The Mechinism Of Thioredoxin Protecting Against Hyperoxia-induced Lung Injury

[Objective] To investigate the changes and potential roles of the expression of apoptosis signal-regulating kinase 1(ASK1), thioredoxin (Trx) and thioredoxin reductase (TrxR) in the pathogenesis of lung injury of premature newborn rats exposed to hyperoxia.[Methods] In the first day after delivery, preterm SD rats were randomly divided into air group and hyperoxia group with 64 rats in each group. The rats in hyperoxia group were exposed to 85% oxygen, and those in air group were exposed to room air. The rats were respectively sacrificed in 1, 4, 7, 10 and 14 days after exposure to air or hyperoxia exposure. Sections of lung were stained with HE to assess lung histologic changes. Total RNA of lung were extracted, Trx mRNA and TrxR mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry was used to detect the expression and distribution of ASK1. Western blot was used to detect changes of expression of ASK1 protein.[Results] (1) Rats in hyperoxia group showed typical lung injury, which was characterized by alveolitis and delay of lung development. (2) Immunohistochemistry detected that ASK1 expressed generally in the cytoplasm of alveolar epithelial cells and vascular endothelial cells; ASK1 protein expression in hyperoxia group at 1th and 4th day were 0.4382±0.0227 and 0.5270±0.0432, higher than those in air group (0.3458±0.02630) and (0.3760±0.0058) (P<0.01), and it was until 7th day that the expression became weaker (0.4343±0.0254), but still increased compared with the air group (0.3473±0.0220)(P<0.01). Compared with the air group, Trx mRNA and TrxR mRNA in the hyperoxia group increased significantly and peaked at 10th day(0.6860±0.0811) and 7th day(2.0351±0.1600) respectively(P<0.05). (3) Western blot showed ASK1 protein expressions had the same trends with the results from the immunohistochemistry.[Conclusion] Hyperoxia induced the typical change of bronchopulmonary dysplasia in premature newborn rats. ASK1 participated in the pathogenesis of hyperoxia-induced lung injury, and in which, Trx and TrxR could be an important protective role.[Objective] To study the effect of hyperoxia on the expression of thioredoxin (Trx) and thioredoxin reductase(TrxR) in rat embryonic typeⅡalveolar epithelial cells(AECⅡ).[Methods] The lung tissue of 19-day fetal rats digested with Trypsin and Collagenase was isolated and purified in different centrifugal forces and different adherences, then the primary fetal rat AECⅡwere cultured in vitro. Cells grew to subconfluence and then were randomly divided into two groups: air group and hyperoxia group. After standing in culture chamber for 30 minutes, the air group was put in the same room air and the hyperoxia group was given 850mL/L oxygen(2L/min) for 10 minutes; then culture flasks of the two groups were enclosed and cultured in culture chamber. After cells were cultured for 12 h, 24 h and 48 h, the effects of hyperoxia on shape and activity of AECⅡwere observed by inverted phase contrast microscope. Cells in the two groups were harvested to detect their reactive oxygen species (ROS), apoptosis by FACS, and the levels of Trx and TrxR mRNA, Trx1 and TrxR1 mRNA were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). At the same time, western blot was used to detect the expression of Trx1 protein in AECⅡ. [Results] Compared with the air group, the growth condition of the hyperoxia group AECⅡwas not well and suspension cells increased. FACS detection showed, compared with air group, in accordance with the generation of excessive ROS, apoptosis percentage of AECII exposed to hyperoxia was increased significantly at each different time interval (P<0.001). RT-PCR showed, compared with appropriate controls, the expressions of both Trx, TrxR mRNA and Trx1, TrxR1 mRNA were significantly increased in AECII exposed to hyperoxia for 12 h and 24 h (P<0.01), respectively. Up to for 48 h, Trx1 and Trx mRNAs as well as TrxR and TrxR1 mRNAs in hyperoxia group became weak but the differences was no significance between two groups(P>0.05). Western blot showed, Trx and Trx1 protein expressions in hyperoxia group had the same trends with the results from the RT-PCR compared with air group.[Conclusion] Exposure to prolonged hyperoxia can produce excessive ROS and induce the injury and death of AECⅡ. The expressions of Trx system mRNA and Trx protein in AECⅡcould be up-regulated by hyperoxia, which might be an important protective mechanism in the development of hyperoxia-induced lung injury.[Objective] The purpose of the present study was to investigate the direct functional effects of suppressing Trx levels on A549 Type II alveolar epithelial cells exposed to moderate hyperoxia including the signaling cascades.[Methods] A549 cells were fed and subcultured in vitro. After transfection with NT siRNA or Trx siRNA for 48 h, A549 cells were divided into air groups and hyperoxia groups. Air groups were cultured in 5% CO2 in a humidified cell culture incubator, and hyperoxia groups were cultured in 60% O2 and 5% CO2 in cell culture incubator. After exposure to oxygen or air for 12 h, we harvested cells and detected intracellular ROS levels and apoptosis cells in A549 human endothelial cells by flow cytometry. We also detected the phosphorylation levels of all three subfamilies of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathways by western blot. In addition, we also detected the changes of ASK1 and EGFR acting as respective key upstream protein of above two signaling pathways.[Results] Compared to hyperoxia control group,①Trx siRNA significantly increased the generation of ROS in A549 cells exposed to 60 % hyperoxia for 12 h ( P <0.05);②Trx siRNA significantly increased apoptosis cells in hyperoxia experiment group A549 cells (P <0.05);③Trx siRNA not only enhanced the expression of ASK1, but also down-regulated the level of EGFR in hyperoxia exposed A549 cells (P <0.01);④In parallel, we observed that p38 and JNK1/2 MAPKs of three subfamilies were further phosphorylated, whereas phosphorylated ERK1/2 and Akt mediating cells survival signaling pathway were weakened (P <0. 01).[Conclusion] Taken together, our results indicate that Trx plays an important role in hyperoxia-induced epithelial lung cells injury and apoptosis via regulating MAPKs and PI3K/Akt signaling pathways.[Objective] To investigate the effects of hyperoxia on human adenocarcinoma of lung A549 cells over-expressed thioredoxin, and explore the protective roles of Trx in hyperoxia induced cells injury.[Methods] A549 cells were passaged and cultured in 5% CO2 culture chamber. When approaching to the condition of subconfluence in CO2 culture chamber, the cells were randomly divided into air control group, air experiment group, hyperoxia control group and hyperoxia experiment group. The two control groups were transfected blank plamid, while the two experiment groups were transfected thioredoxin plasmid. Air groups were cultured in 5% CO2 in a humidified cell culture incubator, and hyperoxia groups were cultured in 60% O2 and 5% CO2 in cell culture incubator. After exposure to oxygen or air for 12 h, we harvested cells and assayed intracellular ROS levels and apoptosis cells by flow cytometry. At the same time, the total proteins of A549 cells were extracted and the phosphorylation levels of all three subfamilies of the mitogen-activated protein kinases and phosphatidylinositol 3-kinase-Akt pathways were detected by western blot. In addition, we also detected the changes of ASK1 and EGFR acting as respective key upstream protein of above two signaling pathways.[Results] Compared to hyperoxia control group,①apoptosis cells and the level of ROS were significantly decreased in hyperoxia experiment group A549 cells over-expressed Trx (P <0.01);②western blot showed, Trx overexpression in A549 cells not only down-regulated the phosphorylation levels of ASK1, but also enhanced the level of EGFR in hyperoxia-exposed A549 cells (P <0.01);③phosphorylation levels of Akt and ERK1/2 in hyperoxia experiment group A549 cells over-expressed Trx were remarkably elevated for 12 h ( P <0.01); phosphorylation levels of JNK1/2 and p38 in A549 cells exposure to hyperoxia for 12 h were significantly decreased (P <0. 05).[Conclusion] Trx over-expression could decrease of apoptotic cells in moderate hyperoxia-induced epithelial cells and protect epithelial cells from hyperoxic injury. The research have confirmed Trx played an important role in protecting lung epithelial cells from hyperoxia induced injury via down-regulating phosphorylation levels of JNK1/2 and p38 MAPKs signaling pathways and up-regulating phosphorylation levels of ERK1/2 and Akt.