The Role Of CGRP On Fetal Rat Alveolar Epithelial Cell Type Ⅱ Exposed To Hyperoxia And Its Signal Transduction Pathway

Part one An enhanced method for isolation,primary culture,and identification of typeⅡalveolar Epithelial cells from fetal ratsObjectiveTo develop the isolation and purification technology for typeⅡalveolar epithelial cells (AECⅡ) of fetal rat, thus providing experimental means for the study of lung development or neonatal lung disease.MethodsLung tissues of 19-day fetal rats were digested with trypsin and collagenase, then purified for AECⅡwith different centrifugal force and repeated attachment. Cell viability was evaluated by typran inclusion dying before plating into six-well flask. Growth status and shape of attached cells were observed with inverted phase contrast microscope. AECⅡwere identified by electron microscope and its percentage was assessed by modified Papanicolaou dying and immunofluorescence ,the latter aiming to detect expression of surfactant protein C(SP-C) in AECⅡ.Results The total amount of cells we harvested from 3 to5 fetal rat was (36±5)×106 with a viability of (98±2)%. Cells were like polygonal and connected like island under microscope.AECⅡwas ascertained by lamellar bodies found in cytoplasma under electron microscope and its percentage was (96±3)% identified by modified Papanicolaou dying, the same as the result by immunofluorescence for SP-C.ConclusionsHighly Purified and viable AECⅡcould be achieved by our methods and the cell model could be used in further study.Part two Protection of calcitonin gene-related peptide in hyperoxia-induced injury of premature rat typeⅡalveolar epithelial cellsObjectiveTo explore the influence of 60% oxygen and the effects of calcitonin gene-related peptide(CGRP) on typeⅡalveolar epithelial cells(AECⅡ) isolated from premature rat lung in vitro.Methods AECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into four groups: air group, hyperoxia group, hyperoxia plus CGRP group, hyperoxia plus CGRP and CGRP8-37 group. AECⅡof air group and hyperoxia group were exposed to 21% or 60% oxygen respectively for 24h while hyperoxia plus CGRP group were added with CGRP and hyperoxia plus CGRP and CGRP8-37 group with CGRP and CGRP8-37(CGRP receptor antagonist) before placed into 60% oxygen. Concentrations of maleic dialdehyde(MDA), superoxide dismutase(SOD) and total antioxidant capacity (TAOC) in culture cells were detected by ultraviolet spectrophotometer. Reactive oxygen species (ROS) and apoptosis rate of AECII were analyzed by flow cytometry and the mRNA level of surfactant associated protein C (SP-C) was measured by RT-PCR.ResultsThe levels of MDA, ROS and apoptosis cell number were increased whereas TAOC, SOD and SP-C mRNA expression declined in hyperoxia group compared with those in air group(P<0.01). Reversely, MDA, ROS and apoptosis rate were significantly lower and levels of TAOC, SOD and SP-C mRNA expression were significantly higher in group hyperoxia plus CGRP group than in hyperoxia group or hyperoxia plus CGRP and CGRP8-37 group.Conclusions Exposure to 60% oxygen for 24h could cause oxidative injury, induce apoptosis and decrease SP-C mRNA level of AECII in vitro in premature rats, while CGRP may play a protective role against hyperoxic lung injury by antioxidant, inhibition of AEC apoptosis and promotion of the SP-C mRNA expression.Part three The effects of calcitonin gene-related peptide on proliferation of premature rat typeⅡalveolar epithelial cells exposed to 60% oxygenObjectiveTo explore the influence of 60% oxygen and the effects of calcitonin gene-related peptide(CGRP) on growth of typeⅡalveolar epithelial cells(AECⅡ) isolated from premature rat lung in vitro.MethodsAECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into six groups: air, air/CGRP, air/CGRP8-37, hyperoxia, O2/CGRP, and O2/CGRP8-37. AECⅡwere exposed to FiO2 21%(air) or 60% (hyperoxia) for 24h respectively. Air/CGRP and O2/CGRP were carried out by adding CGRP into medium and then putting the plates into air or 60% oxygen for 24h. Air/CGRP8-37 and O2/CGRP8-37 were done by adding both CGRP and CGRP8-37 into cultural fluid before placing the plate into air or 60% oxygen. MTT assay was taken to determine the optimal concentration of CGRP by evaluating the effects of different concentration of CGRP(10-10～10-7M) on growth of normal AECⅡ. Cell proliferation ability was determined by MTT assay and cell cycles by flow cytometry. The mRNA levels and protein contents of surfactant protein C (SP-C) in six groups were measured by RT-PCR and immunofluorescence respectively.ResultsThe 10-8M of CGRP was choose in the study for that MTT results showed CGRP promoted growth of AECⅡin a dose-dependent manner in a range of 10-10～10-8M. CGRP in a concentration of 10-8M could also increase the rate of cells subjected to S and G2/M phases in cell cycles and raised the expression of SP-C mRNA and protein in normal culture of AECⅡ. When exposed to 60% oxygen for 24h, AECⅡshowed a decreased viability, enhanced proportion of cell in G0/G1 phase with a corresponding decline of rates in S and G2/M phases, and reduced levels of mRNA and proteins of SP-C. But addition with 10-8M of CGRP prior to hyperoxia treatment promoted AECⅡproliferation, enhanced the cell proportions of S and G2/M phases, and raised the expression of SP-C mRNA and protein.Conclusions Exposure to 60% oxygen for 24h could inhibited AECⅡgrowth, proliferation and differentiation whereas CGRP could partially counteract the inhibition effects of hyperoxia on AECⅡand play a protective role against hyperoxia-induced lung injury.Part four The protection of CGRP on hyperoxia-induced typeⅡalveolar epithelial cell injury mediated by PKCalpha pathwayObjectiveTo explore the mechanism of CGRP in protecting AECⅡfrom hyperoxia-induced injury through the determination of the signal transduction molecular -- PKC alpha and NF -κB.MethodsAECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into six groups: air, air/CGRP, air/CGRP8-37, hyperoxia, O2/CGRP, and O2/CGRP8-37. AECⅡwere exposed to FiO2 21%(air) or 60% (hyperoxia) for 24h respectively. Air/CGRP and O2/CGRP were carried out by adding CGRP into medium and then putting the plates into air or 60% oxygen for 24h. Air/CGRP8-37 and O2/CGRP8-37 were done by adding both CGRP and CGRP8-37 into cultural fluid before placing the plate into air or 60% oxygen. Western blot was taken to detect the fraction of PKCalpha in membrane and cytosol and translocation of NF-κB was observed under laser confocal microscopy.ResultsWhen CGRP was added into the medium in normal cultural condition, AECⅡshow an increased ratio of membrane to cytoplasm fraction of PKCalpha and strong fluorescence of NF-κB in nucleus compared with those in air and air/CGRP8-37(p < 0.01). After exposure to hyperoxia, the ratio of membrane to cytoplasm fraction of PKC declined and the fluorescence of NF-κB in nucleus enhanced compared with those in air. The ratio of PKCalpha and fluorescence of NF-κB were increased in O2/CGRP compared with those in hyperoxia and O2/CGRP8-37. The differences among groups were significant.ConclusionsThe PKC alpha mediated signal transduction of CGRP and participated in the process that CGRP protected AECⅡfrom hyperoxia-induced injury, while NF-κB is a downstream molecular of PKC alpha, executing in part the function of the PKC alpha signal.