Anti-Inflammatory molecular mechanisms of Bo-Ye-Niu- Pi-Xiao (Cynanchum taiwanianum Yamazaki)

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1 Africn Journl of Biotechnology Vol. 11(7), pp , 24 Jnury, 212 Aville online t DOI: /AJB ISSN Acdemic Journls Full Length Reserch Pper Anti-Inflmmtory moleculr mechnisms of Bo-Ye-Niu- Pi-Xio (Cynnchum tiwninum Ymzki) Houi Lee 1, Tzuching Wng 2*, Tchen Lin 3, Jihsin Guo 1, Chiching Yng 1 nd Yingpei Shen 2,4 1 Deprtment of Food Science nd Technology, Ntionl Pingtung University of Science nd Technology, Pingtung, 9121, Tiwn, ROC. 2 Deprtment of Food nd Beverge Mngement, Tzuhui Institute of Technology, Pingtung 92641, Tiwn, ROC. 3 Deprtment of Tourism nd Leisure Mngement, Fortune Institute of Technology, Kohsiung City 8316, Tiwn, ROC. 4 Ph.D Progrm in Mngement of Dyeh University, Chnghu 51591, Tiwn, ROC. Accepted 9 Novemer, 211 Bo-Ye-Niu-Pi-Xio, Cynnchum tiwninum Ymzki (fmily: Asclepidcee) is well-known nd populr her; its rhizome hs een used s folk medicine in Tiwn. Mny therpeutic effects of C. tiwninum Ymz. hd een studied; however, there is still no nti-inflmmtory effect nd mechnism of C. tiwninum Ymz. reported. Besides, it is well-known tht the phosphoryltion of I kpp B-lph (IκBα) to phospho-iκbα (p-iκbα) is decisive step in the nucler fctor kpp B (NF-κB) ctivtion pthwy. In ddition, the induction of cyclooxygense-2 (COX-2) nd inducile nitric oxide synthse (inos) y cytokines, such s interleukin-1β (IL-1β), is well known to e prtly medited y NFκB. Hence, the ojective of this study ws to evlute the ctivity nd distinguish the mechnism of nti-inflmmtion of C. tiwninum Ymz. rhizome on rt kidney cell line NRK-52E induced y IL-1β. In the present study, we investigted the nti-inflmmtory effect nd potentil mechnism of C. tiwninum Ymz. rhizome ethnolic extrct (CTEE) in IL-1β-induced NRK-52E cells. Production of nitric oxide (NO) nd prostglndin E2 (PGE 2 ) y enzyme linked immunosorent ssy. The messenger rionucleic cid (mrna) nd protein expression of inos nd COX-2, phosphoryltion of IκBα, nd ctivtion of NF-κB ws executed y reverse trnscription-polymerse chin rection nd Western lotting. Results show tht the CTEE significntly (P <.5) inhiited NO nd PGE 2 production nd lso significntly (P <.5) ttenuted protein nd mrna expression of inos nd COX-2 in IL-1β-induced NRK-52E cells without ovious cytotoxic effects. Furthermore, the CTEE suppressed the NF-κB nucler trnsloction, in terms of inhiition of IκBα phosphoryltion. The results provided evidence for its folkloric uses nd suggest tht the nti-inflmmtory ctivities of CTEE my result from the inhiition of inflmmtory meditors, such s NO nd PGE 2 nd n upstrem suppression of NF-κB-dependent mechnism might e involved. Key words: Cynnchum tiwninum Ymzki, nti-inflmmtory, cyclooxygense-2 (COX-2), inducile nitric oxide synthse (inos). INTRODUCTION Inflmmtion rection is not only the response of living tissues to infection nd injury ut lso relevnt to diseses including therosclerosis nd cncer (Coussens nd Wer, 22). Severl reports indicted tht over- *Corresponding uthor. E-mil: tcw511@gmil.com. Tel: x 333. Fx: production of nitric oxide (NO) re cytotoxic nd re in reltion to cell injury or tissue dmge in numer of diseses such s inflmmtion nd crcinogenesis (Mordn et l., 1993). Therefore, to find potent nitric oxide synthse (NOS), inhiitor might e n effective therpeutic strtegy for inflmmtion-relted diseses (Koo et l., 21). In inflmmtory tissues, prostglndin E2 (PGE 2 ) is lso one of the mjor meditors nd is well-known oncogenic signl (Nrumiy et l., 1999).

2 Lee et l In the inflmmtion process, two inducile enzymes, tht is, the inducile nitric oxide synthse (inos) nd cyclooxygense-2 (COX-2), re ccountle for the synthesis of NO nd PGE 2 (Sripnidkulchi et l., 29), respectively. Recently, the ctivities of these two enzymes could e inhiited y severl plnt extrcts such s wild itter gourd (Lii et l., 29) nd green te (Nkgw nd Yokozw, 22). At inflmed sites, NO nd PGE 2 hve pleiotropic-effects nd produced respectively y COX-2 nd inos which re induced in response to different stimuli including cytokines (Smith et l., 2). Therefore, the inhiitors of these two enzymes my lso e considered s nti-inflmmtory gents. It is well-known tht the phosphoryltion of I kpp B- lph (IκBα) result in the degrdtion of IκBα nd consequent dissocition of nucler fctor-kpp B (NFκB) from IκBα (Hyden nd Ghosh, 24). Susequently, the free NF-κB trnsloctes into the nucleus nd then ctivtes its trget genes (Vinygmoorthi et l., 25), including inos nd COX-2 (Stylinou et l., 1998) nd then incresing the expression of inflmmtory meditors (L et l., 23). Hence, phosphoryltion of IκBα to phospho-iκbα (p-iκbα) is decisive step in the NF-κB ctivtion pthwy (Yin et l., 1998). In ddition, the induction of COX-2 nd ino synthse y cytokines, such s interleukin-1β (IL-1β), is well known to e prtly medited y NF-κB (Newton et l., 1997). Bo-Ye-Niu-Pi-Xio, Cynnchum tiwninum Ymzki (fmily: Asclepidcee) is well-known nd populr her, which its rhizome hs een used s folk medicine in Tiwn. Although mny therpeutic effects of C. tiwninum Ymz. hd een studied, such s ntiferile, diuretic, ntitussive, expectornt, nodyne, nd tonic ctivities in Chin (Chen et l., 1991) nd heptoprotection in Tiwn (Lin et l., 1995), there is still no nti-inflmmtory effect nd mechnism of C. tiwninum Ymz. reported. Recently, numer of reserchers focused on the chemicl constituents (Lin et l., 1998) nd potentil iologiclly ctive compounds of C. Tiwninum Ymz. (Chen et l., 1991); however, little is studied out its ctul effects nd mechnisms of the forementioned phrmcologicl ctivities. The ojective of this study is to evlute the ctivity nd distinguish the mechnism of nti-inflmmtion of C. tiwninum Ymz. rhizome ethnolic extrct (CTEE) on rt kidney cell line NRK-52E induced y interleukin 1-β (IL-1β). MATERIALS AND METHODS Plnt mteril The C. tiwninum rhizome ws collected from Neimen region, Kohsiung, in Southern Tiwn, in April 28. The plnt ws identified y Dr. Yn of the Ntionl Museum of Nturl Science, Tichung, Tiwn. The voucher specimen of C. tiwninum ws deposited in Deprtment of Mngement nd Utiliztion, Fengshn Tropicl Horticulturl Experiment Brnch. The herrium numer is FTHES-8-1. The clened rhizome ws sujected to slicing, hot ir-drying (5 C) nd then grinding into powder. The ethnolic extrct of C. tiwninum ws prepred s follows. 1 g of the C. tiwninum powder ws soked in 5 ml 95% ethnol t 6 C nd shken for 1 h. The C. tiwninum ethnolic extrct (CTEE) ws concentrted using vcuum concentrtor nd then sujected to freeze-drying. The percentge yield of the ethnolic extrct ws 2.2% (w/w). The CTEE ws filtered through.45 µm filter nd stored t 4 C until use. Chemicls nd regents Anti-serum inos, I-κB, p-iκb nd NF-κB were purchsed from Snt Cruz Biotechnology Inc. (CA, USA). Avidin-horserddish perxidse (Av-HRP) ws otined from BD Phrmingen Inc. (Cliforni, USA). Bovine serum lumin, sodium icronte, 3- (4,5-dimethylthizol-2-yl)-2,5-diphenyl-tetrzolium romide (MTT) formzn, sulfnilmide, N-(1-nphthyl)ethylenedimine dihydrochloride, ethidium romide, 4-(2-hydroxyethyl)-1- piperzineethnesulfonic cid (HEPES), potssium chloride, mgnesium chloride, ethylene dimine tetrcetic cid (EDTA), dithiothreitol (DTT), phenylmethnesulfonyl fluoride (PMSF), leupeptin, protinin, NP-4 lysis uffer (NP-4), 3,3- diminoenzidine (DAB), β-ctin nd glycerol were purchsed from Sigm Chemicl Co. (MO, USA). COX-2, PGE 2 nd PGE 2- cetylcholineesterse conjugte were otined from Cymn Co. (Ann Aror, MI, USA). Isopropnol ws otined from Merck Chem. Inc. (Germny). Esy-BLUE TM RNA extrction kit ws otined from INtRON Biotechnology Inc. (Kore). Fetl ovine serum (FBS), Dulecco s modified egle s medium (DMEM), TRI ZOL regent, penicillin-streptomycin nd trypsin-edta were purchsed from GIBCO-BRL (NY, USA). Interleukin-1β (IL-1β) ws otined from R&D Systems Inc. (MN, USA). Tq DNA polymerse, oligo-dt primer nd dntps were otined from Invitrogen Co. (NY, USA). Cell culture The norml rt kidney cell line NRK-52E (BCRC 686) ws purchsed from Bioresource Collection nd Reserch Center of Food Industry Reserch nd Development Institute in Tiwn nd mintined in DMEM contining 1% FBS, 1 U/ml penicillin, 1 µg/ml streptomycin, nd 1% L-glutmine, t 37 C in 5% CO 2 humidified ir. Cells were preincuted with nd without vrious concentrtion of CTEE (.1,.5, 1. mg/ml) followed y IL-1β (5 ng/ml) 3 min lter. Superntnts were collected fter IL-1β tretment for 18 h for NO/PGE 2 determintion, respectively. Cell viility ssy Cell viility ws evluted y the mitochondril-dependent MTT reduction method. The CTEE-treted cells were wshed with phosphte uffer sline (PBS) twice nd the medium ws replced with DMED without contining phenol red. MTT solution (.5 mg/ml) ws dded to the cell cultures. After incution for 3 h t 37 C, the medium ws removed gin nd the formzn crystls in vile cells were dissolved with 1 ml of isopropnol. After 1 min shking, the solution ws centrifuged t 5 g for 5 min. The sornce of the superntnt of ech smple ws determined t 57 nm. The opticl density of formzn formed in control (untreted) cells ws tken s 1% viility. Stimultion experiment Cells were hrvested y gentle scrping, plted into 24-well pltes

3 1692 Afr. J. Biotechnol. t density of cells per well, nd llowed to dhere for 24 h t 37 C under 5% CO 2 tmosphere. For stimultion, the culture medium ws replced with fresh DMEM contining 1% FBS, 1 µg/ml penicillin, 1 µg/ml streptomycin, nd 1% L-glutmine in the presence or sence of 5 ng/ml IL-1β. To evlute the effects of the extrcts, cells were first incuted with the CTEE t the concentrtions of.1,.5 or 1. mg/ml for 5 h, nd then with or without 5 ng/ml IL-1β s forementioned for 18 h. As reference controls, ssys were lso performed with medium contining PBS nd IL-1β only. Nitrite ssy As n indictor of NO production, we determined the nitrite (NO 2 - ) concentrtion in the culture medium y the Griess rection (Green et l., 1982). 1 µl of ech culture superntnt, ssyed in triplicte, ws rected with n equl volume of Griess regent (1% sulfnilmide nd.1% N-(1-nphthyl)ethylenedimine dihydrochloride in 2.5% phosphoric cid solution) t room temperture for 1 min under drk. Asornce ws mesured t 54 nm ginst clirtion curve with sodium nitrite stndrds. PGE 2 mesurement PGE 2 production ws mesured in culture medium in order to determine COX-2 ctivity. The culture medium of control nd treted cells ws collected, centrifuged, nd stored t -8 C until tested. The level of PGE 2 relesed into culture medium ws quntified y using commercil competitive enzyme immunossy kit (EIA, Cymn Chemicl, Co., Ann Aror, MI, U.S.A.) ccording to the mnufcturer s instruction. Western lotting Whole cell extrcts contining equl quntities of proteins (3 to 5 µg) were electrophoresed in 1% polycrylmide gel. Susequently, the seprted proteins were trnsferred to polyvinylidene fluoride memrne using Semi-Dry Trnsfer Cell (TRANS-BLOT, BIO-RAD, U.S.A.). Briefly, the memrne ws locked for 3 min with locking uffer (5% skim milk in 5 mm Tris-HCl, 2 mm NCl, nd.5% Tween 2, ph 7.5), nd ws incuted with pproprite dilutions of primry ntiodies (ginst inos, COX-2, IκB, p- IκB nd NF-κB) for overnight t 4 C. After wshing twice with the forementioned uffer, the memrne ws further incuted for 2 h with 1:1 dilution of iotin-conjugted got nti-rit or ntimouse ntiody (Biotin Co., Duulin, Irelnd), nd developed with Av-HRP nd DAB solution. In the cse of COX-2, n enhnced chemiluminescence (ECL) method ws used s Western lot detection system (Amershm Biosciences, Inc., Pisctwy, NJ, U.S.A.) ccording to the mnufcturer s instruction. Nucler protein preprtion Cells were preincuted with ech of the CTEE for 3 h efore the ddition of 5 ng/ml IL-1β for 9 min. Cells were then wshed twice nd scrped with cold PBS nd centrifuged. The pellets were resuspended in the hypotonic extrction uffer contining 1 mm HEPES, 1 mm KCl, 1 mm MgCl 2, 1 mm EDTA,.5 mm DTT,.2 mm PMSF, 4 µg/ml leupeptin, 2 µg/ml protinin, nd.5% NP-4 for 15 min on ice nd were then centrifuged t 6 g for 15 min. Nucler proteins were extrcted y gently mixing with 5 µl hypertonic extrction uffer contining 1 mm HEPES,.4 mm KCl, 1 mm MgCl 2, 1 mm EDTA,.5 mm DTT,.2 mm PMSF, 4 µg/ml leupeptin, 2 µg/ml protinin, nd 1% glycerol t 4 C for 3 min. The smples were then centrifuged t 1, g for 15 min. The superntnt fluid contining the nucler proteins ws collected for nucleic protein ssy. RNA extrction nd reverse trnscription-polymerse Chin Rection (RT-PCR) Totl cellulr RNA ws isolted using n esy-blue TM RNA extrction kit ccording to the mnufcturer s instructions. Briefly, totl cellulr RNA ws extrcted with TRI ZOL regent (Gico Lortories, Inc., Grnd Islnd, NY, U.S.A.) ccording to the mnufcturer s instructions. RNA concentrtions were clculted from sornce t 26 nd 28 nm. Totl RNA (2 mg) ws converted to cdna y tretment with 2 units of reverse trnscriptse nd 5 ng of oligo-dt primer in 5 mm Tris-HCl (ph 8.3), 75 mm KCl, 3 mm MgCl 2, 1 mm DTT, nd 1 mm dntps t 42 C for 1 h. The rection ws then stopped y incuting the solution t 7 C for 15 min, fter which 3 ml of the cdna mixture ws used for enzymtic mplifiction. PCR ws then performed using rection mixture comprised of 5 mm KCl, 1 mm Tris-HCl (ph 8.3), 1.5 mm MgCl 2,.2 mm dntps, 2.5 units of Tq DNA polymerse, nd.1 m M ech of primers specific for inos, COX-2, nd β-ctin. The mplifiction conditions were s follows: Denturtion t 94 C for 3 min for the first cycle nd then 35 cycles of 94 C for 45 s, nneling of inos t 56 C for 45 s or nneling of COX-2 t 53 C for 45 s with finl extension t 72 C for 7 min. The PCR products were then electrophoresed on 1.5% grose gel nd stined with ethidium romide. β-ctin which served s n internl control. The primers used in this study were s follows: inos, 5 -AGCCCAACAATACAAATGACCCTA-3 (sense) nd 5 - TTCCTGTTGTTTCTATTTCCTTTGT-3 (ntisense); Cox-2, 5 - CACTCAGTTTGTTGAGTCATTC-3 (sense) nd 5 - GATTAGTACTGTAGGGTTAATG-3 (ntisense); β-ctin, 5 - ATGAAGATCCTGACCGAGCGT-3 (sense) nd 5 - AACGCAGCTCAGTAACAGTCCG-3 (ntisense). Sttisticl nlysis The control nd tretment groups were compred y ANOVA. Duncn s t-test ws used for evluting sttisticl significnce. P <.5 ws considered to e sttisticlly significnt. RESULTS Effect of CTEE on cell viility To exclude possile cytotoxic effect of CTEE on NRK- 52E cells in either the presence or the sence of IL-1β, the MTT ssy ws employed. Cell viilities of NRK-52E cells were not significntly ltered nd the cell viilities were ove 95% up fter 18 h incution with vrious concentrtions of CTEE (.1,.5, nd 1. mg/ml) in the presence or sence of IL-1β (dt not shown). Effect of CTEE on IL-1β-induced NO nd PGE 2 production To estimte the effects of CTEE on IL-1β-induced NO nd PGE 2 production in NRK-52E cells, cells were treted

4 Lee et l (A) 15 Relted NO rtioo, % Relted NO rtio, %. 1 5 d d c f e (B) 15 Relted PGE2 rtioo, % Relted PGE2 rtio, % 1 5 c c d d cd IL-1β (5 ng/ml) Ethnol CTEE (.1 mg/ml) CTEE (.5 mg/ml) CTEE (1. mg/ml) Figure 1. Effects of CTEE on NO (A) nd PGE 2 (B) production in IL-1β-induced NRK-52E cells. NRK- 52E cells were treted with IL-1β (5 ng/ml) lone or with CTEE (.1,.5 or 1 mg/ml, respectively) for 18 h. Dt re the men ± S.D. from three or five independent experiments nd re expressed s the percentge of the PBS vehicle control. Vlues not shring the sme letter re significntly different (P <.5). with.5 ng/ml IL-1β nd vrious concentrtions of CTEE for 18 h. After collecting the culture medi, the nitrite concentrtions, reflecting the NO, within the medi were ssyed. IL-1β stimultion generted noticele ccumultion of nitrite in the culture medium; however, CTEE significntly reduced the IL-1β-induced nitrite production in dose-dependent mnner (Figure 1A). Agreeing with the effect of CTEE on NO production, the CTEE lso generted remrkle decrese in PGE 2 in dose-dependent mnner (Figure 1B). When CTEE concentrtion ws 1 mg/ml, the reduction percentge of IL-1β-induced NO nd PGE 2 were 5. nd 78.%,

5 1694 Afr. J. Biotechnol. respectively. Which oth inhiition of NO nd PGE 2 correlted with the inhiition of inos nd COX-2 ctivities, nd oth inhiition of NO nd PGE 2 correlted with the inhiition of inos nd COX-2 ctivities. Effect of CTEE on IL-1β-induced mrna nd protein expression of inos nd COX-2 For verifying whether the inhiitory effects of CTEE on NO nd PGE 2 production originte from decresed messenger rionucleic cid (mrna) nd protein expression of IL-1β-induced over expression of inos nd COX-2 in NRK-52 cells, the RT-PCR nd Western lot nlysis hve een executed. The expression of the inos nd COX-2 mrna nd protein were significntly (P <.5) lower in unstimulted cells thn those of in stimulted cells; however, these were extremely incresed fter IL-1β-induced tretment. CTEE exhiited significnt nd dose-dependent inhiitory effect on mrna nd protein expression of inos nd COX-2 in IL- 1β-induced NRK-52E cells (Figure 2A nd B). The low concentrtion (.1 mg/ml) of CTEE could not significntly (P >.5) suppressed the mrna expression; however, when CTEE concentrtion ws 1 mg/ml, CTEE significntly (P <.5) decresed protein nd mrna expression of inos nd COX-2 (y 9. nd 64.% for inos nd y 87. nd 85.% for COX-2, respectively). The findings were consistent with the enhnced production of NO nd PGE 2 shown in Figure 1. Effect of CTEE on IL-1β-induced phosphoryltion of IκBα The phosphoryltion of IκBα to p-iκbα cn results in IκBα itself degrdtion nd the relese of NF-κB which then trnsloctes to the nucleus. Accordingly, we inquired further whether CTEE could preclude the phosphoryltion of IκBα induced y IL-1β. Becuse p-iκbα is mrker of NF-κB pthwy ctivtion, we hve determined the expression of p-iκbα in this study. Figure 3 show tht phosphoryltion of IκBα ws remrkly incresed upon exposure to IL-1β lone, nd tht the phosphoryltion ws significntly (P <.5) inhiited in the presence of CTEE in dose-dependent mnner. And the inhiitory effect of CTEE on phosphoryltion of IκBα ws ccounted for the following NF-κB nucler trnsloction. Effect of CTEE on IL-1β-induced nucler trnsloction of NF-κB p65 To elucidte the mechnisms underlying the inhiition of inos nd COX-2 expression in IL-1β-induced NRK-52E cells, we inspected the effects of CTEE on the NF-κB nucler trnsloction. Becuse the NF-κB p65 is the mjor component of the ctivted NF-κB, we hve evluted the levels of NF-κB p65 in nucler extrcts y Western lotting nlysis. CTEE inhiited IL-1β-induced nucler trnsloction of NF-κB p65, in dose-dependent mnner (Figure 4). Tken together, these dt suggest tht the inhiitory effect of CTEE on the IL-1β-induced trnsloction of NF-κB p65 might e involved in the suppression of IκBα phosphoryltion. DISCUSSION The inflmmtory cytokine IL-1β plys s n immunoregultory roles involved in mny pthologicl processes. Severl reports demonstrted tht IL-1β stimultion significntly enhnced the production of NO nd PGE 2 (Tmur et l., 22; Wu et l., 25) y enhncing the expression of COX-2 nd inos (Wu nd Guo, 27); however, the stimultion ws prevented y either trnscriptionl or trnsltionl lockers (Newton et l., 1997). The IL-1β ws employed s stimultor to induce inflmmtion response in NRK-52E cells in this study. The results indicted tht IL-1β successfully induced inflmmtion response nd incresed the production of NO nd PGE 2 in NRK-52E cells. As expected, the CTEE significntly reduced IL-1β induced NO nd PGE 2 production in dose-dependent mnner in NRK-52E cells. The NO production ws regulted y constitutive NOS (cnos) under norml physiologicl conditions (Nkgw nd Yokozw, 22); however, NO ws lso over-produced t inflmed sites y inos in inflmmtory events (Kim et l., 1999). Therefore, regultion of inos in tissues is very importnt for the tretment of inflmmtion nd tumorigenesis (Chn et l., 1998). On the other hnd, COX-2, one of inducile enzymes lso, is crucil in the inflmmtory response, converts rchidonic cid to PGE 2 nd is required in introducing nd sustining rections during the inflmmtory course (Surh et l., 21). Prk et l. (24) noted tht overproduction of PGE 2 medited y COX-2 ws connected to the development of inflmmtion nd crcinogenesis. In this study, we found the expression of inos nd COX-2 protein nd mrna were inhiited y CTEE, confirming the suppressive effect of CTEE on the NO nd PGE 2 production. The present work lso showed tht the inhiitory effect of CTEE on IL-1β-induced inos nd COX-2 expression my hve resulted from the trnscriptionl inhiition of inos nd COX-2 gene, respectively. The present study demonstrtes for the first nlysis detecting NF-κB nd p-iκbα expression in IL-1β-induced NRK-52E cells. Our dt show tht IκBα ws phosphorylted nd ws degrded to p-iκbα nd then the NFκB complex ws presented oth in cytosol nd nucleus. Becuse the phosphoryltion nd degrdtion of IκBα is necessry for NF-κB ctivtion (Henkel et l., 1993)

6 Lee et l (A) (B) inos COX-2 β-ctin inos COX-2 β-ctin 2 2 inos protein intensity, % c inos mrna intensity, % c c c c c 2 2 COX-2 protein intensity, % COX-2 mrna intensity, % Figure 2. Effects of CTEE on inos nd COX-2 protein (A) nd mrna (B) expression in NRK-52E cells. NRK-52E cells were treted with IL-1β (5 ng/ml) lone or with CTEE (.1,.5 or 1 mg/ml, respectively) for 18 h. Protein extrcts from cell pellets were sujected to SDS-PAGE followed y the Western lot nlysis using nti-cox-2 nd nti-inos ntiodies. Totl mrnas were prepred from the cell pellets using TRIzol. The reltive levels of mrnas were ssessed y RT-PCR. Results were normlized to β-ctin. Dt re the men ± S.D. from three or five independent experiments nd re expressed s the percentge of the PBS vehicle control. Vlues not shring the sme letter re significntly different (P <.5). nd NF-κB signling pthwy involved in the regultion of inflmmtory responses (Silvermn nd Mnitis, 21), herein, se on our results, we not only considered tht IL-1β successfully induced the inflmmtion ut lso showed tht CTEE locked the NF-κB signling pthwy in IL-1β-induced NRK-52E cells. In terms of CTEE ntiinflmmtory mechnism, we showed tht CTEE exerted the inhiitory effect on the expression of inos nd COX- 2, s well s the production of NO nd PGE 2 ; moreover, the inhiitory effect ws ccompnied y reduced

7 1696 Afr. J. Biotechnol. 2 IκBα protein intensity, % IκB protein intestity, % p-iκbα protein intensity, % p-iκb proteoin intensity, % IL-1β (5 ng/ml) Ethnol CTEE (.1 mg/ml) CTEE (.5 mg/ml) CTEE (1. mg/ml) Figure 3. Effects of CTEE on IκBα nd p-iκbα protein level on NRK-52E induced y IL-1β. NRK-52E cells were treted with IL-1β (5 ng/ml) lone or with CTEE (.1,.5 or 1 mg/ml, respectively) for 18 h. Results were normlized to β-ctin. Dt re the men ± SD from three or five independent experiments nd re expressed s the percentge of the PBS vehicle control. Vlues not shring the sme letter re significntly different (P <.5). phosphoryltion of IκBα, confirming the inhiition resulted from the lockge of NF-κB pthwy. Recently, one of the newly designed drugs for inflmmtion-relted diseses ws to design n inhiitor to lock the NF-κB signling pthwys (Vitour et l., 25). The C. tiwninum Ymz. might e potentil cndidte s new drug gent. The mechnism of nti-inflmmtory effect of CTEE in IL-1β-induced NRK-52E cells ws elucidted in this study, nmely the suppression of NF-κB ctivtion cused y CTEE ccounted for the inhiition of the expression of inos nd COX-2, s well s the following inhiition of production of NO nd PGE 2.

8 Lee et l Cytosolic NF-κB protein intensity, % IκB protein intestity, % 5 Nucler NF-κB protein intensity, % p-iκb proteoin intensity, % 1 5 IL-1β (5 ng/ml) Ethnol CTEE (.1 mg/ml) CTEE (.5 mg/ml) CTEE (1. mg/ml) Figure 4. Effects of CTEE on cytosolic nd nucler NF-κB protein level on NRK-52E induced y IL- 1β. NRK-52E cells were treted with IL-1β (5 ng/ml) lone or with CTEE (.1,.5 or 1 mg/ml, respectively) for 18 h. Results were normlized to β-ctin. Dt re the men ± SD from three or five independent experiments nd re expressed s the percentge of the PBS vehicle control. Vlues not shring the sme letter re significntly different (P <.5). Mny flvonoids present in numerous dietry plnts (Yoon et l., 25) nd medicine hers (Hung et l., 1994) nd showed nti-inflmmtory ctivities through the inhiition of COX-2 nd inos expression (Jung et l., 27) s well s the suppression of NO production (Kim et l., 25). Flvonoids including kempferol nd quercetin in C tiwninum Ymz. were identified (Chen et l., 1991; Lin et l., 1998). Consistent with these

9 1698 Afr. J. Biotechnol. previous reports, we lso found the presence of quercetin in CTEE (our unpulished dt). Quercetin is one of nturlly occurring dietry flvonoids found in mny plnts nd cn down-regulte the inos protein expression in A549 humn lung denocrcinom cells (Bnerjee et l., 22) nd inhiits IL-1β-induced prolifertion in humn irwy smoothmuscle cells (Kumr- Roiné et l., 29) s well s ttenute NO production in IL-1β-induced heptocytes (Mrtínez-Flórez et l., 25). However, the nti-inflmmtory compound(s) of C. Tiwninum Ymz. nd/or their potentil synergistic ctivtion re uncler t present nd must wit future studies. These works re undertken in our lortory. In conclusion, the suppression of NO nd PGE 2 production y CTEE in NRK-52E cells treted with IL-1β could hve result from the inhiition of protein expression nd mrna trnscription of inos nd COX-2, respectively. Moreover, the cpcity of inhiition of NF-κB nucler protein DNA inding ctivity nd of IκBα phosphoryltion my ccount, t lest in prt, for the ntiinflmmtory mechnism of C. tiwninum Ymz. Arevitions COX-2, Cyclooxygense-2; inos, inducile nitric oxide synthse; CTEE, Cynnchum tiwninum Ymzki rhizome ethnolic extrct; IκBα, I kpp B-lph; p-iκbα, phospho-iκbαl; NF-κB, nucler fctor kpp B; mrna, messenger rionucleic cid; NO, nitric oxide; PGE 2, prostglndin E2. REFERENCES Bnerjee T, Vn der Vliet A, Zioh VA (22). Downregultion of COX-2 nd inos y mentoflvone nd quercetin in A549 humn lung denocrcinom cell line. PLEFA, 66: Chn MM, Hung HI, Fenton MR, Fong D (1998). In vivo inhiition of nitric oxide synthse gene expression y curcumin, cncer preventive nturl product with nti-inflmmtory properties. Biochem. Phrmcol. 55: Chen ZS, Li JS, Kuo YH (1991). The constituents of Cynnchum tiwninum. J. Chin. Chem. Soc. 38: Coussens LM, Wer Z (22). Inflmmtion nd cncer. 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