Effects And Mechanisms Of Exosomes Isolated From Human Umbilical Cord Mesenchymal Stem Cells On Alzheimer's Disease

Background:Alzheimer's disease(AD)is the most common dementia which severely damages memories and cognitive functions of patients.According to the statistical data published by WHO in 2015,more than 47 million people suffered from dementia and this number will triple by 2050.All forms of AD are characterized by excessive amyloid-? peptide(A?)accumulation in the brain,which triggers the neuroinflammation process in the central nervous system(CNS).Neuroinflammation and oxidative stress are both obvious pathological features of neurodegenerative diseases which has been indicated to mediate neuronal damage and also aggravate the backward accumulation of A? in AD.It is urgent to seek an effective strategy to treat this disease based on its pathophysiology.In recent years,cell therapies have been demonstrated as potential approaches for AD treatment.In previous researches,our team investigated the therapeutic effects of hucMSCs on AD in vivo.However,hucMSCs have relative limitations in the maintenance of biological activity,the quantification of bioactive substances,and the logistics of delivery in clinical therapies.Thus,there is an urgent need to find an effective cell-free approach to mimic the functions of hucMSCs and avoid their flaws.A novel member of extracellular vesicles named exosomes has been considered as a promising cell-free therapy because of their multiple biological activities and function of cell-to-cell communication.In recent years,numerous research teams have proved that as a new therapeutic method for many diseases,exosomes isolated from hucMSCs(hucMSC-exosomes)possess their particular morphological and functional characteristics.HucMSC-exosomes are 30-150nm extracellular membrane vesicles CD9,CD63 and CD81 positive.They serve as natural transporters of RNA,cytokines and proteins from hucMSCs to other cells in different tissues to act on cell proliferation,immune-regulation and many other functions.In clinical treatment,hucMSC-exosomes express all of the advantages of hucMSCs and avoid their imperfections.They are also easier to be quantified and maintain their bioactivities in preservation and transport.Moreover,multiple researches have shown that exosomes can cross the blood-brain barrier(BBB),especially under pathological conditions such as AD and other neurodegenerative diseases.They realize the intercellular communication from peripheral circulation to the CNS.In addition,because of the source of human,hucMSC-exosomes seem easier to be accepted in clinic treatment by both doctors and patients in the future.However,whether hucMSC-exosomes can be used in the treatment of AD remains unknown,especially concerning the anti-neuroinflammatory and anti-oxidative stress effects of hucMSC-exosomes.Objective1.To investigate the effects of hucMSC-exosomes-injection A? deposition,and cognitive functions in A?PP/PS1 transgenic AD mouse models.2.To detect the effects of hucMSC-exosomes on inflammatory regulating and A?clearance in vivo and in vitro.3.To investigate the effects of hucMSC-exosomes on oxidative stress and A?generation in vivo and in vitro.Methods:1.We obtained fresh human umbilical cords of full-term births by cesarean section,cultivated hucMSCs to migrate from the Wharton's jelly.HucMSC-exosomes were extracted from the cells culture supernatants by employing ExoQuick-TC(System Bioscience,CA,USA).The electron microscopic images were captured and analyzed by a transmission electron microscope at 80kV.Western blot was used to detect the surface markers CD9,CD63,of HucMSC-exosomes.2.To investigate the potential therapeutic effects of hucMSC-exosomes on AD,we used APP/PS1 mice for the hucMSC-exosomes group and APP/PS1 mice for the control group.In addition wild-type mice(WT)with a C57BL/6 background were used as the proper controls in behavior tests.The hucMSC-exosomes suspension(30?g dissolved in 100?L PBS)or control solution was injected into them through tail veins,every 2 weeks,4 times altogether.Three weeks after the last injection,we evaluated the spatial memory performance of mice by using the modified Morris water-maze(MWM)test.Thioflavin S staining was used to evaluate the number of A? plaques in the cortex and the hippocampus.Furthermore,ELISA was performed to explore soluble A?40 and A?42 quantities in the brains of AD mice.Western blot was used to detect levels IDE,NEP,BACE1,A?PP and CTF?in the two groups.3.To detect the anti-neuroinflammatory effects of hucMSC-exosomes on AD mice,we used Immunofluorescent staining and Quantitative real-time PCR to investigate the activation of microglial cells in the brains of AD mice.ELISA was conducted to analyze the levels of inflammation-related factors transforming growth factor-?(TGF-?),Interleukin-10(IL-10),Tumor necrosis factor-?(TNF-?)and Interleukin-1?(IL-1?)from mice brains and peripheral blood(PB).4.To investigate the influence of hucMSC-exosomes on microglial alternative activation in vitro,A?25-35 pretreated BV2 cells were treated either with hucMSC-exosomes(30?g/ml)or with the reagent of the control group for 24 hours.The assessment of the alternative activation relied on molecule markers Argl and Ym1.Immunofluorescent staining and Quantitative real-time PCR were used to investigate the activation of BV2 cells in vitro.We also used ELISA kits to detect the inflammatory cytokines in the culture medium of BV2 cells5.To detect the anti-oxidative stress effects of hucMSC-exosomes on AD in vivo,the oxidative stress kits of GSH,GSSG,GPX,SOD,MDA were used to evaluate the levels of oxidative stress damage in different groups of AD mice.6.To detect the anti-oxidative stress effects of hucMSC-exosomes on AD in vitro,A?1-42 pretreated SH-SY5Y cells were treated either with hucMSC-exosomes(30?g/ml)or with the reagent of the control group for 24 hours.Then the oxidative stress kits of GSH/GSSG,GPx,SOD,MDA and CCK-8 kit were used to evaluate the levels of oxidative stress damage and cell viability in different groups.Results1.Effects of hucMSC-exosomes-injection on AD mice1.1Injection of hucMSC-exosomes increases spatial learning and memory function of A?PP/PS1 miceThe mice treated with hucMSC-exosomes had significantly shorter mean escape latency than the control group.Compare with WT group,the mice treated with hucMSC-exosomes had a longer mean escape latency in the second and third days but had no significant difference in the fourth and fifth days.The mice spatial memory was evaluated by performing probe trials 24 hours after the last training.The mice treated with hucMSC-exosomes accomplished a larger number of platform location crossing times and a longer time spent in the target quadrant than the mice from the control group but had no significant difference compared with WT group,suggesting that the hucMSC-exosomes injection increased the behavioral performance of the mice.The swimming speed was similar between the two groups.1.2.Effects of HucMSC-exosomes injection on A? deposition and soluble A?quantitiesThe number of A? plaques in the cortex and the hippocampus areas of the brain were significantly lower in the hucMSC-exosomes-injected group than in the control group.Furthermore,hucMSC-exosomes injection reduced both soluble A?40 and A?42 levels in the mice.2.HucMSC-exosomes promote the clearance of A? through regulation neuroinflammation2.1 HucMSC-exosomes increase the level of A? degrading enzymesWe found that the levels of IDE and NEP were higher in the mice treated with hucMSC-exosomes than in the control group which indicated that hucMSC-exosomes could increase the levels of A?-degrading enzymes in vivo2.2 HucMSC-exosomes injection reduces the inflammatory reaction of microglia and induces alternative microglial activation in vivoThe density of Iba-1-positive microglia presented by quantitative image analysis was lower in the hucMSC-exosomes injection group than in the control group.It was demonstrated by the qRT-PCR assay that gene-expression levels of markers for M2-like microglia YM-1,Arg-1,MRC1,FIZZ1,and CD163 in the brains of the mice with hucMSC-exosomes injection were higher than those in the mice treated with the reagent of the control group.The levels of the pro-inflammatory cytokines IL-1? and TNF-a were lower in the hucMSC-exosomes injection group than in the control group in PB and brains respectively.In contrast,the levels of the anti-inflammatory cytokines IL-10 and TGF-? were higher in the hucMSC-exosomes injection group than in the control group.2.3 Immunoregulatory effects of hucMSC-exosomes on BV2 cellsIn our tests of immunofluorescent staining,Ym-1 staining intensity was increased in the hucMSC-exosomes group compared with the control group.Using qRT-PCR the up-regulation of Argl and Ym1 was also determined.A significant increase in Argl and Yml RNA levels was observed after treatment with hucMSC-exosomes.The levels of the proinflammatory cytokines,IL-1? and TNF-a,were lower in the hucMSC-exosomes group than in the normal control group.In contrast,the level of the anti-inflammatory cytokines IL-10 and TGF-? were higher in the hucMSC-exosomes group than in the normal control group.3.HucMSC-exosomes decreased the generation of A? through reducing oxidative stress damage3.1 Injection of hucMSC-exosomes reduced the level of BACE1 and decreased the generation of A?We found that the levels of BACE1 and CTF? were lower in the mice treated with hucMSC-exosomes than in the control group which indicated that hucMSC-exosomes could reduce the levels of APP-cleavage enzymes and decreased the generation of A? in vivo3.2 HucMSC-exosomes injection reduced oxidative stress damage in vivo and in vitroWe found that the levels of GSH/GSSG.GPx and SOD were higher in the groups treated with hucMSC-exosomes than in the control groups,while the level of MDA was lower in the groups treated with hucMSC-exosomes than in the control groups in vivo and in vitro.Conclusion1.Injection of hucMSC-exosomes increases the cognitive functions and decrease the levels of A? deposition and soluble A? quantities in A?PP/PS1 mice2.Injection of hucMSC-exosomes promotes A? degradation and decrease A?generation in A?PP/PS1 mice3.HucMSC-exosomes reduces the inflammatory reaction of microglia and induces alternative microglial activation in vivo and in vitro4.HucMSC-exosomes reduces the oxidative stress damage in vivo and in vitiroSignificanceOur study will help further the understanding of the effects of hucMSC-exosomes on alleviating the cognitive functions and A? deposition in A?PP/PS1 mice,and investigate the mechanisms in neuroinflammation and oxidative stress.Moreover,it will help us advance the understanding of the role of hucMSC-exosomes in therapy of AD.