Objective To explore the expression of thioredoxin-2 (Trx-2) suppressed by small interference RNA (SiRNA) in A549 cells exposed to hyperoxia and its relationship with the lung cell metabolism and apoptosis, to discuss the pathogenesis and the prevention and control of hyperoxia induced lung injury. Methods A549 cells were gained by serial sub cultivation in vitro. A549 cells were transfected with synthetic Trx-2 sequence-specific SiRNA by Lapofectamine 2000 when they were approaching confluence in carbon dioxide (CO2) culture chamber (37℃ , 5% CO2). These cells were randomly divided into four groups, air group without interference, hyperoxia group without interference, air group after interference, and hyperoxia group after interference. Hyperoxia group was continuously exposed to oxygen (92% oxygen, 5% carbon dioxide) , while air group was still in specific room air (5% carbon dioxide). After exposure to oxygen or room air for 12, 24 and 48 hours, RNA of cells was isolated. Trx-2, ATPase 6, 8 and CytC mRNA was detected by reverse transcription polymerase chain reaction ( RT-PCR). Trx-2 protein was detected by Western-blot. Flow cytometry was used to detect the apoptosis of A549 cells suppressed by Trx-2 SiRNA. Results Sequence-specific SiRNA targeted Trx-2 and significantly down-regulated its expression in A549 cells.Compared with air group without interference, the expression of Trx-2 and CytC mRNA were significantly increased after 24 hours exposure (P < 0.05), but the expression of CytC mRNA significantly decreased after 48 hours exposure in hyperoxia group without interference (P < 0.05); the expression of ATPase 6 mRNA was significantly increased after 12 and 48 hours of exposure (P < 0.05), but the expression of ATPase 8 mRNA was significantly increased after 24 and 48 hours of exposure in hyperoxia group without interference (P < 0.05). Compared with air group after interference, the expression of Trx-2 mRNA significantly decreased after 24 and 48 hours of exposure (P < 0.05), but the expression of CytC mRNA was significantly increased after 12 and 24 hours of exposure in hyperoxia group after interference (P < 0.05); the expression of ATPase 6, 8 mRNA significantly decreased after 24 and 48 hours of exposure in hyperoxia group after interference (P < 0.05). The apoptosis rate of A549 cells in hyperoxia group with inferterence was significantly higher than air group with inferterence after 24, 48 hours of exposure (P < 0.05). Conclusions Exposure to high concentrations oxygen can significantly change the expression of Trx-2, ATPase 6, 8 and CytC and this indicates that dysmetabolism and cell apoptosis participate the development of hyperoxia induced lung injury, and Trx-2 likely protects mitochondria of A549 cells in hyperoxia lung injury.%目的 研究小分子干扰RNA (SiRNA)抑制高氧暴露下人肺腺癌A549细胞硫氧还蛋白-2(Trx-2)表达及其与肺细胞代谢和凋亡的关系,探讨高氧肺损伤的发病机制和防治措施.方法 体外传代培养A549细胞系,待其生长至接近亚汇合状态时,将体外化学合成Trx-2序列特异性的SiRNA通过Lipofectamine2000转染A549细胞.将细胞随机分为无干扰空气组、无干扰高氧组、干扰后空气组、干扰后高氧组4组.高氧组持续暴露于常压高浓度氧(92％O2,5％CO2)中；空气组仍置于5％CO2培养箱中.4组分别于高氧或空气暴露12、24、48 h提取A549细胞总RNA.采用半定量RT-PCR测定Trx-2、三磷酸腺苷合成酶6,8(ATPase 6、8)及细胞色素C (CytC)mRNA的表达；Western-blot检测Trx-2蛋白表达；流式细胞术检测高氧和沉默Trx-2对A549细胞凋亡的影响.结果 序列特异性SiRNA可抑制Trx-2表达,SiRNA-1、SiRNA-2、SiRNA-3 3组Trx-2蛋白表达均受到抑制,SiRNA-1组抑制效率最高(79.51％).与无干扰空气组相比,无干扰高氧组12 h CytC和24 h Trx-2 mRNA的表达增高,48 h CytC mRNA的表达降低(P＜0.05).与干扰后空气组相比,干扰后高氧组24、48 h Trx-2 mRNA的表达减弱,但在12、24 h CytC mRNA表达水平升高(P＜0.05).与无干扰空气组相比,无干扰高氧组12、48 hATPase 6 mRNA的表达增高,24、48 h ATPase 8 mRNA的表达增高(P＜0.05).与干扰后空气组相比,干扰后高氧组24、48 h ATPase 6,8 mRNA的表达减弱(P＜0.05).干扰后高氧组24、48 h A549细胞凋亡百分数明显高于干扰后空气组(P＜0.05).结论 高氧暴露和沉默Trx-2导致A549细胞ATPase 6,8表达下调和CytC表达升高,表明能量代谢障碍和细胞凋亡参与高氧肺损伤发病过程,Trx-2对高氧肺损伤线粒体起重要的保护作用.
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