Hyperoxia Induces AECⅡs Apoptosis Via C/EBP Homologous Protein-mediated Pathway

Objective Bronchopulmonary dysplasia(BPD)is a serious threat to the survival rate and long-term survival of premature infants,especially premature infants.High concentration oxygen exposure is one of the important pathogenic factors of BPD in premature infants.Alveolar type II epithelial cell(AECⅡ)is a kind of lung stem cells,which plays an important role in lung development.CCAAT/ enhancer binding protein homologous protein(CHOP)mediated endoplasmic reticulum stress(ERS)related apoptosis pathway has been shown to participate in a variety of diseases,including the occurrence and development of lung disease.The purpose of this study was to investigate the effects of CHOP mediated ERS pathway on the apoptosis of AECⅡ induced by hyperoxia exposure using primary culture of AECⅡ.Methods 19~20 D SD rats were taken out under sterile conditions to take out fetal rats,and the primary culture of AECⅡ was carried out.The AECⅡ cells were randomly divided into air group and hyperoxia group.The hyperoxia group was exposed to 95% O2.Cells were harvested in 24,48 and 72 h,the morphological changes of cells were observed by inverted phase contrast microscope,Annexin V/propidium iodide(PI)double staining followed by flow cytometry analysis was performed to measure the apoptotic fraction of cells.q RT-PCR and western blotting analysis were used to measure the m RNA and protein expression levels of glucose-regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK),activating transcription factor 4(ATF4),the activating transcription factor 6(ATF6),inositol-requiring protein1(IRE1),splicing X-box binding protein-1(XBP1s)and CHOP in the hyperoxia and air control groups.Immunofluorescence analysis was used to characterize the expression and localization of CHOP in the hyperoxia and air control groups..Results(1)AECⅡ primary culture: Primary rat AECⅡ cells were successfully cultured from rat fetal lung tissue.(2)Establish the model of hyperoxic lung injury: AECⅡs showed irregular spreading and vacuolization after 24 h exposure to 95 % oxygen,the cells became evidently flattened and hypertrophied after 48 h exposure.These changes became more evident after 72 hours of exposure.In contrast,cells in air control group grew rapidly,were well arranged,and had no apparent morphological changes.(3)Annexin V/PI double staining and cell apoptosis by flow cytometry: The early apoptosis rate of AECⅡ was low in air group,increased significantly in hyperoxia group,and the differences were statistically significant(all P <0.05).(4)Detect the expression of GRP78,CHOP,PERK,IRE1,ATF6,ATF4,XBP1 and m RNA by q RT-PCR : In each time point,GRP78,CHOP,PERK,IRE1,ATF6,XBP1 and ATF4 m RNA in hyperoxia group were significantly increased compared with air group,the differences were statistically significant(all P <0.05).With the time,the expression of m RNA in the hyperixia group increased gradually,and the difference was statistically significant(all P <0.05).(5)Detect the protein expression of CHOP,PERK,GRP78,IRE1,ATF6,XBP1 and ATF4 by using Western blot: Compared with the air group,the protein expression of CHOP,PERK,GRP78,IRE1,ATF6,XBP1 and ATF4 increased significantly,the differences were statistically significant(all P <0.05).With the time,the expression of protein in hyperixia group increased gradually,and the difference were statistically significant(all P <0.05).(6)The expression of CHOP was detected by immunofluorescence: CHOP was localized within the nucleus as it was co-localized with DAPI.The expression of CHOP in the hyperoxia group was significantly increased in comparison with it in air control group at every time point(all P <0.05).Conclusion(1)Hyperoxia-treated AECⅡs were prone to apoptosis.(2)Hyperoxia initiate ERS to promote AEC IIs apoptosis.(3)CHOP mediated ERS pathway may participate in AECⅡs apoptosis induced by hyperoxia.(4)Three ERS sensors,PERK,ATF6 and IRE1,may play an important role in CHOP activation during AECⅡs apoptosis.