocol [ 13 , 33 ]. Subsequent experiments, including pulmonary histopathological examination, pulmonary function testing and ELISA assays, revealed marked inflammatory cell infiltration, emphysema-like changes and increased collagen fiber deposition in the lung tissue of COPD-NCDs mice. These findings were accompanied by reduced pulmonary ventilation function and elevated levels of inflammatory cytokines in both lung tissue and serum, suggesting enhanced pulmonary and systemic inflammatory responses. These findings were consistent with previous reports and further confirm the successful establishment of the COPD model [ 33 ]. In contrast, following intervention with hUCMSC-Exos, the inflammatory response in the mice’s lung tissue was alleviated, lung function improved, and levels of inflammatory cytokines in both lung tissue and serum decreased. These results suggested that hUCMSC-Exos alleviated COPD-related lung injury and systemic inflammatory responses. Building on the improvements in peripheral inflammation and lung injury, we further evaluated the effects of hUCMSC-Exos on CNS function. Behavioral results showed that mice with COPD-NCDs exhibited a significant decline in spatial learning and memory abilities, alongside increased anxiety-like behavior, suggesting that the COPD-NCDs model had been successfully established. However, these abnormalities were alleviated following treatment with hUCMSC-Exos. Further histological analysis indicated that hUCMSC-Exos intervention alleviated neuronal damage, as evidenced by reduced neuronal nuclear atrophy, increased Nissl bodies, and elevated NeuN expression. These findings further confirmed the neuroprotective effects of hUCMSC-Exos in the COPD-NCDs model. Moreover, the neuroprotective effects of hUCMSC-Exos may be closely related to their regulation of the neuroinflammatory microenvironment. Microglia and astrocytes are key regulatory cells in the inflammatory response of the CNS. Under steady-state conditions, microglia adopt a ramified morphology and continuously monitor the local microenvironment. However, upon inflammatory or injury stimuli, they transition to an activated state, releasing various inflammatory factors that participate in neuroinflammatory processes [ 34 – 36 ]. In addition to providing structural support to neurons, astrocytes are involved in maintaining synaptic homeostasis and regulating the function of the BBB [ 37 – 39 ]. Previous studies have shown that upon release of TNF-α, IL-1α and C1q by activated microglia, astrocytes can be induced to adopt an A1 neurotoxic phenotype. This phenotype is neurotoxic and can further exacerbate damage to neurons and oligodendrocytes [ 14 , 15 ]. Moreover, C1q is the initiating molecule of the classical complement pathway and plays an important role in the CNS not only in innate immune defense but also in synaptic pruning and neuroinflammatory regulation [ 40 – 42 ]. In recent years, numerous studies have confirmed the protective role of hUCMSC-Exos in suppressing neuroinflammation. For example, hUCMSC-Exos could inhibit microglial-mediated inflammatory responses, reduce infarct volume and improve neurological function in models of ischemic stroke [ 27 ]. Moreover, hUCMSC-Exos not only suppressed excessive microglial activation but also promoted neurological recovery in TBI rat models by regulating astrocyte activation states [ 43 ]. Furthermore, hUCMSC-Exos partially inhibited the formation of A1 neurotoxic astrocytes, thereby mitigating the damage they inflicted on neurons and neural tissue [ 44 ]. In vitro and in vivo studies have also demonstrated that hUCMSC-Exos alleviated inflammatory responses associated with intracerebral hemorrhage, improved cerebral oedema and BBB function, and ultimately promoted neurological recovery [ 45 ]. Building on the aforementioned research, this study further evaluated the role of hUCMSC-Exos in regulating neuroinflammation in COPD-NCDs. Consistent with previous findings, this study found that hUCMSC-Exos significantly improved the neuroinflammatory response in mice with COPD-NCDs. Specifically, this was manifested by reduced microglial cell body hypertrophy and an increase in the number of dendritic processes, suggesting a decrease in their activation levels. Simultaneously, levels of inflammatory factors such as TNF-α, IL-1α, C1q, IL-1β and IL-6 in brain tissue were significantly reduced. Further analysis revealed that hUCMSC-Exos suppressed A1 neurotoxic astrocyte phenotype and promoted the increase in A2 neuroprotective astrocyte phenotype. These findings suggest that hUCMSC-Exos may alleviate neuroinflammatory responses by inhibiting excessive microglial activation and regulating astrocyte phenotypic conversion, thereby improving neuronal damage and cognitive dysfunction. To further elucidate the neuroprotective effects described above, this study analyzed their potential molecular mechanisms in conjunction with transcriptomic results. At the mechanistic level, the Wnt signaling pathway plays a crucial role in regulating immune responses and providing neuroprotection within the CNS. It enhances glial cells' anti-inflammatory properties, reduces the production of inflammatory mediators, promotes neuronal survival, and participates in post-injury repair and regeneration by regulating the proliferation and differentiation of neural progenitor cells [ 46 ]. cAMP, as a vital intracellular regulator maintaining microglial homeostasis, can induce microglial activation through negative modulation of inflammatory signaling pathways [ 47 , 48 ]. The PI3K/AKT signaling pathway is also closely associated with neuroinflammation regulation and neural function maintenance. Research indicates that TREM2 activation may mitigate neuroinflammation by modulating PI3K/AKT signaling, thereby improving learning and memory-related impairments[ 49 , 50 ]. Furthermore, p38 MAPK, a stress-activated kinase sensitive to oxidative stress and inflammatory responses, exhibits sustained activation closely linked to neuronal injury, apoptosis, and cognitive decline[ 51 ]. Related studies also suggest that inhibiting p38 MAPK may help alleviate cognitive impairment associated with environmental tobacco smoke [ 52 ]. In this study, transcriptome sequencing revealed alterations in multiple signaling networks related to neuroinflammation following hUCMSC-Exos intervention, suggesting that its mechanism of action in suppressing neuroinflammation in COPD-NCDs mice may involve regulating key signaling pathways such as Wnt, cAMP, PI3K-Akt, and MAPK. In summary, this study successfully established a stable COPD-NCDs mouse model and employed hUCMSC-Exos via tail vein administration for intervention. Results indicated that hUCMSC-Exos attenuated pulmonary and systemic inflammatory responses, ameliorated neuroinflammation and neuronal injury, and alleviated cognitive dysfunction at the behavioral level. Transcriptomic analysis further suggested that the neuroprotective effects of hUCMSC-Exos might involve the coordinated regulation of multiple inflammation-related signaling pathways, providing experimental evidence for its potential application in COPD-NCDs and future mechanistic studies. However, this study remains subject to certain limitations. (1) The proposed signaling pathways were inferred primarily through histological observations, inflammatory cytokine assays, and transcriptomic sequencing analyses, lacking causal validation for key pathways. Whether pathways such as Wnt, cAMP, PI3K-Akt, and MAPK are essential for the efficacy of hUCMSC-Exos requires further confirmation through pathway-specific inhibitor or agonist interventions, alongside strategies including gene knockdown, overexpression, or conditional knockout. (2) This study lacks direct evidence of hUCMSC-Exos delivery and distribution within the brain. Although reduced neuroinflammation and improved neuronal injury were observed, whether hUCMSC-Exos cross the BBB and their enrichment characteristics in brain regions such as the hippocampus and prefrontal cortex require clarification through in vivo tracing, fluorescence imaging, and quantitative analysis. (3) This study primarily focuses on glia-mediated neuroinflammation. However, the development of COPD-NCDs may also be influenced by multiple factors including chronic hypoxia, impaired cerebral microvascular endothelial function, and neurotransmitter metabolic disorders, which have not been systematically evaluated. Future work could incorporate supplementary validation using BBB tight junction proteins, cerebral microvascular function markers, and oxidative stress-related indicators. (4) Regarding the administration protocol, this study employed tail vein injection with fixed dosage and cycle. The effects of different administration routes, dose gradients, timing, and treatment duration on efficacy remain uninvestigated. Longer-term follow-up is also lacking to assess sustained efficacy and safety. (5) At the model and sample level, this study employed a single strain and a fixed experimental time window. Extrapolating results to COPD models with different genetic backgrounds, sexes, or disease severity requires caution. Future work should expand sample sizes and replicate validation under diverse conditions. Future research may advance in the following areas. (1) Subsequent functional validation of key pathways identified through transcriptome sequencing analysis should clarify the association mechanisms between the key active components of hUCMSC-Exos and their receptors and downstream signaling pathways. (2) In vivo tracing should determine the targeted distribution characteristics of hUCMSC-Exos, combined with BBB permeability assessment to elucidate potential pathways for their entry into the brain. (3) Optimize delivery strategies and conduct extended efficacy and safety monitoring to determine optimal intervention windows and dosage regimens. (4) Characterize the role of key exosomal cargoes—such as microRNAs and proteins—in regulating interactions between microglia and astrocytes, thereby providing robust experimental evidence for exosomal therapeutic approaches targeting COPD-NCDs. Abbreviations COPD Chronic obstructive pulmonary disease COPD-NCDs COPD-related neurocognitive disorders hUCMSC-Exos Human umbilical cord mesenchymal stem cell-derived exosomes NC Normal control CS Cigarette smoke BBB Blood-brain barrier CNS Central nervous system MSCs Mesenchymal stem cells Exos Exosomes MSC-Exos MSC-derived exosomes SPF Specific Pathogen Free HUCMSCs Human Umbilical Cord Mesenchymal Stem Cells TEM Transmission Electron Microscope PMSF Phenylmethylsulphonyl fluoride SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis PVDF Polyvinylidene difluoride FEV0.1/FVC Forced expiratory volume in 0.1 s/forced vital capacity RI Airway resistance Cdyn Dynamic compliance TI Inspiratory time TE Expiratory time PIF Peak inspiratory flow PEF Peak expiratory flow Penh Enhanced pause EF50 Expiratory flow at 50% expired volume Rpef The ratio of time to peak expiratory flow to expiratory time MV Minute ventilation VT Tidal volume OFT Open Field Test NOR Novel Object Recognition MWM Morris Water Maze H&E Hematoxylin and eosin ELISA Enzyme-Linked Immunosorbent Assay IF Immunofluorescence SD Standard deviation DEGs Differentially expressed genes BP Biological Process CC Cellular Component MF Molecular Function Declarations Ethics approval All protocols involving animal experiments in this experiment were approved by the Ethics Committee for Animal Experiments of the College of Basic Medical Sciences of Jilin University (Approval No.2025–773), and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Medical Basic Research Innovation Center of Airway Disease in North China, Key Laboratory of Pathobiology, Key Laboratory of Precision Infectious Diseases, Jilin Province (20200601011JC), Engineering Laboratory for Precision Prevention and Treatment of Common Diseases, Jilin Province (2022C036), Department of Science and Technology of Jilin Province: Key Scientific and Technological Research and Development Projects (20230204055YY). Authors’ contributions Conceptualization, F.W. and X.M.; writing—original draft preparation, H.X.; computer graphics, H.X.; writing—review and editing, X.Y., S.B., Y.D., J.C.; supervision, Z.D., X.M.; project administration, Z.D., X.M.; funding acquisition, F.W. All authors reviewed the manuscript. Acknowledgements Thanks to the Key Laboratory of Pathobiology of the Ministry of Education at Jilin University and the Teaching and Research Room of Pathogenic Biology at Jilin University. Authors’ information Authors and Affiliations The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China; Hui Xiao Department of Histology & Embryology, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China；Xiao Yu, Jiayue Cui, Zhiyong Dong, Xiaoting Meng Department of Forensic Medicine, Basic Medical College, Jilin University, Changchun, 130021, China; Shilong Bao, Yiding Dong College of Basic Medical Sciences, Department of Pathogen Biology, the Medical Basic Research Innovation Center of Airway Disease in North China, Ministry of Education, Jilin University, Changchun, 130021, China, Fang Wang Corresponding author Correspondence to Xiaoting Meng and Fang Wang References Christenson SA, Smith BM, Bafadhel M, Putcha N. Chronic obstructive pulmonary disease. Lancet. 2022;399:2227–42. Dobric A, De Luca SN, Spencer SJ, Bozinovski S, Saling MM, McDonald CF, Vlahos R. 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Supplementary Files supplementaryfile.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 06 May, 2026 Editor assigned by journal 23 Apr, 2026 Submission checks completed at journal 23 Apr, 2026 First submitted to journal 19 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9465519","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637983282,"identity":"95eea915-c8af-420d-bfe2-75e0ecee5c03","order_by":0,"name":"Hui Xiao","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Xiao","suffix":""},{"id":637983286,"identity":"254d77d1-a84a-427c-b544-59582d9e6e9c","order_by":1,"name":"Xiao Yu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yu","suffix":""},{"id":637983288,"identity":"ca18865f-d350-42be-902d-2fd2762454ff","order_by":2,"name":"Yiding Dong","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yiding","middleName":"","lastName":"Dong","suffix":""},{"id":637983289,"identity":"ad328bdb-eaf8-4a36-8680-d5a1618ecd69","order_by":3,"name":"Shilong Bao","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Shilong","middleName":"","lastName":"Bao","suffix":""},{"id":637983292,"identity":"d7f5131b-3f4e-466c-8224-aeb6f6e09751","order_by":4,"name":"Jiayue Cui","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jiayue","middleName":"","lastName":"Cui","suffix":""},{"id":637983294,"identity":"32340f75-ee19-4226-bfb0-876776091443","order_by":5,"name":"Zhiyong Dong","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyong","middleName":"","lastName":"Dong","suffix":""},{"id":637983295,"identity":"b50f085d-4932-4321-a30d-024a51047899","order_by":6,"name":"Xiaoting Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYPACG2Y+EMVDvI6ENGY2UrUcZiBei2577+HXvD/Os7NJJDA+eNvGIG9OSIvZmXNpljMSbjMDtTAbzm1jMNzZQEjLjRwzgw8QLWzSvG0MCQYHCGm5/8bMICHhHEgL+2/itNzgMX7wIeEA2BZm4rScyTFjnJGWzMzG87BZcs45CcMNBLUcP2P8mcfGLpmfPfnghzdlNvIEbQECNgkgkczAwNgApCUIqwcC5g9Awo4opaNgFIyCUTAyAQBWmDkqrds9RAAAAABJRU5ErkJggg==","orcid":"","institution":"Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoting","middleName":"","lastName":"Meng","suffix":""},{"id":637983296,"identity":"be763844-0b2b-403b-bbf3-1ab5feda62b2","order_by":7,"name":"Fang Wang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-20 01:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9465519/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9465519/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109435128,"identity":"6490c5a4-875a-40ed-bd15-f3f01d95e0fa","added_by":"auto","created_at":"2026-05-18 05:56:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":350387,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of human umbilical cord mesenchymal stem cells (hUCMSCs) and hUCMSC-derived exosomes (hUCMSC-Exos). (A) Third-passage hUCMSCs exhibited adherent growth with a uniform spindle-shaped morphology arranged in a swirling (whorl-like) pattern (scale bar = 200 μm). (B) After adipogenic induction, hUCMSCs showed distinct lipid droplets by Oil Red O staining (scale bar = 100 μm). (C) After osteogenic induction, hUCMSCs showed prominent calcified nodules by Alizarin Red staining (scale bar = 100 μm). (D) Flow cytometric analysis of hUCMSC surface marker expression. The cells highly expressed CD73 (99.54%), CD90 (99.83%), and CD105 (99.94%), while the hematopoietic markers CD34 (1.22%) and CD45 (1.08%) were expressed at low levels. (E) Particle size distribution of hUCMSC-Exos analyzed by nanoparticle tracking analysis (NTA). The average particle size was 126.2 nm, with a peak at 115.3 nm, and a particle concentration of 2.3 × 10¹⁰ particles/mL. (F) Transmission electron microscopy image showed the morphology of hUCMSC-Exos. Red arrows indicate typical round or cup-shaped exosomes (scale bar = 200 nm). (G) Western blot analysis of hUCMSC-Exos marker proteins. hUCMSC-Exos expressed the exosomal markers CD9, HSP70, and TSG101, but were negative for the endoplasmic reticulum marker Calnexin.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/55c9ec1b7bf77adb6b6bc96f.png"},{"id":109435121,"identity":"b623fc45-a91e-49d7-b22d-2e609f6f4b9f","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72166,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos improved pulmonary ventilation function in mice with COPD-NCDs. (A) Schematic diagram of the experimental design. Mice were exposed to cigarette smoke (CS) for 24 weeks. hUCMSC-Exos or PBS were administered via tail-vein injection once weekly during weeks 20–23. Behavioral and pulmonary function tests were performed in week 24, following completion of all functional assessments, samples were collected. (B) Body weight changes in each group from week 1 to week 24 (n = 10). (C) Body weight of mice in each group at week 24 (n = 10). (D) Forced expiratory volume in 0.1 s/forced vital capacity (FEV0.1/FVC) (n = 5). (E) Airway resistance (RI) (n = 5). (F) Dynamic compliance (Cdyn) (n = 5). (G) Inspiratory time (TI) (n = 5). (H) Expiratory time (TE) (n = 5). (I) Peak inspiratory flow (PIF) (n = 5). (J) Peak expiratory flow (PEF) (n = 5). (K) Enhanced pause (Penh) (n = 5). (L) Expiratory flow at 50% tidal volume (EF50) (n = 5). (M) Ratio of time to peak expiratory flow to expiratory time (Rpef) (n = 5). (N) Minute ventilation (MV) (n = 5). (O) Tidal volume (VT) (n = 5). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/4e62929c5118cc8e15c2fde8.png"},{"id":109435122,"identity":"f2f1e296-7602-403e-8c82-e39066df9741","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307368,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos alleviated lung histopathological injury, pulmonary inflammation, and systemic inflammation in mice with COPD-NCDs. (A) Representative H\u0026amp;E-stained images of lung tissue from each group (scale bar = 50 μm, n = 5). (B) Representative Masson’s trichrome-stained images of lung tissue from each group (scale bar = 50 μm, n = 5). (C–E) ELISA analysis of TNF-α, IL-1β, and IL-6 levels in lung tissue homogenate supernatants (n = 5). (F–H) ELISA analysis of TNF-α, IL-1β, and IL-6 levels in serum. (n = 5). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/ff38c2aa6c2ea91c80ff9ab9.png"},{"id":109759644,"identity":"0acfbdf4-ba10-4c47-add7-9102d285ea0b","added_by":"auto","created_at":"2026-05-22 07:27:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88349,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos alleviated neurocognitive dysfunction in mice with COPD-NCDs. (A) Schematic diagram of the open field test (OFT). (B) Schematic diagram of the novel object recognition (NOR) test. (C) Representative trajectories in the OFT (n = 8). (D) Total distance travelled in the OFT (n = 8). (E) Distance travelled in the central area of the OFT (n = 8). (F) Time spent in the central area of the OFT (n = 8). (G) Discrimination index in the NOR test (n = 8). The discrimination index was calculated as (time spent with the novel object − time spent with the familiar object) / total exploration time. (H) Representative trajectories in the Morris water maze (MWM) test (n = 8). (I) Escape latency in the MWM training phase (days 1–5) (n = 8). (J) Number of platform crossings in the MWM test (n = 8). (K) Time spent in the target quadrant in the MWM test (n = 8). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/aa56a383f76594f0e68d3a14.png"},{"id":109435123,"identity":"061c3640-179b-451e-a814-993e0372b28d","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298629,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos alleviated hippocampal neuronal damage in mice with COPD-NCDs. (A) Representative HE-stained images of the hippocampal DG, CA3, and CA1 regions in each group (scale bar = 20 μm). (B) Representative Nissl-stained images of the hippocampal DG, CA3, and CA1 regions in each group (scale bar = 20 μm). (C) Representative NeuN immunofluorescence images of the hippocampal DG, CA3, and CA1 regions in each group (scale bar = 50 μm). (D–F) Quantitative analysis of the numbers of Nissl-positive neurons (surviving neurons) in the DG, CA3, and CA1 regions of the hippocampus (n = 4). (G–I) Quantitative analysis of mean NeuN fluorescence intensity in the DG, CA3, and CA1 regions of the hippocampus. (n = 3, Mean fluorescence intensity was measured using ImageJ software). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/04bd7922f782b6797f91e2c5.png"},{"id":109435129,"identity":"173873c7-31ac-4530-a6f8-f8a263419c46","added_by":"auto","created_at":"2026-05-18 05:56:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":234354,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos attenuated neuroinflammation by inhibiting microglial activation in mice with COPD-NCDs. (A) Representative IBA1 immunofluorescence images of the hippocampal DG, CA3, and CA1 regions in each group (scale bar = 50 μm). (B–D) Quantitative analysis of mean IBA1 fluorescence intensity in the DG, CA3, and CA1 regions of the hippocampus (n = 3, Mean fluorescence intensity was measured using ImageJ software). (E–I) ELISA analysis of TNF-α, IL-1α, C1q, IL-1β, and IL-6 levels in brain tissue homogenate supernatants (n = 5). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/1f0d7a3007e0cd74e079cb06.png"},{"id":109435125,"identity":"4ebc8895-dbde-44f1-96a4-e674ea5e4168","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":329690,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos inhibited A1 neurotoxic astrocyte activation. (A) Representative immunofluorescence images of GFAP/C3 double staining in the hippocampal DG, CA3, and CA1 regions of each group (scale bar = 50 μm). (B–D) Quantitative analysis of the percentage of C3\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e cells among GFAP\u003csup\u003e+\u003c/sup\u003e cells in the DG, CA3, and CA1 regions. (E) Western blot analysis of C3 protein expression in brain tissue from each group. (F) Quantitative analysis of relative gray values of C3 protein expression normalized to β-actin (n = 3). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/41703e0ae789a4b67fcaae12.png"},{"id":109435126,"identity":"38197ce2-4dfe-485a-9b25-dfb40a813da7","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":367236,"visible":true,"origin":"","legend":"\u003cp\u003ehUCMSC-Exos promoted A2 neuroprotective astrocyte activation. (A) Representative immunofluorescence images of GFAP/S100A10 double staining in the hippocampal DG, CA3, and CA1 regions of each group (scale bar = 50 μm). (B–D) Quantitative analysis of the percentage of S100A10\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e cells among GFAP\u003csup\u003e+\u003c/sup\u003e cells in the DG, CA3, and CA1 regions. (E) Western blot analysis of S100A10 protein expression in brain tissue from each group. (F) Quantitative analysis of relative gray values of S100A10 protein expression normalized to β-actin. (n = 3). Data are presented as mean ± SD. ns, not significant (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/3a0f9af1f86fc8d0aad202db.png"},{"id":109799564,"identity":"e27920a1-913b-49fb-89b7-37dd1410e0c6","added_by":"auto","created_at":"2026-05-22 15:31:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":113168,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular mechanisms underlying hUCMSC-Exos treatment in mice with COPD-NCDs revealed by transcriptome sequencing. (A) Venn diagram showing the distribution and overlap of differentially expressed genes (DEGs) among the NC, COPD-NCDs, and COPD-NCDs + Exos groups (n = 6). (B) Volcano plot of DEGs in the NC group and the COPD-NCDs group. DEGs were identified using |fold change| ≥ 1.5 and \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. (C) Volcano plot of DEGs between the COPD-NCDs group and the COPD-NCDs + Exos group. DEGs were identified using |fold change| ≥ 1.5 and \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. (D) GO classification of DEGs in the NC group and the COPD-NCDs group. (E) GO classification of DEGs between the COPD-NCDs group and the COPD-NCDs + Exos group. (F) KEGG pathway enrichment analysis of DEGs between the NC group and the COPD-NCDs group. (G) KEGG pathway enrichment analysis of DEGs between the COPD-NCDs group and the COPD-NCDs + Exos group. (n = 6).\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/a29159aa4960536e5e795916.png"},{"id":109906512,"identity":"66d01e8a-d96f-4d14-ab30-b38438ea7875","added_by":"auto","created_at":"2026-05-25 06:40:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3501573,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/2ed9b9e1-c3e1-4390-8884-b9954a199cb6.pdf"},{"id":109435120,"identity":"689a031b-d601-466e-a396-8d12705c330c","added_by":"auto","created_at":"2026-05-18 05:56:25","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22088212,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-9465519/v1/e721b00504566738136645c1.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Neurocognitive Disorders in Chronic Obstructive Pulmonary Disease by Suppressing Neuroinflammation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) is a common respiratory disorder characterized primarily by chronic airway inflammation and progressive airflow limitation, exhibiting marked heterogeneity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. COPD has become the third leading cause of death globally, resulting in approximately 3.23\u0026nbsp;million deaths annually and imposing a substantial economic and public health burden on healthcare systems worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Smoking is the primary risk factor, accounting for over 70% of cases in high-income countries and 30\u0026ndash;40% in low- and middle-income countries [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond impaired lung function, COPD frequently presents with multiple extrapulmonary complications, including cardiovascular diseases, anxiety, depression, osteoporosis, and metabolic disorders [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these, cognitive impairment has increasingly drawn attention. Research indicates that up to 61% of COPD patients exhibit cognitive impairment, primarily manifesting as memory decline, impaired executive function, and reduced attention [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, the degree of cognitive decline correlates with disease severity, with patients with severe COPD demonstrating poorer overall cognitive function compared to those with mild or moderate COPD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. COPD-related neurocognitive disorders (COPD-NCDs) not only diminish treatment adherence and pulmonary rehabilitation outcomes but also significantly impair quality of life and prognosis, increasing mortality and disability rates [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, effective interventions for COPD-NCDs remain scarce.\u003c/p\u003e \u003cp\u003eRecent research suggests that persistent pulmonary inflammation and oxidative stress induced by cigarette smoke (CS) exposure may promote the spillover of inflammatory mediators into the systemic circulation. This disrupts the integrity of the blood-brain barrier (BBB), activates microglia and astrocytes within the central nervous system (CNS), and triggers neuroinflammation and neuronal damage, thereby contributing to cognitive impairment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During this process, microglia undergo a transition from a homeostatic surveillance phenotype to an activated phenotype, characterized by cell body hypertrophy and shortened processes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous research has shown that activated microglia release TNF-α, IL-1α and C1q, which further induce astrocytes to adopt an A1-like neurotoxic phenotype and exacerbate neuronal damage through the release of molecules such as complement C3 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Consequently, inhibiting the neuroinflammatory response mediated by the abnormal activation of microglia and astrocytes may represent an important therapeutic strategy for managing COPD-NCDs.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs), owing to their potent immunomodulatory properties, have emerged as a promising candidate for anti-inflammatory therapy. MSCs possess self-renewal capacity, multipotent differentiation potential, and immunomodulatory properties, demonstrating therapeutic promise in various inflammatory and neurological disorders [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The biological effects of MSCs are primarily mediated by their paracrine actions, with exosomes (Exos) serving as key paracrine mediators [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Exos carry bioactive molecules, including proteins, lipids, and nucleic acids, and participate in intercellular communication [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMSC-derived exosomes (MSC-Exos) exhibit multiple biological functions, including immune modulation, angiogenesis induction, promotion of cell proliferation and migration, and acceleration of damaged neuron repair and regeneration [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, they attenuate allogeneic transplant rejection, do not exhibit tumorigenic potential, and have a low embolism risk, representing a promising cell-free therapeutic approach [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos), in particular, have demonstrated significant neuroprotective effects in various CNS disorders [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, their therapeutic efficacy and molecular mechanisms in COPD-NCDs remain understudied.\u003c/p\u003e \u003cp\u003eAgainst this background, this study aims to establish a COPD-NCDs mouse model to evaluate the therapeutic effects of hUCMSC-Exos on cognitive impairment. We hypothesize that hUCMSC-Exos exert neuroprotective effects by modulating microglial activation and astrocytic A1/A2 phenotype conversion, thereby alleviating neuroinflammation. This study may provide novel experimental evidence and a theoretical basis for the treatment of COPD-NCDs.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Laboratory Animals\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice (6 weeks old, weighing 18\u0026ndash;20 grams, Specific Pathogen Free (SPF) grade) were procured from Changchun Yisi Laboratory Animal Technology Co., Ltd. (Changchun, China; Licence No. SYXK(Ji)2023-0010). They were housed at the SPF-grade animal facility of the School of Basic Medical Sciences, Jilin University, maintained at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C with 50% \u0026plusmn; 10% humidity, and provided with ad libitum access to food and water. All animal experimentation protocols in this study were approved by the Animal Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.2025\u0026ndash;773).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) Culture\u003c/h2\u003e \u003cp\u003ehUCMSCs (Sciencell, #7530) were cultured in MSC basal medium (Dakewe, #6114011) supplemented with 10% fetal bovine serum (XP BioMed, #C04400-500) (MSC complete medium). The cells were cultured at 37\u0026deg;C, 5% CO₂ incubator, passaged when confluence reached 80\u0026ndash;90%, and cells from passages 3\u0026ndash;7 were selected for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Phenotypic Characterization of hUCMSCs\u003c/h2\u003e \u003cp\u003eCell phenotypes were validated by flow cytometry. The cells (1 \u0026times; 10⁶/mL in PBS) were incubated with anti-CD34, anti-CD45, anti-CD73, anti-CD90, and anti-CD105 antibodies for 30 minutes at 4\u0026deg;C in the dark (Details of the antibodies are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After washing, the cells were resuspended in 100 \u0026micro;L PBS and analyzed by flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies for flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eManufacturers/Cat.no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConjugate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Biosciences, #561254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Biosciences, #555595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Biosciences, #560839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Biosciences, #560942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Biosciences, #561865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Multilineage Differentiation of hUCMSCs\u003c/h2\u003e \u003cp\u003eTo evaluate the multipotent differentiation potential of hUCMSCs, 1 \u0026times; 10⁴ cells were seeded into poly-L-lysine-coated 6-well plates and cultured in MSC complete medium. When cell confluence reached 80%\u0026ndash;90%, the medium was replaced with either adipogenic differentiation induction medium (Dakewe, #6114531) or osteogenic differentiation induction medium (Dakewe, #6114541), and induction was continued for 18\u0026ndash;21 days, with medium changes carried out regularly according to the manufacturer\u0026rsquo;s instructions. Upon completion of induction, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O staining solution (Dakewe, #4060711) and Alizarin Red staining solution (Dakewe, #4060611), respectively, followed by observation and photography under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Isolation of hUCMSC-Exos\u003c/h2\u003e \u003cp\u003ehUCMSCs at passages 3 to 7 were seeded at a density of 5 \u0026times; 10⁵ cells per 10 cm culture dish and cultured in MSC complete medium. When cell confluence reached 60%\u0026ndash;70%, the original medium was discarded and the cells were washed 2\u0026ndash;3 times with PBS. Then, 10 mL of exosome-depleted medium, comprising MSC basal medium (Dakewe, #6114011) supplemented with 10% exosome-depleted fetal bovine serum (Umibio, #UR50202), was added. The cells were then cultured for a further 48 h to collect conditioned medium. After the culture supernatant was collected, differential centrifugation was performed at 4\u0026deg;C in the following order: 3,000 \u0026times; g for 20 min, 10,000 \u0026times; g for 45 min, filtration through a 0.22 \u0026micro;m filter membrane, and finally 120,000 \u0026times; g for 120 min. The supernatant was discarded, the pellet was resuspended in an appropriate volume of PBS, the protein concentration was determined using the BCA method, and the samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for future use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Transmission Electron Microscope (TEM)\u003c/h2\u003e \u003cp\u003eA total of 20\u0026ndash;30 \u0026micro;L of the hUCMSC-Exos suspension was dispensed onto a 200-mesh copper grid coated with formvar and allowed to adsorb at room temperature for 3\u0026ndash;5 minutes. After excess liquid was removed, the grid was counterstained with 2% phosphotungstic acid (pH 7.0) filtered through a 0.22 \u0026micro;m membrane for 3\u0026ndash;5 minutes, and the excess staining solution was then removed and the grid was allowed to dry. Once the sample was completely dry, the morphology of the hUCMSC-Exos was observed and photographed using a TEM at 80 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western Blot\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from the samples using RIPA lysis buffer containing phenylmethylsulphonyl fluoride (PMSF). The protein samples were mixed with 5\u0026times; protein loading buffer and denatured. Conventional proteins were heated at 100\u0026deg;C for 5 minutes, whereas membrane proteins were incubated at 37\u0026deg;C for 30 minutes. Twenty micrograms of total protein from each sample were loaded, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with a protein-free rapid blocking solution (Servicebio, #G2052-500ml) and then incubated with the primary antibodies overnight at 4\u0026deg;C. The primary antibodies used were anti-CD9 antibody (1:1000, Abcam, #ab263019), anti-HSP70 antibody (1:1000, Abcam, #ab181606), anti-TSG101 antibody (1:5000, Abcam, #ab125011), anti-Calnexin antibody (1:2500, Abcam, #ab133615), anti-C3 antibody (1:2500, Invitrogen, #PA521349), anti-S100A10 antibody (1:2500, Invitrogen, #PA595505), and anti-β-actin antibody (1:2500, Proteintech Group, #20536-1-AP). After washing the membrane, HRP-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody (1:5000, Proteintech Group, #RGAR001) was added and incubated at room temperature for 2 hours. Finally, the bands were visualized using enhanced ECL chemiluminescent substrate (Thermo Scientific, #34580), followed by image acquisition and analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Establishment of COPD-NCDs Model and hUCMSC-Exos Therapy\u003c/h2\u003e \u003cp\u003eMice were acclimatized for one week prior to the start of the experiment and were subsequently randomized into a normal control (NC) group and a CS-exposed group. Mice in the NC group were exposed to ambient indoor air;\u003c/p\u003e \u003cp\u003ethe CS-exposed group was used to establish a COPD-NCDs model using commercially available filtered CS (specifications: 10 mg tar/cigarette; 0.9 mg nicotine/cigarette; 11 mg carbon monoxide/cigarette). Mice were exposed for 5 days per week, at a rate of 9 cigarettes per day, for a total of 24 weeks. Body weight changes were recorded weekly throughout the experiment.\u003c/p\u003e \u003cp\u003eAfter 20 weeks of CS exposure, the model mice were further randomly divided into three groups: the COPD-NCDs group, the COPD-NCDs\u0026thinsp;+\u0026thinsp;PBS group, and the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group. Together with the NC group, four experimental groups were finally included: NC, COPD-NCDs, COPD-NCDs\u0026thinsp;+\u0026thinsp;PBS, and COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos (n\u0026thinsp;=\u0026thinsp;18 per group). The COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group received intravenous tail injections of hUCMSC-Exos from weeks 20\u0026ndash;23, once weekly at a dose of 100 \u0026micro;g per mouse, for 4 consecutive weeks (The dosing frequency in this study was optimised by drawing on previous studies of systematic administration of hUCMSC-Exos in neurological disorders, while taking into account the cycle of the model used in this study and the intervention window [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]). As determined by the BCA assay, the protein concentration of hUCMSC-Exos was 0.5 mg/mL, with a single-dose volume of 200 \u0026micro;L. The COPD-NCDs\u0026thinsp;+\u0026thinsp;PBS group received an equal volume of sterile PBS during the same period.\u003c/p\u003e \u003cp\u003eNeurocognitive and pulmonary function tests were conducted in week 24. Following completion of all functional assessments, samples were collected. Mice were anaesthetized via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg), and blood was collected via the retroorbital venous plexus. The mice were subsequently euthanized under deep anesthesia, and lung and brain tissues were rapidly isolated for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Invasive Pulmonary Function Testing\u003c/h2\u003e \u003cp\u003eAfter deep anesthesia with sodium pentobarbital, the mice were positioned supine and immobilized. Following routine disinfection of the neck skin, a longitudinal incision was made, the muscles were dissected to fully expose the trachea, and a small incision was made in the upper segment of the trachea to insert and secure a tracheal tube. The tube was then connected to the Buxco pulmonary function testing system and a small-animal ventilator. Following system calibration and once respiration had stabilized. The following parameters were measured: forced expiratory volume in 0.1 s/forced vital capacity (FEV0.1/FVC), airway resistance (RI) and dynamic compliance (Cdyn).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Non-invasive Pulmonary Function Testing\u003c/h2\u003e \u003cp\u003eNon-invasive pulmonary function testing was performed on awake, freely moving mice using a whole-body volumetric recording system. After the mice had acclimatized to the environment and the instrument had been calibrated, each mouse was placed individually in a sealed recording chamber for 5\u0026ndash;10 minutes. Data were collected once breathing had stabilized. The following parameters were analyzed: inspiratory time (TI), expiratory time (TE), peak inspiratory flow (PIF), peak expiratory flow (PEF), enhanced pause (Penh), expiratory flow at 50% expired volume (EF50), the ratio of time to peak expiratory flow to expiratory time (Rpef), minute ventilation (MV) and tidal volume (VT), among other respiratory parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Open Field Test (OFT)\u003c/h2\u003e \u003cp\u003eThe OFT apparatus consisted of an open-field chamber (60 cm \u0026times; 60 cm \u0026times; 60 cm) in the form of an opaque black cube, with the floor divided into a central area and a peripheral area. Mice were gently placed in the central area of the open field and allowed to explore freely for 10 minutes. Their behavior was recorded using a video tracking system. After each experiment, the mice\u0026rsquo;s urine and feces were promptly removed, and the apparatus was wiped with 75% ethanol to eliminate odor interference. The following parameters were recorded and analyzed: total distance travelled, distance travelled within the central zone, and time spent in the central zone. A reduction in time spent in the central zone indicated an increase in anxiety-like behavior.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Novel Object Recognition (NOR)\u003c/h2\u003e \u003cp\u003eThe NOR and OFT tests were conducted in the same apparatus. The experiment consisted of three phases: habituation, training and testing. (1) During the habituation phase, on Day 1, the mice were placed in an empty box and allowed to explore freely for 5 minutes. (2) During the training phase, on Day 2, two identical objects were placed diagonally opposite each other within the arena, and the mice were allowed to explore freely for 10 minutes. (3) Test phase: 24 hours after training, one of the familiar objects was replaced with a novel object, and the mice were again allowed to explore freely for 10 minutes. A video tracking system was used to record the time the mice spent exploring the familiar and novel objects. After each experiment, the mice\u0026rsquo;s urine and feces were removed, and the apparatus and object surfaces were wiped with 75% ethanol to eliminate any residual odors that might cause interference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Morris Water Maze (MWM)\u003c/h2\u003e \u003cp\u003eThe MWM was conducted in a quiet environment with stable lighting. The apparatus consisted of a circular tank with a diameter of 120 cm, a water depth of approximately 30 cm, and a water temperature maintained at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. A non-toxic white dye was added to the water to render it opaque, and a hidden platform was fixed at the center of the target quadrant, situated 1\u0026ndash;2 cm below the water surface. The tank was divided into four quadrants, with different visual markers on the tank walls serving as spatial reference cues. The experiment comprised five days of orientation navigation training followed by a spatial exploration test on the sixth day. During the training period, the mice were placed into the water from one of the four different quadrants each day, and their escape latency and swimming trajectories were recorded while they searched for the platform within 60 seconds. If they failed to locate the platform, they were guided to it and held there for 10 seconds. Twenty-four hours after the final training session, the platform was removed. The mice were placed in the water from the side opposite the target quadrant and allowed to swim freely for 60 seconds. The time spent in the target quadrant, the number of platform crossings, and the swimming trajectories were recorded to assess spatial learning and memory abilities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Histological Examination\u003c/h2\u003e \u003cp\u003eFollowing the collection of lung and brain tissue, the specimens were fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. After dewaxing and rehydration, the lung tissue sections were stained with hematoxylin and eosin (H\u0026amp;E) (Beyotime, #C0105S) and Masson\u0026rsquo;s stain (Solarbio, #G1340). Brain tissue sections were dewaxed and rehydrated, followed by staining with H\u0026amp;E (Beyotime, #C0105S) and Nissl staining (Solarbio, #G1430).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eTissue samples were homogenized in pre-chilled PBS at a mass-to-volume ratio of 1:10 (w/v). After centrifugation at 4\u0026deg;C and 3000 \u0026times; g for 5 minutes, the supernatant was collected, aliquoted, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later use. Mouse whole blood was centrifuged at 4\u0026deg;C and 2000 rpm for 20 minutes, and the serum was then collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later use.\u003c/p\u003e \u003cp\u003eThe concentrations of TNF-α, IL-1β, and IL-6 in lung tissue homogenate supernatants and serum, as well as TNF-α, IL-1α, C1q, IL-1β, and IL-6 in brain tissue homogenate supernatants, were determined according to the instructions provided with the ELISA kits (ColorfulGene Biotech). Prior to the experiment, all reagents were allowed to reach room temperature. Standards or samples were added to each well in duplicate, followed sequentially by the enzyme-conjugated antibody and color development substrate, and the plates were incubated at 37\u0026deg;C. After the reaction was terminated, the absorbance at 450 nm was measured using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Immunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eSections were permeabilized with 0.1% Triton X-100. Following blocking with 10% normal goat serum at room temperature for 1 hour, the sections were incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-NeuN antibody (1:200, Abcam, #ab279297), anti-IBA1 antibody (1:500, Synaptic Systems, #234009), anti-GFAP antibody (1:300, Sigma, #MAB3402), anti-C3 antibody (1:500, Invitrogen, #PA521349), and anti-S100A10 antibody (1:500, Invitrogen, #PA595505). The following day, after the sections were equilibrated at room temperature for 30 minutes, they were incubated with Alexa Fluor 488-labelled goat anti-rabbit IgG (1:500, Invitrogen, #A11008), Alexa Fluor 555-labelled goat anti-chicken IgG (1:500, Invitrogen), Alexa Fluor 546-labelled goat anti-rabbit IgG (1:500, Invitrogen, #A11010), and Alexa Fluor 488-labelled goat anti-rat IgG secondary antibodies (1:500, Invitrogen, #A11006) at room temperature for 2 hours. Cell nuclei were counterstained with Hoechst dye. Images were acquired using a fluorescence microscope (Olympus BX53, Japan) and processed using ImageJ (Version 1.8.0) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Mouse RNA Sequencing Analysis\u003c/h2\u003e \u003cp\u003eBrain tissue samples were collected from mice in the NC group, the COPD-NCDs group and the hUCMSC-Exos group, with six samples per group. Total RNA was extracted using the TRIzol method, and its concentration, purity and integrity were assessed. Samples with a RIN\u0026thinsp;\u0026ge;\u0026thinsp;7.0 were selected for library preparation. From each sample, 1 \u0026micro;g of total RNA was taken. Following mRNA enrichment using oligo(dT) magnetic beads, sequencing libraries were constructed and subjected to 150-base paired-end sequencing on the Illumina platform. After quality control, clean reads were obtained from the raw data. These were aligned to the mouse reference genome using Hisat2, and transcript assembly and expression quantification were performed using StringTie. Differential expression analysis was performed using DESeq2, with differentially expressed genes identified based on |log₂Fold Change| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Subsequently, GO and KEGG enrichment analyses were conducted using ClusterProfiler. Bioinformatics analysis was performed using BMKCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.biocloud.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Statistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8.0 was used for statistical analysis and figure generation. Mean fluorescence intensity was measured using ImageJ (Version 1.8.0) software. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was assessed using Levene\u0026rsquo;s test. Comparisons between two groups were performed using the unpaired two-tailed Student\u0026rsquo;s t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. In all statistical analyses, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas ns indicated no statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of hUCMSCs and hUCMSC-Exos\u003c/h2\u003e \u003cp\u003eThe cultured hUCMSCs exhibited typical adherent growth, with uniform cell morphology characterized by a spindle-shaped appearance and a swirling arrangement under phase-contrast microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following adipogenic and osteogenic induction, distinct lipid droplet and calcium nodule formation were observed, respectively, suggesting multipotent differentiation potential (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C). Flow cytometry results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) showed that the cells highly expressed MSC-specific markers CD73 (99.54%), CD90 (99.83%) and CD105 (99.94%), while expression levels of the hematopoietic markers CD34 (1.22%) and CD45 (1.08%) were extremely low. NTA results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) showed that hUCMSC-Exos had an average particle size of 126.2 nm, a peak particle size of 115.3 nm, and a particle concentration of 2.3 \u0026times; 10\u0026sup1;⁰ particles/mL. TEM revealed a typical round or cup-shaped vesicular structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Western blot analysis confirmed the expression of CD9, HSP70 and TSG101, while Calnexin was undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), indicating successful isolation of hUCMSC-Exos.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 hUCMSC-Exos Improved Pulmonary Ventilation Function in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eFollowing 20 weeks of exposure to CS, mice received tail-vein injections of hUCMSC-Exos for four consecutive weeks between weeks 20 and 23. The experimental protocol is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Longitudinal body weight monitoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) showed that body weight gain in the COPD-NCDs group was significantly slowed compared with the NC group. Following treatment with hUCMSC-Exos, the trend in body weight gain improved markedly, while the PBS group exhibited a trend largely consistent with that of the COPD-NCDs group. Analysis of final body weight further indicated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) that at week 24, the body weight of mice in the COPD-NCDs group was significantly lower than that of the NC group (COPD-NCDs group: 20.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36 vs. NC group: 26.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas following treatment with hUCMSC-Exos, body weight was significantly higher than that of the COPD-NCDs group (23.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eMouse pulmonary ventilation function was further assessed via invasive and non-invasive pulmonary function testing. Results of invasive pulmonary function testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited reduced FEV0.1/FVC (COPD-NCDs group: 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs. NC group: 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), increased RI (COPD-NCDs group: 7.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 vs. NC group: 4.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and reduced Cdyn (COPD-NCDs group: 0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 vs. NC group: 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Following treatment with hUCMSC-Exos, the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group showed increased FEV0.1/FVC (0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), decreased RI (5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and increased Cdyn (0.008\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating improved lung function. In contrast, no statistically significant differences were observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eResults from non-invasive pulmonary function testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;O) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited prolonged TI (COPD-NCDs group: 0.069\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 vs. NC group: 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and TE (COPD-NCDs group: 0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 vs. NC group: 0.075\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as well as increased Penh (COPD-NCDs group: 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs. NC group: 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, PIF (COPD-NCDs group: 5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 vs. NC group: 7.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), PEF (COPD-NCDs group: 4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 vs. NC group: 6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), EF50 (COPD-NCDs group: 2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 vs. NC group: 3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Rpef (COPD-NCDs group: 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 vs. NC group: 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), MV (COPD-NCDs group: 40.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3 vs. NC group: 55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and VT (COPD-NCDs group: 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 vs. NC group: 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were all significantly reduced, indicating impaired pulmonary ventilation in the COPD-NCDs group. Following treatment with hUCMSC-Exos, TI (0.055\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), TE (0.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and Penh (0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly lower than in the COPD-NCDs group, while PIF (6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PEF (5.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), EF50 (3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Rpef (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), MV (49.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and VT (0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly increased. These results indicated that hUCMSC-Exos improved pulmonary ventilation function in COPD-NCDs mice. No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 hUCMSC-Exos Alleviated Pulmonary Histopathological Injury, Pulmonary Inflammation, and Systemic Inflammation in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of hUCMSC-Exos on pathological damage in the lung tissue of COPD-NCDs mice, H\u0026amp;E staining was used to observe changes in lung tissue structure, while Masson\u0026rsquo;s staining was employed to assess collagen fiber deposition. The HE staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) showed that the lung tissue structure in the NC group was intact, with regular alveolar arrangement and no obvious inflammatory cell infiltration. In contrast, the COPD-NCDs group exhibited disorganized alveolar structures, thinning or rupture of alveolar septa, enlargement and fusion of alveolar spaces, and marked inflammatory cell infiltration. Following treatment with hUCMSC-Exos, inflammatory cell infiltration was reduced, and alveolar dilatation and tissue damage were significantly improved, whereas no significant differences were observed between the PBS group and the COPD-NCDs group.\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed that the COPD-NCDs group exhibited a significant increase in collagen fiber deposition in the airway walls, indicating aggravated fibrosis. Following treatment with hUCMSC-Exos, the area of collagen deposition decreased, and the thickening of the airway walls and the degree of fibrosis improved markedly. In contrast, no significant improvement was observed in the PBS group.\u003c/p\u003e \u003cp\u003eELISA was further employed to detect the levels of TNF-α, IL-1β and IL-6 in the lung tissue and serum of mice in each group. The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;E) that, compared with the NC group, the levels of the inflammatory factors TNF-α (COPD-NCDs group: 290.97\u0026thinsp;\u0026plusmn;\u0026thinsp;26.33 vs. NC group: 233.69\u0026thinsp;\u0026plusmn;\u0026thinsp;20.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), IL-1β (COPD-NCDs group: 55.62\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27 vs. NC group: 42.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and IL-6 (COPD-NCDs group: 69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11 vs. NC group: 57.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were significantly elevated in the lung tissue of the COPD-NCDs group, suggesting an enhanced inflammatory response. Treatment with hUCMSC-Exos significantly reduced the levels of TNF-α (248.13\u0026thinsp;\u0026plusmn;\u0026thinsp;19.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), IL-1β (44.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.44, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and IL-6 (60.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), thereby alleviating pulmonary inflammation. No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the NC group, the serum levels of TNF-α (COPD-NCDs group: 370.64\u0026thinsp;\u0026plusmn;\u0026thinsp;17.67 vs. NC group: 307.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), IL-1β (COPD-NCDs group: 63.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02 vs. NC group: 51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and IL-6 (COPD-NCDs group: 86.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15 vs. NC group: 63.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the COPD-NCDs group were significantly elevated, indicating an enhanced systemic inflammatory response. There was no statistically significant difference between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Following treatment with hUCMSC-Exos, the levels of TNF-α (325.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.06, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), IL-1β (55.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and IL-6 (72.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were significantly reduced compared with the COPD-NCDs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;H), indicating that hUCMSC-Exos ameliorated the systemic inflammatory response in COPD-NCDs mice.\u003c/p\u003e \u003cp\u003eGiven the observed improvement in pulmonary inflammation and systemic inflammation, we next examined whether hUCMSC-Exos could ameliorate cognitive dysfunction in COPD-NCDs mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4 hUCMSC-Exos Alleviated Neurocognitive Impairment in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo evaluate the neuroprotective effects of hUCMSC-Exos on neurocognitive function in mice with COPD-NCDs, behavioral tests including the OFT, NOR, and MWM were conducted after completion of treatment. Schematic diagrams of the experimental apparatus are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B.\u003c/p\u003e \u003cp\u003eOFT results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;F) showed that, compared with the NC group, mice in the COPD-NCDs group exhibited a significantly reduced total distance travelled (COPD-NCDs group: 604.69\u0026thinsp;\u0026plusmn;\u0026thinsp;70.08 vs. NC group: 2093.4\u0026thinsp;\u0026plusmn;\u0026thinsp;462.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and a greater tendency to remain in the peripheral area, suggesting decreased locomotor activity and increased anxiety-like behavior. Both the distance travelled in the central area (COPD-NCDs group: 130.12\u0026thinsp;\u0026plusmn;\u0026thinsp;42.25 vs. NC group: 321.92\u0026thinsp;\u0026plusmn;\u0026thinsp;90.79, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and the time spent there (COPD-NCDs group: 15.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58 vs. NC group: 52.81\u0026thinsp;\u0026plusmn;\u0026thinsp;6.82, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) were markedly reduced, suggesting decreased spontaneous activity and anxiety-like behavioral changes. Following hUCMSC-Exos treatment, the total distance travelled increased significantly (1568.31\u0026thinsp;\u0026plusmn;\u0026thinsp;242.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and both the distance travelled in the central area (238.17\u0026thinsp;\u0026plusmn;\u0026thinsp;55.6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the time spent there (43.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.87, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increased significantly, whereas the tendency to remain in the peripheral area was reduced. No statistically significant differences were observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eNOR results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) showed that the discrimination index in the COPD-NCDs group was significantly lower than that in the NC group (COPD-NCDs group: 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs. NC group: 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating impaired recognition memory. In the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, the discrimination index increased significantly (0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas no significant improvement was observed in the PBS group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe MWM was further used to evaluate spatial learning and memory. During the training phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), the escape latency on day 5 was significantly prolonged in the COPD-NCDs group compared with the NC group, indicating impaired spatial learning ability. hUCMSC-Exos treatment significantly shortened the escape latency. In the probe trial, trajectory plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) showed that mice in the NC group mainly swam within the target quadrant, whereas mice in the COPD-NCDs group showed reduced swimming activity in the target quadrant. Statistical analysis further showed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u0026ndash;K) that the time spent in the target quadrant (COPD-NCDs group: 25.35\u0026thinsp;\u0026plusmn;\u0026thinsp;8.76 vs. NC group: 58.27\u0026thinsp;\u0026plusmn;\u0026thinsp;9.69, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and the number of platform crossings (COPD-NCDs group: 1\u0026thinsp;\u0026plusmn;\u0026thinsp;1 vs. NC group: 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) were both significantly reduced in the COPD-NCDs group compared with the NC group. In contrast, both indicators were significantly increased in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas no statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHaving established that hUCMSC-Exos improved cognitive function, we next investigated whether these effects were associated with the alleviation of neuroinflammation and neuronal damage in the hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5 hUCMSC-Exos Alleviated Hippocampal Neuronal Damage in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eThe hippocampus is a critical brain region involved in learning, memory, and emotional regulation. To determine whether cognitive impairment in mice with COPD-NCDs was accompanied by hippocampal structural damage and neuronal loss, H\u0026amp;E staining, Nissl staining, and NeuN IF were performed to evaluate hippocampal histopathology and neuronal survival.\u003c/p\u003e \u003cp\u003eH\u0026amp;E staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) showed that neurons in the NC group exhibited intact morphology and were arranged in a dense and orderly manner. In contrast, the COPD-NCDs group showed a reduced number of neurons, disordered arrangement, and obvious pathological changes, including nuclear shrinkage, hyperchromatic nuclei, blurred nuclear membranes, and cytoplasmic vacuolization.\u003c/p\u003e \u003cp\u003eNissl staining results (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;F) showed that the hippocampus of the NC group was rich in Nissl bodies, with clear boundaries, uniform staining, and regular arrangement. Compared with the NC group, the numbers of Nissl-positive neurons in the DG, CA3, and CA1 regions were significantly reduced in the COPD-NCDs group (DG: COPD-NCDs group: 38\u0026thinsp;\u0026plusmn;\u0026thinsp;4 vs. NC group: 81\u0026thinsp;\u0026plusmn;\u0026thinsp;7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; CA3: COPD-NCDs group: 12\u0026thinsp;\u0026plusmn;\u0026thinsp;5 vs. NC group: 52\u0026thinsp;\u0026plusmn;\u0026thinsp;7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; CA1: COPD-NCDs group: 35\u0026thinsp;\u0026plusmn;\u0026thinsp;7 vs. NC group: 83\u0026thinsp;\u0026plusmn;\u0026thinsp;6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), accompanied by sparse distribution, partial dissolution of Nissl bodies, and abnormal neuronal morphology, indicating marked neuronal damage. No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, treatment with hUCMSC-Exos significantly increased the numbers of Nissl-positive neurons in all hippocampal regions (DG: 61\u0026thinsp;\u0026plusmn;\u0026thinsp;7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, CA3: 26\u0026thinsp;\u0026plusmn;\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA1: 59\u0026thinsp;\u0026plusmn;\u0026thinsp;8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and their arrangement tended to return to normal, suggesting that hUCMSC-Exos alleviated hippocampal neuronal damage.\u003c/p\u003e \u003cp\u003eNeuN IF was further used to assess neuronal survival. The results (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;I) showed that, compared with the NC group, the fluorescence intensity of NeuN-positive neurons in the DG, CA3, and CA1 regions was significantly decreased in the COPD-NCDs group (DG: COPD-NCDs group: 19.84\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27 vs. NC group: 49.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; CA3: COPD-NCDs group: 18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 vs. NC group: 42.05\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CA1: COPD-NCDs group: 21.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 vs. NC group: 47\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating impaired neuronal survival in the hippocampus. No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, treatment with hUCMSC-Exos significantly increased NeuN fluorescence intensity in all hippocampal regions (DG: 33.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, CA3: 30.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA1: 36.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eCollectively, these results indicated that mice with COPD-NCDs exhibited significant hippocampal neuronal damage and loss, whereas hUCMSC-Exos markedly improved hippocampal histopathological changes and neuronal survival, thereby exerting a protective effect on hippocampal neurons.\u003c/p\u003e \u003cp\u003eHaving established that hUCMSC-Exos protected hippocampal neurons, we next investigated whether these effects were mediated by the modulation of neuroinflammation, including microglial activation and astrocytic A1/A2 phenotype conversion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6 hUCMSC-Exos Alleviated Neuroinflammation by Suppressing Microglial Activation in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo investigate the role of microglia-mediated neuroinflammation in COPD-NCDs, IBA1 IF staining combined with morphological observation was used to evaluate the activation status of microglia in the hippocampus. The results showed (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D) that microglia in the NC group displayed a typical resting-state morphology, characterized by small cell bodies, slender processes, and abundant branching. In contrast, an increased number of hypertrophic microglia was observed in the hippocampus of the COPD-NCDs group, characterized by markedly enlarged cell bodies, shortened and reduced processes. The mean IBA1 fluorescence intensity was significantly increased in the DG (COPD-NCDs group: 84.16\u0026thinsp;\u0026plusmn;\u0026thinsp;5.79 vs. NC group: 51.54\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), CA3 (COPD-NCDs group: 86.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.87 vs. NC group: 45.04\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and CA1 (COPD-NCDs group: 87.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 vs. NC group: 42.28\u0026thinsp;\u0026plusmn;\u0026thinsp;6.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) regions, indicating microglial activation in the hippocampus. The morphology of microglia in the PBS group was similar to that in the COPD-NCDs group.\u003c/p\u003e \u003cp\u003eHowever, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, microglial morphology in all hippocampal regions was markedly improved, as evidenced by reduced cell body size, more numerous and elongated processes, and decreased mean IBA1 fluorescence intensity (DG: 62.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.16, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA3: 55.75\u0026thinsp;\u0026plusmn;\u0026thinsp;5.26, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, CA1: 64.28\u0026thinsp;\u0026plusmn;\u0026thinsp;8.85, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The overall morphology was similar to that in the NC group, suggesting that hUCMSC-Exos suppressed microglial activation in the hippocampus of mice with COPD-NCDs.\u003c/p\u003e \u003cp\u003eTo further evaluate the regulatory effect of hUCMSC-Exos on neuroinflammation in brain tissue, ELISA was performed to detect the levels of inflammatory and complement-related factors, including TNF-α, IL-1α, C1q, IL-1β, and IL-6, in the brain tissue of mice from each group. The results showed (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;I) that, compared with the NC group, the levels of TNF-α (COPD-NCDs group: 282.54\u0026thinsp;\u0026plusmn;\u0026thinsp;20.38 vs. NC group: 230.91\u0026thinsp;\u0026plusmn;\u0026thinsp;31.89, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), IL-1α (COPD-NCDs group: 51.03\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7 vs. NC group: 30.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), C1q (COPD-NCDs group: 5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 vs. NC group: 3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), IL-1β (COPD-NCDs group: 48.77\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64 vs. NC group: 35.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and IL-6 (COPD-NCDs group: 145.88\u0026thinsp;\u0026plusmn;\u0026thinsp;11.67 vs. NC group: 110.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.61, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) were significantly elevated in the COPD-NCDs group. No statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, the levels of TNF-α (236.92\u0026thinsp;\u0026plusmn;\u0026thinsp;22.76, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), IL-1α (40.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), C1q (4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), IL-1β (41.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and IL-6 (120.25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in brain tissue were significantly reduced compared with the COPD-NCDs group, indicating that hUCMSC-Exos alleviated neuroinflammatory responses in mice with COPD-NCDs. Collectively, these results indicated that hUCMSC-Exos effectively inhibited excessive microglial activation and reduced the levels of inflammatory and complement-related factors in brain tissue, thereby attenuating neuroinflammation.\u003c/p\u003e \u003cp\u003eActivated microglia can release IL-1α, TNF-α, and C1q, which are known to induce neurotoxic A1 astrocytes. We therefore next examined whether hUCMSC-Exos modulated astrocytic A1/A2 phenotype in the hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7 hUCMSC-Exos Inhibited A1 Neurotoxic Astrocyte Activation in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo further investigate the regulatory effects of hUCMSC-Exos on astrocyte reactivity and the A1 neurotoxic phenotype, IF staining was performed to detect the co-expression of the astrocyte marker GFAP and the A1 reactive astrocyte-associated marker C3. Western blot analysis was used to assess C3 protein expression.\u003c/p\u003e \u003cp\u003eIF results (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;D) showed that, compared with the NC group, the number of GFAP/C3 double-positive cells in the DG, CA3, and CA1 regions of the hippocampus was significantly increased in the COPD-NCDs group (DG: COPD-NCDs group: 32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 vs. NC group: 17.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CA3: COPD-NCDs group: 31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 vs. NC group: 19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CA1: COPD-NCDs group: 40\u0026thinsp;\u0026plusmn;\u0026thinsp;4 vs. NC group: 19\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating enhanced A1 astrocyte activation. The PBS group showed similar IF findings to the COPD-NCDs group, with no statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, the number of GFAP/C3 double-positive cells in all hippocampal regions was significantly reduced (DG: 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA3: 22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, CA1: 26.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that A1 astrocyte activation was suppressed. Western blot results further confirmed (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;F) that, compared with the NC group, C3 protein expression was significantly elevated in the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, C3 protein expression was significantly reduced in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group compared with the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no statistically significant difference was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.8 hUCMSC-Exos Promoted A2 Neuroprotective Astrocyte Activation in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo further evaluate the regulatory effect of hUCMSC-Exos on the protective A2 astrocyte phenotype, IF staining was performed to detect the co-expression of the astrocyte marker GFAP and the A2 astrocyte-associated marker S100A10. Western blot analysis was used to assess S100A10 protein expression.\u003c/p\u003e \u003cp\u003eIF results (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;D) showed that, compared with the NC group, the number of GFAP/S100A10 double-positive cells in the DG, CA3, and CA1 regions of the hippocampus was significantly reduced in the COPD-NCDs group (DG: COPD-NCDs group: 14.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52 vs. NC group: 32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.86, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CA3: COPD-NCDs group: 15\u0026thinsp;\u0026plusmn;\u0026thinsp;3 vs. NC group: 27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CA1: COPD-NCDs group: 12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61 vs. NC group: 31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting reduced A2 astrocyte activation. The PBS group showed IF findings similar to those of the COPD-NCDs group, with no statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group, the number of GFAP/S100A10 double-positive cells in all hippocampal regions increased significantly (DG: 25.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA3: 22.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CA1: 22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating enhanced A2 astrocyte activation.\u003c/p\u003e \u003cp\u003eWestern blot results further confirmed (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;F) that, compared with the NC group, S100A10 protein expression was significantly reduced in the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, S100A10 protein expression was significantly increased in the the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group compared with the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No statistically significant difference in S100A10 protein expression was observed between the PBS group and the COPD-NCDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Transcriptome Analysis Revealed the Molecular Mechanisms of hUCMSC-Exos Treatment in Mice with COPD-NCDs\u003c/h2\u003e \u003cp\u003eTo investigate the potential molecular mechanisms underlying the therapeutic effects of hUCMSC-Exos in mice with COPD-NCDs, RNA-seq analysis was performed on brain tissue from the NC, COPD-NCDs, and COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos groups. Differential expression analysis was conducted using DESeq2, with fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the screening criteria.\u003c/p\u003e \u003cp\u003eVenn diagram analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) showed the distribution and overlap of differentially expressed genes (DEGs) among the groups. Compared with the NC group, a total of 494 DEGs were identified in the COPD-NCDs group, including 207 up-regulated and 287 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), indicating marked alterations in the gene expression profile of brain tissue in the COPD-NCDs group. Further comparison between the COPD-NCDs group and the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group identified 671 DEGs, of which 450 were up-regulated and 221 were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). These results suggested that COPD-NCDs markedly altered the transcriptional profile of brain tissue, whereas hUCMSC-Exos intervention further reshaped the expression patterns of relevant genes.\u003c/p\u003e \u003cp\u003eGO enrichment analysis of the DEGs further showed that, in the comparison between the NC and COPD-NCDs groups, DEGs were primarily enriched in Biological Process (BP) categories such as cellular processes, biological regulation, response to stimulus, signaling, and metabolic processes. In Cellular Component (CC) categories, they were mainly enriched in intracellular components and protein complexes. In Molecular Function (MF) categories, they were primarily associated with binding and catalytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). In the comparison between the COPD-NCDs and COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos groups, DEGs were similarly enriched in BP categories including cellular processes, biological regulation, response to stimulus, metabolic processes, signaling, and immune system processes. CC categories were again dominated by intracellular components and protein complexes. MF categories mainly involved binding, catalytic activity, transport, and transcriptional regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eBased on the functional changes suggested by GO analysis, KEGG pathway enrichment analysis was further performed to identify the key signaling pathways involved. The results showed that, compared with the NC group, DEGs in the COPD-NCDs group were mainly enriched in the Wnt, cAMP, PI3K-Akt, TNF, and MAPK signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Compared with the COPD-NCDs group, DEGs in the COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos group were mainly enriched in pathways including MAPK, PI3K-Akt, cAMP, and Wnt (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). These results suggested that the Wnt, cAMP, PI3K-Akt, and MAPK pathways might be involved in the regulatory effects of hUCMSC-Exos on COPD-NCDs-related pathological processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNeurocognitive dysfunction is one of the common extrapulmonary complications of COPD, significantly affecting patients\u0026rsquo; quality of life and disease prognosis. Currently, there is a lack of effective treatment strategies for COPD-NCDs. Previous studies have shown that exposure to CS can induce pulmonary inflammation and exacerbate oxidative stress, facilitating the entry of inflammatory mediators from the lungs into the systemic circulation. This further compromises the integrity of the BBB, activates microglia and astrocytes, and consequently leads to neuroinflammation, neuronal damage and cognitive decline [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, targeting inflammation- and oxidative stress-related pathways may represent a promising therapeutic strategy for COPD-NCDs. Existing research indicates that certain anti-inflammatory or antioxidant drugs hold potential value in this field. For example, ebselen, an organoselenium compound with glutathione peroxidase-like activity, can alleviate CS-induced cognitive impairment to some extent by reducing pulmonary inflammation and maintaining synaptophysin expression in the hippocampus [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, its poor water solubility may limit bioavailability in vivo and increase the risk of potential toxicity, thereby affecting further clinical application [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Given the limitations of existing pharmacological treatments, the development of novel intervention strategies that combine biosafety with neuroprotective potential is of great significance.\u003c/p\u003e \u003cp\u003eMSC-Exos can partially mimic the therapeutic effects of stem cells while avoiding the safety and technical constraints associated with cell transplantation. In comparison, hUCMSC-Exos are considered to have greater clinical translational potential due to their stable source, lower ethical burden and superior biosafety. Previous studies have demonstrated that hUCMSC-Exos can carry various cytokines, growth factors and functional RNA molecules, and are capable of crossing the BBB to exert neuroprotective and immunomodulatory effects in a range of CNS disorders. Furthermore, their low immunogenicity and absence of oncogenic risk further enhance their application prospects. However, there is currently a lack of direct evidence regarding the role of hUCMSC-Exos in COPD-NCDs.\u003c/p\u003e \u003cp\u003eTherefore, this study employed hUCMSC-Exos to treat COPD-NCDs mice and systematically evaluated the therapeutic effects. A COPD-NCDs mouse model was successfully established using a 24-week CS exposure protocol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Subsequent experiments, including pulmonary histopathological examination, pulmonary function testing and ELISA assays, revealed marked inflammatory cell infiltration, emphysema-like changes and increased collagen fiber deposition in the lung tissue of COPD-NCDs mice. These findings were accompanied by reduced pulmonary ventilation function and elevated levels of inflammatory cytokines in both lung tissue and serum, suggesting enhanced pulmonary and systemic inflammatory responses. These findings were consistent with previous reports and further confirm the successful establishment of the COPD model [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In contrast, following intervention with hUCMSC-Exos, the inflammatory response in the mice\u0026rsquo;s lung tissue was alleviated, lung function improved, and levels of inflammatory cytokines in both lung tissue and serum decreased. These results suggested that hUCMSC-Exos alleviated COPD-related lung injury and systemic inflammatory responses.\u003c/p\u003e \u003cp\u003eBuilding on the improvements in peripheral inflammation and lung injury, we further evaluated the effects of hUCMSC-Exos on CNS function. Behavioral results showed that mice with COPD-NCDs exhibited a significant decline in spatial learning and memory abilities, alongside increased anxiety-like behavior, suggesting that the COPD-NCDs model had been successfully established. However, these abnormalities were alleviated following treatment with hUCMSC-Exos. Further histological analysis indicated that hUCMSC-Exos intervention alleviated neuronal damage, as evidenced by reduced neuronal nuclear atrophy, increased Nissl bodies, and elevated NeuN expression. These findings further confirmed the neuroprotective effects of hUCMSC-Exos in the COPD-NCDs model. Moreover, the neuroprotective effects of hUCMSC-Exos may be closely related to their regulation of the neuroinflammatory microenvironment.\u003c/p\u003e \u003cp\u003eMicroglia and astrocytes are key regulatory cells in the inflammatory response of the CNS. Under steady-state conditions, microglia adopt a ramified morphology and continuously monitor the local microenvironment. However, upon inflammatory or injury stimuli, they transition to an activated state, releasing various inflammatory factors that participate in neuroinflammatory processes [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition to providing structural support to neurons, astrocytes are involved in maintaining synaptic homeostasis and regulating the function of the BBB [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Previous studies have shown that upon release of TNF-α, IL-1α and C1q by activated microglia, astrocytes can be induced to adopt an A1 neurotoxic phenotype. This phenotype is neurotoxic and can further exacerbate damage to neurons and oligodendrocytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, C1q is the initiating molecule of the classical complement pathway and plays an important role in the CNS not only in innate immune defense but also in synaptic pruning and neuroinflammatory regulation [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, numerous studies have confirmed the protective role of hUCMSC-Exos in suppressing neuroinflammation. For example, hUCMSC-Exos could inhibit microglial-mediated inflammatory responses, reduce infarct volume and improve neurological function in models of ischemic stroke [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, hUCMSC-Exos not only suppressed excessive microglial activation but also promoted neurological recovery in TBI rat models by regulating astrocyte activation states [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, hUCMSC-Exos partially inhibited the formation of A1 neurotoxic astrocytes, thereby mitigating the damage they inflicted on neurons and neural tissue [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In vitro and in vivo studies have also demonstrated that hUCMSC-Exos alleviated inflammatory responses associated with intracerebral hemorrhage, improved cerebral oedema and BBB function, and ultimately promoted neurological recovery [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding on the aforementioned research, this study further evaluated the role of hUCMSC-Exos in regulating neuroinflammation in COPD-NCDs. Consistent with previous findings, this study found that hUCMSC-Exos significantly improved the neuroinflammatory response in mice with COPD-NCDs. Specifically, this was manifested by reduced microglial cell body hypertrophy and an increase in the number of dendritic processes, suggesting a decrease in their activation levels. Simultaneously, levels of inflammatory factors such as TNF-α, IL-1α, C1q, IL-1β and IL-6 in brain tissue were significantly reduced. Further analysis revealed that hUCMSC-Exos suppressed A1 neurotoxic astrocyte phenotype and promoted the increase in A2 neuroprotective astrocyte phenotype. These findings suggest that hUCMSC-Exos may alleviate neuroinflammatory responses by inhibiting excessive microglial activation and regulating astrocyte phenotypic conversion, thereby improving neuronal damage and cognitive dysfunction.\u003c/p\u003e \u003cp\u003eTo further elucidate the neuroprotective effects described above, this study analyzed their potential molecular mechanisms in conjunction with transcriptomic results. At the mechanistic level, the Wnt signaling pathway plays a crucial role in regulating immune responses and providing neuroprotection within the CNS. It enhances glial cells' anti-inflammatory properties, reduces the production of inflammatory mediators, promotes neuronal survival, and participates in post-injury repair and regeneration by regulating the proliferation and differentiation of neural progenitor cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. cAMP, as a vital intracellular regulator maintaining microglial homeostasis, can induce microglial activation through negative modulation of inflammatory signaling pathways [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The PI3K/AKT signaling pathway is also closely associated with neuroinflammation regulation and neural function maintenance. Research indicates that TREM2 activation may mitigate neuroinflammation by modulating PI3K/AKT signaling, thereby improving learning and memory-related impairments[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Furthermore, p38 MAPK, a stress-activated kinase sensitive to oxidative stress and inflammatory responses, exhibits sustained activation closely linked to neuronal injury, apoptosis, and cognitive decline[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Related studies also suggest that inhibiting p38 MAPK may help alleviate cognitive impairment associated with environmental tobacco smoke [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, transcriptome sequencing revealed alterations in multiple signaling networks related to neuroinflammation following hUCMSC-Exos intervention, suggesting that its mechanism of action in suppressing neuroinflammation in COPD-NCDs mice may involve regulating key signaling pathways such as Wnt, cAMP, PI3K-Akt, and MAPK.\u003c/p\u003e \u003cp\u003eIn summary, this study successfully established a stable COPD-NCDs mouse model and employed hUCMSC-Exos via tail vein administration for intervention. Results indicated that hUCMSC-Exos attenuated pulmonary and systemic inflammatory responses, ameliorated neuroinflammation and neuronal injury, and alleviated cognitive dysfunction at the behavioral level. Transcriptomic analysis further suggested that the neuroprotective effects of hUCMSC-Exos might involve the coordinated regulation of multiple inflammation-related signaling pathways, providing experimental evidence for its potential application in COPD-NCDs and future mechanistic studies.\u003c/p\u003e \u003cp\u003eHowever, this study remains subject to certain limitations. (1) The proposed signaling pathways were inferred primarily through histological observations, inflammatory cytokine assays, and transcriptomic sequencing analyses, lacking causal validation for key pathways. Whether pathways such as Wnt, cAMP, PI3K-Akt, and MAPK are essential for the efficacy of hUCMSC-Exos requires further confirmation through pathway-specific inhibitor or agonist interventions, alongside strategies including gene knockdown, overexpression, or conditional knockout. (2) This study lacks direct evidence of hUCMSC-Exos delivery and distribution within the brain. Although reduced neuroinflammation and improved neuronal injury were observed, whether hUCMSC-Exos cross the BBB and their enrichment characteristics in brain regions such as the hippocampus and prefrontal cortex require clarification through in vivo tracing, fluorescence imaging, and quantitative analysis. (3) This study primarily focuses on glia-mediated neuroinflammation. However, the development of COPD-NCDs may also be influenced by multiple factors including chronic hypoxia, impaired cerebral microvascular endothelial function, and neurotransmitter metabolic disorders, which have not been systematically evaluated. Future work could incorporate supplementary validation using BBB tight junction proteins, cerebral microvascular function markers, and oxidative stress-related indicators. (4) Regarding the administration protocol, this study employed tail vein injection with fixed dosage and cycle. The effects of different administration routes, dose gradients, timing, and treatment duration on efficacy remain uninvestigated. Longer-term follow-up is also lacking to assess sustained efficacy and safety. (5) At the model and sample level, this study employed a single strain and a fixed experimental time window. Extrapolating results to COPD models with different genetic backgrounds, sexes, or disease severity requires caution. Future work should expand sample sizes and replicate validation under diverse conditions.\u003c/p\u003e \u003cp\u003eFuture research may advance in the following areas. (1) Subsequent functional validation of key pathways identified through transcriptome sequencing analysis should clarify the association mechanisms between the key active components of hUCMSC-Exos and their receptors and downstream signaling pathways. (2) In vivo tracing should determine the targeted distribution characteristics of hUCMSC-Exos, combined with BBB permeability assessment to elucidate potential pathways for their entry into the brain. (3) Optimize delivery strategies and conduct extended efficacy and safety monitoring to determine optimal intervention windows and dosage regimens. (4) Characterize the role of key exosomal cargoes\u0026mdash;such as microRNAs and proteins\u0026mdash;in regulating interactions between microglia and astrocytes, thereby providing robust experimental evidence for exosomal therapeutic approaches targeting COPD-NCDs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCOPD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChronic obstructive pulmonary disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCOPD-NCDs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCOPD-related neurocognitive disorders\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ehUCMSC-Exos\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman umbilical cord mesenchymal stem cell-derived exosomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNormal control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCigarette smoke\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBBB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood-brain barrier\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCNS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentral nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMSCs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eExos\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExosomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMSC-Exos\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMSC-derived exosomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSPF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpecific Pathogen Free\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHUCMSCs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Umbilical Cord Mesenchymal Stem Cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission Electron Microscope\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePMSF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhenylmethylsulphonyl fluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSDS-PAGE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium dodecyl sulphate-polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePVDF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyvinylidene difluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFEV0.1/FVC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eForced expiratory volume in 0.1 s/forced vital capacity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAirway resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCdyn\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDynamic compliance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInspiratory time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExpiratory time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePIF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeak inspiratory flow\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePEF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeak expiratory flow\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePenh\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnhanced pause\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEF50\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExpiratory flow at 50% expired volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRpef\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe ratio of time to peak expiratory flow to expiratory time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinute ventilation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTidal volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOFT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOpen Field Test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNOR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNovel Object Recognition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMWM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMorris Water Maze\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH\u0026amp;E\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDEGs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBiological Process\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular Component\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular Function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protocols involving animal experiments in this experiment were approved by the Ethics Committee for Animal Experiments of the College of Basic Medical Sciences of Jilin University (Approval No.2025\u0026ndash;773), and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Medical Basic Research Innovation Center of Airway Disease in North China, Key Laboratory of Pathobiology, Key Laboratory of Precision Infectious Diseases, Jilin Province (20200601011JC), Engineering Laboratory for Precision Prevention and Treatment of Common Diseases, Jilin Province (2022C036), Department of Science and Technology of Jilin Province: Key Scientific and Technological Research and Development Projects (20230204055YY).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, F.W. and X.M.; writing\u0026mdash;original draft preparation, H.X.; computer graphics, H.X.; writing\u0026mdash;review and editing, X.Y., S.B., Y.D., J.C.; supervision, Z.D., X.M.; project administration, Z.D., X.M.; funding acquisition, F.W. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the Key Laboratory of Pathobiology of the Ministry of Education at Jilin University and the Teaching and Research Room of Pathogenic Biology at Jilin University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China; Hui Xiao\u003c/p\u003e\n\u003cp\u003eDepartment of Histology \u0026amp; Embryology, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China；Xiao Yu, Jiayue Cui, Zhiyong Dong, Xiaoting Meng\u003c/p\u003e\n\u003cp\u003eDepartment of Forensic Medicine, Basic Medical College, Jilin University, Changchun, 130021, China; Shilong Bao, Yiding Dong\u003c/p\u003e\n\u003cp\u003eCollege of Basic Medical Sciences, Department of Pathogen Biology, the Medical Basic Research Innovation Center of Airway Disease in North China, Ministry of Education, Jilin University, Changchun, 130021, China, Fang Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xiaoting Meng and Fang Wang\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChristenson SA, Smith BM, Bafadhel M, Putcha N. 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Mar Drugs 2019, 17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chronic obstructive pulmonary disease, Neurocognitive disorders, Human umbilical cord mesenchymal stem cell-derived exosomes, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-9465519/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9465519/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePatients with chronic obstructive pulmonary disease (COPD) frequently suffer from COPD-related neurocognitive disorders (COPD-NCDs), which severely impair their quality of life. Neuroinflammation is a key pathological mechanism, but effective therapies are still lacking. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) have anti-inflammatory and neuroprotective effects in other neurological disorders. However, their efficacy and underlying mechanisms in COPD-NCDs remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice were divided into normal control (NC), COPD-NCDs (model), COPD-NCDs\u0026thinsp;+\u0026thinsp;PBS (vehicle), and COPD-NCDs\u0026thinsp;+\u0026thinsp;Exos (treatment) groups. COPD-NCDs was induced by 24 weeks of cigarette smoke exposure, and hUCMSC-Exos were administered via tail vein during weeks 20\u0026ndash;23. Cognitive function, pulmonary function, lung and hippocampal pathology, microglial activation, astrocytic A1/A2 phenotype, inflammatory cytokines, and hippocampal RNA-seq were assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ehUCMSC-Exos significantly improved lung function, reduced pulmonary inflammation, emphysema, collagen deposition, and systemic inflammation. Importantly, hUCMSC-Exos also improved cognitive function, attenuated hippocampal neuronal damage (increased Nissl-positive neurons and NeuN expression), inhibited microglial activation, and reduced inflammatory cytokine levels in brain tissue. Furthermore, hUCMSC-Exos downregulated the A1 astrocyte marker C3 and upregulated the A2 marker S100A10. RNA-seq suggested modulation of MAPK, Wnt, cAMP and PI3K-Akt signaling pathways.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ehUCMSC-Exos improved cognitive function in COPD-NCDs mice, which was associated with inhibition of microglial activation and a shift from an A1-like to an A2-like astrocytic phenotype. These findings highlight the potential of hUCMSC-Exos as a therapeutic strategy for COPD-NCDs, although the causal relationship between phenotype shift and cognitive improvement requires further mechanistic validation.\u003c/p\u003e","manuscriptTitle":"Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Neurocognitive Disorders in Chronic Obstructive Pulmonary Disease by Suppressing Neuroinflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 05:56:14","doi":"10.21203/rs.3.rs-9465519/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-06T15:37:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-23T17:37:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-23T11:45:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-04-20T01:45:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dfcad77f-be22-4839-87f5-ccb9116f93ac","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"28","date":"2026-05-06T15:37:08+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T05:56:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 05:56:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9465519","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9465519","identity":"rs-9465519","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}