Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin solution were purchased from Corning, Inc. (Corning, NY, USA). LPS from Escherichia coli O111:B4, Griess reagent, Triton X-100 and 2-mercaptoethanol were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit primary antibodies [iNOS (cat. no. 13120), COX-2 (cat. no. 12282), phosphorylated (p)-ERK1/2 (Thr202/Tyr204; cat. no. 4370), p38 (cat. no. 8690), p-p38 (Thr180/Tyr182; cat. no. 4511), JNK/SAPK (cat. no. 9252), p-JNK/SAPK (Thr183/Tyr185; cat. no. 9255), NF-κB p65 (cat. no. 8242) and GAPDH (cat. no. 5174)], horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody (cat. no. 7074) and anti-rabbit IgG (Heavy+Light; H+L), F(ab′)2 fragment (Alexa Fluor^® 488 conjugate; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-ERK1/2 rabbit primary antibody (cat. no. sc-514302) and rabbit normal serum were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). DAPI was purchased from Roche Diagnostics GmbH (Mannheim, Germany) and formaldehyde was bought from Junsei Chemical Co., Ltd. (Tokyo, Japan). ProLong^® Gold Anti-fade Reagent was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Isolated 6FU was provided by Ali et al (12); the isolation process was performed as previously described. Whole A. decursiva powder was refluxed in methanol for 3 h and filtered. Subsequently, the filtrate was dried in a vacuum at 40°C for concentrating, followed by suspension in distilled water. This extract was partitioned by ethyl acetate (EtOAc), and the EtOAc fraction was served to silica gel chromatography using dichloromethane (CH₂Cl₂)-MeOH (10:1→0:1, gradient). Following chromatography, 20 subfractions (F-1 to F-20) were obtained, F-6 was partitioned with a silica gel chromatography using CH₂Cl₂-MeOH column (20:1→0:1, gradient). Following these processes, 6FU was obtained. The presence of a single compound that was 6FU were confirmed by nuclear magnetic resonance studies.

The murine RAW 264.7 macrophage cell line and the 293 human kidney cell line were obtained from American Type Culture Collection (Manassas, VA, USA) and incubated with DMEM containing 10% FBS and 1% penicillin/streptomycin solution at 37°C with 5% CO₂ and a humidified atmosphere.

The cell viability of 6FU in RAW 264.7 and 293 cells were measured by a WST-1 assay (13-15). In total, 1×10⁴ RAW 264.7 and 293 cells were seeded in each well of 96-well cell culture plates. RAW 264.7 cells were cultured for 24 h with or without 1 µg/ml LPS and 50 or 100 µM 6FU. 293 cells were incubated with or without 25, 50 and 100 µM 6FU for 24 h. Following incubation, 10 µl EZ-cytox Cell Viability Assay Solution WST-1^® (Daeil Lab Service Co., Ltd., Seoul, Korea) was added to each well and incubated for 3 h. Subsequently, the absorbance was measured using a micro-plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 460 nm.

The nitrite concentration in the medium was measured with the Griess reaction (13-15). RAW 264.7 cells were seeded in 24-well cell culture plates (5×10⁴ cells/well) and pre-treated with 10, 25 and 50 µM 6FU for 2 h and further incubated with 1 µg/ml LPS for 24 h. The supernatant of each well (100 µl) was transferred to 96-well plates and Griess reagent was added in the dark. Absorbance was measured at 540 nm and the calculated nitrate concentration was considered as an indicator of NO production.

To perform western blot analysis, RAW 264.7 cells were pre-treated with 6FU for 2 h and stimulated with or without LPS (1 µg/ml) for 6 and 24 h. Whole cells were harvested and lysed with the cell lysis buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1% Triton X-100, 1% deoxycholate and 0.1% SDS; Intron Biotechnology, Inc., Seongnam, Korea] and the lysates were subsequently centrifuged at 18,000 × g for 20 min. Separate nuclear and cytoplasmic proteins were obtained using NE-PER^® nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The protein concentration in the cell lysates were measured using Bradford reagent (Biosesang, Inc., Seongnam, Korea). An equal amount (20 µg) of the prepared proteins were separated by 12% SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Pall Life Sciences, Ann Harbor, MI, USA). Following blocking with 1X PBST buffer containing 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with primary rabbit antibodies [1:1,000 with 1X PBS containing 5% bovine serum albumin (BSA; BioShop Canada, Inc., Burlington, ON, Canada) and 0.1% Tween-20] at 4°C. The membranes were washed three times with PBST, followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibodies (1:1,000 with 1X PBS containing 5% BSA and 0.1% Tween-20) for 1 h at room temperature. The membranes were developed on X-ray film for visualization using an enhanced chemiluminescent detection solution (Pierce; Thermo Fisher Scientific, Inc.).

In total, 1×10⁵ cells were plated at cover-glass bottom dishes (SPL Life Sciences, Pocheon, Korea) and pre-treated with 25 µM 6FU for 30 min (16). To investigate the nuclear translocation activity of p-ERK1/2 or NF-κB, LPS was treated for 6 or 24 h respectively. Subsequently, for pre-fixing, cells were stained with 1 µg/ml DAPI diluted in methanol (99.8%) and incubated for 15 min at 37°C, followed by washing with PBS buffer and fixed with 4% formaldehyde for 15 min at room temperature. Following incubation, these cells were blocked with 5% rabbit normal serum containing 0.3% Triton X-100 in 1× PBS for 1 h in the dark and incubated with the anti-ERK1/2 (Thr202/Tyr204) or -NF-κB p65 primary antibody (1:2,000 with 1× PBS containing 0.3% Triton X-100) at 4°C overnight. Following the reaction, the cells were washed with 1× PBS and incubated for 50 min with anti-rabbit IgG (H+L), F(ab′)2 fragment (Alexa Fluor^® 488 conjugate) as secondary antibodies (0.5 µg/ml with 1× PBS containing 0.3% Triton X-100) at room temperature in the dark. Following staining, cells were mounted using ProLong^® Gold Anti-fade Reagent. Stained cells were observed using Carl Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany; magnification, ×400).

For total RNA extraction, RAW 264.7 cells were pre-treated with 6FU (25 and 50 µM) prior to stimulation with LPS (1 µg/ml), subsequently RNA was extracted with 2-mercaptoethanol the and RNeasy plus mini kit, according to the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany). The concentration of total RNA was measured using a nanodrop (Mecasys Co., Ltd., Daejeon, Korea) and 2 µg RNA was synthesized to cDNA using a SuPrimeScript RT Premix (GeNet Bio, Inc., Daejeon, Korea) under the following conditions; 50°C for 60 min and 70°C for 10 min. The cDNA was amplified using Prime Taq Premix (GeNet Bio, Inc.) with specific primers presented in Table I, according to the manufacturer’s protocols (95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, 30 cycles). Amplified PCR products were stained with ethidium bromide (Sigma-Aldrich; Merck KGaA) and visualized in a 2% agarose gel using ImageMaser^® VDS Software version 3.0 in ImageMaster^® VDS GE Healthcare, Chicago, IL, USA).

One-way analysis of variance with Dunnett’s multiple comparison tests were used for determining the statistical significance of differences between experimental and control groups. Analysis was performed using Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Results are presented as the mean ± standard deviation and all experiments were performed in triplicates independently. P<0.05 was considered to indicate a statistically significant difference.

The cell viability of RAW 264.7 and 293 cells was measured by a WST-1 assay. RAW 264.7 cells were treated with or without 6FU (50 and 100 µM) and LPS (1 µg/ml) for 24 h. 293 cells were incubated with or without various concentrations of 6FU (25, 50 and 100 µM) for 24 h. As presented in Fig. 1, 6FU and LPS did not demonstrate any cytotoxicity on RAW 264.7 cells. Additionally, in 293 cells, the cell viability of 6FU treated cells was 99.8, 93.9 and 88.1% at 25, 50 and 100 µM concentrations, respectively (data not shown). Therefore, <100 µM 6FU was used for investigating its anti-inflammatory capacity in the absence of cytotoxicity.

RAW 264.7 cells were pretreated with or without 6FU for 2 h and subsequently stimulated with LPS for 24 h in order to evaluate the NO production level. The level of NO secretion was significantly increased in LPS-stimulated cells compared with non-stimulated cells (Fig. 2A; P<0.01). However, the expression level of NO was decreased by treatment with 6FU in a dose-dependent manner (Fig. 2A). Western blot analysis was performed to investigate whether 6FU has an ability to modulate the expression of pro-inflammatory enzymes, including iNOS and COX-2. The results demonstrated that 6FU downregulated the expression of iNOS and COX-2 in contrast with LPS only-treated cells (Fig. 2B).

As presented in Fig. 3, 6FU significantly suppressed the mRNA expression of pro-inflammatory cytokines, including IL-6, TNF-α and IL-1β, compared with LPS-stimulated RAW264.7 cells (P<0.01).

The LPS-induced phosphorylation level of MAPKs, including p-ERK, p38 and JNK were measured by western blot analysis. In LPS only-treated RAW 264.7 cells, the phosphorylation level of ERK, p38 and JNK were increased. However, only the expression of p-ERK1/2 was markedly decreased in the 6FU-treated LPS-stimulated RAW264.7 cells in a dose dependent manner compared with the phosphorylation level of p38 and JNK (Fig. 4A). In addition, the translocation of phosphorylated ERK1/2 to the nucleus was inhibited following pretreatment with 6FU in LPS-stimulated RAW 264.7 cells by western blotting and IF staining (Fig. 4).

To investigate the activity of 6FU on nuclear translocation of NF-κB, western blot analysis and IF staining were performed. Fig. 5A demonstrated that 6FU decreased the concentration of NF-κB in the nucleus in LPS-stimulated RAW 264.7 macrophages. In contrast, the expression level of NF-κB in the cytoplasm was upregulated by 6FU. Furthermore, 6FU inhibited nuclear translocation activity of NF-κB in LPS-treated cells (Fig. 5B). Therefore, 6FU decreased the expression and nuclear translocation of NF-κB in LPS-stimulated macrophages.