its increase of survival and multi-organic protection from endotoxin shock in vivo. These studies thus identify C12 as a unique anti-inflammatory agent that may be positioned as a therapeutic agent for the treatment of inflammatory diseases. Results C12 inhibited LPS-induced NO production in mouse peritoneal macrophages (MPMs) Previously, we demonstrated that C12 was able to inhibit LPS- induced TNF-a and IL-6 release in a dose-dependent manner in RAW264.7 macrophages [15]. Due to the importance of NO as an inflammatory mediator on inflammation-related physiological and pathological processes [10], we tested the effect of C12 on the nitrite level, an index of NO production, in MPMs stimulated with LPS (1 mg/mL). As shown in Figure 1B, C12 dose-dependently repressed LPS-induced increase in nitrite after 18-hour incubation. C12 treatment at 10 mM reduced nitrite level by 45% compared to the LPS-challenged group (p,0.01). C12 suppressed LPS-induced proinflammatory gene expression in MPMs MPMs were treated with LPS (0.5 mg/mL) for 6 h and examined for the expression of proinflammatory genes in the presence or absence of C12 by real-time quantitative PCR. In the dose- dependent study in our previous work [15], C12 at 2.5 uM exhibited an inhibition of 77.4% against LPS-induced IL-6 expression and an inhibition of 40.3% against TNF-a in macrophages. Therefore, we selected the concentration of 2.5 uM for further RT-qPCR study here. As shown in Figure 2, C12 at 2.5 mM potently inhibited LPS-induced up-regulation of IL-12 (74.3%, p,0.01), IL-1b (91.5%, p,0.01), TNF-a (80.2%, p,0.01), IL-6 (69.6%, p,0.01), and cycloxygenase-2 (COX-2, 40.7%, p,0.05) transcripts in MPMs. C12 inhibited LPS-induced NF-kB activation and ERK/JNK phosphorylation In the LPS-induced inflammatory stress, LPS-TLR4 signal transduction leads to MyD88-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor (NF)-kB, which regulate the transcription of a variety of inflammatory factors and enzymes [16,17]. We investigated whether C12 acts as an anti-inflammatory agent by inhibiting NF-kB and MAPKs. We first evaluated the effect of C12 C12 Exerts Anti-Inflammatory Effects on NF-kB p65 nuclear translocation. As shown in Figure 3A, LPS could strengthen NF-kB p65 nuclear translocation (red point in blue nucleus), while in C12-pretreated cells, LPS-induced nuclear p65 decreased, suggesting that C12, at a concentration of 10 mM, inhibited p65 translocation from cytoplasm to nuclei. IkBa phosphorylation and degradation frees NF-kB p65 from seques- tration, allowing it to translocate to the nucleus, bind to target promoters, and turn-on transcription [12]. We next determined the total IkBa and phosphorylated IkBa in total cell protein extracts by Western blotting. LPS exposure for 1 h induced a 7.4- fold phosphorylation (Figure 3B) and an 87% degradation of IkBa (Figure 3C) in MPMs. Conversely, C12 at 10 mM inhibited LPS-induced IkBa phosphorylation (20% inhibition, Figure 3B) and degradation (4.43-fold increase, Figure 3C) in MPMs, respectively. ERK, p38, and JNK constitute the MAPK family and are important intracellular components of the inflammatory responses to LPS. Especially, phosphorylated JNK and ERK could activate NF-kB signaling by IKKa/b-mediated IkBa phosphorylation and degradation [12,16,17]. Therefore, we tested whether the anti- inflammatory effect of C12 is due to its interference with these pathways. Figure 3D–3F shows that all of three MAPKs were activated by LPS stimulation. Among these kinases, the LPS- induced phosphorylation of ERK and JNK were significantly attenuated by pretreatment with C12 (10 mM). However, C12 did not decrease the amount of phosphorylated p38. Although C12 inhibits LPS-induced ERK phosphorylation in MPMs, it is unknown whether the ERK pathway is required for the inhibitory effects of C12 on proinflammatory responses. To this end, we utilized PD98059, a specific p-ERK inhibitor, to suppress the cellular ERK pathway activity. In the presence of PD98059, the inhibition of LPS-induced TNF-a release by C12 were signifi- cantly abrogated in MPMs (p,0.05, Figure 3G). These results imply that NF-kB activation and ERK phosphorylation might be an important step in mediating the anti-inflammatory effects of C12 in macrophages. C12 inhibited the increases of serum TNF-a, IL-6 and NO levels in LPS-challenged mice C12 was further evaluated its anti-inflammatory benefits in animals. Given the in vitro results and in vivo toxicity test in (Figure S1), mice were administered with C12 at 15 mg/kg (i.p.) or 10 and 30 mg/kg (i.v.) in the following in vivo experiments (Text S1). The serum levels of TNF-a, IL-6 and NO can reflect the degree of Figure 1. The structure and NO-inhibitory activity of C12. A. Chemical structures of C12 and its water-soluble form C12-HCl; B. C12 inhibitd nitrite production. MPMs were pretreated with vehicle (H2O) or C12 (2.5, 5, and 10 mM) for 2 h and then stimulated with 1 mg/mL LPS for 18 h. The nitrite levels in medium were detected as described in Materials and Methods. Bars represent the mean6SD of three independent experiments performed in duplicate, and asterisks indicate significant inhibition (* p,0.05, ** p,0.01). doi:10.1371/journal.pone.0024377.g001 PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24377