TLR4/NMDAR Involved In NLRP3 Inflammasome-mediated Inflammatory Injury After Intracerebral Hemorrhage

Background:ICH(Intracerebral hemorrhage)is a common critical syndrome with high mortality and morbidity.In recent years,we have made great progress in understanding of ICH,but there isn't satisfactory therapies in clinical practice.Increasing evidence indicates that inflammation play a crucial role in secondary brain injury after ICH.However,the exact mechanism of the signaling pathway in the immune system-mediated neuroinflammatory damage after ICH is not fully understood.Microglia,the resident immune cells of the central nervous system,are rapidly activated following ICH,accounting for 10-20% of brain cells.Activated microglial cells are thought to release a series of inflammatory mediators,including cytokine,chemokines,cyclooxygenase-2(COX-2)and reactive oxygen species(ROS).These inflammatory mediators may result in secondary brain damage after ICH.In the past few decades,the mechanism of igniting innate immune system is widely studied.Pathogen associated molecular patterns(PAMP)and danger associated molecular patterns(DAMP)are thought to be the main cause of initiating innate immune and inflammatory responses.PAMP is mainly associated with exogenous infection,in which membrane receptor TLR4 and cytoplasm NLRP3 recognize exogenous pathogen resulting in the release of proinflammatory mediators.Compared with PAMP,DAMP is usually implicated in endogenous dangerous signals.Hemin,a breakdown product of hemoglobin,is the main cause eliciting the secondary brain injury after ICH.With strong oxidability,hemin may be identify as a dangerous signal and thereby activate innate immune system.NMDAR,a type of ionotropic glutamate receptor,has been demonstrated to play a vital role in brain inflammatory insult.However,whether NMDAR participating in ICH-induced neuroinflammation remains uncovered.Objective: To investigate the effect of hemin on expression of microglial TLR4 and NLRP3 inflammasome;To study the relationship of inflammaoty receptor TLR4 and NLRP3;To explore the synergic effect of NMDAR1 in hemin-induced activation of NLRP3 inflammasome.Method:1.Establishment of the hemin injecting in striatum to mimic the ICH models.The expression of TLR4 ? NLRP3 ? ASC and IL-1? were detected by western blot.Double-immunofluorescence staining was performed to assess microglial expression of ASC and IL-1?.2.N9 microglial cells were treated with different concentrations or different time of hemin,the expression of TLR4?NLRP3?ASC?caspase-1and IL-1? were examined by western blot.ELISA was employed to study the secretion of TNF-? and caspase-1.3.The effects of TLR4 on hemin-induced the activation of NLRP3 inflammasome were investigated in N9 and EOC20(defective in TLR4)microglial cells or in wild-type mice?TLR4-/-mice and TLR4d/d mice.Western blot was prepared to detect the expression of NLRP3 and IL-1?.The binding of TLR4 to NLRP3 was measured by immunoprecipitation.4.After administration of NMDAR inhibtor,the expression of NLRP3 and IL-1? were evaluated by western blot.The synergic effect of NMDAR1 in hemin-induced NLRP3 activation was tested by immunoprecipitation.Result:1.Hemin injection into striatum induced microglial activation and subsequently increased the expression of TLR4?NLRP3?ASC and IL-1?.2.Treatment with hemin not only induced TLR4?NLRP3?ASC?caspase-1and IL-1? upregulation,but also enhanced the secretion of TNF-? and caspase-1.3.Defective in TLR4 attenuated the activation of NLRP3 inflammasome.Hemin potentiated the binding of TLR4 to NLRP3.4.Inhibiting NMDAR reduced NLRP3-mediated inflammation.Hemin promoted the combination of NMDAR1 and NLRP3.Conclusion:1.Hemin accelerates microglial revitalite,then activates both TLR4 and NLRP3 signaling pathways and eventually contributes to inflammatory damage after ICH.2.TLR4 plays a momentous role in hemin-induced NLRP3 activation by synergistic effect.3.NMDAR1 participates in hemin-induced NLRP3-mediated inflammatory damage through synergic activation.