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Journal of Ethnopharmacology 125 (2009) 246–251

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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ichroa febrifuga Lour. inhibits the production of IL-1� and IL-6 through blockingF-�B, MAPK and Akt activation in macrophages

un Young Parka, Ga-young Parka, Woo Shin Kob, YoungHee Kima,∗

Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Pusan 609-735, Republic of KoreaDepartment of Oriental Medicine, College of Oriental Medicine, Dongeui University, Pusan 614-054, Republic of Korea

r t i c l e i n f o

rticle history:eceived 13 February 2009eceived in revised form 4 June 2009ccepted 5 July 2009vailable online 14 July 2009

eywords:ichroa febrifuga

L-1�

a b s t r a c t

Aim of the study: The roots of Dichroa febrifuga Lour. have been used as a traditional antimalarial drugand also used in the treatment of productive cough and unstable fever caused by infection in China andKorea. In this study, we evaluated the anti-inflammatory effect and underlying molecular mechanism ofaqueous extract of Dichroa febrifuga (AEDF) in C57BL/6 mouse peritoneal macrophages.Materials and methods: The effect of AEDF on proinflammatory cytokine (IL-1� and IL-6) production wasanalyzed by ELISA and real-time RT-PCR. The effects of AEDF on NF-�B/I�B-�/IKK were measured byreporter assay (in RAW 264.7 cells), EMSA, Western blotting and kinase assay. The effects of AEDF on Aktand MAPKs activity were assayed by Western blotting.

L-6F-�Bktitogen-activated protein kinase (MAPK)

Results: AEDF inhibited the production of IL-1� and IL-6, NF-�B activation, I�B-� degradation, and IKK,Akt, ERK1/2 and JNK activities in LPS-stimulated mouse peritoneal macrophages.Conclusions: These results suggest that AEDF inhibits proinflammatory cytokine (IL-1� and IL-6) pro-duction in LPS-stimulated mouse peritoneal macrophages, and that these effects are mediated by theinhibition of the activity of IKK/I�B/NF-�B and the phosphorylation of Akt, ERK1/2, and JNK. Our resultsprovide a molecular basis for understanding the inhibitory effects of Dichroa febrifuga roots on endotoxin-

mediated inflammation.

. Introduction

The roots of Dichroa febrifuga Lour. (Saxifragaceae) have beensed as a traditional antimalarial drug and also used in the treat-ent of productive cough in China and Korea. This plant has aide application as a complementary therapeutic agent in Korea

or the treatment of unstable fever caused by infection. Febrifug-ne, major constituent of Dichroa febrifuga roots has been reportedo reduce parasitemia in Plasmodium berghei NK65-infected miceMurata et al., 1999; Ishih et al., 2008). One of the constituentsf this plant, changrolin (4-[3,5-bis{(N-purrolidinyl)-methyl}-4-ydroxyanilino]-quinazoline) has been used as an anti-arrhythmicrug (Lu et al., 1995). Halofuginone, an alkaloid isolated from thislant, has been shown to be a potent inhibitor of tissue fibrosisTacheau et al., 2007). Halofuginone is also reported to inhibit NF-�Bnd p38 MAPK in activated T cells (Leiba et al., 2006) and ameliorate

rinitrobenzene sulfonic acid (TNBS)-induced colonic inflamma-ion (Karakoyun et al., in press). According to our previous study,he aqueous extract from Dichroa febrifuga root (AEDF) suppresseshe endotoxin-induced inflammatory responses through inhibiting

∗ Corresponding author. Tel.: +82 51 510 2526; fax: +82 51 513 9258.E-mail address: yheekim@pusan.ac.kr (Y. Kim).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.07.003

© 2009 Elsevier Ireland Ltd. All rights reserved.

the production of NO in vivo and in vitro (Kim et al., 2000). But,the underlying mechanism of AEDF function has remained to becharacterized so far.

Macrophages are immune cells usually dispersed throughoutthe body. They are particularly important in innate immunity asthey are among the first cells responding to microbial infection.They detect pathogenic substances through pattern-recognitionreceptors and subsequently initiate and regulate inflammatoryresponse (Medzhitov and Janeway, 1997) using a wide range ofsoluble proinflammatory mediators. LPS is one of the most pow-erful activators of macrophages, and macrophages and monocytesinduced by LPS are known to be activated through the productionof inflammatory mediators, such as NO and other free radicals, inaddition to numerous cytokines including TNF-�, IL-1� and IL-6.These cytokines lead to a variety of responses including the activa-tion of Th1 response, increased expression of adhesion moleculeson vascular endothelial cells (Luscinskas and Gimbrone, 1996),the induction of acute phase response proteins by the liver (Diehland Rincon, 2002), and are generally involved in the development

of inflammatory diseases (Mahida, 2000). Thus, the inhibition ofexcessive production of these cytokines can be employed as crite-rion to evaluate anti-inflammatory effects of drugs.

NF-�B is one of the most important transcription factors andregulates various cellular genes involved in immune and acute

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hase inflammatory responses, and in cell survival (Li and Verma,002). NF-�B is a heterodimeric transcription factor composed of50 and p65 (RelA), but a variety of other Rel-containing dimersre also known to exist. In unstimulated cells, NF-�B is presentn the cytosol bound to the inhibitory protein I kappa B (I�B). Inesponse to the stimulation such as LPS, I�Bs are rapidly ubiquiti-ated and degraded by 26S proteasome complex. The free NF-�Bimers translocate to the nucleus, bind with high affinity to spe-ific sites in the promoter regions of target genes and stimulate theirranscription. The critical event that triggers the polyubiquitinationnd degradation of I�Bs is their stimulus-dependent phosphoryla-ion at two serine residues (Ser32 and 36) that are located withinheir conserved N-terminal regulatory region (Li and Verma, 2002).he protein kinase that phosphorylates these regulatory sites hasecently been identified as I�B kinase (IKK) complex. IKK consistsf two catalytic subunits, IKK� and IKK�, and regulatory subunits,KK� (DiDonato et al., 1997; Rothwarf et al., 1998). IKK was definedhrough its ability to catalyze the phosphorylation of the N-terminalegulatory serines on I�B-� and I�B-�, as well as its rapid activationn response to cell stimulation by TNF-�, IL-1 and LPS.

In the present study, we investigated the effects of AEDF onPS-induced inflammatory cytokine release and NF-�B activation,nd further explored the possible mechanisms of this inhibitiony AEDF in the macrophages. We examined the effect of AEDF onkt and MAPKs activities which have been known to regulate NF-B. Our results provide a molecular basis for understanding the

nhibitory effects of Dichroa febrifuga roots on endotoxin-mediatednflammation.

. Materials and methods

.1. Preparation of extract

The roots of Dichroa febrifuga Lour. (Saxifragaceae) were pur-hased from a local herb store, Kwang Myoung Dang (Pusan, Korea)n April 2005. The roots were identified and authenticated based onts microscopic and macroscopic characteristics by Professor Woohin Ko who specializes in traditional oriental herb medicine inollege of Oriental Medicine, Dongeui University (Pusan, Korea).voucher specimen (number DF-05-04) has been deposited at theepartment of Molecular Biology, Pusan National University, Pusan,orea. The dry roots (300 g) were extracted with distilled water at00 ◦C for 4 h. The extract was filtered through 0.45 �m filter andreeze-dried (yield, 14 g) and kept at 4 ◦C. The dried extract wasissolved in phosphate buffered saline (PBS) and filtered through.22 �m filter before use.

.2. Macrophage culture

C57BL/6 mice purchased from Dae Han Laboratory Animal Cen-er (Korea) were used 8 to 12 weeks of age (25–30 g). ThioglycollateTG) broth (Brewer, DIFCO, Detroit, MI)-elicited macrophages werearvested 3 days after intraperitoneal injection of 2.5 ml TG intoice and isolated as reported previously (Kim et al., 2000). Murineacrophage RAW 264.7 cells were maintained in Dulbecco’s modi-

ed Eagle’s medium supplemented with glutamine (1 mM) and 10%BS.

.3. ELISA

TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml)ere incubated with various concentrations of AEDF for 1 h, and

hen LPS (1 �g/ml) was treated for 24 h. Following 24 h incubation,L-1� and IL-6 levels were quantified in culture media by ELISA KitKomabiotech, Korea) according to the manufacturer’s instructions.

acology 125 (2009) 246–251 247

2.4. Real-time reverse transcription (RT)-polymerase chainreaction (PCR)

The total cellular RNA was isolated using RNA spin mini RNAisolation kits (GE Healthcare) according to the manufacturer’sinstructions. 1 �g of total RNA was reverse-transcribed usingMaxime RT PreMix (Intron Biotechnology) and anchored oligo-dT15-primers. Real-time PCR was performed in a Chromo4TM

instrument (BIO-RAD) with the SYBR Green Master Mix (AppliedBiosystems, CA). The relative amount of target mRNA wasdetermined using the comparative threshold (Ct) method bynormalizing target mRNA Ct values to those for GAPDH (�Ct)(Livak and Schmittgen, 2001). The primer sequences are as follows:IL-1�-sense (5′-GTTGACGGACCCCAAAAGAT-3′), IL-1�-antisense(5′-CCTCATCCTGGAAGGTCCAC-3′), IL-6-sense (5′-CACGGCCTTCCC-TACTTCAC-3′), IL-6-antisense (5′-TGCAAGTGCATCATCGTTGT-3′),GAPDH-sense (5′-AGGTGGTCTCCTCTGACTTC-3′), GAPDH-anti-sense (5′-TACCAGGAAATGAGCTTGAC-3′).

2.5. Transient transfection and luciferase assay

RAW 264.7 cells were transfected with �B-luc reporter plasmidconsisted of three �B concatamers from the Ig� chain and fireflyluciferase gene (Kim et al., 2004) using Superfect reagent accordingto the manufacturer’s instruction. pSV110-�-galactosidase controlplasmid was cotransfected as an internal control for transfec-tion efficiency. Twenty-four hours after transfection, the cellswere incubated with indicated reagents for 1 h and then treatedwith LPS (1 �g/ml) for 24 h. Luciferase activity was assayed withthe luciferase assay kit (Promega) according to the manufac-turer’s instruction. Luminescence was measured with luminometer(Turner Design, Promega). Luciferase activity was normalized to�-galactosidase activity.

2.6. Preparation of nuclear extract and electrophoretic mobilityshift assay

Nuclear extracts were prepared as described previously (Kimet al., 2004). Electrophoretic mobility shift assay (EMSA) ofnuclear extracts was performed according to the manufacture’sinstructions (Promega, Madison, WI) with some modifications.In brief, the probe consisted of a double-stranded oligonu-cleotide containing the consensus binding sequence for NF-�B(5′-AGTTGAGGGGACTTTCCCAGGC-3′, Promega, Madison, WI) wasend-labeled with [�-32P]-ATP (3000 Ci/mmol at 10 �Ci/ml) usingT4 polynucleotide kinase. Nuclear extracts were incubated withgel shift binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl,0.2 mM EDTA, 2.5 mM DTT, 10% glycerol, 0.05% NP-40, 0.25 mg/mlpoly-dI/poly-dC, protease inhibitor cocktail) for 10 min at roomtemperature and then the mixture was incubated with 32P-labeledprobe for 20 min at room temperature. The incubation mixture wasloaded onto nondenaturating gel and run in 0.25× Tris/borate/EDTAbuffer. Gels were dried and exposed to X-ray film at −70 ◦C.

2.7. Western blot analysis

The cells were washed with phosphate buffered saline (PBS)three times and scraped off and lysed with lysis buffer (1% Tri-ton X-100, 1% deoxycholate). Protein concentration of lysateswas determined and equal amounts of protein were sepa-rated electrophoretically using 10% SDS-PAGE (sodium dodecyl

sulfate-polyacrylamide gel electrophoresis), and then the gel wastransferred to 0.45 �m nitrocellulose paper. The blot was incubatedwith anti-NF-�B p65, I�B-�, IKK�/�, �-tubulin antibody (SantaCruz Biotechnology), anti-Akt, phospho-Akt, phospho-I�B-�, JNK,phospho-JNK, p38, phospho-p38, ERK1/2, phospho-ERK1/2 anti-

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Fig. 1. Suppression of IL-1� and IL-6 release by AEDF. TG-elicited mouse peritoneal6

NF-�B/DNA complex was observed in unstimulated macrophages,while a large quantity of NF-�B/DNA complex was induced byLPS treatment. This increased NF-�B binding affinity was dramati-cally inhibited by AEDF in a dose-dependent manner (Fig. 3B). The

Table 1Effects of AEDF on LPS-induced cytokine mRNA expressiona.

Treatment Cytokine (mRNA content)b

IL-1� IL-6

LPS 8470.3 ± 772.5 3028.0 ± 218.2LPS+AEDF

0.1 mg/ml 5441.5 ± 693.8* 2290.7 ± 231.60.25 mg/ml 2023.3 ± 584.4** 493.4 ± 72.9**

0.5 mg/ml 1219.4 ± 352.4** 305.3 ± 113.2**

1.0 mg/ml 1195.9 ± 146.4** 218.6 ± 154.0**

a TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml) were treated withvarious concentrations of AEDF in the presence of LPS for 6 h. Subsequently, the cellswere harvested and mRNA quantity was assayed by real-time PCR as described inSection 2.

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48 S.Y. Park et al. / Journal of Ethn

ody (Cell Signaling Technology), and secondary antibody and thenetected by the enhanced chemiluminescence detection systemccording to the recommended procedure (Amersham Co.).

.8. IKK assay

IKK was assayed as described previously (Kim et al., 2004).hole cell extracts were lysed with lysis buffer (10% glycerol, 1%

riton X-100, 1 mM EGTA, 5 mM EDTA, 1 mM sodium pyrophos-hate, 20 mM Tris–HCl, pH 7.9, 10 mM �-glycerophosphate, 137 mMaCl, 1 mM PMSF, 10 mM NaF, 1 mM sodium orthovanadate, pro-

ease inhibitor cocktail) for 15 min at 4 ◦C. Equal amounts of totalellular protein were immunoprecipitated with anti-IKK� mono-lonal antibody (Santa Cruz, CA) in TNT buffer (20 mM NaCl, 20 mMris–HCl, pH7.5, 1% Triton X-100, 300 �M sodium orthovanadate,mM PMSF, protease inhibitor cocktail). The IKK�–antibody com-lex was precipitated with protein A/G sepharose beads for 2 h at◦C. The kinase assay was carried out in kinase buffer containing�Ci [�-32P] ATP and GST-I�B-� (1–317) fusion protein (Santa Cruz,A) as substrate and incubated for 30 min at 37 ◦C. Each sample wasixed with Laemmli’s sample buffer, heated for 10 min at 100 ◦C

nd subjected to 10% SDS-PAGE. The gels were dried and visualizedy autoradiography.

.9. Statistical analysis

All results were expressed as means ± S.E. Each experiment wasepeated at least three times. Statistical significances were com-ared between each treated group and analyzed by the pairedtudent’s t-test. Data with p < 0.05 were considered statistically sig-ificant.

. Results

.1. Inhibitory effect of AEDF on proinflammatory cytokineroduction in macrophages

To investigate the anti-inflammatory effect of AEDF, we exam-ned the effect of AEDF on proinflammatory cytokines production.G-elicited mouse peritoneal macrophages were challenged withPS in the presence or absence of AEDF and the levels of IL-1� andL-6 in the medium were measured. Whereas LPS-treated cells pro-uced a large amount of IL-1� and IL-6, AEDF suppressed the releasef IL-1� and IL-6 into culture supernatant in a dose-dependentanner (Fig. 1). To determine whether the decreased cytokine

ecretions are correlated with cytokine expression, we analyzed theRNA quantities of IL-1� and IL-6 by real-time RT-PCR. As shown in

able 1, AEDF consistently down-regulated the LPS-induced expres-ions of IL-1� and IL-6 mRNA. On the other hand, cell viability waseasured by MTT assay to examine whether the effect of AEDF is

ue to its cytotoxicity. As shown in Fig. 2, AEDF did not decreaseell viability but rather protected the toxicity of LPS. These resultsuggest that AEDF inhibits proinflammatory cytokine release byeducing the cytokine expression level without affecting cell via-ility.

.2. Effect of AEDF on NF-�B activity in macrophages

Since NF-�B plays a critical role in LPS-induced cytokinexpression (Li and Verma, 2002), we investigated whether AEDF

uppresses cytokine expression through the regulation of NF-B activity. RAW 264.7 cells were transiently transfected with

uciferase reporter genes driven by NF-�B binding consensus con-atamers (�B-luc) and the effect of AEDF was examined. As shownn Fig. 3A, LPS treatment markedly induced the transactivation of

macrophages (1 × 10 cells/ml) were incubated with various concentrations of AEDFfor 1 h, and then LPS (1 �g/ml) was treated for 24 h. IL-1� (A) and IL-6 (B) in the cul-tured supernatant were measured by ELISA kit, respectively. Values are means ± S.E.of three independent experiments. *p < 0.05 and **p < 0.01 vs LPS-treated group.

NF-�B, while AEDF inhibited LPS-induced transactivation of NF-�Bin a dose-dependent manner.

To clarify if AEDF affects DNA binding activity of NF-�B, EMSAwas performed using a consensus NF-�B oligonucleotide. TG-elicited mouse peritoneal macrophages were incubated with AEDFfor 1 h and stimulated with 1 �g/ml LPS for 30 min. A low level of

Relative content of each mRNA was indicated as folds to control. Values aremeans ± S.E. of three independent experiments.

* The statistical significance between samples with and without AEDF is indicated(p < 0.05 vs LPS-treated group).

** The statistical significance between samples with and without AEDF is indicated(p < 0.01 vs LPS-treated group).

S.Y. Park et al. / Journal of Ethnopharmacology 125 (2009) 246–251 249

Fig. 2. Effect of AEDF on cell viability. TG-elicited mouse peritoneal macrophages(1 × 106 cells/ml) were incubated with various concentrations of AEDF in the absence(closed circle) or presence of 1 �g/ml of LPS (open circle) for 24 h. Then cell viabilitywas measured by MTT assay according to Section 2. Data represent the relative via-bility to control group (no-AEDF and no-LPS) and are expressed as the means ± S.E.of three independent experiments.

Fig. 3. Inhibitory effect of AEDF on NF-�B activation. (A) RAW 264.7 cells(4 × 105 cells/ml) were transfected with �B-luc reporter and luciferase activity wasassayed as described in Section 2. All transfection experiments were performed induplicate in three independent experiments. Data are expressed as the means ± S.E.*p < 0.05 and **p < 0.01 vs LPS-treated group. (B) Effect of AEDF on DNA bindingactivity of NF-�B. TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml) wereincubated with various concentrations of AEDF for 1 h, and then stimulated with LPS(1 �g/ml) for 30 min. Nuclear proteins were extracted and assayed for NF-�B DNAbinding affinity by EMSA as described in Section 2. Lane c represents competitionassay with 25-fold excess of unlabeled oligonucleotide probe. n.s., non-specific band.

Fig. 4. Inhibitory effect of AEDF on LPS-stimulated degradation of I�B-�. (A)TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml) were incubated with0.5 mg/ml AEDF for 1 h, and then stimulated with 1 �g/ml LPS for 15, 30 or 60 min. (B)

Macrophages (1 × 106 cells/ml) were incubated with various concentrations of AEDFfor 1 h, and then stimulated with 1 �g/ml LPS for 15 min. Cells were harvested andequal cytosolic extracts were analyzed by Western blotting. Western blot detectionof �-tubulin was estimated protein-loading control for each lane.

specificity of NF-�B/DNA complex was confirmed by competitionexperiment. A 25-fold excess of unlabeled oligonucleotide probeeffectively blocked binding of labeled probe to the protein complex.

3.3. Suppression of I�B-˛ degradation by AEDF

To analyze whether AEDF affect NF-�B signaling pathway, weinvestigated the effects of AEDF on upstream signaling pathwayof NF-�B, that is, I�B-� degradation. TG-elicited mouse peritonealmacrophages were pretreated with 0.5 mg/ml of AEDF for 1 h andstimulated with LPS (1 �g/ml) for 15, 30 or 60 min, and then cytoso-lic extracts were prepared. As shown in Fig. 4A, LPS led to adegradation of I�B-� at an early time-point (about 15 min) but I�B-� began to be resynthesized at 30 min. However, when pretreatedwith AEDF, the degradation of I�B-� was markedly inhibited. Wealso examined that AEDF suppressed I�B-� degradation in a dose-dependent manner when cells were stimulated with LPS for 15 min(Fig. 4B).

3.4. Inhibition of LPS-induced IKK˛ activity by AEDF

It has been known that the degradation of I�B-� is provoked bytheir stimulus-dependent phosphorylation at two serine residues(Ser32 and 36) located within their conserved N-terminal regula-tory region (Li and Verma, 2002). So we investigated whether theinhibitory action of AEDF was due to its effect on I�B phosphory-lation. TG-elicited mouse peritoneal macrophages were pretreatedwith AEDF for 1 h and stimulated with LPS (1 �g/ml) for 15 min.Then the cytoplasmic levels of phosphorylated I�B-� were exam-ined by Western blot analysis. Incubation of macrophages with LPScaused marked phosphorylation of cytosolic I�B-�, but AEDF sig-nificantly inhibited the phosphorylation of I�B-� (Fig. 5A).

In many mammalian cells, I�B phosphorylation is due to arapid activation of multi-subunit protein kinase, IKK. We stud-ied whether AEDF exhibits inhibitory effects on LPS-induced IKKactivity in peritoneal macrophages. TG-elicited mouse peritonealmacrophages were exposed to AEDF for 1 h and then stimulated

with LPS (1 �g/ml) for 10 min. To directly measure IKK activityin cells, IKK� was immunoprecipitated and kinase activity wasassayed using recombinant GST-I�B-� (1–317) as a substrate. Asshown in Fig. 5B, after stimulation with LPS, GST-I�B-� fusion pro-tein was phosphorylated strongly, indicating stimulation of the IKK

250 S.Y. Park et al. / Journal of Ethnopharmacology 125 (2009) 246–251

Fig. 5. Suppressive effect of AEDF on LPS-stimulated phosphorylation of I�B-� andIKK activity. (A) TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml) wereincubated with various concentrations of AEDF for 1 h, and then stimulated with LPS(1 �g/ml) for 15 min. Cells were harvested and equal cytosolic extracts were analyzedby Western blotting with anti-phospho-I�B-� antibody. Western blot detection of�-tubulin was estimated protein-loading control for each lane. (B) Macrophages(1 × 106 cells/ml) were incubated as described above, and then stimulated with LPS(arw

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Fig. 6. Effect of AEDF on Akt, ERK1/2, p38 and JNK activity in LPS-stimulatedmacrophages. TG-elicited mouse peritoneal macrophages (1 × 106 cells/ml) weretreated with indicated concentrations of AEDF for 1 h and stimulated with 1 �g/mlLPS for 15 min. Equal amount of cell extracts was analyzed by Western blotting

1 �g/ml) for 10 min. Whole cell extracts were immunoprecipitated with anti-IKK�ntibody. The precipitates were incubated with GST-I�B-� (1-317) and [�-32P] ATP,esolved by SDS-PAGE and analyzed by autoradiography. Western blotting for IKKas performed as a loading control (lower panel).

ctivity. AEDF inhibited IKK activity in a dose-dependent manner.estern blot analysis showed that the level of IKK protein was not

hanged by incubation with AEDF.

.5. Inhibitory effect of AEDF on LPS-induced Akt and MAPKctivities

Because the activation of extracellular signal-regulated kinases/2 (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK), PI-3K/Akt path-ay leads to the phosphorylation and activation of the NF-�B (Lee et

l., 2000; Madrid et al., 2001; Hattori et al., 2003; Li et al., 2003; Xiaot al., 2007; Dan et al., 2008; Minhajuddin et al., 2009), we inves-igated the effects of AEDF on the activation of ERK1/2, p38, JNKnd Akt in LPS-stimulated macrophages. Activation of MAPKs andkt requires phosphorylation of serine and threonine residues. TG-licited mouse peritoneal macrophages were treated with indicatedoncentrations of AEDF for 1 h and then stimulated with 1 �g/mlPS for 15 min. Using immunoblot analysis with anti-phospho-pecific antibody, we found that AEDF suppressed LPS-inducedctivation of ERK1/2, JNK and Akt in a dose-dependent mannerFig. 6), while activity of p38 was slightly inhibited by AEDF. Themount of non-phosphorylated kinases was unaffected by eitherPS or AEDF treatment.

. Discussion

In the present study, we found that AEDF impaired LPS-inducedene expression and secretion of proinflammatory cytokines (IL-1�nd IL-6) in mouse peritoneal macrophages. Several inflammatoryytokines, particularly TNF-�, IL-1� and IL-6, are known to playey roles in the induction and perpetuation of inflammation inacrophages. These cytokines play pivotal roles in the induction

f the innate immune response as well as in the determination ofhe magnitude and nature (Th1 vs Th2) of the acquired immuneesponse (Netea et al., 2003). We have previously demonstrated

hat AEDF exhibits anti-inflammatory function by inhibiting theroduction of NO and TNF-� in macrophages (Kim et al., 2000). Ouresults in the present investigation are consistent with our previousata, and these results suggest that AEDF acts as anti-inflammatorygent influencing these proinflammatory cytokines and NO.

with anti-phospho-Akt, anti-phospho-ERK1/2, anti-phospho-p38 or anti-phospho-JNK antibody. Western blot detection of non-phosphorylated kinases was estimatedprotein-loading control for each lane.

The NF-�B pathway is also a therapeutic target in inflam-matory diseases because NF-�B plays an important role inthe transcriptional activation of TNF-�, IL-1�, IL-2, IL-6, IL-8,matrix metalloproteinases, cell adhesion molecules, hematopoi-etic growth factors, acute phase proteins, COX-2, and iNOS (Liand Verma, 2002). Inappropriate regulation of NF-�B is directlyinvolved in a wide range of human disorders, including a varietyof cancers, neurodegenerative diseases, arthritis, asthma, inflam-matory bowel disease, sepsis and numerous other inflammatoryconditions (Blackwell et al., 1996; Pierce et al., 1997; Lewis andManning, 1999; Segain et al., 2000). Therefore, agents that inhibitNF-�B activation would have anti-inflammatory effects (Lewis andManning, 1999). Our study showed that AEDF inhibits the LPS-induced DNA binding activity of NF-�B and transactivation ofreporter gene in macrophages. Negative regulation of NF-�B activ-ity is very complex in that several mechanisms are involved in thetermination of NF-�B activation in response to specific signals. Thecritical inhibitory step is thought to be the suppression of I�B-�degradation. AEDF suppressed not only the LPS-stimulated degra-dation but also phosphorylation of I�B-�. This means that AEDFmight regulate the kinase phosphorylating I�B-�. The phosphory-lation of N-terminal regulatory serines on I�B-� and I�B-� is knownto be catalyzed by IKK. Recently, IKK has been implicated to be a newtarget of anti-inflammatory therapy (Birrell et al., 2005; Cheon et al.,2006; Tas et al., 2006; Lawrence and Bebien, 2007). In the presentstudy, the activity of IKK on the I�B-� was markedly reduced byAEDF in a dose-dependent manner. These results implicate thatAEDF inhibits NF-�B through the suppression of IKK activity, andAEDF could be useful as a therapeutic agent for inflammatory dis-ease.

The activation of NF-�B is regulated by cellular kinases, includ-ing MAPKs and Akt. MAPKs are a family of serine/threonine proteinkinases that are an important part of intracellular signaling path-ways, connecting extracellular signals to intracellular regulatoryproteins. MAPK family members ERK1/2 and p38 MAPK are knownto participate in the regulation of LPS-induced proinflammatorycytokine production (Lee et al., 2000; Guha and Mackman, 2001;

Xiao et al., 2007). The third signal transduction pathway of theMAPK family is the JNK pathway, which is also activated primarilyby cellular stress and cytokines, and its downstream targets includetranscription factors important in cytokine expression (Kyriakis and

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vruch, 1996; Ciallella et al., 2005). In addition, Akt/protein kinase(PKB) is a serine–threonine kinase that is best known for its ability

o inhibit cell death pathways (Hemmings, 1997; Kandel and Hay,999; Weichhart and Saemann, 2008). Activation of Akt by growthactors and cytokines generally occurs via the phosphoinositide--kinase (PI-3K) pathway. Upon stimulation, PI-3K phosphorylatespecific phosphoinositide lipids, which accumulate in the plasmaembrane, creating docking sites for Akt. At the plasma mem-

rane Akt undergoes phosphorylation at two residues, leading tots activation. Although NF-�B and Akt were initially thought toe components of distinct signaling pathways, several studies haveemonstrated convergence of the NF-�B and Akt signaling path-ays (Madrid et al., 2001; Minhajuddin et al., 2009). Indeed, IKK issubstrate of Akt, thus, activation of Akt stimulates NF-�B activ-

ty. Thus, the activations of ERK1/2, p38, JNK and Akt are useds a hallmark of LPS-induced signal transduction in macrophages.herefore, to further confirm the inhibitory mechanism of NF-�Bctivation by AEDF, we investigated the effects of AEDF on Akt andAPKs phosphorylation in macrophages stimulated with LPS for

5 min, and it was found that Akt, ERK1/2 and JNK phosphoryla-ions were suppressed by AEDF in a dose-dependent manner. Theseesults suggest that AEDF inhibits LPS-induced NF-�B activation byown-regulating the phosphorylations of Akt, ERK1/2, and JNK.

In conclusion, we demonstrated that AEDF inhibited proinflam-atory cytokine (IL-1� and IL-6) production in LPS-stimulatedouse peritoneal macrophages, and that these effects were medi-

ted by the inhibition of the activity of IKK/I�B/NF-�B and thehosphorylation of Akt, ERK1/2, and JNK. Our results provide aolecular basis for understanding the inhibitory effects of Dichroa

ebrifuga roots on endotoxin-mediated inflammation. As the effectf AEDF is mediated via the inhibition of NF-�B, Akt and MAPKss well as proinflammatory cytokines, Dichroa febrifuga could beeveloped into potent anti-inflammatory drugs and be used inathological processes in which the induction of NF-�B is involved.

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