| BackgroundIntracerebral hemorrhage(ICH)is associated with high mortality and morbidity and accounts for 10–15% of all strokes.The mortality of ICH is approximately 40% at 1 month,which makes ICH the leading cause of death in China.It has not benefit from the positive therapy for ICH induced primary injury(such as early removal of the hematoma and prevention of hematoma expansion)in clinical trials thus far,therefore,we have focused instead on the secondary injury.Secondary injury following ICH is triggered by the presence of intraparenchymal blood,which subsequently activates cytotoxic,excitotoxic,oxidative,and inflammatory pathways.Increasing evidence has shown that inflammatory injury plays a critical role in ICH-induced secondary brain injury.The use of fingolimod Inhibit peripheral inflammatory cell infiltration can reduce inflammatory damage after ICH.CD4~+CD25~+ regulatory T cells have immunomodulatory function and play an important role in autoimmune tolerance and immunosuppression.In lupus nephritis,ankylosing spondylitis,rheumatoid arthritis and other autoimmune inflammation,Treg cells can delay the progress of the disease and improve the prognosis.In ischemic stroke,knockout Tregs aggravates brain tissue damage,and the transplant of Tregs can reduce the damage.In ICH,the number of peripheral blood Tregs was reduced,and the transplant of Tregs could relieve the secondary inflammation injury of ICH.Although the transplant of Tregs can reduce the inflammation of the central nervous system,the mechanism is not clear.Previous studies have shown that Tregs can reduce the infiltration of inflammatory factors in the central nervous system,reduce the damage of blood-brain barrier,secrete anti-inflammatory factors,regulate antigenic delivery,and regulate inflammatory cell activation.These effects mainly through two pathways,one is through its surface molecules such as LAG-3 or CTLA 4 effect on inflammatory cells,the other is secrete anti-inflammatory cytokine such as IL-10,TGF-? to regulate the function of inflammatory cells and immunosuppression through relevant signal pathways.Increasing evidence suggest that the resident microglia are believed to be the early inflammatory cells in response to the extravascular blood components mediate inflammatory cascades [6,7].But the interesting thing is,activated microglia have two sides and are divided into M1(classicactivation)and M2(alternative activation)according to different functions and surface markers.The M1 phenotype can secrete a large number of pro-inflammatory cytokines such as IL-6 and TNF-?,which mediate inflammatory cascade and aggravate inflammatory damage.The M2 phenotype secrete protective anti-inflammatory cytokines such as IL-10 and TGF-? to restrain inflammation,enhanced phagocysis of necrotic tissue and promote tissue repair.Treg cells can regulate immunosuppression by secretion of IL-10.Recent studies have suggested that Treg cells can induce the M2 phenotype polarization of microglial cells via the IL-10 /GSK3 ?/PTEN signaling pathway in ICH.The NOX2 deficient of TBI mice can induce the M2 phenotype polarization of microglia via the IL-10 /STAT3 signaling pathway to reduce the inflammatory damage.STAT3 is the downstream signaling molecule of the anti-inflammatory cytokine IL-10,therefore,we hypothesized that Tregs can also induce the M2 phenotype polarization via IL-10/STAT3 pathway to reduce the inflammatory damage.This study will explore the role of Treg cells in ICH and try to elucidation the specific mechanism of Treg cells.Part ? Treg significantly ameliorate intracerebral hemorrhage-induced injury in miceObjectiveTo investigate the neuroprotective effect of Treg in ICH mice.MethodsThe mice were randomly divided into Intracerebral hemorrhage group(ICH)and Sham operation group(Sham).The ICH group was injected 20 ul non-anticoagulated autologous blood into the right basal ganglia of the mice,and the Sham operation group was injected the same amount of saline.The ICH group and the sham group were randomly divided into the Tregs treatment group(24h after surgery)and the control group(equivalent saline).Clark scoring was used to evaluate the neurological deficient score of 1 day,4 days and 7 days after surgery.At 96 hours after ICH,the water content of brain tissue was measured by dry-wet weight method,Clark scoring method was used to assess neurological deficient score,MRI and tissue sections were used to assess changes in hematoma and Image-pro Plus were used to calculate hematoma volum.Use ELISA to detects inflammatory cytokines of perihematoma.ResultCompared with the sham group,the neurological function of the ICH group was obvious damaged.Treatment with Treg significantly reduced the neurological deficient score,water content of brain tissue and hematoma volume.ConclusionTreatment with Treg cells reduced inflammatory damage in ICH mice.Part ? Treg Modulated Microglia/Macrophage Polarization Toward M2 Phenotype after ICHObjectiveTo explore the specific mechanism of Tregs how to reduce inflammatory damage of ICH.Methods1.The mice were randomly divided into Intracerebral hemorrhage group(ICH)and Sham operation group(Sham).The ICH group was injected 20 ul non-anticoagulated autologous blood into the right basal ganglia of the mice,and the Sham operation group was injected the same amount of saline.The ICH group and the sham group were randomly divided into the Tregs treatment group(24h after surgery)and the control group(equivalent saline).At 96 hours after ICH,immunofluorescence was used for detecting the specialized protein marker expression of M1(CD16/32)and M2(CD206),perform ELISA to detect M1 phenotype related inflammatory cytokine(IL-6,TNF-?)and M2 phenotype related inflammatory cytokines(IL-10,TGF-?),PCR was used for detecting the signature gene change of M1 polarization related m RNA(CCL3,i NOS)and M2 polarization related m RN(Arg1,Ym1).2.Perform BV2 / Tregs transwell co-culture system.At 72 hours,perform ELISA to detect M1 phenotype related inflammatory cytokine(IL-6,TNF-?)and M2 phenotype related inflammatory cytokines(IL-10,TGF-?),PCR was used for detecting the signature gene change of M1 polarization related m RNA(CCL3,i NOS)and M2 polarization related m RN(Arg1,Ym1).ResultsIn vivo,we used CD16/32 as a marker for M1 phenotype,CD206 was used as a marker for the M2 type,perform the immunofluorescence staining of perihematoma.Compared with the control group,the expression of CD206 was increased and the expression of CD16/32 decreased after treatment with Treg cells.In Treg treated group,Tregs increased M2-related cytokines such as IL-10 ? TGF-? and M2-related m RNA expression including Arg1 and Ym1.Accordingly,Tregs decreased M1-related cytokines such as IL-6 ? TNF-a and M1-related m RNA expression including CCL3 and i NOS.In vitro,Tregs increased the M2-related cytokines such as IL-10?TGF-? and the m RNA levels of Arg 1and Ym 1,and these changes were accompanied by a decrease in the M1-related cytokines IL-6 and TNF-? and the m RNA levels of CCL3 and i NOS.ConclusionThese results indicated that Tregs promoted microglia/macrophage polarization to M2 phenotype after ICH.Part ? IL-10/STAT3 was involved in the microglia polarization modulating effect of TregsObjectiveTo investigate the signaling pathway of Treg cells induce microglia M2 phenotype polarization.MethodsPerform BV2 / Tregs transwell co-culture system.Western blot was used to detect STAT3,p-STAT3 and TGF-? protein levels.PCR was used for detecting the signature gene change of M2 polarization related m RNA(Arg1?Ym1).ResultsCompared with the control group,the co-culture of the Tregs and BV2 increase the expression of p-STAT3,TGF-?,and increase the level of Arg1,Ym1.When IL-10 was neutralized,compared with the Tregs co-culture group,the expression of p STAT3,TGF-? and Arg1,Ym1 were decreased.ConclusionRegulatory T cells can modulate microglia M2 polarization through the IL-10/STAT3 pathway... |
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