Calcitonin Gene Related Peptide Protects Premature Lung Against Oxidative Stress Via Regulating WNT7b/β-Catenin Pathway

Part I Different Oxygen Concentrations and Time Exposureon Premature LungObjective To observe the pulmonary histopathology and RAC ofpremature rats and the survival of AECII exposed to graded oxygendetermining the effect of different oxygen concentrations and timeexposure on lung development.Methods Premature rats were exposed to oxygen for3d,7d,14d at4different concentrations:21%(air),40%,60%or95%. The left lunghomogenate was performed and the right lungs were removed forhistological analyses. The lungs homogenate were assayed for lipidperoxidation and antioxidant status using biochemical methods. PrimaryAECII were exposed to the graded oxygen for6h,12h,24h,48h or72h.The cell death was detected by flow cytometry. The cell proliferationwas measured by MTT, ROS and TAOC was detected by biochemicalmethods.Results Compared with air, there is no difference in weight, survival rate,pulmonary histopathology or oxidant and antioxidantstatus after40%oxygen exposure, the pups exposed to60%oxygen hadlower weight and survival (p<0.05), the lungs manifested inflammationat7d and expansion of alveolar space at14d with RAC reducedsignificantly (p<0.01), at the same time, lipid peroxidation andantioxidant status were disturbed. The lung injury performances ofpremature rats exposed to95%oxygen were even heavier.Conclusion Exposed to more than60%concentrations of oxygencan cause oxidative damage of premature lung leading to lungdevelopment arrest in dose-dependent and time-dependent manner. Part II Calcitonin Gene Related Peptide AttenuatedOxidative Injury of Premature LungObjective To observe the effect of CGRP on pulmonaryhistopathology and RAC of premature rats and the proliferation anddeath of AECII after hyperoxia exposure, exploring the consequence ofCGRP on restoration and regeneration of premature lung afterhyperoxia-induced injury.Methods Premature rats were exposed to95%oxygen for3d,7d, 14d. The pulmonary CGRP content was assayed by radioimmunoassay.The left lung homogenate was performed and the right lungs wereremoved for histological analyses and the lungs homogenate wereassayed for lipid peroxidation and antioxidant status using biochemicalmethods After CGRP8-37administration. Primary AECII were exposed tohyperoxia and CGRP for6h,12h,24h,48h or72h. The cell death wasmeasured by flow cytometry. The proliferation of AECII was detectedby MTT cell proliferation assay, ROS and TAOC was detected bybiochemical methods.Results Compared with air control, the CGRP content of lungexposed to hyperoxia was significantly increased (p<0.01) which reachthe peak at7d. The inflammation and RAC decreased was moresignificant after CGRP8-37administration. The hyperoxia-induced celldeath was notably reduced and cell proliferation was markedly increasedafter CGRP intervention compared to hyperoxia alone (p<0.01).Conclusion CGRP could promote cell survival of AECII exposed tohyperoxia and attenuated oxidative injury of premature lung and lungdevelopment arrest. Part III Calcitonin Gene Related Peptide Protected PrematureLung against Oxidative Stress via Wnt7b/β-catenin PathwayObjective By analyzing the activation of Wnt7b/β-catenin afterhyperoxia and CGRP intervention, the possible signaling transductionpathways involved in CGRP regulation was to be discussed.Methods Premature rats and AECII were exposed to hyperoxia andCGRP treatment. The protein levels of Wnt7b and β-catenin wasassessed by western blot detection and RT-PCR assay of TCF and c-mycmRNA expression.Results The Wnt7b and β-catenin protein levels were remarkablyincreased after hyperoxia exposure (p<0.01vs. air control) and CGRPadministration could enhanced the protein expression (p<0.01). Therewas an increase in either TCF or c-myc mRNA after CGRP treatmentcompare to hyperoxia alone (p<0.01).Conclusion Wnt7b/β-catenin pathway was involved in CGRPprotecting premature lung against hyperoxia-induced injury.