Fig. 4

TSG suppressed microglial ROS production and NADPH oxidase signaling pathway activation. Primary microglia-enriched cultures were treated with TSG for 30 min before LPS stimulation for 15 min. The extracellular superoxide production was detected by SOD-inhibitable reduction of WST-1 and the intracellular ROS level was determined with DCFH-DA assay (a), Superoxide: the two-way ANOVA interactions analysis of showed F = 4.076 and P = 0.089; intracellular ROS: the two-way ANOVA interactions analysis of showed F = 2.451 and P = 0.137). The subcellular fractions were isolated to perform western blotting for p47 and p67 levels in membrane and cytosolic fractions of microglia. β-actin and gp91 were applied as internal cytosolic and membrane controls, respectively (b), Cytosol-p47: the two-way ANOVA interactions analysis of showed F = 2.273 and P = 0.206; Cytosol-p67: the two-way ANOVA interactions analysis of showed F = 3.798 and P = 0.109; Membrane-p47: the two-way ANOVA interactions analysis of showed F = 1.367 and P = 0.283; Membrane-p67: the two-way ANOVA interactions analysis of showed F = 4.084 and P = 0.091). The whole cell levels of microglial phosphorylated p65 (p-p65), IKK (p-IKK), p38 (p-p38) and JNK (p-JNK) compared to total p65, IKK, p38 and JNK were investigated by western blotting (c), p65: the two-way ANOVA interactions analysis of showed F = 1.425 and P = 0.279; IKK: the two-way ANOVA interactions analysis of showed F = 4.615 and P = 0.061; p38: the two-way ANOVA interactions analysis of showed F = 2.784 and P = 0.114; JNK: the two-way ANOVA interactions analysis of showed F = 4.467 and P = 0.066). Data were expressed as a percentage of the control cultures and were the mean ± SEM from 3 independent experiments performed in triplicate. ^#P < 0.05 compared with control cultures; ^*P < 0.05 compared with LPS-treated cultures