[11]. One of these widely used stimuli is LPS, a bacterial endotoxin used to study experimentally induced infection, inflammation, or tissue damage, as well as the biochemistry of inflammatory responses. LPS activates nuclear factor-kB (NF-kB), cyclic AMP responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) family, which are classified into at least three components: extracellular signal- regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK [12] and which have been implicated in the release of immune-related cytotoxic factors such as iNOS, COX-2, and proinflammatory cytokines [7,12]. The mammalian target of rapamycin (mTOR) is a serin/ threonin protein kinase with a central role in the regulation of cell growth and proliferation, as well as of physiological processes such as transcription, mRNA turnover and translation, ribosomal biogenesis, vesicular trafficking, autophagy and cytoskeletal organization [13]. mTOR exists in two functionally distinct complexes called mTORC1 and mTORC2. mTORC1, com- posed of mTOR, mLST8/GbL (G protein b-subunit like protein) and raptor (regulatory associated protein of mTOR) is sensitive to rapamycin, unlike mTORC2 which is composed of mTOR, mLST8/GbL and rictor [14]. In freshly isolated human monocytes and primary myeloid dendritic cells (DCs), mTOR activation inhibits the production of proinflammatory cytokines while it enhances the release of the anti-inflammatory cytokine by blocking NF-kB activation and increasing STAT3 activity [15]. Thus, the mTOR pathway might have opposite roles in the immune system [16]. Recent studies indicated that mTOR selectively controls microglial activation in response to proin- flammatory cytokines and appears to play a crucial role in microglial viability [17], mediates iNOS induction during hypoxia in the BV-2 microglial cell line [18], and reduces microglia/ macrophages activation and increases the number of surviving neurons after brain injury [19], suggesting a key role in the control of microglial functions. Resveratrol (3, 4, 5-trihydroxy-trans-stilbene) is a natural non- flavonoid polyphenolic found in grapes, red wine, mulberries, knotweed, peanuts and other plants (Figure 1) [20]. Although these plants and their extracts have been used for various therapeutic purposes by ancient cultures, resveratrol itself was first described in 1940 as a phenolic component of the medicinal herb hellebore [21]. There are numerous reports in the literature show that resveratrol dampens inflammation in arthritis and immune res- ponsiveness in autoimmune diseases [22,23], suppresses angio- genesis and metastasis in several cancers [24], and inhibits ROS products and platelet aggregation in cardiovascular diseases [25,26]. Resveratrol can penetrate the blood-brain barrier to exert strong neuroprotective effects in vivo [21]. In addition to these beneficial actions, resveratrol has been noted for its anti- inflammatory activities. It can attenuate the activation of immune cells and the subsequent synthesis and release of proinflammatory mediators through the inhibition of the transcriptional factors such as NF-kB and activator protein-1 (AP-1) [27]. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, such as Alzheimer disease, Parkinson disease, and Huntington disease [28], the precise mechanisms for its beneficial effects are still not fully understood. This study examined the effects of resveratrol on LPS- stimulated inflammatory responses in microglia and the potential role of mTOR in this process. To this end, a fuller understanding of the molecular mechanism of microglial activation is clearly desirable in delineating the therapeutic target molecules to reduce the brain inflammation and resulting neuronal injury or death in neurodegenerative diseases. Resveratrol Inhibit LPS-Induced Activation by mTOR Figure 1. Chemical structure of resveratrol. doi:10.1371/journal.pone.0032195.g001 Results Resveratrol and/or rapamycin do not affect the viability of BV-2 cells The cytotoxicity of resveratrol and rapamycin was evaluated in the presence or absence of LPS by MTT assay. Resveratrol and/ or rapamycin did not decrease the viability of the BV-2 microglial cells when they were incubated with or without LPS (1 mg/ml) in the presence or absence of resveratrol (25, 50, and 100 mM) and/ or rapamycin (10 nM) for 18 h. Hence, resveratrol and/or rapamycin exerted no significant cytotoxicity on BV-2 microglial cells (Figure 2). Resveratrol activates the mTOR signaling pathway in BV-2 cells To identify the signaling pathways that are activated by resveratrol in cultured microglial BV-2 cells. We investigated whether resveratrol could activate the mTOR signaling pathway and affect LPS-induced mTOR phosphorylation. Incubation of BV-2 cells with resveratrol (50 mM) significantly induced phos- phorylation of serine residue 2881 of mTOR in time-dependent manner (Figure 3A). Furthermore, when the cells were treated with resveratrol (50 mM) for 1 h before LPS (1 mg/ml) stimulation, mTOR phosphorylation was elevated initially and further in- creased steadily during the 1 h incubation with LPS. The levels of phospho-mTOR were always significantly higher in the cells pretreated with resveratrol compared with the cells treated with LPS only (Figure 3B). PTEN (phosphatase tensin homolog on chromosome 10) and Akt is an effective modulator of mTOR, so we further examined whether resveratrol affects PTEN and Akt phosphorylation induced by LPS. When the cells were treated with resveratrol (25, 50, and 100 mM) for 1 h before LPS (1 mg/ml) stimulation for 40 min, resveratrol significantly increased phosphorylation of PTEN, so it downregulated PTEN activity by its phosphorylation (Figure 3C), and the level of phospho-Akt was always significantly higher in dose-dependent manner in the cells pretreated with resveratrol compared with the cells treated with LPS only (Figure 3D). mTOR is involved in resveratrol-inhibited releases of NO and PGE2 induced by LPS in BV-2 cells Treatment of BV-2 cells with LPS (1 mg/ml) caused a significantly increase in NO and PGE2 releases in comparison with untreated controls by Griess reagent and enzyme-linked immunosorbent assay (ELISA), after 8 h exposure to LPS. Resveratrol (25, 50, and 100 mM) inhibited the LPS-induced production of NO and PGE2 over the concentration range used here. However, following the pretreatment of cells with 10 nM PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32195