The Role And Mechanism Of Umbilical Cord MSC-derived Exosomes In Inflammatory Response Of Microglia After Ischemic Stroke

Background:Stem cell(MSC)transplantation is a research hotspot in the treatment of brain injury(including ischemic stroke)with optimistic effects,but its mechanism of action is not clear.Recent studies have shown that exosomes(Exos)secreted by stem cells have obvious effects on various disease models,suggesting that MSC-Exos may be one of the new mechanisms for stem cell transplantation for ischemic stroke.Microglial cells are known to be mainly involved in the central nervous system(CNS)inflammatory response and are active after brain injury.Therefore,this study intends to use human umbilical cord mesenchymal stem cells(hUMSC)as a cell source for transplantation and the stroke model is a treatment object.Starting from the correlation between umbilical stem cell exosomes(hUMSC-Exos)and microglial cells,the role of hUMSC-Exos and microglial cells in the process of stroke was explored.Objective:To investigate the regulation and mechanism of hUMSC-Exos on microglial-mediated neuroinflammatory response after ischemic stroke.Research content and methods:1.Culture,expansion and identification of hUMSC,and isolation and identification of Exos(1)Detection and identification of MSC surface markers by flow cytometry.(2)100000g ultracentrifugation method was used to isolate exosomes from hUMSC culture supernatant,and electron microscopy,NTA,and WB were used to detect whether Exos met the exosomes identification standards.2.Explore the effect of hUMSC-Exos on post-stroke microglia inflammation(1)Explore the effects of hUMSC-Exos post stroke,inflammatory response,and microglial inflammation in vivo.C57BL/6 mice were divided into sham operation group(Sham group),surgery group+PBS group(Vehicle group),surgery+Exos treatment group(hUMSC-Exos group),and the latter two groups were induced by MCAO stroke model.Intravenous injection of hUMSC-Exos 250 ?l PBS diluted 50?g of Exos into the hUMSC-Exos group,and injection of 250 ?l PBS into the Vehicle group as controls.After 72 hours,the neurological score,TTC staining to detect cerebral infarction volume,HE staining,immunohistochemical analysis of inflammatory protein interleukin 6(IL-6),expression of nuclear factor kB(NF-kB).Immunofluorescence detection of M1 and M2 microglial cells expressing IBA-1+CD16 and IBA-1+CD206,respectively.ELISA to detect the expression of IL-6,tumor necrosis factor(TNF-?)and interleukin 1b(IL-1b)in the injured brain.(2)In vitro experiments explore the effect of hUMSC-Exos on the inflammatory response of microglial cells activated by glucose-oxygen deprivation(OGD)model.Exos was labeled with PKH26,and co-cultured with microglia BV2 for 6 hours to observe the uptake of Exos by microglia.In addition,microglia were divided into the untreated control group(Con group)and experimental model group(OGD group),OGD and hUMSC-Exos co-culture group(OGD-1+hU-MSC-Exos group),qPCR and ELISA methods were used to detect the expression of IL-6,TNF-?,IL-1b in cells and cell supernatants.3.Explored the miR-146a-5p in hUMSC-Exos mediating IRAK/TRAF6 channels on microglial inflammation(1)To explore the effect of miRNAs in Exos on the inflammation response of microglia.Small interfering RNA(siRNA)was used to interfere the synthesis of Drosha,a key protein in miRNAs,then we collected exosomes for in vitro functional experiments.In vitro experiments,microglia are divided into a blank control group(Control group),OGD model group(OGD group),OGD model and si-Drosha blank vector exosomes co-culture control group(Exossi-Control group),OGD model and si-Drosha interference exosomes co-culture group(Exossi-Drosha group),qPCR and ELISA were used to detect the expression of IL-6,TNF-? and IL-1b in the cell transcription level and the supernatant respectively.(2)hUMSC-Exos was subjected to miRNA second-generation sequencing,and miR-146a-5p was selected from the top ten expression vectors for mechanism research.Lentiviral vector-mediated shRNA was used to inhibit miR-146a-5p of hUMSC,and the exosomes were collected and used subsequent cell and animal function experiments.(3)In in vitro experiments,microglial BV2 was divided into control group(Con group),model group(OGD group),microglial OGD model and hUMSC-Exos co-culture group(OGD+Exos group),microglial OGD model and miR-146a-5p knockdown exosomes co-culture group(OGD+miR-146a-5p k/d Exos group)and microglial OGD and miR-146a-5p knockdown vector exosomes co-culture group(OGD+miR-cn Exos group).IL-6,IL-1b and TNF-? and signaling pathways IRAKI,TRAF6 and NF-?B(p-p65)in cells detected by WB.The expression of IL-6,TNF-?,and IL-1b in the supernatant was detected by qPCR and ELISA,respectively.(4)In in vivo experiments,the effects of miR-146a-5p in hUMSC-Exos on stroke,inflammatory response,and microglial inflammatory response were explored.C57BL/6 mice were divided into MCAO model tail vein injection of miR-146a-5p knockdown exosomes group(miR-146a-5p k/d Exos group)and MCAO model tail vein injection of miR-146a-5p knockdown vector control group(miR-CN Exos group);each mouse in the miR-146a-5p k/d Exos group was intravenously injected with miR-146a-5p k/d Exos 250ul containing 50ug of exosomal suspension.The miR-CN Exos group was injected with a volume of miR-CN Exos 250ul containing 50 ug of exosomal suspension as a control.After 72 hours,the neurological scores of each group were compared,TTC staining was used to detect cerebral infarction volume,and immunohistochemical analysis for IL-6,NF-kB expression,immunofluorescence for M1 microglia IBA-1+CD16 expression,WB detection of TRAF6,IRAK1,p-p65 expression,and ELISA detection of injured brain IL-6,TNF-?,IL-1b expression.Results:1.(1)hUMSC streaming results were positive for CD73(100%),CD105(99.96%)and CD90(100%),and CD45(0.06%),HLA-DR(0.50%),CD34(0.06%),CD11b(0.06%)and CD9(0.46%)are negative,suggesting that the hUMSC used in this study meets the MSC identification standards.(2)the exosomes morphology is a typical morphology of the double-layered film wine tray,the size is mainly concentrated in the 30?150 nm,CD9,Alix,and TSG101 exosomal marker proteins were expressed,indicating that hUMSC secretes exosomes.The exosomes collected in this study met the exosomal identification standards and laid the foundation for subsequent research;2.(1)Compared with the in vivo experimental control group,hUMSC-Exos promotes the recovery of neurological and behavioral behavior in mice(p<0.01),reduces cerebral infarct volume(p<0.05),reduces tissue and cell edema,and reduces the expression of IL-6 and NF-kB(p<0.05),reduces M1 microglia(p<0.05),increases M2 microglia(p<0.05),reduces IL-6,TNF-?,IL-Expression of 1b(p<0.05);(2)Uptake of exosomes in microglia was observed;compared with the OGD group in vitro,hUMSC-Exos significantly reduced IL-6,TNF-?,IL-1b transcription level(p<0.01)and the contents of IL-6,TNF-?,and IL-1b in the supernatant(p<0.01);It was revealed that hUMSC-Exos protects stroke,reduces post-stroke inflammatory response,and pro-inflammatory response of microglia.3.(1)Compared with the Exosssi-control control group,exosomes in the Exosssi-Drosha group reduced the transcription levels of IL-6,TNF-?,and IL-lb(p<0.05),and reduced the IL-6,TNF-?,and IL-1b in supernatant(p<0.05);(2)miR-146a-5p is the top ten miRNAs of hUMSC-Exos,molecules of interest;(3)in vitro experiments,compared with OGD+ Exos and OGD+miR-cn Exos group,the expression of IL-6,IL-1b,and TNF-?,and the signaling pathways IRAK1,TRAF6,and NF-?B(p-p65)in microglia both increased(p<0.01),and IL-6,TNF-?,and IL-1b were also significantly increased in the transcription level and in the cell supernatant in the miR-146a-5p k/d Exos group;(4)In the in vivo experiment,compared with the miR-cn Exos group,the miR-146a-5p k/d Exos group had poor neurological and behavioral recovery(p<0.01),severe cerebral infarction(p<0.05),and the expression IL-6 and NF-kB were higher in the injured brain tissue(p<0.05),and M1 microglia marker IBA-1+CD16 positive in the injured brain tissue were more expressed(p<0.05),IRAK1,TRAF6 and p-p65 were also more expressed(p<0.05).IL-6,TNF-? and IL-1b expressed in the injured brain tissue were also more.It was revealed that miR-146a-5p in hUMSC-Exos inhibits IRAK/TRAF6 pathway and reduces microglial inflammation.Conclusion:hUMSC-Exos reduces ischemic stroke damage by miR-146a-5p inhibiting microglial IRAK1/TRAF6 signaling pathway and reducing microglial-mediated inflammatory response,stem cell exosomes replace stem cells to treat CNS damage is possible.