Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway
Fig 1
Lansoprazole activates the Nrf2/ARE pathway in RL34 cells.
A: Representative immunoblot for Nrf2 expression levels in the nuclear and cytoplasmic protein lysates obtained from cells treated with 100 μM of lansoprazole for 3 h; α-tubulin and histone H1 were loading controls for the cytoplasmic and nuclear lysates, respectively (left). Densitometric quantification of the representative immunoblots was performed using ImageJ, and values were normalized to each of the loading control protein level (right). B: Representative immunocytochemical images of the subcellular localization of Nrf2. RL34 cells were treated with 100 μM of lansoprazole for 3 h. Cells were fixed in 4% PFA for 15 min, followed by staining with an anti-Nrf2 antibody and an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. Counterstaining of the nuclei was performed with Hoechst 33342. The scale bar represents 50 μm. C and D: Relative mRNA expression levels of Nrf2-induced genes HO1, NQO1, and GSTA2 in cells treated with 100 μM lansoprazole for 3 h. RNA levels were normalized to those of β-actin (C) or GAPDH (D). E: Induction of the ARE-dependent luciferase reporter activity by lansoprazole. RL34 cells stably expressing an ARE-reporter gene were treated with 100 μM of lansoprazole for 3 h, and the luciferase activity in the cell lysates was measured. F: Degradation rate of the Nrf2 protein after CHX chase. Cells were treated with 100 μM of lansoprazole for 3 h and were then treated with 10 μM of CHX for the indicated periods. Cell lysates were used for immunoblotting analysis with an anti-Nrf2 and anti-β-actin antibody. Intensities of the Nrf2 bands were quantified and plotted on a semilog graph to obtain half-life values. Abbreviations used in the figure: LPZ, lansoprazole; CHX, cycloheximide. Data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical analysis was performed by using Student’s t-test. *: p < 0.05 vs. DMSO-treated cells.