A 95% ethanol extract (code no. PBC345AS) of A. asphodeloides was obtained from the Korea Plant Extract Bank (KPEB Daejeon, South Korea). The concentrated ethanol extract was manufactured according to the manufacturer's instructions. Briefly, the rhizomes of A. asphodeloides were dried at room temperature (RT), treated with ethanol (GR grade), and sonicated numerous times at 45°C. The extracts were filtrated to remove solid substances and concentrated with reduced pressure at 45°C. A stock solution (200 mg/ml) of the extract was prepared in Sigma-Aldrich dimethoxysulfoxide (DMSO; Merck KGaA, Darmstadt, Germany) and this was stored at −20°C before use.

RAW 264.7 macrophages, a mouse monocytic cell line and HEK 293 cells, a human embryonic kidney cell line, were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) containing 10% fetal bovine serum (GE Healthcare Bio-Sciences), 50 U/ml penicillin, and 50 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere containing 5% CO₂. Rabbit anti-inhibitor of κBα (IκBα; cat. no. sc-371) and mouse anti-α-tubulin (cat. no. sc-5286) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti-inducible NO synthase (iNOS; cat. no. 2982), anti-cyclooxygenase-2 (COX-2) (cat. no. 4842), anti-p-IκBα (Ser32/36; cat. no. 9246), anti-p38 (cat. no. 9212), anti-p-p38 (Thr180/Tyr182; cat. no. 9211), anti-extracellular signal-regulated kinase (ERK; cat. no. 9102), anti-c-Jun N-terminal kinase (JNK; 9252), anti-p-JNK (Thr183/Tyr185; cat. no. 9251) and mouse anti-p-ERK (Thr202/Tyr204; cat. no. 9106) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit immunoglobulin G (IgG; cat. no. LF-SA8002) and goat anti-mouse IgG (cat. no. LF-SA8001) secondary antibodies were purchased from AbFrontier (Young In Frontier Co., Ltd., Seoul, South Korea). Ez-cytox solution was purchased from Daeil Lab (Seoul, South Korea). Ready-SET-Go! ELISA kits for the detection of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were obtained from Ebioscience, Inc. (Thermo Fisher Scientific, Inc.).

RAW 264.7 macrophages were pretreated with EAA (10, 20, 50, 100 and 200 µg/ml) for 2 h and further incubated for 24 h at 37°C in the absence or presence of LPS (1 µg/ml). Following incubation, Ez-cytox solution (one-tenth of the culture medium) was added to each well and incubated for 1 h at 37°C. Supernatants were transferred to fresh 96-well plates and the absorbance was measured at a wavelength of 450 nm using a Synergy H1 Microplate Reader.

NO production in activated macrophages was measured according to the method described in our previous study (15). Briefly, cells were seeded in 96-well plates (4.0×10⁴ cells/well) and incubated at 37°C overnight. Cells were pretreated with various concentrations of EAA (10, 20, 50 and 100 µg/ml) for 2 h prior to LPS treatment. Following stimulation with LPS (1 µg/ml) for 24 h, the supernatants (100 µl) were transferred to fresh 96-well plates and 100 µl Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid) was added to each well. Nitrite standard solution (NaNO₂; 2.5, 5, 10, 25, 50 and 100 µM) was used to generate a standard curve for calculating the quantity of NO in the supernatants. The absorbance was measured at a wavelength of 540 nm using a Synergy H1 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were seeded in 96-well plates (4.0×10⁴ cells/well) and incubated at 37°C overnight. Cells were pretreated with various concentrations of EAA (10, 20, 50 and 100 µg/ml) for 2 h prior to LPS treatment. Subsequent to stimulation with LPS (1 µg/ml) for 24 h, the culture supernatants were collected and diluted according to a predetermined dilution rate for each proinflammatory cytokine. The production of proinflammatory cytokines, including IL-6 and TNF-α, was measured using Ready-SET-Go! ELISA kits for each type of cytokine, according to the manufacturer's instructions. Briefly, 96-well plates were coated with coating solution overnight at 4°C, washed with 1X phosphate-buffered saline (PBS)/Tween-20 (PBST) three times, and treated with 1X Assay Diluent for 1 h at RT. The wells were emptied and diluted supernatant and standard solutions were added to each well. Following incubation (2 h) at RT, the plates were washed with 1X PBST three times and detection antibody solution diluted in 1X Assay Diluent was added to each well. The plates was washed after 1 h of treatment, horseradish peroxidase (HRP)-streptavidin solution was added for 30 min and the plates were washed five times with 1X PBST. Tetramethylbenzidine solution was subsequently added to the plates and they were incubated for 10 min in the dark. Additional 1 N H₃PO₄ was added to the wells to terminate the reaction and the absorbance in each well was measured using a Synergy H1 Microplate Reader at a wavelength of 450 nm.

HEK 293 cells were seeded into a 100-mm dish (70% confluence on the day of transfection) and transfected with pNF-κB-luc or pAP-1-luc cis-reporter plasmids (Agilent Technologies, Inc., Santa Clara, CA, USA), which contains the NF-κB or AP-1 promoters, respectively [gWIZ-green fluorescent protein (GFP; Aldevron, Fargo, ND, USA) served as an internal control for transfection efficiency]. Transfected cells were divided into 12-well plates, incubated at 37°C overnight, and treated with various concentrations of EAA (10, 20, 50 and 100 µg/ml) in the presence of phorbol-12-myristate-13-acetate (PMA). After 24 h of incubation at 37°C, cells were lysed using cell culture lysis reagent (Promega Corporation, Madison, WI, USA) and luciferase activity was measured using luciferin as a substrate. The GFP expression levels were determined using the fluorescence change of excitation at 485 nm and emission at 525 nm.

RAW 264.7 macrophages pretreated with EAA were further stimulated with LPS (1 µg/ml) for the optimal duration for detecting target proteins [IκBα and transforming growth factor β-activated kinase 1 (TAK1) for 3 min; mitogen-activated protein kinases (MAPKs) for 15 min; and iNOS and COX-2 for 24 h]. Following stimulation for the indicated durations, cells were washed three times with ice-cold PBS. Lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid, 1% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 1 mM Na₃VO₄) was added to the washed wells and collected into microtubes after 10 min. Following centrifugation at 15,814 × g for 30 min at 4°C, the supernatants were prepared in fresh microtubes.

After boiling the mixture of lysates and sample buffers, aliquots of the samples (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes with transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.8), and 20% methanol (v/v)] at 100 V for 1 h. After blocking with 5% non-fat dried milk, each membrane was incubated overnight at 4°C with the primary antibodies (dilution, 1:1,000). Each membrane was then incubated for an additional 1 h with the secondary peroxidase-conjugated IgG antibodies (1:5,000). Subsequent to washing five times with 1X PBST, the target proteins were detected using enhanced chemiluminescence. Protein levels were quantified by scanning of the immunoblots and analysis with LabWorks software 4.6 (UVP, LLC, Upland, CA, USA).

The data are represented as the mean ± standard error of the mean (SEM). Comparisons between multiple experimental groups were performed using one-way analysis of variance followed by Dunnett's post-hoc test using GraphPad Prism 3.0 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference. The data from nine replicates were analyzed, including three independent experiments with three replicates in each.

As the inhibitory effect of an anti-inflammatory agent should be assessed at concentrations that do not exhibit cytotoxicity, the maximal non-cytotoxic concentration was determined with a cell viability assay. EAA did not exhibit cytotoxicity in concentrations up to 100 µg/ml. However, significant cytotoxicity was observed at 200 µg/ml (Fig. 1A). Thereafter, concentrations of ≤100 µg/ml EAA were used throughout the current study. To evaluate the anti-inflammatory properties of EAA, the effect of EAA on the production of NO, a proinflammatory mediator, was measured in LPS-treated RAW 264.7 cells. As shown in Fig. 1B, EAA inhibited LPS-induced NO production in a dose-dependent manner. The results indicate that EAA inhibits LPS-induced NO production in macrophages.

Subsequently, the effect of EAA on the protein expression levels of iNOS, an NO synthesizing enzyme, and COX-2, an enzyme responsible for the production of PGE₂, was evaluated in order to investigate the transcriptional regulation of proinflammatory mediators. As shown in Fig. 1C and D, the immunoblot analysis with iNOS- and COX-2-specific antibodies revealed an inhibitory effect of EAA on LPS-induced iNOS and COX-2 expression levels in a dose-dependent manner. These data indicate that NO and PGE₂ production are tightly regulated at the protein level by EAA.

As the excessive production of inflammatory mediators, including IL-1β, IL-6 and TNF-α, as well as NO and PGE₂, in macrophages is accompanied by severe inflammation (16), the effect of EAA on the production of proinflammatory cytokines in LPS-treated RAW 264.7 macrophages were measured to investigate the additional anti-inflammatory properties of EAA. As presented in Fig. 2A, EAA inhibited LPS-induced production of IL-6. However, LPS-induced TNF-α production was not alleviated by EAA (Fig. 2B). These results indicate that EAA inhibits the production of IL-6, whereas TNF-α production is not regulated by EAA in LPS-treated macrophages.

To identify which transcription factors are involved in the inhibitory effects of EAA on the production of proinflammatory mediators, the transcriptional activities of NF-κB and AP-1, major transcription factors in the inflammatory responses, were measured using luciferase reporter assays. HEK 293 cells were treated with various concentrations of EAA in the presence of PMA, which induces transcriptional activation of NF-κB- or AP-1-dependent genes. The luciferase reporter gene is placed under the control of NF-κB or AP-1 transcriptional activity. As presented in Fig. 3A and B, PMA-induced luciferase activities, which are regulated by NF-κB or AP-1 transcription factor, respectively, were reduced by EAA treatment. This indicates that EAA exerts anti-inflammatory responses via the inhibition of NF-κB and AP-1 transcriptional activities.

LPS-induced NF-κB activation in macrophages is predominantly mediated by IκBα phosphorylation at Ser-32/36, followed by IκBα degradation and the translocation of released cytoplasmic p50 and p65 complex to the nucleus (17). Phosphorylation in the activation loops of MAPKs leads to their activation, which results in transcriptional activation of AP-1, a transcription factor that binds to the promoter regions of inflammatory mediator genes (4). EAA-mediated IκBα phosphorylation levels in LPS-treated RAW 264.7 cells were measured to assess whether EAA regulates NF-κB activation. The p-IκBα levels were reduced and the IκBα levels were increased by EAA treatment in a dose-dependent manner (Fig. 4A), indicating that EAA inhibits IκBα phosphorylation at Ser-32/36 and, thus, reduces degradation of IκBα. Subsequently, the phosphorylation levels in the activation loops of three MAPKs (ERK, JNK and p38) were measured by EAA in LPS-treated RAW 264.7 cells. As shown in Fig. 4B, EAA selectively inhibits the phosphorylation of p38 without altering the total p38 levels, whereas ERK and JNK phosphorylation was not regulated by EAA. These results indicate that EAA exhibits its anti-inflammatory properties in macrophages by inhibiting the activation of the major inflammatory signaling pathways, NF-κB and p38.