gene expression in liver of mouse models [27,28]). We further surmised that C12 would decrease LPS-induced inflammatory gene expression in liver. Figure 5 presented the evidence that i.v. injection of C12 significantly reduced the mRNA levels of a host of proinflammatory mediators (TNF-a, IL-1b, IL-6, COX-2, iNOS, and PGES) in the LPS-challenged mouse livers. Thus, C12 is able to decrease the production of proinflammatory mediators both in vitro and in vivo. LPS injection further causes a whole-body inflammatory response such as multiple organ failure (MOF), including lung injury. The pulmonary injury is considered to be a direct cause of death by LPS injection, and often occurred in the context of MOF [29]. The lung histological examinations in Figure 6A demonstrated that C12 suppressed LPS-caused severe lung damage remarkably. LPS-induced acute multi-organ failure ultimately causes animal death. We further confirmed that C12 would significantly decrease LPS-induced lethality. Thus, the shift toward an anti-inflammatory cytokine profile by C12 is in agreement with its beneficial effect in the endotoxemic shock model. Of particular interest in this study is the identification of molecular mechanism by which C12 inhibits inflammatory gene expression. LPS binds to TLR4-CD14-MD88 complex on the cell surface to trigger MAP kinase phosphorylation and then activation of NF-kB, a transcription factor normally sequestered in the cytoplasm that upon stimulation translocates to the nucleus to drive transcription of target genes, including cytokines, chemo- kines and cellular adhesion molecules [12,16,17]). The transcrip- tion factor NF-kB is retained in the cytoplasm in an inactive form by the inhibitory protein IkB. Activation of NF-kB requires that IkB be phosphorylated on serines 32 and 36, which results in targeted degradation of IkB. The dissociation of IkB causes the translocation of NF-kB p65 sub-unit from the cytoplasm to nucleus. The latter binds to the DNA site and triggers the inflammatory gene expression. Herein, we showed that C12 inhibited LPS-induced ERK/JNK phosphorylation and NF-kB activation and the subsequent induction of proinflammatory mediators in MPMs (Figure 3). JNK has been demonstrated as an upstream regulator of NF-kB signal [12,16,17]. Although MAPK pathways including ERK, p38, and JNK are all involved in LPS-stimulated inflammation, we did not find that C12 affects p38 activation. Furthermore, the anti-inflammatory effect of C12 was attenuated by ERK-specific inhibitor, and PD98059 also significantly inhibited the LPS-induced TNF-a release (Figure 3G). These results indicate that ERK critically plays a key role to turn on proinflammatory gene expression in LPS-stimulated macro- phages, and at least partly, C12 exerts an anti-inflammatory effect via inhibition of ERK pathway. The partial reversion of p-ERK inhibitor suggests that there exist ERK-independent mechanisms in the anti-inflammatory action of C12. In the other hand, the leading curcumin has been reported to exert anti-inflammatory effects by multi-targeting mechanisms and the direct binding targets of curcumin are still unknown. Therefore, although this work only focuses on the MAPK/NF-kB-mediated inflammation, further studies are necessary to establish such notion as examination of the underlying molecular mechanisms and direct molecular targets of C12. Besides the endotoxin and etiologic agents, some chemicals also induce the production of inflammation. During tissue inflamma- tion, there is normally vasodilatation and transient increases in capillary permeability producing extravasation of plasma proteins and tissue oedema. Generally, these reactions are linked to painful perception or hyperalgesic sensitization [19,30,31]. Our results shown in Figure 7 indicate that pre-treatment with C12 reduced the inflammatory pain in the acetic acid and formalin models, and C12 Exerts Anti-Inflammatory Effects also reduced the paw oedema induced by carrageenan, and C12 can inhibit plasma substance extravasation from the blood vessel. These inflammatory models are mainly related to local production of bradykinin and prostaglandins, such as PGE2 and leukotrienes, both derivatives of arachidonic acid. These prostanoids bind the prostaglandin sub-type receptors, triggering the inflammatory and hyperalgesic pathway in tissues. Bradykinins and related kinins represent a group of potent inflammatory mediator peptides involved in pain and inflammation. Bradykinin receptor B1 is associated with a metabotropic signaling pathway producing vasodilatation, an increase of vascular permeability, and an increase of eicosanoids production, such as PGE2 and NO produced by PGES, COX-2 and iNOS. On the other hand, B1 receptors promote pain stimuli and inflammation by the NF-kB pathway [32,33]. Thus, the inhibitions of inflammatory enzyme expression (Figure 5) and NF-kB activation (Figure 3) may mainly contribute to these beneficial effects of C12. Of course, the results are also partly related with the effects of C12 on inflammatory cellular activation during the recruitment of leukocytes to an injured target. In another inflammatory model of formalin-caused pain, the pain in the first phase (0–10 min after formalin i.p. injection) is induced through formalin directly stimulating C-fibers, whereas in the second phase (15–30 min), the pain is generated by the release of several inflammatory mediators [30,34]. Thus, the licking times of the mice in different intervals reflect the degree of the pain. The higher inhibitory rate of C12 in phase II than phase I also partly indicated that the main mechanism of C12 activity is to inhibit the production of inflammatory mediators. Steroids and cyclooxygenase inhibitors have long been used as major therapeutical anti-inflammatory agents. However, they often cause serious side effects in patients [12,13,14]. Thus, developing unique anti-inflammatory agents is currently in high demand. C12 is a new anti-inflammatory compound designed and synthesized in our lab. In conclusion, C12 effectively suppressed the production of various inflammatory mediators at the levels of both protein and mRNA by suppressing the activation of ERK/ JNK//NF-kB signaling. The evidence from in vivo studies also supports that, with the advantages of water-solubility and low toxicity (Figure S1 and Text S1), C12, as a non-steroidal anti- inflammatory candidate, could provide a therapeutic option in the treatment of topical inflammatory diseases. Materials and Methods Animals Male Institute of Cancer Research (ICR) mice and C57BL/6 mice weighing 18–22 g were obtained from the Animal Center of Wenzhou Medical College (Wenzhou, China). Animals were housed at a constant room temperature with a 12:12 hour light- dark cycle, and fed with a standard rodent diet and water. The animals were acclimatized to the laboratory for at least 3 days before use in experiments. Protocols involving the use of animals were approved by the Wenzhou Medical College Animal Policy and Welfare Committee (Approval documents: 2009/APWC/ 0031). Reagents Lipopolysaccharide (LPS), carrageenan, acetic acid and forma- lin solution were purchased from Sigma (Louis, MO). Dexameth- asone was purchased from Xianju Pharma (Zhejiang, China). Saline was prepared as 0.9% NaCl solution. C12 was synthesized and characterized as described in our previous publication [15,35]. C12 was used in the form of C12-HCl in all of the following experiments. Briefly, C12-HCl (C12) was dissolved in PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24377