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The Mechinism of Thioredoxin Protecting Against Hyperoxia-induced Lung Injury

Author: DanRuiYan
Tutor: ChangLiWen
School: Huazhong University of Science and Technology
Course: Pediatrics
Keywords: High oxygen Premature birth Apoptosis signal -regulated kinase Thioredoxin Lung injury Type Ⅱ alveolar epithelial cells Thioredoxin reductase Apoptosis Small RNA interference Apoptosis signal-regulated kinase-1 Mitogen-activated protein kinase A549 Signaling pathway
CLC: R722.1
Type: PhD thesis
Year: 2011
Downloads: 76
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The first part of the high oxygen thioredoxin system of the the preterm newborn rat lung tissue and apoptosis signal-regulated kinase expression [Objective] premature newborn rats exposed to hyperoxia lung injury animal model to observe the morphology of high oxygen on premature lung tissue of mice learning impact of preterm newborn rats in hyperoxia-induced lung damage lung tissue apoptosis signal-regulated kinase 1 (apoptosis signal-regulating The effect ASK1) and thioredoxin (thioredoxin reductase, Trx) and its reductase (thioredoxin. Expression and significance reductase, TrxR), explore the hyperoxia-induced lung injury in the pathogenesis and prevention measures. 128 [method] premature newborn SD rats after birth 1d were randomly divided into the air group and hyperoxia group (n = 64). The hyperoxia group are continually exposed to the oxygen concentration of 85% of the oxygen inside the air group in the atmospheric air. Were exposed to air or high oxygen 1 d, 4 d, 7 d, 10 d and 14 d, with 10% urethane anesthesia by intraperitoneal injection to make extracted premature lung tissue of mice, using HE staining of lung Histopathological changes; extraction of total RNA and total protein in lung tissue, using a semi-quantitative reverse transcription - polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) detection Trx and TrxR mRNA expression; immunohistochemistry methods detect lung ASK1 protein expression and distribution organization ASK1 protein expression levels of the Western blot assay of lung tissue changes. [Results] (1) lung tissue pathology observed: Compared with control group, hyperoxia group preterm rat lung tissue appears obvious the alveolar inflammatory changes and lung development lag. (2) immunohistochemical staining results in premature rat lung tissue ASK1 widely distributed in alveolar epithelial cells and vascular endothelial cell cytoplasm; exposed to hyperoxia d, 4-d lung tissue ASK1 protein expression were 0.4382 ± 0.0227 and 0.5270 ± 0.0432, significantly higher than the air group (0.3458 ± 0.02630 and 0.3760 ± 0.0058) (P lt; 0.01) 7 d decreased as .4343 ± 0.0254, but still higher than the air group (.3473 ± 0.0220) (P lt; 0.01 ). (3) high-oxygen group Trx and TrxR mRNA expression of strength were significantly higher than the air group, both at 10 d (0.6860 ± 0.0811) and reached a peak at 7 d (2.0351 ± 0.1600) (P lt; 0.05). (4) Western blot assay ASK1 protein levels with immunohistochemical method results consistent. [Conclusion] The high oxygen exposure can induce premature rat lung tissue undergoing extensive inflammatory response and lung development lag; ASK1 involved in the development of hyperoxia-induced lung injury; Trx system may play an important protective role. The second part of hyperoxia on fetal rat alveolar type II epithelial cells thioredoxin reductase expression [Objective] study hyperoxia-exposed fetal rat alveolar type II epithelial cells (type II alveolar epithelial cell AEC Ⅱ) thioredoxin protein (thioredoxin, Trx) reductase (thioredoxin reductase, TrxR) expression and meaning. [] Using trypsin and collagenase digestion 19d fetal rat lung tissue blocks by differential centrifugation and repeated attachment method, sterile separation, purification, primary cultured fetal rat AEC II, let it grow to Asian fusion, were randomly divided into the air group and hyperoxia group; allowed to stand in the incubator for 30 minutes, the air in the same indoor air, the the hyperoxia group through 10 minutes 850mL / L more than pure oxygen; then sealed flasks, set carbon dioxide culture culture in the box. Group were cultured 12h, 24 and 48h, inverted phase contrast microscope observation of high oxygen on cell morphology and activity; flow cytometry ROS changes and cell apoptosis; using semi-quantitative reverse transcriptase polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) was used to detect the two groups at each time point Trx and TrxR mRNA, Trxl and TrxRl mRNA expression; using western blot detection of fetal rat AEC Ⅱ group at each time point Trx and Trx1 expression. [Results] Compared with air, high oxygen exposure at all time points significantly adverse AEC II cell growth state over time, increased suspension cells; each time point cells exposed to hyperoxia ROS generation and apoptotic cells was significantly increased (P lt; 0.01) ; RT-PCR results showed that high oxygen exposure for 12 h and 24 h after the AEC Ⅱ Trx, TrxR mRNA and Trx1 of, TrxRl mRNA expression was increased (P lt; 0.05), and 48 h after the expression of the two groups the difference was not statistically significant (P gt; 0.05); Western blot results showed that Trx and Trx1 protein expression changes in the mRNA of the same trend (P lt; 0.05). [Conclusion] The high oxygen exposure so that preterm birth rat AEC Ⅱ generate excessive ROS leading to AEC Ⅱ cell damage and apoptosis; hyperoxic exposure can promote fetal AEC Ⅱ Trx and TrxR of mRNA expression increased; also can be induced Trx protein expression increased, suggesting that Trx system of has an important protective role upregulation of alveolar type II epithelial cell injury induced by hyperoxia. Third partially inhibit thioredoxin expression [Objective] To study the effects and mechanisms of A549 cells exposed to hyperoxia A549 cells exposed to high oxygen inhibition of human A549 alveolar type II epithelial cells sulfur thioredoxin (Trx) expression the direct impact of the signaling cascade pathway. [Method] A549 cells cultured in vitro passage in the transfection siNT or siTrx 48 h, respectively, were randomly divided into the air group and hyperoxia group. The air two groups placed in the air, high oxygen two groups through high 600mL · L-1 medium concentration oxygen. Harvested cells were cultured for 12 h, and the level of reactive oxygen species and the number of apoptotic cells in flow cytometry of alveolar type II epithelial cells; western blot detection MAPKs signaling pathway the three subfamilies protein phosphorylation degree of and PI3K/Akt signaling pathway protein phosphorylation level, is also detected two signaling pathways upstream protein ASK1 and EGFR phosphorylation levels. [Results] Compared with the control group of high oxygen ① small interfering Trx expression significantly increased hyperoxia-exposed ROS levels in A549 cells (P lt; 0.05); ② of Trx expression inhibition significantly increased the hyperoxia-induced apoptosis ( P lt; 0.05); ③ of Trx expression the suppression significantly enhanced ASK1 of activity reduces the EGFR phosphorylation degree of (P lt; 0.01); ④ of Trx expression inhibit activation of the c-JunN end kinase 1/2 (c-Jun N-terminal protein kinase, JNK1 / 2) the Wo P38 Mitogen Activated Protein Kinase (P38mitogen-activited protein kinase, p38) activity (P lt; 0.01), while inhibiting the extracellular signal-regulated kinase 1/2 (the extracellular signal regulated type kinase ERK1 / 2) and Akt activity (P lt; 0.01). [Conclusion] inhibit the the Trx expression of the high oxygen exposure increase in the number of A549 cells apoptosis; Trx hyperoxic cell damage and apoptosis through MAPKs and PI3K/Akt signaling pathway is one of the mechanisms of injury of lung epithelial cells exposed to hyperoxia part thioredoxin effects and mechanisms of expression in A549 cells exposed to hyperoxia [Objective] thioredoxin protein (thioredoxin, Trx) overexpression of human lung adenocarcinoma A549 cells induced by hyperoxia explore Trx in protective effect of high oxygen cell damage. [Method] In vitro cultured A549 cells, until their grown to confluency state is divided into four groups, the air control group, air experimental group, the control group of the high oxygen and high oxygen experimental group. Transfected with blank plasmid control group, the experimental group, transfected with a plasmid containing TRX objective gene. 48h after transfection, the the high oxygen two groups through to moderate concentrations 600mL · L-1 high oxygen; air two groups placed in the same room air; were cultured 12h, cells were collected using the flow cytometry method to detect groups A549 cells ROS generation and apoptosis quantity; phosphorylation levels; the three subfamily proteins degree of phosphorylation P13K/Akt signal pathway proteins detected by western blot MAPKs signaling pathway also detected two upstream protein phosphorylation levels. [Results] Compared with the control group of high oxygen, ROS production and apoptosis quantity ① high-oxygen experimental group cells decreased significantly (P lt; 0.05); ② Western blot Trx overexpression significantly reduced the ASKl activity, increased EGFR phosphorylation degree of (P lt; 0.01); ③ The by Western blot display, high-oxygen experimental group of ERK1 / 2 and Akt phosphorylation degree of significantly enhanced (P lt; 0.01), of JNK and p--38 phosphorylation degree of significantly diminished (P lt; 0.05 ). [Conclusion] Trx overexpression reduces moderate concentrations of hyperoxia-induced alveolar epithelial cell apoptosis protection against hyperoxia-induced injury of the epithelial cells, through this study, we further prove Trx by raised the level of ERK1 / 2 and Akt activity, reduce JNK1 / 2 and p-38 phosphorylation to minimize hyperoxia-induced apoptosis of A549 cells in alveolar epithelial cells against hyperoxia mechanism to play a role.

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CLC: > Medicine, health > Pediatrics > Newborns, premature children disease > Neonatal disease
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