Figure 5.

p-38 MAPK mediates cyclo(His-Pro)-induced Nrf2 translocation and antioxidant gene expression in PC12 cells. Cells were pre-incubated in the presence of 10 μM of SB203580, a well-known compound proved to be selective for p-38 MAPK at such concentration. After 2 hrs, 50 μM of cyclo(His-Pro) was added, and cells were incubated for further 24 hrs followed by 100 μM H₂O₂ treatment. (A) Involvement of p-38 MAPK in cyto-protection by cyclo(His-Pro), as assumed by Western blotting. Results are given as mean ± S.D. values for n = 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H₂O₂-treated cells; §P< 0.05 versus CHP-treated cells. (B) Nrf2 protein is decreased in the nuclear extracts of cells treated with the p-38 MAPK inhibitor, as assessed by Western blotting. (C) Real-time PCR analyses of the samples revealing changes in the expression of Nrf2 downstream genes, whose values were normalized to (J-actin expression, and presented as 2^−AACt. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). (D) Cell viability (MTT reduction), ROS, NO and glutathione (GSH) were assessed in the SB203580-treated cells incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to H₂O₂ treatment. Control values (100%) are indicated in the Figure 2 legend. Results are given as mean ± S.D. values for n = 3 independent experiments. *P < 0.05 versus CHP+H₂O₂-treated cells.