Effects of hyperoxia exposure and small interfering RNA on the expressions of nuclear factor-erythroid 2-related factor 2 and NAD(P)H quinone oxidoreductase 1 enzyme in A549 cells and their relationship with apoptosis / 中华实用儿科临床杂志

ABSTRACT

Objective:To analyze the effect on the expressions of nuclear factor-erythroid 2-related factor 2(Nrf2) and NAD(P)H quinone oxidoreductase 1 enzyme (NQO1) in A549 cells exposed to hyperoxia and interfered by small interfering RNA, and to investigate the role of Nrf2 and NQO1 in hyperoxia-induced lung injury as well as their relationship with apoptosis.Methods:A549 cells were gained by serial sub cultivation in vitro and then randomly divided into 4 groups the air group without interference ( group Ⅰ), the hyperoxia group without interference (group Ⅱ), the air group transfected with Nrf2 siRNA (group Ⅲ), and the hyperoxia group transfected with Nrf2 siRNA (group Ⅳ). The hyperoxia groups (Ⅱ, Ⅳ group) were continuously exposed to an atmosphere containing a high concentration of oxygen (950 mL/L O 2, 50 mL/L CO 2), while the air groups (group Ⅰ, Ⅲ) were still placed in the incubator with 50 mL/L CO 2. In the pre-experiment, cells were transduced with a mixture of siRNA-1, siRNA-2, and siRNA-3. Then the siRNA with the highest efficiency for repressing Nrf2 expression was used for subsequent experiments. The mRNA and protein expression levels of Nrf2 and NQO1 in the 4 groups were detected by quantitative real-time polymerase chain reaction (qPCR) and Western blot.The distribution of Nrf2, Kelch-like ECH-associated protein 1(Keap1) and antioxidant response element(ARE) proteins in A549 cells after interference with Nrf2 was analyzed by immunofluorescence and confocal laser scanning microscope, and the cell apoptosis of the 4 groups were observed. Results:(1) Nrf2 siRNA significantly down-regulated the mRNA expression of Nrf2 in the groups siRNA-1, siRNA-2 and siRNA-3, and the inhibition efficiency of group siRNA-1 was the highest (80.57%). (2) The re-lative mRNA expression levels of Nrf2 and NQO1 in the group Ⅱ were 4.553±0.498 and 5.866±0.582, respectively.The mRNA and protein expression of Nrf2 and NQO1 and the cell apoptosis rate [(21.67±0.75)%]in the hype-roxia group were significantly higher than those in the group Ⅰ (all P<0.01). (3) The relative mRNA expression levels of Nrf2 and NQO1 in the group Ⅳ were 0.937±0.057 and 0.789±0.058, respectively.Compared with the group Ⅱ, the mRNA and protein expression of Nrf2 and NQO1 in the group Ⅳ were significantly decreased, while the cell apoptosis rate [(35.83±0.42)%]was significantly increased (all P<0.01). Conclusions:The abnormal expression of Nrf2 and NQO1 in A549 cells induced by hyperoxia and siRNA interference suggests that Nrf2 and NQO1 are involved in the pathogenesis of hyperoxia induced lung injury.Nrf2 and NQO1 are possibly protective factors in the hyperoxia induced lung injury and apoptosis.