Substance P’s Effect On Type Ⅱ Alveolar Epithelial Cells Exposed Under Hyperoxia And Its Regulation On Shh Signal Pathway

BackgroundProlonged exposure to high concentration of oxygen (>90%ofoxygen) can lead to either acute or chronic lung injury in former prematureinfants. It has led to the production of free oxygen radicals that exceed thecell defense capacity, giving rise to inflammation, cell damage and geneoverexpression with necrosis and apoptosis phenomenon.The alveolar epithelium is the major target of oxidant injury, and itsrepair to the injury depends on the ability of its stem cells, the alveolarepithelial cells type II, can spread, proliferate and play an important role inthe repair process of alveolar epithelium. It has been confirmed that AECII can determine the pathological turnover of lung injury.Substance P is distributed widely in the airway endothelial cell layer.It was found that SP can trigger an exuberant neuroinflammatory response,and regulate proliferation, migration, and differentiation of the impairedCells. However, the molecular mechanisms that regulate the activities ofAECIIs and attenuate Oxidative Stress injury are poorly understood.Objective(1) To culture the premature rat alveolar type ΙΙ epithelial cells in vitro andestablishment of cellular oxidative model.(2) To observe the Oxidative stress injury of alveolar type ΙΙ epithelialcells Exposure to Hyperoxia and the Effect of SP on AECIIs afterExposure to Hyperoxia.Methods(1) Culture and isolation alveolar type ΙΙ epithelial cells of premature rats，the isolated and purified cells were treated with air (21%oxygen),hyperoxia (95%oxygen), SP+hyperoxia, and SP+L703.606+hyperoxia.(2) The morphologic changes of cells in each group had been observedunder inverted phase contrast microscope.(3) Reactive oxygen species（ROS）were measured by flow cytometry.(4) The survival rates and apoptosis rates were measured by MTT andAnnexin V–FITC. ResultsResults showed that the exposure to95%oxygen for24hsignificantly increased the level of ROS, contributed to the apoptosis andobviously decreased the proliferation of AEC IIs. Compared withhyperoxia exposure, Additional SP treatment diminished the level of ROS,lessened AECII apoptosis and improved the cell survival sequentially. Theprotective effect aforesaid were weakened after the treatment of L703.606.ConclusionsExposure to oxygen for24h could cause oxidative injury, induceapoptosis of AECII in vitro in premature rats, while SP may play aprotective role against hyperoxia induced lung injury by antioxidant,inhibition of AECII apoptosis. BackgroundHyperoxic toxicity involves multiple signaling activities.Sonichedgehog (Shh) pathway is a important signal transduction pathwaysreported to play a role in cell injury and repair.Sonic hedgehog (Shh)produces the biologically active N-terminal fragment (Shh-N) to bind its receptor Patched1(Ptch1). Ptch1and Smoothened (Smo) are multipassmembrane proteins that function as a receptor complex for hedgehogligands. When Shh ligands binds to Ptch1, Smo is released from theinhibitory effect of Ptch1, allowing the Gli transcriptional activators toenter the nucleus and activate specific target genes. Shh signaling proteinplays an important role in a variety of processes, such as embryogenesis，tissue repair， wound healing.Sonic hedgehog regulates the proliferation,differentiation, and migration of enteric neural crest cells，regulates adultneural progenitor proliferation and differentiation in vitro and in vivo.In themouse lung， Sonic hedgehog (Shh) plays a critical role in lungmorphogenesis and lung organogenesis.The goal of this study was to investigate the protective effect of SPon primary AECIIs from premature rats exposure to hyperoxia. Also, theeffect of SP on the Shh pathway was also studied. In view of this, weexpect to illuminate the related regulatory mechanism of SP in the damageto AECIIs after hyperoxia exposure.Objective（1） To culture the premature rat alveolar type ΙΙ epithelial cells in vitroand establishment of cellular oxidative model.（2） To understand the activation of SHH signal pathway in AECⅡunder hyperxia-induced oxidative stress and SP intervention, andinvestigate the regulation of SP intervention to SHH pathway. Methods（1） Culture and isolation alveolar type ΙΙ epithelial cells of prematurerats，the isolated and purified cells were treated with air (21%oxygen), hyperoxia (95%oxygen), SP+hyperoxia, and SP+L703.606+hyperoxia.（2） Real Time RT-PCR were used to detect the mRNA levels of SHHand PTCH1in AECII cells.（3） Western blot analysis were used to detect the mRNA levels of SHHand PTCH1in AECII cells.ResultsThe mRNA and protern expression of SHH in hyperoxia-exposedgroup was increased than in air-exposed group. On the other hand, theexpression of PTCH1in hyperoxia-exposed group was decreased inhyperoxia-exposed group.The expression of SHH and PTCH1inhyperoxia-exposed group was elevated by the stimulation with SP. SP cansignificantly increase the expression of SHH and PTCH1. Theup-regulation effect aforesaid was weakened after treated with L703.606.ConclusionsThe SHH signal pathway mediated signal transduction of SP andparticipated in the process that SP protected AECⅡ from hyperoxia-inducedinjury.