Resveratrol Inhibit LPS-Induced Activation by mTOR Figure 4. mTOR is required for resveratrol-inhibited expression of NO (A) and PGE2 (B) induced by LPS in BV-2 cells. Approximately 16106 cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (25, 50, and 100 mM) for 1 h in the absence or presence of rapamycin (10 nM), then exposed to LPS (1 mg/ml) for 8 h. The values shown are mean 6 SEM of data from three independent experiments. #Significant compared with control alone, p,0.05. *Significant compared with LPS alone, p,0.05. nSignificant compared with resveratrol+LPS, p,0.05. doi:10.1371/journal.pone.0032195.g004 Discussion Over-activation of microglia contributes to neurodegenerative processes through the production of various neurotoxic factors including free radicals and proinflammatory cytokines [29]. Therefore, the inhibition of microglial activation could reduce neuronal cell death. In fact, a number of anti-inflammatory agents, which inhibit microglial activation or production of proinflamma- tory mediators under the central nervous system disease conditions, attenuate neuronal degeneration [30]. The present study was undertaken to examine the pharmacological and biological effects of resveratrol on the production of inflammatory mediators in murine BV-2 microglia stimulated with LPS. To further understand the molecular mechanism of resveratrol activity in microglia, we investigated the effects of resveratrol on the mRNA and protein expression levels of iNOS, COX-2 and cytokines (TNF-a, IL-1b), the activation of transcription factor NF-kB and CREB, the activation of the MAPKs family, and if Figure 5. mTOR is required for resveratrol-inhibited expression of iNOS protein and mRNA induced by LPS in BV-2 cells. Panel A shows the immunofluorenscence images for protein expression of iNOS and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to b-actin mRNA. Note the up-regulated protein and mRNA expression of iNOS by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean 6 SEM of data from three independent experiments. #Significant compared with control alone, p,0.05. *Significant compared with LPS alone, p,0.05. nSignificant compared with resveratrol+LPS, p,0.05. doi:10.1371/journal.pone.0032195.g005 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32195