Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway
Fig 5
Activation of p38 MAPK is required for the lansoprazole-induced ARE/Nrf2 pathway in RL34 cells.
A: Effect of SB203580 on the induction of Nrf2 protein expression by lansoprazole. RL34 cells were treated with 10 μM of SB203580 (a specific p38 MAPK inhibitor) for 30 min, and then exposed to 100 μM of lansoprazole for an additional 3 h. Whole cell protein lysates were analyzed by immunoblotting with an anti-Nrf2 antibody; β-actin served as a loading control. B: Effect of SB203580 on the induction of the ARE-dependent reporter activity by lansoprazole. RL34 cells stably expressing the ARE-reporter gene were pretreated with 10 μM of SB203580 for 30 min, and then exposed to 100 μM of lansoprazole for an additional 3 h. Luciferase activity in the cell lysates was measured. C and D: Total RNA was isolated from the cells. Relative mRNA expression levels of HO1 were measured by quantitative RT-PCR. RNA levels were normalized to β-actin (C) and GAPDH (D) mRNA level. Data represent the mean ± SD of three independent experiments performed in triplicate. Student’s t-test was employed for statistical analysis. *: p < 0.05 vs. untreated cells. E: Representative immunocytochemical images of the subcellular localization of Nrf2. RL34 cells were treated with 10 μM of SB203580 for 30 min and then exposed to 100 μM of lansoprazole for an additional 3 h. Cells were fixed in 4% PFA for 15 min, followed by a staining with 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. Abbreviation: LPZ, lansoprazole.