C12 Exerts Anti-Inflammatory Effects Figure 7. Effects of C12 (5 mg/kg or 15 mg/kg, i.p.) on the chemical-induced inflammatory response. A. Paw oedema models induced by carageenan; B. acetic acid-induced pain model; C. acetic acid-vascular permeability models; D. formalin-induced nociception mice (first- inflammatory phase, left; second-inflammatory phase, right). Control animals received saline (0.9% NaCl). Dexamethasone (5 mg/kg) was used as a positive control. Each value represents the mean6SD of 6 animals, and asterisks indicate significant difference of the paw thickness in relation to the corresponding saline group (* p,0.05, ** p,0.01, *** p,0.001). doi:10.1371/journal.pone.0024377.g007 of cytokines from blood vessels to the injured tissues [21]. To demonstrate if C12 can reduce permeability changes during inflammation, we performed a vascular permeability assay in ICR mice using azovan blue and acetic acid. As shown in Figure 7C, acetic acid increased the permeation of azovan blue from the vascellum to abdominal cavity (4.5-fold, p,0.01), while pre-treated with C12 can significantly reduce the OD value compared to the acetic acid-treated group (5 mg/kg, 49%, p,0.05; 15 mg/kg, 63%, p,0.05), suggesting that C12 is able to inhibit vascular permeability and reduce inflammatory effusion. Moreover, the inhibitory effects of C12 on the formalin-induced inflammatory nociception (both first and second phase) in ICR mice were evaluated. As shown in Figure 7D, the results revealed that i.p. pretreatment of C12 could dose-dependently reduce the licking times of the mice stimulated by formalin, with an inhibition of 35.3% (15 mg/kg, p,0.01) in first phase (0–10 min after formalin injection) and an inhibition of 78.7% (15 mg/kg, p 0.001) in second phase (15–30 min after formalin injection), respectively. These results potently suggested that C12 is able to effectively inhibit the in vivo inflammatory response induced by chamical stimulus such as carrageenan, acetic acid and formalin. Discussion Our previous study has found that a novel compound C12 possessed inhibitory effects on LPS-induced TNF-a and IL-6 release in RAW264.7 macrophages [15]. With another advantage of water-solubility when combined with HCl, C12 has been considered a promising anti-inflammatory candidate. In the present study, we demonstrated anti-inflammatory activities of this compound both in vitro and in vivo, using LPS-stimulated MPMs and several mouse models of topical inflammation, respectively. Macrophages play important roles in a host’s immune defense system during infection as well as in the processes of disease development [22]. Activation of macrophages by stimuli, such as LPS, increases the production of numerous inflammatory mediators, including various cytokines and nitric oxide (NO) [10]. Excessive production of NO has been widely reported to be associated with inflammatory response [23,24]. First, we showed that C12 has a dose-dependently inhibitory activity upon NO synthesis in MPMs (Figure 1B). Strong inhibition of inflammatory cytokine release has been ascribed to down-regulation of NF-kB- dependent gene transcription [25,26]. Our next experiments provided the evidence that C12 significantly inhibits IL-12, IL-1b, TNF-a, IL-6 and COX-2 mRNA transcription in MPMs (Figure 2). In addition, our results shown in Figure 4 demonstrated that C12 significantly inhibits the plasma NO level and serum levels of TNF-a and IL-6 in LPS-stimulated mice. In response to LPS, plasma TNF-a increased at a relatively early stage (1 h after challenge) as a proinflammatory factor and then induced downstream inflammatory reactions, including IL-6 and iNOS expression (6 h after challenge) in a NF-kB-dependent manner. Endotoxin, or more specifically LPS, is an inflammatory stimulus that can exert effects on major organs, such as the liver. Several groups have found that LPS could increase inflammatory PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24377