Inflammation & Cell Signaling 2014; 1: e117. doi: /ics.117; 2014 by Nirmal Verma, et al.

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1 Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. RESEARCH HIGHLIGHT Siliinin meliortes Dextrn Sodium Slt induced colitis in mice nd prevents overexpression of inflmmtory genes in lipopolyscchride ctivted humn mcrophges Nirml Verm *, Jishree Pul * School of life sciences, Jwhrll Nehru University, New Delhi, 1167, Indi Correspondence: Nirml Verm or Jishree Pul E-mil: nir.iotech@gmil.com or jpul33@hotmil.com Received: Mrch 1, 14 Pulished online: June 14, 14 The present work ws undertken to evlute the nti-inflmmtory effect of pure compound siliinin on Dextrn Sulfte Sodium (DSS)-induced mouse model of colitis. The expression levels of inflmmtory mrkers such s IL-8, 5-LOX, COX- nd inos in LPS ctivted humn THP-1 derived mcrophges cells were lso mesured. Swiss lino mice were treted with % DSS in their drinking wter for seven dys followed y one dy with RO wter. Siliinin (1mg nd mg per ody weight) ws dministered dily through orl gvge for seven dys nd susequently they were scrificed nd colon tissue smples were collected. Siliinin significntly ttenuted DSS induced Disese ctivity Index (DAI) scores y shortening of colon length nd decresed tissue Myeloperoxidse (MPO) ctivity tht mnifested s weight loss, dirrhe, rectl leeding, nd resulted in infiltrtions of immune cells. Histologicl exmintion indicted tht siliinin suppressed edem, mucosl dmge, nd the loss of crypts induced y DSS. Expression of inflmmtory genes ws ssyed in LPS ctivted THP-1 derived mcrophges y treting with siliinin for 4 hours when totl RNA ws extrcted. Siliinin dministrtion lso effectively nd dose dependently prevented expression of inflmmtory mrker genes in LPS ctivted THP-1 derived mcrophges. These results suggest tht siliinin hs n ntiinflmmtory effect t colorectl site suggesting its prole therpeutic role in meliorting inflmmtion during colitis. Keywords: Siliinin; Anti-inflmmtory effect; Dextrn Sodium Slt; colitis; inflmmtory mrkers; THP-1 cells To cite this rticle: Verm N, et l. Siliinin meliortes Dextrn Sodium Slt induced colitis in mice nd prevents overexpression of inflmmtory genes in lipopolyscchride ctivted humn mcrophges. Inflmm Cell Signl 14; 1: e117. doi: 1.148/ics.117. Copyright: 14 The Authors. Licensed under Cretive Commons Attriution 4. Interntionl License which llows users including uthors of rticles to copy nd redistriute the mteril in ny medium or formt, in ddition to remix, trnsform, nd uild upon the mteril for ny purpose, even commercilly, s long s the uthor nd originl source re properly cited or credited. Introduction Ulcertive colitis (UC) nd Crohn s disese (CD) re chronic inflmmtory disorders of the intestinl trct. They re summrized s inflmmtory owel disese (IBD). In UC, the inflmmtion occurs in the mucos nd mucosl ulcertion cn develop. UC is minly loclized in the rectum nd spreds to proximl prts of the intestine to different extent. [1] In CD, ny prt of the gstrointestinl trct cn e ffected, however the min site of inflmmtion is the terminl ileum nd inflmmtion cn Pge 1 of 7

2 Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. Figure 1. Moleculr structure of Siliinin. Siliinin hs polyphenolic flvonoid structure. occur segmentl nd discontinuously. In contrst to UC, not only the mucos is ffected ut lso ll lyers of the intestinl wll nd grnulom re formed in CD. The pthogenesis of oth diseses is not yet fully elucidted []. Different fctors such s genetics, immune dysregultion, nd the microil flor in the intestine nd rrier dysfunction of intestinl epithelil cells my led to the pthology of IBD [3, 4]. The pthogenesis of IBD is poorly understood ecuse of the vriility in clinicl mnifesttions nd complexity of the mechnisms of chronic inflmmtion. There is incresing evidence tht IBD tissue injury involves mny cell types present in the owel wll. The epithelil cell hs een trditionlly considered n importnt component of IBD pthogenesis, especilly in UC. Due to the vriility, complexity, nd chronicity of gut inflmmtion in IBD, intestinl epithelil cells (IEC) constntly dpt to the multiple events occurring in the mucosl microenvironment, phenomenon tht hs een long recognized. This dpttion might cuse IEC to undergo chnges in growth nd differentition, metolism, secretry pttern, immune function, nd ntigen expression [5]. Dextrn sulfte sodium (DSS)-induced colitis is one of the most common models of chemiclly induced colitis in mice [6]. DSS dministered orlly to mice initites cute inflmmtory owel disese tht resemles humn ulcertive colitis [7]. Epithelil cell toxicity, incresed intestinl permeility, nd mcrophge ctivtion hve een suggested s possile explntions for the deleterious effects of DSS lthough the mechnisms re not well understood. Intestinl microflor lso plys role in the development of the disese; however the mechnisms y which microflor influence mucosl immune responses re lso uncler. During DSS tretment, the disese is chrcterized y colonic epithelil cell lesions nd cute inflmmtion with infiltrtion of neutrophils nd mcrophges present within dmged segments [8]. This cn e followed fter termintion of DSS tretment y the development of chronic colitis chrcterized y lrge numers of ctivted T cells ner disesed segments. Regenertion of the eroded epithelium occurs over the course of severl dys to weeks fter DSS exposure. Siliinin is polyphenolic flvonoid (flvnone; Fig. 1) isolted minly from the fruits or seeds of milk thistle (Silyum mrinum (L.) Gertn), which elongs to fmily Composite [9]. Milk thistle is eing used from the ncient time in trditionl Europen medicine. Siliinin is the mjor pure ioctive compound in milk thistle extrct with smll mounts of other stereoisomers, such s isosilyin, dihydrosilyin, silydinin nd silychristin. Siliinin is widely used s heptoprotective, nti-inflmmtory nd ntifirotics gent [1]. Siliinin is known to exert heptoprotective effect s n ntiinflmmtory gent oth in vivo nd in vitro, conditions s this inhiits tumor necrosis fctor lph (TNF-α) production y mcrophges nd monocytes [11]. In the current study we evluted whether in vivo siliinin tretment my exert protective effect on the DSS induced colitis model of mice. Different inflmmtory prmeters such s disese ctivity index, myeloperoxidse ctivity, colon length, histologicl chnges were checked in mice fter DSS nd DSS + siliinin tretment. In vitro effect ws studied using LPS ctivted THP-1 derived mcrophges cells. Vrious inflmmtory mrkers such s 5-LOX, COX-, IL-8 nd inos were checked y Rel Time PCR fter siliinin nd LPS tretment in THP-1 differentited mcrophges. Mterils nd methods Animls Swiss lino mle mice, 6-8 weeks old weighing 3-36g were used. Mice were kept on 1/1 h light/drk cycle. The mice were fed stndrd chow formul nd RO wter d litum nd llowed to cclimtize for one week. All the protocols were pproved y the Institutionl Animl Ethics Committee of Jwhrll Nehru University, New Delhi, Indi. Experimentl colitis Experimentl colitis ws induced in mice y dministering % DSS (w/v) solution in RO wter over the experimentl period. Siliinin (Sigm Aldrich, Indi) ws dministered orlly in concentrtion of 1 mg/kg/dy nd mg/kg/dy. Mice were rndomly divided into four groups ech contining 5 mice: (1) nontreted group, () only DSS treted group (3) DSS nd 1mg/kg/dy siliinin treted group (4) DSS nd mg/kg/dy siliinin treted group. Disese ctivity Index (DAI) During the period of 7 dys, dily routine clinicl evlutions included: weight loss, Hemoccult test or rectl leeding nd stool consistency. DAI ws clculted y scoring chnges s descried y Cooper [8] nd shown in Tle 1. DAI score ws grded on scle of to 4. DAI ws Pge of 7

3 Disese ctive index Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. Tle 1. Scoring of Disese ctivity Index (DAI score) Score Percentge of Stool Hemoccult weight loss (%) consistency None Norml Norml Hemoccult± 6-1 Loose stool Hemoccult Hemoccult Dirrhe Rectl leeding clculted y comining scores for weight loss, stool consistency nd rectl leeding divided y 3. All the ove oservtions were mde in linded fshion. Colon length DSS only Siliinin 1 mg/kg/dy Sliinin mg/kg/dy control At the end of the experiment, mice were killed y cervicl disloction, nd lprotomy ws performed. Colon ws excised, freed of dherent dipose tissue. Susequent to wshing in ice-cold.9% sline solution, the colon ws plced on filter ppers to mesure their length. Colon length ws tken from cecum to nus. Myeloperoxidse ssy (MPO ssy) Dy 1 dy dy 3 dy 4 dy 5 dy 6 dy 7 Figure 3. Effect of siliinin on colon length in DSS treted nd untreted mice. Colon length ws found to e decresed less in mice treted with siliinin compred to mice treted with only DSS. Dt represents men (n=5/group) ±SEM, Student t test ws used to clculte significnce level (: significnt chnge from control group, : significnt chnge from DSS treted group). Approximtely 1mg tissues from colon region were snp frozen in liquid nitrogen nd homogenized in 1ml of hexdecyltrimethyl mmonium romide (HTAB) dissolved in potssium phosphte uffer. Tissue prticulte ws discrded y centrifugtion (5rpm, min) nd superntnt ws collected. 1µl of superntnt ws tken in triplicte in 96 wells plte. For lnk 1µl of HTAB uffer ws tken in triplicte. µl of Potssium phosphte uffer (ph 6.) contining.5mm o- dinisidinedihydrochloride (MP Biochemicls Inc., Osk, Jpn) nd.5% hydrogen peroxide ws dded. Opticl density ws mesured immeditely t 45nm t room temperture (5 C). One more reding ws tken etween 3 nd 6 seconds. Averge of two redings ( A -3 nd A 3-6) ws tken nd MPO ws clculted using formul: MPO (U/ gm of tissue) = MPO constnt is 1.13 x 1 -. Histopthology Averge of A 3 nd A3 6 (Time) x (MPO constnt) x (tissue weight in gm) For histopthology nlysis, smples from the mid-prt of the colon were fixed in 4% prformldehyde, emedded in prffin, sectioned (5µm), stined with hemtoxylin nd eosin (H nd E), nd exmined t x mgnifiction. Animl Cell culture The humn monocytic cell line THP-1 (NCCS, Pune, Indi) ws mintined in RPMI 164 medi supplemented with 1% het-inctivted fetl ovine serum (FBS; Gico, Invitrogen), 584mg/L L-glutmine, 45 mg/l glucose, penicillin (5 units/ml) nd streptomycin (5 μg/ml) in fully humidified tmosphere with 5% CO t 37 C. In vitro tretment of siliinin in LPS-ctivted Phorol 1-myristte 13-cette (PMA) differentited humn THP-1 mcrophges To check the nti-inflmmtory effect of siliinin in vitro, THP-1 derived mcrophges were used. Humn THP-1 cells (5 1 5 to 1 6 per ml) were differentited in mcrophges y inducing cells with 5 ng PMA for 48 hours in 6 well pltes. Then THP-1 mcrophges were preincuted for 4 h with siliinin (5μM, 5µM, 1μM nd 15μM) nd further incuted for 6h with 1 μg/ml of LPS in 6-well pltes. Totl RNA ws extrcted nd expression of inflmmtory mrker genes ws nlyzed y RT-PCR. RNA extrction nd Rel Time PCR for IL-8, inos, COX- nd 5-LOX Totl RNA ws extrcted from cells using Trizol regent (Sigm Aldrich, Indi). The concentrtion of RNA ws djusted to 1μg μl -1 with RNse free distilled wter. Reverse trnscription of totl RNA ws performed y mens of the Revert Aid First Strnd cdna Synthesis kit (Ferments, St. Leon Rot, Germny) using 1μg of totl RNA per smple in finl volume of μl. The qulity of cdna ws checked y norml PCR rections. Reversetrnscripted products were utilized for Rel time PCR in 75 Rel time PCR system using SYBR green universl PCR mster mixture from Applied Biosystems s per the instructions of the supplier. Prior to ech quntittive reltime PCR, the cdna ws diluted ppropritely. For ll experiments, RNA of untreted cells were isolted (control) Pge 3 of 7

4 MPO ctivity±se Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. t ll the time points nd the GAPDH normlized vlues ( CT) of the stimulted HT 9 cells were expressed reltive to the normlized vlues of the respective control cells ( CT). We crried out n independent experiment to show tht GAPDH ws not regulted y the pplied meditors used in our study. The CT vlues of ll genes rnged from to 3. To quntify gene expression, the comprtive threshold cycle method for reltive quntifiction ( - C = n fold) ws used. The effects of stimultors were checked y screening t different time points where the effect on expression hd the gretest impct. Sttisticl nlysis All dt were nlyzed using the pired student s t-test. These dt re presented s men±sem, dt were nlyzed y one wy ANOVA test. A level of p<.5 ws considered significnt. These exercises were done with grphpd clcultor ville on com/quickclcs y GrphPd softwre Inc. Results Figure 4. Effect of siliinin on MPO ctivity in DSS treted nd untreted mice. MPO ctivity ws found to e incresed in mice fter colitis development. But mice received siliinin long with DSS showed less increse in MPO ctivity. Dt represents men (n=5/group) ±SEM, Student t test ws used to clculte significnce level (: significnt chnge from control group, : significnt chnge from DSS treted group). Siliinin decresed DAI scores in mice model of colitis Administrtion of % DSS ws found to e ssocited with significnt clinicl chnges, which included weight loss, dirrhe, nd the ppernce of occult fecl lood. Siliinin drug ws dministered orlly. Tretment with siliinin, n herl product (1 nd mg/kg/dy for 7 dys) suppressed the pthologicl conditions in dosedependent fshion nd significntly reduced intestinl inflmmtion y simultneous improvement in DAI score (Fig ). Siliinin prevented shortening of colon To determine whether siliinin hs eneficil effect on DSS-induced colon shortening, we mesured nd compred the colon lengths of untreted control mice, mice with DSS-induced colitis, nd in mice co-treted with siliinin (1 nd mg/kg/dy) + DSS. Significnt shortening of colon length ws oserved in mice with DSSinduced colitis s compred to untreted. Orl dministrtion of siliinin reduced shortening of colon length t oth the concentrtions 1mg/kg/dy nd mg/kg/dy (Fig 3). Siliinin prevented increse in MPO level As previously discussed MPO level is considered s specific iomrker of inflmmtion. We found tht n elevted MPO level correlted with the development of colonic inflmmtion during colitis nd tht the dministrtion of siliinin (1 nd mg/kg/dy) significntly suppressed MPO ccumultion in the colonic tissues of mice with DSS induced colitis (Fig 4). There ws significnt increse in MPO ctivity in only DSS treted mice s compred to control (untreted mice). Further when compred with only DSS treted mice, 1mg/kg/dy (p<.5) nd mg/kg/dy siliinin treted mice (p<.1) showed decresed MPO ctivity. Chnges in histologicl prmeters efore nd fter siliinin tretment Pthologicl exmintions of colons were crried out y hemtoxylin nd eosin (H&E) stining nd representtive results re shown in Fig 5. Tissue sections from colon region showed distortion of epithelium in the crypt region with simultneous infiltrtion of inflmmtory cells s expected in colitis induced mice compred to control mice. However, when the mice were co-treted with siliinin, the level of distortion reduced significntly (Fig 5 c, d). Interestingly, crypts structures were rther well-preserved nd inflmmtory rections were significntly lower in tissue smples from mice treted in comintion with DSS nd siliinin (1 nd mg/kg/dy) thn only DSS treted mice. Chnges in the expression of inflmmtory mrker genes in LPS ctivted humn THP-1 derived mcrophges. Expression of four inflmmtory mrker genes 5-LOX, COX-, IL-8 nd inos (Primers re given in Tle ) ws mesured in THP-1 derived mcrophges nlyzed fter LPS+ siliinin tretment y RT-PCR (Fig 6). All the four Pge 4 of 7

5 Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. Figure 5. H & E stined cross section of colon of mice fter siliinin tretment. - control mice showed norml histologicl structures. - Tretment with DSS cused the loss of epithelil lyer, mucosl thinking nd loss of crypts nd infiltrtion of neutrophils. c nd d- DSS + siliinin tretment prevent the loss of epithelium, crypt structure nd less ccumultion of neutrophils compred to only DSS treted mice. Imges were tken t x. genes 5-LOX, COX-, IL-8 nd inos showed dose dependent decrese in the expression when treted with siliinin oth t low s well s high concentrtion of the compound. Discussion Our study used DSS-induced mice model of colitis for in vivo study nd LPS ctivted THP-1 differentited mcrophges for in vitro study to show nti-inflmmtory effect of siliinin, pure compound. DAI represents verge scores for weight loss, rectl leeding nd stool consistency [11]. All these prmeters re directly proportionl to severity of colitis. Colon length lso decresed with the increse in severity of colitis s DAI s reported erlier [1]. We found tht Siliinin decresed DAI scores nd improved colon length oth t low nd high concentrtions. Therefore siliinin is le to prevent ll the ove colitis prmeters nd therey meliortes colitis. mg/kg ws oserved to e effective compred to 1mg/kg dose. This cn e ttriuted due to strong nti-inflmmtory effect t higher doses. Further study is required to confirm this. It indicted tht siliinin used in specific concentrtion my imprt ntiinflmmtory effect in DSS induced colitis mice. MPO is n enzyme present undntly in monocytes nd increse in its ctivity is directly ssocited with increse in numer of monocytes t prticulr site [13]. As expected, MPO level in mice treted with DSS + siliinin significntly decresed compred to mice treted with DSS only. Here lso it ws oserved tht higher dose mg/kg ws more effective in decresing MPO level compred to low dose. This indicted tht siliinin prevented ccumultion of inflmmtory cells t the site of inflmmtion. Decrese in MPO my e the result of some unknown intrcellulr signling pthwy tht needs to e worked out further. Histologicl chnges such s crypts destruction, mucosl thickening nd epithelil lyer erosion re hllmrk of colitis [14]. Our oservtion further reveled tht siliinin ws lso effective in preventing histopthologicl dmges such s crypt destruction, mucosl thickening nd epithelil lyer dmge. To get n insight into the signling pthwy, we further studied in vitro nti-inflmmtory effect of siliinin y treting the humn THP-1 differentited mcrophges with siliinin nd then ctivted with LPS. The expression of four inflmmtory mrkers IL-8, COX-, 5-LOX nd inos were mesured y RT-PCR. IL-8, pro-inflmmtory cytokine is known to increse during IBD nd cts s powerful neutrophil chemo ttrctnt [15]. Thus increse or decrese of IL-8 showed corresponding increse or decrese in infiltrtion of neutrophils t inflmed site. In our study we found siliinin t ll concentrtions decresed the expression of IL-8. This indicted tht siliinin indirectly reduced neutrophils ccumultion y preventing IL-8 over expression. The cyclooxygense (COX) is key enzyme used in the conversion of rchidonic cid to prostglndins. COX- gets rpidly up regulted y growth fctors nd cytokines nd thus ply role in inflmmtion [16]. Increse in COX- expression leds to incresed formtion of prostglndins. Accumultion of prostglndins finlly leds to inflmmtion. In our study siliinin decresed COX- expression in dose dependent mnner. Therefore we hypothesized tht siliinin decresed COX- expression proly y preventing IL-8 over expression during inflmmtion. The importnt role of the enzyme 5-lipoxygense (5- LOX) is the production of leukotrienes (LTs) during the inflmmtory diseses. IBD is lso cused s result of incresed genertion of LTs in the inflmed mucos [17]. In our study we found siliinin decresed 5-LOX expression. Therefore siliinin my lso prevent inflmmtion due to its 5-LOX inhiitory property. Further study is required to confirm the exct pthwy siliinin dopts tht result in 5- LOX inhiition. Nitric oxide (NO) is considered to e pleiotropic free rdicl messenger molecule. Lrge ody of evidence indicte tht the inducile form of the NO synthse enzyme (inos) tht is responsile for high-output production of NO from l-rginine is up-regulted in vrious forms of Pge 5 of 7

6 n fold chnge in expression±se n fold chnge in expression SE n fold chnge in expression±se n fold chnge in expression±se Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. Tle. Genes with Primer sequences nd product size Gene Product inos IL-8 COX- 5-LOX GAPDH forwrd & reverse primer Forwrd 5 -TGTGCCACCTCCAGTCCAG-3 Reverse 5 - GACCTGCAAGTTAAAATCCC-3 Forwrd 5 -CTGATTTCTGCAGCTCTGTG-3 Reverse 5 -CATCAGAAAGCTTTACAATAATT-3 Forwrd 5 -ACAGCCAGACGCCCTCAGACA-3 Reverse 5 -AGGATTTGCTGTATGGCTGAGC-3 Forwrd 5 -AATATCGATGGATGGAGTGGAA-3 Reverse 5 -ATGAAGCGGTTGATGAACAGGTT-3 F-5 -GCTCCTCCTGTTCGACAGTCA-3 F-5 -GCAACAATATCCACTTTACCAG-3' Size 39p 177p 1p 156p 189p LOX COX Siliinin conc. Siliinin conc. c d IL inos Siliinin conc. Siliinin conc. Figure 6. Effect on inflmmtory genes in THP-1 derived mcrophges. Expression of inflmmtory mrker genes 5-LOX, COX, Figure IL-8 nd 5iNOS fter LPS nd siliinin tretment in humn mcrophges. ) 5-LOX, )- COX- expression, c- IL-8 gene expression nd d)- inos gene expression in LPS+siliinin treted cells. Dt represents men (n=3/group) ±SEM, Student t test ws used to clculte significnce level (: significnt chnge from control group, : significnt chnge from DSS treted group). mucosl inflmmtion. Consistent with this, multiple detection strtegies hve demonstrted tht inos expression, enzymtic ctivity, nd NO production re incresed in humn inflmmtory owel disese tissues. There is lso evidence tht the level of inos-derived NO correltes well with the disese ctivity in ulcertive colitis [18]. We oserved decresed inos gene expression in ll the concentrtions of siliinin used. Thus we cn conclude tht siliinin is n effective compound in preventing DSS induced colitis. Our in vitro study lso showed tht siliinin cts s n nti- Pge 6 of 7

7 Inflmmtion & Cell Signling 14; 1: e117. doi: 1.148/ics.117; 14 y Nirml Verm, et l. inflmmtory gent y preventing over expression of inflmmtory genes such s cytokine IL-8, nd enzyme 5- LOX nd inos. Decrese in IL-8 leds to suppression of COX- nd neutrophils ccumultion t the site of inflmmtion. It will e interesting to check the effect t the protein level. Conflict of interest The uthors hve declred tht no competing interests exist. Acknowledgments NV is thnkful to the Council of Scientific nd Industril Reserch (CSIR), New Delhi for the reserch fellowship for conducting the study. The uthors re lso thnkful to niml fcility of Jwhrll Nehru University. This work ws supported y the funding received y JP from the CSIR, New Delhi nd University Grnt Commission Resource Net Working funds, New Delhi, Indi. References 1. Gionchetti P, Rizzello F, Hl F, Morselli C, Amdini C, Romgnoli R et l. Stndrd tretment of ulcertive colitis. Dig Dis 3; 1: Arhm C, Cho JH. Inflmmtory owel disese. N Engl J Med 9; 361: Kuchrzik T, Mser C, Lügering A, Kgnoff M, Myer L, Trgn S et l. Recent understnding of IBD pthogenesis: implictions for future therpies. Inflmm Bowel Dis 6; 1: Xvier RJ, Podolsky DK. Unrvelling the pthogenesis of inflmmtory owel disese. Nture 7; 448: Zhng YZ, Li YY. Inflmmtory owel disese: Pthogenesis. World J Gstroenterol 14; : Boismenu R, Chen Y. Insights from mouse models of colitis. J Leukoc Biol ; 67: Egger B, Büchler MW, Lkshmnn J, Moore P, Eysselein VE. Mice hroring defective epiderml growth fctor receptor (wved-) hve n incresed susceptiility to cute dextrn sulfte-induced colitis. Scnd J Gstroenterol ; 35: Cooper HS, Murthy SN, Shh RS nd Sedergrn DJ. Clinicopthologic study of dextrn sulfte sodium experimentl murine colitis. L Invest 1993; 69: Singh RP nd Agrwl R. Flvonoid ntioxidnt silymrin nd skin cncer. Antioxidnts & redox signling ; 4: Sller R, Meier R, Brignoli R. The use of silymrin in the tretment of liver diseses. Drugs 1; 61: Al-Anti L, Essid E, Reinehr R, Petzinger E. Siliinin protects OTA-medited TNF-lph relese from perfused rt livers nd isolted rt Kupffer cells. Mol Nutr Food Res 9;53: Hendrickson BA, Gokhle R nd Cho JH: Clinicl spects nd pthophysiology of inflmmtory owel disese. Clin Microiol Rev ; 15: Msoodi I, Kochhr R, Dutt U, Vishnvi C, Prsd KK, Viphei K et l. Evlution of fecl myeloperoxidse s iomrker of disese ctivity nd severity in ulcertive colitis. Dig Dis Sci 1; 57: Geoes K, Riddell R, Öst Ä, Jensfelt B, Persson T, Löferg R. A reproducile grding scle for histologicl ssessment of inflmmtion in ulcertive colitis. Gut ; 47: Oppenheim JJ, Zchrie COC, Mukid N, Mtsushim K. Properties of the novel proinflmmtory supergene 'intercrine' cytokine fmily. Annu Rev Immunol 1991; 9: Prk KS, Kim BH, Chng IM. Inhiitory Potencies of Severl Iridoids on Cyclooxygense-1, Cyclooxygnse- Enzymes Activities, Tumor Necrosis fctor-α nd Nitric Oxide Production In Vitro. Evid Bsed Complement Alternt Med 1; 7: Rsk-Mdsen J, Bukhve K, Lursen LS, Luritsen K. 5- Lipoxygense inhiitors for the tretment of inflmmtory owel disese. Agents Actions 199; Spec No:C Cross RK, Wilson KT. Nitric oxide in inflmmtory owel disese. Inflmm Bowel Dis 3; 9: Pge 7 of 7