Resveratrol Inhibit LPS-Induced Activation by mTOR Figure 2. Effects of resveratrol on the cell viability of BV-2 microglial cells. BV-2 cells were treated with 25, 50, and 100 mM of resveratrol without LPS treatments or with 1 mg/ml LPS or with 10 nM rapamycin treatments for 18 h. BV-2 cells viabilities were measured and expressed as mean 6 SEM for three independent experiments. doi:10.1371/journal.pone.0032195.g002 rapamycin (1 h before), a specific mTOR inhibitor, significantly reversed resveratrol-downregulated levels of NO and PGE2 induced by LPS (Figures 4A and B). mTOR is required for resveratrol-inhibited expression of iNOS and COX-2 proteins and mRNA induced by LPS in BV-2 cells To investigate the effect of resveratrol on iNOS and COX-2, the expression of both proinflammatory enzymes was examined by double immunofluorescence labeling and RT-PCR assay. BV-2 cells were stimulated with LPS (1 mg/ml) for 4 h which resulted in increase of the protein and mRNA levels of iNOS and COX-2. Pre- treatment with resveratrol (25, 50, and 100 mM) inhibited iNOS and COX-2 protein and mRNA levels significantly (Figures 5 and 6), compared with LPS-treated cells. However, following the pretreatment of cells with 10 nM rapamycin (1 h before), resveratrol could not inhibit upregulation of the protein and mRNA levels of iNOS and COX-2 induced by LPS (Figures 5 and 6). mTOR is involved in resveratrol-attenuated expression of the proinflammatory cytokines TNF-a and IL-1b in LPS stimulated BV-2 cells To investigate whether resveratrol represses the production of TNF-a and IL-1b and whether mTOR is involved in this process in BV-2 cells, cells were stimulated with LPS (1 mg/ml) for 4 h in the presence or absence of resveratrol (25, 50, and 100 mM). After treatment with LPS, the protein levels of the cytokines in BV-2 cells were evaluated by immunofluorescence labeling which showed that TNF-a as well as IL-1b immunoexpression was noticeably enhanced by LPS stimulation. Pretreatment with resveratrol resulted in a drastic decrease in cytokine expression. However, in BV-2 cells subjected to pretreatment with 10 nM rapamycin (1 h before), TNF-a and IL-1b immunofluorescence intensity was obviously increased (Figures 7A and 8A). To further investigate whether the inhibitory effect of resveratrol on TNF-a and IL-1b production is due to the reduced expression of cognate genes, the effect of resveratrol on mRNA expression of TNF-a and IL-1b was assessed in LPS-stimulated BV-2 cells. As shown in Figures 7B and 8B, the mRNA expression of these inflammatory mediators was very low or hardly detectable in unstimulated BV-2 cells. However, BV-2 cells expressed high levels of TNF-a and IL-1b mRNA when stimulated with LPS (1 mg/ml; 4 h). More importantly, resveratrol suppressed LPS- induced expression of these genes. In parallel to the double immunofluorescence labeling, pretreatment of BV-2 cells with rapamycin significantly reversed resveratrol-downregulated levels of TNF-a and IL-1b mRNA (Figures 7B and 8B). mTOR pathway is linked to resveratrol-suppressed LPS-induced expression of NF-kB/RelA protein, phosphorylation of IkB-a and CREB, and protects against LPS-induced activation in BV-2 cells To further elucidate the mechanisms of resveratrol on the inhibition of expression of iNOS, COX-2, and proinflammatory cytokines in microglia, we next examined the effect of resveratrol on NF-kB and CREB, the two major transcription factors involved in the expression of these inflammatory mediators. NF-kB activation includes IkB-a’s degradation through phosphorylation and a subsequent nuclear NF-kB translocation. We determined whether the resveratrol inhibitory effect was through the blockade of NF-kB activation in BV-2 cells. As shown in Figure 9, pre- treatment of resveratrol (25, 50, and 100 mM) significantly suppressed the LPS (1 mg/ml; 4 h) induced expression levels of NF-kB/RelA mRNA and protein, as well as phosphorylation of IkB-a in a mTOR-dependent manner (Figure 10A). CREB is the physiological substrate for MAPKs and stress- activated protein kinases-1 (MSK1), which is activated by ERK and p38 MAPK-mediated signaling in response to LPS. We next determined whether resveratrol regulates the phosphorylation of CREB and whether such an effect, if any, is mediated through the mTOR signaling pathway in BV-2 cells. As demonstrated in Figure 10B, CREB activation was markedly stimulated by LPS; additionally the activation was significantly inhibited by treatment with resveratrol at 50 and 100 mM; nonetheless, pre-treatment PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32195