C12 Exerts Anti-Inflammatory Effects Figure 2. C12 inhibited LPS-induced inflammatory mRNA expression in MPMs. Cells were pretreated with compounds at 2.5 mM or vehicle (H2O) control for 2 h and treated with LPS (0.5 mg/mL) for 6 h. The mRNA levels of inflammatory cytokines IL-12, IL-1b, TNF-a, IL-6 and COX-2 were quantified by real-time quantitative PCR. The mRNA value was normalized to internal control b-actin mRNA and was expressed as a ratio to H2O. Bars represent the mean6SD of three independent experiments (* p,0.05, ** p,0.01). doi:10.1371/journal.pone.0024377.g002 inflammatory response and injury in vivo. To evaluate the in vivo anti-inflammatory effect of C12, C57B/L6 (B6) mice administered with LPS (10 mg/kg, i.v.) and C12 (15 mg/kg, i.v.) were sacrificed and the plasma levels of inflammatory mediators were determined by ELISA. The plasma levels of TNF-a, IL-6 and NO increased over the 1–12 h period after LPS injection, and reached peak at 1 h (TNF-a), 6 h (IL-6) and 6 h (NO), respectively (data not shown). Pretreatment with C12 showed a 71.5% (n = 6, p,0.01) inhibition on TNF-a levels and a 53.0% (n = 6, p,0.05) inhibition on IL-6 levels compared to the LPS group (Figure 4A and 4B). Plasma nitrite level also increased at 6 h after LPS administration and pretreatment with C12-inhibited LPS-mediated NO produc- tion by 44.8% (Figure 4C, statistically non-significant between C12+LPS group vs. LPS group). Figure 3. C12 inhibited LPS–induced NF-kB activation and MAPK phosphorylation. A. Cultured MPMs were pretreated with vehicle (H2O) or 10 mM C12 for 2 h and then stimulated with 0.5 mg/mL LPS. After 1 h of treatment, the cells were incubated with p65 antibody and Cy3 fluorescein-conjugated secondary antibody, and nuclei were stained with DAPI. The images (2006) were obtained by fluorescence microscope and overlay. Similar results were obtained with five independent experiments. B–F. MPMs were pretreated with vehicle (H2O) or C12 (10 mM) for 2 h followed by incubation with LPS (0.5 mg/mL) for 1 h. The protein levels of p-IkBa, IkBa, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 were examined by western blot with actin as a loading control (values on the top of western blots represent the mean optical density ratio in three independent experiments, * p,0.05, ** p,0.01, vs LPS group). G. MPMs were pretreated with vehicle (H2O) or C12 (5 mM) for 2 h, and then stimulated with 0.5 mg/ mL LPS for 12 h in the absence or presence of PD98059 (20 mM). The TNF-a level in medium were detected by ELISA as described in Materials and Methods. TNF-a expression is reported as the fold-expression relative to the LPS-treated group (n = 3, * p,0.05, ** p,0.01). doi:10.1371/journal.pone.0024377.g003 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24377