Metronomic capecitabine inhibits liver transplant rejection in rats by triggering recipients’ T cell ferroptosis

Figure 7 5-fluorouracil disturbs the antioxidative/oxidative balance, resulting in CD3⁺ T cells ferroptosis. A: Levels of antioxidant proteins nuclear erythroid 2 p45-related factor 2, heme oxygenase-1, and glutathione peroxidase 4 assessed by western blot, and corresponding graphs showing the statistical comparison between groups; B and C: Reactive oxygen species (ROS) and lipid ROS of rat and human CD3⁺ T cells were detected by FACS using DCFH-DA and C11-BODIPY fluorescent conjugates, BODIPY emission was recorded on oxidized C11 (FITC channel) signal; the graphs display the comparison of mean fluorescence intensity levels in the different groups; D: Proportion of cells with reduced mitochondrial membrane potential assessed by flow cytometry; E and F: Glutathione and malondialdehyde content T cells after treatment for 48 h. CON groups: T cells were treated with DMSO for 48 h; 5-fluorouracil (5-FU) groups: T cells were treated with 5-FU (15 μmol/L) for 48 h; 5-FU+ferrostatin-1 (Fer-1) groups: T cells were treated with 5-FU (15 μmol/L) and Fer-1 (2 μmol/L) for 48 h. Statistical analysis was done by one-way ANOVA, n = 3. ^dP < 0.05 vs the 5-FU group, ^eP < 0.01 vs the 5-FU group, ^fP < 0.001 vs the 5-FU group. Nrf2: Nuclear erythroid 2 p45-related factor 2; HO-1: Heme oxygenase-1; GPX4: Glutathione peroxidase 4; GSH: Glutathione; MDA: Malondialdehyde; CON: Untreated control groups; 5-FU: 5-fluorouracil; Fer-1: Ferrostatin-1; MFI: Mean fluorescence intensity.