COX-2, iNOS and mPGES was suppressed (59.6%, 36.6%, 83.8%, 87.5%, 72.8% and 54.1% inhibition, p,0.05 or p,0.01, respectively) in C12-pretreated mice compared with LPS-treated group. These results suggested that C12 significantly inhibits the LPS-induced increase of hepatic inflammatory gene transcription. Effects of C12 pretreatment on lung histology in mice after LPS administration Acute lung injury (ALI) is one of the most sensitive responses to LPS injection and ultimately leads to alveolar flooding with serum proteins and oedema fluid [18]. After mice were administrated LPS (10 mg/kg, i.v.), histological changes in the B6 mouse lung such as inflammatory cell infiltration, peri-vascular oedema, and marked alveolar hemorrhage, were observed by H&E staining. C12 pretreatment at 15 mg/kg improved such pulmonary damage remarkably (Figure 6A). C12 also amended the LPS-injured tissue structure of pulmonary lobules and LPS-induced expansion of lung alveolus. C12 treatment prolonged survival in an acute inflammatory model To investigate the beneficial effects of C12 on acute inflamma- tory shock, we infected B6 mice with LPS (20 mg/kg, i.v.) and compared survival rates between C12-treated and placebo control mice. All C12-untreated control mice had died at 48 h after LPS injection, while all of C12-treated animals survived the acute phase of inflammation and displayed 100% survival rates until the end of the experiment (n = 12, p,0.0001, Figure 6B). Meanwhile, the C12 Exerts Anti-Inflammatory Effects body weight of C12-treated mice decreased during 0–48 h but regained slowly since by 60 h after LPS treatment (Figure 6C). Thus, C12 treatment prolongs survival in the acute inflammatory shock model. Treatment with C12 reduced carrageenan-induced paw oedema and inhibited inflammation-related reactions in chemically-induced inflammatory models We further tested the beneficial effects of C12 on inflammation- related models challenged by another stimulus with dexametha- sone as a positive control. Paw oedema induced by carrageenan has been widely used as an inflammatory model in vivo [19,20]. ICR mice were initially treated with C12 (5 and 15 mg/kg) by i.p. injection followed by the paw oedema induced by carrageenan (300 mg/paw) for 4 h. As shown in Figure 7A, compared to carrageenan group, pretreatment with C12 reduced the oedema in size in a dose-dependent way, with the inhibitory rate in oedema size of 39.9% (5 mg/kg, p,0.01, v.s. control) and 46.9% (15 mg/ kg, p,0.01, v.s. control), respectively (Figure 7A). We also pretreated ICR mice with C12 (5 and 15 mg/kg, i.p.) 15 min before chemically-induced inflammatory constrictions with acetic acid (0.6%, 200 mL, i.p.). The inflammatory pain and mice writhing response induced by acetic acid were significantly inhibited by C12 pretreatment in a dose-dependent manner with inhibitory rate of 57.4% (p,0.001), and 91.6% (p,0.001), respectively (Figure 7B). Besides inflammatory gene expression, acetic acid-caused pain and carrageenan-caused paw oedema are usually related to the vascular permeability increasing the release Figure 6. Beneficial Effects of C12 against acute inflammatory injury and shock in B6 mice. A. Mice were treated with C12 (15 mg/kg, i.v.) 15 min before LPS (10 mg/kg, i.v.) administration. After 48 h, lung histopathological analysis was performed using H&E staining as described in Materials and Methods (magnification 6200; n = 3; Con, control). B and C. C12 improves survival of mice subjected to a lethal dose of LPS. Mice were pretreated with vehicle (saline) or 15 mg/kg C12 (i.v.) 15 min before the injection of 20 mg/kg of LPS (i.v.). Survival (B) and body weight (C) were recorded for 7 days after the LPS injection at the interval of 12 h. Results from the summary of two different experiments are shown. n = 12 animals in each group. C12 improved survival rate at 12–48 h (*** p,0.0001). doi:10.1371/journal.pone.0024377.g006 PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24377