Non-cultivated CT was collected from the Chiri Mountain in the far south of the Republic of Korea. Collection was made on February 20, 2007, a time when the plants have many thorns and few leaves. The plant materials were identified by Professor Ho-Young Choi at the Herbology Laboratory, College of Oriental Medicine, Kyunghee University, Seoul. Fresh CT was removed using a sickle and dried in a dark, well-ventilated place. Air-dried CT (1 kg) was cut into pieces by using a fodder chopper, and the extract was prepared by incubating with 80% methanol at room temperature for several days. Then the CT methanol extract (CTM) was fractionated using CHCl3, and the CHCl3 extracts were evaporated to afford a CHCl3-soluble (14 g) fraction (CTC), which was dissolved in DMSO (Sigma, St. Louis, MO, USA) to obtain a final concentration of 100 mg/ml, and was stored at −20°C until required. To use, the extract was further diluted to different concentrations by using cell culture media (DMSO < 0.1%).
Male BALB/c mice (6–8 weeks of age) were purchased from the Korean branch of Taconic, Samtaco (Osan, Korea). Mice were kept in a temperature- and humidity-controlled, pathogen-free animal facility at Kyung Hee University and provided standard mouse chow and water ad libitum. All animal care and experimental procedures were complied with university guidelines of by Korean National Veterinary Research and Quarantine Service, World Organization of Animal Health (WOAH) and were approved by the Ethical Committee for Animal Care and the Use of laboratory animals Kyung Hee University (Approval number # KHUASP(SE)-11-039).
Cell culture and sample treatment
RAW 264.7 cells were obtained from the Korean Cell Line Bank (Seoul), and were grown at 37°C in DMEM medium supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 mg/ml) in a humidified 5% CO2 atmosphere. Cells were incubated with CT at increasing concentrations (62, 125, 250, and 500 mg/ml), and then stimulated with LPS (1 or 10 μg/ml) for the indicated times.
Macrophage isolation and culture
Mice were injected intraperitoneally (i.p.) with 2 ml of sterile thioglycollate medium (Becton Dickinson, Franklin Lakes, NJ, USA). Three days later, macrophages were collected by peritoneal lavage with cold Dulbecco’s modified Eagle’s medium (DMEM). After centrifugation of the lavage, red blood cells were removed by lysis for 3 min in lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA). After centrifugation, the cells were resuspended in DMEM supplemented with 1% antibiotic and antimycotic solution (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (WeIGENE, Deagu, Korea), then incubated for 90 min in a humidified atmosphere of 5% CO2 at 37°C. Non-adherent cells were removed by washing, and the adherent cells were harvested and seeded for the various assays.
MTS-tetrazolium salt assay
Cell viability was measured based on the formation of blue formazan metabolized from colorless MTS by mitochondrial dehydrogenases, which are active only in live cells. RAW 264.7 macrophages were plated in 96-well plates at a density of 4 × 103 cells per well for 24 h, and then washed. Cells incubated with various concentrations of CTM and CTC were treated with LPS (E. coli 026:B6; Sigma, MO, USA) for 24 h and then incubated in 0.5 mg/ml MTS solution. Viabilities were determined using colorimetric MTS assay.
Determinations of nitrite concentrations
RAW 264.7 cells were plated at 2.5 × 105 cells/ml in 24 well-plates and then incubated with or without LPS (1 μg/ml) in the absence or presence of various concentrations (62, 125, 250 and 500 μg/ml) of CTC for 24 h. The nitrite accumulated in culture medium was measured as an indicated of NO production based on the Griess reaction. Briefly, 100 μl of cell culture medium was mixed with 100 μl of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphtylethylenediamine-HCl], incubated at room temperature for 10 min, and then the absorbance at 540 nm was measured in a microplate reader (VersaMax™, Molecular Device, USA). Fresh culture medium was used as the blank in all experiments. The amount of nitrite in the samples was measured with the serial dilution standard curve of sodium nitrite.
Western blot analysis
Cellular proteins were extracted from control and CT-treated RAW 264.7 cells. Briefly, cells were collected by centrifugation and the pellets so obtained were washed once with phosphate-buffered saline (PBS). Pellets were then resuspended in extraction lysis buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM Na fluoride and 0.5 mM Na orthovanadate) containing 5 mg/ml leupeptin and 5 mg/ml aprotinin and incubated for 20 min at 4°C. Cell debris was removed by microcentrifugation, and supernatants were rapidly frozen. Protein concentrations were determined using the Bio-Rad protein assay reagent (Hercules, CA), according to the manufacturer’s instructions. Proteins (40 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis, and electroblotted onto nitrocellulose membranes. Immunoblots were incubated overnight with blocking solution (5% skimmed milk) at 4°C, and then for 4 h with a primary antibody. Blots were then washed three times with Tween 20/Tris-buffered saline (TBST), incubated in 1:100 alkaline phosphatase-conjugated secondary antibody for 1 h at room temperature, washed three times with TBST, and finally reacted with BCIP-NBT solution (Nakanai Tesque, Japan). The monoclonal antibodies (iNOS, COX-2 and β-actin), and the peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Density ratios versus β-actin were determined by densitometry analysis (Scion NIH Image 1.63).
Measurement of PGE2 levels
PGE2 levels in culture media were analyzed using PGE2 assay kits (ParameterTM; R&D Systems, MN, USA). The PGE2 standard and the RD5-39 in the kits were used to construct a standard curve. Briefly, 100 μl of the medium was mixed with 50 μl of primary antibody solution and PGE2 conjugate and incubated for 2 h at room temperature on a shaker. Cells were then washed out of wells using 400 μl of 1× washing buffer; then, color reagent (200 μl) was added and, 30 min later, stop solution (50 μl) was added and mixed in. Absorbance was measured at 450/570 nm using a VersaMaxTM microplate reader (Molecular Devices, CA, USA).
Cytokine array assays
Mouse cytokine antibody array membranes coated with 40 specific cytokine antibodies (RayBioTM Mouse Inflammation Antibody Array 1, RayBiotech, Inc.) were probed with isolated total protein samples. The membranes were blocked by incubation in blocking buffer for 30 min at room temperature and then incubated with samples for 1 h at room temperature. Membranes were then washed three times with Wash Buffer I and twice with Wash Buffer II for 5 min at room temperature per wash and then incubated with biotin-conjugated antibodies at 4°C overnight. Membranes were then washed, incubated with HRP-conjugated streptavidin for 1 h at room temperature, incubated with detection buffer for 1 min, and exposed to X-ray film for 40 s (Kodak Inc.). Exposed films were digitized and relative cytokine levels were determined by densitometry (Scion NIH Image 1.63), by subtracting background staining level and normalizing to positive control spots, which were used as internal standards.
The results shown were obtained from at least three independent experiments and are presented as means ± SDs. Statistical analyses were performed by one-way analysis of variance (ANOVA) with Tukey’s and Duncan’s post hoc tests. All statistical analyses were performed using SPSS v12.0 software. P values < 0.05 were considered to indicate statistical significance.