Bay11

Caco2 cells purchased from American Type Culture Collection were maintained in a high-glucose DMEM medium (Gibco, Rockville, MD, United States) supplemented with 10% fetal bovine serum (Gibco, Rockville, MD, United States), 100-U/mL penicillin, 100-mg/mL streptomycin (Sigma, St. Louis, MO, United States), and 5% CO₂ at 37 °C in a humidified atmosphere for 48 h. To mimic hypoxic conditions, the cells were incubated in a microaerophilic system (Thermo Fisher Scientific, Waltham, MA, United States) with 5% CO₂ and 1% O₂ balanced with 94% N₂ for 12 h. Then, the cells were cultured under normal conditions for 0, 12, 24, and 48 h to achieve reoxygenation.
For inhibitor treatment, the cells were incubated with NF-κB inhibitor BAY11 (40 μmol/L) (Beyotime, Nantong, China), AKT activator IGF-1 (20 μmol/L) (Beyotime, Nantong, China), PI3K activator (740 Y-P, 10 μmol/L) (Beyotime, Nantong, China), NF-κB activator Betulinic acid (BA) (20 μmol/L) (Beyotime, Nantong, China), AKT inhibitor GSK690693 (20 μmol/L) (Beyotime, Nantong, China), and PI3K inhibitor LY294002 (10 μmol/L) (Beyotime, Nantong, China) for 1–8 h and then subjected to H/R. The cells were used for various experiments at different times after the H/R treatment.