Bifidobacterium animalis ssp. lactis 420 Protects against Indomethacin-Induced Gastric Permeability in Rats

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1 Hindwi Publishing Corportion Gstroenterology Reserch nd Prctice Volume 2012, Article ID , 9 pges doi: /2012/ Reserch Article Bifidobcterium nimlis ssp. lctis 420 Protects ginst Indomethcin-Induced Gstric Permebility in Rts Ann Lyr, 1 Mrkku Srinen, 1 Heli Putl, 1 Kis Olli, 1 Smpo J. Lhtinen, 1 Arthur C. Ouwehnd, 1 Mri Mdetoj, 2 nd Kirsti Tiihonen 1 1 DuPont Nutrition nd Helth, Kntvik Active Nutrition, Sokeritehtntie 20, Kntvik, Finlnd 2 Toxis, SBW Corp. Ltd., Lemminkäisenktu C, Turku, Finlnd Correspondence should be ddressed to Ann Lyr, nn.lyr@dnisco.com Received 30 December 2011; Revised 30 Mrch 2012; Accepted 2 My 2012 Acdemic Editor: Emidio Scrpellini Copyright 2012 Ann Lyr et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Gstrointestinl (GI) dverse effects such s erosion nd incresed permebility re common during the use of nonsteroidl nti-inflmmtory drugs (NSAIDs). Our objective ws to ssess whether Bifidobcterium nimlis ssp. lctis 420 protects ginst NSAID-induced GI side effects in rt model. A totl of 120 mle Wistr rts were llocted into groups designted s control, NSAID, nd probiotic. The NSAID nd probiotic groups were chllenged with indomethcin (10 mg/kg 1 ; single dose). The probiotic group ws lso supplemented dily with CFU of B. lctis 420 for seven dys prior to the indomethcin dministrtion. The control group rts received no indomethcin or probiotic. The permebility of the rt intestine ws nlysed using crbohydrte probes nd the visul dmge of the rt stomch mucos ws grded ccording to severity. B. lctis 420 significntly reduced the indomethcin-induced increse in stomch permebility. However, the protective effect on the visul mucosl dmge ws not significnt. The incidence of severe NSAID-induced lesions ws, nevertheless, reduced from 50% to 33% with the probiotic tretment. To conclude, the B. lctis 420 supplementtion protected the rts from n NSAID-induced increse in stomch permebility nd my reduce the formtion of more serious GI mucosl dmge nd/or enhnce the recovery rte of the stomch mucos. 1. Introduction Nonsteroidl nti-inflmmtory drugs (NSAIDs) re commonly used to relieve pin nd fever but lso typiclly cuse gstrointestinl side effects such s mucosl injury. The pthophysiology of NSAID-induced injuries is considered to be either prostglndin (PG)-dependent or non-pg dependent mechnism [1]. The PG-dependent mechnism refers to the inhibition of cyclooxygense (COX), leding to decresed mucosl PG. Trditionl nonselective NSAIDs, such s spirin, ketoprofen, indomethcin, nd diclofenc, ffect the expression of COX-1 nd COX-2 present in the gstrointestinl (GI) membrne [2]. The suppression of COX-2 llevites inflmmtion, wheres the simultneous suppression of COX-1 hmpers the prostglndin production essentil for mucin formtion nd functionl epithelil brrier in the GI trct [2, 3]. Thus GI dverse effects such s erosion nd incresed permebility re common during the long-term use of non-selective NSAIDs [2 4]. Nextgenertion NSAIDs tht selectively inhibit COX-2 re less prone to cusing moderte GI side effects [4], lthough complicted side effects re s common mong selective COX-2 inhibitor users s mong trditionl NSAID users [5, 6]. Recent studies on the pthogenesis of NSAID-induced mucosl injury indicte tht NSAIDs inhibit oxidtive phosphoryltion in epithelil cell mitochondri, independently of the PG-dependent mechnism. The resulting mitochondril dysfunction leds to disturbnces in cellulr energy metbolism nd ion regultion, cusing incresed intestinl permebility nd mucosl dmge [7]. The host GI microbiot my enhnce or reduce the risk of NSAID side effects. In 1996, Uejim nd collegues demonstrted connection between 5-bromo-2-(4-fluorophenyl)- 3-(4-methylsulfonylphenyl) thiophene (BFMeT)-induced

2 2 Gstroenterology Reserch nd Prctice ilel ulcertion nd intestinl microbiot in rts [8]. A decrese in Grm-positive rods nd n increse in Grmnegtive rods, including Escherichi coli, Klebsiell, nd Proteus, ws observed due to ulcertion [8]. Moreover, Lctobcillus cidophilus ATCC4356 nd Bifidobcterium dolescentis ATCC15703 were shown to repress ulcer formtion in rts, puttively by inhibiting the growth of Grm-negtive rods [8, 9]. In elderly humn subjects, decrese in lctobcilli nd ctinobcteri due to NSAID use hs been observed [10]. Within ctinobcteri, minly the numbers of Collinsell [10] were reduced, which hs previously been reported for functionl bowel disorder sufferers [11] nd colon cncer ptients [12]. Thus, n intriguing lterntive for protecting humns from NSAID-induced side effects is the prllel use of probiotics [13, 14]. Indeed, certin probiotic [15] strins induce epithelil cell prolifertion nd mucus secretion, thus potentilly beneficilly ffecting NSAIDinduced dverse effects [16], nd re cpble of stbilizing distorted GI microbiot [17]. To dte, limited number of studies hve investigted the potentil protective effect of different probiotic supplements ginst NSAID-induced gstrointestinl dmge with vrying outcome mesures. In vitro studies with Lctobcillus csei DN [18] nd niml studies pplying Lctobcillus csei strin Shirot [19] nd multistrin mixture of humn origin [20] hve yielded promising results. In clinicl trils, Lctobcillus rhmnosus GG (LGG) hs been shown to reduce indomethcin-induced gstric permebility [13] nd the multistrin supplement VSL#3 hs been shown to llevite inflmmtion cused by indomethcin [14]. Moreover, Lctobcillus cidophilus NCFM nd lctitol my protect ginst the GI microbiot ltertions ssocited with NSAID use [21] mong elderly subjects regulrly consuming NSAIDs [22]. Tken in prllel with NSAIDs, probiotics re promising complementry tretment for relieving NSAIDinduced dverse effects. However, few studies ssessing the subject hve been conducted thus fr, especilly s regrds bifidobcteri, nd, s is commonly known s chrcteristic of probiotics, the puttive protective effect ginst NSAID-induced side effects is lso strin-specific [19, 23]. Our objective in the present study ws to nlyse whether Bifidobcterium nimlis ssp. lctis (B. lctis) 420 hs protective effect ginst NSAID-induced GI dmge in n niml model. Since the moleculr bses of NSAIDinduced GI dmge nd the NSAID therpeutic effect re due to COX-1 nd COX-2 suppression, respectively, we chose B. lctis 420 s cndidte probiotic for our study. B. lctis 420 hs been shown to upregulte COX-1 expression nd to suppress COX-2 expression in Cco-2 cells [24, 25] nd to produce fermenttion products cpble of enhncing the epithelil brrier [25, 26]. We conductedthree seprte studies using well-described rt model bsed on indomethcin-induced GI dmge [27]. All studies included identicl control, NSAID nd probiotic groups. Additionl rms were dded to individul studies to llow the testing of the dose-responsiveness of live B. lctis 420 cells nd the effect of B. lctis 420 metbolites. As outcome mesures, we focused on both the permebility of the stomch nd smll intestine, nd on the visul exmintion of gstric mucosl dmge [27, 28]. 2. Mterils nd Methods 2.1. Animls. All niml experiments were conducted t Toxis In vivo Services (SWB Corp. Ltd., Turku, Finlnd) nd pproved by the Ntionl Animl Cre nd Use Committee nd performed ccording to the Guidnce Document on the Recognition, Assessment, nd Use of Clinicl Signs s Humne Endpoints for Experimentl Animls Used in Sfety Evlution (Environmentl Helth nd Sfety Monogrph Series on Testing nd Assessment no 19. OECD 2000) guidelines. In totl, 160 cliniclly helthy mle Wistr rts (HsdBrlHn:WIST, Hrln Netherlnds, Horst, NL) ged between 8 to 9 weeks with n verge weight of 254 g (individul weights devited by less thn 10% from the verge weight per study) were cclimtised to the fcility for 6 dys nd housed in either spen chip-bedded cges of 2 to 3 nimls or seprtely in metbolic cges. Rised bottom grids were used in the niml cges during the fsting period. Theroomtemperturewskeptt21± 3 Cndreltive humidity t 55 ± 15%. Artificil lighting ws pplied in 12 hour shifts. The nimls were provided with both nonsterile Formulb Diet 5008 nd tp wter d libitum. Animls were cliniclly exmined twice dy during the week nd once dy during the weekends. All clinicl signs were reported Chemicls. Sucrose ws from Suomen Sokeri Oy (Kntvik, Finlnd). Sterile wter ws obtined from Bxter (Helsinki, Finlnd). Unless indicted otherwise, ll other regents were from Sigm (Sigm-Aldrich, St. Louis, MO, USA) Supplements. All supplements were given to the test nimls per os (p.o.) using gvge t volume of 10 ml/kg 1 of weight (pproximtely 2.5 ml per niml) or exctly 2.5 ml per niml. All nimls were observed for ny bnorml signs during dosing in the morning nd fter dosing in the fternoon. Live B. lctis 420 cells (DSM 22089; Dnisco, Niebüll, Germny) were dministered t CFU d 1 (high dose) or 10 8 CFU d 1 (low dose) per niml. The cellfree extrct ws prepred by cultivting B. lctis 420 in Mn, Rogos, nd Shrpe (MRS, Oxoid Ltd, Cmbridge, UK) broth nerobiclly t 37 C until OD600 ws 2.0, which roughly corresponded to bcteril cell count of ml 1 s determined by flow cytometer [29]. The bcteril cells were removed by centrifugtion t g for 15 min (Beckmn Coulter Avnti J-20 Xpi, Bre, CA, US) nd the cell-free extrct ws prepred by evporting the superntnt with Rotvpor (Büchi Rotvpor R-200, Flwil, Switzerlnd) t +40 C to 1/13.5 of its originl volume. The extrct ws then diluted to correspond to bcteril density of CFU ml 1. The L-lctic cid supplement ws djusted to the mount of cetic nd lctic cid in the cellfree extrct (52.4 mm). Indomethcin ws supplied t 10 mg/kg 1 in 50 g L 1 sodium bicrbonte solution. The crbohydrte probes;

3 Gstroenterology Reserch nd Prctice 3 sucrose (1 g), lctulose (120 mg), nd mnnitol (80 mg) were given p.o. in 2 ml volume of sterile wter [27]. 10 µl of 10% thymol in isopropnol ws dded to the urine collection tubes to prevent microbil degrdtion of the probes Permebility Probe Quntifiction. Sucrose, lctulose nd mnnitol in rt urine were determined by high ph nion exchnge chromtogrphy fter purifiction of the smples by solid phse extrction (SPE). The SPE crtridges (Bond Elut SCX, 500 mg, Vrin, Plo Alto, CA, USA) were preconditioned with 2 ml of methnol followed by 2 ml of wter. The urine smple (0.5 ml) ws pssed through the SPE crtridge nd the effluent ws collected into test tube. Therefter, the nlytes were eluted with 3 ml of wter into the sme test tube nd the smple ws diluted to 50 ml with wter. The concentrtions were determined using Dionex HPLC system (Sunnyvle, CA, USA) equipped with ED50 pulsed electrochemicl detector (PED), GP50 pump nd AS50 smpler. For determintion of sucrose nd lctulose, CrboPc PA-1 column (precolumn 4 50 mm nd nlyticl column mm) nd grdient elution with mobile phse tht consisted of mixture of (A) wter, (B) 0.2 M NOH, nd (C) 0.2 M NOH nd 0.5 M sodium cette ws used. The grdient ws 0 8 min, A = 84% nd B = 14%; min, A = 44 nd B = 34%; min, A = 84% nd B = 14%. The flow rte ws 1 ml/min 1 nd the column temperture 35 C. A solution of 0.3 M NOH ws dded to the column effluent before the PED cell t flow rte of 0.6 ml/min. For determintion of mnnitol, CrboPc MA1column(precolumn4 50 mm nd nlyticl column mm) nd grdient elution with mobile phse tht consisted of mixture of (A) wter nd (B) 1 M NOH ws used. The grdient ws: min, A = 40%; min, A = 10%; min, A = 40%. The flow rte ws 0.4 ml/min 1 nd the column temperture 35 C. The concentrtions of crbohydrtes in urine were clculted using the externl stndrd method Histologicl Exmintion. Animls were euthnized by crbon dioxide before necropsy. After euthnsi, gross necropsies were performed for ll nimls. The totl dmged re (TDA) of the intestine (mm 2 ) ws clculted by observing visul lesions with 40x mgnifiction from stomch mucos rinsed with physiologicl slt solution (0.9% NCl). The observtions were grded s follows; Grde 0 s norml; Grde I s mild, slight, few, or smll lesions (number of lesions less thn 10); Grde II s moderte in the ppernce, size, or number of lesions (number of lesions 10 to 20); Grde III s severe, mssive, or extensive lesions in terms of the number or size (number of lesions more thn 20). Grde III represented the mximl chnge in the mcroscopic exmintion Experimentl Design of Animl Studies. Three seprte studies, including totl of 160 rts, were conducted. In ssessing the effect of the high probiotic dose, the results concerning the relevnt tretment groups were combined from ll three studies nd presented s Study I. The ctive Control NSAID Probiotic No supplementtion No chllenge No supplementtion NSAID chllenge on dy 8 fter 15 h fsting Permebility probes given 10 h fter chllenge Urine collection for 15 to 16 h Scrifice nd mcroscopic nlysis on dy 9 Supplementtion from dy 1 to dy 7 NSAID chllenge on dy 8 fter 15 h fsting Figure 1: Study protocol outline followed in niml studies. The probiotic group rts were given live Bifidobcterium lctis B420 cells (high dose or low dose), cell-free extrct or pure lctic cid s supplementtion. groups were given live bcteril cells (high dose or low dose), cell-free extrct, or lctic cid (Figure 1). The control nd indomethcin groups were given sterile wter s plcebo supplement. Active nd plcebo tretments were dministered for seven dys prior to the indomethcin chllenge on dy eight. After 15- to 16-hour fsting period, indomethcin ws given p.o. to induce GI dmge. Ten hours lter crbohydrte probes (sucrose, lctulose, nd mnnitol) were given to determine the permebility of the stomch nd the smll intestine, respectively [27]. Therefter, feces nd urine were collected seprtely for 15 to 16 hours, using rt metbolic cges (Tecniplst, Brinz, Itly). The urine smples were stored t 20 C until nlysis. On dy nine, the nimls were euthnized nd gross necropsy ws performed to evlute mcroscopic dmge in the stomch nd the discchride concentrtions were mesured from the rt urine. The setup of the seprte studies is presented below. Study I: Protective Effectof B. lctis420. The protective effect of live B. lctis 420 ws tested in three trils with n identicl protocol nd three identicl equl size tretment groups consisting of totl of 40 mle Wistr rts in ech group

4 4 Gstroenterology Reserch nd Prctice (control, indomethcin chllenge, nd probiotic high dose; n = 120). The probiotic group received the high dose of B. lctis B420. The results from ll three trils were combined for nlysis. Study II: Dose-Responsiveness of B. lctis 420. For ssessing the dose-responsiveness of B. lctis 420, four tretment groups of 10 mle Wistr rts ech (n = 40) designted (1) control, (2) indomethcin chllenge, (3) probiotic low dose, nd (4) probiotic high dose, were nlysed. Study III: Effectiveness of B. lctis 420 Cell-Free-Extrct nd Lctic Acid. The effectiveness of B. lctis 420 metbolites for mediting gstroprotective effect during NSAID use ws tested in comprison to live cells nd pure lctic cid. Ech tretment group consisted of 15 mle Wistr rts (n = 75); (1) control, (2) indomethcin chllenge, (3) probiotic high dose,(4)cell-freeextrct,nd(5)lcticcid Sttisticl Anlyses. All numericl dt re presented s men vlues with stndrd devitions (SDs). The gstric nd smll intestinl permebility were expressed s the urinl mount of sucrose nd the urinl lctulose : mnnitol-rtio, respectively. For TDA, nlyses were lso performed with vlues weighted ccording to the degree of mucosl dmge (1/10, 3/10, nd 6/10 for dmge res of Grdes I, II, nd III, resp.). One-wy ANOVA with Tukey s multiple comprison test ws used for permebility mesurements nd nonprmetric Kruskl-Wllis test followed by Dunn s multiple comprison test were used for TDA. The nlyses were performed with Prism 5 Version 5.01 (GrphPd Softwre, Inc., Sn Diego, USA). 3. Results 3.1. Animl Trils. In the niml trils, ll except one rt remined live during the intervention nd no significnt difference ws detected between the weights in the different tretment groups (dt not shown). The deth of one rt in the tril nlysing the effect of B. lctis 420 metbolites nd lctic cid ws exmined nd found not to be due to ny supplement given within the study. Hyperemi in the stomch of test nimls ws detected in ll studies in ll tretment groups including the control group receiving no indomethcin or probiotic nd ws therefore not nlysed s n outcome mesure Protective Effect of Live B. lctis 420. In the combined nlysis (Study I), indomethcin cused significnt increse in mucosl permebility nd TDA (Figure 2). Sucrose ws given s mrker to evlute gstric permebility nd lctulose nd mnnitol were given to evlute smll intestinl permebility. Gstric permebility ws significntly incresed in the NSAID group, wheres the smll intestine remined unffected. In the B. lctis 420-supplemented group (high dose), the sucrose levels remined comprble with the control group, regrdless of the NSAID chllenge, implying protectiveeffect ginst NSAID-induced incresed gstric Tble 1: Totl dmged re of the rt stomch mucos. Group N Men (mm 2 ) SD Weighted men b Weighted SD Control NSAID c c 4.43 Probiotic c c 5.00 The tretment groups were control, indomethcin-chllenged group (NSAID), nd probiotic group supplemented dily with colony forming units of Bifidobcterium lctis 420 for seven dys prior to indomethcin chllenge. b The weighted vlues were clculted by multiplying the detected TDA vlues of incresing severity with 1/10, 3/10, nd 6/10, respectively. c ANOVA P < when compred with control. Tble 2: Prevlence of gstric lesions. Group/lesion grde None Any grde Grde II or III Grde III Control NSAID b Probiotic b The tretment groups were control, indomethcin-chllenged group (NSAID), nd probiotic group supplemented dily with colony forming units of Bifidobcterium lctis 420 for seven dys prior to indomethcin chllenge. The lesion Grdes I, II, nd III represent mild, moderte, nd severe lesions, respectively. b ANOVA P < 0.05 when compred with control. permebility. The lctulose : mnnitol levels in the probiotic group lso remined comprble with the control group. The stomch mucos of ll rts ws visully nlysed for lesions which were grded ccording to severity. Significnt dmge to the stomch mucos ws detected (Figure 2(c)), whether nlysed by weighted or nonweighted TDA vlues (Tble 1). However, despite the positive direction of the effects, the protective effect of B. lctis 420 on the stomch mucos ws not significnt ccording to the TDA vlues (Figure 2(c), Tbles 1 nd 2). No significnt difference ws seen in the distribution of the most severe type of lesions (Grde III) between the B. lctis 420-supplemented group nd the group only chllenged with indomethcin (Tble 2). The men sucrose vlues mesured from rt ure did not correlte with the presence of lesions on the stomch mucos, but ppered reltively stble within ech tretment group (Tble 3) Dose-Responsiveness nd Effect of Metbolites. In the individul rt studies ssessing dose-responsiveness (Study II) nd the effect of metbolites (Study III) of B. lctis 420, only the gstric permebility ws significntly ffected by the NSAID chllenge, but the TDA vlues remined t low level even within the indomethcin chllenge group not dministered by the probiotic (Tble 4). On the other hnd, in the metbolite study the TDA vlues showed significnt increse due to indomethcin, but the permebility of neither the stomch nor the TDA vlues were ffected (Tble 5). None of the tretments (high or low dose of live B. lctis 420, cell-free extrct supplementtion, or lctic cid) resulted in sttisticlly significnt protective effect in the individul studies, Studies II nd III (Tbles 4 nd 5).

5 Gstroenterology Reserch nd Prctice (mg/smple) Lctulose : mnnitol-rtio Control NSAID Probiotic 0 Control NSAID Probiotic () (b) (mm 2 ) Control NSAID Probiotic Grde I Grde II Grde III (c) Figure 2: Protective effect of Bifidobcterium lctis 420 ginst indomethcin-induced side effects. Quntities of permebility probes sucrose () nd lctulose : mnnitol (b) in rt urine smples nd the totl dmge res (TDA) of gstric mucos (c) combined from three individul studies (n = 119). The tretment groups were control group, n indomethcin-chllenged group (NSAID), nd probiotic group supplemented dily with colony forming units of Bifidobcterium lctis 420 for seven dys prior to indomethcin chllenge. Sucrose mesures gstric permebility nd the lctulose : mnnitol-rtio reflects smll intestinl permebility. TDA vlues include Grde I (mild, slight, few, or smll), Grde II (moderte ppering, size, or number), nd Grde III (severe, mssive, or extensive in number or size) lesions. The verticl lines in figures () nd (b) represent men vlues. Significnt (P <0.05) differences between tretment groups re denoted by n sterisk. Nevertheless, these results still contributed to the positive effects of the pooled dt set (Study I). 4. Discussion Despite the GI-relted side effects, such s ulcertion nd incresed epithelil permebility, occsionl nd long-term NSAID use is common. Specific probiotic strins hve nti-inflmmtory effects including protection from nd enhnced heling of ulcertion in colitis nd the cpbility of enhncing the GI epithelil brrier [16, 30]. Therefore, probiotics provide n intriguing lterntive s protective dietry supplement during NSAID consumption. The probiotic strin selected for the present study, B. lctis 420, hs previously been shown to ffect the COX-1 nd COX- 2 expression profile in Cco-2 cells in mnner opposite to tht of the expected effect of NSAIDs [24, 25]. Moreover, B. lctis 420 is cpble of enhncing the intestinl epithelil cell brrier in Cco-2 cell monolyer [25, 26]. In the current study, identiclly treted groups from three independent studies were combined (3 tretment groups, n = 119) in order to ssess the protective effect of B. lctis 420 t CFU d 1.TheB. lctis 420 supplementtion, dministered for seven dys prior to 10 mg/kg 1 single dose of indomethcin chllenge, protected rts from

6 6 Gstroenterology Reserch nd Prctice Tble 3: Urinry sucrose levels of rts grouped ccording to lesion sttus. Group Lesion sttus N Urinry sucrose levels Men (mg/smple) SD No lesions Control Lesions All No lesions NSAID Lesions All No lesions Probiotic Lesions All The tretment groups were control, indomethcin-chllenged group (NSAID), nd probiotic group supplemented dily with colony forming units of Bifidobcterium lctis 420 for seven dys prior to indomethcin chllenge. Tble 4: Dose-responsiveness of Bifidobcterium lctis 420 ginst indomethcin-induced side effects. Group Quntities of permebility probes b (mg/smple ± SD) TDA c (mm 2 ± SD) Sucrose Lctulose : mnnitol Control ± 4.08 d 0.87 ± ± 0.00 NSAID ± 8.94 d 0.85 ± ± 5.13 Probiotic high dose ± ± ± 2.68 Probiotic low dose ± ± ± 3.15 The tretment groups were control, indomethcin-chllenged group (NSAID), nd probiotic groups supplemented dily with colony forming units (high dose) or 10 8 colony forming units (low dose) colony forming units of Bifidobcterium lctis 420 for seven dys prior to indomethcin chllenge. b Sucrose mesures gstric permebility, nd the lctulose : mnnitol-rtio reflects smll-intestinl permebility. c TDA stnds for totl dmged re. d Significnt (ANOVA; P < 0.05) differences between tretment groups re denoted pir wise by superscript letters. n indomethcin-induced increse in gstric permebility. However, no sttisticlly significnt increse due to the NSAID chllenge ws seen in smll intestinl permebility, lthough the pplied dose of indomethcin hs previously been shown to be efficient in incresing both gstric nd smll intestinl permebility [27]. Bifidobcteri hve, however, been previously linked with reduced ilel ulcertion due to BFMet chllenge in rts, with the NSAID-chllenged control group displying ulcertions within the ileum between 18 to 72 hours fter NSAID dministrtion [9]. The effectof B. lctis 420 on the NSAID-induced mucosl dmge ws not significnt, lthough fewer nimls in the probiotic group tended to hve the most severe type Grde III lesions in comprison with the NSAID-chllenged nimls not given B. lctis 420 (33% versus 50%; Tble 2). Possibly the B. lctis 420 supplementtion protected ginst more serious GI mucosl dmge or enhnced the recovery rte of the stomch mucos, tht is, Grde III lesions were llevited to Grde II lesions before necropsy. Moreover, the effect of NSAID chllenge on TDA vlues ws inconsistent; while in the first tril NSAID induced cler increse in TDA vlues, in the second nd the third trils, the effect of NSAID ws either very smll or modest, providing limited scope for improvement. Although the initil study ( substudy of Study I) hd too few rts to show significnt effect, we conducted seprte studies for ssessing the dose-responsiveness of B. lctis 420 (four tretment groups, n = 40) nd the effect of B. lctis 420cell-freeextrctndlcticcid(fivetretment groups, n = 74) with the dditionl gin of elevting the count of nimls ssyed s in Study I. In the dose-response study, neither of the doses (10 8 CFU d 1 nd CFU d 1 ) showed protective effect (Tble 4). Due to high vrince, the beneficil trend mong the verge sucrose permebility vlues ws not sttisticlly significnt. According to TDA vlues, the indomethcin chllenge ws not sufficient in the dose-response study. Nevertheless, for the combined nlysis of the control, NSAID nd probiotic groups, the dt from the dose-response study ws lso included since the gstric permebility hd incresed significntly with NSAID dministrtion. When testing the effect of B. lctis 420 metbolites nd lctic cid, the gstric permebility mesurements filed to show dequte NSAID chllenge, wheres the lctulose : mnnitol-rtio nd the TDA vlues indicted the NSAID chllenge to be dequte, but none of the supplementtions (live cells, cell-free extrct, or lctic cid) were protective. Wtnbe nd coworkers [19] hve previously found less concentrted (3 to 15 mm) L-lctic cid supplement to protect ginst mucosl dmge in the smll intestine of indomethcin-chllenged rts. Although specultive, our results indicte the opposite for the effect of 52.4mM L-lctic cid supplementtion on the gstric mucos. In our study, the concentrtion of lctic cid ws djusted ccording to the

7 Gstroenterology Reserch nd Prctice 7 Tble 5: Effect of Bifidobcterium lctis 420 cell-free extrct nd lctic cid ginst indomethcin-induced side effects. Group Quntities of permebility probes b (mg/smple ± SD) TDA c (mm 2 ± SD) Sucrose Lctulose : mnnitol Control ± ± 0.28 d,e 0.70 ± 2.44 f,g,h NSAID ± ± 0.45 d 6.28 ± 6.85 f Live cells ± ± 0.53 e 2.84 ± 3.12 g Cell free extrct ± ± ± 2.41 Lctic cid ± ± ± 9.21 h The tretment groups were control group, n indomethcin-chllenged group (NSAID), nd groups supplemented dily with colony forming units of Bifidobcterium lctis B420 (live cells), B. lctis 420 cell-free extrct (cell-free extrct), or lctic cid for seven dys prior to indomethcin chllenge. b Sucrose mesures gstric permebility nd the Lctulose : mnnitol-rtio reflects smll-intestinl permebility. c TDA stnds for totl dmged re. d h Significnt (ANOVA; P < 0.05) differences between tretment groups re denoted pir wise by superscript letters. cetic cid nd lctic cid concentrtions in the B. lctis 420 cell-free extrct. The results from the metbolite study were lso included in the combined nlysis. The three rt studies were conducted in strict ccordnce with identicl timing nd dosing procedures to void vrition. Nevertheless, the effect of the indomethcin chllenge on the gstric mucos vried between both the individul studies nd within ech study s there ws interindividul vrition mong the rts within ech tretment group. In ddition to interindividul differences in rt physiology, the subjective visul severity scoring nd re estimtion pplied in the TDA method my hve introduced dditionl vrition to the TDA vlues, lthough ll TDA nlyses were performed by the sme exminer. The indomethcin btch used in the dose-response study ws lso different from the one used in the other studies, possibly explining the low TDA vlues. A higher indomethcin dose could hve reduced the vrition in indomethcin-induced GI dmge between the studies. However, in previous study ssessing the protective effects of Lctobcillus csei strin Shirot, 10 mg/kg 1 single dose ws sufficient for gstric ulcertion formtion in rts [19] nd 25 mg/kg 1 lredy effectively induced gstric ulcertion in ll rts it ws dministered to [31]. Moreover, n indomethcin-induced dely in gstric emptying [32] nd the rpid heling of the gstric epithelium [31] my hmper the correltion between gstric permebility nd TDA mesurements. The sugr permebility vlues lso showed high interindividul vrition, but were more comprble between the seprte studies. In ll rt studies, the nimls were given permebility probes 10 hours fter indomethcin chllenge followed by 15 to 16 hours of urine collection nd euthnized lterntely from ech tretment group strting from 25 hours fter indomethcin chllenge. Thus the niml study protocol results in n unvoidble, slightly erlier ssessment of sugr permebility thn the ssessment of mucosl dmge, which my llow for some degree of heling to occur nd therefore ffectthe TDA results. Kunes et l. lso showed tht the mximl smll intestinl dmge is reched in the smll intestine of indomethcin-chllenged rts only t 48 to 72 hours fter indomethcin dosing [31], which is why the TDA of the smll intestine ws not nlysed in this study. Indeed, the protective effect of B. lctis 420 ws significnt only regrding the permebility of the stomch, for which the pplied protocol time-scle is optiml (Figures 1 nd 2()). Moreover, the gstric permebility, tht is, the mount of sucrose quntified from the rt urine smples, nd the presence of mucosl dmge in the stomch epithelium did not correlte (see Tble 3), lthough both hve previously been reported to increse with indomethcin dministrtion [27]. In recent study conducted by Senol nd collegues, probiotic mixture including 13 strins of humn origin ws effective ginst spirin-induced gstric mucosl dmge in rts ccording to mcroscopic exmintion with only ten rts in ech tretment group, lthough the histologicl nlysis showed no effect [20]. In ddition to pplying different NSAID thn in the present study, their study protocol included both longer prophylctic probiotic supplementtion (14 dys) nd fsting period prior to the chllenge with spirin (14 hours), while the nimls were euthnized just 3 hours fter the dministrtion of spirin. An erlier evlution of the gstric mucos could indeed be more efficient in evluting the gstric mucosl dmge s some of the vrition my be due to the heling process being lredy underwy nd becoming effective. This would, however, hmper the permebility nlysis, which ws prioritized in our study. Gottelnd nd coworkers [13] hve successfully pplied permebility probes to evlute the protective effect of probiotic in clinicl study. They supplemented live nd het-killed LGG cells to 16 humn subjects consuming indomethcin. The intestinl permebility ws ssessed using gstric (sucrose) nd smll intestinl (lctulose : mnnitol) permebility mrkers which showed significnt protective effect ginst incresed gstric permebility with live LGG cells [13]. Tken together, even though substntil interindividul vrition ws seen in the mnifesttion of dverse effectsfter indomethcin chllenge between individul rts, B. lctis 420 supplementtion significntly reduced indomethcininduced gstric permebility in rts. Bsed on the results of the present study nd previous in vitro studies [24 26], testing B. lctis B420 in clinicl intervention would be justified.

8 8 Gstroenterology Reserch nd Prctice Authors Contributions All uthors contributed to the design of the study, interprettion of the results, nd writing of the pper. M. Srinen quntified the permebility probes, H. Putl nd K. Olli coordinted the niml studies, M. Mdetoj ssessed the visul mucosl dmge, nd A. Lyr nlysed the dt nd compiled the pper. Acknowledgments Brit Mäki, Luri Nski, nd Jn Oksnen re grtefully cknowledged for their skillful technicl ssistnce. Hnnele Kettunen, Tuoms Slusjärvi, nd Jussi Nurmi re cknowledged for prticipting in the coordintion of the niml studies. Jnne Kskinoro t Toxis Ltd., conducted the niml studies. Sgnit Kulthinl t AkrNumero ws consulted s bioinformticin. References [1] H. Mtsui, O. Shimokw, T. Kneko, Y. Ngno, K. Ri, nd I. 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