The lung tissues were homogenized with the tissue lysis buffer (Beyotime). The tissue homogenate was lysed on ice for 15 min and treated with centrifugation (4000 g, 4 °C, 15 min). According to the protocol, MPO activity in the supernatant was determined using a MPO assay kit (A044-1-1, Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd, Nanjing, China). Then, cells were lysed primarily. Cell lysate was centrifuged at 600 g and 4 °C for 10 min to collect the supernatant. According to the protocols, MDA content and SOD activity were measured using a MDA commercially available kit (A003-1-2, Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd) and a SOD assay kit (A001-3-2, Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd).
Following the manufacturer's instructions, ROS level was determined using a ROS assay kit (S0033S, Beyotime). For in vivo analysis, lung homogenate was incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 30 min. The change in fluorescence intensity at 500/530 nm was analyzed using a fluorescence microplate (Bio-Rad). For in vitro analysis, cells were incubated with DCFH-DA at 37 °C for 20 min and washed with serum-free medium. The fluorescence intensity at 488/525 nm was determined and results were shown as percentages.
Following the manufacturer's instructions, ROS level was determined using a ROS assay kit (S0033S, Beyotime). For in vivo analysis, lung homogenate was incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 30 min. The change in fluorescence intensity at 500/530 nm was analyzed using a fluorescence microplate (Bio-Rad). For in vitro analysis, cells were incubated with DCFH-DA at 37 °C for 20 min and washed with serum-free medium. The fluorescence intensity at 488/525 nm was determined and results were shown as percentages.