Fig. 4. (+)-CLA attenuates APAP-induced cytotoxicity through the activation of Keap1–Nrf2 pathway.

a, b Hepa RG cells were pre-incubated with (+)-CLA (5 μM) for 1 h, and then incubated with APAP (10 mM) for another 24 h. a The protein expressions of Keap1 and Nrf2 were analyzed by western blot (upper panel) and the band intensities relative to β-actin were quantified using image J software (bottom panel). b The mRNA level of Nrf2 was analyzed by qRT-PCR. c The influence of Nrf2 on the protective effect of (+)-CLA on APAP-induced cytotoxicity was determined. After transfaction with control siRNA (no silencing) or Nrf2 siRNA for 6 h, cells were incubated with (+)-CLA (5 μM) for 1 h, and then treated with APAP (10 mM) for another 24 h. The cell viability was measured by the MTT assay. d The interaction between Keap1 and Nrf2 was determined by Co-IP assay. HepG2 cells, transfected with Nrf2 and Keap1 plasmids, were treated with or without (+)-CLA for 6 h, and immunoprecipitated proteins were subjected to western blot analysis with Nrf2 and Keap1 antibodies. e, f (+)-CLA promoted the nuclear translocation of Nrf2. Cells were incubated with or without indicated doses of (+)-CLA for 18 h. The protein levels of Nrf2 in the nucleus and cytoplasm were determined using e western blot analysis and f immunofluorescence. Scale bar = 10 μm. g–i The effect of (+)-CLA on Keap1–Nrf2 pathway was investigated in the liver tissues of APAP-treated mice. g The protein expressions of HO-1, Keap1, and Nrf2 were analyzed by western blot. h The protein expressions of Nrf2 in the nucleus and cytoplasm were analyzed by western blot. i The mRNA levels of HO-1, NQO1, and GCLM were analyzed by qRT-PCR. Data are expressed as mean ± SD of three independent experiments and the statistical differences were analyzed by one-way or two-way ANOVA (a–c, e and i, two-way ANOVA; d one-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. APAP group.