Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway
Fig 3
Nrf2 is essential for the cytoprotective activity of lansoprazole.
A and B: Knockdown efficiency of siRNAs targeting Nrf2. RL34 cells were transfected with one of the two siRNAs targeting different sites in the rat Nrf2 mRNA (5 nM) or with control siRNA (5 nM) for 24 h. Nrf2 mRNA levels were measured using quantitative RT-PCR and were normalized to those of β-actin (A) or GAPDH (B). Results are expressed as a percentage of the mRNA levels of the samples treated with control siRNA. C and D: Effects of Nrf2 knockdown on the expression of the lansoprazole-induced gene HO1 mRNA. Cells were transfected with 5 nM of control or pooled Nrf2 siRNA and, 24 h later, were treated with 100 μM of lansoprazole for 3 h. HO1 mRNA levels were measured using quantitative RT-PCR; RNA levels were normalized to the β-actin (C) or GAPDH (D) mRNA level. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using Student’s t-test. *: p < 0.05 vs. DMSO-treated control siRNA-transfected cells. E: Effects of Nrf2 knockdown on the expression of lansoprazole-induced Nrf2 and HO1 protein. Cells were transfected with 5 nM of control or pooled Nrf2 siRNA and then 24 h later were treated with 100 μM of lansoprazole for 3 h. Nrf2 and HO1 protein levels were measured using immunoblotting. β-actin served as a loading control. F: Effect of siRNA depletion of Nrf2 on the lansoprazole-mediated cytoprotection. siRNA-transfected cells were treated with 100 μM of lansoprazole for 3 h and then exposed to 20 μM of cisplatin for an additional 24 h. Cell viability was quantified using an MTS assay. Data are expressed as a percentage of viability when compared with DMSO-treated control siRNA-transfected cells. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed by using ANOVA and Dunnett’s test. *: p < 0.01 vs. cisplatin treatment group. Abbreviations: LPZ, lansoprazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.