British Journal of Pharmacology (2003) 139, & 2003 Nature Publishing Group All rights reserved /03 $

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1 British Journl of Phrmcology (23) 139, 11 2 & 23 Nture Publishing Group All rights reserved /3 $25. Inhibition of lipopolyscchride-inducible nitric oxide synthse, TNF- nd COX-2 expression by suchinone effects on I-jB phosphoryltion, C/EBPnd AP-1 ctivtion 1 Ae Kyung Lee, 1 Sng Hyun Sung, 1 Young Choong Kim & *,1 Sng Geon Kim 1 College of Phrmcy nd Reserch Institute of Phrmceuticl Sciences, Seoul Ntionl University, Seoul , South Kore Keywords: Abbrevitions: 1 Suchinone, lignn isolted from Sururus chinensis (Sururcee), is distereomeric lignn with cytoprotective nd ntioxidnt ctivities in cultured heptocytes. The effects of suchinone on the inducible nitric oxide synthse (inos), tumor necrosis fctor- (TNF-) nd cyclooxygense 2(COX- 2) gene expression nd on the ctivtion of trnscription fctors, nucler fctor-kb (NF-kB), CCAAT/ enhncer-binding protein (C/EBP), ctivtor protein-1 (AP-1) nd camp-response element-binding protein (CREB) were determined in Rw264.7 cells s prt of the studies on its nti-inflmmtory effects. 2 Expression of the inos, TNF- nd COX-2genes ws ssessed by Northern nd Western blot nlyses. NO production ws monitored by chemiluminescence detection using NO nlyzer. To identify the trnscriptionl fctors ffected by suchinone, the extents of NF-kB, C/EBP, AP-1 nd CREB ctivtion were mesured. Activtion of the trnscription fctors ws monitored by gel mobility shift ssy, wheres p65 nd I-kB were nlyzed by immunocytochemicl nd immunoblot nlyses. 3 Suchinone inhibited the induction of inos, TNF- nd COX-2by lipopolyscchride () (IC5p1 mm) with suppression of the mrnas. 4 Suchinone (1 3 mm) inhibited -inducible nucler NF-kB ctivtion nd nucler trnsloction of p65, which ws ccompnied by inhibition of I-kB phosphoryltion. 5 -inducible increse in the intensity of C/EBP binding to its consensus sequence ws lso inhibited by suchinone. The AP-1, but not CREB, DNA binding ctivity ws wekly inhibited by suchinone. 6 These results demonstrte tht suchinone inhibits -inducible inos, TNF- nd COX-2 expression in mcrophges through suppression of I-kB phosphoryltion nd p65 nucler trnsloction nd of C/EBP nd/or AP-1 ctivtion, which my constitute nti-inflmmtory effects of the lignn. British Journl of Phrmcology (23) 139, doi:1.138/sj.bjp Nucler fctor-kb; inos; TNF-; COX-2; C/EBP CREB, camp-response element-binding protein; COX-2, cyclooxygense 2; FBS, fetl bovine serum; inos, inducible nitric oxide synthse; I-kB, Inhibitor-kB;, lipopolyscchride; NF-kB, nucler fctor-kb; PI, propidium iodide; SDS, sodium dodecyl sulfte; SSC, stndrd sline citrte; TNF-, tumor necrosis fctor- Introduction Sururus chinensis hs been trditionlly used for the tretment of heptitis in Orientl folk medicine (Chung & Shin, 199). The queous frction of the Sururus herbs lso induces humorl chnges implicted with hypertension nd symptomticlly relieves edem (Chung & Shin, 199). Distereomeric lignns including suchinone, suchinone A nd 1 -epi-suchinone hve been isolted from the n-hexne frction of S. chinensis (Lour.) Bill. (Sururcee). Suchinone ws identified s biologiclly ctive lignn (Figure 1). Previous studies hve shown tht suchinone protects heptocytes ginst the injury induced by toxicnts, s evidenced by both the inhibition of crbon tetrchloride-induced cell deth *Author for corresponding t: College of Phrmcy, Seoul Ntionl University, Sillim-dong, Kwnk-gu, Seoul , South Kore; E-mil: sgk@snu.c.kr. nd the restortion of cellulr glutthione nd ntioxidnt enzymes (Sung et l., 2). Lipopolyscchride () is n endotoxin, which induces septic shock syndrome nd stimultes the production of inflmmtory meditors such s NO, tumor necrosis fctor- (TNF-), interleukins, prostnoids nd leukotrienes (Hewett & Roth, 1993; Wtson et l., 1999; Kubes & McCfferty, 2). NO is rdicl produced from L-rginine vi nitric oxide synthse (NOS) nd lso n importnt cellulr second messenger. NO plys dul role s both beneficil nd detrimentl molecule in the process of inflmmtion. Inducible NOS (inos) is cpble of producing high output of NO during inflmmtion, wheres constitutively expressed NOS is physiologiclly criticl. It hs been proposed tht inosmedited high output production of NO cuses cell injury through genertion of potent rective rdicls such s

2 12 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone inhibited by suchinone in prllel with the suppression of inflmmtory gene expression. Methods Regents Figure 1 A chemicl structure of suchinone. peroxynitrite. Since NO is one of the mjor contributing fctors during the inflmmtory process, we first studied the effects of suchinone on nucler fctor-kb (NF-kB)-medited inos expression nd NO production in mcrophges exposed to. TNF- is the principl meditor of the responses to nd my ply role in innte immune responses. High concentrtions of cuse tissue injury nd shock, in which TNF- is one of the principl meditors. As prt of the studies on suchinone s effects ginst cute inflmmtion, we designed to study the effect of suchinone on -inducible TNF- expression. Cyclooxygense 2(COX-2) is induced by, certin serum fctors, cytokines nd growth fctors, nd is predominnt cyclooxygense t sites of inflmmtion. Development of COX-2inhibitors represents mjor dvnce in the therpy of inflmmtory processes nd their use includes prevention or tretment of disorders ssocited with the induction of this enzyme (e.g. colon cncer). In view of the observtion tht suchinone hs cytoprotective nd ntioxidnt effects in cultured heptocytes, we further evluted the effect of suchinone on -inducible COX-2gene expression in mcrophges. NF-kB, which is ctivted by the inflmmtory responses during virl nd bcteril infections (Grilli & Memo, 1999; Kim et l., 2), is involved in the expression of inos nd TNF- genes (Wtson et l., 1999). In ddition to the potentil role of NF-kB response element in the expression of COX-2, the regultory region for the gene includes CCAAT/enhncerbinding protein (C/EBP) nd camp-response element (CRE)/ E-box elements. Activtion of ctivtor protein-1 (AP-1) consisting of Jun, Fos nd Fr homodimers or heterodimers is ssocited with the cellulr oxidtive stress nd the ltered redox stte, nd the trnscriptionl fctor regultes expression of the ssocited genes including inos nd TNF- (Dieter et l., 1999; Zhou et l., 21). Since NF-kB, C/EBP, AP-1 nd cyclic-amp-response element-binding protein (CREB) re commonly or individully involved in the regultion of inflmmtory genes, ltertions in the ctivtion of these trnscription fctors by suchinone were determined s prt of the mechnistic studies. The effects of suchinone on -induced ctivtion of NFkB nd degrdtion of I-kB nd inos gene expression were monitored by gel mobility shift ssy nd immunoblot nlysis. The DNA binding ctivities of C/EBP, AP-1 nd CREB were lso monitored to identify the trnscriptionl fctors ffected by suchinone in ssocition with the suppression of TNF- nd COX-2. We found tht ctivtion of NF-kB, C/EBP nd AP-1, but not tht of CREB, ws Suchinone, distereomeric lignn, ws isolted from the n- hexne frction of S. chinensis by successive silic gel chromtogrphy nd reverse-phse high-pressure liquid chromtogrphy. The chemicl structure ws confirmed by vriety of spectroscopic nlyses (Figure 1) (Sung & Kim, 2; Sung et l., 2). Alkline phosphtse-conjugted got nti-mouse nd nti-got IgGs were purchsed from KPL (Githersburg, MD, U.S.A.). Anti-c-Rel (p65), nti-p5, nti-i-kb, nti-c/ebp (, b, d, e) nd p3 ntibodies were supplied from Snt Cruz Biotechnology (Snt Cruz, CA, U.S.A.). Antimurine inos ntiserum ws obtined from Trnsduction Lbortories (Lexington, KY, U.S.A.). [- 32 P]dCTP (3 mci mmol 1 ) nd [g- 32 P]ATP (3 mci mmol 1 ) were obtined from Amershm (Arlington Heights, IL, U.S.A.). The consensus sequence of NF-kB, C/EBP, AP-1 nd CREB, nd rndom-prime nd 5 -end lbeling kits were supplied from Promeg Co. (Mdison, WI, U.S.A.). Most regents for the moleculr studies were obtined from Sigm Chemicl Co. (St. Louis, MO, U.S.A.). Cell culture Rw264.7 cells, murine mcrophge cell line (Americn Type Culture Collection, Mensss, VA, U.S.A.), were cultured in Dulbecco s modified Egle s medium contining 1% fetl bovine serum (FBS), 1 U ml 1 penicillin nd 1 mgml 1 streptomycin. Rw264.7 cells were plted t density of ml 1 nd preincubted for 24 h t 371C. Cells were mintined t 371C in humidified tmosphere contining 5% CO 2. For ll experiments, cells were grown to 8 9% confluency, nd were subjected to no more thn 2 cell pssges. Rw264.7 cells were incubted with 1 mgml 1 (Escherichi coli 26:B6; Difco, Detroit, MI, U.S.A.) to ctivte NF-kB, C/EBP, AP-1 nd CREB, nd to stimulte the inos, COX-2nd TNF- gene expression. Cells were incubted in the medium without 1% FBS for 12h nd then exposed to or +suchinone for the indicted time periods (1 18 h). Suchinone s dissolved in dimethylsulfoxide ws dded to the incubtion medium 1 h prior to the ddition of. Dimethylsulfoxide (vehicle) lone ws ineffective. Assy of nitrite production NO production ws monitored by mesuring the nitrite content in culture medium. This ws performed by mixing the smples with Griess regent (1% sulfnilmide,.1% N-1- nphthylenedimine dihydrochloride nd 2.5% phosphoric cid). Absorbnce ws mesured t 54 nm fter incubtion for 1 min. Northern blot nlysis Totl RNA ws isolted from Rw264.7 cells ccording to the modified method of Chomczynski & Scchi (1987). Totl

3 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone 13 RNA (2 mg) ws resolved by electrophoresis in 1% grose gel contining 2.2 M formldehyde nd trnsferred to nitrocellulose pper by cpillry trnsfer. The nitrocellulose pper ws bked in vcuum oven t 81C for 2h. Blots were incubted in hybridiztion buffer contining 5% deionized formmide,.1% sodium dodecyl sulfte (SDS), 2 mgml 1 of sonicted slmon sperm DNA, 6 stndrd sline/phosphte/edta (1 stndrd sline/phosphte/edta contins.15 M NCl, 1 mm NH 2 PO 4, nd 1 mm N 2 EDTA, ph 7.4), nd 5 Denhrdt s solution [.1% Ficoll,.1% polyvinylpyrrolidine nd.1% bovine serum lbumin (Pentex frction V)] t 421C for 1 h without probe. Specific cdna probes for TNF nd COX-2genes were mplified by reverse trnscriptionpolymerse chin rection (RT PCR) using the selective primers nd cloned in TA vector (Promeg, Mdison, WI, U.S.A.). The primers used re s follows, COX-2, sense primer: 5 -TCTCCAACCTCTCCTACTAC-3, ntisense primer: 5 -GCACGTAGTCTTCGATCACT-3 (624 bp); nd TNF-, sense primer: 5 -TACTGAACTTCGGGGTGATTGGTCC-3, ntisense primer: 5 -CAGCCTTGTCCC-TTGAAGAGAA- CC-3 (295 bp). Hybridiztion ws performed t 421C for 18 h with het-dentured probe, which ws rndom-prime lbeled with [- 32 P]dCTP. Filters were wshed in 2 stndrd sline citrte (SSC) (1 SSC contins.15 M NCl nd.15 M sodium citrte, ph 7.4) nd.1% SDS for 1 min t room temperture twice nd in.1 SSC nd.1% SDS for 1 min t room temperture twice. Filters were finlly wshed in the solution contining.1 SSC nd.1% SDS for 6 min t 61C. The stripped membrnes were hybridized with lbeled probe complementry to 18S rrna to quntify the mount of RNA loded onto the grose gel nd trnsferred to the nitrocellulose pper. Films were exposed t 71C for h using intensifying screens. Ech dt point represents men7s.d. from independent mesurements of three or four different experiments. RT PCR nlysis RT PCR ws performed with totl RNA (.5 mg) obtined from the liver using the selective primers [inos, sense primer: 5 -ATGTCCGAAGCAAACATCAC-3, ntisense primer: 5 - TAATGTCCAGGAAGTAGGTG-3, 499 bp; glycerldehyde-3-phosphte dehydrogense (GAPDH), sense primer: 5 -TCGTGGAGTCTACTGGCGT-3, ntisense primer: 5 - GCCTGCTTCACCACCTTCT-3, 51 bp]. PCRs were conducted using the following conditions for 38 cycles: denturtion t 941C for.5 min, nneling t 491C for.5 min, nd elongtion t 721C for 1 min. Bnd intensities of the mplified DNAs were compred fter visuliztion on n UV trnsillumintor. Immunoblot nlysis Cells were lysed in the buffer contining 2 mm Tris Cl (ph 7.5), 1% Triton X-1, 137 mm sodium chloride, 1% glycerol, 2 mm EDTA, 1 mm sodium orthovndte, 25 mm b-glycerophosphte, 2mM sodium pyrophosphte, 1 mm phenylmethylsulfonylfluoride nd 1 mg/ml leupeptin. Cell lystes were centrifuged t 1, g for 1 min to remove debris. Expression of inos nd COX-2ws immunochemiclly monitored in the lyste frction of Rw264.7 cells using ntimouse inos nd COX-2ntibodies, respectively. Polyclonl nti-i-kb ntibody ws used to ssess I-kB protein in cytosol. Polyclonl nti-c/ebpb nd C/EBPd ntibodies were used to ssess C/EBPb nd C/EBPd proteins in the nucler frction. The secondry ntibodies were lkline phosphtseconjugted nti-mouse nd nti-got ntibodies. The bnds of inos nd COX-2proteins were visulized using 5-bromo-4- chloro-3-indolylphosphte nd 4-nitroblue tetrzolium chloride, or ECL chemiluminescence detection kit. Enzyme-linked immunosorbent ssy (ELISA) Rw264.7 cells were preincubted with 3 3 mm suchinone for 1 h nd then further incubted with (1 mgml 1 ) for 6 h. The level of TNF- in the culture medium ws mesured by ELISA using nti-mouse TNF- ntibody nd biotinylted secondry ntibody (Endogen, Woburn, MA, U.S.A.). Preprtion of nucler extrcts Nucler extrcts were prepred essentilly ccording to Schreiber et l. (199). Briefly, dishes were wshed with icecold PBS, scrped nd trnsferred to microtubes. Cells were llowed to swell by dding 1 ml of lysis buffer [1 mm HEPES (ph 7.9), 1 mm KCl,.1 mm EDTA,.5% Nonidet-P4, 1 mm dithiothreitol nd.5 mm phenylmethylsulfonylfluoride]. Tubes were vortexed to disrupt cell membrnes. The smples were incubted for 1 min on ice nd centrifuged for 5 min t 41C. Pellets contining crude nuclei were resuspended in 5 ml of the extrction buffer contining 2mM HEPES (ph 7.9), 4 mm NCl, 1 mm EDTA, 1 mm dithiothreitol nd 1 mm phenylmethylsulfonylfluoride, nd then incubted for 3 min on ice. The smples were centrifuged t 15,8 g for 1 min to obtin the superntnt contining nucler extrcts. Gel retrdtion ssy A double-strnded DNA probe for the consensus sequence of NF-kB (5 -AGTTGAGGGGACTTTCCCAGGC-3 ), C/EBP (5 -TGCAGATTGCGCAATCTGCA-3 ), AP-1 (5 -CGCTT- GATGACTCAGCCGGAA-3 ) nd CREB (5 -AGAGATTG- CCTGACGTCAGAGAGCTAG-3 ) were used for gel shift nlyses fter end lbeling of the probes with [g- 32 P]ATP nd T 4 polynucleotide kinse. The rection mixtures contined 2 ml of 5 binding buffer contining 2% glycerol, 5 mm MgCl 2, 25 mm NCl, 2.5 mm EDTA, 2.5 mm dithiothreitol,.25 mg ml 1 poly di-dc nd 5 mm Tris-Cl (ph 7.5), 2 mg of nucler extrcts nd sterile wter in totl volume of 1 ml. Rections were initited by the ddition of 1 ml probe (1 6 cpm) following 1 min preincubtion nd continued for 2 min t room temperture. The specificity of protein binding to the DNA ws confirmed by competition rections, in which 2- fold molr excess of unlbeled oligonucleotides ws dded to ech rection mixture before the ddition of rdiolbeled probe. In some experiments, the specificity of NF-kB binding to the DNA consensus sequence ws confirmed by supershift nlysis using specific ntibodies directed ginst p65 or p5 (2 mg ech). Also, the specificity of C/EBP binding to the DNA consensus sequence ws confirmed by the inhibition of C/EBP binding nd supershift using nti-c/ebp, nti-c/ebpb, nti- C/EBPd, nti-c/ebpe or nti-p3 ntibody. Smples were

4 14 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone loded onto 4% polycrylmide gels t 14 V. The gels were removed, fixed nd dried, followed by utordiogrphy. Immunocytochemistry of p65 Stndrd immunocytochemicl method ws used to detect nucler trnsloction of p65 subunit of NF-kB (Cho et l., 22). Counter stining with propidium iodide (PI) verified the loction nd integrity of nuclei. After wshing, stined cells were exmined using lser scnning confocl microscope. Scnning densitometry Scnning densitometry ws performed with Imge Scn & Anlysis System (Alph-Innotech Corportion, Sn Lendro, CA, U.S.A.). One-wy nlysis of vrince (ANOVA) procedures were used to ssess significnt differences mong tretment groups. For ech significnt effect of tretment, the Newmn Keuls test ws used for comprisons of multiple group mens. The criterion for sttisticl significnce ws set t Po.1. Results Inhibition of -inducible inos expression The potentil toxicity of suchinone to Rw264.7 cells ws ssessed by MTT ssy fter 24 h incubtion. Cell vibility ws not chnged by the presence of suchinone up to the concentrtion of 1 mm. Thus, 1 3 mm concentrtions of suchinone were chosen in subsequent experiments. We determined whether suchinone inhibited the induction of inos by. NO production ws monitored in Rw264.7 cells stimulted by in the presence or bsence of suchinone for 18 h. (1 mgml 1 ) incresed the level of nitrite nd nitrte (i.e. NO) in culture medium by 2-fold s compred to control. Suchinone (1 3 mm) significntly inhibited -inducible NO production (i.e. 35 6%) (Figure 2). Western blot nlysis confirmed tht (1 mgml 1, 18 h) induced inos protein nd tht suchinone t the concentrtion of 1 mm mrkedly suppressed the induction of inos by (Figure 2b). Expression of inos ws 48% inhibited by suchinone. Studies were extended to determine whether the expression of inos protein prlleled tht of its mrna. mximlly incresed the inos mrna t 6 12h (Tkhshi et l., 2), which ws confirmed by our previous study (Cho et l., 22). RT PCR nlysis showed tht suchinone inhibited -inducible increse in the inos mrna (6 h) (Figure 2c). Inhibition of -inducible TNF- expression Production of TNF- ws mesured in the medium of Rw264.7 cells cultured with (1 mgml 1 ) in the presence or bsence of suchinone for 6 h (Figure 3). Suchinone t the concentrtions of 3 nd 3 mm inhibited TNF- production in -treted cells by 4 nd 5%, respectively. Northern blot nlysis ws used to verify whether the inhibition of TNF- production by suchinone ccompnied suppression of TNF- mrna. Suchinone lso inhibited the increse in TNF- mrna by (Figure 3b). NO Production (ng/ml) b c Reltive inos Levels (% of lone) inos mrna GAPDH Inhibition of -inducible COX-2 expression The expression of COX-2protein ws monitored in Rw264.7 cells exposed to (1 mgml 1, 12h). Suchinone (1 3 mm) effectively suppressed the induction of COX-2by (Figure 4). (1 mgml 1, 6 h) lso incresed the COX-2 mrna, which ws 49% inhibited by the presence of Su (µm) (µm) Su (µm) Su Figure 2 Effects of suchinone (Su) on the induction of inos by. () Inhibition of NO production by Su in Rw264.7 cells. Rw264.7 cells were treted with vrious concentrtions of Su dissolved in dimethylsulfoxide for 1 h prior to the ddition of (1 mgml 1 ), nd the cells were further incubted for 18 h. cells were incubted with vehicle lone. The concentrtions of nitrite nd nitrte in culture medium were monitored s described in the Methods section. (b) Inhibition of -inducible inos protein expression by Su. The level of inos protein ws monitored 18 h fter tretment of cells with (1 mgml 1 ) with or without Su pretretment (i.e. 1 h before ). The reltive inos protein levels were mesured by scnning densitometry of the bnd intensities. (c) The effects of Su on the inos mrna level in Rw264.7 cells stimulted with (1 mgml 1 ). RT PCR ws performed to determine inos mrna in totl RNA frctions (4 mg ech) isolted from cells treted with in the presence or bsence of Su. Cells were pretreted with Su for 1 h followed by the ddition of, nd the inos mrna level ws ssessed 6 h fter the ddition of. The mount of RNA loded in ech lne ws confirmed by RT PCR of GAPDH mrna.

5 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone 15 TNF-α (ng/ml) b Reltive TNF-α mrna Levels (% of lone) suchinone (Figure 4b). Hence, suchinone ws ctive in suppressing the expression of the genes implicted with inflmmtion. Effect of suchinone on -inducible NF-kB ctivtion NF-kB is ctivted in cells chllenged with nd other inflmmtory stimuli nd involved in the trnscriptionl ctivtion of responsive genes (Bldwin, 1996). Gel shift nlysis ws conducted to determine whether suchinone chnged NF-kB DNA binding ctivity. Previous studies hve shown tht NF-kB ws ctivted t 3 min 1 h fter tretment (Kim et l., 2). (1 mgml 1, 1 h) incresed the binding ctivity of nucler extrcts to the NF-kB DNA consensus sequence. Tretment of mcrophges with suchinone (3 mm) for 1 h prior to the ddition of significntly (o5%) inhibited -inducible increse in the bnd intensity of NF-kB binding (Figure 5, left). We chose the concentrtion of 3 mm for the gel shift nlysis becuse preliminry study showed tht suchinone notbly inhibited NF-kB ctivtion t the concentrtion. Addition of nti-p65 ntibody (µm) Su (µm) Su TNF-α 18S rrna Figure 3 The effect of suchinone (Su) on -inducible TNF- expression. () The level of TNF-. Production of TNF- ws mesured in the medium of Rw264.7 cells cultured with (1 mgml 1 ) in the presence or bsence of Su for 6 h. (b) The expression of TNF- mrna. TNF- mrna ws monitored by Northern blot nlysis in cells exposed to with or without Su for 3 h. The mount of RNA loded in ech lne ws confirmed by rehybridiztion of the stripped membrne with 32 P-lbeled probe complementry to 18S rrna. Ech br represents the men7s.d. from four seprte experiments. One-wy ANOVA ws used for comprisons of multiple group mens followed by Newmn Keuls test (significnt s compred to lone, Po.1). Reltive COX-2 Levels (% of lone) b Reltive COX-2 mrna Levels (% of lone) (µm) COX-2 18S rrna to the rection mixture obtined from -treted cells cused supershift of the NF-kB DNA binding, wheres nti-p5 ntibody wekly shifted the retrded bnd. The presence of both nti-p65 nd nti-p5 ntibodies lso supershifted the NF-kB bnd (Figure 5, right). Since p65 ws the mjor component of NF-kB ctivted by in mcrophges, we determined the trnsloction of p65 into the nucleus by immunocytochemistry (Figure 5b). Rw264.7 cells were incubted with in the presence or bsence of 3 mm suchinone for 1 h, fixed nd permebilized. p65 protein ws locted predominntly in the cytoplsm of control cells. In contrst, p65 protein moved into the nucleus t 1 h fter tretment. The p65 protein ws detected predominntly in the cytoplsm of cells exposed to in combintion with suchinone, which verified tht suchinone inhibited nucler locliztion of p65 protein. Nucler integrity ws confirmed by PI stining of the identicl cells (Figure 5b). Trnsloction of NF-kB to the nucleus is preceded by proteolytic degrdtion of I-kB subunit. To ssess whether suchinone could directly ffect I-kB in mcrophge cells, the level of I-kB protein ws immunochemiclly ssessed in Rw264.7 cells incubted with or without suchinone (Figure 5c). (1 mgml 1 ) reduced the I-kB level t Su (µm) Figure 4 Inhibition of -inducible COX-2expression by suchinone (Su). () COX-2protein expression. COX-2expression ws mesured in Rw264.7 cells cultured with 1 mgml 1 with or without vrious concentrtions of Su for 12h. (b) The level of COX-2mRNA. COX-2mRNA ws ssessed by Northern blot nlysis in Rw264.7 cells exposed to with or without Su. The mount of RNA loded in ech lne ws confirmed by rehybridiztion of the stripped membrne with 32 P-lbeled probe complementry to 18S rrna. Ech br represents the men7s.d. from four seprte experiments. One-wy ANOVA ws used for comprisons of multiple group mens followed by Newmn Keuls test (significnt s compred to lone, Po.1). Su

6 16 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone NF-κB +Su Su +nti-p5 Ab +nti-p65 Ab +nti-p5/p65 Ab +NF-κB 2x SS b Immunocytochemistry p65 PI +Su c d I-κBα Actin p-i-κbα +Su Su Actin +Su Su Figure 5 Inhibition of -inducible NF-kB ctivtion nd I-kB phosphoryltion by suchinone (Su). () Gel shift nlysis of nucler extrcts using the consensus sequence of NF-kB. Nucler extrcts were isolted 1 h fter tretment (1 mgml 1 )of Rw264.7 cells, nd were subjected to gel shift nlysis. Ech lne contined 5 mg of nucler extrcts. The specificity of NF-kB binding ws confirmed by supershift nlysis using the ntibodies directed ginst p65 nd/or p5 protein. The rrow (left) shows NF-kB complex nd SS indictes supershift of the retrded NF-kB bnd. The specificity of NF-kB binding ws lso confirmed by the ddition of n excess mount of free probe (2 ). (b) Immunofluorescence subcellulr locliztion of p65 protein. Rw264.7 cells were treted with 1 mgml 1 of for 1 h, nd p65 protein locliztion ws immunochemiclly detected using nti-p65 ntibody. cused p65 protein to migrte to the nucleus t 1 h. Su (3 mm) prevented -induced nucler trnsloction of p65 protein. The sme fields were counter stined with PI for loction of nuclei. Results were confirmed by repeted experiments. (c) The effect of Su on -induced I-kB degrdtion. The effect of on I-kB degrdtion ws immunochemiclly ssessed in Rw264.7 cells. Degrdtion of I-kB protein ws significntly inhibited by tretment of cells with 3 mm Su for 1 h. Equl loding of proteins ws verified by ctin immunoblotting. (d) Western blot nlysis of phosphorylted I-kB. Representtive immunoblot shows the inhibition of I-kB phosphoryltion by Su in Rw264.7 cells. Phosphorylted I-kB protein ws monitored t 15 min. Equl loding of proteins ws verified by ctin immunoblotting. Results were confirmed by repeted experiments. 3 min, resulting in 25% of control. Suchinone significntly recovered the level of I-kB protein (Figure 5c). Phosphoryltion of I-kB precedes degrdtion of I-kB. Suchinone inhibited -inducible I-kB phosphoryltion (Figure 5d). Thus, suchinone inhibited nucler NF-kB binding through prevention of I-kB phosphoryltion nd subsequent nucler trnsloction of p65 protein. Effect of suchinone on -inducible C/EBP ctivtion Expression of the COX-2gene depends on the C/EBP element present in the upstrem region of the gene (Thoms et l., 2). To test whether suppression of COX-2 gene induction by suchinone ws medited by inctivtion of C/EBP, electrophoretic mobility shift for C/EBP binding ctivity ws

7 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone 17 performed with the nucler extrcts of cells exposed to in the presence or bsence of suchinone using rdiolbeled C/EBP consensus oligonucleotide. Tretment of cells with for 3 h resulted in n increse in C/EBP binding compred to control. Suchinone inhibited -inducible C/EBP binding (Figure 6, left). Addition of nti-c/ebpb or nti-c/ebpd ntibody to the nucler extrcts obtined from -treted cells cused supershift of the C/EBP DNA binding. Anti- C/EBP, nti-c/ebpe or nti-p3 ntibody filed to ffect the mobility of the retrded bnd (Figure 6, right). Next, we ssessed whether the levels of nucler C/EBPb nd C/EBPd were chnged by suchinone in -treted cells. The increse in nucler C/EBPb by ws completely inhibited by suchinone (Figure 6b, upper). However, the level of +Su Su C/EBP +nti-c/ebp ε Ab +nti-c/ebp δ Ab +nti-c/ebp β Ab +nti-p3 Ab +C/EBP 2x +nti-c/ebp α Ab SS C/EBPd protein ws not chnged by the presence of suchinone (Figure 6b, lower). These results verified tht suchinone inhibits the nucler trnsloction of C/EBPb nd C/EBPb binding to the consensus DNA oligonucleotide. Effect of suchinone on -inducible AP-1 nd CREB ctivtion Studies were extended to determine whether suchinone ffected the ctivtion of AP-1. Gel shift retrdtion nlysis reveled tht nucler AP-1 trnscription complex ws ctivted by (1 h) in mcrophges. The specificity of the DNA probe to -ctivted AP-1 binding complex ws supported by competition for binding to rdiolbeled AP-1 probe with n excess of unlbeled AP-1 oligonucleotide, nd the bnd intensity of the migrting complex with the AP-1 consensus oligonucleotide ws decresed by the specific ntibodies ginst Jun D (Cho MK & Kim SG, unpublished dt). Pretretment of cells with 3 mm suchinone wekly inhibited -inducible AP-1 ctivtion with slight decrese in gel retrdtion (Figure 7). It hs been shown tht muttion of the CRE site in the COX-2gene brogted COX-reporter ctivity nd tht expression of CREB substntilly repressed dependent COX-reporter ctivity presumbly through CRE site(s) (Wdleigh et l., 2). We monitored the effect of suchinone on -inducible CREB binding ctivity. In cells exposed to (6 h), suchinone (3 mm) filed to chnge the bnd intensity of CREB DNA binding (Figure 7b). AP-1 b CREB +Su Su +Su Su b C/EBP β Actin +Su Su C/EBP δ Actin +Su Su Figure 6 Activtion of C/EBP trnscription complex. () Gel shift nlysis of C/EBP. Nucler extrcts were prepred from Rw264.7 cells cultured with (1 mgml 1 ) in the presence or bsence of suchinone (Su, 3 mm) for 3 h. All lnes contined 5 mg of nucler extrcts nd 5 ng of lbeled C/EBP consensus sequence. The specificity of C/EBP binding ws confirmed by supershift nlysis using the ntibodies directed ginst C/EBP (, b, d, e) nd p3 proteins. The rrow (left) shows C/EBP complex nd SS indictes supershift of the retrded NF-kB bnd. The specificity of C/EBP binding ws confirmed by the ddition of n excess mount of free probe (2 ). (b) The levels of C/EBPb nd d proteins in the nucler frction. Suchinone (3 mm) inhibited the increse in the level of nucler C/EBPb. The level of C/EBPd in the nucler frction ws not notbly chnged. Equl loding of proteins ws verified by ctin immunoblotting. Figure 7 The effect of suchinone (Su) on AP-1 nd CREB binding ctivity. () AP-1 binding ctivity. Gel shift nlysis ws crried out with nucler extrcts from control cells or cells treted with in the presence or bsence of 3 mm Su for 1 h. Ech rection contined 5 mg of nucler extrcts nd 5 ng of rdio-lbeled AP-1 consensus sequence. Results were confirmed by repeted nlyses. (b) Gel shift nlysis of nucler extrcts using the consensus sequence of CREB. Nucler extrcts were isolted 6 h fter tretment in the presence or bsence of Su (3 mm). CREB binding ctivity ws ssessed s described in (). Results were confirmed by repeted experiments.

8 18 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone Discussion NO is free rdicl generted from L-rginine by NOS. Incresed expression of inos is ssocited with inflmmtory responses nd lso with serious disorders such s septic shock nd rheumtoid rthritis (Slerno et l., 22). In view of the involvement of inos in inflmmtory process, we monitored the inos gene expression in mcrophges exposed to suchinone, biologiclly ctive lignn derived from S. chinensis. Northern nd Western blot nlyses reveled tht suchinone inhibited the induction of inos. Suppression of inos expression by suchinone ws in prllel with the comprble inhibition of NO production. Both NF-kB nd AP-1 binding sites hve been identified on the murine inos promoter nd ply role in -medited induction of inos in Rw264.7 cells (Figure 8). The present study demonstrted tht suchinone prevents ctivtion of p65/nf-kb by in mcrophge cells nd effectively inhibits nucler trnsloction of p65. NF-kB is ctivted by oxidtive stress s well s by inflmmtion. Activtion of the NF-kB complex is lso relted with the cellulr redox stte (Hirot et l., 1999). The intrcellulr thiol level chnges the expression of severl genes following erly ctivtion of NF-kB (Prmentier et l., 2). The ctivtion of NF-kB cn be blocked by thiol compounds such s N-cetylcysteine nd cysteine (Shrivstv & Aggrwl, 1999). An dditionl study showed tht N-cetylcysteine (1 mm, 18 h) filed to chnge the inhibitory effect of suchinone on -inducible inos expression. Hence, the inhibition of NF-kB ctivtion by suchinone my not be relted with the chnge in the cellulr redox stte. The current study showed tht phosphoryltion of I-kB (i.e. degrdtion of I-kB), which is required for NF-kB ctivtion ws inhibited by suchinone. Members of the protein tyrosine kinse fmily ply roles in mcrophge ctivtion in response to (Geng et l., 1993). Phosphoryltion of I-kB in cells stimulted by is considered to be medited with the NF-kB-inducing kinse nd the subsequent I-kB kinse complexed with other proteins in the plsm membrne (Stncovski & Bltimore, 1997). This is supported by the observtion tht the inhibition of the NF-kB-inducing kinse nd the subsequent I-kB kinse reduced the expression of inos by in mcrophges (Mtusushim et l., 21). NF-kB becomes ctivted by I-kB degrdtion following phosphoryltion of I-kB t serine residues (Michel & Snker, 1998). The cellulr level of I-kB ws monitored to infer if suchinone blocked degrdtion process of I-kB. Phosphoryltion of I-kB, I-kB degrdtion nd nucler trnsloction of p65 protein by were ll significntly bolished in cells pretreted with suchinone. The recovery of I-kB protein in Rw264.7 cells provided strong evidence tht suchinone inhibited ctivtion of NF-kB s consequence of the inhibition of I-kB phosphoryltion. This supports tht suchinone my inhibit the upstrem cellulr kinse(s), but not NF-kB binding to the DNA sequence. The pthwys of NFkB-inducing kinse nd MEKK1 regulte the phosphoryltion of I-kB vi IKK (Pn et l., 2). It is likely tht the inhibition of I-kB phosphoryltion by suchinone is medited with the suppression of IKK. Additionl experiments were crried out to ssess whether suchinone chnged the nucler binding ctivity of AP-1 in response to. We lso found tht incresed the AP-1 binding ctivity in mcrophges. Previous studies hve shown tht the AP-1 complex ctivted by includes Jun fmily members including Jun D, c-jun nd Jun B (Grnger et l., 2). The migrtion of -induced retrded AP-1 bnd ppered to be fster in the presence of suchinone, indicting tht one or more components involved in the ctivtion of AP- 1 complex were ffected by the gent. It is highly likely tht inhibition of -induced AP-1 ctivtion by suchinone lso Figure 8 The cis-cting response elements present in the promoter regions of the murine inos, TNF- nd COX-2genes. The NFkB nd AP-1 binding sites on the inos promoter ply role in the induction of inos. Wheres the upstrem promoter region of the TNF- gene contins binding sites for the ctivtors including NF-kB, C/EBPb nd c-jun, tht of the COX-2gene comprises NF-kB, C/EBP nd CREB binding sites.

9 A.K. Lee et l Inhibition of inflmmtory gene expression by suchinone 19 contributes to the inhibition of inos expression. The ctivtion of AP-1 is lso involved in the induction of inos (Cho et l., 22). In ddition to the inhibition of the NF-kB ctivtion by suchinone, slight decrese in the AP-1 ctivtion in this study my lso contribute to the inhibition of inos induction. TNF- is toxic cytokine, which is involved in inflmmtion nd other pthologicl processes such s rheumtoid rthritis nd infections. Also, mcrophges re the principl source of TNF-. TNF- promoter reporter ssy reveled tht ctivtion of NF-kB lrgely contributes to the induction of TNF- expression mong the NF-kB (kb3), C/EBPb nd c- Jun binding sites present in the 12 bp promoter region of humn TNF- gene (Figure 8) (Liu et l., 2). In the ctivtion of NF-kB, the cellulr NF-kB p65 subunit ws identified s dominnt trnscription fctor responsible for the induction of the TNF- gene (Liu et l., 2). In the present study, the ctivtion of the bnd retrded s complex of p65 nd p5 ws inhibited by suchinone, which ws consistent with blockge of the nucler trnsloction of p65 subunit. Hence, the inhibitory effect of suchinone on TNF- expression my lso result from the inhibition of NF-kB ctivtion nd of I-kB phosphoryltion. It hs been reported tht neither c-jun nor C/EBP ctivtion ffected the expression of TNF- gene (Liu et l., 2). Hence, it is unlikely tht the inhibition of AP-1 nd C/EBP ctivtion by suchinone contribute to tht of TNF- expression. We found tht the extent of TNF- suppression by suchinone ws less thn tht of inos expression. This reflects tht only the inhibition of NF-kB by suchinone contributes to TNF- expression. Mcrophges secrete inflmmtory meditors including lipid metbolites (e.g. prostglndins (PGs)) nd other cytokines. COX-2ctlyzes the inducible production of PGs, which clerly represents n importnt step in the inflmmtory process (Wdleigh et l., 2). The production of PGs by in mcrophges is primrily becuse of the trnscriptionl ctivtion of the COX-2gene (Lee et l., 1992; Reddy & Herschmn, 1994). The cis-cting elements identified on the promoter region of murine COX-2include NF-kB, C/EBP nd CREB (Figure 8) (Civno et l., 2). Although the NF-kB binding site is present in the regultory region of COX-2gene, the puttive NF-kB is not required for the induction of COX-2 by in murine mcrophges, s shown by dominntnegtive inhibition of NF-kB nd COX-2-reporter gene ctivity (Wdleigh et l., 2). More importntly, the C/ EBP element is believed to ply criticl role in the induction of COX-2in mcrophges. In prticulr, ctivtion of C/EBPb leds to the induction of COX-2(Thoms et l., 2; Wdleigh et l., 2). If C/EBPb is inctivted, the expression of COX-2in response to is impired (Gorgoni et l., 21). In the present study, we found tht C/EBPb, constitutively expressed in Rw264.7 cells, ws incresed fter tretment of cells with nd the bnd intensity of inducible C/EBP binding returned to tht of control by tretment of cells with suchinone. The complex consisting of C/EBPb homodimer is primrily involved in the ctivtion of C/EBP response element in mcrophge cells exposed to (Grnger et l., 2). Hence, the suppression of COX-2 by suchinone my result from its inhibition of -inducible ctivtion of C/EBPb. We lso observed tht the inducible C/EBP protein complex binding to the C/EBP DNA consensus sequence comprised C/EBPd, but not C/EBP nd C/EBPe. However, the level of nucler C/EBPd ws not incresed by in the present study. Activtion of CREB lone ws not sufficient to stimulte COX-2expression (Civno et l., 2), lthough the initil phse of COX-2expression by involved CREB (Civno et l., 21). In ddition, the expression of CREB repressed -dependent COX-reporter ctivity. 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