Fig. 4

Andrographolide induces HO-1 expression through p38 MAPK- and ERK-dependent regulation of Nrf2. Effects of andrographolide on a p38 MAPK and b ERK activation. Primary astrocytes were treated with increasing concentrations of andrographolide for 6 h and processed for immunobloting. Bar graphs showing mean ± S.E.M. fold changes in optical density (OD, with vehicle-only “0 μM” group set as 1) of phospho-protein normalized to total protein, ***p < 0.001; significantly different from vehicle-only group (one-way ANOVA with Dunnett’s post hoc tests). c Effects of p38 MAPK and ERK inhibition on HO-1 mRNA expression. Primary astrocytes were pretreated with or without SB202190 (p38 MAPK inhibitor) or PD98059 (MEK/ERK inhibitor) for an hour followed by 4 h of incubation with andrographolide (with presence of inhibitors) before processing for RT-PCR. Bar graphs are mean ± S.E.M. fold change in transcript level, with untreated group set as 1. Raw transcript values were normalized to mean expression of housekeeping genes (see the “Methods” section) prior to conversion to fold-change values. *p < 0.05, **p < 0.01, and ***p < 0.001; significantly difference from untreated group (one-way ANOVA followed by Dunnett’s post hoc tests.). ^# p < 0.05, ^## p < 0.01; significantly different from 30 μM andrographolide (one- way ANOVA followed by Bonferroni’s post hoc tests). d, e Effects of p38 MAPK and ERK inhibition on Nrf2 in the cytosolic and nuclear compartments, respectively. Primary astrocytes were pretreated with PD98059 or SB202190 for 1 h followed by another 1 h of incubation with 30 μM andrographolide (in the presence of inhibitors). Cytoplasmic and nuclear fractions were separated and processed for immunoblotting. Bar graphs show immunoreactivities (mean ± S.E.M. fold changes in optical densities, OD, with untreated group set at 1) of Nrf2 normalized to β-actin (cytoplasmic) or to lamin B1 (nuclear). *p < 0.05, **p < 0.01, ***p < 0.001; significant pairwise difference and n.s. = not significant (p > 0.05) using one-way ANOVA with Bonferroni’s post hoc tests. All data were from three to four independent experiments