Anti-in flammatory effects of Lacto-Wolfberry in a mouse model of experimental colitis

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1 Online Submissions: doi:1.3748/wjg.v18.i World J Gstroenterol 1 October 14; 18(38): ISSN (print) ISSN (online) 1 Bishideng. All rights reserved. ORIGINAL ARTICLE Anti-in flmmtory effects of Lcto-Wolfberry in mouse model of experimentl colitis Dvid Philippe, Virl Brhmbhtt, Frncis Fot, Yen Sudn, Ptrick Serrnt, Stephnie Blum, Jlil Benycoub, Krine Vidl Dvid Philippe, Virl Brhmbhtt, Frncis Fot, Yen Sudn, Ptrick Serrnt, Stephnie Blum, Jlil Benycoub, Krine Vidl, Deprtment of Nutrition nd Helth, Nestlé Reserch Center, CH-1 Lusnne, Switzerlnd Author contributions: Philippe D, Blum S, Benycoub J nd Vidl K designed the experiments; Philippe D, Fot F, Sudn Y nd Serrnt P performed the reserch; Philippe D, Brhmbhtt V, Benycoub J nd Vidl K nlyzed the dt nd wrote the mnuscript;; ll uthors pproved the finl mnuscript. Correspondence to: Virl Brhmbhtt, MBBS, PhD, R nd D Specilist, Deprtment of Nutrition nd Helth, Nestlé Reserch Centre, Vers-chez-les-Blnc 6, PO Box 44, CH-1 Lusnne, Switzerlnd. virlvishnuprsd.brhmbhtt@rdls.nestle.com Telephone: Fx: Received: Mrch 1, 1 Revised: July 1, 1 Accepted: July 18, 1 Published online: October 14, 1 Abstrct AIM: To investigte the nti-in flmmtory properties of Lcto-Wolfberry (LWB), both in vitro nd using mouse model of experimentl colitis. METHODS: The effects of LWB on lipopolyscchride (LPS)-induced rective oxygen species (ROS) nd interleukin (IL)-6 secretion were ssessed in murine mcrophge cell line. in vitro ssessment lso included chrcterizing the effects of LWB on the ctivtion of NF-E relted pthwy nd inhibition of tumor necrosis fctor- (TNF- )-induced nucler fctor- B (NF- B) ctivtion, utilizing reporter cell lines. Following the in vitro ssessment, the nti-inflmmtory efficcy of n orl intervention with LWB ws tested in vivo using preclinicl model of intestinl in flmmtion. Multiple outcomes including body weight, intestinl histology, colonic cytokine levels nd nti-oxidtive mesures were investigted. RESULTS: LWB reduced the LPS-medited induction of ROS production [+LPS vs 1% LWB + LPS, 159 ± reltive luminescence units (RLU) vs 389 ± 5.9 RLU, P <.1]. LWB ws more effective thn wolfberry lone in reducing LPS-induced IL-6 secretion in vitro (wolfberry vs.5% LWB, 15% ± 7.8% vs 64% ± 5%, P <.1). In ddition, LWB incresed reporter gene expression vi the nti-oxidnt response element ctivtion (wolfberry vs LWB, 73% ± 6.9% vs 148% ± 8.3%, P <.1) nd inhibited the TNF- -induced ctivtion of the NF- B pthwy (milk vs LWB, 1% ± 6.7% vs 35% ± 3.3%, P <.5). Furthermore, orl supplementtion with LWB resulted in reduction of mcroscopic ( vs, 5.39 ±.61 vs 3.66 ±.59, P =.445) nd histologicl scores ( vs, 5.44 ±.3 vs 3.66 ±.59, P =.87) in colitic mice. These effects were ssocited with significnt decrese in levels of inflmmtory cytokines such s IL-1 ( vs, 57 ± 45 g/l vs 89 ± 38 g/l, P =.16), kertinocyte-derived chemokine/growth regulted protein- ( vs, 184 ± 49 g/l vs 75 ± g/l, P =.44), IL-6 ( vs, 318 ± 99 g/l vs 117 ± 18 g/l, P =.315) nd other pro-inflmmtory proteins such s cyclooxygense- ( vs,.95 ±.1 AU vs.36 ±.11 AU, P =.36) nd phosphorylted signl trnsducer nd ctivtor of trnscription-3 ( vs,.51 ±.15 AU vs.1 ±.4 AU, P =.57). Moreover, ntioxidnt biomrkers, including expression of gene encoding for the glutthione peroxidse, in the colon nd the plsm nti-oxidnt cpcity were signi ficntly incresed by supplementtion with LWB ( vs, 1. ±.1 mmol/l vs.1 ±.19 mmol/l, P =.95). CONCLUSION: These results demonstrte the ntiinflmmtory properties of LWB nd suggest tht the underlying mechnism is t lest in prt due to NF- B inhibition nd improved nti-oxidtive cpcity. 1 Bishideng. All rights reserved October 14, 1 Volume 18 Issue 38

2 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry Key words: Lcto-Wolfberry;; Colitis;; Nutrition;; Inflmmtion;; Wolfberry;; In flmmtory bowel disese;; Crohn s disese Peer reviewers: Dr. Ferenc Sipos, nd Deprtment of Internl Medicine, Semmelweis University, Szentkirályi 46, 188 Budpest, Hungry;; Dr. Tedros Bezbeh, Institute for Biodignostics, Ntionl Reserch Council, 435 Ellice Avenue, Winnipeg R3B 1Y6, Cnd;; Je Hee Cheon, Professor, Deprtment of Internl Medicine, Yonsei University College of Medicine, 5 Seongsnro, Seodemun-gu, Seoul 1-75, South Kore Philippe D, Brhmbhtt V, Fot F, Sudn Y, Serrnt P, Blum S, Benycoub J, Vidl K. Anti-inflmmtory effects of Lcto- Wolfberry in mouse model of experimentl colitis. World J Gstroenterol 1; 18(38): Avilble from: URL: DOI: INTRODUCTION Inflmmtory bowel disese (IBD), which includes Crohn s disese (CD) nd ulcertive colitis (UC), represents group of chronic disorders chrcterized by inflmmtion of the gstrointestinl trct, typiclly with relpsing nd remitting clinicl course. They ffect between.5%-1% of the Western world s popultion [1]. The primry focus of IBD therpy is to induce remission of cute inflmmtory flre ups nd to mintin the stte of remission. The innte immune system plys centrl role in the cute inflmmtory process. As prt of the innte immune response, neutrophils re one of the erly responders to locl injury. Both, the circulting levels nd ctivtion of neutrophils, re incresed in IBD ptients with ctive disese [,3]. Activted neutrophils nd monocytes relese plethor of meditors including rective oxygen species (ROS), eicosnoids nd proinflmmtory cytokines. In fct, the therpeutic benefit of depleting grnulocytes in CD ptients hs been demonstrted [4]. Aprt from neutrophils, monocytes nd mucosl mcrophges ply n importnt role in the development of IBD s shown by n increse of the number of recruited monocytes nd ctivted mcrophges in the inflmed gut of ptients with IBD [5]. Indeed during ctive inflmmtion, neutrophils recruit nd ctivte monocytes which themselves secrete pro-inflmmtory meditors such s tumor necrosis fctor (TNF- ), interleukin (IL)-1 nd IL-6 [6]. A vst body of literture supports the role of nutritionl therpy in IBD, prticulrly in CD (reviewed in [7,8] ). While enterl nutrition is not s effective s steroid therpy in induction of remission in CD, the benefit to the ptient is well estblished [9]. Thus, identifiction nd chrcteriztion of novel nti-inflmmtory foods my id in improving the currently vilble nutritionl formultions. A vriety of functionl nutrients, such s glycosides [1], lkloids [11] nd blck te extrcts [1], hve been shown to exert their beneficil effects through inhibition of Nucler fctor- B (NF- B) ctivtion. NF- B is one of the most importnt regultors of pro-inflmmtory cytokine expression nd reducing its ctivity my hve beneficil effects under cute inflmmtory conditions [13]. Besides NF- B, phytochemicls re lso known to ctivte NF-E relted (Nrf) pthwy through the nti-oxidnt response element cusing n increse in the nti-oxidtive enzymes such s ctlse (CAT), superoxide dismutse (SOD) nd glutthione peroxidse [14]. Wolfberry, the fruit of Lycium brbrum-lso clled s Goji (Gouqi or Gou QZ in Romnized Chinese), is sweet red berry, which hs been trditionlly used in Chinese medicine. It is lso one of the richest sources of zexnthin, n ntioxidnt tht hs been postulted to improve visul cuity [15]. Aprt from ntioxidnt ctivity, wolfberry juice hs lso been demonstrted to hve immunomodultory effects [16]. However, lrge prt of the supporting evidence is derived either from in vitro experiments or niml studies wherein, wolfberry extrcts were delivered prenterlly. We believe tht this might be due to reduced biovilbility of ctive ingredients when given enterlly. Therefore, to improve biovilbility of its nti-oxidnt components, wolfberry ws processed with skimmed milk nd freeze-dried to generte Lcto- Wolfberry (LWB), wter-dispersible powder [17]. This novel preprtion, which contins pproximtely 5% wolfberry nd 5% skimmed milk, hs been cliniclly demonstrted to improve the biovilbility of zexnthin [17]. Subsequently, the immune-enhncing properties of LWB in both, young-dult nd ged mice, hve been chrcterized [18]. Recent studies hve demonstrted tht dietry supplementtion with LWB enhnces immune response to flu vccine [19] nd plsm oxidtive cpcity in elderly []. The im of this study ws to chrcterize the nti-inflmmtory nd nti-oxidtive properties of LWB. We first demonstrte tht LWB inhibits lipopolyscchride (LPS)-induced ROS nd IL-6 production in murine mcrophge cell line. Next, using reporter cell lines we show tht LWB ctivtes Nrf pthwy, while inhibiting the NF- B pthwy. Finlly, using mice model of colitis, we demonstrte tht LWB reduces the severity of colitis by mediting reduction in pro-inflmmtory cytokines, nmely IL-6, IL1 nd kertinocyte-derived chemokine/growth regulted protein- (KC/GRO- ). MATERIALS AND METHODS Inflmmtory response in LPS-chllenged RAW cells The murine mcrophge cell line RAW 64.7 (ATCC, United Sttes) ws mintined in Dulbecco s modified Egle medium (DMEM, Amimed, Bioconcept, Switzerlnd) supplemented with 1% het-inctivted fetl-clf serum (FCS, Amimed) t 37 in 5 ml/l CO/ir incubtor. Intrcellulr ROS ws mesured using ROS-sensitive fluorescent dye,,7 -dichlorofluorescin dicette (DCFH-DA, Sigm, United Sttes). Cells ( October 14, 1 Volume 18 Issue 38

3 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry cells/well in 96 well pltes) were incubted overnight with LPS, from Escherichi coli serotype 55:B5 (Sigm, United Sttes) t.5 mg/l, either in the bsence or presence of LWB (.1% or 1% finl concentrtion). Control cells in the bsence of LPS were lso included. Cells were then treted with 1 mol DCFH-DA for 3 min t 37 nd wshed twice with phosphte-buffered sline (PBS). Fluorescence ws mesured t 485 nm excittion nd 538 nm emission by Fluoroskn enzyme linked immunosorbent ssy plte reder (Lbsystems Oy, Finlnd) t the indicted time points. For experiments mesuring IL-6, RAW 64.7 cells were seeded in 96 well pltes t 1 4 cells/well. After 3 d (pproximtely 8% of confluence), cells were stimulted with LPS t.5 mg/l nd incubted in the presence of either LWB (1% finl concentrtion), wolfberry (.5%) or skimmed milk (.5%) for 4 h t 37. Cell culture superntnts were then hrvested nd IL-6 secretion ws quntified using commercil enzyme linked immunosorbnt ssy (ELISA) kit ccording to mnufcturer s protocol (Murine IL-6 Eli-pir, Diclone, Frnce). Cell vibility ws determined by CellTiter-Glow Luminescent ssy (Promeg, United Sttes) ccording to mnufcturer s instructions. It should be noted tht the sme lots of wolfberry nd skimmed milk tht were used in the preprtion of LWB were used for ll experiments described. NF- B inhibition ssy The humn colonic denocrcinom cell line, HT-9 (ATCC, United Sttes), ws stbly trnsfected with the plsmid pnf- B-SEAP-NPT. The plsmid ws kind gift from Prof. Kim (Nturl Products Reserch Institute, Seoul). It contins secreted lkline phosphtse (SEAP) encoding sequence downstrem of four tndem copies of NF- B binding sites. Stbly trnsfected cells (HT-9 clone 34) were mintined in high glucose (4.5 g/l) DMEM contining 1% L-glutmine, 1% het-inctivted FCS, 1% penicillin/streptomycin, 5 mg/l G418 (Invitrogen, Switzerlnd) nd 1 mg/l Normocin (Invivogen, Frnce) t 37 in 5 ml/l CO/ir incubtor. For the NF- B inhibition ssy, HT-9 clone 34 cells were seeded t 1 4 cells/well in 96-flt bottom well pltes. After 3-4 d of culture (pproximtely 8% confluence), cells were wshed with PBS nd stimulted with recombinnt TNF- (1 g/l, RD systems, Englnd) in the bsence or the presence of either LWB (1% finl concentrtion), wolfberry (.5%) or skimmed milk (.5%) for 4 h t 37. SEAP relese ws ssessed in the superntnts using the Phosph-Light TM System (Applied Biosystems, United Sttes) ccording to mnufcturer s protocol. Nrf ctivtion ssy AREc3 (CXR biosciences, United Kingdom) is reporter cell line tht stbly expresses the nti-oxidnt response element (ARE)-driven luciferse gene [1]. These cells were cultured t 1 cells/well in 96 well pltes (Nunc) t 37 nd 5 ml/l CO/ir incubtor in DMEM supplemented with 1% FCS. After 1 d, cells were wshed (LWB-supplemented) (n = 9) Lcto-Wolfberry t 1% in the chow diet (control group) (n = 9) Chow diet LWB by gvge (5 mg) PBS Food intke nd body weight mesurement once dily Dys D-7 D-6 D-5 D-4 D-3 D- D-1 D D1 D D3 D4 Colitis induction Scri fice Figure 1 Animl study design. Body weight nd food intke ws monitored throughout the durtion of the experiment (i.e., D-7 to D4). Colitis ws induced on D. For the Lcto-wolfberry (LWB) fed group, diet ws supplemented with 1% LWB from D-7 until D4 nd 5 mg of LWB ws lso gvged from D to D4. Control nimls were fed regulr diet for the sme period nd gvged with equl volume of control solution from D to D4. PBS: Phosphte buffered solution. with PBS nd treted, in the bsence of serum, with either, LWB (1% finl concentrtion), wolfberry (.5%) or skimmed milk (.5%) for 4 h t 37. Luciferse ctivity ws mesured using the Luciferse Assy (Promeg, United Sttes) following mnufcturer s instructions. Experimentl colitis model Mle mice (C57BL/6J), ged 7 wk, were obtined from Chrles River Lbortories Inc. (Frnce) nd housed five per cge in temperture-controlled room with free ccess to food nd wter. The overll study design is provided in Figure 1. Mice (n = 9 per group) were rndomly ssigned to either the control (LWB, chow fed) or the LWB-supplemented group (, 1% in the diet) 7 d prior to colitis induction (D-7), which ws induced on D, s previously described []. Chemiclly induced colitis ws performed s described erlier [3]. Briefly, colitis ws induced with n intrrectl dministrtion of,4,6-trinitrobenzene sulfonic cid (TNBS) t 15 mg/kg dissolved in 5% ethnol solution. In order to compenste for reduced food intke fter colitis induction, mice were lso supplemented by dily gvge with 5 mg of LWB from D to D4. Control mice were gvged with n equl volume of PBS. Food intke nd body weight mesurements were tken once dily from D-7 to D4. Animls were scrificed on D4. All experimentl protocols were conducted in ccordnce with Swiss lw nd Nestlé policy on ethics nd niml welfre. Mcroscopic nd histologicl ssessment The distl colon tissue ws wshed with PBS nd mcroscopic scoring ws performed using the system of Wllce et l [4]. Smples of the inflmed tissues (1 cm bove the nl cnl) were collected for histologicl nlysis. The tissues were fixed in 4% prformldehyde t October 14, 1 Volume 18 Issue 38

4 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry for 4 h. Sections were prepred, stined with hemtoxylin nd eosin, nd grded ccording to Ameho et l [5]. mrna expression nlysis Colon tissue homogeniztion, RNA extrction nd reverse trnscription were performed s described erlier []. Custom-mde Low Density Arry (LDA) crds were purchsed from Applied Biosystems (United Sttes) nd used ccording to mnufcturer s instructions. Briefly, mixes (1 L), contining 1 ng of cdna, X TqMn Mix nd nuclese-free wter, were prepred nd loded onto the LDA crd. The LDA crds were then processed using n utomted fluorometer ABI Prism 79HT. Gene expression ws clculted using the reltive quntifiction method with SDS.. softwre. Electrophoresis nd Western blotting nlysis Colon tissue homogeniztion, protein extrction, electrophoresis nd Western blotting nlysis were performed s described erlier []. Briefly, fter tissue homogeniztion, protein ws quntified using the RC DC Protein Assy (Bio-Rd, United Sttes). Proteins were loded nd seprted on 4%-1% bis-tris gel (Invitrogen). The blot ws probed with ntibodies ginst murine cyclooxygense- (COX-) (Cymn, United Sttes), signl trnsducer nd ctivtor of trnscription-3 (STAT-3) nd phosphorylted STAT-3 (pstat-3, Cell Signlling Technology, United Sttes) nd -Actin (Sigm, United Sttes). Reltive quntittion of bnds ws determined using Scion Imge Densitometry System (Scion Corp., United Sttes), with normliztion to -Actin. Myeloperoxidse ssy Protein levels for myeloperoxidse (MPO) were mesured in colon protein extrcts by ELISA following the mnufcturer s instructions (Hycult biotechnology, The Netherlnds). Cytokine nlysis IL-1, IL-6, IL-1, IL-1p7, KC/GRO-, interferon- (IFN- ) nd TNF- were mesured in the colon protein extrcts using multiplex ssy kits (Meso Scle Discovery, United Sttes) ccording to mnufcturer s protocol. Cytokine concentrtions were determined with Discovery Workbench 3. softwre, using curve 4-PL s suggested by the mnufcturer. Plsm nti-oxidnt cpcity Totl nti-oxidnt cpcity of plsm ws performed using n ssy, which mesures inhibition of, -zino-di- (3-ethylbenzthizoline sulphonte) (ABTS ) to ABTS + by metmyoglobin s Trolox equivlents (Cymn, United Sttes), ccording to mnufcturer s protocol. Sttisticl nlysis Dt were nlyzed by mens ± SE either the Mnn- Whitney test or where pproprite, two-wy nlysis of vrince with Bonferroni post test. P vlues of less thn 5% were considered s significnt. RESULTS Effects of LWB in vitro Anti-oxidnt effects: ROS production from LPS-stimulted RAW 64.7 cells ws evluted in the presence nd bsence of LWB (Figure A). As expected, LPS incresed ROS production, in time dependent mnner, s compred to the untreted controls (-LPS). Interestingly, LWB ws ble to reduce LPS-induced ROS production. The inhibitory effect of LWB ws significnt from the h time point t both.1% nd 1% finl concentrtion. At the 6 h time point, LWB reduced the mount of LPSinduced ROS production by bout 5% nd 75%, t the concentrtions of.1% nd 1%, respectively (P <.1 for both concentrtions). Next, the effects of LWB on Nrf ctivtion, using stble ARE-driven reporter gene expressing cell line, AREc3 [1], were evluted (Figure B). The dt show tht LWB t 1% finl concentrtion incresed Nrf ctivity by pproximtely %, wheres its individul components, i.e., wolfberry (.5%) nd milk (.5%), induced only mild or lmost no increse in Nrf ctivity, respectively. Anti-inflmmtory effects: Finlly, the effects of LWB (1%), wolfberry (.5%) nd milk (.5%) on LPS-induced IL-6 production (Figure C) nd TNF- -induced NF- B ctivity (Figure D) were ssessed. As shown, LWB inhibited LPS-induced IL-6 production in RAW 64.7 cells by pproximtely 8% nd TNF- -induced NF- B ctivity by pproximtely 35%. These were significntly different from the vlues obtined for milk, pproximtely % nd 1%, respectively (P <.1 nd P <.5, respectively). However, no significnt differences were observed from wolfberry, which inhibited IL-6 production by pproximtely 65% nd NF- B ctivtion by 1%. Anti-inflmmtory effects of LWB in vivo The nti-inflmmtory effects of LWB were chrcterized in chemiclly-induced colitis model s described in mterils nd methods section. LWB ttenutes colitis-induced body weight loss: Body weight nd food intke of ech mouse ws monitored dily. Following colitis induction t D, the percentge men chnge in body weight of control mice ws -6.4, -9.5, -8.9 nd -7.6 t D1, D, D3 nd D4, respectively (Figure 3, LWB). The mice fed with LWB hd percentge men chnge in body weight of -4.7, -4.6, -1.9 nd -.5 t D1, D, D3 nd D4, respectively (Figure 3, ). Thus, while both mice hve reduction in body weight t D1, the reduction in body weight loss of the LWB fed mice were significntly lower from D-D4 (P <.5 t D nd P <.1 t D3 nd D4). The totl food intke between the two groups did not chnge (dt not shown) October 14, 1 Volume 18 Issue 38

5 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry A ROS production 1 (RLU) LPS +LPS LWB.1% + LPS LWB 1% + LPS B % increse in Nrf ctivity , c t /h LWB Wolfberry (.5%) Milk (.5%) C % inhibition of IL-6 production D % inhibition of NF B ctivity LWB Wolfberry (.5%) Milk (.5%) LWB Wolfberry (.5%) Milk (.5%) Figure Anti-in flmmtory nd nti-oxidtive effects of Lcto-Wolfberry in vitro. A: The effect of Lcto-Wolfberry (LWB) on lipopolyscchride (LPS)-induced rective oxygen species (ROS) production in RAW 64.7 cells ws mesured s described in the methods section. Results re expressed in reltive luminescence units (RLU). P <.5 t the given time points nd dose s compred to LPS-stimulted ROS production (+LPS); B: Effects of given concentrtions of LWB, milk nd wolfberry on NF-E relted ctivity in AREc3 cells. Results re expressed s percentge of increse compred to control conditions. P <.5 vs milk; c P <.5 vs wolfberry; C: Effects of given concentrtions of LWB, milk nd wolfberry on LPS-induced interleukin-6 production in RAW 64.7 cells. Results re expressed s percentge of inhibition compred to induction fter LPS stimultion. P <.5 vs milk; D: Effects of given concentrtions of LWB, milk nd wolfberry on tumor necrosis fctor- -induced nucler fctor- B ctivtion in reporter cell line. Results re expressed s percentge of inhibition compred to induction fter TNF- stimultion. All results re depicted s men ± SE. P <.5 vs milk. % chnge in body weight Dys fter TNBS Figure 3 Lcto-Wolfberry ttenutes colitis-induced body weight loss. Percentge chnge in body weight ws clculted from rtio of body weight mesured t ech dy fter colitis induction nd body weight t D. Results re depicted s men ± SE. P <.5 vs -Lcto-Wolfberry (LWB) t the respective time points. TNBS:,4,6-trinitrobenzene sulfonic cid. LWB reduces colonic inflmmtion: Supplementtion with LWB significntly reduced the colonic inflmmtion s judged by mcroscopic (Figure 4A) nd histologicl (Figure 4B, C nd D) evlution of intestinl inflmmtion. Mcroscopic lesions were ssessed s delineted by Wllce et l [4]. Colons of control mice () presented significntly higher scores compred to mice fed with LWB, 5.39 ±.61 nd 3.66 ±.47, respectively (Figure 4A, P <.5). In greement with the mcroscopic ssessment, histologicl evlution showed lower inflmmtory infiltrtes nd better mucosl integrity in mice fed with LWB (, Figure 4D) s compred to the control (, Figure 4C). This difference ws reflected in the histologicl score, 5.44 ±.3 nd 3.66 ±.59, for the control nd LWB-fed mice, respectively, which ws significntly different (Figure 4B, P <.1). The 7% reduction of MPO content in the LWB treted group () s compred to control mice () provides further support to reduced neutrophil infiltrtion in the LWB group (Figure 4E, P <.5). LWB reduces pro-inflmmtory effector proteins: To delve deeper into the effect of LWB, levels of vrious effector proteins were mesured in the colon tissue. As shown in Tble 1, levels of IL-1, IL-6 nd KC/GRO- were significntly reduced in colitic mice fed with LWB () compred to the control (). Moreover IL-1, IFN-, TNF- nd IL-1p7 levels were lso re October 14, 1 Volume 18 Issue 38

6 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry A Mcroscopic score B Histologicl score C D E 3 5 m 5 m MPO 1 3 (ng/mg totl protein) 1 Figure 4 Lcto-Wolfberry reduces colonic inflmmtion. A: Mcroscopic evlution of the colon ws performed ccording to Wllce criteri. Results re expressed in individul vlues nd the men vlue is represented by the horizontl blck line; B: Histologicl evlution nd scoring of the colon ws performed ccording to the Ameho criteri. The individul scores of the mice in the two groups re provided, with the men vlue being represented by the horizontl line; C: Representtive section of score 6 from the control fed mice [-Lcto-Wolfberry (LWB)]; D: Representtive section of score 1 from LWB fed mice (); E: Colonic myeloperoxidse (MPO) levels were mesured by enzyme linked immunosorbnt ssy. The vlues re represented s men ± SE. P <.5 vs. Tble 1 Cytokine levels in colon smples (men ± SE) Cytokines ( g/l) Reduction (%) P vlue TNF- 8 ± 4 31 ± IL-1 57 ± ± IL ± ± KC/GRO- 184 ± ± IL-1p7 415 ± ± IFN- 18 ± 6 7 ± IL-1 48 ± ± LWB: Lcto-Wolfberry; TNF- : Tumor necrosis fctor- ; IL-1 : Interleukin-1 ; IL-6: Interleukin-6; KC/GRO- : Kertinocyte-derived chemokine/growth regulted protein- ; IL-1p7: Interleukin-1p7; IFN- : Interferon- ; IL-1: Interleukin-1. duced by more thn 5%, however these were not sttisticlly significnt. COX- nd pstat3 levels were ssessed semi- quntittively by Western blotting nlysis nd densitometry. As shown in Figure 5A, colon of mice fed with LWB hd pproximtely 65% reduction of COX- levels (P <.1). Mice fed with LWB lso demonstrted n 8% reduction in pstat3 expression (Figure 5B) in the colon. However, this difference didn t rech sttisticl significnce (P =.57). LWB improves nti-oxidtive cpcity: Finlly, chnges in mrna expression of Nrf trget genes, such s CAT, SOD nd glutthione peroxidse (GPx1) were exmined in the colon tissue. GPx1 mrna expression ws higher by more thn % in mice fed with LWB () compred to control () (Figure 6A, P <.). No difference of gene expression ws detected for CAT nd SOD (dt not shown). Finlly, the mesures of ntioxidnt cpcity in the plsm demonstrted tht mice supplemented with LWB () hd more thn 7% increse in nti-oxidtive cpcity compred to control mice () (Figure 6B, P <.1). DISCUSSION The im of the study ws to investigte the nti-inflmmtory properties of LWB in vitro nd in n niml model of intestinl inflmmtion. Initil experiments showed tht LWB reduces LPS-induced ROS genertion. The nti-oxidnt effects of wolfberry re well chrcterized [16]. Moreover, LWB hs lso been proposed to hve ROS scvenging ctivity [6]. Thus, this finding ws not surprising. However, phytochemicls hve lso been shown to ctivte the Nrf pthwy [14]. Nrf is redox-sensitive trnscription fctor, which regultes the expression of ARE-driven nti-oxidnt enzymes [14]. Thus, the effect of LWB on Nrf ctivtion ws tested. Interestingly, LWB 5356 October 14, 1 Volume 18 Issue 38

7 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry A 1. B.7.6 COX- expression (AU) pstat-3 expression (AU) Figure 5 Effect of Lcto-Wolfberry on cyclooxygense- nd phosphorylted- signl trnsducer nd ctivtor of trnscription-3 levels. A: Cyclooxygense- (COX-) levels were ssessed semi-quntittively by Western blotting nd densitometry. COX- levels were normlized to -ctin. Results re expressed in men ± SE. P <.5 vs -Lcto-Wolfberry (LWB); B: Phosphorylted-signl trnsducer nd ctivtor of trnscription-3 (pstat3) levels were ssessed by Western blotting nd densitometry. The results re expressed s rtio of pstat3 to STAT3 (men ± SE). P =.57 vs group. A 1 3 GPx1 mrna rel expression mm Trolox equivlent 1 Figure 6 Anti-oxidnt effect of Lcto-Wolfberry in murine colitis. A: mrna expression of glutthione peroxidse 1 (GPx1) ws mesured s described in methods section. Results re expressed s men ± SE; B: Anti-oxidnt bility in plsm ws mesured in mmol/l Trolox equivlent. Results re expressed in men ± SE. P <.5 vs -Lcto-Wolfberry (LWB). hd two fold increse in Nrf ctivtion, however this effect ws not fully replicted by its mjor components tested seprtely, i.e., either, wolfberry or milk. Hence, this could suggest synergistic effect between the two mjor components of LWB resulting from the LWB mnufcturing process. Finlly, LWB demonstrted wolfberry-equivlent inhibition of LPS-induced IL-6 secretion nd TNF- -induced NF- B ctivtion. The beneficil effects observed in vitro prompted further exmintion of LWB in n niml model of colitis. The pro-inflmmtory roles of ROS production, NF- B ctivtion nd cytokines, such s IL-1, IL-6, IL-8 nd TNF-, hve been firmly estblished in IBD pthology [13,7,8]. Thus, the effects of LWB were tested in murine model of colitis. Firstly, LWB supplementtion ttenuted colitis-induced body weight loss. Secondly, both colonic prmeters, mcroscopic nd microscopic, confirmed reduction in the severity of colitis fter LWB intervention. In further support, supplementtion with LWB reduced neutrophil infiltrtion in the colon tissue. In this experimentl model, the secretion of Th1 cytokines, nmely, IL-1, IL-6, TNF- nd IFN-, ply n importnt role in the propgtion of colitis [9]. As NF B ctivtion controls expression of most of these genes [13] nd considering the in vitro inhibitory effects of LWB on NF- B ctivtion, the cytokine levels were mesured to gin mechnistic insight. Supplementtion with LWB resulted in reduced levels of mjority of these cytokines, while significntly reducing the levels of IL-1 nd IL-6. Further, LWB supplementtion reduced not just the levels of IL-6, but lso the downstrem signling vi STAT-3 ctivtion. Interestingly however, there ws only non significnt trend observed in the reduction of TNF-. It is possible tht this could be either due to technicl vribility or due to different kinetics of this cytokine. Moreover, the significnt reduction in KC/GRO-, n estblished chemokine involved in neutrophil chemotxis [3], cn explin the reduction in neutrophil infiltrtion. Thus, modultion of cytokine levels, perhps vi decrese in NF- B ctivtion, is responsible for the nti-inflmmtory effects of LWB in this study. The increse of Nrf ctivtion in vitro s well s n increse in the plsm nti-oxidtive cpcity nd upregultion of GPx1 fter LWB supplementtion in colitic mice lso suggests possibility of n nti-oxidnt mechnism underlying the nti-inflmmtory effects of LWB. However, the lck of upregultion of other Nrf trget genes, such s, CAT nd SOD mens tht the nti-oxidtive properties explins only prt of 5357 October 14, 1 Volume 18 Issue 38

8 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry the overll effects observed. Wolfberry is believed to contin t lest three different biologiclly ctive components: (1) Lycium Brbrum polyscchrides (LBP);; () zexnthin diplmitte;; nd (3) -O- -D-glucopyrnosyl-L-scorbic cid ( Vitmin C nlogue) [16]. The nti-oxidtive properties of ll three ctive ingredients of wolfberry re well documented [16]. LWB is prepred by milk bsed extrction process of wolfberry, which is believed to increse biovilbility of its ctive ingredients, s demonstrted for zexnthin [17]. In ddition, the in vitro ssys suggest tht the nti-inflmmtory ctivity of LWB is ttributble to wolfberry rther thn its milk component. However, to the best of our knowledge, wolfberry hs not been shown to reduce cytokine levels under inflmmtory conditions, s observed in our present study. In fct, wolfberry hs been shown to up-regulte cytokine expression [31] nd both LWB nd wolfberry hve demonstrted immune-enhncing effect [18,19,3-34]. On the other hnd, the nti-inflmmtory properties of milk components re well estblished [35,36]. Overll, it seems tht depending on the physiologicl environment LWB my provide support to recover homeostsis nd/or immune competence. In the present study, we cn speculte tht synergistic effect between the nti-oxidtive ingredients of wolfberry nd the ntiinflmmtory components in milk cn lso be potentil mechnism of the benefits observed in our model. In tht respect, further studies re required to identify the ctive nti-inflmmtory ingredients in LWB. Nutritionl therpies re n effective nd sfe form of intervention to induce remission in ctive stte of CD [7,8]. Despite this profile of effectiveness nd sfety, they hve not gined widespred usge, prticulrly in the re of dult IBD. One of the resons for this could be tht while they re effective, review of the clinicl trils compring the efficcy of nutritionl therpies to steroids concluded tht, they re not s effective s steroids in induction of remission [9]. Thus, clerly more needs to be done in this re. Our findings suggest tht ddition of LWB to enterl diet formultions might help improve disese outcomes in IBD ptients. However, it should be noted tht further work ddressing efficcy in different colitis models nd in-depth confirmtion of mechnism of ction is necessry before cliniclly relevnt reserch cn be undertken. ACKNOWLEDGMENTS Lcto-Wolfberry ws kindly provided by Wng J (Nestlé Reserch Center, Beijing);; the uthors kindly cknowledge the technicl ssistnce provided by Genevieve Perruisseu. COMMENTS Bckground Inflmmtory bowel disese (IBD) consists of group of disorders, such s Crohn s disese (CD) nd ulcertive colitis (UC). The incidence of IBD is incresing throughout the world. Both, CD nd UC re chrcterized by relpsing-remitting disese progression. Currently, there is no known cure for IBD nd the vrious vilble therpies re only pllitive. Herein, the uthors hve identi fied the nti-in flmmtory properties of nutritionl ingredient in preclinicl model of colitis. Reserch frontiers Due to the chronic nture of IBD nd the dverse effects of existing therpies, nutritionl ingredients with nti-in flmmtory properties my bene fit the ptient in the long term. With this in mind, the chrcteriztion of nti-inflmmtory properties of novel or trditionl food ingredients is n importnt field of reserch. Innovtions nd brekthroughs Previously, the uthors hve chrcterized the benefits of Lcto-Wolfberry (LWB) on the dptive immune system. In this rticle, they hve chrcterized the nti-inflmmtory properties of LWB. The uthors first demonstrte the nti-in flmmtory nd nti-oxidtive properties of LWB in cellulr models nd subsequently show tht LWB cn meliorte chemiclly-induced colitis. Applictions The identi fiction of the nti-in flmmtory properties of LWB rises new possibilities of developing novel nutritionl solutions for ptients with IBD. Terminology LWB is skimmed milk extrct of the trditionl Chinese ingredient, wolfberry, speci ficlly developed to increse the biovilbility of its ctive ingredients. Peer review In the originl rticle, the uthors exmined the complex nti-in flmmtory effect of LWB dministrtion in,4,6-trinitrobenzene sulfonic cid induced colitis nd in selected cell lines. The study is well designed nd the results nd conclusions re cler nd logicl. REFERENCES 1 Hnuer SB. Inflmmtory bowel disese: epidemiology, pthogenesis, nd therpeutic opportunities. In flmm Bowel Dis 6; 1 Suppl 1: S3-S9 Hni H, Tkeuchi K, Iid T, Kshiwgi N, Snibdi AR, Mtsushit I, Sto Y, Ksug N, Nkmur T. Reltionship between fecl clprotectin, intestinl in flmmtion, nd peripherl blood neutrophils in ptients with ctive ulcertive colitis. Dig Dis Sci 4; 49: McCrthy DA, Rmpton DS, Liu YC. 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9 Philippe D et l. Anti-in flmmtory effects of Lcto-Wolfberry 1 Song YA, Prk YL, Kim KY, Chung CY, Lee GH, Cho DH, Ki HS, Prk KJ, Cho SB, Lee WS, Kim N, Ahn BW, Joo YE. Blck te extrct prevents lipopolyscchride-induced NF- B signling nd ttenutes dextrn sulfte sodium-induced experimentl colitis. BMC Complement Altern Med 11; 11: Krrsch T, Jobin C. NF-kppB nd the intestine: friend or foe? In flmm Bowel Dis 8; 14: Surh YJ, Kundu JK, N HK. Nrf s mster redox switch in turning on the cellulr signling involved in the induction of cytoprotective genes by some chemopreventive phytochemicls. Plnt Med 8; 74: Richer SP, Stiles W, Grhm-Hoffmn K, Levin M, Ruskin D, Wrobel J, Prk DW, Thoms C. Rndomized, double-blind, plcebo-controlled study of zexnthin nd visul function in ptients with trophic ge-relted mculr degenertion: the Zexnthin nd Visul Function Study (ZVF) FDA IND #78, 973. Optometry 11; 8: e6 16 Pottert O. Goji (Lycium brbrum nd L. chinense): Phytochemistry, phrmcology nd sfety in the perspective of trditionl uses nd recent populrity. Plnt Med 1; 76: Benzie IF, Chung WY, Wng J, Richelle M, Bucheli P. Enhnced biovilbility of zexnthin in milk-bsed formultion of wolfberry (Gou Qi Zi; Fructus brbrum L.). Br J Nutr 6; 96: Vidl K, Benycoub J, Snchez-Grci J, Fot F, Segur- Roggero I, Serrnt P, Moser M, Blum S. Intke of milkbsed wolfberry formultion enhnces the immune response of young-dult nd ged mice. Rejuvention Res 1; 13: Vidl K, Bucheli P, Go Q, Moulin J, Shen LS, Wng J, Blum S, Benycoub J. Immunomodultory effects of dietry supplementtion with milk-bsed wolfberry formultion in helthy elderly: rndomized, double-blind, plcebocontrolled tril. Rejuvention Res 1; 15: Bucheli P, Vidl K, Shen L, Gu Z, Zhng C, Miller LE, Wng J. Goji berry effects on mculr chrcteristics nd plsm ntioxidnt levels. Optom Vis Sci 11; 88: Wng XJ, Hyes JD, Wolf CR. Genertion of stble ntioxidnt response element-driven reporter gene cell line nd its use to show redox-dependent ctivtion of nrf by cncer chemotherpeutic gents. Cncer Res 6; 66: Philippe D, Fvre L, Fot F, Adolfsson O, Perruisseu- Crrier G, Vidl K, Reuteler G, Dyer-Schneider J, Mueller C, Blum S. Bi fidobcterium lctis ttenutes onset of in flmmtion in murine model of colitis. World J Gstroenterol 11; 17: Philippe D, Dubuquoy L, Groux H, Brun V, Chuoï-Mriot MT, Gveriux-Ruff C, Colombel JF, Kieffer BL, Desreumux P. Anti-in flmmtory properties of the mu opioid receptor support its use in the tretment of colon in flmmtion. J Clin Invest 3; 111: Wllce JL, McNughton WK, Morris GP, Beck PL. Inhibition of leukotriene synthesis mrkedly ccelertes heling in rt model of in flmmtory bowel disese. Gstroenterology 1989; 96: Ameho CK, Adjei AA, Hrrison EK, Tkeshit K, Moriok T, Arkki Y, Ito E, Suzuki I, Kulkrni AD, Kwjiri A, Ymmoto S. Prophylctic effect of dietry glutmine supplementtion on interleukin 8 nd tumour necrosis fctor lph production in trinitrobenzene sulphonic cid induced colitis. Gut 1997; 41: Feng Z, Ji H, Li X, Bi Z, Liu Z, Sun L, Zhu Z, Bucheli P, Bllèvre O, Wng J, Liu J. A milk-bsed wolfberry preprtion prevents prentl stress-induced cognitive impirment of offspring rts, nd inhibits oxidtive dmge nd mitochondril dysfunction in vitro. Neurochem Res 1; 35: Bouguen G, Chevux JB, Peyrin-Biroulet L. Recent dvnces in cytokines: therpeutic implictions for inflmmtory bowel diseses. World J Gstroenterol 11; 17: Strober W, Fuss IJ. Proin flmmtory cytokines in the pthogenesis of inflmmtory bowel diseses. Gstroenterology 11; 14: Boismenu R, Chen Y. Insights from mouse models of colitis. J Leukoc Biol ; 67: Bozic CR, Gerrd NP, von Uexkull-Guldenbnd C, Kolkowski LF, Conklyn MJ, Breslow R, Showell HJ, Gerrd C. The murine interleukin 8 type B receptor homologue nd its lignds. Expression nd biologicl chrcteriztion. J Biol Chem 1994; 69: Gn L, Zhng SH, Liu Q, Xu HB. A polyscchride-protein complex from Lycium brbrum upregultes cytokine expression in humn peripherl blood mononucler cells. Eur J Phrmcol 3; 471: 17-3 Chen Z, Kwong Hut Tn B, Chn SH. Activtion of T lymphocytes by polyscchride-protein complex from Lycium brbrum L. Int Immunophrmcol 8; 8: Chen Z, Soo MY, Srinivsn N, Tn BK, Chn SH. Activtion of mcrophges by polyscchride-protein complex from Lycium brbrum L. Phytother Res 9; 3: Chen Z, Lu J, Srinivsn N, Tn BK, Chn SH. Polyscchride-protein complex from Lycium brbrum L. is novel stimulus of dendritic cell immunogenicity. J Immunol 9; 18: de Medin FS, Dddou A, Requen P, Cpitán-Cñds F, Zrzuelo A, Dolores Suárez M, Mrtínez-Augustin O. New insights into the immunologicl effects of food bioctive peptides in niml models of intestinl in flmmtion. Proc Nutr Soc 1; 69: Wlker A. Brest milk s the gold stndrd for protective nutrients. J Peditr 1; 156: S3-S7 S- Editor Gou SX L- Editor A E- Editor Zhng DN 5359 October 14, 1 Volume 18 Issue 38