Resveratrol Inhibit LPS-Induced Activation by mTOR Figure 3. Resveratrol activates the Akt/mTOR signaling pathway in BV-2 microglial cells. Panel A shows that BV-2 cells were treated with resveratrol (50 mM) for the indicated times, Panel B shows that BV-2 cells were pre-treated with resveratrol (50 mM) for 1 h, then exposed to LPS (1 mg/ml) for the indicated times and Panels C and D shows that BV-2 cells were pre-treated with resveratrol (25, 50, and 100 mM) for 1 h, then exposed to LPS (1 mg/ml) for 40 min. Various treated BV-2 cell lysates (50 mg protein) were prepared and subjected to Western blot analysis by using antibodies specific for PTEN and phospho-PTEN, Akt and phospho-Akt, mTOR and phospho-mTOR as described in the methods. The relative protein levels were quantified by scanning densitometry and normalized to total Akt. 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.g003 with rapamycin at 10 nM had increased the phosphorylation of CREB most drastically. Inhibition of MAPKs signaling by resveratrol is mTOR- dependent during BV-2 cells activation by LPS The effect of resveratrol on MAPKs, which are upstream signaling molecules in inflammatory reactions, was examined in LPS-stimulated BV-2 cells. Western blot analysis was carried out using the phospho- or total forms of antibodies against the three MAPKs, including ERK1/2, JNK, and p38 MAPK. It was observed that resveratrol at different concentrations 25, 50, and 100 mM significantly decreased the LPS-stimulated phosphorylation of ERK1/2 at 30 min, respectively, whereas it had no effect on the expression level of ERK1/2 in LPS- stimulated BV-2 cells (Figure 10C). Likewise, resveratrol at all concentrations significantly suppressed the phosphorylation of JNK and p38 MAPK, respectively, but did not affect the expression levels of JNK and p38 MAPK in LPS-stimulated BV- 2 cells (Figures 10D and E). To determine the upstream regulators of MAPKs signaling in resveratrol-inhibited activation of BV-2 cells by LPS, BV-2 cells were pretreated with 10 nM rapamycin (1 h before) and, very interestingly, this reversed the effect of resveratrol-inhibited phosphorylation of ERK1/2, JNK and p38 MAPK (Figures 10C, D and E). PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32195