Chemicals and reagents
XH-14 was synthesized by using a Sonogashira reaction as previously reported [9, 12]. LPS derived from Escherichia coli and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Hyclone (Logan, UT, USA). The final concentrations of DMSO never exceeded 0.1%, which did not affect the assay systems. The antibodies (Abs) used were: anti-iNOS rabbit polyclonal, anti-COX-2 monoclonal Ab (mAb), anti-inhibitor of NF-κB (IκBα) mAb, anti-phospho-c-Jun, anti-C-Jun, anti-phospho-c-Fos, anti-c-Fos, anti-phospho-JNK rabbit polyclonal, anti-ERK1/2 rabbit polyclonal, anti-phospho-p38 rabbit polyclonal, anti-p38 rabbit polyclonal, (Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin mAb (Sigma-Aldrich).
Cell culture and cell viability assay
RAW 264.7 murine macrophages obtained from the Korean Cell Bank (Seoul, Korea) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2. The effects of XH-14 on cell viability were tested using the CellTiter 96® AQueous One Solution Assay of cell proliferation (Promega, Madison, WI, USA). RAW 264.7 cells were plated at a density of 2 × 104 cells in a 96-well flat-bottom plate, and XH-14 were added to each plate at indicated concentrations. After a 24 h incubation period, the number of viable cells was counted according to the manufacturer's instructions. This assay is based on the reduction of a tetrazolium compound, MTS, to formazan, which has an optimum absorption at 490 nm. Thus, the quantity of the product in the cell culture is indicated by the optical density of formazan at 490 nm, which is directly proportional to the number of living cells.
Measurement of nitrite, prostaglandin E2 (PGE2) and cytokines
The amount of nitrite, PGE2 interleukin (IL)-1β and IL-6 produced by the mouse macrophages was measured in RAW 264.7 cell culture supernatant. RAW 264.7 cells were plated at a density of 2.5 × 105 cells in a 48-well cell culture plate with 500 μl of culture medium and incubated for 12 h. They were then treated with indicated concentrations of XH-14 plus LPS (100 ng/ml) and incubated for another 24 h. The amount of nitrite and PGE2 produced was measured using the Griess reagent system (Promega) and an enzyme-linked immunosorbent assay (ELISA) kit (ENZO Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instruction, respectively. IL-1β and IL-6 were measured using an ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions.
Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from RAW 264.7 cells and reverse-transcribed into cDNA using TriZol Reagent (Invitrogen, Carlsbad, CA, USA) and CCC TAC CAA GT-3’, R 5’- CAC CCA AAG TGC TTC AGT CA-3’; murine COX-2: F 5’-AAG ACT TGC CAG GCT GAA CT-3’, R 5’-CTT CTG CAG TCC AGG TTC AA -3’; murine IL-1β: F 5’-TTC TCC ACA GCC ACA ATG AG-3’, R 5’-ACG GAC CCC AAA AGA TGA AG-3; murine IL-6: F 5’-CAT CCA GTT GCC TTC TTG GGA-3’, R 5’- CCA GTT TGG TAG CAT CCA TC -3’; GAPDH: F 5’-TCT TGC TCA GTG TCC TTG C-3’, R 5’-CTT TGT CAA GCT CAT TTC CTG G-3’. Real-time PCR assay was carried out with LightCycler (Roche Diagnostics, Germany) using LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics). Transcripts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a housekeeping gene, were quantified as endogenous RNA of reference to normalize each sample. Relative quantities were estimated by the delta-delta-Ct method. The results were normalized as relative expression in which the average value of the iNOS, COX-2, IL-1β and IL-6 mRNA was divided by the average value of GAPDH mRNA.
Western blotting analysis
Whole cell extracts, cytoplasmic and nuclear proteins (30 μg protein/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electrophoretically transferred onto nitrocellulose membranes. Immunoreactive bands were detected by incubating the samples with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using a WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology, Seongnam-Si, Korea).
The data are depicted as the mean ± SEM. Student t-test was performed using the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA) and P < 0.05 was considered as statistically significant.