Figure 6

Microglial PHOX is the target of FLZ's inhibition in LPS-induced neurotoxicity. (A): The role of PHOX in FLZ's protective effect. PHOX^+/+ and PHOX^-/- mouse neuronal-glial cultures were pretreated with vehicle or with 10 μM FLZ for 1 h, followed by 10 ng/ml LPS treatment. Neurotoxicity was assessed by [³H]-DA uptake 7 days later. (B): The effect of FLZ on LPS-induced gp91 mRNA expression. Enriched microglia cells were treated with vehicle or with different concentrations of FLZ for 1 h before the addition of 2 ng/ml LPS. For mRNA analysis, total RNA was harvested 3 h after LPS treatment, followed by real-time reverse transcription-PCR analysis using specific primers. (C): Effect of FLZ on cytosolic p47^phoxprotein translocation. HAPI cells were pretreated with vehicle or with 10 μM FLZ for 1 h followed by 10 ng/ml LPS treatment for 10 min. Subcellular fractions were isolated to perform western blot analysis. C = cytosolic extract; M = membrane extract. GAPDH and gp91^phoxare used as internal cytosolic and membrane controls, respectively. The data are expressed as percentages of control culture values and represent the mean ± S.E.M. for three independent experiments, each performed with triplicate samples. ## p < 0.01 and ### p < 0.001 compared with vehicle-treated control group; * p < 0.05 and ** p < 0.01 compared to LPS treatment group.