Fig. 5: Ibuprofen inhibits HCC by blocking IRG1/itaconate-regulated immune evasion.

a Analysis of Irg1 mRNA and protein levels in RAW264.7 cells treated with ibuprofen after Hepa 1-6 supernatant (CM1) stimulation. b Schematic diagram of CD8⁺ T cells cocultured with different RAW264.7 cell-derived supernatants (left panel). RAW264.7 cells were treated with Hepa 1-6 cell supernatant (conditional medium 1, CM1), followed by treatment with or without ibuprofen, which was then replaced with fresh medium (the collected supernatants were denoted as CM3 or CM4). RAW264.7 cell-derived untreated supernatant was denoted as CM2. Finally, CD8⁺ T cells were cultured with CM2, CM3, or CM4 and analyzed by flow cytometry. Representative flow cytometry data and summary plot showing the percentages of EOMES⁺, PD-1⁺, and TIM-3⁺ cells among CD8⁺ T cells that were cocultured with RAW264.7 cells previously stimulated with Hepa 1-6 cell supernatant (right panel). c Schematic diagram of WT mice treated with ibuprofen or ibuprofen combined with 4-OI. d The liver images are shown, and the ratio of liver/body weight was determined. n = 6 mice per group. e UPLC‒MS/MS analysis of the abundance of itaconate in the liver tissues of (c). f Representative flow cytometry data and summary plot showing the percentages of EOMES⁺, PD-1⁺, and TIM-3⁺ cells among CD8⁺ T cells in (c). n = 6 mice per group. g Schematic diagram of WT mice treated with ibuprofen or anti-PD-1 antibody. h Tumor images are shown (left panel), and the liver/body weight were measured (right panel). n = 6 mice per group. i Representative flow cytometry data and summary plot showing the percentages of EOMES⁺, PD-1⁺, and TIM-3⁺ cells among CD8⁺ T cells in (g). n = 6 mice per group. All data represent mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-test (d, e) and one-way ANOVA (a, b, f, h, i). Data are representative of three independent experiments with similar results (a, b). Source data are provided as a Source Data file.