Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells Lian-Mei Zhong1,2., Yi Zong3., Lin Sun4., Jia-Zhi Guo 3 , Wei Zhang 2 , Ying He 3 , Rui Song 4 , Wen-Min Wang 2 , Chun-Jie Xiao1*, Di Lu3* 1 School of Life Science, Yunnan University, Kunming, Yunnan, China, 2 Department of Neurology, the First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China, 3 Department of Anatomy, Kunming Medical University, Kunming, Yunnan, China, 4 Department of Cardiology, the Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China Abstract Background: Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. Methodology/Principal Findings: BV-2 cells were treated with resveratrol (25, 50, and 100 mM) and/or LPS (1 mg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor kB-a (IkB-a) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and nuclear factor-kB (NF-kB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IkB-a, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N- terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). Conclusion and Implications: This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-kB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol. Citation: Zhong L-M, Zong Y, Sun L, Guo J-Z, Zhang W, et al. (2012) Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells. PLoS ONE 7(2): e32195. doi:10.1371/journal.pone.0032195 Editor: Joao B. Calixto, Universidad Federal de Santa Catarina, Brazil Received October 10, 2011; Accepted January 24, 2012; Published February 21, 2012 Copyright: 2012 Zhong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants (30860336 and 30560170) from the National Natural Sciences Foundation of China, grants (2008CC007 and 2009CI033) from the Department of Science and Technology of Yunnan Province, and grants (2008CD016, 2010CD156 and 2011FB177) from the Joint Special Funds for the Department of Science and Technology of Yunnan Province-Kunming Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ludi20040609@126.com (DL); xiao2009cj@yahoo.com.cn (CJX) . These authors contributed equally to this work. Introduction Microglia, the resident immune cells in the brain, serves the first line of defense when injury or disease occurs and play a homeostatic role in the central nervous system (CNS) [1]. Although activated microglia scavenge dead cells from the CNS and secrete different neurotrophic factors for neuronal survival [2,3], it is believed that severe activation causes various autoimmune responses leading to neuronal death and brain injury [4,5]. Activation of microglia and consequent release of proinflammatory and/or cytotoxic factors such as tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), nitric oxide (NO), prostaglandin E2 (PGE2), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), and cyclooxygenase- 2 (COX-2) are believed to contribute to neuronal damage, particularly in neurodegenerative diseases [6,7,8,9]. Subsequently, the damaged neurons release toxic soluble factors, which in turn induce microglial activation termed as reactive microgliosis [10]. It has recently been suggested that the activation of microglia can increase neurotoxicity through the production of proinflam- matory and cytotoxic factors in neuron-glia cultures treated with lipopolysaccharide (LPS), b-amyloid, glutamate, and arachidonate PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32195