| Background and objectivesStroke is a serious neurological disorder with high mortality and disability rates,and is classified into Hemorrhagic stroke(HS)and Ischemic stroke(IS).The epidemiological data show that stroke has become the second most common cause of death in the world,and the first cause of death in China.IS is the most common type of stroke,accounting for 75% of all stroke.IS is a disease of brain necrosis caused by insufficient blood supply to brain tissue due to narrowing or occlusion of arteries supplying blood to brain tissue.Despite the fact that the diagnosis and treatment of IS have received attention and importance worldwide,the treatment options for IS patients,especially for acute large vessel occlusive stroke,still seem to be deficient nowadays.Except for acute thrombolysis or thrombectomy,no effective strategies that can significantly improve the prognosis of IS patients have emerged,and the short time window for acute thrombolysis or thrombectomy results in the fact that the vast majority of IS patients are unable to receive timely treatment and thus develop irreversible ischemic damage to brain tissue.Therefore,exploring new strategies that can effectively treat IS remains a hot issue in clinical practice and basic research.After IS,neuroinflammation plays an important role in post-ischemic injury of brain tissue,and previous studies have shown that modulating the development of neuroinflammation in the early period after IS can alleviate neurological impairment.Microglia,which are intrinsic immune cells in the brain,are cells that firstly respond to injury signals released after ischemic damage,rapidly being activated and secreting proinflammatory cytokines and chemokines.Macrophages are recruited by chemokines to enter the ischemic brain tissue after the integrity of the blood-brain barrier(BBB)is disrupted after IS,and they are also rapidly activated to collaborate with microglia to promote inflammation and exacerbate brain injury.Therefore,microglia and macrophages play a key role in the progressive changes of neuroinflammation after ischemic stroke.Mesenchymal stem cells(MSCs),also known as mesenchymal stromal cells,are easier to be obtained than embryonic stem cells,and are an important type of adult stem cells involved in medical research.They have properties such as immunomodulation and homing,which can affect the microenvironment of damaged tissues and enhance tissue repair,providing a broad prospect for X the development of cellular therapies.Human amniotic MSCs(MSCs)are a special type of MSCs derived from the placenta,with low immunogenicity and strong proliferative properties,which are easy to be cultured under suitable culture conditions and are suitable for stable preparation in vitro.In addition,the high plasticity and multidirectional differentiation potential,strong immunomodulatory ability,as well as the low risk of tumorigenicity after transplantation from human amniotic membrane MSCs,these advantages make human amniotic membrane MSCs have a wide range of prospects for clinical applications,and applications have been carried out in the fields of cardiac repair,neural reconstruction,bone remodeling,and liver regeneration,while less experience has been gained for the application of human amniotic membrane MSCs in the neurological disorders.Extracellular vesicles(EVs)are small membrane vesicles released from cells.They can carry a variety of biomolecules,such as proteins,nucleic acids,lipids,etc.,and transfer information between cells,which play a very important role in both normal physiological and pathological processes.Therefore,EVs have a vast range of applications in the areas of disease diagnosis,therapy and drug delivery.Recent studies have shown that the survival rate of MSCs after transplantation to animal is not high,and the number of MSCs in the lesion is small,so it is hypothesized that the therapeutic effect on brain tissue injury may be more derived from the effect of EVs rather than the effect of MSCs in the lesion itself.Studies have shown that the use of EVs in the treatment of brain tissue injury has similar therapeutic effects to those of MSCs,and also avoids the drawbacks of stem cell transplantation,such as embolization of microvessels by stem cell clusters or potential tumorigenicity.However,whether human amniotic MSC-derived extracellular vesicles(MSC-EVs)can exert neuroprotective effects in the treatment of ischemic stroke and whether they can modulate neuroinflammation after ischemic stroke and regulate the inflammatory state of microglia and macrophages after IS is still unclear and requires further investigation.The aim of this study is to explore whether MSC-EVs could exert neuroprotective effects in a rat model of acute ischemic stroke using high-quality MSC-EVs derived from human amniotic membrane MSCs,to validate the safety of in vivo application of MSC-EVs,and to investigate the modulation of neuroinflammatory damage by MSC-EVs in vivo and in vitro.The potential mechanism of regulating inflammation will be clarified by transcriptomic analysis,and the effector proteins of MSC-EVs regulating neuroinflammation will be screened by proteomics,which will provide further theoretical support for the translation of MSC-EVs to the clinical practice.MethodsThis study is divided into the following three parts:Part Ⅰ: Extraction and characterization of MSC-EVs and neuroprotective effects on ischemic stroke(1)Human amniotic MSCs were cultured and MSC-EVs were extracted with high quality using tangential flow filtration and size exclusion chromatography,and extracellular vesicles characterization was performed by transmission electron microscopy,nanoparticle tracking analysis(NTA)and Exo View(?) extracellular vesicle surface marker protein analysis;(2)The MCAO/R model was constructed,and the experimental animals were divided into the sham-operated group(Sham group),the MCAO/R model with PBS administration group(MCAO-PBS group)and the MCAO/R model with MSC-EVs administration group(MCAOEV group).(3)The neuroprotective effect of MSC-EVs was evaluated by observing the size of infarcted area by TTC staining.The integrity of blood-brain barrier was evaluated by bloodbrain barrier permeability test.The alteration of tissue structure and neuronal damage was evaluated by HE staining and Nissl staining,and the neurological function damage and recovery was evaluated by Longa score,rotarod test,open field test,new object recognition test and water maze test.The safety of MSC-EVs was verified by recording body weight and HE staining of organs other than brain tissue.(4)The effects of MSC-EVs on the repair of brain tissue damage were evaluated by observing neurons,astrocytes,myelin sheaths,blood vessels and cell proliferation in ischemic brain tissues after IS using immunofluorescence.The safety of MSC-EVs was verified by recording body weight and HE staining of other organs outside the brain tissue.Part Ⅱ: Modulation of neuroinflammation by MSCs-EVs in the MCAO/R model(1)Inflammatory cytokine levels in brain tissue were measured using CBA multifactor flow assay to assess the severity of neuroinflammation and the ability of MSCs-EVs to modulate inflammation.(2)Inflammatory cytokine levels within the blood were measured using CBA multifactor flow assay to assess the ability of MSC-EVs to regulate inflammation within the blood after IS.XII(2)The pro-inflammatory phenotype and anti-inflammatory phenotypic changes of microglia within brain tissue after ischemic stroke were observed by immunofluorescence on tissue sections in the acute phase(4 days)and chronic phase(30 days)after IS.(4)Pro-inflammatory phenotypic changes of macrophages in brain tissue and blood after IS detected by flow cytometry.Part Ⅲ: Regulation of the inflammatory state of microglia and macrophages in vitro by MSCs-EVs and the underlying mechanisms(1)We constructed an in vitro microglia LPS model,and detected the effects of MSCsEVs on the secretion of inflammatory factors,morphology,early apoptosis,cell migration ability,cell phagocytosis,and macrophage chemotaxis ability of microglial cells in the in vitro LPS model by CBA multifactor assay,flow cytometry,cell scratch assay,and transwell cell chemotaxis assay.(2)We constructed an in vitro macrophage LPS model,and detected the effects of MSCsEVs on the secretion of inflammatory factors,morphology,early apoptosis,cell migration ability,and microglia chemotaxis ability of macrophage in the in vitro LPS model by CBA multifactor assay,flow cytometry,cell scratch assay,and transwell chemotaxis assay.(3)Screening the differential gene expression between LPS+MSC-EV group and LPS group by transcriptome sequencing and bioinformatics analysis,and performing GO functional enrichment analysis and KEGG pathway enrichment analysis to explore the potential mechanism of MSCs-EVs inhibiting cellular inflammatory activation.We screened MSC-EVs and human fibroblast-derived extracellular vesicles(HSF-EVs)for differential proteins by extracellular vesicle microproteomics and bioinformatics analyses,and performed GO functional enrichment and KEGG pathway enrichment analyses,to validate the ability of proteins in MSC-EVs to regulate cellular inflammation and to screen potential key inflammation-regulating proteins of MSC-EVs.ResultsThe results of the first part of the study: MSC-EVs were identified by transmission electron microscopy,NTA detection and Exo View(?) extracellular vesicle surface marker protein analysis.The results of the Exo View(?) extracellular vesicle surface marker protein analysis provided the characteristics of the subpopulation of extracellular vesicles used in this study.The validation of the effectiveness of MSC-EVs on ischemic stroke treatment was carried out after constructing the MCAO/R model.MSC-EVs can reduce the area of cerebral infarction,alleviate the disruption of the integrity of blood-brain-barrier,reduce the destruction of brain tissue structure,interstitial edema,neuronal degeneration and necrosis.The behavioral test of MCAO/R model treated with MSC-EVs showed had a neuroprotective effect.The effects of MSC-EVs on the long-term repair of post-infarcted brain tissue were observed by immunofluorescence in tissue slices showed that MSC-EVs could reduce the damage of neurons in the brain tissue caused by ischemia,promote the migration or extension of astrocytes and their protrusions into the infarcted tissues,promote the repair and regeneration of myelin sheaths in the damaged area,promote the generation of neovasculature in the area of ischemic injury,and accelerate the proliferation of damaged cells.The good biosafety of MSC-EVs was confirmed.The second part of the research results: MSC-EVs could significantly reduce the increase of pro-inflammatory cytokines such as IL-1β,IL-6,TNF,and immune cell chemotactic factor MCP-1 in the brain induced by ischemic injury of brain tissue.Meanwhile,MSC-EVs could significantly reduce the increase of IL-6,TNF and MCP-1 in the blood induced by ischemic injury of brain tissue.MCAO/R model could significantly promote the migration of microglia into the ischemic brain tissue in the acute stage,and MSC-EVs could inhibit the high expression of CD86 in microglia in ischemic brain tissue in MCAO/R model and promote the high expression of Arg1 in microglia of ischemic brain tissue.In contrast,more microglia Iba1 signals could be observed in the ischemic injury area of the MCAO/R model in the chronic phase,and there was no significant difference in the expression of CD86 and Arg1 in the chronic phase between the MCAO-EV group and the MCAO-PBS group.MSC-EVs treatment of the MCAO/R model was able to inhibit the pro-inflammatory state of macrophage alterations within the blood and in brain tissues that entered ischemic injury areas through the BBB.The third part of the research results: MSC-EVs could significantly inhibit the increase of TNF secretion from microglial cells after LPS stimulation after 12 hours of MSC-EVs application.After 24 hours,MSC-EVs were able to significantly inhibit the increase of IL-6and TNF secretion from microglia after LPS stimulation.And PCR of microglial cells after 24 hours revealed that MSC-EVs could significantly inhibit the increase of IL-1β,IL-6,IL-10 and TNF m RNA expression in microglial cells after LPS stimulation.Meanwhile,MSC-EVs could inhibit the enlargement of microglia volume and intracellular granularity induced by LPS stimulation,and inhibit the high expression of CD86 and phagocytosis of microglia induced by LPS stimulation.MSC-EVs were found to inhibit the pro-apoptotic effect of LPS on microglia,XIV the pro-migratory effect of LPS on BV2 cells,and the enhancement of microglia chemotaxis towards macrophages induced by the stimulation of LPS.MSC-EVs were able to significantly inhibit the increase of IL-6 and MCP-1 secretion from macrophages after LPS stimulation after12 hours.At 24 hours after the application of MSC-EVs,MSC-EVs can significantly inhibited the increase of IL-1β,IL-6,IL-10,TNF and MCP-1 secretion from macrophages after LPS stimulation.And PCR of macrophages with MSC-EVs applied for 24 hours revealed that MSCEVs could significantly inhibit the increase of IL-1β,IL-6,IL-10 and TNF m RNA expression in macrophages after LPS stimulation.Meanwhile,MSC-EVs were able to inhibit the enlargement of macrophage volume and intracellular granularity induced by LPS stimulation MSC-EVs were found to inhibit the pro-apoptotic effect of LPS on macrophages,the promigratory effect of LPS on macrophages,and the enhancement of macrophage chemotaxis towards microglia induced by LPS stimulation.The results of transcriptomics analysis provided information on the differential gene expression between the LPS+MSC-EV group and the LPS group,and further GO and KEGG enrichment analyses revealed the potential mechanism by which MSC-EVs regulate inflammation.Extracellular vesicle microproteomics provided information on the differential proteins between MSC-EVs and HSF-EVs,and verified the ability of proteins within MSC-EVs to regulate cellular inflammation by GO functional enrichment analysis and and KEGG pathway enrichment analysis,and screened 20 potential key proteins upregulated in comparison with HSF-EVs that may play a role in the regulation of inflammation by MSC-EVs.Conclusion(1)MSC-EVs can effectively alleviate the ischemic brain injury caused by MCAO/R model,reduce the infarct area,reduce the structural damage of brain tissue,maintain the integrity of the blood-brain barrier,improve the neurological function damage caused by IS.Meanwhile,MSC-EVs have good biological safety in therapeutic dose.(2)MSC-EVs can reduce the secretion of pro-inflammatory cytokines in the brain and blood of the MCAO/R model and play an anti-inflammatory role.MSC-EVs inhibited the activation of microglial cells in the brain tissue towards pro-inflammatory direction in the acute phase,and inhibited the activation of macrophages in the blood and brain tissue of MCAO/R model towards pro-inflammatory direction.(3)In vitro,MSC-EVs were further verified to regulate the inflammatory state of microglia and macrophages.MSC-EVs were able to inhibit the pro-inflammatory cytokine secretion and gene expression of microglia and macrophages stimulated by LPS and exerted antiinflammatory effects.MSC-EVs inhibited LPS-stimulated morphological changes,proinflammatory activation,apoptosis,cell migration and chemotaxis ability of microglia and macrophages.(4)MSC-EVs can play a role in regulating inflammation through multiple inflammationrelated pathways.Comparison of MSC-EVs with HSF-EVs screened out 20 up-regulated potential key proteins of MSC-EVs that may exert function of inflammation regulation. |
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