| More recently, stroke has become one of the major causes of death worldwide, while more than80%of all strokes are ischemic cerebrovascular disease. Intravenous thrombolytic therapy is the only proven effective treatment in the acute setting. However, reperfusion therapy is a double-edged sword:on one hand, it can rescue ischemic brain tissue and improve clinical symptom; on the other hand, some patient condition become more deteriorating than before, it is known as cerebral ischemia/reperfusion injury. Experiments show that a variety of pathological processes can cause ischemia/reperfusion injury. Reperfusion injury mechanism provides important theoretical basis for the treatment of brain diseases and prevention.The innate immune system regulates initial responses through a set of conserved pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). Studies have highlighted the role of NLRP3, one member of NLR family, forming an intracellular complex with active caspase-1, known as an inflammasome, to create a platform for regulating secretion of IL-1β and IL-18. More recently, triggers of the NLRP3inflammasome have provided a mechanistic understanding for Alzheimer’s disease, type2diabetes, atherosclerosis, inflammatory bowel disease. However, so far it is unknown the contribution of NLRP3to the pathogenesis of ischemic stroke. Therefore, in the present study, we used an in vivo middle cerebral artery occlusion (MCAO) model and in vitro cell cultures by oxygen glucose deprivation (OGD) for the first time to investigate the expression and function of NLRP3in ischemic brains.OBJECTIVE:1. To study the expression and role of NLRP3pathway in mice brain after ischemia/reperfusion injury.2. To explore the mechanism that microglia and endothelial cells enhanced ischemia/reperfusion injury mediated by NLRP3pathway.3. To investigate that NADPH oxidase contributes to NLRP3pathway in cerebral ischemia injury.METHODS:PART1:THE EXPRESSION AND ROLE OF NLRP3PATHWAY IN MICE BRAIN AFTER ISCHEMIA/REPERFUSION INJURY1. Induction of cerebral ischemia and neurological scoring.1.1Different groups were allocated in a randomized manner, the ischemia model was induced by occlusion of the middle cerebral artery (MCAO) with a microfilament in wild-type mice.1.2Neurological scoring and TTC staining were used to evaluate the damage to central nervous system.2. The expression and distribution of NLRP3in mice brain after MCAOTo explore the expression and distribution of NLRP3in mice brain after ischemia/reperfusion by Real time PCR, Western Blot, Immunofluorescence staining.3. In vitro, mRNA level of NLRP3was detected in primary cultured neurons, astrocytes, BV2, and bEND.3by RT-PCR.4. NLRP3deficiency ameliorated stroke outcome4.1Identification of genotype, grouping and induction of cerebral ischemia4.2To analyze infarct, serial stroke assessment MRI was performed. HE staining and nissl staining were used to determine the neural morphology in the cerebral cortex and hippocampus. TUNEL staining was performed to detect neuron apoptosis.4.3Morphological and functional assessment of neurovascular unit4.4The expression of Caspase-1and cytokines were examined by Western Blot and ELISA, respectively. 4.5Western Blot and gelatin zymography were performed to analysis for the expression and activity of MMP9after MCAO.PART2:THE MECHANISM THAT MICROGLIA CELLS ENHANCED ISCHEMIA/REPERFUSION INJURY MEDIATED BY NLRP3PATHWAY.1. Cells were treated by the model of oxygen-glucose deprivation (OGD)The expression of NLRP3, Caspase-1, NOX2were detected after BV2subjected to OGD treatment by Western Blot. NADPH oxidase activity was determined by measurement of superoxide (O2-) production using a modified DHE fluorescent spectrometric assay2. ShRNA-NLRP3and shRNA-NOX2were used to knock down NLRP3and NOX2level of BV2, respectively. The expression of NLRP3related molecules were detected after OGD.2.1ShRNA-NLRP3and shRNA-NOX2were used to knock down NLRP3and NOX2level of BV2cells, respectively.2.2After BV2cells transfected with shRNA-NLRP3were subjected to OGD treatment, the expression of Caspase-1was detected by Western Blot2.3After BV2cells transfected with shRNA-NOX2were subjected to OGD treatment, the expression of NLRP3was detected by Western Blot2.4Cytokines in cells were measured with enzyme-linked immunoabsorbent assay kits.3. Transwell co-cultureMicroglia in the upper inserts were incubated overnight and then subjected to OGD with or without shRNA-NLRP3transfection. Neuron were grown on a coverslip in the bottom well of the chamber. After exposure to microglia in the transwell chambers, apoptosis of neuron was quantified by counting TUNEL.PART3:THE MECHANISM THAT ENDOTHELIAL CELLS ENHANCED ISCHEMIA/REPERFUSION INJURY MEDIATED BY NLRP3PATHWAY.1. Cells were treated by the model of oxygen-glucose deprivation (OGD)The expression of NLRP3、Caspase-1, MMP2/9, ZO-1, TJP-2were detected after bEND.3subjected to OGD treatment by Western Blot. Caspase-1activity assay kit was used to determine Caspase-1activity. 2. ShRNA-NLRP3was used to knock down NLRP3expression of bEND.3. The expression of NLRP3related molecules were detected after OGD.2.1ShRNA-NLRP3was used to knock down NLRP3level of bEND.3cells.2.2The expression of MMP2/9, ZO-1, TJP-2were detected by Western Blot after bEND.3transfected with shRNA-NLRP3were subjected to OGD treatment.2.3Cytokines in cells were measured with enzyme-linked immunoabsorbent assay kits.3. Cell permeability assayEndothelial cells were seeded onto the collagen-coated inserts and incubated for48hours until a monolayer was formed. Brain endothelial cells were treated with OGD and endothelial cells permeability was assessed by the passage of FITC-conjugated dextran.4. Role of IL-1β in the endothelial cells permeabilityTo investigate the role of IL-1β in the endothelial cells permeability, bEND.3cells were cultured by different concentration of IL-1β,after that, bEND.3cells permeability and expression of MMP2/9, ZO-1, TJP-2were detected.PART4:NADPH OXIDASE CONTRIBUTES TO NLRP3PATHWAY IN CEREBRAL ISCHEMIA INJURY1. The expression and activity of NOX2were determined after wild-type mice subjected to MCAO.2. The assessment of morphology was investigated after wild-type and NOX2-/-mice subjected to MCAO.MRI was performed to analyze infarct volume. Calculations of edema and neurological scoring were also performed after MCAO.3. Western Blot was used to analyze the expression of NLRP3after MCAO.4. StatisticsRESULTS:PART1:THE EXPRESSION AND ROLE OF NLRP3PATHWAY IN MICE BRAIN AFTER ISCHEMIA/REPERFUSION INJURY1. Induction of cerebral ischemia and neurological scoring.Overall,15%animals used in this study were excluded because of insufficient neurological scoring or died during surgery.2. The expression and distribution of NLRP3in mice brain after MCAOBy real-time RT-PCR and Western Blot analyses, we found that NLRP3expression was markedly enhanced in the ischemic cerebral hemisphere with a peak expression of NLRP3at24hours after reperfusion. We further found that NLRP3was mainly expressed in microglia and endothelial cells by confocal Immunofluorescence staining. Relative levels of proinflammatory cytokines and Caspase-1in the ischemic cerebral hemisphere from WT mice at different time points after reperfusion were markedly enhanced.3. In vitro, mRNA level of NLRP3was detected in primary cultured neurons, astrocytes, BV2, and bEND.3by RT-PCR.NLRP3mainly expressed in microglia and endothelial cells was confirmed by mRNA detection in vitro by RT-PCR.4. NLRP3deficiency ameliorated stroke outcomeUsing MRI, we observed the reduced infarction volume, edema formation, in NLRP3-/-ischemic mice, which was consistent with neurologic scoring. HE staining, Nissl staining and TUNEL further indicated that NLRP3deficiency protected against ischemia-induced neuron injury and death.BBB permeability reduced in NLRP3-/-ischemic mice by Evans blue permeability assays. Furthermore, cerebromicrovessel exhibited capillary integrity with normal endothelial cells and basal lamina in sham-operated group by electron microscopy analysis. In ischemic WT mice, the endothelial cells were swollen and deformed, and the integrity of BBB was destroyed, However, NLRP3deficiency significantly blocked ischemia-induced BBB disruption.In consistent with reduced cerebral injuries, ischemia enhanced expression and cleavage of caspase-1, MMP9and the production of inflammatory cytokines were markedly attenuated in the ischemic brain from NLRP3-/-mice.PART2:THE MECHANISM THAT MICROGLIA CELLS ENHANCED ISCHEMIA/REPERFUSION INJURY MEDIATED BY NLRP3PATHWAY.1. Cells were treated by the model of oxygen-glucose deprivation (OGD)In vitro, we found that OGD significantly enhanced microglia NLRP3and NOX2 expression in a time-dependent manner accompanied by enhanced expression and cleavage of caspase-1.2. ShRNA-NLRP3and shRNA-NOX2were used to knock down NLRP3and NOX2level of BV2, respectively. The expression of NLRP3pathway was detected after OGD.2.1Gene silencing of NLRP3and NOX2by shRNA-NLRP3and shRNA-NOX2were observed by Western Blot.2.2Studies showed that gene silencing of NLRP3by shRNA-NLRP3transfection significantly attenuated OGD-induced caspase-1cleavage and caspase-1activity.2.3We found that gene silencing of NOX2inhibited OGD-induced NLRP3expression.2.4By ELISA, we found that both shRNA-NLRP3and shRNA-NOX2blocked OGD-induced increases in cytokine levels3. Transwell co-cultureThe role of microglial NLRP3was determined by using a two chamber Transwell system for neuron-microglial cocultures. Microglia mediated neuron damage was associated with OGD induced microglial NLRP3signaling. Gene silencing of microglial NLRP3or NOX2attenuated neuron death by OGD-treated microglia.PART3:THE MECHANISM THAT ENDOTHELIAL CELLS ENHANCED ISCHEMIA/REPERFUSION INJURY MEDIATED BY NLRP3PATHWAY.1. Cells were treated by the model of oxygen-glucose deprivation (OGD)We found that in brain microvessel endothelial cells, OGD significantly enhanced expression of NLRP3, caspase-1, and MMP2/9in a time dependent manner, and reduced expression of tight junction proteins such as ZO-1and TJP-2.2. ShRNA-NLRP3was used to knock down NLRP3level of bEND.3. The expression of NLRP3pathway was detected after OGD.Gene silencing of NLRP3significantly attenuated OGD induced, MMP2/9, cytokine expression and recovered ZO-1and TJP-2expression.3. Cell permeability assayWe found that the increased endothelial cell permeability was attenuated by NLRP3knockdown. 4. Role of IL-1β in the endothelial cells permeabilityAfter direct IL-1β treatment, we found that bEND.3cells permeability was enhanced accompanied by the enhanced MMP2/9expression and reduced expression of ZO-1and TJP-2.PART4:NADPH OXIDASE CONTRIBUTES TO NLRP3PATHWAY IN CEREBRAL ISCHEMIA INJURYOur results showed that the infarction volume (MRI) and edema formation was significantly reduced in NOX2-/-mice compared with those of WT ischemic mice, which was consistent with the decreased neurologic score. Furthermore, we found that NOX2deficiency reduced NLRP3expression, suggesting that NOX2mediated cerebral injury in ischemic stroke was associated with NLRP3signaling.CONCLUSION:1. In vivo, we found that NLRP3deficiency ameliorated cerebral injury in mice after ischemic stroke by reducing infarcts and blood brain barrier (BBB) damage.2. We further showed that the contribution of NLRP3to neurovascular damage was associated with an autocrine/paracrine pattern of NLRP3mediated IL-1β release as evidenced by increased brain microvessel endothelial cell permeability and microglia-mediated neurotoxicity.3. We found that NADPH oxidase deficiency improved outcomes after ischemic stroke by mediating NLRP3signaling. |
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