Abstract： Objective To examine the correlation between the hyperoxia-induced reactive oxygen species (ROS) production and Toll-like receptors (TLRs) expression in cultured alveolar epithelial cells in order to understand the role of TLRs signaling in inflammatory response during acute lung injury.Methods Three groups of cultured human lung adenocarcinoma cell line A549 were exposed to:①air,②hyperoxia (95 ％O2 + 5 ％CO2 ) and ③ N-acetylcysteine (NAC) pretreatment + hyperoxia.The level of intracellular ROS,TLR2/4 mRNA,TLR2/4 protein and cytokin interleukin-6/8 (IL-6/8) concentrations in the culture supernatant were measured by flow cytometry,reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) in cells harvested at different time points into the treatment.Results A549 cells were found to have a baseline (mainly intracellular) TLR2/4 expression.Continued exposure to hyperoxia caused ①a progressive increase in intracellular ROS,which became significantly higher than the air treated cells after 2 hours of exposure (11.820±3.123 vs.7.223± 1.170,P＜0.01 ),and reached a peak after 48 hours of exposure (113.837±5.247,P＜0.01); ② an increase in intracellular TLR2/4 mRNA which peaked after 2 hours of exposure (TLR2 mRNA:1.820± 0.056 vs.1.263±0.023; TLR4 mRNA:2.080±0.220 vs.1.317±0.107,both P＜0.01)； ③ significant increase in TLR2/4 protein expression (mainly intracellular),both peaked after exposure for 6 hours (TLR2 protein:8.370±1.548 vs.3.930±0.277; TLR4 protein:25.803±5.783 vs.8.867±2.230,both P＜0.01); ④ a progressive increase in culture supernatant IL-6 (ng/L),IL-8 (ng/L) concentration,both peaked at 48 hours (IL-6:2 213.41±69.99 vs.9.76±1.47; IL-8:11 520.38±429.93 vs.159.56±20.80,both P＜ 0.01).NAC pre-treatment significantly supressed the hyperoxia induced intracellular ROS (14.050±1.257 vs.31.180 ± 2.336,P ＜0.01) production,the hyperoxia induced expression of TLR2/4 (TLR2 mRNA:1.270±0.061 vs.1.683±0.025; TLR4 mRNA:1.587±0.058 vs.1.650±0.139; TLR2 protein:3.458± 0.258 vs.8.371 ± 1.548; TLR4 protein:11.611 ± 3.403 vs.25.803 ± 5.783,all P＜0.05),and the hyeroxia induced increase in IL-6 and IL-8 in supernatant level (IL-6:8.42±0.70 vs.73.51±16.70; IL-8:134.94±5.19 vs.772.82± 96.05,both P＜0.05),as seen in the hyperoxia treated groups.Conclusion Hyperoxia induced intracellular ROS production can up-regulate TLR2/4 expression in A549 cells,leading to the release pro-inflammatory cytokines IL-6 and IL-8 from these cells.