| Part Ⅰ The role and underlying mechanism of IL-33 in mice intracerebral hemorrhage modelObjective: Interleukin-33(IL-33)is a recently identified member of the IL-1 family that exerts biologic functions by binding to a heterodimer composed of IL-1 receptor-related protein ST2 L and IL-1RAc P.However,the role of IL-33 and whether IL-33 accounts for inflammation,apoptotic,and autophagic neuropathology after intracerebral hemorrhage(ICH)are not clear.Here,we established a mouse ICH model in this study,to determine the role of IL-33 and explore the underlying mechanism.Methods: A total of 456 male mice(ICR),each weighing 25 to 30 g,were used for all experiments.Male mice were subjected to an infusion of type IV collagenase into the left striatum to induce ICH model.The time course of endogenous IL-33 and ST2 L expressions in ipsilateral basal ganglia was detected by western blot at different time points(1 h,3 h,6 h,12 h,24 h,48 h and 72 h),and the phenotypes of the IL-33 and ST2 L immune-reactive cells in brain were detected by double Immunofluorescent Staining.To investigate the effect of IL-33 on ICH,the behavior tests(Garcia and corner turn test(CTT))and water content of ipsilateral basal ganglia was assessed by injecting intracerebroventricularly(i.c.v.)with 100 ng/mouse of recombinant mouse IL-33 30 min before ICH.In addition,in order to investigate the role of IL-33 in ICH-induced cell death,the PI and TUNEL staining were performed to assess for loss of plasmalemma integrity and apoptotic cells in the core and peripheral,as well as combined peripheral and core fields,respectively.To further explore the underlying mechanism of IL-33 after ICH,the levels of pro-inflammatory cytokines such as TNF-a and IL-1β expressions in the ipsilateral basal ganglia tissue were detected at 24 h and 72 h after ICH.In parallel,apoptosis related proteins such as Bcl-2 and CC-3,and autophagy related proteins such as Beclin-1,LC3-Ⅱ and P62 were examined.Results:(1)The results showed that endogenous IL-33 protein was highly expressed in sham group,whereas,IL-33 was decreased within 6 hours and then reached the valleys at 6 h and 72 h after ICH(P<0.05).We also examined the intracerebral expression of ST2 L,a transmembrane receptor for IL-33,which significantly was upregulated at 6 h,then reached two peaks at 6 h and 24 h,and remained elevated for up to 72 h following ICH(P<0.05).(2)Double immunofluorescence results confirmed that IL-33 was co-expressed mainly in GFAP-positive astrocytes and Iba-1-positive microglia.However,few IL-33+/MAP2+ merged cells were detected after ICH.In addition,ST2 L was co-labeled with GFAP-positive astrocytes and Iba-1-positive microglia,respectively.Whereas,the localization of ST2 L expression are not positioned in the MAP2 positive cells after ICH.(3)ICH led to a significant increase in the percentage of water content at 24 h and 72 h after ICH,compared with sham group(P<0.01).Conversely,IL-33 pretreatment significantly reduced the percentage of water content(P<0.05).However,no significant differences were found among all groups,in contralateral and ipsilateral cortex,contralateral basal ganglia,or in the cerebellum.(4)The behavior results indicated ICH induced severe neurobehavioral deficits in the Garcia test and corner turn test(P<0.01).In contrast,IL-33 pretreatment attenuated significantly neurobehavioral function at 24 h and 72 h after ICH(P <0.05).(5)Comparison with ICH group,pretreatment with IL-33 can significantly reduce the number of PI-and TUNEL-positive cells in the peripheral,core,and combined peripheral and core fields(P<0.05).(6)ELISA results indicated that ICH resulted in higher levels of TNF-a and IL-1β,while IL-33 pretreatment obviously reduced the level of TNF-a and IL-1β(P<0.05).Furthermore,at 72 h after ICH,pretreatment by IL-33 with dosage of 300 ng significantly reduced the levels of TNF-a and IL-1β,compared with IL-33 with dosage of 100 ng(P<0.05),suggesting that the anti-inflammation effect of IL-33 is dose-dependent at 72 h post-ICH(P<0.05).(7)Western blot results suggested that down-regulation of Bcl-2 expression and up-regulation of CC-3 expression were detected after ICH,while,IL-33 pretreatment significantly inhibited down-regulation of Bcl-2 expression and up-regulation of CC-3 expression,compared with vehicle group at 24 h and 72 h after ICH(P <0.05).However,s ST2(an inhibitor of IL-33)treatment could obviously increase the CC-3 expression but reduce Bcl-2 expression at 24 h and 72 h after ICH(P <0.05).(8)Western blot analysis results suggested that IL-33 pretreatment suppressed Beclin-1 expression,up-regulated the LC3-Ⅱ/LC3-Ⅰ ratio and increased p62 expression.Additionally,IL-33 pretreatment down-regulated remarkably the Beclin-1/Bcl-2 ratio after ICH(p<0.05).However,s ST2 treatment obviously elevated the level of LC3-Ⅱ,beclin-1 expression and Beclin-1/Bcl-2 ratio,but reduced P62 expression(p<0.05,)Conclusion:(1)IL-33 significantly alleviated ICH-induced brain edema,suppressed cellular plasmalemma integrality disruption and cell apoptosis,and improved neurobehavioral deficits.(2)In addition,IL-33 provides neuroprotection through suppressing inflammation,apoptotic,and autophagic activation in collagenase-induced ICH model.Part Ⅱ The effect and mechanism of IL-33 in a rat model of recurrent neonatal seizureObjective: IL-33 is a novelly identified chromatin-associated cytokine of IL-1 family cytokines that signals through a heterodimer comprised of ST2 L and IL-1RAcp,and has a crucial role in many diseases.However,very little is known about the effect and underlying mechanisms of IL-33 on recurrent neonatal seizure(recurrent neonatal seizure,RNS).The objectives of this study were to characterize the role of IL-33 and underlying mechanisms in RNS.Methods: On postnatal day(P7),36sprague-dawley(SD)rats were assigned randomly to three groups: RNS plus IL-33 group,RNS plus PBS and Sham group.From P7,rats in the RNS+IL-33 and RNS+PBS group were subjected to recurrent seizures induced by volatile flurothyl two times each day for consecutive 7 days,with an interval time of 30 minutes once.While rats in the Sham group were placed into the container for an equal amount of time to their counterpart without exposuring to flurothyl or IL-33.The levels of NF-κB,Bcl-2,CC-3,Beclin-1,LC3-Ⅱ and P62 protein in cerebral cortex and hippocampus were determined by western blot at P14.Neurological behavioral parameters of brain damage(forelimb suspension test,negative geotactic test and cliff avoidance test)were observed at P23 and P28.Morris water maze(MWM)test was performed during P31–P35.Results:(1)The results showed that RNS contributed to significant reduction in IL-33 and ST2 L expression in cortex,while,in hippocampus,RNS induced an increase in IL-33 and ST2 L evidently,compared with Sham group(P <0.05).However,after injection with IL-33,a remarkable increase in total IL-33 was detected in both brain cortex and hippocampus,implying that IL-33 has been arrived at the site of the injured brain parenchyma.Furthermore,a striking increase in ST2 L in cortex and an evident reduction in ST2 L in hippocampus were induced by IL-33 treatment(P <0.05).(2)Double immunofluorescence results confirmed that IL-33 co-localized with GFAP+ astrocytes,and mainly displayed nuclear staining.Novelty,IL-33 was expressed exclusively in cytoplasm of the Iba-1+ microglia.In parallel,IL-33 was also present in the cytoplasm of IL-33+/Neu N + merged cells.Furthermore,ST2 L expressed mainly in the membrane of GFAP+ astrocytes,Iba-1+ microglia and Neu N+ neurons,respectively.(3)RNS group had an evident delay or reduction of forelimb suspension test,negative geotactic reaction test and cliff avoidance test,compared with Sham group at P23 and P28.In contrast,these behavioral deficits were reversed by IL-33 treatment,compared with RNS group(P <0.05).Besides,we observed that a significant reduction in BWG was induced by RNS at P7,however,IL-33 treatment obviously promoted BWG from P7 to P13(P <0.05).(4)ELISA results indicated that IL-1β and TNF-α expression were increased significantly in RNS group,compared with Sham group.On the contrary,IL-1β and TNF-α increase in serum,brain cortex and hippocampal tissues homogenates were reversed by IL-33 treatment,compared with the RNS group(P <0.05).(5)Western blot results suggested that a significant increase of NF-κB activity in brain cortex,while a significant reduction in hippocampus,compared with Sham group.However,IL-33 strikingly suppressed NF-κB activity in both cortex and hippocampus tissues,compared with RNS group(P <0.05).(6)Western blot analysis results suggested that RNS group significantly inhibited the expression of Bcl-2 and promoted CC-3 expression,compared with Sham group.Conversely,IL-33 treatment obviously increased Bcl-2,and reduced CC-3 expression in both cortex and hippocampus(P <0.05).(7)Western blot analysis results suggested that higher amounts of LC3-Ⅱ/LC3-Ⅰ and Beclin-1,and lower amounts of P62 in RNS group,compared with Sham group.However,IL-33 treatment decreased the ratio of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 expression and maintained P62 at normal level in both brain cortex and hippocampus after RNS(P <0.05).Conclusion: IL-33 offers neuroprotection through suppressing apoptotic,autophagic and NF-κB-mediated inflammatory pathways in a rat model of recurrent neonatal seizure Part Ⅲ The role and underlying mechanism of IL-33 in mice traumatic brain injury modelObjective: IL-33 is a novelly recognized member of IL-1 family,which can act both as a cytokine and as a nuclear factor.Moreover,IL-33 plays an important role in diverse central nervous system(CNS)diseases.However,the role and underlying mechanism of IL-33 in traumatic brain injury(TBI)have never been elucidated.The aims of this study were to determine the role of IL-33 and explore the underlying mechanism.Methods: Male mice(ICR),each weighing 25 to 30 g,were used for all experiments.Mice TBI model was established by weight drop device in adult mice based on procedures previously reported.The expression changes and lo-calization of endogenous IL-33 and ST2 L were detected at 24 h after TBI by western blot and double immunofluorescent staining,respectively.In order to investigate the neuroprotective role of IL-33 in TBI,the behavior tests(motor test and Morris water maze)and water content of brain tissue were evaluated at a concentrations(50ng/mouse)of IL-33 after TBI.To further explore the neuroprotective mechanism of IL-33 after TBI,the levels of the two pro-inflammatory cytokines expression such as TNF-a and IL-1β in serum were detected at 24 h after TBI.Furthermore,mice was treated with IL-33,salubrinal(SAL)or SAL plus IL-33,and brain tissues were obtained for specific protein analysis.apoptosis and autophagy related proteins were examined.Results:(1)The results showed that TBI contributed to significant increase in IL-33 in cortex,while,in hippocampus,TBI induced a reduction in IL-33 evidently,compared with Sham group.However,after injection with IL-33,SAL or IL-33 plus SAL,a remarkable increase in total IL-33 was detected in both brain cortex and hippocampus.Additionally,a striking increase in ST2 L in cortex and hippocampus was induced by TBI.However,a remarkable reduction in ST2 L was detected by IL-33,SAL or IL-33 plus SAL in both brain cortex and hippocampus(P<0.05).(2)Double immunofluorescence results confirmed that IL-33 was co-expressed in GFAP-positive astrocytes,and mainly displayed nuclear staining.Novelty,IL-33 was also expressed in the oligodendrocytes.(3)TBI led to an obvious increase in the percentage of water content at 24 h after TBI,when compared with sham group(P<0.01).Conversely,IL-33 treatment with dosage of 50 ng/mouse showed the most obvious improvement in cerebral edema,compared with TBI group(p<0.05).However,no significant differences were found among all groups,in contralateral hemisphere and cerebellum.(4)The behavior results indicated TBI induced severe neurobehavioral deficits,when compared to sham group.In contrast,IL-33 treatment attenuated motor deficits and improved spatial memory acquisition significantly(p<0.05).(5)ELISA results indicated that IL-1β and TNF-α expression were increased significantly in TBI group,compared with Sham group.On the contrary,IL-1β and TNF-α increase in serum were reversed by IL-33,SAL or IL-33 plus SAL treatment,compared with the TBI group(p<0.05).(6)Western blot analysis results suggested that TBI significantly inhibited the expression of Bcl-2 and promoted CC-3 expression,compared with Sham group.Conversely,IL-33,SAL or IL-33+SAL treatment obviously increased Bcl-2,and reduced CC-3 expression in both cortex and hippocampus(P<0.05).(7)Western blot analysis results suggested that TBI induced significantly higher amounts of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and lower amounts of P62 in RNS group,compared with Sham group.However,IL-33,SAL or IL-33+SAL treatment decreased Beclin-1 expression,the ratio of LC3-Ⅱ/LC3-Ⅰ and maintained P62 at normal level in both brain cortex and hippocampus after RNS(P<0.05).Conclusion:(1)IL-33 significantly alleviated TBI-induced brain edema and improved neurobehavioral deficits.(2)IL-33 provides neuroprotection through suppressing inflammation,apoptotic,and autophagic activation in our mouse TBI model... |
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