The Effects And Molecular Mechanisms Of Wnt Signaling In The Protection Of Neuropeptide Substance P On The Oxidative/Antioxidant Status In Hyperoxia Alveolar Type Ⅱ Epithelial Cells

Part1The effect of Substance P on the oxidative/antioxidantstatus in hyperoxia typeⅡ alveolar epithelial cellsBackgroundIn the new born premature,due to mechanical ventilation repeatedexcessive expansion alveolar and alveolar catheter, prolonged exposure tohigh concentration of oxygen to produce a large number of oxygen freeradicals, thus cause lung oxidative stress property damage. The alveolarepithelium cell is the major target of Oxidative stress property damage.Bec-ause of alveolar epithelial cellsI（AECI） have high differentiation and losetheir regenerative capacity, so the alveolar epithelial cells repair dependentirely on the alveolar epithelial cells Ⅱ（AEC Ⅱ）,which can proliferateand transdifferente to AECI cells,but high oxygen exposure can lead toAEC Ⅱ apoptosis, inhibiting AEC Ⅱcell proliferation, cause lung damagerepair difficulty or appear fibrosis and other abnormal repair. Neuropeptide substance P （SP） is composed of11amino acid,whichhas a wide range of biological effectsIt was found that In the lung injurycaused by myocardial ischemia and burns,SP can cause airwayinflammation.But also some people found that SP can Stimulate epithelialcell growth factor and receptor expression,regulate epidermalstem cellmigration and differentiation,and in the end,promote epithelial repaired.Ho-wever, the effect of Substance P on the oxidative/antioxidant status inhyperoxia type Ⅱalveolar epithelial cells is poorly understood.Objective（1） Use the different speed adherent technology for culture andisolation AECⅡ c ellsfrom19days rat, and then establish the cellularoxidative model.（2） To observe the effection of alveolar type Ⅱepithelial cellsexposured to Hyperoxia and the function of SP on the oxidative/antioxidant status in hyperoxia alveolar type Ⅱ epithelial cells.Methods（1）AEC Ⅱ were isolated from the lung of19-day rat fetus,and theywere cultured in six-well plates for24h.And then the cells were randomlydivided into four groups：1、air group；2、high oxygen group；3、high oxygen+SP group；4、high oxygen+SP+SP antagonist group. Air group andhyperoxia group were exposed to21%and95%oxygen for24hours,respectively;SP was added into hyperoxia sp group before oxygen exposure,while SP and its receptor antagonists （L703.606） were added intohyperoxia SP receptor antagonist group simultaneously before oxygenexposure，then both of the two groups were exposed to95%oxygen for24hours.（2） The morphologic and adherence status of AECⅡ cells wereobserved under inverted microscope.（3） Flow cytometry were used to detect ability of cell proliferationand cell cycle.（4） The levels of intracellular superoxide anion（O₂-） were measuredby SpectraMax M5/M5e and confocal microscopy.（5）JC-1probe method to detect the mitochondrial membrane potentiallevel.（6）The concentrations of MDA、TAOC and SOD were detected byMicroplate Reader.ResultsCompared with air Group, the cell ratio of G2and S,the level of TAOCand SOD,the apoptosis cell number were decreased, while the level ofMDA,the mitochondrial membrane potential level and the intracellularsuperoxide anion （O₂-） concentration were increased in hyperoxia group （P<0.01）.In contrast，the cell ratio of G2and S，the level of TAOC and SOD，the apoptosis cell number were significantly higher and the level ofMDA,the mitochondrial membrane potential level and the intracellular superoxide anion （O₂-） concentration were significantly lower in hyperoxiaSP group then those in high oxygen group （P <0.01）.The differences in6parameters above between hyperoxia group and hyperoxia plus SP andSP（L703.606）group were not statistically significant. The above effect canbe weakened after the treatment of L703.606.ConclusionsSP can reduce the high oxygen induced AEC Ⅱcell damage anddeath,enhance the ability of oxidation resistance and promote cellproliferation. Part2The effects and molecular mechanisms of Wnt signalingin the protection of neuropeptide substance P in hyperoxiaexposed alveolar type Ⅱ epithelial cellsBackgroundWnt signaling pathway is a classic signal transduction pathway inevery organisms,which play an important role on embryonic development,cell damage repair and tumorigenesis.And the Wnt classic signalingpathway is an signal transduction pathway which act on AECⅡproliferation,lung development and the lung space conformation.When Wntsignal is activated, Wnt protein combined with Dally/FRPs,and then combined with Frizzled/LRP（the protein complexes with cell surfacereceptor）, the beta catenin were gatherd in the cells,and then transfer intothe corethe to control the gene expression of downstream targe.In part one,we have found that SP can reduce the high oxygen inducedAEC Ⅱ cell damage and death,enhance the ability of oxidation resistanceand promote cell proliferation.The protection of SP is related to improveingthe oxidation/antioxidant imbalance.However, the ability of oxidationresistancewhether or not releated to Wnt signal classical channel, it is notcleared.In view of this, we expect to detect the expression of Wnt7b andβ-catenin of SP in the damage to AECⅡs after hyperoxia exposure.In theend,we can further discuss the pathogenesis of BPD.ObjectiveTo understand the oxidative/antioxidant effect of SP intervention ofhigh oxygen exposure AECⅡ i s related to activate the Wnt classic signalingpathway,observe the Wnt7b and β-catenin signaling molecule in Wntclassic signaling pathway.Methods（1） AECⅡ wasisolated and cultured from the lung of19-day ratfetus,and they were cultured in six-well plates for24h.And then the cellswere randomly divided into four groups：1、air group；2、high oxygengroup；3、high oxygen+SP group；4、high oxygen+SP+SP antagonistgroup. Air group and hyperoxia group were exposed to21%and95% oxygen for24hours,respectively;SP was added into hyperoxia sp groupbefore oxygen exposure,while SP and its receptor antagonists （L703.606）were added into hyperoxia SP receptor antagonist group simultaneouslybefore oxygen exposure,then both of the two groups were exposed to95%oxygen for24hours.（2）The mRNA levels of Wnt7b and β-catenin were detected by RealTime PCR.（3）The protein levels of Wnt7b and β-catenin were detected byWestern blot.ResultsHyperoxia exposure resulted in significant decrease of the mRNA andprotern expression of Wnt7b and β-catenin,while SP treatment reversed theexpression of Wnt7b and β-catenin in hyperoxia-exposed groupsignificantly.But after treated with L703.606,the effect of SP was decreasedsignifiently.ConclusionsWnt signaling pathway is changed after high oxygen exposure in AECⅡ,the oxidative/antioxidant effect of SP intervention of high oxygenexposure AECⅡ i s related to activate the Wnt classic signaling pathway.