Diarctigenin, a Lignan Constituent from Arctium lappa, Down-Regulated Zymosan-Induced Transcription of Inflammatory Genes through Suppression of DNA Binding Ability of Nuclear Factor-κB in Macrophages

Arctium lappa, also called burdock, is a perennial herb in the family of Compositae. The seeds of A. lappa are used as an alternative medicine in Korea for the treatment of inflammatory disorders. Arctiin and its aglycone arctigenin are major lignan constituents of A. lappa. Arctiin has antitumor-promoting effects in animal models such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary carcinogenesis and diethylnitrosamine-induced hepatocarcinogenesis (Hirose et al., 2000). Arctiin down-regulates cyclin D1 expression, inhibiting the growth of human tumor cells (Matsuzaki et al., 2008). Arctiin and arctigenin prevent glutamate-induced neurotoxicity in cortical cells (Jang et al., 2002). Arctigenin has anti-inflammatory properties in animal models, including carrageenin-induced paw edema and arachidonic acid-induced ear edema (Kang et al., 2008). Arctigenin inhibits tumor necrosis factor (TNF)-α and nitric oxide (NO) production and suppresses the activation of nuclear factor (NF)-κB in macrophages (Cho et al., 1999, 2002). Diarctigenin is bis-5′,5′-arctigenin, a butyrolactone lignan dimer (Fig. 1A). We previously isolated diarctigenin from A. lappa as an inhibitor of NO production in macrophages (Park et al., 2007). However, no other pharmacological properties of diarctigenin have been demonstrated.

Zymosan is a reagent prepared from Saccharomyces cerevisiae that is used as a model substance for analyzing acute inflammatory responses and the development of anti-inflammatory agents. LPS is a glycolipid component of the outer membrane of Gram-negative bacteria. Water-insoluble zymosan substances are recognized by toll-like receptor (TLR)-2 or Dectin-1 on immune cells and LPS by TLR-4 and its accessory protein MD-2, which can trigger signal cascade for NF-κB activation (Nagai et al., 2002; Ikeda et al., 2008).

NF-κB is a transcription factor consisting of homodimeric or heterodimeric subunits of the Rel protein family (Baeuerle and Baltimore, 1988). In normal cells, NF-κB is sequestered in the cytoplasm as an inactive complex bound to inhibitory κB(IκB) proteins, including IκBα, IκBβ, and IκBϵ (Baeuerle and Baltimore, 1988). Exposure to LPS or zymosan activates the cellular IκB kinase (IKK) complex through TLR/MyD88- and Dectin-1/Card9-Bcl10-Malt1 complex-dependent pathways (Karin and Ben-Neriah, 2000; Nagai et al., 2002; Ikeda et al., 2008). The IKK complex consists of catalytic subunits of IKKα and IKKβ and a regulatory subunit of IKKγ. Almost all inflammatory stimuli require IKKβ to phosphorylate cytoplasmic IκB proteins (Li et al., 1999). This phosphorylation is essential for subsequent ubiquitination followed by proteasome-mediated degradation of IκB proteins (Karin and Ben-Neriah, 2000). NF-κB, after unmasking from IκB proteins, moves into the nucleus and binds to the κB motifs to regulate the expression of immune and inflammatory genes, including inducible NO synthase (iNOS), cyclooxygenase (COX)-2, TNF-α, and interleukin (IL)-1β and IL-6 (Tian and Brasier, 2003).

The goal of this study was to understand how diarctigenin inhibited NO production and also evaluate its anti-inflammatory potential. We demonstrated that diarctigenin could down-regulate inflammatory gene expression at the transcription level through inhibition of the DNA binding ability of NF-κB in zymosan- or LPS-activated macrophages.

Materials and Methods

Chemicals. Diarctigenin (>97% purity) was isolated from A. lappa as described in our previous work (Park et al., 2007). Fetal bovine serum (FBS) and other culture materials were purchased from Invitrogen (Carlsbad, CA). All other chemicals, including zymosan (S. cerevisiae) and LPS (Escherichia coli 055:B5), were otherwise purchased from Sigma-Aldrich (St. Louis, MO). In this study, zymosan was washed several times with sterile waters, concentrating water-insoluble substances responding to TLR-2 or Dectin-1 on immune cells (Ikeda et al., 2008), before use for activation of macrophages. Diarctigenin was relatively water-insoluble, difficult to make up correct concentration, and thus its varied concentrations (0.6–30 μM) were used in this study.

Antibodies and Plasmids. Antibodies against iNOS, IκBα, IκBβ, IκBϵ, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and those against phosphor (p)-IκBα and NF-κB p65 were from Cell Signaling Tech (Danvers, MA). Promoter-dependent luciferase reporter plasmids of piNOS (-1592/+183)-Luc, pTNF-α (-1260/+60)-Luc, pIL-1β (-1856/+1)-Luc, or pIL-6 (-250/+1)-Luc, an expression vector encoding NF-κB p65, and an NF-κB-dependent reporter plasmid of pNF-κB-secretory alkaline phosphatase (SEAP)-neomycin phosphotransferase (NPT) have been described previously (Hiscott et al., 1993; Lowenstein et al., 1993; Zhang et al., 1994; Yao et al., 1997; Moon et al., 2001; Kim et al., 2008).

Cell Culture. RAW 264.7 macrophages were purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's media supplemented with 10% FBS, benzylpenicillin potassium (143 U/ml), and streptomycin sulfate (100 μg/ml) under 37°C and 5% CO₂ atmosphere. RAW 264.7 cells harboring pNF-κB-SEAP-NPT construct were grown under the same conditions, with the exception of supplement of Geneticin (G-418; 500 μg/ml) to the media. Primary macrophages were prepared from female C57BL/6 mice (Daehan Biolink Co., Eumseong, Korea) as described previously (Sud et al., 2008). In brief, peritoneal cavities of mice were flushed with phosphate-buffered saline. After centrifugation, cell pellets were resuspended in RPMI 1640 media supplemented with 10% FBS, benzylpenicillin potassium (143 U/ml), and streptomycin sulfate (100 μg/ml) and incubated under 37°C and 5% CO₂ atmosphere. This animal study was carried out, in accordance with all ethical regulations for experimental animal care.

Nitrite Quantification. RAW 264.7 cells or primary macrophages from mice were pretreated with various concentrations of diarctigenin for 2 h and stimulated with zymosan or LPS for 24 h, in which zymosan was used at 0.3 mg/ml and LPS was used at 1 μg/ml for substantially full induction (Ikeda et al., 2008; Kim et al., 2008). Aliquots of the culture media were mixed with the same volume of 0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine, and then absorbance values were measured at 540 nm with sodium nitrite as a standard.

Enzyme-Linked Immunosorbent Assay of Prostaglandin and Cytokines. The cells were pretreated with diarctigenin for 2 h and stimulated with zymosan (0.3 mg/ml) for 24 h. Amounts of PGE₂, TNF-α, IL-1β, or IL-6 in the culture media were measured using appropriate ELISA kits (R&D Systems, Minneapolis, MN).

Cell Proliferation Assay. The cells were incubated with various concentrations of diarctigenin for 24 h. They were exposed to a water-soluble WST-1 of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (Dojindo Lab., Kumamoto, Japan) for 3 h, and then absorbance values were measured at 450 nm.

Immunoblotting Analysis. The cells were pretreated with diarctigenin for 2 h, stimulated with zymosan (0.3 mg/ml) for the indicated times, and then disrupted in a lysis buffer (20 mM HEPES, pH 7.9, 1% Triton X-100, 20% glycerol, 1 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 mM Na₃VO₄, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). Cell extracts were resolved on SDS-acrylamide gel by electrophoresis and then transferred to a polyvinylidene difluoride membrane. Either 5% nonfat milk in phosphate-buffered saline containing Tween 20 or 5% bovine serum albumin in Tris-buffered saline containing Tween 20 was used as the blocking buffer. The blots were usually incubated at 4°C overnight with primary antisera. The antisera (dilution) were anti-iNOS (1:1500), anti-IκBα (1:500), anti-IκBβ (1:500), anti-IκBϵ (1:500), anti-p-IκBα (1:200), and anti-GAPDH (1:2000). The blots were then incubated with secondary antisera, horseradish peroxidase-labeled anti-rabbit IgG (1:1000) at room temperature for 2 to 5 h. The blots for anti-iNOS antibody were washed with a stripping buffer (63 mM Tris, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol) at 50°C and then reacted with anti-GAPDH antibody for normalization of the iNOS signal. Immune complexes on the blots were finally visualized by exposure to X-ray film after reacting with an enhanced chemiluminescence reagent (GE Healthcare, Chalfont St Giles, UK).

Reverse Transcription-Polymerase Chain Reaction of iNOS Transcript. The cells were pretreated with diarctigenin for 2 h and stimulated with zymosan (0.3 mg/ml) for 6 h. Total RNA was subjected to a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using an RNA PCR kit (Bioneer Co., Daejeon, Korea). In brief, total RNA was reverse-transcribed at 42°C and then subjected to 25 cycles of PCR consisting of 30-s denaturation at 94°C, 30-s annealing at 55°C, and 90-s extension at 72°C. Sequences of primers for RT-PCR and sizes of the amplified products are as follows: iNOS, sense 5′-GTCAACTGCAAGAGAACGGAGAC-3′, antisense 5′-GAGCTCCTCCAGAGGGTAGGCTTG-3′, 457 bp; and β-actin, sense 5′-CACCACACCTTCTACAATGAGCTGC-3′, antisense 5′-GCTCAGGAGGAGCAATGATCTTGAT-3′, 745 bp. RT-PCR products were resolved on agarose gels by electrophoresis and then visualized by staining with ethidium bromide.

Promoter-Dependent Luciferase Reporter Assay. The 264.7 cells were transiently transfected with each luciferase reporter construct of piNOS (-1592/+183)-Luc, pTNF-α (-1260/+60)-Luc, pIL-1β (-1856/+1)-Luc, or pIL-6 (-250/+1)-Luc using Lipofectamine (Invitrogen) according to the manufacturer's recommendations. In brief, the cells (2 × 10⁶) in 1.8 ml of Opti-MEM I (Invitrogen) were incubated for 2 h and then gently mixed with the reporter construct (2 μg)-Lipofectamine (6 μl) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 × 10⁵ cells per well. The transfected cells were pretreated with diarctigenin for 2 h and stimulated with zymosan (0.3 mg/ml) for 16 h. Luciferase activity was measured with cell extracts using an assay kit (Promega, Madison, WI). Protein levels were measured using a dye-based assay kit (Bio-Rad, Hercules, CA). In another experiment, the cells were transiently transfected with piNOS (-1592/+183)-Luc reporter construct, in combination with an expression vector encoding NF-κB p65. In brief, the cells (2 × 10⁶) in 1.8 ml of Opti-MEM I were incubated for 2 h and then gently mixed with the reporter construct (2 μg) expression vector (1 μg)-Lipofectamine (9 μl) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 × 10⁵ cells/well. The transfected cells were treated with diarctigenin for 16 h and then subjected to the luciferase reporter assay.

NF-κB-Dependent SEAP Reporter Assay. RAW 264.7 cells harboring pNF-κB-SEAP-NPT construct were pretreated with diarctigenin for 2 h and stimulated with zymosan (0.3 mg/ml) for 16 h. SEAP expression was measured as described previously (Moon et al., 2001). In brief, aliquots of the culture media were heated at 65°C for 5 min and then were reacted with 4-methylumbellifery phosphate (500 μM) in the dark. SEAP activity was measured as relative fluorescence units (RFU) with emission at 449 nm and excitation at 360 nm. In another experiment, RAW 264.7 cells harboring pNF-κB-SEAP-NPT construct were transiently transfected with an expression vector encoding NF-κB p65. In brief, the cells (2 × 10⁶) in 1.8 ml of Opti-MEM I were incubated for 2 h and then gently mixed with the expression vector (1 μg)-Lipofectamine (3 μl) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 × 10⁵ cells per well. The transfected cells were treated with diarctigenin for 16 h and then subjected to the SEAP reporter assay.

Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay. The cells were pretreated with diarctigenin for 2 h, stimulated with zymosan (0.3 mg/ml) for 1 h, and then disrupted in a lysis buffer (10 mM HEPES, pH 7.9, 2 mM MgCl₂, 10 mM KCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenymethylsulfonyl fluoride). After centrifugation, pellets were resuspended in an extraction buffer (20 mM HEPES, pH 7.9, 50 mM MgCl₂, 420 mM KCl, 20% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenymethylsulfonyl fluoride) and incubated on ice for 30 min. After centrifugation, supernatants were used as the sources of nuclear extracts. A double-stranded oligonucleotide of 5′-AGTTGAGGGGACTTTCCCAGGC-3′ was used as a probe, with the NF-κB motif underlined. The ³²P end-labeled oligonucleotide was reacted with nuclear extracts in a binding buffer [10 mM Tris, pH 7.5, 1 mM MgCl₂, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 μg/ml poly(dI-dC), and 4% glycerol] on ice for 30 min. The resulting DNA complexes were resolved on 6% nondenaturing acrylamide gels by electrophoresis and then subjected to autoradiography. In another experiment, nuclear extracts were prepared from RAW 264.7 cells stimulated with zymosan (0.3 mg/ml) alone for 1 h and then subjected to DNA binding reactions in the presence of diarctigenin.

Confocal Fluorescence Microscopy. RAW 264.7 cells were pretreated with diarctigenin for 2 h and stimulated with zymosan (0.3 mg/ml) for 1 h. These cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and then blocked in 1% bovine serum albumin in phosphate-buffered saline. For immunostaining, the cells were incubated with anti-NF-κB p65 antibody for 2 h, and then stained with Alexa Fluor 568-labeled secondary antibody for 1 h. For nuclei staining, the cells were incubated with 4′,6-diamidino-2-phenylindole solution.

Statistical Analysis. Data are expressed as means ± S.E.M., and were subjected to the one-way analyses of variance followed by the Dunnett's test. Values of p < 0.05 were considered as significantly different.

Results

Here, we investigated the effects of diarctigenin (Fig. 1A) on the expression of inflammatory genes, including iNOS and cytokines, and on the activation of transcription factor NF-κBin macrophages. We focused on zymosan- and LPS-induced gene transcription, considering the fact that inflammatory responses challenged by these stimuli were well characterized.

Diarctigenin Efficiently Inhibited Inflammatory Mediator Production in Macrophages. We first quantified NO in RAW 264.7 macrophages. Nitrite, a stable metabolite of NO, was quite low in normal cells (7–8 μM) but markedly increased to 25 to 28 μM upon exposure to zymosan or LPS alone (Fig. 1, B and C). Pretreatment of the cells with diarctigenin dose-dependently (linear contrast, p < 0.05) inhibited zymosan-induced nitrite production with an IC₅₀ value of 9.6 μM (Fig. 1B) and LPS-induced nitrite production with an IC₅₀ value of 8.8 μM (Fig. 1C). Moreover, diarctigenin obviously inhibited zymosan- or LPS-induced nitrite production in primary macrophages from mice (Fig. 1D).

PGE₂ and cytokine levels were measured using ELISA in the culture media of zymosan-activated RAW 264.7 macrophages. Upon exposure to zymosan alone, the cells released pronounced amounts of PGE₂ (2162 ± 74 pg/ml) over the basal levels (238 ± 68 pg/ml) (Table 1). Diarctigenin inhibited zymosan-induced PGE₂ production with an IC₅₀ value of 8.4 μM (Table 1). In a parallel experiment, diarctigenin also inhibited zymosan-induced productions of TNF-α, IL-1β, and IL-6 (Table 1). Diarctigenin at the effective concentrations did not affect the proliferation of RAW 264.7 macrophages (Fig. 1E), excluding its nonspecific cytotoxicity. Therefore, diarctigenin is an efficient inhibitor of inflammatory mediator production in zymosan- and LPS-activated macrophages.

Diarctigenin Down-Regulated Zymosan-Induced Transcription of iNOS and Cytokine Genes in Macrophages. To understand whether the effect of diarctigenin on NO production was due to zymosan-induced iNOS expression, we performed Western blot analysis using cell extracts from RAW 264.7 macrophages exposed to zymosan in the absence or presence of diarctigenin. The iNOS signal was barely detectable in normal macrophages but markedly increased upon exposure to zymosan alone (Fig. 2A). Diarctigenin inhibited the zymosan-induced synthesis of iNOS protein with an IC₅₀ value of 9.6 μM (Fig. 2A). However, housekeeping GAPDH levels were not significantly affected by treatment with zymosan or diarctigenin (Fig. 2A).

To understand whether diarctigenin inhibited iNOS transcription, semiquantitative RT-PCR was performed using total RNA from zymosan-activated RAW 264.7 cells. The iNOS transcripts were hardly detectable in normal cells but markedly increased upon exposure to zymosan alone (Fig. 2B). Diarctigenin attenuated zymosan-induced synthesis of iNOS transcripts with an IC₅₀ value of 12.3 μM (Fig. 2B). However, housekeeping β-actin levels were not changed at all by treatment with zymosan or diarctigenin (Fig. 2B).

The transcriptional regulation of iNOS gene by diarctigenin was further delineated using a promoter activity assay. We transfected RAW 264.7 cells with piNOS-Luc, a construct encoding iNOS promoter (-1592/+183) fused to the luciferase reporter gene (Lowenstein et al., 1993). Upon exposure to zymosan alone, the transfected cells increased luciferase expression up to ∼8-fold over the basal levels (Fig. 2C). Diarctigenin inhibited the zymosan-increased luciferase expression with an IC₅₀ value of 10.4 μM (Fig. 2C).

In a parallel experiment, RAW 264.7 cells were transfected with pTNF-α (-1260/+60)-Luc, pIL-1β (-1856/+1)-Luc, or pIL-6 (-250/+1)-Luc constructs (Hiscott et al., 1993; Zhang et al., 1994; Yao et al., 1997). Upon exposure to zymosan alone, the cells harboring pTNF-α (-1260/+60)-Luc construct had an ∼3.5-fold increased luciferase expression over the basal levels (Table 2). Diarctigenin also inhibited zymosan-induced luciferase expression, a reporter of TNF-α promoter activity (Table 2). Likewise, zymosan-induced promoter activities from pIL-1β (-1856/+1)-Luc and pIL-6 (-250/+1)-Luc constructs were also inhibited by treatment with diarctigenin (Table 2). Therefore, diarctigenin could down-regulate the expression of zymosan-inducible inflammatory genes at the transcription level.

Diarctigenin Blocked NF-κB Activation in Zymosan- or LPS-Stimulated Macrophages. NF-κB plays a pivotal role in the transcription of inflammatory genes, including iNOS and cytokines (Tian and Brasier, 2003). Because diarctigenin down-regulated zymosan-induced transcription of inflammatory genes (Fig. 2; Table 2), we tested whether the compound could affect NF-κB activation using RAW 264.7 cells harboring the pNF-κB-SEAP-NPT construct, which contains four copies of an NF-κB-responsive κB motif fused to the SEAP reporter gene (Moon et al., 2001). The cells were pretreated with diarctigenin and then exposed to zymosan or LPS for induction of NF-κB-dependent reporter gene expression. Basal SEAP levels were quite low in the cells and significantly increased SEAP expression upon exposure to zymosan alone (Fig. 3A), indicating that cellular NF-κBis functional. Diarctigenin dose-dependently (linear contrast, p < 0.05) blocked zymosan-induced SEAP expression in the cells with an IC₅₀ value of 9.1 μM (Fig. 3A), similar to its potency to modulate the production of inflammatory mediators (Fig. 1B; Table 1). Moreover, diarctigenin inhibited LPS-induced SEAP expression in the cells with an IC₅₀ value of 8.4 μM (Fig. 3B). Therefore, diarctigenin was an efficient inhibitor of NF-κB activation in zymosan- or LPS-stimulated macrophages.

Diarctigenin Inhibited the Zymosan-Induced Signal Cascade for NF-κB Activation in Macrophages. NF-κB transcriptional activity in macrophages is preceded by the DNA binding activity of nuclear NF-κB and proteolytic degradation of cytoplasmic IκB proteins (Karin and Ben-Neriah, 2000). We determined whether diarctigenin affected the DNA binding ability of NF-κB in zymosan-activated RAW 264.7 macrophages. The cells were treated with zymosan in the absence or presence of diarctigenin. Nuclear extracts of the cells were reacted with a κB-specific ³²P end-labeled oligonucleotide and then subjected to an electrophoretic mobility shift assay. Upon exposure to zymosan alone, DNA binding ability of NF-κB was significantly increased over the basal levels (Fig. 4A). Diarctigenin inhibited this DNA binding ability of NF-κB in the cells (Fig. 4A).

To understand whether diarctigenin prevented the DNA binding ability and transcriptional activity of NF-κBbyits inhibitory action on the degradation of cytoplasmic IκB proteins, we performed Western blot analysis with cell extracts from RAW 264.7 macrophages stimulated with zymosan in the absence or presence of diarctigenin. Upon exposure to zymosan alone, cellular IκBα was dramatically degraded within 30 to 45 min (Fig. 4B), and cellular IκBβ or IκBϵ was degraded within 45 to 60 min (data not shown). Pretreatment with diarctigenin (25 μM) neither inhibited the IκBα degradation in a time course study (Fig. 4B) nor affected the IκBβ or IκBϵ degradation in zymosan-activated RAW 264.7 cells (Fig. 4C). For the proteasome-dependent degradation, IκBα is phosphorylated on Ser32 and Ser36 residues, and IκBβ and IκBϵ are phosphorylated on similar conserved Ser residues by the IKK complex (DiDonato et al., 1996). Upon exposure to zymosan alone for 10 min, RAW 264.7 cells significantly increased IκBα phosphorylation, in which IκBα degradation had not yet started (Fig. 4D). Diarctigenin did not affect zymosan-induced IκBα phosphorylation significantly (Fig. 4D).

Diarctigenin Suppressed Expression Vector NF-κB p65-Elicited NF-κB Transcriptional Activity and iNOS Promoter Activity. Diarctigenin inhibited the DNA binding ability and transcriptional activity of nuclear NF-κB but did not affect the phosphorylation or degradation of cytoplasmic IκB proteins in zymosan-activated macrophages (Figs. 3 and 4). To delineate whether diarctigenin affected the NF-κB-activating signal cascade downstream of IκB degradation, we transfected RAW 264.7 cells harboring the pNF-κB-SEAP-NPT reporter construct, with an expression vector encoding NF-κB p65. Transfection with NF-κB p65 vector resulted in a significantly increased SEAP expression as a reporter of NF-κB transcriptional activity, which was suppressed by treatment with diarctigenin (Fig. 5A).

Because diarctigenin down-regulated iNOS expression at the transcription level and inhibited NF-κB activation in zymosan-stimulated macrophages (Figs. 2 and 3), we tested whether diarctigenin could inhibit NF-κB-regulated transcription of iNOS gene. RAW 264.7 cells were transfected with the piNOS (-1592/+183)-Luc reporter construct, in combination with an expression vector encoding NF-κB p65. Transfection with NF-κB p65 vector increased luciferase expression, as a reporter of iNOS promoter activity (Fig. 5B). Diarctigenin obviously inhibited NF-κB p65 vector-elicited luciferase expression (Fig. 5B).

Diarctigenin Targeted the DNA Binding Ability of NF-κB. To elucidate the target event downstream of IκB degradation, we studied confocal fluorescence microscopy. RAW 264.7 cells were pretreated with diarctigenin for 2 h and stimulated with zymosan for 1 h. Upon exposure to zymosan alone, most of NF-κB was translocated from the cytoplasm to the nucleus (Fig. 6). Diarctigenin (10–30 μM) did not affect zymosan-induced nuclear import of NF-κBin the cells significantly (Fig. 6). DNA binding ability of NF-κB was inhibited when RAW 264.7 cells were treated with zymosan in the presence of diarctigenin (Fig. 4A). To verify that diarctigenin directly affected the DNA binding ability of NF-κB, nuclear extracts were treated with diarctigenin and then subjected to DNA binding reactions. Diarctigenin also inhibited this DNA binding ability of NF-κB in vitro (Fig. 7).

Discussion

The seeds of A. lappa are used as a traditional medicine in Korea for the treatment of inflammatory disorders. In our previous work to screen anti-inflammatory agent, methanol extracts of the seeds of A. lappa were subjected to phytochemical analysis, identifying diarctigenin as an active constituent that inhibits NO production in macrophages (Park et al., 2007). However, its molecular basis remains undefined.

In the present study, we observed that diarctigenin could down-regulate the expression of inflammatory genes at the transcription level through suppression of NF-κB activation, directly targeting the DNA binding ability of NF-κB in macrophages. Consistently, diarctigenin inhibited zymosan- or LPS-induced NO production in macrophages and also other inflammatory mediators, such as PGE₂, TNF-α, IL-1β, and IL-6. Immune cells such as macrophages produce NO by iNOS where the free radicals facilitate the killing of invading microorganism; however, excessive production of the radicals can cause septic shock and tissue injury (Zamora et al., 2000). PGE₂ is also produced at inflamed sites by COX-2 and gives rise to pain and swelling (Simon, 1999). TNF-α, IL-1β, and IL-6 are NF-κB-inducible proinflammatory cytokines and also induce the transcription of iNOS and COX-2 genes (Feldmann et al., 1998; Tian and Brasier, 2003). In this study, diarctigenin attenuated zymosan-induced mRNA synthesis of iNOS and inhibited promoter activities of iNOS, TNF-α, IL-1β, and IL-6 genes, indicating that diarctigenin down-regulated the expression of inflammatory genes at the transcription level.

NF-κB serves as an important regulator for immune homeostasis in the host and also plays a fundamental role in the initiation and amplification of inflammatory responses, mainly triggering the transcription of inflammatory genes (Tak and Firestein, 2001). NF-κB-responsive κB motifs have been identified in the promoter regions of inflammatory genes: iNOS with two sites at -8287/-8270 and -119/-102 relative to the transcription start; COX-2 with one site at -223/-214; TNF-α with three sites at -594/-577, -217/-200, and -103/-86; IL-1β with two sites at -2800/-2720 and -296/-286; and IL-6 with one site at -72/-63 (Hiscott et al., 1993; Lowenstein et al., 1993; Zhang et al., 1994; Yao et al., 1997).

Prolonged or imbalanced activation of NF-κB, however, is implicated in the pathogenesis of inflammatory disorders, such as asthma, arthritis, and inflammatory bowel disease (Bacher and Schmitz, 2004; Park and Christman, 2006; Atreya et al., 2008). There is no doubt that NF-κB signaling cascade constitutes a promising therapeutic target for inflammatory and autoimmune disorders (Uwe, 2008). In this study, diarctigenin inhibited zymosan- or LPS-induced NF-κB-transcriptional activity in macrophages. Furthermore, diarctigenin inhibited the DNA binding ability of NF-κB but did not affect the phosphorylation or degradation of IκB proteins in zymosan-activated macrophages, indicating that diarctigenin could interrupt the signal cascade for NF-κB activation downstream of IκB degradation. This hypothesis was clarified by inhibition of diarctigenin on cellular NF-κB transcriptional activity or iNOS promoter activity elicited by transfecting an expression vector of NF-κB p65.

To elucidate the primary target of diarctigenin, we further investigated the signaling events immediately downstream of IκB degradation in macrophages. Diarctigenin did not affect the nuclear import of NF-κB p65 but inhibited the DNA binding ability of NF-κB when the cells were treated with zymosan in the presence of diarctigenin. To verify that diarctigenin directly blocked the DNA binding ability of NF-κB, nuclear extracts were prepared from zymosan aloneactivated macrophages and then reacted with NF-κB-specific oligonucleotide probe in the presence of diarctigenin. This in vitro DNA binding ability of NF-κB was also inhibited by diarctigenin. Therefore, diarctigenin inhibited the NF-κB activating pathway, directly targeting the DNA binding ability of NF-κB in zymosan-activated macrophages.

Diarctigenin is a dimeric structure of arctigenin, covalently linking each carbon at 5′ position. This kind of carbon-carbon linkage in the herbal lignans is usually recalcitrant to hydrolytic cleavage, generating the monomeric structure, in mammalian cells (Nose et al., 1992). Arctigenin has been assigned to show anti-inflammatory properties, inhibiting LPS-induced NO production in macrophages as well as NF-κB activation through dually targeting IκBα phosphorylation and nuclear import of NF-κB (Cho et al., 2002). Therefore, molecular target of diarctigenin on NF-κB activation in this study was different from those of its monomeric structure, arctigenin.

Several lignan structures have been reported to inhibit NF-κB activation. Very similar to anti-inflammatory mechanism of diarctigenin in this study, 7,7′-dihydroxybursehernin from Geranium thunbergii inhibits the DNA binding ability of NF-κB and down-regulates the expression of iNOS and COX-2 genes in LPS-activated RAW 264.7 cells (Pokharel et al., 2007). Nordihydroguaiaretic acid, also known as a lipoxygenase inhibitor, suppresses NF-κB activation by dually targeting IκB phosphorylation and the DNA binding ability of NF-κB in TNF-α-activated E6.1 Jurkat cells (Brennan and O'Neill, 1998). Magnolol from Magnolia officinalis inhibits IKK complex-mediated IκB phosphorylation in U937 histiocytic lymphoma cells (Tse et al., 2007). Magnolol also inhibits TNF-α-induced NF-κB activation by directly targeting the nuclear import of NF-κB in embryonic kidney cells (Tanaka et al., 2007). Sauchinone and manassantins from Saururus chinensis directly inhibit the transcriptional activity of NF-κB and suppress NF-κB-regulated antiapoptotic genes in TNF-α-stimulated HT-1080 fibrosarcoma cells and inflammatory genes in LPS-activated RAW 264.7 cells (Hwang et al., 2003; Lee et al., 2003). Other lignan compounds of sesamin and podophyllotoxin derivatives also inhibit NF-κB activation, but their molecular targets remain to be defined (Jeng et al., 2005; Vasilev et al., 2005).

In summary, diarctigenin is a small-molecule inhibitor of the DNA binding ability of NF-κB, resulting in prevention of zymosan-induced NF-κB activation in macrophages. This mechanism of action contributes to suppressive effect of diarctigenin on NF-κB-regulated transcription of inflammatory genes. Taken together, this study suggests a pharmacological potential of diarctigenin in the NF-κB-associated inflammatory disorders.