Research Paper Volume 12, Issue 18 pp 17990—18007

Figure 1. Activation of SIRT1 suppresses neuroinflammatory responses in microglia. (A) BV-2 microglial cells were incubated with various concentrations (5 or 7.5 μM) of SIRT1 activator (CAY) or SRT1720 (SRT; 1 μM)—for 24 h. Whole-cell lysate proteins were extracted and assessed using a fluorogenic SIRT1 assay kit to detect SIRT1 activity. (B) BV-2 microglial cells were pretreated with SIRT1 activator (5 μM) for 30 min, followed by stimulation with LPS (100 ng·mL⁻¹) for 6 h. Relative mRNA levels of IL-1β were analyzed by real-time PCR and normalized with the levels of β-actin mRNA. (C) BV-2 microglia were pretreated with SIRT1 activator CAY compound (5 μM) or SRT (1 μM) for 30 min before stimulation with either LPS (100 ng·mL⁻¹) or PGN (10 μg·mL⁻¹) for 24 h. (D) Adult mouse microglia (IMG) were pretreated with CAY compound (5 μM) for 30 min before stimulation with LPS (100 ng·mL⁻¹) for 24 h. (E) IMG cells were transfected with empty vector or wild-type SIRT1 for 24 h before stimulation with LPS (100 ng·mL⁻¹) for 24 h. Whole-cell lysate protein was extracted and iNOS and COX-2 protein levels were assessed by western blot analysis. Culture media from BV-2 (F) or IMG (G and H) microglial cells were harvested to determine the nitrite content by the Griess reaction. The results represent the mean ± SEM of n = 3–4. * p < 0.05; compared with the control group, # p < 0.05; compared with LPS or PGN treatment groups.