mTOR signaling pathway might be involved in resveratrol’s action on activated BV-2 cells. The results of this study indicated that resveratrol activated the mTOR signaling pathway and induced mTOR phosphorylation in a time-dependent manner in BV-2 cells. Similar to resveratrol, LPS also induced slightly mTOR phosphorylation in BV-2 cells. However, the levels of phospho- mTOR significantly increased 40 min after the addition of LPS. Thus, it appears that the delay in mTOR activation initially allows LPS to activate MAPKs and induce an acute inflammatory response. In contrast, when the cells were treated with resveratrol before LPS stimulation, mTOR phosphorylation was elevated initially and further increased during LPS exposure. Hence, the proinflammatory response to LPS was suppressed by resveratrol from the beginning, resulting in inhibition of LPS-induced MAPKs activity and activation of transcription factor NF-kB and CREB. PTEN is originally identified as tumor suppressor gene mutated in a large percentage of human cancers [31]. It is considered to be a key negative regulator of the Akt/mTOR signaling, which is well known as a prosurvival pathway, suggesting perspective molecular target for anti-cancer research [32]. Moreover, several reports have suggested anti-inflammatory activity of PTEN [33,34]. Here we further identified that resveratrol inactivates PTEN protein by its phosphorylation in LPS-stimulated BV-2 cells. Moreover, the presence of resveratrol significantly increased phosphorylation of Akt in LPS-stimulated BV-2 cells. Thus, we suggest that the modulatory effect of resveratrol on PTEN/Akt/mTOR signaling is critical for its inhibition of proinflammatory mediators and cytokines expression in LPS-activated BV-2 cells. COX-1 is constitutively expressed in most tissues, while COX-2 is induced by an array of stimuli including cytokines, LPS, and growth factor in microglia and astrocytes [7]. COX-2 is the key enzyme in the formation of PGE2 which appear to be an important source of PGE2 during inflammatory conditions [35]. Microglial cells in the healthy brain do not express iNOS, but they become activated to produce iNOS and to release a large amount of NO following ischemic, traumatic, neurotoxic or inflammatory damage [36]. Therefore, any substance that can attenuate expression of iNOS and COX-2 could be beneficial for delaying the progression of neurological disorders. In the present study, resveratrol (25, 50, and 100 mM) significantly inhibited the production of NO, PGE2, iNOS and COX-2 in a dose-dependent manner in LPS-stimulated BV-2 microglial cells, suggesting possible beneficial effects of resveratrol by attenuation of activation of microglial cells and subsequent inflammatory neurotoxins. More importantly, we have shown that pretreatment of BV-2 cells with rapamycin (10 nM), a mTOR inhibitor, effectively reversed the protective effects of resveratrol as evidenced by the reduced expression levels of NO, PGE2, iNOS and COX-2 that were comparable to that induced by LPS alone. These results suggest that mTOR plays an important role in the inhibition of NO, PGE2, iNOS and COX-2 following resveratrol-protection against LPS-induced microglial activation. TNF-a and IL-1b are two main proinflammatory cytokines that are produced by activated microglia during CNS inflammation. In the CNS, a number of stimuli, such as LPS, amyloid-b and traumatic brain injury have been shown to abundantly produce TNF-a and IL-1b [37,38]. Overproduction of proinflammatory cytokines from activated microglial cells has a detrimental effect on neuronal cells. This study investigated whether resveratrol inhibits LPS-induced production of proinflammatory cytokines in BV-2 cells. Resveratrol inhibited LPS-induced production of TNF-a and IL-1b in a dose-dependent manner. Meanwhile, Pre-treatment with resveratrol significantly inhibited TNF-a mRNA levels, and Resveratrol Inhibit LPS-Induced Activation by mTOR resveratrol notablely inhibited dose-dependently IL-1b mRNA levels, compared with LPS-treated control. Furthermore, we have shown that pretreatment of cells with rapamycin followed by LPS+resveratrol, TNF-a and IL-1b protein and mRNA expres- sion levels were markedly increased compared with that of the cells treated with LPS+resveratrol. The findings support that mTOR participates in resveratrol-inhibited expression of proinflammatory cytokines. The transcriptional regulation of NO, PGE2, iNOS, COX-2 and inflammatory cytokines, such as TNF-a and IL-1b, is a tightly controlled event. A variety of transcription factors, including NF- kB and CREB, is known to be involved in the transcriptional regulation of these inflammatory mediators [39]. In un-stimulated cells, NF-kB is retained in the cytoplasm by binding to IkB-a. The processes of NF-kB activation include degradation through phosphorylation and a subsequent nuclear translocation of the RelA subunit of NF-kB. The molecular mechanisms underlying anti-inflammatory effect of resveratrol, which showed the most potent anti-inflammatory activity, were further studied. The present results have shown that NF-kB/RelA level, markedly increased by LPS stimulation in BV-2 cells, was effectively reversed by resveratrol treatment in a mTOR-dependent manner. Concomitantly, pre-incubation of LPS stimulated cells with rapamycin reversed the resveratrol-induced phosphorylation of IkB and CREB. These results indicated that the inhibition of resveratrol on the expression of NO, PGE2, iNOS, COX-2 and proinflammatory cytokines is partially through the suppression of NF-kB/RelA expression, the phosphorylation of IkB-a and CREB in a mTOR-dependent manner in LPS-stimulated BV-2 cells. MAPKs family has been shown to play important roles in LPS- induced iNOS, COX-2, and proinflamatory cytokines expression in many types of cells [40,41,42]. It also has been reported that LPS-induced proinflammatory cytokines expression is mediated by MAPKs signal transduction pathway in BV-2 cells [43]. Therefore, we investigate the effect of resveratrol on activation (phosphorylation) of three MAPKs induced by LPS in BV-2 cells. The results of this study indicate that resveratrol inhibits LPS- increased activation of MAPKs, including ERK1/2, JNK, and p38 MAPK, within 30 min after stimulation, whereas resveratrol decreased LPS-induced activation of MAPKs, which was accom- panied by alterations in NO, PGE2, iNOS, COX-2, and pro- inflammatory cytokines. This result is inconsistent with a recent study [27]. The discrepancy may be due to differences in cell origin and experimental conditions (Our data indicate that resveratrol-inhibited activation of MAPKs occurs at higher concentrations). Additionally, rapamycin partly reversed the effect of resveratrol-inhibited phosphorylation of ERK1/2, JNK and p38 MAPK. In conclusion, this investigation demonstrates that resveratrol significantly attenuates overactivation of microglial cells by re- pressing expression levels of neurotoxic proinflammatory mediators and cytokines via activation of mTOR signaling pathway. These results suggest the potential of resveratrol as an anti-inflammatory drug candidate. Methods Cells and treatments The mouse microglial cell line BV-2 was developed in the laboratory of Dr Blasi at the University of Perugia and was a generous gift of Dr Cheng-gang Zou (School of Life Science, Yunnan University, Kunming, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL, Gaithersburg, MD, USA) containing 2% fetal bovine serum PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32195