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

Diarctigenin was previously isolated as an inhibitor of nitric oxide (NO) production in macrophages from the seeds of Arctium lappa used as an alternative medicine for the treatment of inflammatory disorders. However, little is known about the molecular basis of these effects. Here, we demonstrated that diarctigenin inhibited the production of NO, prostaglandin E2, tumor necrosis factor, and interleukin (IL)-1 and IL-6 with IC50 values of 6 to 12 M in zymosanor lipopolysaccharide(LPS) activated macrophages. Diarctigenin attenuated zymosaninduced mRNA synthesis of inducible NO synthase (iNOS) and also inhibited promoter activities of iNOS and cytokine genes in the cells. Because nuclear factor (NF)B plays a pivotal role in inflammatory gene transcription, we next investigated the effect of diarctigenin on NFB activation. Diarctigenin inhibited the transcriptional activity and DNA binding ability of NFB in zymosan-activated macrophages but did not affect the degradation and phosphorylation of inhibitory B (I B) proteins. Moreover, diarctigenin suppressed expression vector NFB p65-elicited NFB activation and also iNOS promoter activity, indicating that the compound could directly target an NFBactivating signal cascade downstream of I B degradation and inhibit NFB-regulated iNOS expression. Diarctigenin also inhibited the in vitro DNA binding ability of NFB but did not affect the nuclear import of NFB p65 in the cells. Taken together, diarctigenin down-regulated zymosanor LPS-induced inflammatory gene transcription in macrophages, which was due to direct inhibition of the DNA binding ability of NFB. Finally, this study provides a pharmacological potential of diarctigenin in the NFB-associated inflammatory disorders. 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 antitumorpromoting effects in animal models such as 2-amino-1methyl-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)and nitric oxide (NO) production and suppresses the activation of nuclear factor (NF)B in macrophages (Cho et al., 1999, 2002). Diarctigenin is bis-5 ,5 -arctigenin, a butyrolactone lignan This work was supported by Grant KRF-2005-005-J15001 from the Korean Government (MOEHRD, Basic Research Promotion Fund) and other research funds from Chungbuk BIT Research-Oriented University Consortium and from Research Center for Bioresource and Health. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.140145. ABBREVIATIONS: TNF, tumor necrosis factor; NO, nitric oxide; IL, interleukin; LPS, lipopolysaccharide; iNOS, inducible nitric-oxide synthase; NF, nuclear factor; I B, inhibitory B; TLR, toll-like receptor; IKK, I B kinase; COX, cyclooxygenase; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEAP, secretory alkaline phosphatase; NPT, neomycin phosphotransferase; PG, prostaglandin; ELISA, enzymelinked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; Luc, luciferase; bp, base pair; p, phosphor. 0022-3565/08/3272-393–401$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 327, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 140145/3396260 JPET 327:393–401, 2008 Printed in U.S.A. 393 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from 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 NFB activation (Nagai et al., 2002; Ikeda et al., 2008). NFB is a transcription factor consisting of homodimeric or heterodimeric subunits of the Rel protein family (Baeuerle and Baltimore, 1988). In normal cells, NFB is sequestered in the cytoplasm as an inactive complex bound to inhibitory B (I B) proteins, including I B , I B , and I Bε (Baeuerle and Baltimore, 1988). Exposure to LPS or zymosan activates the cellular I B kinase (IKK) complex through TLR/MyD88and 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 IKK and IKK and a regulatory subunit of IKK . Almost all inflammatory stimuli require IKK to phosphorylate cytoplasmic I B proteins (Li et al., 1999). This phosphorylation is essential for subsequent ubiquitination followed by proteasome-mediated degradation of I B proteins (Karin and BenNeriah, 2000). NFB, after unmasking from I B proteins, moves into the nucleus and binds to the B motifs to regulate the expression of immune and inflammatory genes, including inducible NO synthase (iNOS), cyclooxygenase (COX)-2, TNF, and interleukin (IL)-1 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 NFB in zymosanor 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 Fig. 1. Effect of diarctigenin on nitrite production. A, chemical structure of diarctigenin (DAGN). RAW 264.7 cells were pretreated with DAGN for 2 h and stimulated with zymosan (B) or LPS (C) for 24 h. Amounts of nitrite in the culture media were determined using sodium nitrite as a standard. D, primary macrophages from mice were pretreated with DAGN for 2 h and stimulated with zymosan (0.3 mg/ml) or LPS (1 g/ml) for 24 h. Effects of diarctigenin on nitrite production were represented as percentage of the control, zymosan, or LPS alone-treated group. E, RAW 264.7 cells were incubated with zymosan (0.3 mg/ml) in the presence of various concentrations of DAGN for 24 h. Proliferation of the cells was analyzed by WST-1 method. Data are means S.E.M. from three to five separate experiments. #, p 0.05 versus paired groups. , p 0.05 versus zymosan or LPS alone-treated group. 394 Kim et al. at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from 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 M) were used in this study. Antibodies and Plasmids. Antibodies against iNOS, I B , I B , I Bε, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and those against phosphor (p)-I B and NFB p65 were from Cell Signaling Tech (Danvers, MA). Promoter-dependent luciferase reporter plasmids of piNOS ( 1592/ 183)-Luc, pTNF( 1260/ 60)-Luc, pIL-1 ( 1856/ 1)-Luc, or pIL-6 ( 250/ 1)-Luc, an expression vector encoding NFB p65, and an NFBdependent reporter plasmid of pNFB-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 g/ml) under 37°C and 5% CO2 atmosphere. RAW 264.7 cells harboring pNFB-SEAP-NPT construct were grown under the same conditions, with the exception of supplement of Geneticin (G-418; 500 g/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 g/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 g/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, IL-1 , 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-(2,4disulfophenyl)-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 g/ml leupeptin, and 1 g/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-I B (1:500), anti-I B (1:500), anti-I Bε (1:500), anti-p-I B (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 -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( 1260/ 60)-Luc, pIL-1 ( 1856/ 1)-Luc, or pIL-6 ( 250/ 1)-Luc using Lipofectamine (Invitrogen) according to the manufacturer’s recommendations. In brief, the cells (2 10) in 1.8 ml of Opti-MEM I (Invitrogen) were incubated for 2 h and then gently mixed with the reporter construct (2 g)-Lipofectamine (6 l) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 10 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 NFB p65. In brief, the cells (2 10) in 1.8 ml of Opti-MEM I were incubated for 2 h and then gently mixed with the reporter construct (2 g) expression vector (1 g)-Lipofectamine (9 l) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 10 cells/well. The transfected cells were treated with diarctigenin for 16 h and then subjected to the luciferase reporter assay. NFB-Dependent SEAP Reporter Assay. RAW 264.7 cells harboring pNFB-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 M) 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 pNFBSEAP-NPT construct were transiently transfected with an expression vector encoding NFB p65. In brief, the cells (2 10) in 1.8 ml of Opti-MEM I were incubated for 2 h and then gently mixed with the expression vector (1 g)-Lipofectamine (3 l) complex in 0.2 ml of Opti-MEM I. After incubation for 6 h, transfection reactions were pooled and then redistributed at 5 10 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 reDown-Regulation of Inflammatory Genes by Diarctigenin 395 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from suspended 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 NFB motif underlined. The P 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 g/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-NFB 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