The Role Of NMDA Receptor And Its Mechanisms On Alveolization Impairment And Lung Collagen Deposition In Neonatal Rats Exposed To Hyperoxia

Background Oxygen therapy is an important measure to improvethe state of hypoxia. But prolonged inhalation of high concentrations ofoxygen (hyperoxia) will lead to varying degrees of acute and chronic lunginjury. Neonatal period is alveolar mature critical period, long-terminhalation of high concentrations of oxygen could lead to diffuse alveolardamage associated with infiltration of inflammatory cells characterizedby acute lung injury and alveolization impairment and fibrosis mainlycharacterized chronic lung injury.CLD is generally believed that the incidence of prematurity andintrauterine infection, inflammation, oxygen toxicity, and barotrauma,nutrition, and other related factors. Inhalation of high concentrations ofoxygen-induced oxidative stress is closely related with CLD. Thepathogenesis of CLD is not yet clear, but the role of cytokines mediatedinflammatory reaction occurred in CLD has been confirmed by a largenumber of clinical and animal experiments. The expression of cytokinesinduced by hyperoxia may be related to nuclear factor NF-κB activation.NF-κB is a category with a variety of gene promoter sitesκB loci and topromote specific binding protein transcription. Activated NF-κB DNAcombination with specific lociκB initiating and participating in theregulation of immune and inflammatory responses of gene transcriptionplays an important role in the immune response, inflammation, cellgrowth and development, and participates in hyperoxia-induced lunginjury. In vivo and in vitro studies have shown that high oxygen through aseries of chain reactions activates NF-κB which can control the geneexpression of a variety of cytokines, inflammatory mediators, adhesion molecules, NO, apoptosis protein, further to promote the inflammatoryresponse and cell damage which caused hyperoxia-induced lung injuryand lung development impaired.TGF-βis a type of important growth regulator factor in theorganization, development and rehabilitation and plays an important rolein repairing alter injury. Activation of TGF-β1 expression in lung tissuehas a very significant impact on pathological changes, especially inducedpulmonary fibrosis, bFGF is regarded as another cytokine related with thelung fibrosis, because it promoting fibroblast proliferation and collagensynthesis by fibroblasts. Experimental have proved that the high oxygeninduced the increased releasing of TGF-β1 and bFGF, and they may playan important role in the hyperoxia-induced alveolitis and the late alveolardevelopment and pulmonary fibrosis, but need to be further proved.It remains indistinct of the NMDA receptor effects on the alveolardevelopment and lung collagen deposition in prolonged hyperoxicexposure, and what relationship between NMDA receptor and the changeof NF-κB activity, TGF-β1 and bFGF content in the hyperoxia-exposedlung. Our investigation could provide a new way to solve thiscontradictive clinical trouble.Section One The protective effects of MK-801 on prolongedhyperoxia-induced lung injury in neonatal ratsObjective By observing the protective effect of Glutamate NMDAreceptor antagonist MK-801 on the alveolization impairment and lungcollagen deposition in prolonged hyperoxia-induced lung injury.Methods The 22-day gestation full term rats in 12 hours alter birthwere randomly marked with a diferent number and assigned to fourgroups: air control group, air+MK-801group, hyperoxia group and hyperoxia+MK-801 group. Hyperoxia group and hyperoxia+MK-801group were exposed to≥95ï¼…O₂ for 7 to 14 days. Air control group andair+MK-801 group were placed in room air. Air+MK-801 group andhyperoxia+MK-801group received MK-801 (0.05mg/kg) intraperi-toneally every day. Air control group and hyperoxia group recieved thesame volume of saline. After 7, 14 days of continuous exposure to highconcentration oxygen, the neonatal rats were sacrificed with an overdoseof intra-peritoneal pentobarbital, and exsanguinated by severing theabdominal aorta. The counts of inflammatory cell in BALF; the contentof protein, MDA and LDH in bronchoalveolar lavage fluid (BALF); theratio of lung wet weight to lung dry weight (W/D); the content ofhydroxyproline in the Lung homogenates; the change of mRNA ofprocollagenâ… ,â…¢and histological changes, including staining of HE andmasson, and radical alveolar count (RAC) of hyperoxia-induced lunginjury were measured respectively.Results 1. lung W/D and BALF analyse: After having 7days ofexposure, the hyperoxia group has been saw a significant increase in lungW/D (P＜0.01) and BALF leukocytes and neutrophils counts (P＜0.01,respectively), protein and LDH,MDA content (P＜0.01, respectively),After having 14days of exposure, above- mentioned index have declinedand lower than the 7th day (P＜0.01, respectively), but still higher thanair control group. 2. The content of HYP in lung homogenates haveincreased after 7days of exposure compared with the air control group (P＜0.01,respectively), with the days of exposure increasing, the contentcontinue increased and much higher than air control group(P＜0.01,respectively). 3.The precollagenâ…¢mRNA expressions have increasedafter 7days of exposure compared with the air control group (P＜0.01).with the days of exposure increasing, the precollagenâ…¢mRNA hasdeclined,but was still higher than air group.(P＜0.01). Whereas the procollagenâ… mRNA expression has a trend to increase but failed toreach significance until having 14days of exposure (P＜0.01). 4. Lunghistology: The hyperoxia group showed dilated and congestive capillaryvessels, erythrocyte extravasation, and leukocyte infiltration in alveolar,pulmonary interstitial inflammation cell infiltration and a little collagendeposition. The pathologic changes were more and more severe with daysof exposure increasing that broadened pulmonary interstitial tissue, cellsand collagen deposition in Masson, and disruption of the alveolarstructure were saw in the lung tissures. At the sametime RAC hasdecreased dramatically in the hyperoxia group (P＜0.01).When MK-801(0.05mg/kg) was administrated to neonatal rats in hyperoxia, thesusceptibility to hyperoxia was diminished. MK-801 adminstrationshowed a significant decrease in the elvation of lung W/D, (P＜0.05,P＜0.01),BALF leukocyte, and neutrophil counts (P＜0.01, respectively),lung tissue HYP content(P＜0.05, respectively), and BALF protein andLDH and MDA content (P＜0.01, respectively),and procollagenâ… ,â…¢mRNA expressions (P＜0.05,P＜0.01) induced by hyperoxia in neonatalrats. and pathological injury changes, RACinduced by hyperoxia werealleviated by MK-801 (0.05 mg/kg) (P＜0.01, respectively).ConclusionNeonatal rats after inhalation sustained high concentration of≥95ï¼…oxygen can cause acute inflammatory lung tissue damage, and aggravatedwith the days of exposure increasing, newborn rats oxygen-induced lunginjury by early alveolitis acute injury to the alveolization impairment andlung tissue fibrosis with a lot of collagen deposition. NMDA receptorantagonist MK-801 can alleviated hyperoxia-induced lung injury inneonatal rats in prolonged hyperoxia exposure. Section Two The mechanisms of NMDA receptor on alveolizationimpairment and lung collagen deposition in neonatal rats exposed tohyperoxiaObjective By observing the influence of MK-801 on the changeof NF-κB activity, the realeasing of TGF-β1and bFGF in thehyperoxia-exposed lung, further to elaborate the role and mechanism ofNMDA receptor in alveolization impairment and lung collagen depositionafter hyperoxia-induced lung injury , and to explore new therapy inhyperoxia-induced chronic lung disease.Methods The 22-day gestation full term rats in 12 hours after birthwere randomly marked with a diferent number and assigned to fourgroups: air control group, air+MK-801group, hyperoxia group andhyperoxia+MK-801 group. Hyperoxia group and hyperoxia+MK-801group were exposed to≥95ï¼…O₂ for 7 to 14 days. Air control group andair+MK-801 group were placed in room air. Air+MK-801 group andhyperoxia+MK-801group received MK-801 (0.05mg/kg) intraperit-oneally every day. Air control group and hyperoxia group recieved thesame volume of saline. After 7, 14 days of continuous exposure to highconcentration oxygen, the neonatal rats were sacrificed with an overdoseof intraperitoneal pentobarbital, and exsanguinated by severing theabdominal aorta. The TGF-β1,bFGF mRNA expressions were measuredby real-time polymerase chain reaction. The content of TGF-β1andbFGF in the Lung homogenates were measured by enzyme linkedimmunosorbent assay (ELISA) and The expression of NF-κB in the lungtissues were detected by immunohistochemistry.Results The expressions of TGF-β1 mRNA in lung have increasedafter 7days of exposure compared with the air control group (P＜ 0.01,respectively), with the days of exposure increasing, the contentcontinue increased and much higher than air control group(P＜0.01,respectively). The content of TGF-β1 in lung homogenates has the samechangement as the mRNA.The expressions of bFGFmRNA has a trend toincrease but failed to reach significance until having 14days of exposure(P＜0.01),and so was the content of bFGF in lung homogenates. TheNF-κB expression has increased in lung by immunohistochemistry on the7th day of exposure (P＜0.01) and the stain of NF-κB immuno-histochemistry was more stronger on the 14th day of exposure (P＜0.01).MK-801 adminstration showed a significant decrease in the elvation ofthe mRNA and content of TGF-β1(P＜0.01 respectively),bFGF(P＜0.05,P＜0.01) and the expression of NF-κB(P＜0.01 respectively).ConclusionHyperoxia exposure can lead to the alveolization impairment andlung collagen deposition by promoting the expression of NF-κB andup-regulation TGF-β1 and bFGF. MK-801 adminstration can decrease inthe elvation of the mRNA and content of TGF-β1,bFGF and theexpression of NF-κB. It suggests that the mechanism of the MK-801alleviates the impaired alveolization and lung collagen deposition afterthe hyperoxia lung injury may related to it inhibition the activation ofNF-κB and down reglulation the transcription of TGF-β1 and bFGF.