| Part 1 The expression of CYP1A1,Nrf2 and NQO1 in A549 cells exposed to hyperoxiaObjective To observe the expression of cytochrome P4501A1（CYP1A1）,nuclear transcription factor E2-related factor 2（Nrf2）and NAD（P）H quinone oxidoreductase 1enzyme（NQO1）in A549 cells exposed to hyperoxia for different time,and explore the correlation between CYP1A1,Nrf2,NQO1 and bronchopulmonary dysplasia（BPD）.Methods A549 cells were randomly divided into two groups:air group and hyperoxia group.The hyperoxia group was continuously exposed to oxygen（95%O2,5%CO2）,while the air group remained in incubator with 5%CO2.Total RNAs were extracted from two groups at 12h,24h and 48h,and detected the expression of CYP1A1,Nrf2and NQO1 by using quantitative real time polymerase chain reaction（q RT-PCR）and Western blot（WB）,and one-way ANOVA were used to analyze results.Results（1）Compared with the air group,the expression of CYP1A1 in the level of m RNA and protein in the hyperoxia group were significanctly up-regulation especially at 24h（p<0.05）.（2）Compared with the air group,the expression trend of Nrf2 and NQO1 after hyperoxia exposure in the level of m RNA and protein were similar to that of CYP1A1,which increased significantly especially at 24h（p<0.05）.Conclusions The expression of CYP1A1,Nrf2 and NQO1 in A549 cells exposed to hyperoxia were significantly abnormal,and the difference were significant at 24h,suggesting that CYP1A1,Nrf2 and NQO1 may be involved in the hyperoxia induced lung injury of BPD.Part 2 The expression of Nrf2 and NQO1 after FICZ enhanced/sh RNA inhibited AHR in A549 cells exposed to hyperoxia and their relationship with apoptosisObjective To observe the expression of Nrf2 and NQO1 after 6-formylindolo [3,2-b] carbazole（FICZ）enhanced/short hairpin RNA（sh RNA）inhibited aryl hydrocarbon receptor（AHR）in A549 cells exposed to hyperoxia and their relationships with apoptosis,explore the effect of AHR,Nrf2 and NQO1 in the development of BPD.Methods The expression of AHR were enhanced by FICZ/inhibited by sh RNA in A549 cells respectively,the dimethyl sulfoxide（DMSO）hyperoxia group and the control sh RNA hyperoxia group were set as control groups.A549 cells were randomly divided into six groups: group1 was air group without intervention,group2 was hyperoxia group without intervention,group3 was FICZ enhanced hyperoxia group,group4 was control sh RNA hyperoxia group,group5 was AHR sh RNA inhibited hyperoxia group,group6 was DMSO hyperoxia group.The hyperoxia groups were continuously exposed to oxygen（95% O2,5% CO2）,while the air group remained in incubator with 5% CO2.Total RNAs were extracted from six groups at 24 h,and detected the expression of CYP1A1,Nrf2 and NQO1 by using q RT-PCR and WB,and the activation level of AHR was represented by the expression of CYP1A1.Morphological changes of cells as well as nuclear translocations of AHR and Nrf2 were observed,reactive oxygen species（ROS）production were detected,and apoptosis of A549 cells in each group were detected by flow cytometry,one-way ANOVA were used to analyze results.Results（1）The expression of AHR were significantly down-regulated by AHR sh RNA.The AHR protein expression in the groups AHR sh RNA1,AHR sh RNA2 and AHR sh RNA3 were down-regulation,and the inhibition efficiency of group AHR sh RNA2 was highest（64.49%）.（2）Compared with group1（air group without intervention）,the A549 cells in the group2（hyperoxia group without intervention）were atrophic,the expression of CYP1A1,Nrf2 and NQO1 were significantly up-regulation with the protein of AHR and Nrf2 were mainly distributed in the nucleus,and the production of ROS increased,the apoptosis rate of cells significantly increased as well（p<0.05）.（3）Compared with group6（DMSO hyperoxia group）,the A549 cells in the group3（FICZ enhanced hyperoxia group）were atrophic mildly,the expression of CYP1A1,Nrf2 and NQO1 were significantly up-regulation with more protein of AHR and Nrf2 located in the nucleus,and the production of ROS decreased,the apoptosis rate of cells significantly decreased as well（p<0.05）.（4）Compared with group4（control sh RNA hyperoxia group）,the A549 cells in the group5（AHR sh RNA inhibited hyperoxia group）were atrophic significantly,the expression of CYP1A1,Nrf2 and NQO1 were significantly down-regulation with less protein of AHR and Nrf2 were located in the nucleus,and the production of ROS increased,the apoptosis rate of cells significantly increased as well（p<0.05）.Conclusions AHR-Nrf2-NQO1 pathway is activated by hyperoxia exposure.Nrf2 and NQO1 are the downstream targets of AHR,AHR-Nrf2-NQO1 plays a protective role in BPD by reducing ROS production as well as cell injury and apoptosis. |
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