Isothiocyanate-enriched moringa seed extract alleviates ulcerative

Transcription

1 1 Isothiocynte-enriched moring seed extrct llevites ulcertive colitis symptoms in mice 3 4 Short title: Moring Seed Extrct Allevites Ulcertive Colitis Youjin Kim 1,,* Alex G. Wu 3, Ash Jj-Chimedz, Brittny L. Grf, Crrie Wtermn 4, Michel P. Verzi 3, Ily Rskin Nutrsor, LLC., Freehold, New Jersey, United Sttes of Americ Deprtment of Plnt Biology, Rutgers, The Stte University of New Jersey, New Brunswick, New Jersey, United Sttes of Americ 3 Deprtment of Genetics nd the Humn Genetics Institute of New Jersey, Rutgers, The Stte University of New Jersey, Pisctwy Township, New Jersey, United Sttes of Americ 4 Deprtment of Nutrition, University of Cliforni-Dvis, Dvis, Cliforni, United Sttes of Americ * Corresponding uthor: E-mil: youjin@nutrsor.com (YK) 3 4 1

2 5 Astrct Moring (Moring oleifer Lm.) seed extrct (MSE) hs nti-inflmmtory nd ntioxidnt ctivities. We investigted the effects of MSE enriched in moring isothiocynte-1 (MIC-1), its puttive ioctive, on ulcertive colitis (UC) nd its nti-inflmmtory/ntioxidnt mechnism likely medited through Nrf-signling pthwy. Dextrn sulfte sodium (DSS)-induced cute (n=8/group; 3% DSS for 5 d) nd chronic (n=6/group; cyclic rottions of.5% DSS/wter for 3 d) UC ws induced in mice tht were ssigned to 4 experimentl groups: helthy control (wter/vehicle), disese control (DSS/vehicle), MSE tretment (DSS/MSE), or 5-minoslicyic cid (5-ASA) tretment (positive control; DSS/5-ASA). Following UC induction, wter (vehicle), 15 mg/kg MSE, or 5 mg/kg 5-ASA were orlly dministered for 1 or wks. Disese ctivity index (DAI), spleen/colon sizes, nd colonic histopthology were mesured. From colon nd/or fecl smples, pro-inflmmtory iomrkers, tight-junction proteins, nd Nrf-medited enzymes were nlyzed t protein nd/or gene expression levels. Compred to disese control, MSE decresed DAI scores, nd showed n increse in colon lengths nd decrese in colon weight/length rtios in oth UC models. MSE lso reduced colonic inflmmtion/dmge nd histopthologicl scores (modestly) in cute UC. MSE decresed colonic secretions of pro-inflmmtory kertinocyte-derived cytokine (KC), tumor necrosis fctor (TNF)-α, nitric oxide (NO), nd myeloperoxidse (MPO) in cute nd chronic UC; reduced fecl lipoclin- in cute UC; downregulted gene expression of pro-inflmmtory interleukin (IL)-1, IL-6, TNF-α, nd inducile nitric oxide synthse (inos) in cute UC; upregulted expression of cludin-1 nd ZO-1 in cute nd chronic UC; nd upregulted GSTP1, n Nrf-medited phse II detoxifying enzyme, in chronic UC. MSE ws effective in mitigting UC symptoms nd reducing UC-induced colonic pthologies, likely y suppressing pro-inflmmtory iomrkers nd incresing tightjunction proteins. This effect is consistent with Nrf-medited nti-inflmmtory/ntioxidnt signling

3 47 48 pthwy documented for other isothiocyntes similr to MIC-1. Therefore, MSE, enriched with MIC-1, my e useful in prevention nd tretment of UC. 3

4 49 Introduction Ulcertive colitis (UC), type of inflmmtory owel disese (IBD), is chronic intestinl disorder chrcterized y recurrent inflmmtion of the gstrointestinl trct. It ffects over 1.6 million people in the United Sttes [1-3]. The etiopthogenesis of IBD involves complex interply of genetic, environmentl, microil, nd immune fctors, which result in ltered rrier properties of the mucous nd epithelil lyers. These ltertions cn llow luminl toxins, pthogens, nd ntigens to penetrte the intestinl mucos nd elicit n overproduction of pro-inflmmtory cytokines nd trfficking of effector leukocytes into the intestinl mucos. This cn ultimtely led to uncontrolled nd exggerted intestinl inflmmtion [4]. Phrmcologicl intervention is one of the key pproches for UC tretment. Anti-inflmmtory drugs such s corticosteroids, 5-minoslicyic cid (5-ASA) derivtives, nd immune-suppressnts cn reduce mucosl inflmmtion nd UC-relted symptoms [5]. While these therpies re often effective, their long-term use is limited y significnt side effects including immune suppression [6]. Hence, there is need to discover new therpeutic options for UC tht re sfe nd cple of sustining clinicl remission, while improving gut mucosl heling. In this respect, dietry nd medicinl plnts contining ioctive phytochemicls re emerging s promising lterntives to drug therpies for prevention nd tretment of UC [7]. Moring (Moring oleifer Lm.) is fst growing, drought-tolernt tree tht thrives in tropicl nd sutropicl regions. It elongs to the Moringcee fmily within the order Brssicles which includes roccoli nd other cruciferous vegetles of the Brssiccee fmily. Trditionlly, moring is renowned for its nutritionl vlue nd medicinl enefits. All prts of the plnt (i.e., leves, seeds, roots, rk, flowers) re edile nd used for tretment of numer of cute nd chronic ilments such s inflmmtion, infection, hypertension, nd dietes [8]. Recently, medicinl enefits of moring hve 4

5 een confirmed in multiple in vitro nd in vivo studies [9-11]. Oserved phrmcologicl effects of moring, linked to its nti-inflmmtory, nti-cteril, nd ntioxidnt properties, re ttriuted to the presence of phytoctive compounds such s flvonoids, phenolic cids, nd, most notly, its unique glucosinoltes [1]. Moring glucosinoltes cn e converted, vi myrosinse, to highly ioctive nd stle moring isothiocyntes (MICs) [9, 1], of which MIC-1 (4-[(α-L-rhmnosyloxy)enzyl] isothiocynte) is the most undnt MIC in moring seeds. Isothiocyntes from Brssiccee plnts, txonomiclly relted to moring, re well-known nti-inflmmtory compounds tht likely ct vi ctivtion of nucler fctor erythroid -relted fctor (Nrf), key trnscription fctor involved in ntiinflmmtory nd ntioxidnt responses [13, 14]. Studies hve shown tht MIC-1 inhiited nucler fctor-kpp B (NF-κB) expression nd melnom growth in nude mice [15], nd ttenuted gene expression of inducile nitric oxide synthse (inos) nd interleukin-1β (IL-1β), s well s production of nitric oxide (NO) nd tumor necrosis fctor-α/β (TNF-α/β) in RAW mcrophges [1, 16]. An MIC-1- enriched moring lef extrct lso reduced IL-1β nd TNF-α levels in plsm nd vrious tissues, nd meliorted weight gin, insulin resistnce, nd heptic gluconeogenesis in oese mice [17]. Furthermore, MIC-1 is known to ctivte NAD(P)H quinone oxidoreductse 1 (NQO1) [18], one of the phse II detoxifying enzymes tht re directly regulted y Nrf [13]. Nrf-deficient mice re susceptile to UC nd colorectl cncer, which ws correlted with downregultion of ntioxidnt/phse II detoxifying enzymes nd upregultion of pro-inflmmtory cytokines nd relted iomrkers [19]. Therefore, Nrf signling hs een suggested to ply role in mediting the inflmmtory response involved in UC. To exmine the in vivo efficcy of potentil therpeutic gents, vrious colitogenic sustnces, such s dextrn sulfte sodium (DSS), di-/tri-nitroenzene sulfonic cid, oxzolone, or cetic cid, hve een used to estlish chemiclly-induced UC models []. Of the experimentl UC models, DSS-induced UC in mice is most commonly used due to its simplicity, ffordility, high degree of uniformity, nd 5

6 reproduciility [1]. The DSS model generlly mimics the common clinicl fetures of inflmmtion nd histopthology oserved in humn UC []. In this experimentl model, chnges in concentrtions nd durtions of DSS, delivered in drinking wter, cn mimic cute or chronic UC [3]. Erlier, our lortory developed moring lef extrct enriched in severl MICs, verging 3-5% (w/w), y extrcting fresh moring leves in wter nd utilizing endogenous myrosinse to convert the precursor glucosinolte to MICs, nd reported iologicl ctivities of this extrct nd stility of MICs contined [1, 17-18]. Susequently, we developed method for producing 4-5% (w/w) MIC-1- enriched moring seed extrct (MSE) nd iochemiclly chrcterized this extrct [S1 File]. This compnion pper provides evidence tht MIC-1 is the min nti-inflmmtory ioctive in MSE responsile for reductions of crrgeenn-induced edem in vivo nd inflmmtory responses in lipopolyscchride (LPS)-stimulted RAW mcrophges in vitro [S1 File]. Here we investigte the potentil of MSE to relieve UC symptoms in DSS-induced cute nd chronic UC models in mice

7 Mterils nd methods Chemicls nd regents DSS (moleculr weight: 36-5 kd) ws purchsed from MP Biomedicls, Inc. (Solon, OH), nd 5-ASA (known s meslmine) ws from TCA Americ (Portlnd, OR). Roswell Prk Memoril Institute (RPMI) 164 medium, het-inctivted fetl ovine serum (HI-FBS), nd ntiiotics (1 U/ml penicillin + 1 µg/ml streptomycin) were purchsed from Thermo Fisher Scientific, Inc. (Wlthm, MA). All other chemicls were purchsed from Sigm-Aldrich Co. (St. Louis, MO) unless otherwise noted Formultion of MSE MSE ws prepred following the protocol developed in our lortory [S1 File]. Briefly, moring seeds (otined from Jmic Moring Frmers Assocition, Kingston, Jmic) were ground to fine powder using lender (VitMix 5; Clevelnd, OH) nd mixed with mient temperture Millipore wter in rtio of seeds (1 g) to wter (3 ml). The mixture ws then plced on shker nd incuted for h t 37 C. After incution, ethnol ws dded to the mixture in rtio of seeds (1 g) to ethnol (1 ml), stirred, nd filtered. Susequently, solvents were removed from the extrct y rotry evportion nd lyophiliztion efore eing stored t - C. MSE powder used in this study contined 47% (w/w) of MIC Animls nd experimentl protocols All niml protocols used in this study were pproved y the Institutionl Animl Cre nd Use Committee t Rutgers, The Stte University of New Jersey (No ), nd followed federl nd stte lws. Mle C57BL/6J mice t 7 wks of ge (verge weight: - g) were purchsed from Jckson 7

8 Lortories (Br Hror, ME) nd cclimtized to the niml fcility for 1 wk with d liitum ccess to drinking wter nd rodent chow diet (Purin 51; LDiet, St. Louis, MO) prior to experiments. Mice were initilly housed 4 nimls per cge, nd mintined under stndrdized conditions ( ± C, 1-h light/drk cycle, nd 5-6% humidity). Experiments egn with mice t 8 wks of ge, rndomly ssigned to the experimentl groups, ecuse this ge is optiml for successful nd reproducile UC induction y DSS [4]. For this study, two types of DSS-induced UC mouse models with fetures of cute nd chronic UC conditions were utilized. UC induction protocols nd experimentl designs for ech UC model re descried elow nd summrized in Tle 1. DSS-induced cute UC model After cclimtion, 3 mice were rndomly divided into 4 experimentl groups s follows sed on their ody weights (n=8/group; 4 mice/cge): helthy control (wter/vehicle), disese control (DSS/vehicle), MSE tretment (DSS/MSE), or positive control (DSS/5-ASA). Acute UC ws induced in mice (except helthy control) y replcing their drinking wter with utoclved wter contining freshly prepred 3.% (w/v) DSS (d liitum) for 5 d. Mice in helthy control were littermtes tht received utoclved wter insted, nd housed nd processed identiclly during UC induction. Following UC induction, tretments were dministered dily to ll mice vi orl gvge in volume of 5 ml/kg ody weight (w) with niml feeding needles (G x 1. ; Cdence, Inc., Stunton, VA) for 7 consecutive d. Tretments included vehicle (utoclved wter), 15 mg/kg w of MSE, or 5 mg/kg w of 5-ASA. Mice were llowed free ccess to utoclved drinking wter nd chow pellets during the time. DSS-induced chronic UC model After cclimtion, 4 mice were ssigned to 4 weight-lnced experimentl groups (n=6/group; 3 mice/cge) s descried ove. Chronic UC ws induced in mice y 4 cycles of.5% (w/v) DSS in drinking wter for 3 d, which ws lternted with 6 d of utoclved wter (d liitum) in etween, resulting in totl of 3 d for the entire induction period. Susequently, tretments (vehicle, MSE, or 5-ASA) were 8

9 orlly delivered to mice dily for 14 consecutive d, nd mice hd free ccess to utoclved wter nd chow during the time Tle 1. Experimentl designs nd protocols Acute UC model Chronic UC model UC induction y DSS 3.% (w/v) DSS (5 d) 4 cycles of.5% (w/v) DSS for 3 d, with lternte utoclved wter for 6 d in etween (3 d in totl) Dosing method Orl gvge Orl gvge Tretment period 7 d 14 d 16 Experimentl design Modified sed on Dey et l., 1 [5] Group 1) Wter/vehicle (helthy control) Group ) DSS/vehicle (disese control) Group 3) DSS/MSE (MSE tretment; 15 mg/kg w of MSE) Group 4) DSS/5-ASA (positive control; 5 mg/kg w of 5-ASA) Throughout the entire experimentl periods with oth UC models, w nd food consumption were recorded dily or every d. Intkes of DSS-contining wter were lso monitored dily to confirm tht mice received sufficient mounts of DSS to induce UC. At the end of ech experiment, mice were nesthetized with CO nd euthnized y decpittion Disese ctivity index (DAI) ssessment DAI ws scored following the criteri descried in Tle in order to vlidte the progression of UC in oth models. The DAI score ws clculted s sum of the scores for % w chnge, intestinl leeding, stool consistency, nd rectl prolpse. % w chnge ws clculted s: [(w ech d initil w) / initil w] x 1. To score intestinl leeding, loody stool ws confirmed y either visile oservtion or the fecl occult leeding test (FOBT) using commercilly ville kit (Sure-Vue ; Thermo Fisher Scientific, Inc., Wlthm, MA). 9

10 Tle. Disese ctivity index (DAI) for clinicl evlution of DSS-induced UC in mice Score % w chnge Intestinl leeding Stool consistency Rectl prolpse (FOBT) Gin, loss < 1% No color chnge Norml None 1 Loss 1 to <5% Loss 5 to <1% Color chnge Loose Oserved prolpse 3 Loss 1 to <15% Loss 15% Gross leeding Dirrhe Extensive prolpse Modified from Dey et l., 1 [5] Fecl occult leeding test Tissue isoltion, mesurements, nd preprtions At the time of scrifice in cute nd chronic UC experiments, the spleen nd colon were dissected from ech mouse nd mesured within 5-1 min postmortem. Individul spleen tissues were weighed (mg). The colons, connected to the cecum, were isolted y seprting them from the ileocecl junction of the smll intestine nd the distl rectum of the nus, nd thoroughly flushed with cold PBS (ph 7.4; supplemented with 1% ntiiotics) using ll tip dosing needle ttched to 1 ml syringe to remove feces nd lood. After pushing out remining colon contents with lunt forceps, the colons were strightened nd mesured in length (cm). The colons were then seprted from cecum nd weighed. Susequent to these mesurements, the colons were opened y longitudinl incision, nd wshed three times in cold PBS (ph 7.4; supplemented with 1% ntiiotics) for complete removl of fecl mtter, from which only middle-to-distl colon segments were used for further nlyses. These colon segments were further cut into 3 pieces longitudinlly, ech of which ws individully processed for orgn culture, quntifiction of gene expression, nd histopthologicl ssessments Ex vivo colon orgn culture Approximtely 1/3 of wshed middle-to-distl colon segments were kept in cryogenic tues filled with cold RPMI 164 medium (supplemented with 1% ntiiotics) on ice until delivery to the cell culture 1

11 fcility. On culturing, ech colon specimen ws trnsferred into seprte well of 48-well microtiter plte contining 1 ml/well of RPMI 164 medium (supplemented with 1% FBS nd 1% ntiiotics), nd incuted t 37 C with 5% CO for 18 h. Superntnts were collected, centrifuged t,5 rpm for 1 min t 4 C, nd stored t -8 C until nlyses of colonic pro-inflmmtory iomrkers. 199 Histopthologicl evlution of UC Another 1/3 of middle-to-distl colon segments were sujected to histopthologicl ssessment. The colonic sections were fixed in 4% prformldehyde overnight t 4 C, which were then wshed with PBS (ph 7.4), dehydrted through ethnol grdients, nd prffin emedded. The emedded locks were sectioned (5 µm), nd the smples were then stined with hemtoxylin nd eosin (H&E). Susequently, ll colonic sections were scored in n investigtor-linded mnner relting to the severity of colonic dmge nd inflmmtion using light microscopy with Retig 13 CCD cmer (Q-imging; Burny, BC, Cnd) nd n Eclipse E8 microscope (Nikon Instruments Inc., Lewisville, TX) with QC-Cpture imging softwre under x mgnifiction nd ir for x/.75 N.A. The severity of UC ws evluted using the histopthologicl scoring system with totl score rnge from to 11 (Tle 3), ccording to the mtrices previously defined y Lroui et l. (1) [6]

12 19 Tle 3. Histopthologicl scoring system Score Severity of inflmmtion Crypt dmge Ulcertion Rre inflmmtory cells in the Intct crypt foci of ulcertion lmin propri 1 Incresed numers of Loss of sl 1/3 of crypt 1- foci of ulcertion grnulocytes in the lmin propri Confluent inflmmtory cells Loss of sl /3 of crypt 3-4 foci of ulcertion extended to sumucos 3 Trnsmurl extension of the inflmmtory infiltrtion Loss of entire crypt Confluent or extensive ulcertion 4 - Chnge in epithelil surfce - cused y erosion 5 - Confluent erosion - Lroui et l., 1 [6] 1 Quntifiction of colonic pro-inflmmtory iomrkers From the superntnt of ex vivo colon cultures, pro-inflmmtory chemokine, kertinocyte-derived chemokine (KC), nd pro-inflmmtory cytokine, TNF-α, were quntified using the mouse CXCL1/KC DuoSet ELISA kit nd the mouse TNF-α Quntikine ELISA kit, respectively. Myeloperoxidse (MPO), mrker for neutrophil infiltrtion in colonic epithelil tissues, ws lso mesured using the mouse MPO DuoSet ELISA kit. All ELISA kits were purchsed from R&D Systems, Inc. (Minnepolis, MN), nd utilized ccording to mnufcturer s protocols. NO production ws nlyzed in duplictes using the Griess Regent System (Promeg Corp., Mdison, WI) following the mnufcturer s protocol. Nitrite levels (µm) in individul smples were determined t 5 nm y comprison with stndrd curve of nitrite (.1 M NNO ; to 1 µm). Throughout these nlyses, sornce ws red using Synergy HT microplte reder (BioTek Instruments, Inc., Winooski, VT), nd individul concentrtions otined from ech colon smple were normlized y the mesured colon tissue weight (g) Fecl collection nd quntifiction of fecl lipoclin- (Lcn-) 1

13 At the end of tretment periods of cute nd chronic UC experiments, fecl smples were collected y tking individul mice out of their cges nd collecting 7 fecl pellets per mouse in sterile cryogenic vils, which were snp-frozen on dry ice immeditely, nd stored t 8 C until further processing. Quntifiction of fecl Lcn- ws performed s descried y Chssing et l. (1) [7] with slight modifictions. Briefly, 1 mg of frozen fecl smples were reconstituted in 1 ml of PBS (ph. 7.4) supplemented with.1% (v/v) Tween, which were homogenized for 3 min using 16 MiniG homogenizer (SPEX SmplePrep, Inc., Metuchen, NJ) to otin homogenous fecl suspension. The suspension ws centrifuged t 1, rpm t 4 C for 1 min nd the cler superntnt ws used to quntify Lcn- levels using the mouse Lcn- DuoSet ELISA kit (R&D Systems, Minnepolis, MN) following mnufcturer s instructions. The level of fecl Lcn- ws expressed s Lcn- (ng)/feces (g) Totl RNA isoltion, purifiction, nd cdna synthesis Individul colon tissue smples (~5 mg) were homogenized for 3 min with.8 mm stinless steel eds nd Qizol regent (Qigen Inc., Vlenci, CA) using 16 MiniG homogenizer (SPEX SmplePrep, Inc., Metuchen, NJ). From the colon homogentes, totl RNA ws isolted nd purified using n RNesy Plus Universl mini kit (Qigen Inc., Vlenci, CA) ccording to mnufcturer s protocols. The purity nd concentrtion of totl RNA ws mesured spectrophotometriclly t 6 nm using the NnoDrop ND- 1 (NnoDrop Technologoes, Inc., Wilmington, DE), nd only smples with sufficient purity (A 6/8.) were sujected to the next step. cdnas were synthesized from 5 µg RNA (per 5 µl rection) using High-Cpcity cdna Reverse Trnscription kit (Applied Biosystems, Foster City, CA) with RNse inhiitor (Applied Biosystems, Foster City, CA), ccording to the mnufcturer s protocol. The therml cycler progrm ws set on Mstercycler epgrdient (Eppendorf North Americ, Huppuge, NY) s follows: 1 min t 5 C, 6 min t 37 C, 6 min t 37 C, 5 sec t 85 C, nd finl hold t 4 C. 13

14 59 Quntittive rel-time PCR (qpcr) The synthesized cdnas were diluted 5-fold or 1-fold, ppropritely, nd. µl of the dilution ws used for qpcr with 5. µl of TqMn Fst Universl PCR Mster Mix (x, No AmpErse UNG; Applied Biosystems, Foster City, CA),.5 µl of ech primer, nd.5 µl of nuclese-free H O (Thermo Fisher Scientific, Inc., Wlthm, MA) to finl rection volume of 1. µl. Quntittive PCR mplifictions were performed on QuntStudio 3 Rel-Time PCR System (Applied Biosystems, Foster City, CA) with the following therml cycler profile: one cycle for sec t 95 C, followed y 4 cycles of two-step procedure consisting of denturtion for 1 sec t 95 C nd nneling extension for sec t 6 C. Inventoried TqMn primer sets (Thermo Fisher Scientific, Inc., Wlthm, MA) were used for nlyses of IL-1β (Mm4348_m1), IL-6 (Mm44619_m1), TNF-α (Mm44358_m1), inos (Mm445_m1), zonul occludens-1 (ZO-1; Mm493699_m1), cludin-1 (Mm591_m1), heme oxygense 1 (HO1; Mm5165_m1), NQO1 (Mm153561_m1), nd glutthione S trnsferse pi 1 (GSTP1; Mm413618_gH), using either hydroxymethylilne synthse (HMBS; Mm _m1) or glycerldehyde-3-phosphte dehydrogense (GAPDH; Mm _g1) s endogenous controls. All experimentl smples were run in duplictes. For individul qpcr experiments, mens of duplicte smples were tken, nd reltive quntifiction of trget gene expression ws normlized with respect to the verge Ct vlue of the housekeeping gene, either HMBS or GAPDH, ccordingly. Dt were nlyzed ccording to the comprtive Ct method ( -ΔΔCt ), where helthy control (wter/vehicle) served s clirtor nd ws ssigned vlue of 1.. Consequently, ll dt re expressed s reltive fold chnge etween helthy control nd other experimentl groups Sttisticl nlysis 14

15 All sttisticl nlyses were performed using SPSS sttisticl softwre pckge (version 11.5; SPSS Inc., Chicgo, IL). Sttisticl significnce mong different groups ws evluted y one-wy nlysis of vrince (ANOVA), followed y post-hoc test: Duncn s test or Tukey s test. Dt re expressed s men ± SEM. A p vlue of <.5 ws considered sttisticlly significnt. 15

16 85 86 Results MSE llevites UC symptoms No nimls died during cute or chronic UC periods. DAI score ws clculted y comining scores of the following prmeters: % w chnge, intestinl leeding, stool consistency, nd rectl prolpse (see Tle ). Rectl prolpse ws not oserved in mice tested in either UC phse. Generlly, mice receiving DSS mnifested similr clinicl symptoms seen in humn UC, such s severe w loss, dirrhe, nd intestinl leeding. By the end of UC induction, grdul increses of DAI scores were oserved in cute (~7.75 times increse t d 5 induction) nd chronic (~5.55 times increse t d 3 induction) UC models compred to helthy control (Fig 1A nd 1B)

17 Disese Activity Index (DAI) Disese Activity Index (DAI) A wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA *** *** * * * * * ** ** * UC induction (3.% DSS for 5 d) Tretment (7 d) * * * 3 Experimentl periods (d) B * UC induction Tretment (14 d) [4 cycles x {.5% DSS (3 d), followed y lternte wter (6 d) in etween} = totl 3 d] Experimentl periods (d) ** * * * * Fig 1. Effects of MSE on DAI scores in DSS-induced UC. (A) Acute UC (n=8/group): dt re shown t dily intervls throughout UC induction nd tretment periods. (B) Chronic UC (n=6/group): dt re shown t 3-d intervls during UC induction nd -d intervls during the tretment period. Experimentl designs nd disese induction procedures re defined in Tle 1 nd DAI scoring criteri in Tle. Dt re expressed s men ± SEM. * P<.5, ** P<.1, nd *** P<.1 compred to disese control (DSS/vehicle). 5-minoslicylic cid (5-ASA); disese ctivity index (DAI); dextrn sulfte sodium (DSS); moring seed extrct (MSE); ulcertive colitis (UC)

18 In chronic UC, prticulrly, fluctution of DAI scores ws shown following the lterntion of DSS nd wter, reflecting the relpse-remission pttern of UC symptoms (Fig 1B). In cute UC, oth MSE nd 5- ASA significntly decresed DAI scores compred to disese control for 6 consecutive d with decrese strting from d 1 (-9.% y MSE; -47.6% y 5-ASA) (Fig 1A). At d 7, however, no significnt differences were found in DAI scores mong ny experimentl groups, possily due to prtil nturl recovery from DSS-induced UC (Fig 1A). In chronic UC, MSE showed strong trend towrds reducing clinicl symptoms compred to disese control strting from d (-73.3%). The remission persisted throughout the tretment. At the end of tretment (d 14), the DAI score dropped lmost to tht of the helthy control (-93%) (Fig 1B). While 5-ASA nd MSE showed similr trends in relieving UC symptoms, the 5- ASA effect ws only significnt t d (Fig 1B), while the MSE effect ws significnt t d, 1, 1 nd 14 (Fig 1B) MSE restores colon integrity in DSS-induced UC models Bw loss, rectl leeding, nd inflmmtion exerted during UC re ssocited with colon shortening nd spleen enlrgement [4, 8]. In this study, spleen weights were not ffected y either DSS or tretments (MSE or 5-ASA) in oth cute nd chronic UC mice (Fig A)

19 Colon weight/length rtio (mg/cm) Colon length (cm) Spleen weight (mg) A 1 1 wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA Acute UC Chronic UC B 1 8 c c Acute UC Chronic UC C Acute UC Chronic UC Fig. Effects of MSE on spleen nd colon in DSS-induced UC. (A) Spleen weight (mg), (B) colon length (cm), nd (C) colon weight/length rtio (mg/cm) were mesured t necropsy of cute UC (n=8/group) nd chronic UC (n=6/group) experiments. Experimentl designs nd disese induction procedures re defined in Tle 1. Dt re expressed s men ± SEM. Different letters indicte significnt differences etween groups (p<.5). 5-minoslicylic cid (5-ASA); dextrn sulfte sodium (DSS); moring seed extrct (MSE); ulcertive colitis (UC). 19

20 However, mice in disese control hd significntly shorter colons compred to helthy control (6.6 ±. cm vs. 7.5 ±. cm for cute UC; 6.3 ±.4 cm vs. 7.6 ±.4 cm for chronic UC). MSE showed positive trend in reversing colon shortening (6.9 ±.1 cm for cute UC, not significnt t p<.5; nd 7.3 ±.3 cm for chronic UC). 5-ASA did not recover colon shortening in either model (6.4 ±.3 cm for cute UC; 6.6 ±. cm for chronic UC) (Fig B). The colon weight/length rtio, n indictor of UC-ssocited colonic edem nd inflmmtion [9], ws significntly higher in disese control (49.5 ± 3.1 for cute UC; 48.7 ±.1 for chronic UC) nd following 5-ASA tretment (48.7 ±.1 for cute UC; 55.9 ± 5. for chronic UC) compred to helthy control (34.9 ±.1 for cute UC; 39.5 ±.9 for chronic UC). MSE tretment reduced the rtio compred to disese control in oth UC models (41.9 ±.7 for cute UC, not significnt t p<.5; nd 41.5 ± 1. for chronic UC) (Fig C) MSE modertely lessens histopthologies ssocited with DSS- induced UC The histopthologicl nlysis ws performed in H&E-stined distl colon sections of mice with DSSinduced cute nd chronic UC. Microscopic imges (x mgnifiction) of representtive colonic sections were tken for ech group, nd histopthologicl scores were ssessed (Tle 3). In oth UC models, the helthy control groups showed intct epithelium with undmged crypts nd no ulcertive foci (Fig 3A nd 3B)

21 Histopthologicl score Low severity High severity A B 373 wter/vehicle DSS/MSE wter/vehicle DSS/MSE B DSS/vehicle DSS/5-ASA DSS/vehicle DSS/5-ASA 378 C 8 8 ulcertion crypt dmge inflmmtion 6 c 6 c 4 4 wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA Acute UC Chronic UC Fig 3. Effects of MSE on colon histopthology in DSS-induced UC. Representtive histologicl oservtions of H&E-stined colonic sections of (A) cute UC mice (n=8/group), nd (B) chronic UC mice (n=6/group). Experimentl designs nd disese induction procedures re defined in Tle 1. (C) Associted colonic histopthologicl scores clculted s sum of scores of ech prmeter consisting of histopthologicl scoring system s descried in Tle 3. Histologicl imges were cquired y x mgnifiction (Scle r = 5 m). Dt re expressed s men ± SEM. Different letters indicte significnt differences etween groups (p<.5). 5-minoslicylic cid (5-ASA); dextrn sulfte sodium (DSS); hemtoxylin nd eosin (H&E); moring seed extrct (MSE); ulcertive colitis (UC). 1

22 In disese control mice, colonic sections exhiited severe crypt loss, extensive mucosl dmge, nd extended inflmmtory cell infiltrtion into mucosl nd sumucosl comprtments, s is commonly reported in the DSS-induced UC model. Extensive erosion nd sumucosl edem were lso presented in the distl colon s the indiction of n inflmmtory phenotype in this group (Fig 3A nd 3B). On the contrry, tretment with either MSE or 5-ASA resulted in less severe inflmmtion (reltively less extensive epithelil erosion) nd development of few regenerting foci compred to disese control in cute UC (Fig 3A). In chronic UC, neither MSE nor 5-ASA tretments mesurly rescued chronic inflmmtion, s extensive mucosl dmge nd confluent ulcertive erosion were similrly oserved in treted nd disese control groups (Fig 3B). In the ssessment of histopthologicl scores, ll mice in DSS-treted groups showed mrked increse of the scores, which ws significntly reduced y 5-ASA. MSE showed trend in reducing histopthologicl scores in cute UC, which ws not significnt t p<.5 (Fig 3C) MSE decreses secretion of colonic pro-inflmmtory iomrkers nd fecl Lcn- levels Cytokines nd chemokines re importnt meditors of the mucosl inflmmtion nd immune responses during UC. In cute UC, compred to disese control, colonic production of KC, TNF-α, nd NO were decresed y MSE (not significntly t p<.5), wheres 5-ASA significntly decresed levels of KC (-48.1%) nd NO (-7.4%) (Fig 4A-4C)

23 Lcn- [ng/(g) feces] NO [µm per (g) colonic tissue] MPO [ug/ml per (g) colonic tissue] KC [pg/ml per (g) colonic tissue] TNF-α [pg/ml per (g) colonic tissue] A 15 1 wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA c B 6 4 c 5 41 C 1 8 Acute UC Chronic UC D 3 Acute UC Chronic UC E 8 Acute UC Chronic UC Acute UC Chronic UC 6 4 c c 414 Acute UC Chronic UC Fig 4. Effects of MSE on colonic pro-inflmmtory iomrkers nd fecl Lcn- levels in DSS-induced UC. Anlyses of (A) KC, (B) TNF-α, (C) NO, nd (D) MPO were performed in colon culture superntnts of cute UC (n=8/group) nd chronic UC (n=6/group) mice. All concentrtions were normlized to 1 g of the colon tissue weight. Fecl levels of (E) Lcn- were normlized to 1 g of the fecl weight. Experimentl designs nd disese induction procedures re defined in Tle 1. Dt re expressed s men ± SEM. Different letters indicte significnt differences etween groups (p<.5). 5-minoslicylic cid (5-ASA); dextrn sulfte sodium (DSS); kertinocyte-derived cytokine (KC); lipoclin- (Lcn-); 3

24 43 44 myeloperoxidse (MPO); moring seed extrct (MSE); nitric oxide (NO); tumor necrosis fctor-α (TNF-α); ulcertive colitis (UC) The levels of MPO were significntly lowered y oth MSE (-35.7%) nd 5-ASA (-57.%) in the cute UC model (Fig 4D). In chronic UC, reductions in these mrkers were more ovious, showing tht, compred to disese control, colonic productions of ll tested pro-inflmmtory mrkers were significntly lowered y MSE (-.3% for KC; -6.4% for TNF-α; -41.8% for NO; -67.1% for MPO). The effect of 5-ASA ws negligile in chronic UC, only showing significnt reduction in MPO (-51.9%) (Fig 4A-4D). Mice receiving DSS showed drmtic increse of the fecl Lcn- level in oth cute nd chronic UC, which, in cute UC, ws significntly reduced y MSE (-7.7%), ut not y 5-ASA, compred to disese control (Fig 4E) MSE downregultes colonic expression of pro-inflmmtory cytokines nd inos Pthologicl process of UC is closely relted to the incresed level of pro-inflmmtory cytokines tht cn lso ctivte inos. We exmined the effect of MSE on colonic expression of pro-inflmmtory cytokines including IL-1β, IL-6, nd TNF-α, s well s inos. In cute UC, compred to helthy control, ll these pro-inflmmtory mrker genes were upregulted y DSS (up to ~15 times for inos). However, in the cute model, the expression of these pro-inflmmtory mrker genes ws, in most cses, mrkedly lowered y MSE (-73.% for IL-1β; -97.1% for IL-6; -43.4% for TNF-α, not significnt t p<.5; - 73.% for inos) compred to disese control (Fig 5A-5D)

25 GSTP1 / GAPDH Cludin-1 / HMBS ZO-1 / HMBS TNF-α / HMBS inos / HMBS IL-1β / HMBS IL-6 / HMBS A C E Acute UC Acute UC wter/vehicle DSS/vehicle DSS/MSE DSS/5-ASA Chronic UC Chronic UC D B F Acute UC Acute UC Chronic UC Chronic UC 448 Acute UC Chronic UC Acute UC Chronic UC G 1 1 HNQO1 / GAPDH HO1 / GAPDH Acute UC Chronic UC Acute UC Chronic UC I Acute UC Chronic UC 5

26 Fig 5. Effects of MSE on colonic expression of pro-inflmmtory mrkers, tight-junction proteins, nd Nrf-medited enzymes in DSS-induced UC. Gene expression of (A) IL-1β, (B) IL-6, (C) TNF-α, (D) inos, (E) cludin-1, (F) ZO-1, (G) GSTP1, (H) NQO1, nd (I) HO1 ws nlyzed y qpcr in colon tissues of cute UC (n=8/group) nd chronic UC (n=6/group) mice. All dt re expressed s reltive fold chnge compred to helthy control (vlue = 1.). Experimentl designs nd disese induction procedures re defined in Tle 1. Dt re expressed s men ± SEM. Different letters indicte significnt differences etween groups (p<.5). 5-minoslicylic cid (5-ASA); dextrn sulfte sodium (DSS); glycerldehyde-3- phosphte dehydrogense (GAPDH); glutthione-s-trnsferse pi 1 (GSTP1); hydroxymethylilne synthse (HMBS); heme oxygense 1 (HO1); interleukin-1β (IL-1β); interleukin-6 (IL-6); inducile nitric oxide synthse (inos); moring seed extrct (MSE); NAD(P)H quinone oxidoreductse 1 (NQO1); tumor necrosis fctor-α (TNF-α); ulcertive colitis (UC), zonul occludens-1 (ZO-1) ASA, similr to MSE, downregulted the expression of the pro-inflmmtory genes, except for TNF-α (Fig 5A-5D). In chronic UC, neither MSE nor 5-ASA showed significnt effects on pro-inflmmtory mrker genes (Fig 5A-5D) MSE elevtes colonic expression of tight-junction proteins nd Nrf- medited phse II detoxifying enzymes UC is ssocited with incresed intestinl permeility nd decresed tight-junction protein expression, such s cludin-1 nd ZO-1, in the inflmed mucos [9]. DSS normlly suppresses cludin-1 gene expression; however, MSE incresed cludin-1 expression in oth UC models nd the effect ws highly significnt in the cute model (558%) (Fig 5E). A similr result ws oserved for ZO-1, showing decresed gene expression y DSS, which ws upregulted y MSE, ut not y 5-ASA, in cute (148%; not 6

27 significnt t p<.5) nd chronic UC (148%) compred to disese control (Fig 5F). Gene expression of phse II detoxifying enzymes, including GSTP1, NQO1, nd HO1, re directly regulted y the Nrf trnscription fctor. Nrf cn lso modulte NF-κB-dependent pro-inflmmtory signls [19]. In chronic UC, MSE led to upregulted gene expression of GSTP1 (16%), NQO1 (144%), nd HO1 (18%), compred to disese control; however, only GSTP1 upregultion ws significnt (Fig 5G-5I). In ddition, the expression of NQO1 nd HO1 genes ws significntly upregulted y MSE compred to helthy control (Fig 5H nd 5I). In cute UC, DSS showed trend towrds reducing the Nrf-regulted gene expression tht ws not ffected y MSE or 5-ASA (Fig 5G-5I). 48 7

28 483 Discussion This study imed to investigte the efficcy nd mechnisms of ction of MIC-1-enriched MSE in lleviting UC symptoms. Dt indicte tht orl dministrtion of MSE t 15 mg/kg w mitigted UC symptoms nd ssocited iomrkers in oth DSS-induced cute nd chronic UC mouse models y modulting inflmmtory nd ntioxidnt response systems. Acute UC is chrcterized y rpid onset of severe clinicl symptoms within reltively short induction period, wheres chronic UC represents less severe nd more sustined inflmmtion developed through slower ut long-lsting induction period with possile flre-up nd remission symptomtic cycles. DSS disrupts the integrity of the mucosl rriers [], leding to incresed permeility of luminl toxic sustnces into the lmin propri nd sumucosl comprtments, resulting in secondry inflmmtion in the intestine [4]. In our study, two DSS-induced UC models were successfully estlished, with more severe UC symptoms oserved in the cute thn chronic UC model, reflected y higher DAI scores (~7x vs. ~5x higher DAI score in cute vs. chronic UC, respectively) compred to DSS-untreted mice. Orl delivery of MSE (15 mg/kg w for 1 or wks) promptly reduced the severity of UC symptoms in oth UC models, including improvements in w loss, stool consistency, nd intestinl leeding. MSE improved the integrity of colon tissues y correcting colon shortening, likely due to loss in crypt structure, nd colon thickness, which is due to edem formtion [3]. MSE lso reduced the severity of colonic inflmmtion developed during UC, diminished epithelil erosion nd regenerted few foci in colonic tissues in cute UC. However, MSE filed to ttenute these histopthologicl signs of UC in the chronic UC model. This is, nevertheless, consistent with previous report tht complete heling of owel ulcertion my not e concomitnt with clinicl remission of UC symptoms, nd with recommendtions from previous clinicl studies to mesure mucosl restitution fter 8 wks of tretments [31]. 5-ASA, n nti-inflmmtory drug for tretment of mild-to-moderte 8

29 severity of UC in humns, ws used s positive control with recommended dose of 5 mg/kg w [31]. 5-ASA produced comprle DAI score to tht of MSE in cute UC, ut ws less efficcious thn MSE in chronic UC. Moreover, the impct of 5-ASA on colonic histopthologicl signs of UC ws only evident in cute UC, nd could not improve errnt chrcteristics of colon tissues, like colon shortening, cused y DSS. Our oservtions in chronic UC mice corroorte previous findings tht 5-ASA, leit widely used s first-line therpy for UC, is lrgely ineffective in chronic UC [3]. Our findings re consistent with pulished reports on powerful nti-inflmmtory effects of vrious isothiocyntes [1, 15, 16] nd previous oservtions tht orlly dministered phenethyl isothiocynte (PEITC; ~75 mg/kg w) or sulforphne (5 mg/kg w), isothiocyntes from Brssic vegetles tht hve the sme phrmcophore (N=C=S) s MIC-1, could lso relieve cute nd chronic symptoms of DSS-induced UC in mice [14, 5]. Our compnion pper pulished in this journl [S1 File] demonstrtes tht MIC-1 is the min nti-inflmmtory component of MSE. MIC-1 ws more effective thn curcumin (one of the most potent nti-inflmmtory phytochemicls [33]) in reducing the production of NO nd expression of inos, IL-1β, nd IL-6 in LPS-stimulted RAW mcrophges. Additionlly, MIC-1 significntly upregulted the expression of Nrf-medited phse II detoxifiction genes (GSTP1, NQO1, nd HO1) in these cells more effectively thn curcumin. MSE ws more potent thn curcuminoid-enriched turmeric extrct in reducing crrgeenn-induced pw edem in rts [S1 File]. Tken together with our current oservtions, the ville dt suggests tht MIC-1 is the ioctive component of MSE responsile for its therpeutic effects in UC. Imlnced production of cytokines is prt of UC pthophysiology. These cytokines form complex network tht medites mucosl/sumucosl inflmmtion nd influences the integrity of epithelium [34, 35]. An increse of pro-inflmmtory cytokines, including IL-1β, IL-6, TNF-α, nd interferon-γ (IFN-γ), is ssocited with disese ctivity nd development of ll UC [36-38]. Menwhile, KC serves s chemottrctnt for neutrophils [39], wheres MPO is direct indictor of neutrophil 9

30 infiltrtion into the colon nd serves s surrogte mrker of UC-relted inflmmtion [3]. Administrtion of nti-kc ntiodies reduced neutrophil trfficking into the colons of mice with DSSinduced UC [4], indicting key role of KC in colonic recruitment of neutrophils. Lcn-, lso referred to s neutrophil geltinse-ssocited lipoclin (NGAL) or sideroclin, is cteriosttic protein minly relesed from neutrophils t sites of inflmmtion [41], nd its level in serum, colon, nd fecl smples is elevted in multiple models of UC nd in humn IBD [4, 43]. inos-generted NO is signling molecule in inflmmtion, infection, nd cncer [44]. inos knockout mice exhiited sustntil reduction of DSS-induced colonic injury compred to wildtype [44]. A positive correltion etween NO nd incresed pro-inflmmtory cytokines (TNF-α, IL-6, IL-17, IL-1, nd IFN-γ) is descried in IBD ptients [45]. We lso oserved incresed production nd/or gene expression of pro-inflmmtory mrkers (IL-1, IL-6, TNF-α, inos, NO, KC, MPO, nd Lcn-) in oth UC models. MSE tretment generlly reduced these mrkers, lthough the effect of MSE differed in cute nd chronic UC models. Similrly, previous study showed tht PEITC downregulted colonic IL-1β gene expression in DSS-induced UC mice [5]. These results re consistent with the well-documented nti-inflmmtory effects of vrious isothiocyntes, which re medited, t lest in prt, y Nrf ctivtion [45]. Tight-junction proteins, such s ZO-1, occludins, nd cludins, form physiologiclly ctive rriers in intestinl epithelil cells tht cn e disrupted in IBD [9, 46, 47]. For exmple, the loss of ZO- 1 protein ws oserved in DSS-induced UC mice [9]. Our dt showed tht, while DSS tretment led to some downregultion of cludin-1 nd ZO-1 genes, MSE restored or upregulted these genes, consistent with its oserved therpeutic effects in UC. Oxidtive stress is oth consequence of UC [48] nd one of its contriuting fctors [49]. Our dt suggest tht MSE protects ginst chronic colonic inflmmtion y lowering oxidtive stress through upregultion of ntioxidnt/phse II detoxifying enzymes, in prticulr, GSTP1. GSTP1 is the most undnt isoform of glutthione S trnsferse (GST) in the colon [49], ctlyzing the conjugtion of 3

31 xenoiotics to glutthione [5], nd DSS-induced UC mice exhiited downregultion of GST gene expression [5]. We suggest tht the upregultion of ntioxidnt enzymes oserved in our study is the result of MIC-1-medited ctivtion of Nrf, similr to tht cused y sulforphne in DSS-induced UC [14]. Studies hve shown tht isothiocyntes such s sulforphne nd PEITC ctivte Nrf, which susequently reduces oxidtive stress nd inflmmtion [13, 51]. Nrf knockout ttenuted the ntiinflmmtory effects of PEITC nd curcumin in mcrophges ex vivo [5]. Therefore, MIC-1 nd other isothiocyntes re likely responsile for interctions with the Nrf/Kep1 complex nd further downstrem processes [53]. In conclusion, MSE, enriched with the MIC-1, its min ioctive, meliorted pthologicl events ssocited with cute nd chronic UC. Anti-inflmmtory nd ntioxidnt ctivities of MSE re likely ssocited with Nrf-medited nti-inflmmtory/ntioxidnt signling pthwy. This study supports the therpeutic potentil of MSE for prevention nd tretment of UC nd should e further explored in humn studies

32 568 Acknowledgements 569 The uthors wnt to thnk Peter Kuhn, Sr Fghni, nd Hetl Klriy for their technicl ssistnce

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