The Effect Of CEBPA On Alveolar Surfactant Protein And Cell Proliferation And Apoptosis After AECⅡ Exposed To Hyperoxia

ObjectiveBronchopulmonary dysplasia（BPD）is one of the common chronic pulmonary disease in premature infants.The occurrence of Bronchopulmonary dysplasia（BPD）is closely related to the mechanical ventilation and long-term inhalation of high concentration of oxygen.CCAAT enhancer binding protein alpha（C/EBPα）plays an important role of key transcription factors in lung development and maturation.This study firstly used of Alveolar typeⅡEpithelial Cell（AECⅡ）in premature rat,to explore the effect of hyperoxia on expression of C/EBPαand pulmonary surfactant protein（SP-A,SP-B,SP-C,SP-D）and their correlations.At the same time,used of AECⅡcell lines,to investigate the effects of C/EBPαoverexpression on proliferation,apoptosis and surfactant protein-C in AECⅡcells after hyperoxia exposure.MethodsSD male and female rats mated,female rats were pregnant for 20-21 days after the abdominal injection of anaesthesia,dissection of the uterus removed fetal rats,AECⅡwas isolated and purified,and then pr imary culture the AECⅡ.The hyperoxia-induced cell model was constructed using 95%O₂ as a follow-up experiment for the hyperoxia group.The AECⅡs were randomly divided into air group and hyperoxia group.The two groups of cells were collected at 24 h,48 h and72 h after exposure to air and hyperoxia.Inverted phase contrast microscopy was used to observe the morphological changes of each group of cells,real-time quantitative PCR and Western blot were performed to measure the mRN A and protein expression of C/EBPαand SP-A,SP-B,SP-C and SP-D.Cell Counting K it-8（CCK-8）was applied to detect the proliferation of AECⅡs.Use of AECⅡcell lines,C ultured AECⅡcells were transfected with pcDNA3.1（+）-C/EBPαor pc DNA3.1（+）-empty vector plasmid by Lipofectamine2000 transient transfection.The cells were randomly divided into air group,air+pc DNA3.1-C/EBPαgroup,air-empty vector group,hyperoxia group,hyperoxia+pcDNA3.1-C/EBPαgroup,and hyperoxia-empty vector group.Real-time quantitative PCR and Western blot were performed to measure the mRNA and protein expression of C/EBPαand SP-C.Cell Counting Kit-8（CCK-8）was applied to cell proliferation.PI staining flow cytometry was used as assays for cell cycle,Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis.Results（1）In the air group,with the prolongation of the incubation time,the AECⅡcells grew well and the morphology was normal,and no dead cells that obviously floated were observed.And then the air group showed a significantly decrease mRN A and protein expression of C/EBPα,and signifcantly increased mRNA and protein expression of SP-A,SP-B,SP-C,SP-D and proliferation of AECⅡs.However,in the hyperoxia group,the morphology of AECⅡcells changed,gradually became irregular,and floating dead cells gradually increased.At 48 hours,the expression of C/EBPαand SP-A,SP-B,SP-C,SP-D and proliferation reached a peak.And then compared with the air group,the hyperoxia group showed a significantly increased mRNA and protein expression of C/EBPαand SP-A,SP-B,SP-C,SP-D and proliferation of AECⅡs at 48 hours.In the hyperoxia group,the protein expression of C/EBPαwas positively correlated with the protein expression of SP-A,SP-B,SP-C,SP-D and proliferation of AECⅡs.（2）After AECⅡ cells were cultured until the fusion rate was over 50%,the pcDNA3.1（+）-C/EBPαplasmid was transfected into AECⅡcells.After transfection for 48 h,the expression mRNA and protein of C/EBPαin the transfection group was significantly higher than that in other groups.（P<0.05）.（3）Compared to the air group,decreased cell proliferation,increased apoptosis,decreased SP-C expression,decreased percentage of cells in G₁ phase,and increased percentage of cells in the S and G₂ phase was observed in the hyperoxia group and hyperoxia-empty vector group（P<0.05）.while opposite effects were observed in the hyperoxia+pc DNA3.1-C/EBPαgroup as compared to the hyperoxia group and hyperoxia-empty vector group（P<0.05）.No significant changes were observed in cell proliferation,SP-C expression,and apoptosis rates in the air+pc DNA3.1-C/EBPαgroup group as compared to the air group and air-empty vector group.Conclusion（1）Under short-term exposure to hyperoxia,C/EBPαcan promote the secretion of pulmonary surfactant protein to participate in the body’s protective regulation.However,with the prolonged exposure hyperoxia,C/EBPαlosed compensatory protection.（2）C/EBPαoverexpression could significantly up-regulates the expression of SP-C,promotes cell proliferation,and inhibits apoptosis in AECⅡcells after exposure to hyperoxia.These data suggested that C/EBPαoverexpression may reverse the damage and exert a protective role in hyperoxia-induced lung cell injury.