Human umbilical cord mesenchymal stem cells achieve neuroprotection via exosome-mediated anti-inflammation and blood-brain barrier recovery

Human umbilical cord mesenchymal stem cells achieve neuroprotection via exosome-mediated anti-inflammation and blood-brain barrier recovery | 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 cells achieve neuroprotection via exosome-mediated anti-inflammation and blood-brain barrier recovery Jiren Zhang, Ge Xu, Dongman Zhao, Jian Yang, Shilei Qi, Lei Xie, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032340/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ischemia-reperfusion can aggravate cerebral damage. Mesenchymal stem cells have gained attention to improve the outcome of ischemia-reperfusion injury. This study aims to investigate the effects and mechanisms of human umbilical cord mesenchymal stem cells (hUC-MSCs) on cerebral ischemia-reperfusion injury in rats. Methods A middle cerebral artery occlusion-reperfusion (MCAO/R) model was successfully established, and hUC-MSCs or hUC-MSC-derived exosomes (hUC-MSC-exos) were injected into rats via stereotactic brain injection or the tail vein. Neurological functions were evaluated using Garcia, foot-fault, adhesive removal, and forepaw grip strength tests. In addition, we detected the expression of IL-1β, IL-10 and MMP9 in brain tissue using enzyme-linked immunosorbent assay. Immunofluorescence experiments detected the express of CD68 in the M1- microglia, the express of CD206 in the M2-microglia and the expression of Nrf2 in brain tissue. Western blotting experiments detected the expression of occludin, ZO-1, HO-1, and Nrf2 in brain tissue. Results HUC-MSCs significantly reduced the error rate and time to sense in the foot-fault test and in adhesive removal test. Similarly, hUC-MSCs can also significantly reduced infarct size and brain water content. HUC-MSCs improved morphology of brain tissue and cellular structure, including an increase in number of neurons containing Nissl bodies. And T2-weighted imaging revealed a reduction in high signal areas within the ischemic hemisphere in the cell group (hUC-MSCs). Further findings demonstrated that hUC-MSC-exos also improved neurological function and ameliorated the brain injury and morphological changes. In addition, hUC-MSC-exos decreased the contents of IL-1β, MMP-9, and the expression of CD68 (M1-microglia) r, augmented the expression of IL-10, ZO-1, Occludin, Nrf2, HO-1, and the expression of CD206 (M2-microglia). Conclusion These results indicated that human umbilical cord mesenchymal stem cells may excert neuroprotective effects in cerebral ischemia-reperfusion injury by inhibiting inflammation and protecting blood-brain barrier integrity via exosome-mediated Nrf2/HO-1 signaling pathway. Cerebral ischemia-reperfusion injury Human umbilical cord mesenchymal stem cell Exosomes Neuroinflammation Blood-brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ischemia-reperfusion, known as the restoration of blood supply to an ischemic area, may result in tissue injury [ 1 ]. Cerebral ischemia-reperfusion injury is a common condition in the treatment of ischemic stroke [ 2 ]. When cerebral blood flow resumes after a prolonged blockage, the ischemic brain tissue undergoes a series of complex cellular and molecular events, leading to degenerative damage. During reperfusion the inflammatory cascade reaction will be triggered which lead to secondary neuronal injury and death following the initial ischemic attack [ 3 ]. Excessive activation of endogenous neuroinflammatory processes leads to hypoxic tissue destruction, occurrence of apoptosis, and so on, in turn affects neurological function [ 4 , 5 ]. The treatment strategies for cerebral ischemia-reperfusion injury include free radical scavenging, inflammation suppression, mitochondrial/cellular protection, microcirculation maintenance, etc. A range of drug and interventional therapies have emerged that can alleviate cerebral ischemia-reperfusion injury [ 6 ]. Stem cell therapy and stem cell-derived exosomes have become a recent focus [ 7 , 8 ]. Stem cell therapy has shown significant potential in treating central nervous system diseases [ 9 ]. Stem cells, due to their ability to self-renew and differentiate, show excellent results in tissue repair and regeneration [ 10 ]. By secreting anti-inflammatory factors and promoting neuronal regeneration, stem cells can reduce inflammation and improve neurological function [ 11 ]. Stem cell therapy has demonstrated positive effects in treating cerebral ischemia, traumatic brain injury, and neurodegenerative diseases [ 12 , 13 ]. Recent studies have shown that human umbilical cord mesenchymal stem cells (hUC-MSCs) exhibit positive effects in treating neurodegenerative diseases and central nervous system injuries [ 14 ]. The advantages of hUC-MSCs include their wide availability, low immunogenicity, and ease of isolation and cultivation. Exosomes, which are vesicles secreted by stem cells, contain bioactive molecules such as proteins and nucleic acids. Stem cell-derived exosomes play a crucial role in intercellular communication, which is associated with various physiological and pathological processes. Increasing evidence suggests that stem cell-derived exosomes possess anti-inflammatory, antioxidant, angiogenic, and tissue repair properties, offering new perspectives for the treatment of cerebral ischemia [ 15 , 16 ]. However, the therapeutic effects and mechanisms of hUC-MSCs and their exosomes on cerebral ischemia-reperfusion injury remain unclear. This study aims to explore the role and potential mechanisms of hUC-MSCs in cerebral ischemia-reperfusion injury. Materials and methods Preparation of human umbilical cord mesenchymal stem cells HUC-MSCs were purchased from Shandong Qilu Cell Therapy Engineering Technology Co., Ltd. (Shandong, China). The hUC-MSCs were cultured in mesenchy stem cell basal medium (MSCQLXB 01-500, Shandong, China) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. HUC-MSCs were digested with trypsin after 4 to 5 passages, then transferred to 15 mL centrifuge tubes, centrifuged at 4°C and 1200 g for 5 minutes, the supernatant was discarded, and the cell concentration was adjusted to 3.3 × 10⁴ cells/µL with PBS for subsequent use. Preparation of human umbilical cord mesenchymal stem cell-derived exosomes HUC-MSC-exos (Lot No. EXO-03-KA240321) were provided by Shandong Qilu Cell Therapy Engineering Technology Co., Ltd. (Shandong, China). The specific method involves collecting the supernatant from hUC-MSCs, centrifuging at 4°C at 300 g for 30 minutes, then centrifuging at 16,500 g for 30 minutes to remove cell debris. The supernatant was transferred to a new centrifuge tube and ultracentrifuged at 100,000g for 70 minutes at 4°C. The supernatant was removed, and the precipitate was resuspended in phosphate-buffered saline (PBS) to obtain the hUC-MSC-exos. Subsequently, Then, dilute it to a protein concentration of 100 mg/mL [ 17 ]. Identification of Exosomes The morphology of exosomes was observed by TEM (Hitachi, HT-7800). Then the size distribution of hUC-MSC-exos was analyzed by a laser scattering microscope for NTA. Finally, exosomes were identified by expression of CD9 (20597-1-AP, Proteintech), CD81 (27855-1-AP, Proteintech), Alix (ab275377, abcam) and GM130 (11308-1-AP, Proteintech). Rat models of MCAO/R All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20240322). Male Sprague-Dawley (SD) rats (weighing 250 to 300 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). The rats were housed at 22.0 ± 1.0°C, 55.0 ± 2.0% humidity, a 12/12 h light/dark cycle, with free access to food and water. The animals were anesthetized with 2% sodium pentobarbital (30 mg/kg). MCAO/R model was established according to previous method [ 18 ]. The rat was placed in a supine position, and a 1.5 cm midline incision was made on the neck to expose the external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA). Hemostatic forceps were used to occlude the distal CCA. A minimal incision was made on the proximal CCA, and a filament was inserted into the artery. The suture was tightened to secure the filament. After 1.5 hours of ischemia, the filament was gently withdrawn, and a microvascular clip was placed near the cardiac side of the proximal CCA ligation. The suture was removed, and the incision was flushed with saline. The CCA was sutured using 11 − 0 microsurgical sutures. After blood flow was restored by removing the microvascular clips, the suture site was compressed, and the CCA was observed for bleeding before closing the neck incision. Human umbilical cord mesenchymal stem cells injection SD rats were randomly divided into three groups: sham group, model group, cell group. After 1.5 hours of cerebral ischemia, cell suspension was injected using a stereotaxic apparatus. The coordinates: AP + 1.0 mm, ML -3.0 mm, DV -4.0 mm. A microinjection pump was used to administer 3 µL of a suspension containing 1×10 5 hUC-MSCs at a rate of 0.5 µL/min. The sham and model groups were injected with an equivalent volume of PBS. Human umbilical cord mesenchymal stem cell-derived exosomes injection SD rats were randomly divided into three groups: sham group, model group, exos group. The exos group were injected with hUC-MSC-exos (100 mg/rat) by tail vein. The sham and model groups were injected with an equivalent volume of PBS. Garcia test At 24 h after reperfusion, Garcia test evaluates spontaneous activity, symmetry in the movement of the four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch [ 19 ]. The score of 18 points indicates normal neurological function. And 3 points indicates the most severe impairment of neurological function. Foot-fault test The rats were placed on a grid with the size of 1.0 × 2.0 m. The total number of steps (30 steps excluding the first 5 and last 5 steps) and the number of foot faults were recorded. The limb falling from the grid was regarded as foot fault. The error rate = (Number of foot faults/Total steps) × 100%. Adhesive removal test Adhesive tape (1.0×1.0cm square) was applied to the contralateral forepaw of the ischemic hemisphere [ 20 ]. The time to sense was recorded. Each rat was given 60s as the maximum observation period. Forepaw grip strength test The forelimb of rats grasped the platform of the grip strength meter (YLS-13A, Jinan Yiyan Technology, China). Subsequently, the tail of rats was pulled away. The maximum grip strength of the forelimb was measured [ 21 ]. 2,3,5-triphenyltetrazolium chloride staining At 24 h after cerebral ischemia-reperfusion, rats were deeply anesthetized with 4% isoflurane and euthanized. The brains were collected and cut into five sections with 2.0 mm thick. The sections were incubated in 2% 2,3,5-triphenyltetrazolium chloride (T8877, Sigma, Germany) at 37°C for 20 min. The tissue with infarction was gray. And the normal brain tissue was red. Images were taken using a digital camera (NIKON P7000) and the infarct size was analyzed with Image J software (Image J, USA). Magnetic resonance imaging MRI scans were performed. Rats were anaesthetized by inhaling 2% isoflurane, and respiratory rate was between 40 and 60 beats/min. Rats were mounted in a 3.0 T MRI scanner (GE Discovery MR750) with the heads being fixed by a rat-specific coil. The parameters for T2WI examination: frequency, FOV 4.0, slice thickness 1.0, spacing 0.2, NEX 2.00. Hematoxylin and eosin and Nissl stainings Rats were euthanized by administering a lethal dose of isoflurane. The brains were harvested and immersed in 10% formalin for 2 d. Specimens were then exposed to serial dilutions of alcohol for dehydration and xylene for transparency. Subsequently, the brain samples were embedded in paraffin wax and cut into sections with 5 µm in thickness using a vibrating microtome. Two serial sections were stained respectively with Hematoxylin and Eosin (C0105M, Beyotime Biotechnology, Shanghai, China) and Nissl (C0117, Beyotime Biotechnology, Shanghai, China) stainings. Histological evaluation was conducted with light microscopy (CX33, Olympus, Tokyo, Japan) by an experimenter who was blinded to the groups. Brain water content Brain water content was determined using a wet-dry weight method. Rats were euthanized by overdose of anesthetic. The ischemic hemisphere of brain was harvested and weighed to obtain the wet weight. The hemisphere was then placed in a 60°C incubator for 24 h to get dry weight. The water content was calculated using the formula: (Wet weight - Dry weight)/Wet weight×100%. Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay kits were from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China) and Shanghai Future Industrial Co., Ltd. (Shanghai, China). The levels of IL-1β (SEKR-0002, Solarbio; JL20884, Shanghai Future Industrial), IL-10 (SEKR-0006, Solarbio; JL13427, Shanghai Future Industrial), and MMP-9 (SEKR-0027, Solarbio) in the ischemic hemisphere of brain were assayed according to the manufacturer’s instructions. Immunofluorescence Immunofluorescence staining was performed to evaluate M1 microglia (CD68), M2 microglia (CD206), and Nrf2 nuclear translocation. Rats were anesthetized with isofurane and perfused with 0.9% saline and 4% paraformaldehyde (PFA). The brain was harvested and immersed in 4% PFA overnight at 4 ℃. The samples were dehydrated with 20% and 30% sucrose. The brains were cut into 20 µm thick sections using the Leica-1950 cryostat (Leica Instruments, Germany). The sections were permeabilized with Triton X-100 (P0096; Beyotime, Shanghai, China) for 15 min and subsequently blocked with QuickBlock™ immunostaining blocking solution (P0260; Beyotime, Shanghai, China) at room temperature for 1 h. Then, the brain sections were incubated with the following primary antibodies overnight at 4℃: rabbit anti-CD68 (1:200, Cell Signaling Technology, E307V), rabbit anti-CD206 (1:200, Cell Signaling Technology, E6T5J), goat anti-iba1 (1:100, Abcam, ab5076) and rabbit anti-Nrf2 (1:80, Abmart, TA0639M). The next day, the primary antibodies were removed and sections were washed with PBST for three times. Then, the sections were incubated in Alexa Flour 488 conjugated goat anti-rabbit IgG (1:500, Beyotime, Shanghai), CY3 conjugated goat anti-rat IgG 1:500, Beyotime, Shanghai), for 1h at 37°C in the dark. The sections were rinsed and mounted on microscope slides with PBST, and then counterstained with DAPI for 5 min. The fluorescent images were acquired using the Olympus FV3000 confocal microscope (Olympus, Japan). Western blot Brain tissues were lysed with RIPA buffer (Beyotime, Shanghai, China). The concentration of protein was measured using the BCA assay kit (Beyotime, Shanghai, China). Protein (20 µg) was separated using SDS-PAGE gel electrophoresis and then transferred onto PVDF membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with non-fat milk for 2 h, incubated at 4°C overnight with the primary antibodies: anti-ZO-1 (AF5145, Affinity, 1:1000), anti-Occludin (DF7504, Affinity, 1:2000), anti-Nrf2 (TA0639M, Abmart, 1:2000), or anti-HO-1 (ab13243, Abcam, 1:1000). Then the membranes were washed three times with TBST. They were incubated with secondary antibody: goat anti-rabbit (A0208, Beyotime, 1:1000), goat anti-mouse (A0216, Beyotime, 1:1000) for 1 h at room temperature. The membranes were washed according to the procedure mentioned above. Immunoreactive bands were detected using an enhanced chemiluminescence reagents (BL520B, Biosharp). The relative intensities of protein bands were normalized to β-actin (GB15001, Servicebio,1:1000), GAPDH (AF0006, Beyotime, 1:1000) or Lamin B 1 (ab16048, Abcam, 1:2000). The relative density of bands was analyzed by Image J software (Image J, USA). Statistical analysis All data analyses and graph generation were performed using GraphPad Prism 10.0 (San Diego, CA) and ImageJ. Results are presented as the Mean ± Standard Deviation (SD). The statistical process is as follows: First, a normality test is conducted. If the data follow a normal distribution, the data was assessed by one-way of variance (ANOVA) followed by Fisher's Least Significant Difference (LSD) test. The data of non-normal distribution were analyzed with the Kruskal-Wallis test followed by uncorrected Dunn's test. A significance level of P < 0.05 was considered statistically significant for all comparisons. Results HUC-MSCs attenuated cerebral ischemia-reperfusion injury and exerted neuroprotective effects The design of the animal experiment is displayed in Fig. 1 a. To assess neurological function, the foot-fault and adhesive removal test were measured. In the foot-fault test, model group showed more limb coordination errors versus sham group. The error rate was significantly reduced in the cell group (hUC-MSCs) versus the model group (Fig. 1 b). In adhesive removal test, model group exhibited significantly prolonged time to sense versus sham group. And cell group displayed significant reductions in time to sense versus the model group (Fig. 1 c). To assess the cerebral ischemic injuries, infarct size was measured by TTC (triphenyltetrazolium chloride) staining. As shown in Fig. 1 d and 1 e, the model group exhibited a significant increase versus the sham group, the cell group exhibited a significant reduction in the infarct size in middle cerebral artery occlusion/reperfusion (MCAO/R) rats versus the model group. We used hematoxylin and eosin (HE), Nissl staining, and magnetic resonance imaging (MRI) to exhibit the neuronal damage and tissue pathological changes. As shown in Fig. 1 f- 1 g, HE stainning revealed that there were no morphological changes in the brain of the sham group, the model group exhibited neuronal degeneration, necrosis, disordered cell arrangement, and irregular morphology, however the cell group significantly improves the morphology of brain tissue and cellular structure versus the model group. As shown in Fig. 1 h- 1 i, in the sham group, Nissl-positive cell was well-organized with clearly visible Nissl bodies, the model group, Nissl-positive cells decreased, with partial pyramidal neurons exhibiting dissolved Nissl bodies, blurred contours, and lighter staining intensity. Conversely, in the cell group, there was significantly increase in the number of neurons containing Nissl bodies versus the model group. As shown in Fig. 1 j, in the sham group, the brain tissue was no abnormalities in the T2-weighted imaging (T2WI), in the model group, T2WI revealed high signal areas in the cortical, hippocampal, and partial striatal regions of ischemic hemisphere. However, the cell group exhibited a significance decrease in the high signal areas of the ischemic hemisphere, including the cortex, hippocampus, and striatum versus the model group. To assess the severity of blood-brain barrier (BBB), brain water content and tissue swelling were measured by a wet-dry weight method. In Fig. 1 k, Model group showed a significant increase in brain water content versus the sham group. However, the cell group demonstrated a marked reduction in brain water content versus the model group. Identification of hUC-MSC-derived exosomes The hUC-MSC-derived exosomes (hUC-MSC-exos) has been identified. transmission electron microscopy (TEM) revealed that the exosomes had a double-layered structure (Fig. 2 a). Nanoparticle tracking analysis (NTA) showed that the particle size of most hUC-MSC-exos was below 100 nm (Fig. 2 b). In addition, Western blotting (WB) showed that the enrichment of exosomal marker proteins CD9, CD81, and Alix, while the Golgi protein GM130 was undetectable (Fig. 2 c). HUC-MSC-exos attenuated cerebral ischemia-reperfusion injury and exerted neuroprotection In line with the results of hUC-MSCs therapy for the MCAO/R. The design of the animal experiment is displayed in Fig. 3 a. In Fig. 3 b and Fig. 3 e, versus the sham group, the scores, forelimb force of rats in the model group significantly decreased. And versus the model group, the scores, forelimb force of rats in exos group (hUC-MSC-exos) increased significantly. In Fig. 3 c- 3 d, versus the sham group, the error rate, time of sense in the model group significantly elevated. And versus the model group, the error rate, time of sense of rats in the exos group significantly reduced. In Fig. 3 f- 3 g, the brains of rats in model group displayed obviously necrosis versus the sham group. However, hUC-MSC-exos significantly reduced cerebral infarct size versus the model group. In Fig. 3 h- 3 i, HE staining indicated that the brains of the sham group showed no significant pathological alterations. Compared to the sham group, brain tissues of the model group exhibited pronounced neuronal degeneration and necrosis, disordered cell arrangement, and irregular morphology. Conversely, the exos group displayed a marked improvement in tissue structure and morphology versus the model group. Similarly, in Fig. 3 j- 3 k, in the model group, Nissl-positive cells significantly decreased and neurons had fewer cytoplasmic Nissl bodies and exhibited weaker staining, some pyramidal cells showed dissolved Nissl bodies with blurred contours, versus the sham group. In contrast, the exos group displayed a significant improvement of cellular morphology and increased number of neurons containing Nissl bodies. HUC-MSC-exos restored BBB function The assessment of BBB permeability is based on four indicators: brain water content, MMP-9, ZO-1, and Occludin. In Fig. 4 a, versus the sham group, the brain water content of the model group was significantly increased. However, the exo group showed a significant reduction in brain water content versus the model group. In Fig. 4 b, the MMP-9 in model group was significantly increased versus the sham group. And the MMP-9 in the exos group significantly decreased versus the model group. In Fig. 4 c- 4 d, the expression of occludin and ZO-1 was significantly decreased in the model group versus the corresponding sham group, respectively. And the expression of occludin and ZO-1 were significantly increased in the the exos group versus the corresponding model group, respectively. Restoring the function of BBB by anti-inflammation The restoration of BBB may be achieved through anti-inflammation including cytokines changes and microglial polarization. In Fig. 5 a, the IL-1β level in model group was significantly increased versus the sham group. The IL-1β level in the exos group significantly decreased versus the model group. In Fig. 5 b, the IL-10 level in model group was significantly increase versus the sham group. The IL-10 level in the exos group significantly increased versus the model group. In Fig. 5 c, the expression of CD68 (M1-microglia) was significantly reduced in the exos group versus the model. In Fig. 5 d, the expression of CD206 (M2-microglia) was significantly elevated in the exos group versus the model group. HUC-MSC-exos activate the Nrf2/HO-1 pathway of anti-inflammation HUC-MSC-exos exerted anti-inflammation by activating Nrf2 and downstream molecular HO-1. In Fig. 6 a, the levels of nuclear Nrf2 significantly increased in the exos group versus the model group. And in Fig. 6 b, the levels of HO-1 were also significantly increased in the exos group versus the model group. The results in Fig. 6 c are similar with Fig. 6 a, the nuclear Nrf2 significantly increased in the exos group versus the model group. Discussion This study demonstrates that hUC-MSCs exert neuroprotective effects on cerebral ischemia-reperfusion injury through their exosomes. This effect is achieved by activating the Nrf2/HO-1 signaling pathway, which reduces neuroinflammatory responses and maintains the integrity of the BBB. The study shows that stereotactic injection of hUC-MSCs can improve neurological function, reduce the ischemic brain volume, and exert neuroprotective effects on cerebral ischemia-reperfusion injury. Previous studies have shown that the neuroprotective effects of stem cells can be realized through their exosomes [ 22 , 23 ]. Therefore, we further investigated the functions of the exosomes secreted by hUC-MSCs. The results were consistent with our predictions, showing that hUC-MSC-exos exhibited similar neuroprotective effects to hUC-MSCs in improving neurological function and reducing ischemic brain volume. Moreover, after the exosome treatment, the expression of MMP-9 decreased, while the expression of ZO-1 and Occludin increased, and the brain water content decreased, indicating that the integrity of the BBB was restored. This suggests that hUC-MSC-exos may improve the integrity of the BBB by regulating the expression and enzyme activity of the MMP-9 gene, thereby exerting neuroprotective effects. Similarly, it has also been found that hUC-MSCs can inhibit the upregulation of MMPs [ 24 ] and affect the permeability of BBB caused by ischemic stroke [ 25 ]. Inflammatory responses are a significant factor affecting the integrity of the BBB [ 26 , 27 ]. The pathogenesis of cerebral ischemia-reperfusion injury is complex, with inflammatory responses playing a crucial role in this condition [ 28 ]. Inflammation not only exacerbates brain tissue damage and promotes the expansion of the infarcted area but also disrupts the BBB, further deteriorating neurological function. This study shows that after exosome action, the levels of pro-inflammatory cytokine IL-1β [ 29 ] decrease, while the levels of anti-inflammatory cytokines IL-10 [ 30 ] increase, indicating a reduction in neuroinflammatory responses. Microglia are resident macrophages in the brain. Under ischemic stimulation, microglia can differentiate into two phenotypes: M1 and M2. Increasing evidence suggests that during the development of cerebral ischemia-reperfusion injury, M1 and M2 microglia can dynamically switch [ 31 ]. Therefore, interventions that promote the transition of microglia from an inflammatory M1 phenotype to a reparative M2 phenotype may have potential therapeutic value for cerebral ischemia-reperfusion injury 7 . M2 macrophages play a crucial role in the recovery process following nerve injury. By inhibiting the expression of matrix metalloproteinase-9 (MMP-9) [ 32 ], they help maintain the integrity of the BBB, thereby reducing the increased permeability caused by inflammation [ 33 ]. The current results showed that hUC-MSC-exos treatment promoted the transition of microglia from the M1 phenotype to the M2 phenotype, also suggesting a reduction in neuroinflammatory responses. As cytokines and microglial polarization reflective of neuroinflammatory response can influence the permeability of the BBB [ 34 , 35 ]. Our results suggest that the restorative effect of exosomes on BBB integrity may be achieved through their anti-inflammatory action. HO-1 is a crucial cellular protective molecule that plays a vital role in suppressing inflammatory responses. Numerous studies have shown that HO-1 can promote the development of macrophages into an anti-inflammatory M2 phenotype [ 36 ]. This process leads to the secretion of anti-inflammatory cytokines and neurotrophic factors, thereby facilitating tissue repair and nerve regeneration. Nrf2, a key transcription factor for HO-1, plays a significant role in regulating inflammatory responses [ 37 ]. Activating Nrf2 can reduce tissue damage and promote functional recovery [ 38 , 39 ]. In addition to its role in inflammation, the activation of Nrf2 in stem cells and their exosomes has become a critical mechanism for these cells to exert anti-inflammatory and protective effects, particularly in ischemic injuries [ 40 ]. In this study, compared to the model group, after hUC-MSC-exos administration, the levels of HO-1 and nuclear Nrf2 were significantly elevated, and the Nrf2/HO-1 signaling pathway was activated, which regulated the polarization of microglia towards a reparative M2 phenotype, thereby exerting an anti-inflammatory effect [ 41 ]. This suggests that hUC-MSC-exos can alleviate neuroinflammatory responses by activating the Nrf2/HO-1 signaling pathway, maintaining the integrity of the BBB, and exerting neuroprotective effects. This study has several limitations. Firstly, we only evaluated the short-term therapeutic effects of hUC-MSCs and their exosomes on cerebral ischemia-reperfusion injury, without exploring their long-term efficacy or sustained impact. Future studies should extend the observation period to assess the long-term effects of hUC-MSCs and their exosomes on neurological recovery. Secondly, while this study primarily focused on the anti-inflammatory effects of hUC-MSCs and their exosomes in cerebral ischemia-reperfusion injury, it did not address their role in oxidative stress, despite the significant roles of Nrf2 and HO-1 in antioxidant defense. Lastly, the potential of hUC-MSCs to differentiate into neurons and replace lost neurons remains unexplored. To validate the role of hUC-MSCs in neural repair, future research should focus on evaluating the differentiation potential of hUC-MSCs, particularly their contributions to neuronal reconstruction and functional recovery following cerebral ischemia-reperfusion injury [ 42 ]. Conclusion Our findings indicate that hUC-MSCs offer neuroprotective effects on rats with cerebral ischemia-reperfusion injury. Moreover, the mechanism of hUC-MSCs' action may involve activating the Nrf2/HO-1 signaling pathway through exosomes it secretes (Fig. 7 ). The exosomes from hUC-MSCs promote the nuclear transfer of Nrf2, which in turn inhibits inflammatory responses and regulates microglia to shift from an inflammatory M1 phenotype to a reparative M2 phenotype. This leads to a reduction in pro-inflammatory factors and an increase in anti-inflammatory factors, thereby enhancing the repair of the BBB and exerting neuroprotective effects against cerebral ischemia-reperfusion injury. Declarations Ethics approval, And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20240322). Acknowledgments This work was supported by the Hebei Province Introducing Foreign Intelligence Project and the Natural Science Foundation of Hebei Province (H202220601) and Capital Health Development Research Special Fund (2022-1-2041). Author contributions P.Z. and J.Z. conceptualized the project. T.W. J.Z. and G.X. designed the experiments. J.Z. G.X. and A.L. conducted the experiments and drafted the manuscript. P.Z. and J.Z. revised the manuscript. D.Z. and J.Y. provided technical advice. J.Z. and G.X. analyzed the data. All authors have read and approved the final manuscript. Availability of data and materials All data generated or analyzed for this study are included in this published article. Competing interests The authors declare no competing interests. References Stegner D, Klaus V, Nieswandt B. Platelets as Modulators of Cerebral Ischemia/Reperfusion Injury. Front Immunol. 2019;10:2505. Xu M, et al. Protective effect and mechanism of Qishiwei Zhenzhu pills on cerebral ischemia-reperfusion injury via blood-brain barrier and metabonomics. Biomed Pharmacother. 2020;131:110723. Xue J, et al. Sphingomyelin Synthase 2 Inhibition Ameliorates Cerebral Ischemic Reperfusion Injury Through Reducing the Recruitment of Toll-Like Receptor 4 to Lipid Rafts. J Am Heart Assoc. 2019;8:e012885. Gan Y, et al. Ischemic neurons recruit natural killer cells that accelerate brain infarction. 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J Neuroinflammation. 2022;19:141. Li Y, Li J, Yu Q, Ji L, Peng B. METTL14 regulates microglia/macrophage polarization and NLRP3 inflammasome activation after ischemic stroke by the KAT3B-STING axis. Neurobiol Dis. 2023;185:106253. Hilliard A, Mendonca P, Russell TD, Soliman KF. A. The Protective Effects of Flavonoids in Cataract Formation through the Activation of Nrf2 and the Inhibition of MMP-9. Nutrients 12 (2020). Feng J, et al. Protective effect of cynaroside on sepsis-induced multiple organ injury through Nrf2/HO-1-dependent macrophage polarization. Eur J Pharmacol. 2021;911:174522. Kurmann L et al. Transcryptomic Analysis of Human Brain-Microvascular Endothelial Response to -Pericytes: Cell Orientation Defines Barrier Function. Cells 10 (2021). Chen X, Ren Y, Xie P, Lei Q, Lu W. GM130-silencing may aggravate blood-brain barrier damage and affect microglia polarization by down-regulating PD-L1 expression after experimental intracerebral hemorrhage. Mol Biol Rep. 2024;51:919. Tsai CF et al. Regulatory Effects of Quercetin on M1/M2 Macrophage Polarization and Oxidative/Antioxidative Balance. Nutrients 14 (2021). Ma H, et al. Sevoflurane protects the liver from ischemia-reperfusion injury by regulating Nrf2/HO-1 pathway. Eur J Pharmacol. 2021;898:173932. Wang L et al. Nrf2 Regulates Oxidative Stress and Its Role in Cerebral Ischemic Stroke. Antioxid (Basel) 11 (2022). Lin QM, et al. Mesenchymal stem cells transplantation suppresses inflammatory responses in global cerebral ischemia: contribution of TNF-α-induced protein 6. Acta Pharmacol Sin. 2013;34:784–92. Zhou Y, et al. Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway. J Ovarian Res. 2024;17:80. Wang L, et al. Koumine ameliorates neuroinflammation by regulating microglia polarization via activation of Nrf2/HO-1 pathway. Biomed Pharmacother. 2023;167:115608. Liu J, et al. Mesenchymal stem cells and their microenvironment. Stem Cell Res Ther. 2022;13:429. Cite Share Download PDF Status: Posted Version 1 posted 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-7032340","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496572059,"identity":"249bd388-850f-463d-b752-dd33d3cd5127","order_by":0,"name":"Jiren Zhang","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiren","middleName":"","lastName":"Zhang","suffix":""},{"id":496572060,"identity":"10017f4d-115b-4af4-aaad-7744ddebc48f","order_by":1,"name":"Ge Xu","email":"","orcid":"","institution":"The First Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Xu","suffix":""},{"id":496572061,"identity":"62725dc5-c94c-42a0-b1ef-8ba84affe0ee","order_by":2,"name":"Dongman Zhao","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dongman","middleName":"","lastName":"Zhao","suffix":""},{"id":496572062,"identity":"df65cd92-0476-429f-bb1f-fce6cdda3a17","order_by":3,"name":"Jian Yang","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Yang","suffix":""},{"id":496572063,"identity":"f129b115-ee1b-4926-9818-6c0b4ffdb81a","order_by":4,"name":"Shilei Qi","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shilei","middleName":"","lastName":"Qi","suffix":""},{"id":496572064,"identity":"2eeea570-5f07-4959-86ec-f8eca119ddbb","order_by":5,"name":"Lei Xie","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Xie","suffix":""},{"id":496572065,"identity":"7c92472d-6c1b-46e6-9639-3b9dd44ed4c5","order_by":6,"name":"Mingming Jia","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingming","middleName":"","lastName":"Jia","suffix":""},{"id":496572066,"identity":"a1ce1e28-ce27-44fa-bf13-28598ed2579b","order_by":7,"name":"Anxin Lv","email":"","orcid":"","institution":"Baotou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Anxin","middleName":"","lastName":"Lv","suffix":""},{"id":496572067,"identity":"b488ab42-24f8-46cf-94fb-16230cff7cd3","order_by":8,"name":"Jing Zhang","email":"","orcid":"","institution":"The First Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":496572068,"identity":"7e756a62-ce78-404c-9823-53392149497e","order_by":9,"name":"Wang Tian","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Tian","suffix":""},{"id":496572069,"identity":"7661e187-57c8-45c9-b18f-d7fdea873d1f","order_by":10,"name":"Pinyuan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACNvnzDx9++GEjx8/eQKQWPgkeZmPJnjQgPkCkFjkJHjYgOpxocCOBWIdJ9x6QkOBJSzC4+XjjDYYam2jCWmTOJRgUWNjkSd5OK7ZgOJaW20BQC0OCQQLQlmK+2zlmEowNh4nTcgDkl4abZ4jVIpFj2ADSMuEGD7FaeI4lM0MCGeiXBGL8It/efPwnJCoPb7zxocaGsBZkYCCRQIpyiBZSdYyCUTAKRsHIAAA+IT6YoiYYDgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0002-6789-7193","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Pinyuan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-02 20:03:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7032340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7032340/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88777014,"identity":"1963f8b7-7398-4ad3-bf4b-a53b20b5cf6f","added_by":"auto","created_at":"2025-08-11 10:10:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749238,"visible":true,"origin":"","legend":"\u003cp\u003eHuman umbilical cord mesenchymal stem cells (hUC-MSCs) therapy attenuated cerebral ischemia-reperfusion injury and exerted neuroprotective effects. (a) The design of the animal experiment; (b) The analysis of the error rate in the foot-fault test; (c) The analysis of time to sense in the adhesive removal test; (d) Infarct size was measured by TTC staining; (e) Statistical analysis of cerebral infarction size; (f) HE staining reflected the morphological changes in the brain; (g) The number of pyknotic cells; (h) Nissl staining reflected the neuronal damage; (i) The number of Nissl-positive cells; (j) Magnetic resonance imaging (MRI) measures cerebral ischemic injury; (k) Analysis of brain water content. Data are presented as the Mean ± Standard Deviation (SD), n=3-17 *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e sham, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e sham, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e model,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e model.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/ecf992e6a055cbe9989822bd.jpg"},{"id":88777012,"identity":"72a313ae-cfcb-4c30-9467-13c73c602ed2","added_by":"auto","created_at":"2025-08-11 10:10:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":558919,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of hUC-MSC-derived exosomes (hUC-MSC-exos). (a) The structure of exosomes was revealed by transmission electron microscopy (TEM); (b) The particle size of hUC-MSC-exos was measured by nanoparticle tracking analysis (NTA); (c) The marker proteins CD9, CD81, and Alix, and GM130 of hUC-MSC-exos were measured.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/a15b01261c5dcd41e9d50272.jpg"},{"id":88777019,"identity":"6a50b133-d869-48e8-b8dc-6599911f2c91","added_by":"auto","created_at":"2025-08-11 10:10:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":771644,"visible":true,"origin":"","legend":"\u003cp\u003ehUC-MSC-exos attenuated cerebral ischemia-reperfusion injury and exerted neuroprotective effects. (a) The design of the animal experiment; (b) The analysis of the Garcia scores in the Garcia test; (c) The analysis of the error rate in the foot-fault test; (d) The analysis of time to sense in the adhesive removal test; (e) The analysis of the force in the forepaw grip strength tests; (f) Infarct size was measured by TTC staining; (g) Statistical analysis of cerebral infarction size; (h) HE staining reflected the morphological changes in the brain; (i) The number of pyknotic cells; (j) Nissl staining reflected the neuronal damage; (k) The number of Nissl-positive cells. Data are presented as the Mean ± Standard Deviation (SD), n=5-10; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e sham, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003esham, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e model,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e model.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/ff2f4f1e07700b2dfe27c1c4.jpg"},{"id":88779026,"identity":"3a1b12ea-a3be-4405-8c2e-f1b440150cdc","added_by":"auto","created_at":"2025-08-11 10:26:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":332937,"visible":true,"origin":"","legend":"\u003cp\u003ehUC-MSC-exos restored BBB function. (a) Analysis of brain water content; (b) The expression of matrix metalloproteinase-9 (MMP-9); (c) The expression of occluding; (d) The expression of zonula occludens (ZO-1). Data are presented as the Mean ± Standard Deviation (SD), n=3-8; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e sham, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e sham, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs \u003c/em\u003emodel,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e model.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/ee52870044a8bd6ea4c1bd1a.jpg"},{"id":88779027,"identity":"40b8b187-793b-4812-b55b-8e4fbf92bb15","added_by":"auto","created_at":"2025-08-11 10:26:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":635560,"visible":true,"origin":"","legend":"\u003cp\u003eRestoring the function of BBB by anti-inflammation. (a) The expression of interleukin-1 beta (IL-1β); (b) The expression of interleukin-10 (IL-10); (c) The representative image of M1-microglia (CD68); (d) The representative image of M2- microglia (CD206). Data are presented as the Mean ± Standard Deviation (SD), n=3-8; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e sham, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e sham, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e model, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e model.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/73910043da617debbc278e29.jpg"},{"id":88777024,"identity":"f37fbd33-b2da-4c46-bbb7-c132319fc2b4","added_by":"auto","created_at":"2025-08-11 10:10:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":618858,"visible":true,"origin":"","legend":"\u003cp\u003eHUC-MSC-exos activate the Nrf2/HO-1 pathway of anti-inflammation. (a) The expression of nuclear factor erythroid-related factor (Nrf2); (b) The expression of nuclear Heme oxygenase-1 (HO-1); (c) The representative image of Nrf2. Data are presented as the Mean ± Standard Deviation (SD), n=3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs \u003c/em\u003esham, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e sham, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003emodel.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/2b53703f3a5394a19b746c79.jpg"},{"id":88777021,"identity":"7a72f4ee-a421-4ddd-8873-4c9cddaaa04e","added_by":"auto","created_at":"2025-08-11 10:10:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":539213,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of neuroprotection. HUC-MSCs excerted neuroprotective effects in rats with cerebral ischemia-reperfusion injury by inhibiting inflammation and protecting blood-brain barrier via exosome-mediated Nrf2/HO-1 signaling pathway.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/4cfc360ef182e84e1022b8e4.jpg"},{"id":93998295,"identity":"f9fab39b-5b47-4d91-bea9-0304a4504f93","added_by":"auto","created_at":"2025-10-21 07:29:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5117273,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7032340/v1/4367182d-8284-4ed4-bf89-dd4cb30bc6ba.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eHuman umbilical cord mesenchymal stem cells achieve neuroprotection via exosome-mediated anti-inflammation and blood-brain barrier recovery\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemia-reperfusion, known as the restoration of blood supply to an ischemic area, may result in tissue injury [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cerebral ischemia-reperfusion injury is a common condition in the treatment of ischemic stroke [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When cerebral blood flow resumes after a prolonged blockage, the ischemic brain tissue undergoes a series of complex cellular and molecular events, leading to degenerative damage. During reperfusion the inflammatory cascade reaction will be triggered which lead to secondary neuronal injury and death following the initial ischemic attack [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Excessive activation of endogenous neuroinflammatory processes leads to hypoxic tissue destruction, occurrence of apoptosis, and so on, in turn affects neurological function [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe treatment strategies for cerebral ischemia-reperfusion injury include free radical scavenging, inflammation suppression, mitochondrial/cellular protection, microcirculation maintenance, etc. A range of drug and interventional therapies have emerged that can alleviate cerebral ischemia-reperfusion injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Stem cell therapy and stem cell-derived exosomes have become a recent focus [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Stem cell therapy has shown significant potential in treating central nervous system diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Stem cells, due to their ability to self-renew and differentiate, show excellent results in tissue repair and regeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. By secreting anti-inflammatory factors and promoting neuronal regeneration, stem cells can reduce inflammation and improve neurological function [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Stem cell therapy has demonstrated positive effects in treating cerebral ischemia, traumatic brain injury, and neurodegenerative diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies have shown that human umbilical cord mesenchymal stem cells (hUC-MSCs) exhibit positive effects in treating neurodegenerative diseases and central nervous system injuries [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The advantages of hUC-MSCs include their wide availability, low immunogenicity, and ease of isolation and cultivation. Exosomes, which are vesicles secreted by stem cells, contain bioactive molecules such as proteins and nucleic acids. Stem cell-derived exosomes play a crucial role in intercellular communication, which is associated with various physiological and pathological processes. Increasing evidence suggests that stem cell-derived exosomes possess anti-inflammatory, antioxidant, angiogenic, and tissue repair properties, offering new perspectives for the treatment of cerebral ischemia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the therapeutic effects and mechanisms of hUC-MSCs and their exosomes on cerebral ischemia-reperfusion injury remain unclear. This study aims to explore the role and potential mechanisms of hUC-MSCs in cerebral ischemia-reperfusion injury.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePreparation of human umbilical cord mesenchymal stem cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHUC-MSCs were purchased from Shandong Qilu Cell Therapy Engineering Technology Co., Ltd. (Shandong, China). The hUC-MSCs were cultured in mesenchy stem cell basal medium (MSCQLXB 01-500, Shandong, China) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. HUC-MSCs were digested with trypsin after 4 to 5 passages, then transferred to 15 mL centrifuge tubes, centrifuged at 4\u0026deg;C and 1200 g for 5 minutes, the supernatant was discarded, and the cell concentration was adjusted to 3.3 \u0026times; 10⁴ cells/\u0026micro;L with PBS for subsequent use.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of human umbilical cord mesenchymal stem cell-derived exosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHUC-MSC-exos (Lot No. EXO-03-KA240321) were provided by Shandong Qilu Cell Therapy Engineering Technology Co., Ltd. (Shandong, China). The specific method involves collecting the supernatant from hUC-MSCs, centrifuging at 4\u0026deg;C at 300 g for 30 minutes, then centrifuging at 16,500 g for 30 minutes to remove cell debris. The supernatant was transferred to a new centrifuge tube and ultracentrifuged at 100,000g for 70 minutes at 4\u0026deg;C. The supernatant was removed, and the precipitate was resuspended in phosphate-buffered saline (PBS) to obtain the hUC-MSC-exos. Subsequently, Then, dilute it to a protein concentration of 100 mg/mL [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of Exosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe morphology of exosomes was observed by TEM (Hitachi, HT-7800). Then the size distribution of hUC-MSC-exos was analyzed by a laser scattering microscope for NTA. Finally, exosomes were identified by expression of CD9 (20597-1-AP, Proteintech), CD81 (27855-1-AP, Proteintech), Alix (ab275377, abcam) and GM130 (11308-1-AP, Proteintech).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRat models of MCAO/R\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20240322). Male Sprague-Dawley (SD) rats (weighing 250 to 300 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). The rats were housed at 22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C, 55.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0% humidity, a 12/12 h light/dark cycle, with free access to food and water. The animals were anesthetized with 2% sodium pentobarbital (30 mg/kg). MCAO/R model was established according to previous method [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The rat was placed in a supine position, and a 1.5 cm midline incision was made on the neck to expose the external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA). Hemostatic forceps were used to occlude the distal CCA. A minimal incision was made on the proximal CCA, and a filament was inserted into the artery. The suture was tightened to secure the filament. After 1.5 hours of ischemia, the filament was gently withdrawn, and a microvascular clip was placed near the cardiac side of the proximal CCA ligation. The suture was removed, and the incision was flushed with saline. The CCA was sutured using 11\u0026thinsp;\u0026minus;\u0026thinsp;0 microsurgical sutures. After blood flow was restored by removing the microvascular clips, the suture site was compressed, and the CCA was observed for bleeding before closing the neck incision.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman umbilical cord mesenchymal stem cells injection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSD rats were randomly divided into three groups: sham group, model group, cell group. After 1.5 hours of cerebral ischemia, cell suspension was injected using a stereotaxic apparatus. The coordinates: AP\u0026thinsp;+\u0026thinsp;1.0 mm, ML -3.0 mm, DV -4.0 mm. A microinjection pump was used to administer 3 \u0026micro;L of a suspension containing 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e hUC-MSCs at a rate of 0.5 \u0026micro;L/min. The sham and model groups were injected with an equivalent volume of PBS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman umbilical cord mesenchymal stem cell-derived exosomes injection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSD rats were randomly divided into three groups: sham group, model group, exos group. The exos group were injected with hUC-MSC-exos (100 mg/rat) by tail vein. The sham and model groups were injected with an equivalent volume of PBS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGarcia test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 24 h after reperfusion, Garcia test evaluates spontaneous activity, symmetry in the movement of the four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The score of 18 points indicates normal neurological function. And 3 points indicates the most severe impairment of neurological function.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFoot-fault test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rats were placed on a grid with the size of 1.0 \u0026times; 2.0 m. The total number of steps (30 steps excluding the first 5 and last 5 steps) and the number of foot faults were recorded. The limb falling from the grid was regarded as foot fault. The error rate = (Number of foot faults/Total steps) \u0026times; 100%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdhesive removal test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdhesive tape (1.0\u0026times;1.0cm square) was applied to the contralateral forepaw of the ischemic hemisphere [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The time to sense was recorded. Each rat was given 60s as the maximum observation period.\u003c/p\u003e\u003cp\u003e\u003cb\u003eForepaw grip strength test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe forelimb of rats grasped the platform of the grip strength meter (YLS-13A, Jinan Yiyan Technology, China). Subsequently, the tail of rats was pulled away. The maximum grip strength of the forelimb was measured [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e2,3,5-triphenyltetrazolium chloride staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 24 h after cerebral ischemia-reperfusion, rats were deeply anesthetized with 4% isoflurane and euthanized. The brains were collected and cut into five sections with 2.0 mm thick. The sections were incubated in 2% 2,3,5-triphenyltetrazolium chloride (T8877, Sigma, Germany) at 37\u0026deg;C for 20 min. The tissue with infarction was gray. And the normal brain tissue was red. Images were taken using a digital camera (NIKON P7000) and the infarct size was analyzed with Image J software (Image J, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMagnetic resonance imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMRI scans were performed. Rats were anaesthetized by inhaling 2% isoflurane, and respiratory rate was between 40 and 60 beats/min. Rats were mounted in a 3.0 T MRI scanner (GE Discovery MR750) with the heads being fixed by a rat-specific coil. The parameters for T2WI examination: frequency, FOV 4.0, slice thickness 1.0, spacing 0.2, NEX 2.00.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHematoxylin and eosin and Nissl stainings\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRats were euthanized by administering a lethal dose of isoflurane. The brains were harvested and immersed in 10% formalin for 2 d. Specimens were then exposed to serial dilutions of alcohol for dehydration and xylene for transparency. Subsequently, the brain samples were embedded in paraffin wax and cut into sections with 5 \u0026micro;m in thickness using a vibrating microtome. Two serial sections were stained respectively with Hematoxylin and Eosin (C0105M, Beyotime Biotechnology, Shanghai, China) and Nissl (C0117, Beyotime Biotechnology, Shanghai, China) stainings. Histological evaluation was conducted with light microscopy (CX33, Olympus, Tokyo, Japan) by an experimenter who was blinded to the groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBrain water content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBrain water content was determined using a wet-dry weight method. Rats were euthanized by overdose of anesthetic. The ischemic hemisphere of brain was harvested and weighed to obtain the wet weight. The hemisphere was then placed in a 60\u0026deg;C incubator for 24 h to get dry weight. The water content was calculated using the formula: (Wet weight - Dry weight)/Wet weight\u0026times;100%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme-linked immunosorbent assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEnzyme-linked immunosorbent assay kits were from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China) and Shanghai Future Industrial Co., Ltd. (Shanghai, China). The levels of IL-1β (SEKR-0002, Solarbio; JL20884, Shanghai Future Industrial), IL-10 (SEKR-0006, Solarbio; JL13427, Shanghai Future Industrial), and MMP-9 (SEKR-0027, Solarbio) in the ischemic hemisphere of brain were assayed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescence staining was performed to evaluate M1 microglia (CD68), M2 microglia (CD206), and Nrf2 nuclear translocation. Rats were anesthetized with isofurane and perfused with 0.9% saline and 4% paraformaldehyde (PFA). The brain was harvested and immersed in 4% PFA overnight at 4 ℃. The samples were dehydrated with 20% and 30% sucrose. The brains were cut into 20 \u0026micro;m thick sections using the Leica-1950 cryostat (Leica Instruments, Germany). The sections were permeabilized with Triton X-100 (P0096; Beyotime, Shanghai, China) for 15 min and subsequently blocked with QuickBlock\u0026trade; immunostaining blocking solution (P0260; Beyotime, Shanghai, China) at room temperature for 1 h. Then, the brain sections were incubated with the following primary antibodies overnight at 4℃: rabbit anti-CD68 (1:200, Cell Signaling Technology, E307V), rabbit anti-CD206 (1:200, Cell Signaling Technology, E6T5J), goat anti-iba1 (1:100, Abcam, ab5076) and rabbit anti-Nrf2 (1:80, Abmart, TA0639M). The next day, the primary antibodies were removed and sections were washed with PBST for three times. Then, the sections were incubated in Alexa Flour 488 conjugated goat anti-rabbit IgG (1:500, Beyotime, Shanghai), CY3 conjugated goat anti-rat IgG 1:500, Beyotime, Shanghai), for 1h at 37\u0026deg;C in the dark. The sections were rinsed and mounted on microscope slides with PBST, and then counterstained with DAPI for 5 min. The fluorescent images were acquired using the Olympus FV3000 confocal microscope (Olympus, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBrain tissues were lysed with RIPA buffer (Beyotime, Shanghai, China). The concentration of protein was measured using the BCA assay kit (Beyotime, Shanghai, China). Protein (20 \u0026micro;g) was separated using SDS-PAGE gel electrophoresis and then transferred onto PVDF membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with non-fat milk for 2 h, incubated at 4\u0026deg;C overnight with the primary antibodies: anti-ZO-1 (AF5145, Affinity, 1:1000), anti-Occludin (DF7504, Affinity, 1:2000), anti-Nrf2 (TA0639M, Abmart, 1:2000), or anti-HO-1 (ab13243, Abcam, 1:1000). Then the membranes were washed three times with TBST. They were incubated with secondary antibody: goat anti-rabbit (A0208, Beyotime, 1:1000), goat anti-mouse (A0216, Beyotime, 1:1000) for 1 h at room temperature. The membranes were washed according to the procedure mentioned above. Immunoreactive bands were detected using an enhanced chemiluminescence reagents (BL520B, Biosharp). The relative intensities of protein bands were normalized to β-actin (GB15001, Servicebio,1:1000), GAPDH (AF0006, Beyotime, 1:1000) or Lamin B\u003csub\u003e1\u003c/sub\u003e (ab16048, Abcam, 1:2000). The relative density of bands was analyzed by Image J software (Image J, USA).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data analyses and graph generation were performed using GraphPad Prism 10.0 (San Diego, CA) and ImageJ. Results are presented as the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation (SD). The statistical process is as follows: First, a normality test is conducted. If the data follow a normal distribution, the data was assessed by one-way of variance (ANOVA) followed by Fisher's Least Significant Difference (LSD) test. The data of non-normal distribution were analyzed with the Kruskal-Wallis test followed by uncorrected Dunn's test. A significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant for all comparisons.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHUC-MSCs attenuated cerebral ischemia-reperfusion injury and exerted neuroprotective effects\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe design of the animal experiment is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. To assess neurological function, the foot-fault and adhesive removal test were measured. In the foot-fault test, model group showed more limb coordination errors versus sham group. The error rate was significantly reduced in the cell group (hUC-MSCs) versus the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In adhesive removal test, model group exhibited significantly prolonged time to sense versus sham group. And cell group displayed significant reductions in time to sense versus the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the cerebral ischemic injuries, infarct size was measured by TTC (triphenyltetrazolium chloride) staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, the model group exhibited a significant increase versus the sham group, the cell group exhibited a significant reduction in the infarct size in middle cerebral artery occlusion/reperfusion (MCAO/R) rats versus the model group.\u003c/p\u003e\u003cp\u003eWe used hematoxylin and eosin (HE), Nissl staining, and magnetic resonance imaging (MRI) to exhibit the neuronal damage and tissue pathological changes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, HE stainning revealed that there were no morphological changes in the brain of the sham group, the model group exhibited neuronal degeneration, necrosis, disordered cell arrangement, and irregular morphology, however the cell group significantly improves the morphology of brain tissue and cellular structure versus the model group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, in the sham group, Nissl-positive cell was well-organized with clearly visible Nissl bodies, the model group, Nissl-positive cells decreased, with partial pyramidal neurons exhibiting dissolved Nissl bodies, blurred contours, and lighter staining intensity. Conversely, in the cell group, there was significantly increase in the number of neurons containing Nissl bodies versus the model group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, in the sham group, the brain tissue was no abnormalities in the T2-weighted imaging (T2WI), in the model group, T2WI revealed high signal areas in the cortical, hippocampal, and partial striatal regions of ischemic hemisphere. However, the cell group exhibited a significance decrease in the high signal areas of the ischemic hemisphere, including the cortex, hippocampus, and striatum versus the model group.\u003c/p\u003e\u003cp\u003eTo assess the severity of blood-brain barrier (BBB), brain water content and tissue swelling were measured by a wet-dry weight method. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek, Model group showed a significant increase in brain water content versus the sham group. However, the cell group demonstrated a marked reduction in brain water content versus the model group.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of hUC-MSC-derived exosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe hUC-MSC-derived exosomes (hUC-MSC-exos) has been identified. transmission electron microscopy (TEM) revealed that the exosomes had a double-layered structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Nanoparticle tracking analysis (NTA) showed that the particle size of most hUC-MSC-exos was below 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In addition, Western blotting (WB) showed that the enrichment of exosomal marker proteins CD9, CD81, and Alix, while the Golgi protein GM130 was undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHUC-MSC-exos attenuated cerebral ischemia-reperfusion injury and exerted neuroprotection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn line with the results of hUC-MSCs therapy for the MCAO/R. The design of the animal experiment is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, versus the sham group, the scores, forelimb force of rats in the model group significantly decreased. And versus the model group, the scores, forelimb force of rats in exos group (hUC-MSC-exos) increased significantly. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, versus the sham group, the error rate, time of sense in the model group significantly elevated. And versus the model group, the error rate, time of sense of rats in the exos group significantly reduced.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, the brains of rats in model group displayed obviously necrosis versus the sham group. However, hUC-MSC-exos significantly reduced cerebral infarct size versus the model group.\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, HE staining indicated that the brains of the sham group showed no significant pathological alterations. Compared to the sham group, brain tissues of the model group exhibited pronounced neuronal degeneration and necrosis, disordered cell arrangement, and irregular morphology. Conversely, the exos group displayed a marked improvement in tissue structure and morphology versus the model group. Similarly, in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, in the model group, Nissl-positive cells significantly decreased and neurons had fewer cytoplasmic Nissl bodies and exhibited weaker staining, some pyramidal cells showed dissolved Nissl bodies with blurred contours, versus the sham group. In contrast, the exos group displayed a significant improvement of cellular morphology and increased number of neurons containing Nissl bodies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHUC-MSC-exos restored BBB function\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe assessment of BBB permeability is based on four indicators: brain water content, MMP-9, ZO-1, and Occludin. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, versus the sham group, the brain water content of the model group was significantly increased. However, the exo group showed a significant reduction in brain water content versus the model group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, the MMP-9 in model group was significantly increased versus the sham group. And the MMP-9 in the exos group significantly decreased versus the model group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, the expression of occludin and ZO-1 was significantly decreased in the model group versus the corresponding sham group, respectively. And the expression of occludin and ZO-1 were significantly increased in the the exos group versus the corresponding model group, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRestoring the function of BBB by anti-inflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe restoration of BBB may be achieved through anti-inflammation including cytokines changes and microglial polarization. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the IL-1β level in model group was significantly increased versus the sham group. The IL-1β level in the exos group significantly decreased versus the model group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, the IL-10 level in model group was significantly increase versus the sham group. The IL-10 level in the exos group significantly increased versus the model group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the expression of CD68 (M1-microglia) was significantly reduced in the exos group versus the model. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, the expression of CD206 (M2-microglia) was significantly elevated in the exos group versus the model group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHUC-MSC-exos activate the Nrf2/HO-1 pathway of anti-inflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHUC-MSC-exos exerted anti-inflammation by activating Nrf2 and downstream molecular HO-1. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, the levels of nuclear Nrf2 significantly increased in the exos group versus the model group. And in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, the levels of HO-1 were also significantly increased in the exos group versus the model group. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec are similar with Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, the nuclear Nrf2 significantly increased in the exos group versus the model group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that hUC-MSCs exert neuroprotective effects on cerebral ischemia-reperfusion injury through their exosomes. This effect is achieved by activating the Nrf2/HO-1 signaling pathway, which reduces neuroinflammatory responses and maintains the integrity of the BBB.\u003c/p\u003e\u003cp\u003eThe study shows that stereotactic injection of hUC-MSCs can improve neurological function, reduce the ischemic brain volume, and exert neuroprotective effects on cerebral ischemia-reperfusion injury. Previous studies have shown that the neuroprotective effects of stem cells can be realized through their exosomes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we further investigated the functions of the exosomes secreted by hUC-MSCs. The results were consistent with our predictions, showing that hUC-MSC-exos exhibited similar neuroprotective effects to hUC-MSCs in improving neurological function and reducing ischemic brain volume. Moreover, after the exosome treatment, the expression of MMP-9 decreased, while the expression of ZO-1 and Occludin increased, and the brain water content decreased, indicating that the integrity of the BBB was restored. This suggests that hUC-MSC-exos may improve the integrity of the BBB by regulating the expression and enzyme activity of the MMP-9 gene, thereby exerting neuroprotective effects. Similarly, it has also been found that hUC-MSCs can inhibit the upregulation of MMPs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and affect the permeability of BBB caused by ischemic stroke [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInflammatory responses are a significant factor affecting the integrity of the BBB [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The pathogenesis of cerebral ischemia-reperfusion injury is complex, with inflammatory responses playing a crucial role in this condition [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Inflammation not only exacerbates brain tissue damage and promotes the expansion of the infarcted area but also disrupts the BBB, further deteriorating neurological function. This study shows that after exosome action, the levels of pro-inflammatory cytokine IL-1β [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] decrease, while the levels of anti-inflammatory cytokines IL-10 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] increase, indicating a reduction in neuroinflammatory responses.\u003c/p\u003e\u003cp\u003eMicroglia are resident macrophages in the brain. Under ischemic stimulation, microglia can differentiate into two phenotypes: M1 and M2. Increasing evidence suggests that during the development of cerebral ischemia-reperfusion injury, M1 and M2 microglia can dynamically switch [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, interventions that promote the transition of microglia from an inflammatory M1 phenotype to a reparative M2 phenotype may have potential therapeutic value for cerebral ischemia-reperfusion injury\u003csup\u003e7\u003c/sup\u003e. M2 macrophages play a crucial role in the recovery process following nerve injury. By inhibiting the expression of matrix metalloproteinase-9 (MMP-9) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], they help maintain the integrity of the BBB, thereby reducing the increased permeability caused by inflammation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The current results showed that hUC-MSC-exos treatment promoted the transition of microglia from the M1 phenotype to the M2 phenotype, also suggesting a reduction in neuroinflammatory responses. As cytokines and microglial polarization reflective of neuroinflammatory response can influence the permeability of the BBB [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our results suggest that the restorative effect of exosomes on BBB integrity may be achieved through their anti-inflammatory action.\u003c/p\u003e\u003cp\u003eHO-1 is a crucial cellular protective molecule that plays a vital role in suppressing inflammatory responses. Numerous studies have shown that HO-1 can promote the development of macrophages into an anti-inflammatory M2 phenotype [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This process leads to the secretion of anti-inflammatory cytokines and neurotrophic factors, thereby facilitating tissue repair and nerve regeneration. Nrf2, a key transcription factor for HO-1, plays a significant role in regulating inflammatory responses [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Activating Nrf2 can reduce tissue damage and promote functional recovery [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In addition to its role in inflammation, the activation of Nrf2 in stem cells and their exosomes has become a critical mechanism for these cells to exert anti-inflammatory and protective effects, particularly in ischemic injuries [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, compared to the model group, after hUC-MSC-exos administration, the levels of HO-1 and nuclear Nrf2 were significantly elevated, and the Nrf2/HO-1 signaling pathway was activated, which regulated the polarization of microglia towards a reparative M2 phenotype, thereby exerting an anti-inflammatory effect [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This suggests that hUC-MSC-exos can alleviate neuroinflammatory responses by activating the Nrf2/HO-1 signaling pathway, maintaining the integrity of the BBB, and exerting neuroprotective effects.\u003c/p\u003e\u003cp\u003eThis study has several limitations. Firstly, we only evaluated the short-term therapeutic effects of hUC-MSCs and their exosomes on cerebral ischemia-reperfusion injury, without exploring their long-term efficacy or sustained impact. Future studies should extend the observation period to assess the long-term effects of hUC-MSCs and their exosomes on neurological recovery. Secondly, while this study primarily focused on the anti-inflammatory effects of hUC-MSCs and their exosomes in cerebral ischemia-reperfusion injury, it did not address their role in oxidative stress, despite the significant roles of Nrf2 and HO-1 in antioxidant defense. Lastly, the potential of hUC-MSCs to differentiate into neurons and replace lost neurons remains unexplored. To validate the role of hUC-MSCs in neural repair, future research should focus on evaluating the differentiation potential of hUC-MSCs, particularly their contributions to neuronal reconstruction and functional recovery following cerebral ischemia-reperfusion injury [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings indicate that hUC-MSCs offer neuroprotective effects on rats with cerebral ischemia-reperfusion injury. Moreover, the mechanism of hUC-MSCs' action may involve activating the Nrf2/HO-1 signaling pathway through exosomes it secretes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The exosomes from hUC-MSCs promote the nuclear transfer of Nrf2, which in turn inhibits inflammatory responses and regulates microglia to shift from an inflammatory M1 phenotype to a reparative M2 phenotype. This leads to a reduction in pro-inflammatory factors and an increase in anti-inflammatory factors, thereby enhancing the repair of the BBB and exerting neuroprotective effects against cerebral ischemia-reperfusion injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval, And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20240322).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hebei Province Introducing Foreign Intelligence Project and the Natural Science Foundation of Hebei Province (H202220601) and Capital Health Development Research Special Fund (2022-1-2041).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.Z. and J.Z. conceptualized the project. T.W. J.Z. and G.X. designed the experiments. \u0026nbsp;J.Z. G.X. and A.L. conducted the experiments and drafted the manuscript. P.Z. and J.Z. revised the manuscript. D.Z. and J.Y. provided technical advice. J.Z. and G.X. analyzed the data. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed for this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStegner D, Klaus V, Nieswandt B. Platelets as Modulators of Cerebral Ischemia/Reperfusion Injury. Front Immunol. 2019;10:2505.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu M, et al. 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Stem Cell Res Ther. 2022;13:429.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cerebral ischemia-reperfusion injury, Human umbilical cord mesenchymal stem cell, Exosomes, Neuroinflammation, Blood-brain barrier","lastPublishedDoi":"10.21203/rs.3.rs-7032340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7032340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eIschemia-reperfusion can aggravate cerebral damage. Mesenchymal stem cells have gained attention to improve the outcome of ischemia-reperfusion injury. This study aims to investigate the effects and mechanisms of human umbilical cord mesenchymal stem cells (hUC-MSCs) on cerebral ischemia-reperfusion injury in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eA middle cerebral artery occlusion-reperfusion (MCAO/R) model was successfully established, and hUC-MSCs or hUC-MSC-derived exosomes (hUC-MSC-exos) were injected into rats via stereotactic brain injection or the tail vein. Neurological functions were evaluated using Garcia, foot-fault, adhesive removal, and forepaw grip strength tests. In addition, we detected the expression of IL-1β, IL-10 and MMP9 in brain tissue using enzyme-linked immunosorbent assay. Immunofluorescence experiments detected the express of CD68 in the M1- microglia, the express of CD206 in the M2-microglia and the expression of Nrf2 in brain tissue. Western blotting experiments detected the expression of occludin, ZO-1, HO-1, and Nrf2 in brain tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eHUC-MSCs significantly reduced the error rate and time to sense in the foot-fault test and in adhesive removal test. Similarly, hUC-MSCs can also significantly reduced infarct size and brain water content. HUC-MSCs improved morphology of brain tissue and cellular structure, including an increase in number of neurons containing Nissl bodies. And T2-weighted imaging revealed a reduction in high signal areas within the ischemic hemisphere in the cell group (hUC-MSCs). Further findings demonstrated that hUC-MSC-exos also improved neurological function and ameliorated the brain injury and morphological changes. In addition, hUC-MSC-exos decreased the contents of IL-1β, MMP-9, and the expression of CD68 (M1-microglia) r, augmented the expression of IL-10, ZO-1, Occludin, Nrf2, HO-1, and the expression of CD206 (M2-microglia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThese results indicated that human umbilical cord mesenchymal stem cells may excert neuroprotective effects in cerebral ischemia-reperfusion injury by inhibiting inflammation and protecting blood-brain barrier integrity via exosome-mediated Nrf2/HO-1 signaling pathway.\u003c/p\u003e","manuscriptTitle":"Human umbilical cord mesenchymal stem cells achieve neuroprotection via exosome-mediated anti-inflammation and blood-brain barrier recovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 10:10:35","doi":"10.21203/rs.3.rs-7032340/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3046b857-790b-4ba1-bac9-4b92f905be83","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-21T07:21:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 10:10:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7032340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032340","identity":"rs-7032340","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}