Figure 5 Alleviated hydrogen peroxide-induced ferroptosis in IAR-20 cells co-cultured with BMMSCs. (A) Relative vitality of IAR-20 cells in different groups, which was significantly higher in the R group and RB group than that in the ROS group (R group vs ROS group, P=0.035; RB group vs ROS group, P<0.001; RB group vs R group, P<0.001). (B) Intracellular concentrations of free Fe2+ in IAR-20 cells of different groups, which were significantly lower in the R group and RB group than that in the ROS group (R group vs ROS group, P=0.010; RB group vs ROS group, P<0.001; RB group vs R group, P=0.003). (C, D) Intracellular concentrations of ROS (graded by the fluorescent intensity) in IAR-20 cells of different groups, which were significantly lower in the R group and RB group than that in the ROS group (R group vs ROS group, P=0.036; RB group vs ROS group, P<0.001; RB group vs R group, P=0.002). (E) Western blot of GPX4 and PTGS2 in IAR-20 cells of different groups. The expression levels of GPX4 in the RB group were significantly higher than those in the R group and ROS group (R group vs ROS group, P=0.001; RB group vs ROS group, P<0.001; RB group vs R group, P=0.011). The expression levels of PTGS2 in the RB group were significantly lower than those in the R group and ROS group (R group vs ROS group, P=0.002; RB group vs ROS group, P<0.001; RB group vs R group, P=0.005). (F) The ultrastructure of IAR-20 cells in different groups. The Mock group showed intact cellular morphology, with normal mitochondrial structure. The ROS group showed destroyed intracellular mitochondria, with twisted and broken cell ridges, and a large number of autophagy vacuoles in the cytoplasm. Morphology of the IAR-20 cells in the RB group was significantly better than that of the R group and ROS group, with significant fewer intracellular autophagy vacuoles and better mitochondrial morphology. * P<0.05 vs the ROS group, # P<0.05 vs the R group. Mock group – normal IAR-20cells; ROS group – hydrogen peroxide-induced injury at 45 min; R group – recovered by resuming normal culture for 6 h after injury; RB group – recovered by resuming normal culture and co-cultured with BMMSCs for 6 h after injury. ROS – reactive oxygen species; GPX4 – glutathione peroxidase 4; PTGS2 – prostaglandin-endoperoxide synthase 2; FITC – fluorescein isothiocyanate isomer.