Study On The Contribution Of NOD-like Receptor Containing Pyrin Domain 1 To Pyroptosis In Early Brain Injury After Subarachnoid Hemorrhage

Background and Purpose:Subarachnoid hemorrhage(SAH)is a critical emergency of central nervous system diseases,with high rates of morbidity and mortality.Growing evidence has demonstrated that early brain injury(EBI)is the primary cause to results in poor outcome in patients suffering from SAH.Pyroptosis is recently found as a sort of the programmed cell death model.Pyroptosis is triggered by activation of caspase-1,which results in cell death rapidly and induces inflammation correspondingly.Inflammation after SAH has been considered to play an important role in the pathogenesis of EBI.Both experimental and clinical studies have shown that the level of caspase-1 was significant up-regulated after SAH.Based on these evidences,we propose the hypothesis that pyroptosis could play a vital role in the pathophysiological mechanism of EBI.Inflammasome,a multiprotein complex,is a key component of the pyroptosis process.Upon inflammasome activation,caspase-1 is activated as cleaved caspase-1.Among the inflammasomes,NOD-like receptor containing pyrin domain 1(NLRP1)that was found in neurons could result in the activation of proinflammatory cytokines,as well as the pyroptosis of neurons,which are effector cells of EBI.The purpose of this study is to figure out the role and the mechanism of pyroptosis and NLRP1 inflammasome in the EBI after SAH,respectively.Methods:First,after SAH models in vivo and in vitro were established,the changes of expression and location of active caspase-1 were assessed through Western blotting,immunohistochemistry and immunofluorescene assay.The degree of neurocytes pyroptosis was detected by immunofluorescene assay and flow cytometry.Ac-YVAD-CMK,a specific antagonist of caspase-1,was used to study the role of pyroptosis in EBI.Second,the role and the mechanism of NLRP1 inflammasome were studied in vivo and in vitro.Carbenoxolone and NLRP1 siRNA were used to suppress the activation of NLRP1 inflammasome in vivo and in vitro,respectively.The changes of expression of NLRP1 inflammasome proteins,cleaved IL-1β,and cleaved IL-18 was assessed through Western blotting,or immunofluorescene assay.Co-immunoprecipitation was used to detect the protein composition of NLRP1 inflammasome.Immunofluorescene assay,flow cytometry,cell counting kit 8 assay,Nissl staining,brain water content,Evans blue extravasation,and neurosurgical scores were used to assess the degree of pyroptosis and brain injury.Last,cerebrospinal fluid(CSF)were collected from the patients undergoing artificial hip arthroplasty(as control),and from SAH patients,between 24 hours and 72 hours post-SAH.The changes of levels of inflammasome proteins in the CSF,including NLRP1,ASC,and caspase-1 were analyzed through Western blotting.The levels were correlate with both the degree of EBI and the neurologic outcome.The degree of EBI was measured by Hunt-Hess grade,Fisher grade,cerebral edema on CT scans,and hydrocephalus on CT scans.Glasgow outcome scale(GOS)was used to assess clinical outcome.Results:Our study indicated that the expression of active caspase-1 was significantly elevated after SAH in vivo and in vitro,and the increased expression of active caspase-1 could be detected in the cytoplasm of neurocytes.Meanwhile,SAH induced neurocyte pyroptosis,inflammation,and brain injury could be reduced by the specific antagonist of caspase-1.Further,we demonstrated that the expression of apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)was significantly elevated after SAH in vivo and in vitro,and the increased expression of ASC could be detected in the cytoplasm of neurocytes.NLRP1 was found to participate in the assemble and the activation of inflammasome.Suppressing the activation of NLRP1 inflammasome could reduce SAH-induced pyroptosis,inflammation,and brain injury.Clinical study confirmed elevation of NLRP1 inflammasome proteins in the CSF of SAH patients.Elevated levels of NLRP1,ASC and caspase-1(p20)were associated with severe SAH(Hunt-Hess grade Ⅲ-Ⅴ and Fisher grade 4),CT manifestations of cerebral edema,acute hydrocephalus,and poor outcomes.High level of NLRP1 was the independent risk factor of poor outcome after SAH.Conclusion:Our study provides evidence that,for the first time,SAH induce neurocyte pyroptosis.On the other hand,suppressing the pyroptotic neurocyte death following SAH could alleviate EBI.Pyroptosis maybe play a vital role in the pathophysiological mechanism of EBI following SAH.The expression level of NLRP1 inflammasome was associated with the degree of brain injury,as well as the clinical outcome of SAH patients.SAH-induced pyroptosis,inflammation,and brain injury could be reduced through suppressing the activation of NLRP1 inflammasome.Our results indicated that NLRP1 inflammasome might be an important component during pyroptosis process of neurocyte,in EBI following SAH.