Cell culture and cell viability assay
Raw 264.7 cell line was obtained from cell bank, Institute of Biochemistry and Cell Biology (Shanghai, China). Raw 264.7 cells were cultured in DMEM with 10% fetal bovine serum, in an incubator at 37°C, 5% CO2 and 95% humidity. The cyctotoxic effects of WEL were evaluated in absence or presence of LPS by MTT assay. WEL was dissolved in 10% dimethyl sulfoxide (DMSO) and added directly to culture media before the addition of LPS. The final concentration of DMSO never exceeded 0.1%.
Measurement of NO levels
The nitrite concentration in the culture medium was measured by a Griess reaction test. Cells were plated as a density of 2 × 106 cells/well in 24-well culture plates and pretreated with or without indicated concentrations of WEL (0.1, 1, 10 μM) or N-nitro-L-arginine methyl ester (L-NAME) (100 μM) for 12 h, and then incubated with LPS (1 μg/ml). 100 μM L-NAME, an inhibitor of NO, was used as a positive control. After 20 h incubation, cells were washed three times to remove non-adherent cells. Then, 100 μl of the Griess reagent was mixed with an equal volume of cell supernatant, the optical density at 540 nm was measured and the concentration of nitrite was calculated according to the standard curve generated from known concentrations of sodium nitrite.
Measurement of PGE2 levels
RAW 264.7 macrophages were subcultured in 24-well plates and pretreated with or without indicated concentrations of WEL (0.1, 1, 10 μM) for 12 h or DX (0.1 μM) for 1 h, then incubated with LPS (1 μg/ml) for 20 h. The accumulated PGE2 in the culture medium was measured using ELISA Kit (Cayman Chemical Company, Michigan, USA) according to the manufacturer’s instructions. 0.1 μM DX was used as a positive control.
Measurement of TNF-α levels
The effects of WEL on the production of TNF-α were measured by ELISA. 2 × 106 RAW 264.7 cells (1% serum starved) were seeded on 24-well plate at a density of 2 × 106 per well for over-night. Cells were pre-incubated with WEL (0.1, 1, 10 μM) or DX (0.1 μM) for 1 h, then stimulated with 1 μg/ml LPS for another 20 h. The cytokine concentrations were calculated according to the standard curve using recombinant cytokines in each ELISA kits. All measurements above were performed in triplicate.
Transient transfection and luciferase reporter assay
NF-κB reporter constructs were purchased from Clontech Laboratories, Inc. (Palo Alto, CA, USA). For the reporter assay, cells were seeded into 24-well plates at a density of 5 × 105 cells per well in 500 μl of DMEM without antibiotics and incubated overnight. The cells in each well were transiently transfected with 200 ng of luciferase reporter construct and 50 ng of internal control plasmid of the pCMV-β-galactosidase reporter plasmid or empty expression vector pcDNA3 using lipofectamine TM 2000 reagent according to the manufacturer’s procedures (Invitrogen, Carlsbad, CA, USA). Six hours after transfection, the cells were washed with phosphate-buffered saline to remove LiptofectamineTM 2000 complexes and then supplied with fresh medium (supplemented with fetal calf serum and phenol red free) and treated with WEL (0.1, 1 and 10 μM) for 12 h before stimulation with LPS (1 μg/ml) for 20 h. Subsequently, luciferase activities were measured in cell lysates using Dual Luciferase Reporter reagents following manufacturer’s instruction (Promega, Madison, WI, USA).
Western blotting analysis
After treatment with various concentrations of WEL in presence or absence of 1 μg/mL LPS, cells were analyzed by immunoblotting. The treated cells were washed and scraped into cold phosphate-buffered saline (PBS) and centrifuged at 500 × g at 4°C. The cell pellets were resuspended in lysis buffer and centrifuged to yield whole-cell lysates [23]. 20 μg protein for each sample was separated by SDS-polyacrylamide gels with electrophoresis (Bio-Rad, Hercules, CA, USA) and the gel was transferred to PVDF membrane. The membrane was blocked with 10% skim milk for 1 h and then incubated overnight at 4°C with 1:2000 dilution of the corresponding primary antibody. After washing, the membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. The membrane was immersed in the enhanced chemiluminescence solution for 60 sec. The gel images were visualized using Chem-Doc (Bio-Rad, Hercules, CA) and densitometric analysis was performed with Quantity One 1-D Analysis software (Bio-Rad). The results are representative of three independent experiments.
Drugs and solutions
WEL (Purity ≥ 95%, by HPLC) (Xidian Pharmaceutical Co., Ltd., Jilin, China) HEPES, LPS, N-nitro-L-arginine methyl ester and lipopolysaccharide (L-NAME) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and bovine serum albumin (BSA) (Gibco BRL, Gaithersburg, MD, USA). Griess reaction kit for Nitric Oxide (NO) (Jiancheng Co., Ltd., Nanjing, China). ELISA kits for detecting TNF-α (R&D Systems, Inc., USA). PGE2 ELISA Kit was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Trizol reagent (Invitrogen, Carlsbad, CA, USA). Antibodies specific for COX-2, iNOS, phospho-IκBα, NF-κBp65, phospho-ERK1/2 and glyceraldehydes 3-phosphate dehydrogenase (GADPH) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Antibodies specific for MAPK family proteins (ERK1/2, phospho-p38, p38, phospho-JNK, JNK) (Cell Signaling Technology, Inc., Beverly, MA, USA). All other reagents were of analytical grade.
Statistical analysis
The results were expressed as mean ± standard error of the mean (SEM) with the indicated number (n) of experiments. Differences between groups for continuous variables were evaluated with analysis of variance (ANOVA) and differences between two groups were analyzed using unpaired Student’s t- test (SPSS version 10.0; Chicago, IL). Statistical significance was set as p < 0.05.
