The Role And Mechanism Of NLRP2 In Cerebral Ischemic Injury

ObjectiveStroke is the leading cause of long-term disability and death in the world in recent years.It has the characteristics of high morbidity,high disability,high recurrence rate and high mortality rate.Ischemic stroke commonly accounts for approximately 80%of all stroke cases which includes ischemic stroke and hemorrhagic stroke.Ischemic stroke which is also called cerebral ischemia is due to occlusion of certain cerebral artery and thus blood flow to the relative brain area is interrupted which will finally lead to tissue necrosis.The pathophysiological processes following cerebral ischemia are complex and extensive,Which include oxidative stress,excitatory amino acid toxicity,bioenergetic failure,loss of cellular ion homeostasis,reactive oxygen species-mediated toxicity and cytokine-mediated cytotoxicity.Recent studies have shown that the inflammatory response of the innate immune system is closely related to the pathogenesis and progression of cerebral ischemia.The innate immune response is the first line of defense to limit harm to the host.The inflammatory response is initiated by the detection of acute damage via extracellular and intracellular pattern recognition receptors(PRRs).Nucleotide binding oligomeriza-tiondomain(NOD)-like receptors(NLRs)are a family of intracellular PRRs,playing an important role in the inflammatory response.Fourteen of the 22 Human NLRs contain a Pyrin domain and forms an NLRP subtype(NLRPs).A few members have been studied in depth.NLRP1,NLRP3,and NLRP6 are found to activate the inflammasome through the recruitment of the adaptor protein ASC to the Pyrin domain,which in turn activates caspase-1 leading to the produce and release of IL-1? and IL-18 and elicit inflammation.A recent study showed that human astrocytes express NLRP2 inflammasome in vitro.While NLRP2,as the second member of NLRPs was less studied,especially on its roles in CNS in vivo and neurological diseases.Whether NLRP2 is expressed in the central nervous system and whether it is related to neurological diseases such as cerebral ischemia remains to be further explored.In this experiment,we used a middle cerebral artery occlusion(MCAO)model in vivo and an oxygen glucose deprivation(OGD)model in vitro to study the expression and function of NLRP2 in cerebral ischemic injury.Methods1 The expression of NLRP2 in mice after cerebral ischemia1.1 Establishment of cerebral ischemia and neurological score of C57BL/6 wild-type mouse1.1.1 Selection of mice and construction of the ischemic modelMale wild type C57BL/6 mice with body weight of about 23g were selected in this study.Different groups were allocated in a randomized manner.The middle cerebral artery occlusion model was established as the following procedure.After cutting the skin along the neck midline incision,we bluntly isolated the left common carotid artery,internal carotid artery and external carotid artery under a dissecting microscope.Then we inserted the nylon bolus carefully into the common carotid artery.When the cerebral blood flow meter detects a sudden drop in blood flow,we stopped further insertion of the nylon bolus and started timing.The mice were sacrificed after 12,24,48 hours.1.1.2 Evaluating the damage to central nervous system via Neurological score1.2 The expression and distribution of NLRP2 in the brain of mice after cerebral lischemia1.2.1 To explore the expression of NLRP2 in the tissues after cerebral ischemia by Western blotting(WB),Real-time PCR and immunohistochemistry(IHC).1.2.2 To explore the distribution in the brain using double immunofluorescence(IF)for NLRP2 and various cell markers in the brain by immunofluorescence.1.3 Construction of in vitro model and detection of NLRP2Culturing the primary astrocytes in vitro and building OGD model,different cell groups were allocated in a randomized manner,OGD0.5,1,1.5,2h respectively.To explore the expression of NLRP2 in the primary astrocytes after OGD by western blotting(WB),immunofluorescence(IF)and Real-time PCR.2 The role of NLRP2 in mice after cerebral ischemia injury 2.1 Injecting the adeno-associated virus(AAV)into mouse brain to knockdown NLRP2 geneThe AAV vectors carrying the NLRP2-shRNA were injected into certain sites of the brains with a stereotaxic frame to knockdown NLRP2 gene.2.2 Comparison of morphological damage in mice injected with empty virus and mice injected with virus carrying NLRP2-shRNA.At different time-points after cerebral ischemia,the mice either injected with empty virus or with virus carrying NLRP2-shRNA were sacrificed,and brains were taken out for further detection.Neurological scoring and TTC staining were used to evaluate the damage to central nervous system.2.3 Detection of pro-inflammatory factors in mice cerebral ischemia model Detecting the content of pro-inflammatory factors in brain homogenate of mice injected with empty virus or with virus carrying NLRP2-shRNA using CBA and Real-time PCR methods.2.4 The effects of NLRP2 on OGD-induced apoptosis of primary astrocytes2.4.1 si-NLRP2 were used to knockdown NLRP2 in astrocytes2.4.2 Detection the apoptosis of primary astrocytesAstrocytes transfected with si-NLRP2 were subjected to OGD treatment and then stained with Annexin-V/PI,followed by flow cytometric assay to detect the effect NLRP2 on the apoptosis of OGD-treated primary astrocytes.3 The regulation of NLRP2 pathway in cerebral ischemic injuryWB was used to detect the levels of caspase-1,ASC,IL-1? and other related proteins in mice brains injected with empty virus or with virus carrying NLRP2-shRNA.The effects of NLRP2 on the expression of caspase-1,p-p65,IL-1? and other related proteins in astrocytes transfected with or without si-NLRP2 were also detected by WB.Results1 The expression and role of NLRP2 and its downstream molecules in the brain after ischemia in mice1.1 Establishment of cerebral ischemia and model evaluation of C57BL/6 wild-type mouseIn this study,15%animals were excluded due to insufficient neurological scoring or died during surgery.1.2 The expression and distribution of NLRP2 in the brain of mice after cerebral ischemia1.2.1 To explore the expression of NLRP2 in the tissues after cerebral ischemia by western blotting(WB),Real-time PCR and immunohistochemistry(IHC).Results of WB and Real-time PCR showed that NLRP2 expression was significantly increased after cerebral ischemia and reached a peak at 24 hours,and this result was consisted with immunohistochemistry results.1.2.2 There is also little expression in neurons and almost no expression in microglial cells.1.3 Construction of in vitro OGD model and detection of NLRP2Cultured primary astrocytes were subjected to OGD treatment for 0.5,1,1.5 or 2 hours,and collected at certain time-points for detection of NLRP2 level by western blot and Real-time PCR.We found that the expression of NLRP2 increased significantly after OGD and reached the peak at 1.5 hours after OGD.Results of immunofluorescence also showed that the expression of NLRP2 was increased after OGD 1.5 hours.2 The role of NLRP2 in mice after cerebral ischemia injury2.1 Evaluation of the efficacy of injecting the adeno-associated virus(AAV)into mouse brain to knockdown NLRP2 gene4 weeks after injection of the virus,brain tissues of mice were taken out and embedded with OCT.And then we checked the range of infection under the microscope and found that most part of the injected hemisphere was infected including the area of middle cerebral artery(MCA)territory.2.2 Comparison of morphological damage in mice with injection of empty virus or virus carrying NLRP2-shRNAResults of TTC staining showed that the infarct size of mice was significantly decreased by knockdown of NLRP2 gene.Neurological scores also significantly reduced.2.3 Detection of pro-inflammatory factors in mouse cerebral ischemia modelBoth mice injected empty virus or virus carrying NLRP2-shRNA was subjected to MCAO treatment.Results of CBA and Real-time PCR showed that the silencing of NLRP2 by Sh-NLRP2 transfection significantly weaken the ischemia-induced the increase of IL-1?,IL-18,IL-6,TNF-a and MCP-1.2.4 The effects of NLRP2 on OGD-induced apoptosis of primary astrocytesResults of flow cytometry showed that astrocytes transfected with si-NLRP2 were treated with OGD and apoptotic astrocytes were significantly reduced.3 Regulation of NLRP2 pathway in cerebral ischemic injury Results of WB showed that the expression of NLRP2,caspase-1,ASC,IL-1? and other related proteins increased after cerebral ischemia in mice.Compared with mice injected with empty virus,the expression of NLRP2 was markedly reduced in the mice injected with virus carrying NLRP2-shRNA.And the silencing of NLRP2 by sh-NLRP2 transfection also significantly weakens the increase of ischemia-induced caspase-1,ASC,IL-1? and other related proteins.We got similar results in cultured cells in vitro.Results of WB showed that the expression of NLRP2 in si-NLRP2 astrocytes was significantly reduced compared with control astrocytes.And the decrease of NLRP2 expression by transfected with si-NLRP2 could weaken the increase of OGD-induced caspase-1,p-p65 and IL-1?.ConclusionThere is a basal expression of NLRP2 in mice brain and NLRP2 is statistically induced in models of ischemic stroke both in vivo and in vitro,which induces the expression of a series of inflammatory factors and promotes the ischemia-related inflammatory response.This will deepen our understanding of the pathophysiological mechanism of ischemic stroke and may provide a new target for the treatment of this type of cerebrovascular diseases.