The Role Of Thioredoxin In Hyperoxia-induced Lung Injury

Part I The Effect of Hyperoxia on the Expression of Thioredoxin System[Objective] To set up the animal model of hyperoxic lung injury and investigate the expression levels of thioredoxin system.[Methods] In the first day after delivery, the preterm SD rats were randomly divided into two groups:Air group and Hyperxia group. The rats in hyperoxia group were exposed to 85% oxygen and those in air group were exposed to room air. The rats were killed in 1, 4,7 and 14 days respectively. Total RNA of lung was isolated and Trx and TrxR mRNA expression levels were detected by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry was used on lung sections to detect the distribution and expression of thioredoxin. Western blot was used to detect the expression of thioredoxin protein level in lung tissue. The sections of lung were stained with HE in order to assess lung histologic changes and examine lung radical alveolar counts (RAC).[Results]â‘ Rats in hyperxia group showed typical lung injury, which was characterized by alveolitis and delay of lung development. Compared with air group RACs were significantly decreased in hyperxia group (p<0.05).â‘¡RT-PCR results showed Trx and TrxR mRNA expression levels in Hyperxia group were increased markedly, and Trx protein level was increased correspondingly.â‘¢Immunohistochemistry detected that Trx expressed generally in the cytoplasm of alveolar epithelial cells and vascular endothelial cells.[Conclusions] Hyperoxia induced the typical change of bronchopulmonary dysplasia. Trx and TrxR expression levels could be induced by hyperoxia, which might play importantly protective role in the development of hyperxia-induced lung injury. Partâ…¡Recombinant Human Thioredoxin Protects Fetal Typeâ…¡Epithelial Cells from Hyperoxia-induced Injury[Objective] The aim of this study was to determine the role of Trx in the pathogenesis of hyperoxia-induced alveolar epithelial cell injury.[Methods] Alveolar typeâ…¡epithelial cells from fetal rat lung were exposed to hyperoxia in vitro in the presence or absence of recombinant human Trx. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Apoptosis and levels of reactive oxygen species (ROS) were measured by flow cytometry. Activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathways were detected by Western blotting. We also investigated the effects of rhTrx on the following antioxidants (superoxide dismutase, catalase, glutathione peroxidase).[Results] Trx significantly reduced hyperoxia-induced cell death and increased cell viability. In addition, ROS generation in typeâ…¡cells was inhibited by rhTrx under hyperoxic conditions. We demonstrated that rhTrx protected typeâ…¡cells against hyperoxic injury via sustaining the extracellular signal regulated kinase and PI3K activation, and decreasing of c-Jun N-terminal protein kinase and p38 activation. The results also showed manganese superoxide dismutase and glutathione peroxidase activities were increased by rhTrx in typeâ…¡cells exposed to hyperoxia.[Conclusions] Taken together, these results demonstrate that rhTrx administration markedly attenuates hyperoxia-induced typeâ…¡cell injury through reduction of ROS generation, elevation of antioxidant activities and regulation of both MAPK and PI3K-Akt signaling pathways. Part III Research on the Mechanism of Thioredoxin Protects Preterm Rat from Hyperoxia-induced Lung Injury[Objective] To set up the animal model of hyperoxic lung injury, investigate the effect of the recombinant human thioredoxin (rhTrx) on hyperoxic lung injury and explore it's mechanism of action.[Methods] Preterm Sprague-Dawley rats delivered by hysterotomy at 21-d gestation were allowed to recover in room air for 24 h. After one day of birth, pups were randomized to three groups. Group 1 pups were kept in room air and served as controls. Group 2 and 3 pups were oxygen exposed animals maintained in a transparent Plexiglas chambers with 85% oxygen, in which the oxygen concentration was monitored regularly with an oxygen sensor. Pups in group 2 received daily an intraperitoneal injection of 2 mg/kg/d of rhTrx, whereas pups in groups 1 and 3 received only its vehicle (PBS). The body weight of all pups was measured every day. All lung tissues of premature rat pups were collected at 14d after birth. The sections of lung were stained with HE in order to assess lung histologic changes and examine lung radical alveolar counts (RAC). The levels of VEGF,VEGFR,TLR2 and TLR4 mRNA were detected by real-time reverse transcription polymerase chain reaction (RT-PCR). The protein expression levels of p-ERK 1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38,p38,p-Akt,Akt and caspase 3 were determined by western blot.[Results]â‘ The pups in air group did not dead; the mortality of pups in hyperoxia was 28.6%; compared with hyperoxia group rhTrx pretreatment can decreased the mortality to 11.1%.â‘¡Rats in hyperxia group showed typical lung injury, which was characterized by alveolitis and delay of lung development. Compared with air group RACs were significantly decreased in hyperxia group. rhTrx pretreatment improved the hyperoxia induced changes of lung morphometry.â‘¢Real-time PCR results showed:exposure to hyperoxia resulted in the levels of mRNA for VEGF and VEGFR were decreased, TLR4 was increased, but TLR2 mRNA expression level did not change. Rat pups treated with rhTrx from the hyperoxic environment expressed significantly high levels of mRNA for VEGF,VEGFR and TLR4 than the hyperoxic control pups.â‘£Wssren blot results showed: the levels of active ERK1/2, JNK1/2, p38 and caspase 3 after exposure to hyperoxia were higher than air control group, and Akt activity level was decreased.The levels of active JNK1/2, p38 and caspase 3 were decreased markedly after rhTrx treatment in hyperoxia exposure rat pups, but active ERK1/2 and Akt were increased with the same rhTrx treatment.[Conclusions] Trx had a protective effect on hyperoxic lung injury by which decrease active levels of JNK, p38 and caspase 3, increase active levels of ERK and Akt, subsequently increase the expression of VEGF, VEGFR and TRL4.