Effect of SN on LPS-induced production of pro-inflammatory factors and their mRNA expression. Enriched microglia cells were pretreated with vehicle or SN (10-5, 10-10, and 10-14 M) for 30 min before LPS (10 ng/ml) stimulation. Effects of SN are shown on LPS-induced production of superoxide (A, expressed as % of control); and on intracellular ROS (B, expressed as absolute absorbance). Extracellular superoxide was measured as SOD-inhibitable reduction of WST-1, and intracellular ROS was determined by probe DCFH-DA. Supernatant was collected at 24 h for nitrite assay (C), at 3 h for TNF-α assay (D), and at 24 h for PGE2 assay (E). RNA were extracted at 3 h after LPS stimulation; the effect of SN, at sub-picomolar concentrations, on LPS-induced iNOS, TNF-α, and COX-2 mRNA expression (C-E respectively, open bars) are shown. Results are expressed as mean ± SE for three independent experiments performed in triplicate. *P < 0.05, **P < 0.01 compared with the LPS-treated cultures.