extract inhibits the release of inflammatory mediators from LPS-stimulated mouse macrophages

Available online at Journal of Ethnopharmacology114(2007)439–445Wen-Pi-Tang-Hab-Wu-Ling-San extract inhibits the release ofinflammatory mediators from LPS-stimulated mouse macrophagesHyo Won Jung a,Cheol-Ho Yoon b,Young Ho Kim c,Yong Chool Boo d,Kwon Moo Park e,Yong-Ki Park a,∗a Department of Herbology,College of Oriental Medicine,Dongguk University,Gyeongju780-714,Republic of Koreab Department of Internal Medicine,College of Oriental Medicine,Dongguk University,Gyeongju780-714,Republic of Koreac College of Pharmacy,Chungnam National University,Daejeon305-764,Republic of Koread Department of Molecular Medicine,School of Medicine,Kyungpook National University,Daegu700-422,Republic of Koreae Department of Anatomy,School of Medicine,Kyungpook National University,Daegu700-422,Republic of KoreaReceived30November2006;received in revised form17August2007;accepted22August2007Available online25August2007AbstractAim of the study:Wen-Pi-Tang is a traditional herbal prescription that has been used traditionally for the treatment of various inflammatory diseases, including chronic renal failure,renal injury,renal tubular cell damage and diabetic nephropathy.In this study,we investigated the pharmacological activity of modified Wen-Pi-Tang,Wen-Pi-Tang-Hab-Wu-Ling-San(WHW)extract.Materials and methods:Production of tumor necrosis factor-alpha(TNF-␣),interleukin-1beta(IL-1␤),IL-6and NO in supernatant,mRNA expression of TNF-␣,IL-1␤,IL-6and iNOS,protein expression of iNOS,phosphorylation of mitogen-activated protein kinases(MAPKs)and activation of nuclear factor-kappa B in the extract were assayed.Results:We found that WHW extract had potent anti-inflammatory effects in LPS-stimulated RAW264.7cells and primary peritoneal macrophages. WHW extract strongly inhibited the excessive production of inflammatory mediators,nitric oxide(NO),TNF-alpha(TNF-␣),interleukin1-beta (IL-1␤)and IL-6in LPS-stimulated macrophages.The inhibition of inducible nitric oxide synthase(iNOS)and these cytokines resulted from the reduced expressions of mRNAs of iNOS and these cytokines,respectively.WHW extract attenuated the phosphorylation of mitogen-activated protein kinases(MAPKs),extracellular signal-regulated kinases1and2(ERK1/2)and c-Jun N-terminal kinase(JNK),as well as the activation of nuclear factor-kappa B(NF-␬B)in LPS-stimulated RAW264.7cells.Conclusions:These data suggest that WHW extract may exhibit anti-inflammatory effects through the modulation of MAPK and the NF-␬B-dependent pathway involved in inflammation.©2007Elsevier Ireland Ltd.All rights reserved.Keywords:Anti-inflammation;Herbal medicine;Inflammatory cytokines;Macrophages;Mitogen-activated protein kinases;Nuclear factor-kappa B1.IntroductionInflammation is a multi-step process that is mediated by acti-vated inflammatory and immune cells,including macrophages/ monocytes(Minagar et al.,2002).In the presence of stimuli such as TNF-␣,interferon-gamma(IFN-␥)and lipopolysaccha-ride(LPS),activated macrophages induce the overproduction of proinflammatory cytokines such as TNF-␣,IL-1,macrophage inflammatory proteins(MIP)-1␣,and inflammatory media-∗Corresponding author.Tel.:+82547702661;fax:+82547702661.E-mail address:yongki@dongguk.ac.kr(Y.-K.Park).tors such as oxygen species(ROS),nitric oxide(NO),and prostaglandin E2(PGE2),and mediate many inflammatory diseases(Nathan,1992;Guha and Mackman,2001).The devel-opment of effective herbal medicines has recently sparked renewed interest in the development of novel therapeutic strate-gies to suppress the production of proinflammatory cytokines and NO using macrophages(Chi et al.,2003;Barak et al.,2004; Genovese,2005).Wen-Pi-Tang(On-Pi-To)is a traditional herbal prescrip-tion that is used to treat a variety of inflammatory diseases, including moderate/chronic renal failure(Yokozawa et al., 1996;Takizawa et al.,2005),renal injury(Rhyu et al.,2002), renal tubular cell damage(Yokozawa et al.,2003),diabetic0378-8741/$–see front matter©2007Elsevier Ireland Ltd.All rights reserved. doi:10.1016/j.jep.2007.08.035440H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445nephropathy(Yokozawa et al.,2006)and lung damage resulting from infection with the influenza virus(Yokozawa et al.,2004). It has been suggested that Wen-Pi-Tang could be a useful therapeutic approach to the treatment of inflammatory diseases, but its actual role in the inflammatory process remains unclear. Wen-Pi-Tang is used with Wu-ling-san to treat the patients with chronic renal failure(Lee et al.,2007)in Korean clinics;this combination is referred to as Wen-Pi-Tang-Hab-Wu-Ling-San (WHW).Recently,we reported that WHW extract inhibits the renalfibrosis in kidney cells(Lee et al.,2007),and protects kidney against ischemia/reperfusion injury in mice and(Seok et al.,2007).In this study,we evaluated the potential of WHW as a therapeutic modality for inflammation in RAW264.7 mouse macrophage cells and primary peritoneal macrophages. Our results showed that WHW extract largely inhibited the excessive production of inflammatory mediators such as NO, TNF-␣,IL-1␤and IL-6through the down-regulation of their transcription factors and through the modulation of the MAPK and NF-␬B-dependent pathway.2.Materials and methods2.1.Preparation of WHW extractThe voucher specimens(OB05-1)in the Herbarium of DUCOM,and method used for the WHW extract have been described previously(Lee et al.,2007;Seok et al.,2007).The lyophilized powder of WHW was dissolved in saline prior to use.2.2.Cell cultureRAW264.7mouse macrophage cells(American Type Culture Collection)were maintained in Dulbecco’s modified Eagle’s medium(DMEM)supplemented with10%heat-inactivated fetal bovine serum(FBS;HyClone,Rogan,HT),100U/mL of penicillin and100␮g/mL of streptomycin(GibcoBRL,Grand Island,NY)at37◦C in a5%CO2incubator.Primary macrophages were collected from the peritoneal cavities of mice(8-week-old female C57BL/6)after an intraperi-toneal(i.p.)injection of3mL of1%thioglycolate broth(Sigma, St.Louis,MO)3days before harvesting.The peritoneal macrophages were washed with1×PBS(Ca2+-and Mg2+-free) and plated with RPMI-1640containing10%FBS,100U/mL of penicillin and100␮g/mL of streptomycin overnight for the experiments.2.3.MTT assayA commercially-available cell viability assay was employed to evaluate the cytotoxic effect of WHW extract using the MTT-based colorimetric assay(Roche,Mannheim,Germany). RAW264.7cells(5×104cells/well)and primary macrophages (1×105cells/well)were plated with a variety of concentrations of WHW extract in96-well microtiter plates(Nunc,Roskillde, Denmark),and were then cultured for24h at37◦C in a5%CO2 incubator.At the termination of cultures,10␮L of the MTT solu-tion was added to each well,and the cells were then cultured for4h at37◦C in a5%CO2incubator.One hundred micro-liters of solubilized solution was added to each well;the plate was allowed to stand overnight in the incubator after evalua-tion for complete solubilization of the purple formazan crystals and the measurement of the optical density(OD)at550nm by a microplate reader(GENios,TEKAN Instruments,Inc.,Austria).2.4.Measurement of nitric oxide productionAfter pre-incubation of the RAW264.7cells(1×105cells/ mL)and primary macrophages(5×105cells/mL)for16h,a variety of concentrations of WHW extract,either with or with-out LPS(1␮g/mL),were incubated for24h at37◦C in a5%CO2 incubator.Nitric oxide was measured in culture supernatant by reaction with Griess reagent(1%sulfanilamide and0.1%%N-[1-naphthy]-ethylenediamine dihydrochloride in5%phosphoric acid;Roche)to100␮L of culture supernatant for15min at room temperature in the dark.The absorbance at540nm was deter-mined using a Spectramax250microplate reader(GENios).A standard curve was generated in the same fashion using NaNO2.2.5.Measurement of cytokine levelsRAW264.7cells(1×105cells/mL)and primary macro-phages(5×105cells/mL)were pretreated with a variety of concentrations of WHW extract for30min,and were then stim-ulated with1␮g/mL of LPS.The culture supernatants were collected at24h after LPS stimulation,and the concentrations of TNF-␣,IL-1␤and IL-6were measured by enzyme-linked immunosorbent assay(ELISA)according to the protocol of the manufacturer(eBioscience,California,CA).2.6.Reverse transcription polymerase chain reaction(RT-PCR)RAW264.7cells(1×105cells/mL)and primary macro-phages(5×105cells/mL)were pretreated with0.5mg/mL of WHW extract for30min,and were then stimulated with 1␮g/mL of LPS or without LPS for2h.Total RNA from each cell was prepared by adding TRIzol reagent according to the pro-tocol of the manufacturer(GibcoBRL).The total RNA solution was stored at−70◦C until use.Total RNA was incubated with oligo-dT primer at70◦C for 5min,mixed with a5×first-strand buffer(Promega,Madison, USA),0.5mM of dNTP,3mM MgCl2,RNase inhibitor and Improm-II TM reverse transcriptase(2U),and further incubated at25◦C for5min and42◦C for1h.The reaction was termi-nated after10min at70◦C,and total RNA was depleted by the addition of RNase H.The PCR reaction was conducted with the incubation mixture[2␮L cDNA,4␮M5 and3 primers,a10×buffer(10mM Tris–HCl,pH8.3,50mM KCl,0.1%Triton X-100,25mM MgCl2;Takara Bio Ltd.,Shiga,Japan),250␮M of dNTP and1U of Tag polymerase under the following incuba-tion conditions:30s at94◦C for denaturation,30s at60◦C for annealing,1min for extension and10min forfinal extension at the end of35cycles.The primers used in this experiment were: iNOS[Fw;5 -GAC CAG TAT AAG GCA A GC AC-3 ;and Rv;5 -CTT GTC TTT GAC CCA GTA GC-3 ],TNF-␣[Fw;5 -H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445441TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3 ; and Rv;5 -GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3 ],IL-1␤[Fw;5 -GAA GCT GTG GCA GCT ACC TAT GTC T-3 ;and Rv;5 -CTC TGC TTG TGA GGT GCT GAT GTA C-3 ],IL-6[Fw;5 -GAG GAT ACC ACT CCC AAC AGA CC-3 ;and Rv;5 -TTC ACA GAG GAT ACC ACT CC-3 ],and GAPDH[Fw;5 -C TCG TGG AGT CTA CTG GTG T-3 ;and Rv;5 -GTC ATC ATA CTT GGC AGG TT-3 ].GAPDH was used as an internal control to evaluate the relative expressions of iNOS,TNF-␣,IL-1␤and IL-6.2.7.Western blot analysisRAW264.7cells(1×105cells/mL)and primary macro-phages(1×105cells/mL)were pretreated with0.5mg/mL of WHW extract for30min and stimulated with1␮g/mL of LPS or without for24h.Cells were lysed with0.1mL of50mM Tris–HCl,pH7.2,containing1%sodium deoxycholate,0.1% sodium dodecyl sulfate(SDS),0.15M NaCl and1%NP-40. The cell lysates were assayed for protein concentration by the Bradford staining method,and equal amounts of protein (20␮g/mL)were then electrophoresed on8–10%density SDS-acrylamide gels.After electrophoresis,the proteins were transferred from the gel to a nitrocellulose(NC)membrane using an electric transfer system.Non-specific binding was blocked with3%skim milk in TBS-T buffer(5mM Tris–HCl, pH7.6,136mM NaCl,0.1%Tween-20)for1h.Blots were incubated at room temperature for1h each with primary anti-body against iNOS(1:3000dilution;Santa Cruz Biotechnology, Santa Cruz,CA),actin(1:8000;Sigma),and phosphor-forms of pERK1/2,p38MAPK and JNK(1:1000;Cell Signaling Technology,Charlottesville,V A)added,and were then washed three times with1×TBS-T.Blots were incubated for1h at room temperature(RT)with a1:10,000dilution of horseradish peroxidase(HRP)-labeled anti-mouse IgG(1:1000,Santa Cruz Biotechnology),and were then washed three times with1×TBS-T.Blots were developed with the ECL Western detection reagents(Amersham Bioscience,Piscataway,NJ).2.8.DNA binding assay of nuclear factor-κB(NF-κB)RAW264.7cells(1×105cells/mL)and primary macro-phages(5×105cells/mL)were pretreated with0.5mg/mL of WHW extract for30min and stimulated with1␮g/mL of LPS or without LPS for30min.Nuclear extracts were prepared by NE-PER nuclear and cytoplasmic extraction reagents(Pierce Biotechnology,Inc.,Rockford,IL)according to the protocol of the manufacturer.In brief,the cells in six wells were washed with ice-cold PBS.The cells were then scraped,transferred to micro-tubes,and allowed to swell after the addition of cytoplasmic extraction reagent(CER).The lysates were incubated for10min on ice and centrifuged at12,000rpm for5min at4◦C.Pellets containing crude nuclei were resuspended in nuclear extraction reagent(NER)solution and incubated in ice for1h.The samples were centrifuged at12,000rpm for10min to obtain nuclear frac-tions.The nuclear fractions were assayed for protein concentra-tion by using the Bradford staining method,and equal amounts of protein were then electrophoresed on10%SDS-acrylamide gels.After electrophoresis,the proteins were transferred from the gel to an NC membrane using an electric transfer system. Non-specific binding was blocked with3%skim milk in TBS-T buffer(5mM Tris–HCl,pH7.6,136mM NaCl,0.1%Tween-20)for1h.Blots were incubated at room temperature for1h each with primary antibody against p65NF-␬B(1:5000dilu-tion;Santa Cruz Biotechnology)and actin(1:8000;Sigma),and were then washed three times with1×TBS-T.Blots were incu-bated for1h at RT with a horseradish peroxidase(HRP)-labeled anti-rabbit IgG(1:1000;Santa Cruz Biotechnology),and were then washed three times with1×TBS-T.Blots were developed with ECL Western detection reagents(Amersham Bioscience).2.9.Statistical analysisStatistical analysis was performed to assess the difference between two groups under multiple experimental conditions by one-way ANOV A using PRISM statistical analysis software (GraphPad Software,Inc.,San Diego,CA).3.Results3.1.Cell toxicity of WHW extractTo exclude the possibility that reductions of the levels of NO and inflammatory cytokines from the cells were due todirect Fig.1.Cytotoxic effect of WHW extract in macrophages.The cytotoxic effects of WHW extract in RAW264.7cells(A)and primary macrophages(B)were determined MTT assay.Three independent experiments were performed,and data shown represent the mean±S.D.442H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445toxicity of WHW extract to the cells,we evaluated cell toxicity at a variety of concentrations of WHW extract(0.05–2mg/mL) using the MTT assay(Fig.1).WHW extract-induced cell tox-icity was negligible at concentrations of0.05–0.5mg/mL in both RAW264.7cells(Fig.1A)and primary macrophage cells (Fig.1B).3.2.WHW extract inhibits LPS-induced NO production in macrophagesBecause NO is known to be a proinflammatory mediator in inflammatory diseases(Nathan,1992),we investigated whether the WHW extract inhibits NO production from the macrophages that were activated with LPS.As shown in Fig.2,the production of NO was increased after LPS stimulation in both RAW264.7 cells(Fig.2A)and primary macrophages(Fig.2B).Following pretreatment with the WHW extract,the LPS-induced produc-tion of NO was significantly inhibited in both cell types in a dose-dependent manner.3.3.WHW extract inhibits LPS-induced iNOS expression in macrophagesTo ensure that WHW extract-mediated inhibition of LPS-induced NO production resulted from the inhibition of iNOS activity and not to its expression,RT-PCR and Western blot anal-ysis of iNOS mRNA and protein were performed.Pretreatment with WHW extract greatly suppressed LPS-induced mRNA and protein expression of iNOS in RAW264.7cells(Fig.3A and C)and primary macrophages(Fig.3B and D).Consistent with thefindings shown in Fig.2,WHW extract had a very strong inhibitory effect on iNOSexpression.Fig.2.Effect of WHW extract on LPS-induced NO production in macrophages. RAW264.7cells(A)and primary macrophages(B)were each incubated with different concentrations of WHW extract in the presence of LPS(1␮g/mL)for 24h.NO was measured by the Griess reaction.Three independent experiments were performed,and data represent are the mean±S.D.;*p<0.05,**p<0.01 and***p<0.001compared with LPS alone.3.4.WHW extract reduces LPS-induced production ofproinflammatory cytokines in macrophagesTo investigate the effect of the WHW extract on the produc-tion of proinflammatory cytokines,including TNF-␣,IL-1␤and Fig.3.Effect of WHW extract on LPS-induced mRNA and protein levels of iNOS in macrophages.RAW264.7cells(A and C)and primary macrophages(B and D) were incubated with WHW extract(0.5mg/mL)in the absence or presence of LPS(1␮g/mL)for2h and24h.The mRNA(A and B)and protein levels of iNOS(C and D)was determined by RT-PCR and Western blot.H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445443Fig.4.Effect of WHW extract on LPS-induced production of proinflammatory cytokines in macrophages.RAW264.7cells(A–C)and primary macrophages(D–F) were incubated with WHW extract(0.5mg/mL)in the presence of LPS(1␮g/mL)for24h.The levels of proinflammatory cytokines were measured by ELISA. Three independent experiments were performed,and data shown represent the mean±S.D.IL-6,RAW264.7cells and primary macrophages were cultured with WHW extract in the presence of LPS.The concentration of each proinflammatory cytokine was measured in culture super-natant using ELISA.Fig.4shows that treatment with the WHW extract led to a marked decrease in the increasing levels of TNF-␣,IL-1␤and IL-6from both LPS-stimulated RAW264.7 cells(Fig.4A–C)and primary macrophages(Fig.4D–F);these changes occurred in a dose-dependent manner.We also investigated whether WHW extract suppressed the mRNA expressions of TNF-␣,IL-1␤and IL-6in RAW264.7 cells and primary macrophages.LPS-induced mRNA expression of TNF-␣and IL-6was greatly reduced by pretreatment with WHW extract in RAW264.7(Fig.5A)and primary macrophage cells(Fig.5B).WHW extract also weakly reduced the mRNA expression of IL-1␤in both of the LPS-activated cell types. These data suggest that the WHW extract inhibits a variety of inflammatory mediators,not only NO,but also proinflam-matory cytokines,through the regulation of their transcription factors.3.5.WHW extract reduces LPS-induced phosphorylation of MAPK molecules and NF-kappa B p65in macrophages LPS-induced phosphatidylinositol3-kinase(PI3-kinase)/Ark and MAPKs can activate a variety of transcription fac-tors,including NF-␬B,which coordinates the induction of many inflammatory cytokines(Guha and Mackman,2001). We investigated whether the WHW extract modulatesthe Fig.5.Effect of WHW extract on LPS-induced mRNA expression of proinflammatory cytokines in macrophages.RAW264.7cells(A)and primary macrophages (B)were each incubated with WHW extract(0.5mg/mL)in the absence or presence of LPS(1␮g/mL)for2h.The mRNA expression of proinflammatory cytokines was analyzed by RT-PCR.444H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445Fig.6.Effect of WHW extract on LPS-induced phosphorylation of MAPK molecules and NF-␬B p65in RAW264.7cells.Cells were incubated with WHW extract(0.5mg/mL)in the absence or presence of LPS(1␮g/mL)for30min. The phosphorylation of MAPK molecules(A)and NF-␬B p65protein(B)were analyzed by Western blot.inflammation-mediated signal pathway in RAW264.7cells using Western blot analysis(Fig.6).Treatment with WHW extract inhibited the phosphorylation of the MAPK molecules, including pERK1/2and JNK in LPS-stimulated RAW264.7cells (Fig.6A).The WHW extract also attenuated the LPS-elicited nuclear translocation of NF-␬B p65protein in RAW264.7cells (Fig.6B).Thesefindings suggest that the WHW extract was associated with down-regulation or degradation of NF-␬B p65 protein in LPS-stimulated macrophages.4.DiscussionWen-Pi-Tang has previously been reported to have antioxi-dant effects in renal dysfunction(Rhyu et al.,2002),to regulate the cell cycle by G2/M phase arrest in renal tubular cells (Yokozawa et al.,2003),to improve pathological conditions of the lungs infected with influenza virus(Yokozawa et al.,2004), and to reduce the risk of the development of diabetic nephropathy (Yokozawa et al.,2006).Despite the identified effects in several diseases,the pharmacological activities of the WHW extract have not been fully explored with regard to its immunological effects.Therefore,in this study,we prepared WHW extract for treatment of the patients with inflammatory diseases,including chronic renal failure,rheumatoid arthritis and neurodegenera-tive diseases,and then identified its anti-inflammatory effects in LPS-activated mouse macrophages.NO is produced by activated macrophages as a result of induc-tion by several stimuli,including TNF-␣,IFN-␥and LPS,and may contribute to the pathological process in various acute and chronic inflammatory conditions(Kilbourn and Belloni,1990; Nathan,1992;Murakami et al.,2003).Therefore,the reduction of NO production,including inhibition of iNOS gene activity, depletion of arginine substrate by arginase,and transcriptional down-regulation of iNOS gene expression by endogenous or exogenous manipulation,may present a useful strategy for the treatment of a variety of inflammatory diseases,including some neurological disorders(Albina and Reichner,1998;Boucher et al.,1999;Lee et al.,2006).In this study,we demonstrate that WHW extract significantly inhibited NO production(Fig.2)in LPS-stimulated macrophages without showing any cell toxicity (Fig.1).Furthermore,the inhibitory action of WHW extract on LPS-induced NO production appears to involve the inhibition of iNOS gene expression(Fig.3).Recently,new approaches to the use of Chinese herbal plants to prevent and treat inflammatory responses by inhibiting inflam-matory cytokines such as TNF-␣,IL-1␤,IL-6(Kim et al.,2005) and IL-8(Barak et al.,2004)has become an important area of investigation,as have their associated complications.Here,we confirmed that WHW extract could remarkably suppress the pro-duction of inflammatory cytokines,including TNF-␣and IL-6 from LPS-activated RAW264.7cells and primary macrophages (Fig.4).Furthermore,WHW extract may inhibit the mRNA expressions of TNF-␣and IL-6at the pre-translational level in both cell types(Fig.5).Although no reports have yet addressed the anti-inflammatory effect of Wen-Pi-Tang or On-Pi-To extract through the modulation of inflammatory molecules,the WHW extract appears to have an anti-inflammatory function based on our study results.LPS stimulation of macrophages activates several extracellu-lar signaling pathways,including the I␬B kinase(IKK)-NF-␬B pathway and three MAPK pathways,ERK1/2,JNK and p38 MAPK(Kim et al.,2005).Proinflammatory cytokines that are released from macrophages are also involved in many aspects of inflammatory events,including expressions of iNOS and COX-2involved the mitogen-activated protein kinase3(MKK3)/p38 MAPK and NF-␬B pathways(Wu et al.,2004;Wang et al., 2006).These signaling pathways activate the induction of many genes that encode inflammatory mediators(Baldwin,1996). Therefore,these signaling molecules may represent novel tar-gets for the treatment of patients with inflammatory diseases (Guha and Mackman,2001).Despite several reports describ-ing the pharmacological activities of Wen-Pi-Tang in several inflammatory diseases(Rhyu et al.,2002;Yokozawa et al., 2003,2006,2004;Yokoazwa et al.,2006),little is know about its molecular mechanism.Thus,to understand the molecular mechanisms underlying the suppression of NO,TNF-␣and IL-6,we investigated the effects of WHW extract on LPS-induced activation of the MAPK and NF-␬B pathways.We showed that the anti-inflammatory effect of WHW extract was associated with decreases in LPS-induced phosphorylation of pERK1/2and JNK MAPKs(Fig.6A)and nuclear transloca-tion of NF-␬B p65(Fig.6B)in activated macrophages.These results indicate the potent activity of WHW extract as a modu-H.W.Jung et al./Journal of Ethnopharmacology114(2007)439–445445lator of the LPS-mediated MAPK/NF-␬B pathway in activated macrophages.In conclusion,we demonstrate here that WHW extract is a potent suppressor of secretion of an inflammatory media-tor,NO,and proinflammatory cytokines,TNF-␣and IL-6,in LPS-activated macrophages.Thus,WHW extract may have therapeutic potential for the modulation and regulation of macrophage activation,and may provide safe and effective treat-ment options for a variety of inflammation-mediated diseases.AcknowledgmentThis study was supported by a grant of the Oriental Medicine R&D Project,Ministry of Health&Welfare,Rep.of Korea (B070044to Y.K.Park).ReferencesAlbina,J.L.,Reichner,J.S.,1998.Role of nitric oxide mediation of macrophage cytotoxicity and apoptosis.Cancer Metastasis Review17,39–53. Baldwin Jr.,A.S.,1996.The NF-kappa B and I kappa B proteins:new discoveries and insights.Annual Review of Immunology14,649–683.Barak,V.,Kalickman,I.,Halperin,T.,Birkenfeld,S.,Ginsburg,I.,2004.PADMA-28,a Tibetan herbal preparation is an inhibitor of inflammatory cytokine production.European Cytokine Network15,203–209. Boucher,J.L.,Moali,C.,Tenu,J.P.,1999.Nitric oxide biosynthesis,nitric oxide synthase inhibitors and arginase competition for L-arginine utilization.Cell Molecular Life Science55,1015–1028.Chi,Y.S.,Lim,H.,Park,H.,Kim,H.P.,2003.Effects of wogonin,a plant flavone from Scutellaria radix,on skin inflammation:in vivo regulation of inflammation-associated gene expression.Biochemical Pharmacology66, 1271–1278.Genovese,M.C.,2005.Biologic therapies in clinical development for the treat-ment of rheumatoid arthritis.Journal of Clinical Rheumatology11,S45–S54. Guha,M.,Mackman,N.,2001.LPS induction of gene expression in human monocytes.Cellular Signal13,85–94.Kilbourn,R.G.,Belloni,P.,1990.Endothelial cell production of nitric oxide in response to interferon-␥in combination with tumor necrosis fac-tor,interleukin-1,or endotoxin.Journal of National Cancer Institute82, 772–776.Kim,S.J.,Jeong,H.J.,Moon,P.D.,Lee,K.M.,Lee,H.B.,Jung,H.J.,Jung,S.K., Rhee,H.K.,Yang,D.C.,Hong,S.H.,Kim,H.M.,2005.Anti-inflammatory activity of gumiganghwaltang through the inhibition of nuclear factor-kappaB activation in peritoneal macrophages.Biological and Pharmaceutical Bul-letin28,233–237.Lee,H.J.,Hyun,E.A.,Yoon,W.J.,Kim,B.H.,Rhee,M.H.,Kang,H.K.,Cho, J.Y.,Yoo,E.S.,2006.In vitro anti-inflammatory and anti-oxidative effects of Cinnamomum camphora extracts.Journal of Ethnopharmacology103, 208–216.Lee,S.I.,Kim,H.J.,Baek,M.C.,Park,K.M.,Park,Y.,Yoon, C.H., Boo,Y.C.,2007.Wen-Pi-Tang-Hab-Wu-Ling-San,an oriental herbal prescription,attenuates epithelial mesenchymal transdifferentiation stim-ulated by TGF-␤1in kidney cells.Phytotherapy Research21,548–553.Minagar,A.,Shapshak,P.,Fujimura,R.,Ownby,R.,Heyes,M.,Eisdorfer,C.,2002.The role of macrophage/microglia and astrocytes in the patho-genesis of three neurologic disorders:HIV-associated dementia,Alzheimer disease,and multiple sclerosis.Journal of Neurological Sciences202,13–23.Murakami,A.,Takahashi,D.,Hagihara,K.,Koshimizu,K.,Ohigashi,H.,2003.Combinatorial effects of nonsteroidal anti-inflammatory drugs and food constituents on production of prostaglandin E2and tumor necrosis factor-alpha in RAW264.7murine macrophages.Bioscience Biotechnology and Biochemistry67,1056–1062.Nathan,C.,1992.Nitric oxide as a secretory product of mammalian cells.The FASEB Journal6,3051–3064.Rhyu, D.Y.,Yokozawa,T.,Choa, E.J.,Park,J.C.,2002.Prevention of peroxynitrite-induced renal injury through modulation of peroxynitrite pro-duction by the Chinese prescription Wen-Pi-Tang.Free Radical Research 36,1261–1269.Seok,Y.M.,Kim,J.,Choi,K.C.,Yoon,C.H.,Boo,Y.C.,Park,Y.,Park, K.M.,2007.Wen-Pi-Tang-Hab-Wu-Ling-San attenuates kidney ischemia/ reperfusion injury in mice.A role for antioxidant enzymes and heat-shock proteins.Journal of Ethnopharmacology112,333–340.Takizawa,Y.,Morita,T.,Fujitsuka,N.,Kido,A.,Shindou,S.,Tomisawa,H.,Kase,Y.,Takeda,S.,Aburada,M.,Miyamoto,K.,2005.Pharmacoki-netics of TJ-8117(Onpi-to),a drug for renal failure(II):effects of food, repeated administration and renal failure to plasma concentration of[3H]-(-)epicatechin3-O-gallate in rats.European Journal of Drug Metabolism and Pharmacokinetics30,175–180.Wang,X.M.,Kim,H.P.,Song,R.,Choi,A.M.,2006.Caveolin-1confers anti-inflammatory effects in murine macrophages via the MKK3/p38MAPK pathway.American Journal of Respiratory Cell and Molecular Biology34, 434–442.Wu,M.J.,Wang,L.,Ding,H.Y.,Weng,C.Y.,Yen,J.H.,2004.Glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-induced NF-kappa B.Journal of Biomedical Science11,186–199.Yokozawa,T.,Chung,H.Y.,He,L.Q.,Qura,H.,1996.Effectiveness of green tea tannin on rats with chronic renal failure.Bioscience Biotechnology and Biochemistry60,1000–1005.Yokozawa,T.,Rhyu,D.Y.,Cho,E.J.,2003.Protection by the Chinese pre-scription Wen-Pi-Tang against renal tubular LLC-PK1cell damage induced by3-morpholinosydnonimine.Journal of Pharmacy and Pharmacology55, 1405–1412.Yokozawa,T.,Satoh,A.,Nakagawa,T.,Yamabe,N.,2006.Attenuating effects of Wen-Pi-Tang treatment in rats with diabetic nephropathy.American Journal of Chemical Medicine34,307–321.Yokozawa,T.,Sekiya,M.,Cho,E.J.,Kurokawa,M.,Shiraki,K.,2004.Effect of Wen-Pi-Tang extract on lung damage by influenza virus infection.Phy-tomedicine11,625–632.。