The murine RAW 264.7 macrophage cell line and the 293 cell line (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Little Chalfont, UK) supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) throughout the study. Cells were maintained and treated at 37°C in a humidified 5% CO₂ atmosphere.

A 95% ethanol extract of L. japonicum spores (cat no. PBC417A) was obtained from Korea Plant Extract Bank (KPEB, Daejeon, Korea). A stock solution (200 mg/ml) of the extract was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and stored at −20°C until use. Rabbit polyclonal anti-inhibitor of κBα (IκBα; cat no. sc-371), anti-IκB kinase (IKK) α/β (cat no. sc-7607) and mouse monoclonal anti-α-tubulin (cat no. sc-5286) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit polyclonal anti-inducible NO synthase (iNOS; cat no. 2982), anti-cyclooxygenase (COX)-2 (cat no. 4842), monoclonal anti-phosphorylated (p)-IκBα (Ser32/36; cat no. 9246), polyclonal anti-p38 MAPK (cat no. 9212), anti-p-p38 (Thr180/Tyr182; cat no. 9211), anti-extracellular signal-regulated kinase (ERK) 1/2 (cat no. 9102), anti-c-Jun N-terminal kinase (JNK; cat no. 9252), anti-p-JNK (Thr183/Tyr185; cat no. 9251), monoclonal anti-p-IKKα/β (Ser176/180; cat no. 2697), polyclonal anti-p-MAPK kinase (MKK) 3/6 (Ser189/207; cat no. 9231), anti-MKK6 (cat no. 9264), anti-transforming growth factor β-activated kinase 1 (TAK1; cat no. 4505), monoclonal anti-p-TAK1 (Thr184/187; cat no. 4508) and mouse monoclonal anti-p-ERK1/2 (Thr202/Tyr204; cat no. 9106) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). EZ-Cytox Cell Viability, Proliferation & Cytotoxicity Assay kit was purchased from Daeil Lab Service, Co., Ltd. (Seoul, Korea). Ready-SET-Go! ELISA kits for the detection of IL-6 (cat no. 88-7064) and TNF-α (cat no. 88-7324) were obtained from eBioscience (Thermo Fisher Scientific, Inc.). Accuzol Total RNA Extraction solution was from Bioneer Corporation (Daejeon, Korea) and TOPscript cDNA Synthesis kit was from Enzynomics, Co., Ltd. (Daejeon, Korea). iTaq Universal SYBR Green Supermix was from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

RAW 264.7 macrophages were seeded in 96-well plates at a density of 4.0×10⁴ cells/well and incubated at 37°C overnight. Subsequently, cells were pre-treated with various concentrations of ELJ (50, 100, 200 and 300 µg/ml) or vehicle (DMSO) at 37°C for 2 h prior to stimulation with LPS (Sigma-Aldrich; Merck KGaA) at 37°C. Following stimulation with LPS (1 µg/ml) for 24 h, the supernatants were collected by centrifugation at 1,500 × g for 1 min at room temperature and transferred to 96-well plates. After that, 100 µl Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid) was added to each well at room temperature for 1 min. NaNO₂ solutions (2.5, 5, 10, 25, 50 and 100 µM) were used to construct a standard curve based on the least squares method to calculate the quantity of NO in the supernatant samples. The absorbance of each sample was measured at 540 nm using a Synergy H1 Hybrid Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).

RAW 264.7 macrophages were pre-treated with ELJ (50, 100, 200, 300 and 400 µg/ml) for 2 h, followed by an additional 24 h of incubation at 37°C in the absence or presence of LPS (1 µg/ml). Subsequently, 25 µl EZ-Cytox solution was added to each well in 250 µl medium and cells were incubated for 1 h at 37°C. Supernatants were transferred to new 96-well plates and cell viability was measured by calculating the absorbance at 450 and 650 nm (A₄₅₀-A₆₅₀) using Synergy H1 Hybrid Microplate Reader.

RAW 264.7 macrophages were treated with various concentrations ELJ (50, 100, 200 and 300 µg/ml) or vehicle (DMSO) for 2 h, followed by stimulation with LPS (1 µg/ml) for 24 h. Subsequently, the supernatants were collected after centrifugation at 1,500 × g for 1 min at room temperature and diluted according to the predetermined dilution ratio for each proinflammatory cytokine. The production of the proinflammatory cytokines IL-6 and TNF-α was measured using commercially available ELISA kits according to the manufacturer's protocol. Briefly, a 96-well plate was coated with the coating solution at 4°C overnight, washed 3 times with 1X PBS containing 0.05% Tween-20 (PBST), and treated with 1X Assay Diluent for 1 h at room temperature. The solution was removed, and the supernatants (diluted as appropriate) and standard solutions were added to the wells. Following 2 h of treatment at room temperature, the plate was washed 3 times with 1X PBST and the detection antibody diluted in 1X Assay Diluent was added to the plate for 1 h at room temperature. The plate was then washed with 1X PBST, and 1X horseradish peroxidase (HRP)-streptavidin solution diluted in 1X Assay Diluent was added for 30 min at room temperature, followed by washing 5 times with 1X PBST. The 3,3′,5,5′-tetramethylbenzidine solution was then added to the plate and incubated for 10 min at room temperature in the dark. Subsequently, 1 N H₃PO₄ was added to the plate to stop the reaction, and the absorbance of each well was measured using the Synergy H1 Hybrid Microplate Reader at 450 nm.

RAW 264.7 macrophages were seeded into a 12-well plate at a density of 8×10⁵ cells/well and incubated at 37°C overnight. Cells were pre-treated with ELJ (50, 100, 200 and 300 µg/ml) for 2 h and then incubated with LPS (1 µg/ml) for 3 h. Total RNA was extracted from cells using Accuzol Total RNA Extraction solution and was reverse transcribed into cDNA for 1 h at 37°C using the TOPscript cDNA Synthesis kit, according to the manufacturer's protocol.

PCR amplification of cDNA was performed using iTaq Universal SYBR Green Supermix according to the manufacturer's protocol. Thermocycling conditions were as follows: Initial denaturation at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 5 sec and annealing/extension at 60°C for 30 sec using a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.). Using the 2^−ΔΔCq method (20), gene expression was quantified and normalized to the reference genes β-actin and GAPDH; gene expression was expressed as a ratio over the expression in the LPS treated group (defined as 100%). The primers used for PCR are as follows, in accordance with our previous study (21): iNOS, sense 5′-TGGCCACCAAGCTGAACT-3′, antisense 5′-TCATGATAACGTTTCTGGCTCTT-3′; COX-2, sense 5′-GATGCTCTTCCGAGCTGTG-3′, antisense 5′-GGATTGGAACAGCAAGGATTT-3′; TNF-α, sense 5′-CTGTAGCCCACGTCGTAGC-3′, antisense 5′-TTGAGATCCATGCCGTTG-3′; IL-6, sense 5′-TCTAATTCATATCTTCAACCAAGAGG-3′, antisense 5′-TGGTCCTTAGCCACTCCTTC-3′; IL-1β, sense 5′-TTGACGGACCCCAAAAGAT-3′, antisense 5′-GATGTGCTGCTGCGAGATT-3′; β-actin, sense 5′-CGTCATACTCCTGCTTGCTG-3′, antisense 5′-CCAGATCATTGCTCCTCCTGA-3′; and GAPDH, sense 5′-GCTCTCTGCTCCTCCTGTTC-3′ and antisense 5′-ACGACCAAATCCGTTGACTC-3′.

HEK 293 cells were seeded into a 100-mm dish. When 70% confluent, they were transfected using polyethylenimine (Polysciences, Inc., Warrington, PA, USA) as the transfection reagent for 6 h at 37°C with 4.5 µg pNF-κB-luc or pAP-1-luc cis-reporter plasmids (Agilent Technologies, Inc., Santa Clara, CA, USA), which contained the NF-κB or activator protein 1 (AP-1) promoter, respectively and the luciferase reporter gene. The gWIZ-green fluorescent protein (GFP) plasmid was used as an internal control for transfection efficiency. Transfected cells were seeded into 12-well plates, incubated overnight at 37°C, and treated with various concentrations of ELJ (50, 100, 200 and 300 µg/ml) in the presence of phorbol 12-myristate 13-acetate (PMA), as an activator of NF-κB. Following incubation for 24 h, cells were lysed with Cell Culture Lysis Reagent (Promega Corporation, Madison, WI, USA). For determination of luciferase activity, the Luciferase Assay System (cat no. E1500; Promega Corporation) was used. GFP expression was determined by measuring the fluorescence at 525 nm following excitation at 485 nm and was used as a control for luciferase activity. The luminescence and fluorescence were measured using Synergy H1 Hybrid Microplate Reader and analyzed using Gen5 software version 1.11.5 (BioTek Instruments, Inc.).

RAW 264.7 macrophages were seeded into a 6-well plate (2×10⁶ cells/well). Then, cells were pre-treated with ELJ (50, 100, 200 and 300 µg/ml) for 2 h at 37°C and stimulated with LPS (1 µg/ml) for the detection of the target proteins: For IκBα and TAK1 for 3 min; for MAPKs for 15 min; and for iNOS and COX-2 for 24 h. Following stimulation for the indicated times, cells were washed 3 times with ice-cold PBS and lysed with lysis buffer, containing 0.5% octylphenoxypolyethoxyethanol, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 1 mM Na₃VO₄, for 10 min at 4°C. Following centrifugation at 15,814 × g for 30 min at 4°C, the supernatants were collected.

Total protein was quantified using a Bradford protein assay, and the mixture of lysates and 5X Laemmli sample buffer (Bio-Rad Laboratories, Inc.) was boiled for 5 min at 100°C. Then, equal amounts of extracted protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes using transfer buffer (192 mM glycine, 25 mM Tris-HCl pH 8.8 and 20% v/v methanol). Membranes were blocked for 1 h at room temperature with 5% non-fat dried milk, followed by incubation overnight at 4°C with the following primary antibodies (1:1,000 dilution): anti-IκBα, anti-IKK α/β, anti-α-tubulin, anti-iNOS, anti-COX-2, anti-p-IκBα (Ser32/36), anti-p38 MAPK, anti-p-p38 (Thr180/Tyr182), anti-ERK1/2, anti-JNK, anti-p-JNK (Thr183/Tyr185), anti-p-IKKα/β (Ser176/180), anti-p-MAPK kinase (MKK) 3/6 (Ser189/207), anti-MKK6, anti-TAK, anti-p-TAK1 (Thr184/187) and anti-p-ERK1/2 (Thr202/Tyr204). Subsequently, membranes were incubated for an additional 1 h at room temperature with HRP-conjugated goat anti-mouse (1:5,000; cat no. LF-SA8001A; Abfrontier; Young In Frontier Co., Ltd., Seoul, Korea) or goat anti-rabbit (1:5,000; cat no. LF-SA8002A; Abfrontier; Young In Frontier Co., Ltd.) secondary antibodies. After washing 5 times with 1X PBS containing 0.5% Tween-20, the immunoreactive bands were visualized using enhanced chemiluminescence. Blots were semi-quantified by densitometry using VisionWorksLS Analysis software (UVP, LLC, Upland, CA, USA).

Data are presented as the mean ± standard error of the mean of 3 independent experiments with 3 replicates each. The statistical significance of the differences between groups was assessed using one-way analysis of variance followed by a post hoc Dunnett's test for multiple comparisons. Statistical analysis was performed using GraphPad Prism software version 3.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Since the cell-based evaluation of potential anti-inflammatory agents should be performed under non-cytotoxic conditions, the maximal non-cytotoxic concentration of ELJ was initially determined in murine RAW 264.7 macrophages. Macrophages were treated with various concentrations of ELJ in the presence or absence of stimulation with LPS. Cell viability was determined based on the ability of cells to metabolically reduce a tetrazolium salt to a formazan dye: As presented in Fig. 1A, ELJ did not exert cytotoxic effects at concentrations <300 µg/ml, regardless of LPS stimulation. However, cell viability was significantly reduced at a high concentration of ELJ (400 µg/ml; Fig. 1A). Therefore, ELJ concentrations <300 µg/ml were selected for subsequent experiments.

Since NO serves a key role in inflammatory responses (22), the present study investigated the effects of ELJ on LPS-induced NO production. The levels of NO in RAW 264.7 macrophages were significantly decreased following treatment with ELJ in a dose-dependent manner (Fig. 1B). In addition, the mRNA and protein expression levels of iNOS in ELJ-treated macrophages were analyzed using RT-qPCR and western blot analysis, respectively, since iNOS is a key regulator of NO production during inflammatory responses (23). As presented in Fig. 1C and D, treatment with ELJ resulted in a dose-dependent downregulation in iNOS mRNA and protein expression levels in LPS-stimulated macrophages. These findings suggested that ELJ may counteract the LPS-induced increase in NO production through suppressing iNOS expression in macrophages in vitro.

COX-2 is a proinflammatory enzyme that regulates the production of PGE₂ (24). RT-qPCR and western blot analysis revealed that treatment with ELJ did not affect the mRNA and protein expression levels of COX-2 in LPS-stimulated macrophages (Fig. 2A and B). These results suggested that ELJ may selectively suppress the expression of iNOS but not COX-2 in macrophages in vitro.

Proinflammatory cytokines, including IL-1β, IL-6 and TNF-α, are induced following LPS stimulation and serve pivotal roles during LPS-mediated inflammatory responses (3). Therefore, the effects of ELJ on the expression of proinflammatory cytokines were investigated at the mRNA and protein level. As presented in Fig. 2C, the production of TNF-α was significantly inhibited by ELJ in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. In addition, treatment with high concentrations of ELJ (200–300 µg/ml) was revealed to suppress the production of IL-6 (Fig. 2D). Furthermore, RT-qPCR demonstrated that ELJ downregulated the mRNA expression of proinflammatory cytokines (Fig. 2E). These findings suggested that ELJ may exert anti-inflammatory effects, via inhibiting the production of proinflammatory cytokines in vitro.

To investigate whether the regulation of transcription may be involved in the inhibitory effects of ELJ on proinflammatory mediator production, the transcriptional activity of NF-κB and AP-1 was assessed in vitro. NF-κB and AP-1 are major transcription factors implicated in the inflammatory response; AP-1 is phosphorylated and thus activated by MAPKs (25). A luciferase reporter assay was used in HEK 293 cells treated with PMA, which induces the transcriptional activation of NF-κB- and AP-1-dependent genes (5); the luciferase reporter gene was placed under the control of NF-κB or AP-1 transcriptional activity. As presented in Fig. 3A and B, the NF-κB- and AP-1-regulated luciferase activity was significantly induced following treatment with PMA. However, luciferase activity was revealed to be suppressed following ELJ treatment in PMA-stimulated cells. These findings suggested that ELJ may exert anti-inflammatory effects through the inhibition of NF-κB and AP-1 transcriptional activity.

Since the production of proinflammatory mediators is regulated by NF-κB and MAPK signaling pathways in LPS-stimulated macrophages (4,5), the effects of ELJ on NF-κB and MAPK signaling were evaluated in the present study. IκBα binds NF-κB and stabilizes it in its inactive state, whereas the phosphorylation of IκBα at Ser32/36 is the prerequisite for its dissociation from NF-κB and its subsequent recognition by ubiquitin conjugating enzyme E2 D3, leading to IκBα polyubiquitination and degradation (26,27). Western blot analysis demonstrated that ELJ suppressed the LPS-induced phosphorylation and degradation of IκBα in a dose-dependent manner (Fig. 3C). In addition, the effects of ELJ on the phosphorylation of p38, ERK and JNK, which are major mediators of MAPK signaling, were assessed. MAPK phosphorylation at the phosphorylation sites in their activation loops results in their activation, and the subsequent activation of AP-1, which binds to the promoter of inflammatory mediator genes and induces their transcription (28,29). As presented in Fig. 3D, treatment with ELJ inhibited the phosphorylation of p38 in LPS-stimulated macrophages in vitro; however, it exerted no effects on the phosphorylation of ERK and JNK, as indicated by western blot analysis. The phosphorylation status of MAPKs is directly related to their kinase activity; therefore, these results suggested that ELJ may suppress the production of proinflammatory mediators via inhibiting NF-κB and p38 signaling pathways.

To further investigate the roles of ELJ in the regulation of NF-κB and p38 signaling, its effects on the phosphorylation of IKKα/β and MKK3/6 were explored; IKKα/β and MKK3/6 are upstream kinases of IκBα and p38, respectively (30,31). As demonstrated in Fig. 4A, treatment with ELJ inhibited the LPS-induced phosphorylation of IKKα/β and MKK3/6, without affecting the total expression levels of the proteins. However, the LPS-induced phosphorylation of TAK1, which is an upstream kinase of NF-κB and MAPKs, did not appear to be affected following ELJ treatment (Fig. 4B). These findings suggested that ELJ may target factors located downstream of TAK1 and upstream of IKKα/β and MKK3/6 in the regulation of NF-κB and p38 signaling (Fig. 5).