H2O and the solution was filtered through 0.22 mm microporus filters (Carrigtwohill, Co.Cork, Ireland) before use. The structures of C12 and its chloride are shown in Figure 1. Primary cell preparation and culture For peritoneal macrophage preparation, ICR mice were stimulated by intraperitoneal (i.p.) injection of 3 ml thioglycollate solution (Beef extract (0.3 g), tryptone (1 g), sodium chloride (0.5 g), and soluble starch (6 g) were dissolved and boiled in 100 ml water. Before used, the solution was filtrated with 0.22 mm filter.) per mouse and kept in pathogen-free conditions for 3 days before peritoneal macrophage isolation. Total peritoneal macro- phages were harvested by washing the peritoneal cavity with PBS containing 30 mM of EDTA (8 mL per mouse), centrifuged, then the pellet was resuspended in RPMI-1640 medium (Gibco/BRL life Technologies, Eggenstein, Germany) with 10% FBS (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL streptomycin. Peritoneal macrophages were cultured on 60 mm plates (1.26106 cells in 3 ml media per plate) and maintained at 37uC in a 5% CO2-humidified air. Nonadherent cells were removed by washing with medium at 3 h after seeding. Experiments were undertaken after the cells adhered firmly to the culture plates. Nitrite assay A quantity of 100 mL of cell culture supernatant (or plasma in animal experiments) was collected and combined with 50 mL 1% sulfanilamide in 5% H3PO4 and 50 mL 0.1% N-(1-naphthyl)ethy- lenediamine dihydrochloride (Sigma, Louis, MO) in water, in a 96-well plate, followed by spectrophotometric measurement at 550 nm, using a microplate reader. Nitrite concentration in the supernatant was determined by comparison with a sodium nitrite standard curve. Experiments were performed at least three times and in duplicate. Western Immunoblot analysis The treated cells were collected and lysated, then 30 mg of the whole cell lysates were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane. Each membrane was preincubated for 1 h at room temperature in Tris-buffered saline, pH 7.6, containing 0.05% tween 20 and 5% non-fat milk. Each nitrocellulose membrane was incubated with specific antibodies against p-p38, p38, p-ERK, ERK, p-JNK, JNK (Santa Cruz, CA), and p-IkBa, IkBa, Actin (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were then detected by incubating with secondary antibody conjugated with horseradish peroxidase and visualized using enhanced chemiluminescence reagents (Bio- Rad, Hercules, CA). Assay of cellular NF-kB p-65 translocation The cells were immunofluorescence-labeled according to the manufacturer’s instructions using a Cellular NF-kB p-65 Translo- cation Kit (Beyotime Biotech, Nantong, China). Briefly, after washing and fixing, cells were incubated with a blocking buffer for 1 h to suppress non-specific binding. Next, cells were incubated with the primary NF-kB p65 antibody for 1 h, followed by incubation with a Cy3-conjugated secondary antibody for 1 h, then with DAPI for 5 min before observation. P65 protein and nuclei fluoresce red and blue, respectively, and can be simultaneously viewed by fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 350 nm for DAPI, and 540 nm for Cy3. To create a two-color image, the red and blue images were overlaid, producing purple fluorescence in areas of co-localization [36]. C12 Exerts Anti-Inflammatory Effects Treatment of mice with C12 and LPS Male B6 mice weighing 18–22 g were pretreated with C12 solution (15 mg/kg) by intravenous (i.v.) injection for 15 min and then 200 mL of LPS (10 mg/kg), dissolved in saline (0.9% NaCl), was i.v. injected. Negative control animals received only a similar volume (200 mL) of saline. Mice were anesthetized with diethyl ether and sacrificed at the indicated time after LPS injection. Blood samples were taken from the right ventricle orbital veniplex using a heparinized syringe with a needle. Liver and plasma were flash frozen in liquid nitrogen and stored at 280uC until analysis. Determination of TNF-a and IL-6 After treatment of mice with C12 and LPS, the TNF-a and IL-6 levels in plasma were determined with an enzyme-linked immunosorbent assay (ELISA) kit (Bioscience, San Diego, CA) according to the manufacturer’s instructions. The total amount of the inflammatory factor in the media was normalized to the total protein quantity of the viable cell pellets. Real-time quantitative PCR Cells or liver tissues (50–100 mg) were homogenized in TRIZOL (Invitrogen, Carlsbad, CA) or prepared with a PARIS kit (Ambion, Austin, TX) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative PCR were carried out using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Eppendorf Master- cycler ep realplex detection system (Eppendorf, Hamburg, Germany) were used for q-PCR analysis. The primers of genes including iNOS, COX-2, TNF-a, IL-6, prostaglandin synthetase (PGES), IL-1b and b-actin were synthesized by Invitrogen. The primer sequences used are shown as follows: mouse TNF-a sense primer: 59-TG- GAACTGGCAGAAGAGG-39, Mouse TNF-a antisense primer: 59-AGACAGAAGAGCGTGGTG-39; mouse IL-6 sense primer: 59- GAGGATACCACTCCCAACAGACC-39, mouse IL-6 antisense primer: 59-AAGTGCATCATCGTTGTTCATACA-39; mouse COX-2 sense primer: 59-TGGTGCCTGGTCTGATGATG-39, mouse COX-2 antisense primer: 59-GTGGTAACCGCTCAGGT- GTTG-39; mouse PGES (mPEGS) sense primer: 59-ACACTGC- TGGTCATCAAG-39, mouse PGES antisense primer: 59-GGTCA- CTCCTGTAATACTGG-39; mouse iNOS sense primer: 59- CAGCTGGGCTGTACAAACCTT-39; mouse iNOS antisense primer: 59-CATTGGAAGTGAAGCGTTTCG-39; mouse IL-1b sense primer: 59-ACTCCTTAGTCCTCGGCCA-39, mouse IL-1b antisense primer: 59-CCATCAGAGGCAAGGAGGAA-39; mouse b-actin sense primer: 59-TGGAATCCTGTGGCATCCAT- GAAAC-39, mouse b-actin antisense primer: 59-TAAAACG- CAGCTCAGTAACAGTCCG-39. The amount of each gene was determined and normalized by the amount of b-actin. Lung histopathology Two days after LPS injection (10 mg/kg, i.v.), the lungs of B6 mice C12-pretreated (15 mg/kg, i.v.) or control were distended by infusion of 10% formalin into the trachea at a pressure of 15 cm H2O for 2 h, and fixed in 10% formalin for 24 h. Formalin-fixed lungs were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). To estimate the extent of damage, the specimen was observed under a light microscope. LPS-induced inflammatory mortality in B6 mice Male B6 mice weighing 18–22 g were pretreated with C12 (15 mg/kg) by i.v. injection 15 min before the i.v. injection of LPS (20 mg/kg). Control animals received a similar volume (10 mL/kg) of saline. Body weight change and mortality were recorded for 7 days. PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24377