European Journal of Pharmacology 602 (2009) 422–431

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European Journal of Pharmacology

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Immunopharmacology and Inflammation

Desmethylanhydroicaritin inhibits NF-κB-regulated inflammatory gene expression bymodulating the redox-sensitive PI3K/PTEN/Akt pathway

Ji-Hee Kim a,b,1, Gwangsoo Lee a,b,1, Young-Lai Cho a,b, Chun-Ki Kim a,b, Sanghwa Han c,Hansoo Lee c, Jae Sue Choi d, Jongseon Choe a, Moo-Ho Won e, Young-Guen Kwon f,Kwon-Soo Ha a,b, Young-Myeong Kim a,b,⁎a Vascular System Research Center, Kangwon National University, Chuncheon, 200-701, Republic of Koreab Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, 200-701, Republic of Koreac Division of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon, 200-701, Republic of Koread Division of Food Science and Biotechnology, Pukyong National University, Busan, 608-737, Republic of Koreae Department of Anatomy, School of Medicine, Hallym University, Chuncheon, 200-702, Republic of Koreaf Department of Biochemistry, College of Sciences, Yonsei University, Seoul, 120-749, Republic of Korea

⁎ Corresponding author. Department of Molecular andMedicine, Kangwon National University, Chuncheon, 200-250 8831; fax: +33 244 3286.

E-mail address: ymkim@kangwon.ac.kr (Y.-M. Kim).1 First two authors contributed equally to this work.

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.10.062

a b s t r a c t

a r t i c l e i n f o

Article history:

We investigated the effect Received 3 June 2008Received in revised form 16 October 2008Accepted 31 October 2008Available online 9 November 2008

Keywords:NF-κBInflammationCytokineReactive oxygen speciesAntioxidant

of desmethylanhydroicaritin (DMAI), a major compound of the Chinese herbalmedicine Epimedium, on inflammatory gene expression and the NF-κB signaling pathway. We found thatDMAI suppressed the expression of NF-κB-responsive genes, such as inducible nitric oxide synthase,cyclooxygenase-2, interleukin-1β, and tumor necrosis factor-α, in lipopolysaccharide (LPS)-stimulatedmacrophages and endotoxemic mice as well as protected mice against LPS-induced lethality. DMAI inhibitedNF-κB activation through the inhibition of IκB kinase (IKK) activation, IκB phosphorylation and degradation,and NF-κB nuclear translocation in LPS-stimulated macrophages. This compound inhibited in vitro and invivo LPS-induced phosphatidylinositol 3-kinase (PI3K) activation, phosphatase and tensin homolog deletedon chromosome 10 (PTEN) oxidation, and Akt phosphorylation, which are upstream modulators of IKKactivation. Moreover, treatment with DMAI was not observed to affect the interaction between the Toll-likereceptor 4, MyD88, and TRAF6 as well as mitogen-activated protein kinase activation. DMAI also suppressedintracellular H2O2 accumulation, hydroxyl radical production, and glutathione oxidation without affectingsuperoxide generation and accumulation by NADPH oxidase. Moreover, DMAI inhibited redox-sensitiveactivation of the PI3K/PTEN/Akt pathway and NF-κB activation in macrophages treated with H2O2. Theseresults indicate that DMAI negatively regulates canonical NF-κB-regulated inflammatory gene expression byfunctioning as an inhibitor of the NF-κB pathway through the suppression of redox-based PI3K activation andPTEN inactivation and therefore can be considered as a potential drug for inflammatory diseases.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Macrophages play a key role in both specific and non-specificinflammatory responses and are involved in a variety of inflammatorydiseases (Pierce, 1990). Immune-activated macrophages generatereactive oxygen species, such as superoxide and H2O2, and expressvarious genes such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) as well as other inflammatory mediators including nitricoxide (NO) and prostaglandin E2 (PGE2), which are synthesized byinducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2),

Cellular Biochemistry, School of701, Republic of Korea. Tel.: +33

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respectively. These mediators are involved in the pathogenesis of manyinflammation-associated human diseases (Lee et al., 2003). Therefore,pharmacological inhibitors that regulate reactive oxygen speciesproduction and inflammatory gene expression can be used astherapeutic drugs for inflammatory diseases.

Nuclear factor κB (NF-κB), a transcription factor, plays a central rolein the expression of many inflammatory genes. NF-κB lies dormant inthe cytoplasm through an association with the inhibitory proteinsIκBα/β in resting cells (Li and Verma, 2003; Yamamoto and Gaynor,2001). Stimulation of macrophages with lipopolysaccharide (LPS)triggers IκB kinase (IKK) activation and subsequent phosphorylationof IκB, resulting in the ubiquitin-mediated proteasomal degradation ofIκB (Deng et al., 2000). The liberated NF-κB translocates to the nucleusand binds to κB motifs in the promoters of several inflammatorygenes, including TNF-α, IL-1β, iNOS, and COX-2, leading to theinduction of their mRNA expression. Therefore, regulation of NF-κBactivation is an important therapeutic target for the treatment of

423J.-H. Kim et al. / European Journal of Pharmacology 602 (2009) 422–431

inflammatory diseases (Li and Verma, 2003; Yamamoto and Gaynor,2001).

Reactive oxygen species generated by NADPH oxidase in immune-activated macrophages and neutrophils were initially recognized asdefense molecules against invading organisms (Nathan, 2003) and havebeen increasingly documented as secondary messengers or modulatorsin signal transduction (Dröge, 2002). Several lines of evidence suggest arole of reactive oxygen species as a common and critical modulator forvarious NF-κB-activating signals including LPS, indicating that animportant cellular target of reactive oxygen species is NF-κB. Reactiveoxygen species can promote NF-κB activation via the interaction withredox-sensitive modulators of the NF-κB signal pathway, leading to anincrease in theexpressionof inflammatorygenes. Antioxidantshavebeenshown to suppress NF-κB-dependent inflammatory gene expression andthe pathological process of chronic inflammatory diseases such asrheumatoid arthritis and atherosclerosis (Lee et al., 2003; Rahman et al.,2006). Although reactive oxygen species have been known to beimportant for modulating NF-κB-dependent inflammatory processes,its regulatory mechanism of NF-κB activation is largely unknown.

Desmethylanhydroicaritin (DMAI, Fig. 1A) is a major flavonoidisolated from Epimedium, which has been long used as a traditionalChinese herbal medicine for therapeutic applications in severalinflammatory diseases (Wu et al., 2003). This compound is knownas a potent in vitro free radical and peroxynitrite scavenger (Jung et al.,2005). Although some derivatives of DMAI have demonstrated potentinhibitory activity for the expression of inflammatory genes in LPS-stimulated macrophages (Kim et al., 2007; Kowalski et al., 2005), theanti-inflammatory mechanism of DMAI has not been elucidated.

In this study, we examined the anti-inflammatory effects of DMAIand its underlying mechanism in LPS-treated macrophages as well asthe functional role of reactive oxygen species in the NF-κB activationpathway. DMAI was shown to possess scavenging activity for H2O2

Fig. 1. Effects of DMAI on NF-κB-regulated gene expression in LPS-treated RAW264.7 mac(100 ng/ml) in the presence or absence of various concentrations of DMAI. (B) After 8 h stimuanalysis. Actin was used as an internal control. (C) After 12 h, cells were harvested, washedproteins (30 μg) were separated on SDS-PAGE, and the protein levels of iNOS, COX-2, IL-1β, anitrite (D), PGE2 (E), IL-1β (F), and TNF-α (G) were determined in the culture media using GPb0.01 versus LPS alone.

and hydroxyl radicals and suppress LPS-induced inflammation in vitroand in vivo by inhibiting Akt-dependent NF-κB activation by blockingboth H2O2-induced oxidative inactivation of phosphatase and tensinhomolog deleted on chromosome 10 (PTEN) and direct activation ofPI3K. Thus, these results suggest that DMAI exerts suppressive effectson the production of inflammatory mediators by inhibiting the redox-dependent NF-κB activation pathway.

2. Materials and methods

2.1. Chemicals and reagents

Dulbecco's modified Eagle's medium (DMEM), penicillin, andstreptomycin were purchased from Life Technology Inc. (Rockville,MD, USA). ELISA kits for TNF-α, IL-1β, and PGE2 were procured fromR&D Systems. Antibodies for NF-κB p65, COX-2, IL-1β, TNF-α, IKKα/β,PTEN, IκBα, iNOS, Toll-like receptor 4 (TLR4), MyD88, TRAF6, p-JNK,and actin were obtained from Santa Cruz Biotechnology (Santa Cruz,CA). Antibodies for p-Akt, p-IKKα (thr23), p-p38, p-IκBα, and p-ERKwere procured from Cell Signaling Technology. DMAI was purified aspreviously described (Jung et al., 2005). 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was purchased from Alexis Biochemicals (San Diego,CA), vacuum-distilled twice, and stored under nitrogen at −80 °C untilneeded. Other chemicals were obtained from Sigma (St. Louis, MO)unless indicated otherwise.

2.2. Cell culture and peritoneal macrophage isolation

The murine macrophage cell line RAW264.7 and mouse peritonealmacrophages (Lee et al., 2003) were cultured in DMEM (2 mM L-glutamine,100 units/ml penicillin,100 μg/ml streptomycin) plus 5% fetalbovine serum (HyClone Labs, Logan, UT). After cultured in 6-well plates

rophages. (A) Chemical structure of DMAI. RAW264.7 cells were stimulated with LPSlation, the levels of iNOS, COX-2, IL-1β, and TNF-αmRNAs were determined by RT-PCRtwice with ice-cold PBS, and lysed by three cycles of freezing and thawing. Cytosolic

nd TNF-α were measured byWestern blot analyses. After 16 h stimulation, the levels ofriess reagents and ELISA kits. Data shown are the mean±S.D. (n=4). ⁎, Pb0.05 and ⁎⁎,

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(3×106 cells/well) for 24 h, cells were stimulated with LPS (1 μg/ml) inthe presence or absence of DMAI.

2.3. Gene expression analysis

RAW264.7 cells were treated with LPS in the presence or absenceof 10 μMDMAI for 12 h. Total RNAs were extracted from the cells usinga Trizol reagent kit (Life Technology Inc., Rockville, MD). Microarrayexperiments were performed using Sentrix Mouse-8 ExpressionBeadChips, analyzing over 24,000 known genes and gene candidatesaccording to the manufacturer's instructions (Illumina, San Diego, CA,USA). A 500 ng aliquot of total RNA from each sample was amplified tocDNA using an RNA amplification kit. In vitro transcription reaction ofcDNA to cRNA was performed overnight including biotin-11-UTP forlabeling the cRNA product. Each sample cRNA (750 ng) was hybridizedto BeadChip arrays at 55 °C overnight. Hybridized biotinylated cRNAwas detected with 1 μg/ml Cyanine3-streptavidine. BeadChips werescanned with Illumina BeadArray Reader. Microarray data wereanalyzed using “Avadis” software (Strand Life Sciences, USA).

2.4. Measurements of NO, TNF-α, IL-1β, and PGE2

The accumulation of nitrite, a stable oxidized product of NO, wasdetermined in the cell culture media by Griess reagents as previouslydescribed (Lee et al., 2003). The plasma level of nitrite plus nitrate(NOx) was determined using a nitrate reductase-based colorimetricassay kit (R&D Systems). The concentrations of TNF-α, IL-1β, and PGE2were measured using ELISA kits (R&D Systems) according to themanufacturer's protocol.

2.5. Western blot analysis

Cells were harvested, washed twice with ice-cold PBS, suspended in10 mM Tris–HCl (pH 7.4), and lysed by three cycles of freezing andthawing (Lee et al., 2003). Cell extractswere obtainedbycentrifugation at12,000 ×g at 4 °C for 10 min. Cytosolic proteins (40 μg) wereelectrophoretically separated on SDS-PAGE and transferred onto nitro-cellulose membranes. The membranes were blocked with 5% nonfat-dried milk and hybridized with horseradish peroxidase-conjugatedantibodies against target proteins as previously described (Lee et al.,2003). Proteinbandswere visualizedby incubatingmembranes for 2minwith ECL solution followed by exposure to X-ray film.

2.6. Analyses of NF-κB activation and promoter activity

For promoter activity assay, RAW264.7 cells were co-transfectedwith 0.5 μg plasmids of mouse iNOS promoter (piNOS-Luc) and pNF-κB-Luc (Startagene, CA) and0.5 μgβ-galactosidase expression vector aspreviously described (Lee et al., 2006). After 12 h recovery, cells weretreated with LPS in the presence or absence of 10 μM DMAI for 12 h.Luciferase activities were assayed and normalized to β-galactosidaseactivities to correct differences in transfection efficiencies. Forelectrophoretic mobility shift assay (EMSA), nuclear extracts wereprepared as previously described (Lee et al., 2006). A double strandedNF-κB-specific oligonucleotide labeled with [γ-32P]ATP was incubatedwith the nuclear extracts (10 μg of protein) at room temperature for30 min. NF-κB-DNA binding was analyzed following native polyacry-lamide gel electrophoresis. Nuclear translocation of the p65 subunitwas assessed by immunocytochemical analysis using an anti-p65/RelAantibody (Na et al., 2006). IKKβ activity wasmeasured using an in vitrokinase system after immunoprecipitation of IKKα/β complex asdescribed previously (Lee et al., 2003).

2.7. Co-immunoprecipitation assay

Cells were suspended in 0.5 ml of NP-40 lysis buffer (25 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5 mM NaF, 100 μM Na3VO4,and a protease inhibitor cocktail and stored on ice for 20min. Immunecomplexes were precipitated by incubation for 4 h at 4 °C withpolyclonal MyD88 antibody, which had been pre-coupled to proteinG-Sepharose. The immune complexes were washed three times withPBS containing 1 mM PMSF, separated by SDS-PAGE, and thentransferred to a nitrocellulose membrane. Interaction between TLR4,MyD88, and TRAF6 was measured by Western blot analysis.

2.8. Detection of oxidized PTEN

PTEN oxidationwas determined as described in a previous method(Lee et al., 2002). In brief, cells (1×106 cells in 1 ml DMEM) treatedwith LPS or H2O2 were scraped into 0.2 ml of ice-cold 50%trichloroacetic acid and transferred to a microfuge tube. The cellsuspensions were sonicated and centrifuged at 2000×g for 5 min. Thesupernatants were removed, and the pellets were washed withacetone and then solubilized in 0.2 ml of 100 mM Tris–HCl (pH 6.8)buffer containing 2% SDS and 40 mM N-ethylmaleimide. Portions(5 μl) were subjected to SDS-PAGE under non-reducing conditions andtransferred to a nitrocellulose membrane. The protein band of PTENwas determined by Western blot analysis.

2.9. PI3K assay

Cells were lysed in Tris–HCl (20 mM, pH 8.0), 137 mM NaCl, 1 mMMgCl2, 1 mM CaCl2, 1% NP-40, 10% glycerol, 2 mM Na3VO4, 10 μg/mlaprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride. The lysates were clarified, and equal amounts of proteins(400 μg) were immunoprecipitated with an antibody against the p85subunit of PI3K (Upstate Biotechnology). The immune complexes werewashed twice with 1% NP-40, 1 mM Na3VO4, and PBS (pH 7.4); twicewith 100 mM Tris–HCl (pH 7.5), 500 mM LiCl, and 1 mM Na3VO4; andtwice with 150 mM NaCl and 50 mM Tris–HCl (pH 7.2). The kinasereaction was initiated by adding 5 mg/ml L-α-phosphatidylinositol in20 mM HEPES (pH 7.4), 5 mM MnCl2, 10 μM ATP, 10 μCi of γ-[32P]ATP,and 2.5 mM EGTA. After 20-min incubation, the reactions werequenched by adding 1 M HCl. The phospholipids were extracted usinga 1:1 mixture of chloroform and methanol, and separated by thin-layer chromatography on TLC Silica Gel 60 plates in chloroform/methanol/water/ammonium hydroxide (60:47:11.3:2, v/v).

2.10. Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total cellular RNAs were extracted from cells using the Trizolreagent kit (Life Technology Inc). Five μg of mRNA was converted tocDNA by treatment with 200 units of reverse transcriptase and 500 ngof oligo-dT primer in 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mMMgCl2, 10 mM DTT, and 1 mM dNTPs at 42 °C for 1 h. The reactionwasstopped by heating at 70 °C for 15min. One μl of the cDNAmixturewasused for enzymatic amplification. PCR was performed in 50 mM KCl,10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 units ofTaq DNA polymerase, and 0.1 μM each of primers for iNOS, COX-2, IL-1β, and TNF-α. The amplification conditions and primer sequenceswere the same as previously described (Lee et al., 2003).

2.11. Animal experiment

Female BALB/c mice (6–8 weeks old) were obtained from DaehanBiolink (Daejeon, Korea) and maintained at the specific pathogen-freehousing facility at the School of Medicine, Kangwon NationalUniversity (Chuncheon, Korea). All procedures performed on theseanimals were in accordance with the guidelines of the University

Table 1Typical inflammation-associated genes responsive to LPS that are suppressed by DMAI

Category Accessionno

Symbol Gene name Folda

LPS LPS+DMAI

Inflammatoryenzymes

NM010927 NOS2 Nitric oxide synthase 2 8.92 5.02NM011198 COX2 Cyclooxygenase 2 4.42 2.16

Cytokines NM013693 TNF Tumor necrosis factor 51.76 21.24NM008361 IL1B Interleukin-1β 387.48 39.94NM009971 CSF3 Colony stimulating factor 3 377.14 107.64NM031168 IL6 Interleukin 6 2.42 1.20NM008360 IL18 Interleukin 18 2.97 1.18

Chemokines NM011333 CCL2 Chemokine (CC) ligand 2 13.52 1.24NM013652 CCL4 Chemokine (CC) ligand 4 10.66 6.45NM013653 CCL5 Chemokine (CC) ligand 5 97.08 44.96NM011332 CCL17 Chemokine (CC) ligand 17 7.51 2.32NM009140 CXCL2 Chemokine (CXC) ligand 2 67.05 8.55

aFold change was calculated from the different intensity between treated group anduntreated control.

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Animal Care and Use Committee. Mice were i.p. injected twice withDMAI (10 mg/kg) or saline at 12 h time intervals. One h later, micewere i.p. injected with 4 mg/kg LPS or saline. After 12 h, whole bloodsamples and liver tissues were obtained. Serum was prepared bycentrifugation at 12,000 ×g for 20 min at 4 °C and kept at −20 °C untiluse. For the mortality study, mice were intraperitoneally (i.p.) injectedwith LPS (30 mg/kg) for 1 h following administration with 12 mg/kgDMAI. Mice were also i.p. administered with DMAI at 12 h intervals.Mortality was monitored every 6 h.

2.12. Measurements of reactive oxygen species and glutathione (GSH)

After treatment with 1 μg/ml LPS for 40 min or 10 μM H2O2 for10min in the presence or absence of 10 μMDMAI, RAW264.7 cellswerefurther incubated for 20minwith DCFH2-DA (10 μM) as a fluorescence

Fig. 2. DMAI inhibits inflammatory gene expression in LPS-treated mouse peritoneal macroph(100 ng/ml) in the presence or absence of various concentrations of DMAI. After 16 h, the leveusing Griess reagents and ELISA kits. Data shown are the mean±S.D. (n=4). ⁎, Pb0.05 and ⁎⁎,separated on SDS-PAGE. iNOS, COX-2, IL-1β, and TNF-α proteins were detected byWestern blor with 10 μM DMAI for 10 h. The levels of iNOS, COX-2, IL-1β, TNF-α, and β-actin mRNAs

probe for H2O2, and cells were washed 3 times with PBS. Theintracellular level of reactive oxygen specieswasmeasured by confocalmicroscopy (Na et al., 2006). To quantify intracellular H2O2 accumula-tion, the fluorescence images were processed for densitometricdetermination at the single-cell level. For superoxide productionassay, cells were incubated in 1mlHBSSwith phorbol 12-myristate 13-acetate (2 μM) and ferric cytochrome c (80 μM) in the presence orabsence of 100 units superoxide dismutase and 100 units catalase for90 min (Kim et al., 1994). Superoxide concentration was calculated byapplying ΔE550 nm=21 mM−1·cm−1. In vitro hydroxyl radical generationwas measured in the reaction mixture of 10 μM H2O2, 10 μM Fe2SO4,100 mM DMPO and 10 μM DMAI using an electron paramagneticresonance (EPR) spectrometer. The reaction was initiated by theaddition of H2O2, and DMPO-OH adduct was recorded. EPR spectro-meter conditions were as follows: microwave frequency=9.418 GHz,microwave power=2.10 mW, modulation frequency=100 kHz, andfield center=339.7 mT. GSH and total GSH were assayed spectro-photometrically using GSH assay kits from Calbiochem (cat # 354102)and Cayman Chemical (cat# 703002) as per manufacturers'instructions.

2.13. Data analysis

The data are presented as mean±S.D. from more than threeindependent experiments. Statistical comparisons between groupswere performed using 1-way ANOVA followed by the Student's t test.

3. Results

3.1. Regulatory effect of DMAI on LPS-dependent gene expression inRAW264.7 macrophages

We first examined whether DMAI regulated the gene expressionprofile in LPS-stimulated RAW264.7 macrophages using microarray

ages. Peritoneal macrophages isolated from female C57BL/6 mice were treated with LPSls of nitrite (A), PGE2 (B), IL-1β (C), and TNF-α (D) were determined in the culture mediaPb0.01 versus LPS alone. (E) Cytosolic lysates prepared from 12 h-stimulated cells wereot analyses. (F) Total mRNAs were isolated frommacrophages stimulated with LPS alonewere determined by RT-PCR analysis.

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analysis. Stimulation with LPS resulted in over a 2-fold induction of539 genes after 12 h (data not shown). This substantial programmingof gene expression resembles previously published LPS- and patho-gen-induced transcriptional gene programs in human macrophages(Björkbacka et al., 2004; Nau et al., 2002). Among the most highlyresponsive genes to LPS in our data set were inflammation-associatedgenes, including TNF-α, IL-1β, iNOS, COX-2, as well as somechemokines (Table 1), which are well-characterized markers of NF-κB-responsive inflammation (Sharif et al., 2007) and involved in thepathogenesis of many human diseases (Lee et al., 2003; Lee et al.,2006). LPS-mediated increases in these gene expressions weresignificantly suppressed by co-treatment with DMAI. No cytotoxiceffect of DMAI was observed under the same experimental conditionas measured by lactate dehydrogenase release and crystal violetstaining (data not shown). We further examined the effect of DMAI onthe expression of TNF-α, IL-1β, iNOS, and COX-2 as well as theproduction of NO and PGE2 in LPS-stimulated RAW264.7 cells usingRT-PCR analyses, Western blot, and biochemical analyses to verify themicroarray results (Table 1). RT-PCR analyses revealed that DMAIsignificantly inhibited the mRNA levels of these inflammatory genes(Fig.1B). In addition,Western blot analyses showed that LPS-mediatedincreases in these protein levels were suppressed in a dose-dependentmanner by DMAI treatment (Fig. 1C). Cells stimulated with LPS

Fig. 3. DMAI inhibits the NF-κB signal pathway, but not MAPK activation. RAW264.7 cells wpromoter (B) by a lipofectamine method and stimulated with 100 ng/ml LPS alone or with 1Data shown are the mean±S.D. (n=3). ⁎⁎, Pb0.01. (C) Nuclear NF-κB activity in cells treated wan excess amount of cold probe (CP) or antibody for the NF-κB p65 subunit (Ab). (D) Nuclear twith DMAI for 1 h by an immunocytochemical method. (E) Cells were treated with LPS alontreated with 0.5 μMMG-132, a proteosome inhibitor. IκBα degradation as well as IκBα (Thr 2(F) IKKβ activity was determined in an in vitro kinase assay system using immunoprecipitatwith LPS alone or with DMAI for 20min. P13 K activity, Akt phosphorylation, and PTEN oxidaand p85 subunit of PI3K in the samples were compared byWestern blot analysis. (H)With thWestern blot analysis. (I) Interaction between TLR4, MyD88, and TRAF6 was determined by

increased the accumulation of nitrite (a stable oxidized product of NO)and PGE2 in the culture medium while control cells did not, and thisincreasewas significantly reduced in a dose-dependentmanner by co-treatment with DMAI with an IC50 value of ~5 μM (Fig. 1D and G). Wealso examined the effect of DMAI on NF-κB-regulated cytokineproduction in LPS-stimulated RAW264.7 cells. DMAI treatmentinhibited LPS-induced secretions of IL-1β and TNF-α into the culturemedium in a dose-dependent manner (Fig. 1E and F).

3.2. DMAI inhibits inflammatory gene expression in mouse peritonealmacrophages

To examine the anti-inflammatory effects of DMAI on NF-κB-regulated gene expression in primary cultured murine peritonealmacrophages, cells were stimulated with LPS in the presence orabsence of DMAI and the levels of NF-κB-dependent gene productswere determined. DMAI inhibited increases in the production of NOand PGE2 as well as the secretion of TNF-α and IL-1β into the culturemedia of LPS-stimulated mouse peritoneal macrophages in a dose-dependent manner (Fig. 2A–D). DMAI treatment inhibited LPS-induced increases in cellular protein levels of iNOS, COX-2, TNF-α,and IL-1β in a dose-dependent manner as well (Fig. 2E). Moreover,DMAI significantly blocked the LPS-mediated increases in the mRNA

ere transfected with plasmids containing NF-κB-binding motifs (A) and a murine iNOS0 μM DMAI for 12 h. Luciferase activity in cell lysates was measured by a luminometer.ith LPS alone or with DMAL for 1 h was analyzed by EMSA in the presence or absence ofranslocation of the NF-κB p65 subunit was determined in cells treated with LPS alone ore or with DMAI for 30 min. For measurement of IκBα phosphorylation, cells were co-

3) and IKKα/β (Ser 180/181) phosphorylationwere determined byWestern blot analysis.ed IKK complex from lysates of cells treated with LPS for 20 min. (G) Cells were treatedtionwere determined as described in the section of Materials and methods. Levels of Akte same experimental conditions, phosphorylation of ERK, JNK, and p38 were detected byWestern blot analysis following immunoprecipitation with a MyD88 antibody.

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levels of these proteins (Fig. 2F). These results indicated that DMAIinhibits the production of inflammatory mediators through thesuppression of NF-κB-regulated gene expression at the transcriptionalstep in LPS-stimulated primary macrophages.

3.3. DMAI inhibits the NF-κB pathway, but not the MAPK pathway

To investigate whether the reduced production of inflammatorymediators by DMAI is due to the suppression of NF-κB activation, weexamined the effects of DMAI on NF-κB activation through reportergene assays. Transient transfection of RAW264.7 cells was performedwith two luciferase gene constructs consisting of NF-κB bindingsequences and a murine iNOS promoter with a NF-κB-bindingelement at −85 bp (Xie et al., 1994) by lipofection. Stimulation oftransfected cells with LPS resulted in significant increases in luciferaseactivity in both experimental conditions, and these increases weresuppressed by DMAI treatment (Fig. 3A and B). This compoundrevealed an inhibitory effect on NF-κB-DNA binding activity in thenuclear extract from macrophages co-treated with LPS (Fig. 3C). Thespecific interaction between DNA and NF-κB was demonstrated by acompetitive assay with an excess of cold probe and a supershiftanalysis of NF-κB using an antibody for the NF-κB p65 subunit. SinceNF-κB activation requires nuclear translocation following IκB phos-phorylation and proteolytic degradation (Karin, 1998), we furtherexamined whether DMAI regulates the nuclear translocation of NF-κBby immunohistochemistry. LPS treatment significantly increased thenuclear translocation of NF-κB, while this protein was mostly presentin the cytosol in control cells, and this translocationwas suppressed bytreatment with DMAI (Fig. 3D). We further measured IκBα degrada-tion following treatment of LPS in the presence or absence of DMAI byWestern blot analysis. LPS treatment significantly decreased the IκBα

Fig. 4. DMAI inhibits intracellular H2O2 accumulationwithout affecting NADPH oxidase activand intracellular H2O2 accumulation was measured using confocal microscopy using the fl

displayed as numerical values. (C) Cellular GSH levels weremeasured using a GSH assay kit. Topresented as the ratio of [GSH]/[GSH]+2[GSSG]. (D) Cells were incubated with 10 μM DMcytochrome c (80 μM) for 90 min. Superoxide generation was determined by spectrophotom10 μM DMAI for 1 h, followed by incubation with 10 μM DCFH2-DA for 20 min. Intracellular Hmeasured by EPR spectroscopy. a: H2O2 alone; b: H2O2 Fe2SO4; c: H2O2 Fe2SO4 DMAI; d: H2

protein level compared with control, and this decrease was blocked byDMAI treatment (Fig. 3E). DMAI treatment also suppressed the LPS-induced increase in phosphorylation of IκBα, IKKα (threonine 23), andIKKβ (serines 180/181) compared with control cells (Fig. 3E). DMAIalso remarkably suppressed the LPS-induced increase in IKK activity asdetermined by measurement of in vitro IκBα phosphorylation (Fig.3F), which is responsible for IκB phosphorylation. We next examinedthe effect of DMAI on the PI3K/PTEN/Akt pathway, which is a criticalupstream signal mediator of IKK activation (Ozes et al., 1999;Romashkova and Makarov, 1999). Treatment with DMAI blockedLPS-induced increases in PI3K activity, Akt phosphorylation, and PTENoxidation (Fig. 3G), however, this compound did not affect the LPS-mediated phosphorylation of ERK, JNK, and p38 (Fig. 3H) as well as theinteraction between TLR4, MyD88, and TRAF6 in the cytoplasmicmembrane (Fig. 3I). These results indicate that DMAI blocks the NF-κBsignal pathway through the inhibition of PI3K/PTEN/Akt-dependentNF-κB activation, without affecting MAPK activation and apicalsignalosome formation.

3.4. DMAI inhibits intracellular H2O2 accumulation and restores cellularredox potential in LPS-stimulated macrophages

Activation of TLR4 by LPS is implicated in NADPH oxidase-mediatedgeneration of reactive oxygen species (Li et al., 2006), which play animportant role as a second messenger for NF-κB activation (Li et al.,2006; Sulciner et al., 1996). RAW294.7 cells treated with LPS increasedintracellular accumulation of reactive oxygen species, particularly H2O2,as determined using the H2O2-sensitive fluorescent probe, DCFH2-DA,and this increase was completely suppressed by co-treatment withDMAI (Fig. 4A and B). Since intracellular reactive oxygen species readilyinteracts with GSH, a major cytosolic reductant, which controls

ity. (A) RAW264.7 cells were treated with LPS in the presence or absence of 10 μMDMAI,uorescence probe for H2O2, 10 μM DCFH2-DA. (B) The intracellular H2O2 levels weretal GSH level was 28.5 nmoles/mg protein in control cells and the relative GSH level wasAI for 1 h and then treated with phorbol 12-myristate 13-acetate (2 μM) and ferricetry. (E) Cells were treated with H2O2 (10 μM) for 10 min following pre-treatment with2O2 level was determined by confocal microscopy. (F) Hydroxyl radical production wasO2 DMAI. Data shown in B to D are the mean±S.D. (n=4). ⁎, Pb0.05 and ⁎⁎, Pb0.01.

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intracellular redox status, we measured the intracellular level of GSH.LPS decreased the intracellular GSH level in RAW264.7 cells comparedwith control and this decrease was blocked by co-treatment with DMAI(Fig. 4C). However, DMAI did not significantly affect the total GSH (GSHGSSG) level. NADPH oxidase initially produces superoxide from NADPHand dismutates to H2O2 by superoxide dismutase or a spontaneousprocess.Wenext examinedwhether DMAI regulates the production andaccumulation of superoxide as determined by the reduction ofcytochrome c. DMAI did not affect the production and accumulation ofsuperoxide in phorbol 12-myristate 13-acetate-stimulated RAW264.7cells (Fig. 4D), indicating that DMAI does not inhibit NADPH oxidaseactivity and remove superoxide. However, DMAI effectively suppressedthe intracellular H2O2 accumulation in the cells exposed to exogenousH2O2 (Fig. 4E) aswell as inhibited the production of hydroxyl radicals bythe in vitro reaction of H2O2 with ferrous iron (Fig. 4F). These resultssuggest that DMAI blocks the LPS-induced decrease in intracellularredox potential by scavenging H2O2 and hydroxyl radicals withoutaffecting the production and accumulation of superoxide in LPS-stimulated macrophages.

3.5. DMAI inhibits H2O2-mediated Akt/NF-κB activation via regulation ofPI3K and PTEN activities

We examined whether DMAI inhibits H2O2-mediated NF-κBactivation by modulating the PI3K/Akt pathway and PTEN activity.Treatment of macrophages with H2O2 promoted the phosphorylation

Fig. 5. DMAI inhibits H2O2-induced NF-κB activation. (A) RAW264.7 cells wereincubated with 10 μM H2O2 in the presence or absence of 10 μM DMAI or 10 μM N-acetylcysteine (NAC) for 15 min. Phosphorylation of Akt and IKK was determined byWestern blot analysis using antibodies against target proteins. (B) Cells were stimulatedwith 10 μM H2O2 in the presence or absence of 10 μM DMAI or 10 μM N-acetylcysteine(NAC) for 20 min. IκBα phosphorylationwas determined byWestern blot analysis. IκBαdegradationwas measured in the same condition except for the addition of 0.5 μMMG-132 to culture media. (C) Nuclear extracts were prepared from RAW264.7 cells treatedwith 250 μMH2O2 in the presence or absence of DMAI for 40min. Nuclear NF-κB activitywas analyzed by EMSA in the presence or absence of an excess amount of cold probe(CP) or antibody for the NF-κB p65 subunit (Ab). (D) Cells were incubated with either10 μMH2O2 for 10 min in the presence or absence of DMAI. PI3K activity was assayed byincubation of immunoprecipitated PI3K with its substrates and separation of thereaction mixture on a TLC Silica Gel 60 plate. Levels of the p85 subunit in samples werecompared by Western blot analysis. PTEN oxidation was analyzed by separation ofalkylated PTEN on non-reducing SDS-PAGE followed by immunoblot analysis withantibodies to PTEN.

of Akt, IKKα (threonine 23), and IKKα/β (serines 180/181), and thesebiochemical events were significantly inhibited by treatment withDMAI comparable to the antioxidant N-acetylcysteine (NAC) (Fig. 5A).Both DMAI and NAC also inhibited H2O2-induced IκBα phosphoryla-tion and degradation (Fig. 5B). In addition, DMAI suppressed the H2O2-mediated binding activity of NF-κB to DNA in the nuclear extract asdetermined by EMSA, in which specificity was confirmed using anexcess of cold probe and a NF-κB p65 antibody (Fig. 5C). These resultsindicate that DMAI regulates H2O2-mediated NF-κB activation byinhibiting the Akt/IKK pathway. We next examined whether DMAIregulates redox-dependent PI3K activation and PTEN inactivation.PI3K activitywas increased inmacrophages treatedwithH2O2, and thiseffect was significantly inhibited by co-treatment with DMAI (Fig. 5Dtop). In addition, DMAI blocked the oxidative inactivation of PTEN byH2O2 (Fig. 5D bottom). These results indicate that DMAI reducesintracellular H2O2 accumulation and thus inhibits the redox-mediatedNF-κB activation pathway.

3.6. Anti-inflammatory effect of DMAI in experimental endotoxemia

We further investigated whether DMAI regulates the production ofinflammation-associated mediators and gene products in an endotoxe-mic animal model. Mice were i.p. injected with LPS or a combination ofDMAI, and plasma levels of inflammatory mediators were determined.The levels of nitrite plus nitrate (NOx) and PGE2 were significantlyelevated by LPS injection compared with those of control mice injectedwith saline or DMAI alone, and these increases were suppressed bytreatment with DMAI (Fig. 6A and B). Increased serum levels of TNF-αand IL-1β were observed in LPS-injected mice compared with controls,and these increases also were significantly suppressed by co-adminis-trationwithDMAI (Fig. 6C andD). Furthermore, co-treatmentwithDMAIinhibited elevated levels of iNOS, COX-2, IL-1β, and TNF-α proteins andmRNAs in livers from LPS-injectedmice (Fig. 6E).We next examined theeffect of DMAI on the PI3K/PTEN/Akt pathway in vivo. DMAI treatmentblocked LPS-induced PI3K activation, Akt phosphorylation, and PTENoxidation in vivo (Fig. 6F).We further investigated theprotective effect ofDMAI on LPS-induced lethality. Injection of a LPS dose of 30 mg/kgresulted in a 24 h lethal rate of 70%; however, co-treatment with DMAIsignificantly reduced LPS-induced mortality (Fig. 6G). These resultsdemonstrate that DMAI can inhibit the in vivo production of inflamma-tory mediators and prevented mortality in LPS-induced endotoxemicmice, probably by inhibiting redox-based NF-κB activation.

4. Discussion

This study was designed to investigate the inhibitory effect andmolecular mechanism of DMAI on NF-κB-regulated inflammatory geneexpression. We found that DMAI possessed scavenging activity againstH2O2 and hydroxyl radicals, without affecting superoxide productionand accumulation, thereby preventingGSH oxidation. DMAI suppressedexpression of the inflammation-associated genes, TNF-α, and IL-1β,iNOS, and COX-2, as well as inhibited the production of NO and PGE2 inLPS-stimulated macrophages and endotoxemic mice, resulting in theprevention of lethal endotoxin shock in mice. We further demonstratedthat DMAI inhibited LPS-induced NF-κB activation by attenuating IKKactivation, IκB phosphorylation and degradation, and p65 nucleartranslocation. Our data also showed that this compound inhibitedPI3K activation and PTEN inactivation by reduction of intracellular H2O2

accumulation in macrophages treated with LPS and H2O2, resulting insuppression of the Akt-mediated NF-κB signal pathway. These resultssuggest that DMAI can potentially suppress NF-κB-responsive inflam-matory gene expression by inhibiting the redox-based activation of thePI3K/PTEN/Akt pathway (Fig. 7).

LPS, a strong immune stimulant, activates macrophages andpromotes the production of various inflammatory cytokines andmediators by activating the TLR4/MyD88-dependent NF-κB signal

Fig. 6. DMAI suppresses NF-κB-regulated gene expression in vivo. Mice were i.p. administered twice with DMAI (10 mg/kg) or saline at 12 h time intervals. At 1 h following the secondinjection, mice were i.p. injected with LPS (4 mg/kg) or saline. Twelve hours later, whole blood and liver were obtained by cardiac puncture and abdominal surgery. Plasma levels ofnitrite plus nitrate (NOx) (A), PGE2 (B), TNF-α (C), and IL-1β (D) were determined using a nitrate reductase-based colorimetric assay kit and ELISA kits. Data shown are the mean±S.D.(n=8). ⁎⁎, Pb0.01 versus LPS alone. (E) The levels of iNOS, COX-2, TNF-α, and IL-1β proteins (left) andmRNAs (right) were determined in liver lysates byWestern blotting and RT-PCR,respectively. (F) Akt phosphorylation, PI3K activity, and PTEN oxidationwere determined in liver lysates with the samemethods as described in Fig. 5. (G) Mice were injected i.p. withLPS (30 mg/kg) 1 h after pretreatment with DMAI (12 mg/kg). Animals were further administered with DMAI (12 mg/kg) or saline every 12 h, and mortality was subsequentlydetermined. Each experimental group consisted of 10 mice.

Fig. 7. Possible regulatory mechanism of DMAI in the redox-sensitive activation of theNF-κB pathway. Reactive oxygen species generated by LPS induces oxidative inactiva-tion of PTEN, an endogenous PI3K inhibitor, as well as direct activation of PI3K, resultingin Akt-dependent NF-κB activation. Both pathways can be suppressed by theantioxidant DMAI.

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pathway (Medzhitov et al., 1998; Kawai et al., 1999). NF-κB activationelevates the expression of various inflammatory genes and therefore iscritically involved in several aspects of the pathogenesis of humaninflammatory diseases (Li and Verma, 2003; Yamamoto and Gaynor,2001). Thus, inhibitors of the NF-κB signal pathway have been used astherapeutic drugs in clinical applications for regulating inflammation-associated human diseases. Our data shows that DMAI inhibited NF-κB activation, thus reducing the expression of iNOS, COX-2, IL-1β, andTNF-α as well as the production of NO and PGE2 in LPS-stimulatedmacrophages (Figs. 1 and 2). This in vitro anti-inflammatory effect ofDMAI was further confirmed by an in vivo demonstration in whichincreases in the serum levels of these inflammatory mediators in LPS-administered mice were reduced by co-administration with DMAI(Fig. 6). These results suggest that DMAImay be an inhibitor of the NF-κB signal pathway, which is critically involved in the development ofseveral inflammatory diseases.

NF-κB activation is generally mediated by canonical and non-canonical pathways (Pomerantz and BaltiMore, 2002; Baeuerle andHenkel, 1999). The canonical signal pathway which activates the NF-κB/RelA:p50 heterodimer involves the stimulus-responsive phosphor-ylation of the three inhibitors IκBα/β/ε by the NEMO and IKKβcontaining kinase complex, which tags them for degradation via theubiquitin-proteosome pathway (Baeuerle and Baltimore, 1988; Ghoshet al., 1998). The noncanonical pathway activates the NF-κB/RelB:p52dimer via a precursor processingmechanism. This pathway is requiredfor NIK and IKKα-dependent phosphorylation of RelB p100 (Dejardinet al., 2002; Basak et al., 2007), which triggers proteosomal processingto generate nuclear p52-containing NF-κB activity. Recent studieshave clearly demonstrated another NF-κB activation pathway viaactivation of the serine/threonine kinase Akt, which undergoesphosphorylation-dependent activation of IKKα and IKKβ (Ozes et al.,1999; Romashkova and Makarov, 1999; Bhattacharyya et al., 2004).Akt is activated in response to various stimuli by PI3K activation

(Franke et al., 1997; Wang et al., 2000). Activated PI3K convertsphosphatidylinositol-4,5-phosphate into phosphatidylinositol-3,4,5-phosphate in the membrane (Du and Tsichlis, 2005). These membranechanges allow docking of the lipid kinases PDK1 and Akt, leading toAkt activation via the phosphorylation of Ser473 and Thr308 by PDK1and possibly PDK1-related kinase-2 (McCubrey et al., 2006). We hereshow that DMAI effectively inhibited LPS-induced increases in PI3Kactivity, Akt phosphorylation, NF-κB activation, and intracellular H2O2

accumulation without affecting NADPH oxidase activity and super-oxide scavenging (Figs. 3 and 4). This suggests that its antioxidantactivity is critically involved in the inhibitory effect of the PI3K/Akt/NF-κB signal pathway. Indeed, DMAI blocked exogenous H2O2-

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induced increases in PI3K/Akt activation, IKK phosphorylation, andNF-κB activation by directly scavenging H2O2. These results indicatethat reactive oxygen species, particularly H2O2, generated by NADPHoxidase play an important role in PI3K/Akt-dependent NF-κB activa-tion. Thus, the antioxidant activity of DMAI is critically involved in theinhibition of inflammatory gene expression by modulating NF-κBactivation via the suppression of H2O2-mediated PI3K activation.

The lipid phosphatase, PTEN, removes the phosphate attached tothe 3'-hydroxyl group of the phosphoinositide inositol ring inphosphatidylinositol-3,4,5-phosphate synthesized by PI3K. Thus,PTEN acts as an endogenous negative regulator of the PI3K/Aktpathway, resulting in the suppression of Akt-mediated signal path-ways, including NF-κB activation. PTEN contains a redox-sensitivecysteine residue (Cys124) in the catalytic site, suggesting that itscatalytic activity can be regulated by oxidizing agents. It has beenshown that H2O2 reversibly inactivates PTEN by forming a disulfidebond between Cys124 and Cys71 (Lee et al., 2002), indicating thatH2O2 can promote NF-κB activation through the activation of the PI3K/Akt signal pathway. We here show that DMAI suppressed the H2O2-induced oxidative inactivation of PTEN and promoted the bindingactivity of NF-κB to DNA in the nuclear extract fromH2O2-treated cells.Furthermore, DMAI blocked LPS-induced in vitro and in vivo increasesin PTEN inactivation through the clearance of intracellular H2O2

accumulation, resulting in the inhibition of NF-κB-regulated geneexpression.

H2O2 (Tu et al., 2002) and receptor-mediated reactive oxygenspecies (Kim et al., 2008) were demonstrated to cause Akt-dependentNF-κB activation in cells. Akt is activated by a dual mechanisminvolving redox-based PI3K activation (Kim et al., 2008; Du andTsichlis, 2005; Shaw et al., 1998) and PTEN inactivation (Lee et al.,2002). We have previously shown that the NADPH oxidase inhibitordiphenylene iodonium inhibited NF-κD-dependent iNOS expressionand NO production in LPS-treated macrophages (Bai et al., 2005).Although not shown here, we found that iNOS-dependent NOproduction was significantly lowered in gp91phox (a component ofNADPH oxidase)-deficient peritoneal macrophages compared withnormal macrophages. These data indicate that a low level of reactiveoxygen species stimulates the NF-κB signal pathway by promotingAkt-dependent IKK activation (Ha et al., 2004; Meyer et al., 1993). Ourprevious studies have showed that the antioxidants, astaxanthin andβ-carotene, inhibited the expression of inflammatory enzymes andcytokines by suppressing NF-κB activation (Lee et al., 2003; Bai et al.,2005), suggesting that naturally occurring antioxidants may bedisease-preventing and health-promoting compounds. Similarly,DMAI inhibited NF-κB activation and inflammatory gene expressionin macrophages treated with LPS and H2O2. In addition, DMAI showeda strong antioxidant activity as demonstrated by the suppression ofcellular H2O2 accumulation, hydroxyl radical production, and GSHoxidation without affecting NADH oxidase activity. The antioxidantactivity of DMAI is responsible for the modulation of redox-basedincreases in PI3K activity and inactivation of PTEN in macrophagestreated with H2O2 and LPS as well as in the liver of endotoxemic mice.H2O2 attenuates PTEN activity by oxidation of redox-sensitive cysteinein its catalytic site (Lee et al., 2002). However, themechanismbywhichH2O2 directly increases PI3K activity is largely unknown and should befurther investigated. It is, however, worth noting that H2O2 canstimulate PI3K-dependent activation of Akt in intact cells by recruit-ment of PI3K to its substrate site in the cytoplasmicmembrane, therebyenabling PI3K to maximize its catalytic efficiency (Qin and Chock,2003). These results indicate that DMAI inhibits NF-κB activation bymodulating the redox-based cascade of the PI3K/PTEN/Akt pathwayand subsequently inhibits the production of inflammatory geneexpression.

TLR4 is commonly expressed in innate immune cells especiallymacrophages and dendritic cells. Once activated by LPS, TLR4-dependent signals converge on the adapter protein MyD88, which

binds to the cytoplasmic receptor domain (Kawai et al., 1999). MyD88then recruits members of the IL-1R-associated kinase (IRAK) family tothe receptor complex, which exclusively binds to TRAF6. TRAF6triggers a cascade of cellular signals, culminating in the eventualactivation of MAPKs (O'Neill and Bowie, 2007) as well as activatesNADPH oxidase via a Rac1-dependent manner, resulting in theelevation of intracellular H2O2 accumulation (Ha et al., 2004;Asehnoune et al., 2004). We showed that H2O2 can act as a biologicalsecond messenger for NF-κB activation by undergoing redox-dependent PI3 K activation and PTEN inactivation, leading to Akt-dependent NF-κB activation and inflammatory gene expression. Theantioxidant activity of DMAI effectively inhibited this redox-based NF-κB signal pathway, resulting in suppression of the transcriptional up-regulation of inflammatory genes. However, this compound did notdirectly inhibit NADPH oxidase activity and formation of thesignalsome of MyD88 with TRAF6 on the cytoplasmic domain ofTLR4. These evidences indicate that TRAF6-mediated reactive oxygenspecies generation plays a critical role in inflammatory geneexpression by stimulating Akt-dependent NF-κB activation throughthe redox-sensitive PI3 K activation and PTEN inactivation and thatDMAI inhibits the NF-κB signal pathway by reduction of cellular H2O2

levels (Fig. 7).Taken together, DMAI suppressed the activation of the canonical

NF-κB signal pathway by blocking both H2O2-induced increases inPI3 K activity and PTEN inactivation. These inhibitory effects aredirectly associated with the suppression of inflammatory geneexpression in LPS-stimulated macrophages and endotoxemic mice.Thus, our results suggest that DMAI should be considered as apotential therapeutic drug for inflammatory diseases including sepsis.

Acknowledgments

This work is supported by the Korea Food and Drug Administrationthrough the National Center for the Standardization of HerbalMedicine and the Vascular System Research Center grant from KOSEF.

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