SCM-198 inhibited microglia via c-Jun N-terminal kinase (JNK) and NF-κB pathways. BV-2 cells were pretreated with 1 μM SCM or 10 μM SP (a specific inhibitor of JNK) for 2 hours, followed by 24-hour incubation with 1 μg/ml LPS. Nitric oxide (NO) (a) and TNF-α (b) in the cell culture medium were measured by Griess reaction and ELISA assay, respectively. The inhibitory effect of SCM on NO and TNF-α production could be mimicked by SP. BV-2 cells and primary microglia were both pretreated with 1 μM SCM, 100 μM ibuprofen (IBU) or 20 μM donepezil (DON) for 2 hours, and stimulated with 1 μg/ml lipopolysaccharide (LPS) (c) or Aβ1-40
(d) for 30 minutes; NF-κB p65 translocation was observed using fluorescence microscope (c, left panel of each group, BV-2 cells, scale bar, 100 μM) or confocal microscope (c-d, right panel of each group, primary microglia, scale bar, 10 μM). Primary microglia were stained using CD11b antibody (d, microglial marker, red) and 4',6-diamidino-2-phenylindole (DAPI) (nuclear staining, blue), purity of primary microglia is more than 95%. Data represent mean ± SEM of more than three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s test versus only LPS-treated group; #P < 0.05, ##P < 0.01, ###P < 0.001, Tukey’s test versus control group.