Phosphorylated p65

To identify the proteins and signaling pathways within the lung tissue, the right lung was homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich). The total protein content of each sample was determined by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 30 μg protein was electrophoresed by using 10% SDS-polyacrylamide gel and then transferred to a polyvinyl difluoride membrane. The membrane was blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: p-65 (diluted 1:1000, Abcam), phosphorylated-p65 (diluted 1:1000, Abcam), iNOS (diluted 1:1000, Abcam), HO-1 (diluted 1:1000, Cell Signaling, Beverly, MA, USA), Nrf2 (diluted 1:1000, Cell Signaling), β-actin (diluted 1:1000, Cell Signaling), lamine B1 (diluted 1:1000, Abcam). After washing three times with tris-buffered saline (TBS) that was supplemented with Tween 20, the horseradish peroxidase (HRP)-conjugated secondary antibody was diluted to 1:3000 and incubated for 1 h. Membranes were washed again, and binding was detected by using an enhanced chemiluminescence kit (Thermo-Scientific, Waltham, MA, USA). The density of each protein band was evaluated by using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).

Ferric sulfate and ferrous sulfate heptahydrate were purchased from Energy Chemical Technology Co., Ltd. (Shanghai, China). Ferric citrate was purchased from Sigma‐Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle's Medium (DMEM) was obtained from Sigma (Yuzhong District, Chongqing, China). Antibodies against p21 and C646, a p300/CREB‐binding protein (CBP) inhibitor, were purchased from Abcam Trading Co. Ltd. (Shanghai, China). N‐acetyl‐l‐cysteine (NAC), dihydroethidium (DHE), and a ROS Detection Kit were purchased from Beyotime Company (Jiangsu, China). Prussian Blue staining solution was acquired from Leagene Biological Technology Co. Ltd. (Beijing, China). Antibodies against p53, acetylated p53, CD86, and CD206 were from Cell Signaling Technology (Danvers, MA), and IL‐1, IL‐10, TGF‐β, and TNF‐α were purchased from GeneTex (San Antonio, TX). p21, p300, CBP, p65, phosphorylated p65, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, MA). FITC‐CD86 and APC‐CD206 were purchased from Thermo Fisher Scientific (Waltham, MA).