Research Of Alveolar Macrophage Secretion Caused By NMDA Receptor Activation And The Related Mechanisms

Chapter 1 Expression of NMDA receptors in macrophages and the effect of hyperoxiaOBJECTIVE:The N-methyl-D-aspartate receptor(NMDAR)is an ionotropic receptor for glutamate(Glu).NMDA receptor-dependent neurotoxicity as a final common pathway for neurologic disorders plays an important role in acute and chronic brain diseases.Relatively little attention has been paid to the functional expression ofthese molecules in peripheral non-neuronal tissues.Evidence is emerging for the expression of NMDA receptor subtypes in lung but their physiological and pathological significant remains to be determined.Hyperoxia-induced lung injury is characterized by an intensive inflammatory response initiated and exacerated by the activation of alveolar macrophages(AMs)and neutrophils and the increased proinflammatory cytokines,high levels of NO and reactive oxygen species(ROS).We earlier showed that hyperoxia exposure induces the release of intrinsic Glu,the increased expression of NMDA receptor in neonatal rat lung and increment of proinflammatory cytokines in BALF. Shen Li reported that the activation of NMDA receptor is conductive to acute lung injury in vivo.As reported by Said et al,NMDA can induce excitotoxic lung injury with the occurrence of high-permeability pulmonary edema in isolated perfused rat lung and NMDAR1 and NMDAR2D subfamilies express in the alveolar macrophages.In this study,we searched for evidence of NMDA receptor expression in neonatal rat alveolar macrophage and macrophage cell line RAW264.7 and the change of NMDA receptor expression in neonatal rat alveolar macrophage exposured to hyperoxia.METHODS:1.Collection and isolation adult and neonatal rat alveolar macrophage in primary culture via BAL.2.Expression of NMDAR 1 was analyzed by immunocytochemistry.3.Expression of NMDARlmRNA was examined by reverse transcriptase-polymerase chain reaction(RT-PCR)4.Expression of NMDAR1 and the four known NMDAR2 subtypes (A,B,C and D)were examined by Real-time PCR.RESULTS:1.Immunocytochemistry indicated an increased expression of NMDAR1 in neonatal rat AM compared with in adult rat AM,and the level further elevated via hyperoxia exprosure.Meanwhile,the expression of NMDAR1 was no significant difference between adult rat AM and RAW264.7 cell.2.RT-PCR showed an elevated expression of NMDAR1mRNA in neonatal rat AM compared with adult rat AM and the level further elevated via hyperoxia exprosure.The expression of NMDAR1 mRNA was no significant difference between adult rat AM and RAW264.7 cell.3.Real-time PCR detected the expression of NMDAR1 and the four NMDAR2 subtypes(A,B,C and D)mRNA in adult rat AM, neonatal rat AM and RAW264.7 cell.NMDAR2D was the dominant subtype,NMDAR1 was moderately expressed,NMDAR2C,2B and 2A expression were weakly detectable.The expression of NMDA receptor subtypes in neonatal rat AM were higher than those in adult rat AM,and the levels further promoted via hyperoxia exprosure. CONCLUSIONS:1.NMDAR1 and the four NMDAR2 subtypes(A,B,C and D)all express in adult rat AM,neonatal rat AM and RAW264.7 cell line.2.The expression of NMDARs in rat AM is age-dependent.3.Hyperoxia exposure upregulates the expression of NMDARs in neonatal rat AM.Chapter 2 Effect of NMDA receptor activation on nitric oxide secretion of alveolar macrophageOBJECTIVE:Evidences have been presented for a key mediator role of nitric oxide(NO)in NMDA-induced neurotoxity.Said et al reported that NMDA receptor channel blocker MK-801 and NOS inhibitor L-NAME can attenuate NMDA-induced high-permeability pulmonary edema.This suggested that NMDA-induced lung injury also was NO-dependent.Previous data indicated that NO and inducible nitric oxide synthase(iNOS)play important roles in the pathogenesis of hyperoxic lung injury.In the present study the effect of NMDA receptor activation on NO secretion of rat alveolar macrophage was further explored.We hypothesized that the activation of NMDA receptor might contribute to NO secretion in rat alveolar macrophage and perpetuate the development of hyperoxic lung injury.METHODS:1.Dose-dependent and course-dependent NO levels induced by NMDA were determined using commercial kits.2.Cell viability was determined by metylthiazoletetrazolium(MTT) assay.Lactate dehydroase(LDH)and maleic dialdehyde(MDA)were measured using commercially available kits.3.Biochemical analysis of the effect of NMDA(NMDA receptor agonist)on iNOS activity and NO levelS of alveolar macrophage using commercial kits.4.Real-time PCR detected the effect of NMDA on iNOSmRNA expression of alveolar macrophage.RESULTS:1.The treatment with NMDA led to an increment of NO level in dose dependent manner in rat alveolar macrophage.On 1μmol/L NO level reached the maximum while NMDA concentration ranging from 0.001μmol/L to 10μmol/L,then decreased significantly.The administration of NMDA also produced elevated NO level in course-dependent manner. NO production peaked when cultures were treated with NMDA(1 tool/L)for 6h,then decreased significantly on 8h.2.1μmol/L NMDA was chosen as the optimal concentration to incorporate with alveolar macrophage for 6h.MTT assay showed that the OD values were not significantly different between control and NMDA group.Both LDH activity and MDA content were not significantly higher in the NMDA group than those in control group.These evidences indicated that the optimal NMDA exposure did not bring alveolar macrophage injury.3.Expression of iNOS mRNA and activity,NO level in NMDA group were significantly higher than those in control group.MK-801(10μmol/L)pretreatment could attenuate iNOS mRNA and activity,NO level induced by NMDA.CONCLUSION:The activation of NMDA receptor promote NO secretion in alveolar macrophage.This suggests that alveolar macrophage may be the major cellular source of the increased production of NO in hyperoxia-induced lung injury.Chapter 3 Effect of NMDA receptor activation on proinflammatory cytokines secretion of alveolar macrophageOBJECTIVE:Proinflammatory cytokines play important role in the pathogenesis of hyperoxic lung injury.Our previous evidences showed that hyperoxia exposure can increase TNF-α,IL-1β,IL-6 and MIP-2 mRNA expression and secretion in bronchoalveolar lavage fluid(BALF). MK-801 could attenuate these proinflammatory cytokines mRNA expression and secretion.This suggests that the activation of NMDA receptor play an important role in proinflammatory cytokine secretion in vivo.The present study was determined to investigate the effect of NMDA receptor activation on TNF-α,IL-1β,IL-6 and MIP-2 secretion in rat alveolar macrophage to prove the hypothesis that the activation of NMDA receptor might promote proinflammatory cytokines secretion and alveolar macrophage might be the cellular source of proinflammatory cytokines over-secretion in hyperoxic lung injury.METHODS:1.ELISA determined TNF-α,IL-1β,IL-6 and MIP-2 protein levels in culture supernatants.2.Real-time PCR detected TNF-α,IL-1β,IL-6 and MIP-2mRNA expressions. RESULTS:NMDA treatment promoted TNF-α,IL-1β,IL-6 and MIP-2 mRNA expressions and protein levels of rat alveolar macrophage and MK-801 pretreatment could suppress TNF-α,IL-1β,IL-6 and MIP-2 gene transcription and secretion induced by NMDA.CONCLUSION:The activation of NMDA receptor facilitate proinflammatory cytokines secretion of alveolar macrophage and this effect can be alleviated by NMDA receptor antagonist MK-801.Chapter 4 Research of mechanisms of the elevation alveolar macrophage secretion caused by NMDA receptor activationOBJECTIVE:The previous chapter only examined that the activation of NMDA receptor facilitate proinflammatory cytokines transcription and secretion of rat alveolar macrophage,but its signal pathway remains obscure.In the development of hyperoxic lung injury,NF-κB participates the activation of alveolar macrophage and modulates many proinflammatory cytokines gene transcription.NO plays an critical role in many diverse biological processes.The cross-talk between NO and proinflammatory cytokines aggravates the pathogenesis of hyperoxic lung injury.In this study,we investigated the effect of NO and NF-κB on proinflammatory cytokines secretion of rat alveolar macrophage induced by the activation of NMDA receptor to prove the hypothesis that NF-κB might be an important signaling pathway of proinflammatory cytokines secretion of rat alveolar macrophage induced by the activation of NMDA receptor. METHODS:1.Biochemical analysis determined alveolar macrophage iNOS activity and NO level in culture supernatants;ELISA determined TNF-α, IL-1β,IL-6 and MIP-2 protein levels in culture supernatants;Real-time PCR detected TNF-α,IL-1β,IL-6,MIP-2 and iNOS mRNA expression. These results indicated the effect of NO and NF-κB on proinflammatory cytokine secretion of alveolar macrophage induced by NMDA.2.Detection of NF-κB P65,Ⅰ-κBαprotein by Western-Blot Analysis3.Measurement of NF-κB activity by EMSARESULTS:1.Both AG and SN50 can attenuate the elevation of NO level,iNOS activity,TNF-α,IL-1β,IL-6,MIP-2 protein and mRNA levels of NMDA-induced AMs.SN50 also can abolish iNOS mRNA expression but AG can not.2.NMDA augmentes the degradation ofⅠ-κBαand NF-κB nuclear location of RAW264.7 cells.MK-801,AG,SN50 can inhibit the degradation ofⅠ-κBαand NF-κB nuclear location of NMDA-induced RAW264.7 cells.3.NMDA enhances NF-κB activity of RAW264.7 cells.MK-801, AG,SN50 can inhibit NF-κB activity of NMDA-induced RAW264.7 cells.CONCLUSION:NF-κB may be an important signaling pathway of proinflammatory cytokines secretion of alveolar macrophage induced by NMDA.NO can enhance proinflammatory cytokines secretion by promoting the activation of NF-κB.