C12 Exerts Anti-Inflammatory Effects Figure 4. Inhibition of plasma TNF-a (A), IL-6 (B) and NO (C) by C12 in LPS-injected mice. C12 (15 mg/kg, i.v.) was given to B6 mice 15 min before LPS (10 mg/kg, i.v.) administration. Mice were killed and blood samples were collected 1 or 6 h after administration of LPS. Plasma TNF-a, IL-6 and NO concentrations were measured as described in Materials and Methods. Data point represent the mean6SD (n = 6). (* p,0.05, ** p,0.01, vs LPS group). doi:10.1371/journal.pone.0024377.g004 C12 pretreatment reduced inflammatory gene expression in mouse liver after LPS challenge Real-time qPCR was used to verify the profiles of selected genes TNF-a, IL-1b, IL-6, COX-2, iNOS and mPGES that were involved in inflammatory response in mouse liver after LPS challenge. A dose of LPS (10 mg/kg, i.v.) and C12 (15 mg/kg, i.v.) was chosen for these studies. The mRNA levels of these six genes in the liver were significantly induced by LPS administration, while they peaked at different time points (1 to 6 h), respectively (data not shown). TNF-a, IL-1b, and IL-6 were determined at 1 h, 3 h and 6 h after LPS injection, respectively. As illustrated in Figure 5, the hepatic mRNA expression of TNF-a, IL-1b, IL-6, Figure 5. Inhibitory effects of C12 pretreatment (15 mg/kg, i.v., 15 min before LPS injection) on the induction of inflammatory gene mRNAs in B6 mouse livers 1 or 6 h after LPS administration (10 mg/kg, i.v.). Liver mRNA from vehicle control, LPS, and LPS+C12-treated mice was reverse transcribed for quantitative real-time PCR analysis of the expression of TNF-a, IL-6, IL-1b, COX-2, iNOS, and PGES. Expression is normalized to actin and reported as the fold-expression relative to one of samples in the vehicle control group. Values shown are the mean 6 SD of 3 mice from the saline control group (white), and 4–6 mice each from the LPS (black)- and LPS+C12 (yellow)- treated groups. (* p,0.1, ** p,0.05, compared to LPS group). doi:10.1371/journal.pone.0024377.g005 PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24377