Mitochondrial Respiratory Chain And Hyperoxia-induced Lung Injury

Part 1 An Investigation of the relationship of functional impairmentof mitochondrial respiration chain induced by hyperoxia andhyperoxia induced lung injuryExperiment 1 Effects of Hyperoxia on Mitochondrial MultienzymeComplexâ…¢andâ…¤in the Premature Newborn Rat Lung【Objective】The aim of this study is to investigate the effects of hyperoxia onmitochondrial multienzyme complexâ…¢(cytochrome, Cytb) andâ…¤(ATPase6, 8)in premature newborn rat lung. To explore the relationship of functional impairmentof mitochondrial respiration chain induced by hyperoxia and hyperoxia-inducedlung injury.【Methods】Based on the animal model of hyperoxia induced lung injury set upby our preliminary research, the 1-day-old preterm SD rats were randomly assignedto hyperoxia group and air group. The rats in hyperoxia group were continuouslyexposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day(s)of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6,8 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR).Immunohistochemistry was used to detect the distribution and expression of Cytb protein on lung sections. Western blot was used to detect the expression of Cytbprotein in lung tissue.【Results】(1) Compared with air group, Cytb mRNA expression wassignificantly increased after 1, 4 day(s) of exposure (P＜0.05). The general tendencydecreased after 7 days, and its expression became weak but the difference of CytbmRNA expression was not significant between the two groups (P＞0.05).(2) Western blot showed that Cytb protein expression was increased after 1,4day(s) of exposure, but the difference between the two groups was not significant.The general tendency was decreased after 7 days, and its expression became weakbut difference was not significant after 7, 10 days of exposure (P＞0.05). At day 14its expression became significantly weak (P＜0.05).(3) ATPase6 mRNA expression was significantly increased after 1 day ofexposure (P＜0.05) and did not show any significant change after 4, 7, 10 days ofexposure (P＞0.05). At the 14th day, ATPase6 mRNA expression was significantlyincreased (P＜0.05). ATPase8 mRNA expression did not show any significant changeafter 1, 4, 10 day(s) of exposure (P＞0.05). At the 7th and 14th day, ATPase8 mRNAexpression was significantly increased (P＜0.05).【Conclusion】We are led to conclude that exposure to high concentrations ofoxygen can significantly change the expression of Cytb and ATPase6,8 in prematurerat lung, which results in uncoupling of oxidative phosphorylation in mitochondrialrespiration chain, oxidative stress caused by ROS from "electron leakage", energymetabolism impairment, this indicated that these changes may participate in thephysiological and pathology processes of hyperoxia-induced lung injury. Functionalimpairment of mitochondrial respiration chain induced by hyperoxia palys animportant role in the development of hyperoxia-induced lung injury. Experiment 2 Effects of Hyperoxia on Mitochondrial MultienzymeComplexâ… andâ…£in human adenocarcinoma of lung A549 cell【Objective】The aim of this study is to investigate the effects of hyperoxia onmitochondrial multienzyme complexâ… (NADH dehydrogenase subunit 1, ND1) andâ…£(cytochrome oxidaseâ… , COXâ… )in human adenocarcinoma of lung A549 cell.To explore the relationship of functional impairment of mitochondrial respirationchain induced by hyperoxia and hyperoxia-induced lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber(37℃, 5% CO₂),the cells were randomly divided into air group and hyperoxia group. Hyperoxiagroup was continuously exposed to oxygen (oxygen＞90%, carbon dioxide=5%)While air group still in specific room air (carbon dioxide=5%). After exposure tooxygen or room air for 12, 24 and 48 hours, those cells 'RNA was isolated, ND1 andCOXâ… mRNA were detected by reverse transcription polymerase chainreaction(RT-PCR).【Results】(1) Compared with air group, the expression of ND1 mRNA in A549cell was increased after 12 hours of exposure, but the difference of the two groupswas not significant (P＞0.05). the expression of ND1 mRNA significantly decreasedafter 24 hours of exposure (P＜0.05), ND1 mRNA expression decreased after 48hours of exposure, but the difference of the two groups was not significant (P＞0.05).(2) Compared with air group, the expression of COXâ… mRNA in A549 cellwas increased after 12 hours of exposure, but the difference of the two groups wasnot significant (P＞0.05). the expression of COXâ… mRNA was significantlyincreased after 24, 48 hours of exposure (P＜0.05).【Conclusion】Exposure to high concentrations of oxygen can significantlychange the expression of ND1 and COXâ… in A549 Cell, which results in functionalimpairment of mitochondrial respiration chain induced by hyperoxia palys animportant role in the development of hyperoxia-induced lung injury. Part 2 Effects of Hyperoxia on the Expression of Thioredoxin-2 andCytochrome c in A549 Cell Exposure to Hyperoxia【Objective】To investigate the effects of the expression of thioredoxin-2 andcytochrome c in human adenocarcinoma of lung A549 cell from human alveolarepithelial cellâ…¡exposure to high concentrations of oxygen. And to explore thatwhat Trx-2 and Cytc have changed plays an important role in hyperoxia lung injury,Trx-2 likely protects mitochondria of A549 cell in hyperoxia lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber(37℃,5% CO₂),the cells were randomly divided into air group and hyperoxia group. Hyperoxiagroup was continuously exposed to oxygen (oxygen＞90%, carbon dioxide=5%)While air group still in specific room air (carbon dioxide=5%). After exposure tooxygen or room air for 12, 24 and 48 hours, those cells 'RNA and protein wereisolated, Trx-2 and Cytc mRNA were detected by reverse transcription polymerasechain reaction (RT-PCR). Western-blot was used to detect Trx-2 protein in A549cells.【Results】(1) Compared with air group, Trx-2 mRNA expression became weakafter 12h of exposure (P＞0.05), Trx-2 mRNA expression was significantlyincreased after 24 hours of exposure (P＜0.05); its expression became weak after 48hours of exposure, but both were not significantly different (P＞0.05)(2) Compared with air group, Cytc mRNA expression was significantlyincreased after 12 hours of exposure (P＜0.05); its expression became weak after 24hours of exposure, but both were not significantly different (P＞0.05). itsexpression was significantly decreased after 48 hours (P＜0.05).(3) Compared with air group, Trx-2 protein expression was significantly increased after 12 hours of exposure (P＜0.05); the general tendency increasedafter 24 hours, but both were not significantly different (P＞0.05).its expressionwas significantly decreased after 48 hours (P＜0.05).【Conclusion】Exposure to high concentrations oxygen can significantly changethe expression of Trx-2 and Cytc in A549 cells, which indicates that Cytc plays animportant role in the process of cell apoptosis in hyperoxic lung injury, and Trx-2likely protects mitochondria of A549 cell in hyperoxic lung injury.Part 3 Correlation between Expression of Thioredoxin-2 Suppressedby Small Interference RNA in A549 Cells Exposed to Hyperoxia andthe Lung Cell Metabolism and Apoptosis【Objective】The aim of this study was to explore the expression ofthioredoxin-2 suppressed by SiRNA in A549 cells exposed to hyperoxia, and thecorrelation between with the lung cell metabolism and apoptosis, and to discuss thepathogenesis and control measures of hyperoxia induced lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber(37℃,5% CO₂),A549 cells were transfected with synthetic Trx-2 sequence -specific siRNA byLipofectamine 2000. These cells were randomly divided into four groups:â… airgroup,â…¡hyperoxia group,â…¢air group with small interference RNA, andâ…£hyperoxia group with small interference RNA. Hyperoxia group was continuouslyexposed to oxygen (oxygen=95%, carbon dioxide=5%) While air group still inspecific room air (carbon dioxide=5%). After exposure to oxygen or room air for 12, 24 and 48 hours, those cells 'RNA and protein were isolated, Trx-2, ATPase6,8 andCytc mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR),Trx-2 protein was detected by Western blot, Flow cytometry was used todetect the apoptosis of A549 cells suppressed by Trx-2 SiRNA.【Results】(1)Sequence-specific SiRNA targeting Trx-2 significantly down-regulatedits expression in A549 cells, the results of Western blot assay revealedTrx-2 protein levels were inhibited after transfection in different groups (SiRNA-1group,SiRNA-2 group,SiRNA-3 group), and the highest inhibitory rate wasSiRNA-1 group (79.51%).(2)Compared with air group, the expression of Trx-2 and Cytc mRNA weresignificantly increased after 24 hours exposure (P＜0.05), but the expression of CytcmRNA became weak significantly after 48 hours exposure in hyperoxia group (P＜0.05).Compared with air group with small interference RNA, the expression of Trx-2mRNA became weak significantly after 24,48 hours exposure (P＜0.05), but theexpression of Cytc mRNA was significantly increased after 12,24 hours exposure inhyperoxia group with small interference RNA (P＜0.05)Compared with hyperoxia group, the expression of Trx-2 mRNA wassignificantly decreased after 48 hours exposure, but the expression of Cytc mRNAwas significantly increased after 24 hours exposure in hyperoxia group with smallinterference RNA (P＜0.05)(3)Compared with air group, the expression of ATPase6 mRNA wassignificantly increased after 12,48 hours exposure (P＜0.05), but the expression ofATPase8 mRNA was significantly increased after 24,48 hours exposure in hyperoxiagroup (P＜0.05).Compared with air group with small interference RNA, the expression ofATPase6,8 mRNA became weak significantly after 24,48 hours exposure in hyperoxia group with small interference RNA (P＜0.05)Compared with hyperoxia group, the expression of ATPase6 mRNA wassignificantly decreased after12, 48 hours exposure, but the expression of ATPase8mRNA was significantly increased after 12, 48 hours exposure in hyperoxia groupwith small interference RNA (P＜0.05).(4) Compared with hyperoxia group, The apoptosis percent of A549 cells inhyperoxia group with small interference RNA was signifcantly higher after 24 hoursof exposure(P＜0.05), they were(31.78±4.52)% VS (13.57±0.69)% respectively.The apoptosis percent of A549 cells in hyperoxia group with small interferenceRNA was signifcantly higher than air group with small interference RNA after 24,48 hours of exposure (P＜0.05),they were (31.78±4.52) % VS (15.47±0.57)%, (38.51±4.08) %VS (25.84±1.01) %respectively.【Conclusion】Exposed to high concentrations oxygen can significantly changethe expression of Trx-2,ATPase6,8 and Cytc, the apoptosis percent of 549 cells wassignificantly higher, which indicates that dysmetabolism and cell apoptosisparticipate the development of hyperoxia induced lung injury, Trx-2 likely protectsmitochondria of A549 cells in hyperoxia lung injury. Protection of mitochondrialDNA (mtDNA) may be an important mechanism of anti-hyperoxic lung injury bythioredoxin system.