The Neuroprotective Effect Of IL-33/ST2 Signaling Pathway On White Matter Injury After Ischemic Stroke

Background:Ischemic stroke is a disease with high rate of disability and death.Inflammation plays indispensable role in the pathophysiology of stroke.The activation of inflammatory cells in the central nervous system,such as microglia,and the overexpression of inflammatory cytokines after ischemic stroke lead to severe brain injury and neurological deficits.Therefore,it may be a potential therapy to inhibit the inflammatory response after ischemic stroke and promote the recovery of neurological deficits.Ischemic stroke elicits destruction of both gray and white matter.A large number of studies have shown that white matter injury is negatively correlated with functional prognosis in patients with stroke.Inflammatory response plays an important role in the process of white matter injury.Microglia,the innate immune cells of the central nervous system,are involved in the inflammatory response after stroke.Factors affecting the polarization of microglia,such as low temperature,high glucose and disease,can influence the repair of white matter damage.Mature oligodendrocytes which form myelin in the white matter can be generated from oligodendrocyte precursor cells(OPCs).White matter injury occurs after ischemic stroke,subsequently,OPCs proliferate and differentiate into mature oligodendrocytes to restore white matter injury.Previous studies have shown that the polarization of M1-type microglia into M2 after traumatic brain injury promoted the recovery of white matter injury and the differentiation and maturation of OPCs.In the mice demyelination model,the phenomenon that microglia transformed from Ml-type into M2-type microglia was observed during the regeneration of myelin sheath,and M2-type microglia could promote the differentiation of OPCs in vitro.M2-type microglia promoted the regeneration of oligodendrocytes,while M1-type microglia inhibited the generation of oligodendrocytes when deleting Ml-type or M2-type microglia.The activation of M1-type microglia led to the death of oligodendrocytes after microglia oxygen-glucose deprivation.These suggest that microglia may be involved in white matter damage after ischemic stroke.IL-33 is one of the interleukin-1(IL-1)family members,which mediates inflammation by binding its specific receptor ST2.Serum IL-33 levels were significantly increased in patients with ischemic stroke.ST2 knockout expanded brain infarcts volume and aggravated neurological deficits after tMCAO and dMCAO.IL-33 deficiency resulted in a decrease in the M2 specific protein expression following ischemia-reperfusion injury,which was alleviated by exogenous IL-33 delivery.Besides,intracerebroventricular injection of IL-33 facilitated the polarization of microglia from M1 to M2 phenotype and mitigated white matter injury after intracerebral hemorrhage in rats.Therefore,exploring the effect of IL-33/ST2 on the polarization of microglia and the effect of different types of microglia on the maturation of OPCs and white matter injury after ischemic stroke is significant.And up-regulation of M2 microglia by IL-33 injection is expected to provide a new treatment strategy for ischemic stroke.Objective:1.Explore the role of IL-33/ST2 signal pathway in white matter injury after ischemic stroke.2.Explore the role of different types of microglia in white matter injury after ischemic stroke.3.Further explore the mechanism of IL-33/ST2 signal pathway on white matter injury after ischemic stroke by promoting microglia polarization.Methods:In vivo,tMCAO model was perfomed in mice.Intraperitoneal injection of IL-33 was used to investigate the effect of IL-33 on white matter damage after ischemic stroke.Intraperitoneal injection of IL-33 and anti-ST2 antibody was used to investigate the effect of IL-33/ST2 signaling pathway on white matter damage after ischemic stroke.Recorded and analyzed body weight in mice.Rotarod test and mNSS score were performed to evaluate sensorimotor function in all animals.Infarct volume was examined by TTC staining.Immunofluorescence was used to measure numbers of newly generated OPCs and oligodendrocytes.Western Blot and immunofluorescence were used to identify the degree of white matter injury.Numbers of M1 and M2 microglia were counted by immunofluorescence.M1 and M2 microglia markers mRNA expression were identified by RT-PCR.In vitro,microglia were co-cultured with OPCs,and lipopolysaccharide(LPS)was used to activate microglia.Microglia were respectively cultured in normal culture medium,IL-33 conditioned culture medium(CM),IL-33+anti-ST2 CM.The differentiation and maturation of OPCs and M1 and M2 microglia markers mRNA expression were detected by RT-PCR.Results:1.TTC staining showed that exogenous administration of IL-33 reduced the infarct volume after tMCAO(P=0.011).Neurological functions were impaired in mice after tMCAO,which were defected by mNSS score and the drop latency in rotarod test.Compared with the vehicle group,since the 7th day,mNSS score of IL-33 group was significantly lower(P<0.001).In the rotarod test,since the 3rd day,the drop latency of IL-33 group was significantly longer than the vehicle group(P=0.032).2.NF200 and MBP were marked by immunofluorescence.The ratio of NF200/MBP in the striatum after tMCAO was higher than that in the sham group(P=0.007),and the ratio of NF200/MBP in the IL-33 group was lower than that in the vehicle group(P=0.035).Western blot showed that the expression of MBP in the brain tissues decreased after tMCAO(P<0.001),and the expression of MBP in the IL-33 group was higher than that in the vehicle group(P=0.007).3.OPCs and oligodendrocytes were marked by immunofluorescence.It showed that numbers of newly generated OPCs and oligodendrocytes in ischemic penumbra after tMCAO were increased.Compared with the vehicle group,numbers of newly generated OPCs(P=0.019)and oligodendrocytes(P=0.041)of IL-33 group were higher.4.Ml and M2 types microglia were labeled by immunofluorescence,CD 16,CD206 and Iba-1 were marked respectively.The results showed that the number of M1 microglia in ischemic penumbra after tMCAO was increased,but the number of M2 type microglia was decreased.Compared with the vehicle group,the number of M1 microglia of IL-33 group was fewer(P=0.002),type M2 microglia was higher(P=0.009).The expression of mRNA of M1 and M2 microglia were measured by RT-PCR.The results showed that the expression of mRNA of M1-type microglia CD16,CD32 and iNOS increased after tMCAO,while the expression of M2-type microglia CD206,TGF-?and IL-10 decreased.CD16(P=0.005),CD32(P=0.038)and iNOS(P=0.040)were lower in the IL-33 group than in the vehicle group,while CD206(P=0.006),TGF-?(P=0.002)and IL-10(P=0.018)were higher.5.Compared with the IL-33 group,TTC staining showed that the infarct volume of the IL-33+anti-ST2 group increased(P=0.036).Since the 7th day,mNSS score was significantly higher(P=0.004).Immunofluorescence showed that the NF200/MBP ratio of the IL-33+anti-ST2 group was higher than that of the IL-33 group(P=0.035).Western blot showed that the expression of MBP decreased in the IL-33+anti-ST2 group(P=0.004).Immunofluorescence showed that the number of Ml-type microglia was higher in the IL-33+anti-ST2 group(P=0.001)and the number of M2-type microglia was lower(P=0.013).RT-PCR showed that the expressions of CD16(P=0.015),CD32(P=0.033)and iNOS(P=0.010)mRNA were higher in the IL-33+anti-ST2 group,while the expressions of CD206(P=0.017),TGF-?(P=0.002)and IL-10(P=0.010)mRNA were lower.6.In vitro,compared with the control group,RT-PCR showed that the expression of mRNA of M1-type microglia was higher,the mRNA of M2-type microglia was lower,and the expression of MBP(P<0.001),MOG(P<0.001)and PLP(P<0.001)mRNA in OPCs were lower in the LPS group.Compared with the LPS group,the expression of mRNA in M1-type microglia was lower,the expression of mRNA in M2-type was higher,and the expression of MBP(P=0.037)and PLP(P=0.033)mRNA in OPCs were higher in LPS+IL-33 group.Compared with the LPS+IL-33 group,the expression of mRNA in M1-type microglia was higher,the expression of mRNA in M2-type was lower,and the expression of MBP(P=0.034)and PLP(P=0.033)mRNA were lower in the LPS+IL-33+anti-ST2 group.Conclusions:1.IL-33/ST2 signal pathway has a protective effect on white matter injury after ischemic stroke.2.The polarization of microglia to M2 type protects white matter injury after ischemic stroke.3.IL-33/ST2 signal pathway protects the white matter damage after ischemic stroke through promoting the polarization of microglia into M2 type.