Extracellular Vesicles Derived From Adipose Stem Cells Promote Microglial M2 Polarization And Improve Neurological Recovery After Cerebral Ischemia In Mice

Recent studies have demonstrated that adipose-derived stem cells(ADSCs)and their extracellular vesicles(ADSC-EVs)exhibit tremendous therapeutic potential in cerebral ischemia/reperfusion injury.However,the molecular mechanism of ADSC-EVs in the treatment of cerebral ischemic injury is still poorly understood.In this study,I aimed to explore the role of intravenously injected ADSC-EVs and its micro RNA(mi RNAs)contents in the treatment of cerebral ischemia in mice and the underlying mechanisms.In this study,I first isolated and expanded ADSCs and purified ADSCEVs through ultracentrifugation.Then,ADSC-EVs were intravenously injected to mice during 1-7 days after t MCAO,the therapeutic effects of ADSC-EVs were detected by modified neurological severity score(m NSS),rotarod tests,hanging wire tests,T-maze and step through tests.Brain atrophy was examined by cresyl violet staining.Uptake of PKH-26 labeled ADSC-EVs by microglia were confirmed by Iba1 immunofluorescence staining.Polarization of microglia,angiogenesis and white matter injury were detected by immunofluorescence staining of M1 marker CD16,M2 marker Arg1,vascular endothelial cell marker CD31,and mature oligodendrocyte marker MBP.In vitro,the effects of ADSC-EVs on the polarization of microglia under normal condition,LPS induction or OGD injury were examined by q PCR.In order to explore the contribution of mi RNAs contained in ADSC-EVs in regulating microglia polarization,we performed mi RNA sequencing of ADSC-EVs,and analyzed the relationship between the upregulated mi RNAs and microglia polarizationrelated proteins via IPA.Our results showed that ADSC-EVs administration significantly reduced brain atrophy volume and improved neuromotor function and spatial memory after ischemic stroke in mice.Further studies revealed that ADSC-EVs regulated the polarization of microglia after cerebral ischemia,promoted M2 phenotype and decreased pro-inflammatory M1 phenotype,resulting in increased brain repair.The number of blood vessels,as well as newly proliferated endothelial cells in peripheral ischemia area of mice in ADSC-EVs treatment group were significantly higher than that in the PBS group.In addition,MBP loss in ADSC-EVs treatment group was significantly lower compared with the PBS group.In vitro experiments further confirmed the uptake of ADSC-EVs by primary microglia.Under LPS stimulation or OGD injury,ADSC-EVs treatment significantly upregulated the expression of microglia M2 marker Arg1 and inhibited the expression of microglia M1 marker CD16,IL-1β,TNFα,and i NOS.Notably,the upregulation of Arg1 was positively correlated with the dose of ADSC-EVs.Finally,mi RNAs sequencing of ADSC-EVs versus Fibroblast-EVs revealed 164 up-regulated mi RNAs in ADSC-EVs.After abundance and conservation screening,14 up-regulated mi RNAs were selected and their relationships with 64 microglial polarization-related proteins were explored via IPA analysis.STAT1 and PTEN were highlighted as two downstream proteins targeted by most mi RNAs.Further studies verified that ADSC-EVs treatment indeed significantly inhibited the expression of STAT1 and PTEN in cultured microglia under LPS or OGD injury.Our results demonstrated that ADSC-EVs could regulate the polarization of microglia both in vivo and in vitro.mi RNAs in ADSC-EVs may contribute to its function on microglia polarization.