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GASTROENTEROLOGY 2007;132:1791–1803

he Role of PPAR� on Restoration of Colonic Homeostasis Afterxperimental Stress-Induced Inflammation and Dysfunction

NGEL PONFERRADA,* JAVIER R. CASO,‡ LUIS ALOU,§ ARTURO COLÓN,‡ DAVID SEVILLANO,§ MARÍA A. MORO,‡

GNACIO LIZASOAIN,‡ PEDRO MENCHÉN,* MARÍA L. GÓMEZ–LUS,§ PEDRO LORENZO,‡ ENRIQUE COS,*UAN C. LEZA,‡ and LUIS MENCHÉN*

Servicio de Aparato Digestivo, Hospital General Universitario “Gregorio Marañón,” Madrid; and Departamentos de ‡Farmacología and §Microbiología I, Facultad de
edicina, Universidad Complutense, Madrid, Spain
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ackground & Aims: Psychological stress has beenmplicated in the clinical course of several gastroin-estinal diseases, but the mechanisms implicated andhe effects of stress on the normal colon are not yetully understood. Methods: Male Wistar rats werexposed to various immobilization periods as a stressaradigm. Colon was processed to assess myeloper-xidase activity, nitric oxide synthase 2, cyclooxygen-se 2, and peroxisome proliferator-activated receptoramma (PPAR�) expression and production of pros-aglandins. Colonic permeability, bacterial transloca-ion, tight junctions ultrastructure, and immunoglob-lin (Ig) A levels were also evaluated. Results:xposure to acute (6 hours) immobilization stress pro-uced an increase in myeloperoxidase activity and nitricxide synthase 2 and cyclooxygenase 2 expression. Allhese parameters remained increased after 5 days ofepeated stress exposure, showing a trend to normalizefter 10 days. Levels of the anti-inflammatory eico-anoid 15-deoxy-�12,14-prostaglandin J2 (15d-PGJ2) andxpression of PPAR� run parallel with these changes.olonic epithelial barrier was altered after stress expo-

ure, and a significant decrease in colonic IgA levelsfter acute stress exposure was observed. Pretreatmentith PPAR� agonists 15d-PGJ2 and rosiglitazone pre-

ented colonic inflammation and barrier dysfunction asell as the decrease of IgA production induced aftercute stress; PPAR� specific antagonist T0070907 re-erted these effects. Conclusions: Activation of PPAR�n rat colon in vivo seems to counteract colonic inflam-

ation and dysfunction induced by stress. On the otherand, PPAR� ligands may be therapeutically useful inonditions in which inflammation and barrier dysfunc-ion takes place in colon after exposure to stress.

he relationship between psychosocial stress and sev-eral gastrointestinal disorders is widely accepted by

oth patients and physicians, although the rational dem-nstration of a casual nexus remains difficult because ofethodological aspects and interpretation of clinical

tudies and the shortage of experimental approaches.
tress influences subjective perception of gastrointestinal
ymptoms by patients1 and has been shown to trigger thenset and modify the clinical course of certain gastroin-estinal diseases.2 Irritable bowel syndrome and func-ional dyspepsia are the most prevalent among the gas-rointestinal disorders that have been related withsychological factors.3 It has been reported that patientsith irritable bowel syndrome and functional dyspepsiaad been exposed to stressful events more frequentlyhan control individuals and that life stress was highlyredictive of symptom intensity.4 Stress has been associ-ted also with inflammatory activity of ulcerative coli-is5,6 and with an increased risk of disease exacerba-ion.5,7,8 The high response to placebo frequentlybserved in inflammatory bowel disease clinical trials9,10

lso supports the concept that psychological factors playrole in the clinical course of these diseases. There is

rowing evidence from experimental studies supportinghe ability of psychosocial stress to (1) induce physiologicbnormalities in the gut such as increased motility, al-ered ion secretion, increased permeability, and entericeuron dysfunction,11–14 (2) increase the severity of ex-erimental colitis,15 and (3) even reactivate a quiescentolitis.16

Previous findings of our group showed that stressnduces macroscopic and histologic colonic inflamma-ion, increasing inducible nitric oxide synthase (NOS-2)ctivity and membrane lipid peroxidation, after a lowose of trinitrobenzene sulfonic acid unable to induceuch changes in nonstressed animals.17 Also, it has beenhown that stress increases colonic permeability, facili-ating the entry of luminal contents that activate previ-usly sensitized CD4� T cells in the colon,16 initiating orerpetuating an inflammatory response, but the exactechanisms of the association are still poorly under-

tood.

Abbreviations used in this paper: COX, cyclooxygenase; 15d-PGJ2,5-deoxy-�12,14-prostaglandin J2; NF-�B, nuclear factor �B; NOS, nitricxide synthase; MPO, myeloperoxidase; PPAR, peroxisome prolifera-or-activated receptor; TJ, tight junction.

© 2007 by the AGA Institute0016-5085/07/$32.00

doi:10.1053/j.gastro.2007.02.032

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1792 PONFERRADA ET AL GASTROENTEROLOGY Vol. 132, No. 5

A question of special relevance is the possibility thatome of the changes observed in stress would be a featureot of the deleterious effects of stress but of some po-entially reversible type of adaptive plasticity, as proposedy McEwen.18 Most species of higher multicellular organ-

sms have evolved complex homeostatic and defenseechanisms that allow cells to overcome stressful stim-

li. One such mechanism that has received considerablexperimental attention as a possible anti-inflammatoryathway is peroxisome proliferator-activated receptors

PPARs), a group of 3 ligand-regulated transcription fac-ors originally implicated in adipocyte differentiationnd glucose homeostasis. One of its isoforms, PPAR�,xerts an anti-inflammatory effect by interfering with thectivity of inflammatory transcription factors, such asctivator Protein-1 (AP-1), Signal Transducers and Acti-ators of Transcription (STAT), and nuclear factor �BNF-�B).19 Three isoforms, as a result of alternative splic-ng and differential promoter usage within the sameene, are expressed in humans; PPAR�1, the predomi-ant isoform, is expressed in a broad range of tissues,

ncluding small bowel and colon.20 Therefore, activationf PPAR� by natural agonists, such as 15-deoxy-�12,14-rostaglandin J2 (15d-PGJ2),21 or synthetic agonists cane considered as a pathway to halt the inflammatoryesponse. Indeed, PPAR� ligands have been shown toeduce markedly colonic inflammation in both dextranodium sulfate and trinitrobenzene sulfonic acid colitis

odels in mice,22,23 probably through inhibition ofF-�B activity and decrease of proinflammatory cytokineroduction.To further investigate the possible mechanisms in-

olved in stress-induced inflammation and dysfunctionn the normal colon and the role of PPAR� as a possibleomeostatic mechanism in the colon of stressed animals,e decided to explore whether this may serve as a new

arget for protective pharmacologic maneuvers. We usedstress paradigm that has been reported to cause accu-ulation of oxidative mediators in the gut.17 Acute or

epeated immobilization stress is an easy, reproducible,nd widely used model to study pathophysiologic aspectsf the stress response and to study possible pharmaco-

ogic manipulations to decrease the negative effects oftress exposure on gastrointestinal, cardiovascular, orentral nervous systems.24 We used a pharmacologic ap-roach using natural and synthetic PPAR� agonists (theeoxy-prostaglandin 15d-PGJ2 and the thiazolidinedionerug rosiglitazone, respectively) as well as the specificPAR� antagonist T0070907. Rosiglitazone is a well-ocumented high-affinity and selective ligand forPAR�25 and has been shown to activate PPAR� in in-estinal epithelial cells both in vitro26 and in vivo.27 15d-GJ2 has also been shown to be a high-affinity ligand forPAR�.28,29 On the other hand, T0070907 has previouslyeen shown to inhibit PPAR� activity both in vitro30 and

n vivo.31 b

Materials and MethodsAnimals and Stress ProtocolYoung adult male Wistar rats (Animal Care Facil-

ty of the Complutense University, Madrid, Spain) weigh-ng 210 –230 g were used in this study. All experimentalrotocols adhered to the guidelines of the Animal Wel-are Committee of the Universidad Complutense follow-ng European legislation (2003/65/EC). The rats wereoused individually under standard conditions of tem-erature and humidity and a 12-hour light/dark cycle

lights on at 8 AM) with free access to standard pelletedormula and tap water. All animals were maintainednder constant conditions for 4 days before study. Theestraint was performed using a plastic rodent restrainerhat allowed for a close fit to rats in their home cages.32

tress consisted of 6 hours of immobilization starting atAM for 1, 5, or 10 consecutive days.Control animals were not subjected to stress but were

andled at 9 AM for a few seconds. Immediately afterestraint (still in the restrainer), animals were weighednd killed using intraperitoneally (IP) administered so-ium pentobarbital (320 mg/kg; Vetoquinol, Madrid,pain). Blood for plasma determinations was collected byardiac puncture and anticoagulated in the presence ofrisodium citrate (3.15% wt/vol, 1 vol citrate per 9 vollood).

The colon was removed, and after assessment of mac-oscopic damage, the distal 8-cm portion of the colonas removed and cut longitudinally for the determina-

ions described in the following text. For biochemicaleterminations, tissue was frozen at �80°C until as-ayed. For image studies, tissue was processed as de-cribed in the following text. A separate group of animalsas used for colonic paracellular permeability assay im-ediately after the stress sessions for the in vivo lumen-

o-blood ratio after 51Cr-EDTA instillation.33,34

Various groups of animals were IP injected with ros-glitazone maleate (3 mg/kg, dissolved in saline; Alexisorp, San Diego, CA) or 15d-PGJ2 (120 �g/kg, dissolved

n dimethyl sulfoxide 10%; Cayman Chemical, Ann Arbor,I). The doses of both drugs were chosen based on

revious findings showing maximum inhibitory activityn NOS-2 expression and tumor necrosis factor � release

n a model of stress-induced brain damage.35,36 None ofhe parameters studied were modified in saline-treatedats when compared with noninjected animals. The drugsere injected at the onset of the daily stress session.nimals receiving saline at the onset of stress (either incute or in repeated exposure) were used as the controlroup in Results and the figures. Finally, 2 groups ofonstressed animals received 3 mg/kg rosiglitazone IP or20 �g/kg 15d-PGJ2 IP at 9 AM.

On the other hand, various groups of rats were IPnjected with T0070907 (2-chloro-5-nitro-N-4-pyridinyl-
enzamide; Cayman Chemical), a specific PPAR� antag-

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May 2007 PPAR� AND COLONIC DYSFUNCTION 1793

nist. The drug was injected IP 1 hour before the onset ofhe stress and the injection of PPAR� agonists.

Each experimental group contained at least 8 animals.

Myeloperoxidase ActivityImmediately after the animals were killed and

issected, colonic samples were minced on ice and ho-ogenized (glass/glass) in 0.5% hexadecyltrimethylam-onium bromide, 0.5% Nonidet P40 (Boehringer, Mann-

eim, Germany) in 20 mmol/L phosphate buffer, pH 6.0.he homogenates were then centrifuged for 20 minutest 12,000g. Tissue levels of myeloperoxidase (MPO) activ-ty were determined on supernatants using hydrogeneroxide as substrate for the enzyme. A unit of MPOctivity was defined as that converting 1 �mol of hydro-en peroxide to water in 1 minute at 40°C.37

ProstaglandinsColonic PGE2 and PGJ2 levels were determined

sing enzyme immunoassay kits (Biolink, Barcelona,pain, and Amersham, Buckinghamshire, England, re-pectively). Samples were purified using Amprep minicol-mns (Amersham). After homogenizing colons by soni-ation (Branson Sonifier 250; American Lab Trading,iantic, CT) in the same buffer used for Western blot

nalysis (see following text), the fraction needed for en-yme immunoassay isolation was obtained and PG quan-ification was performed following the manufacturer’snstructions.

Western Blot AnalysisTo determine the levels of NOS-2, cyclooxygenase

COX)-1, COX-2, and PPAR�, tissues were homogenizedt 4°C in 5 vol of buffer containing 320 mmol/L sucrose,

mmol/L DL-dithiothreitol, 10 �g/mL leupeptin, 10g/mL soybean trypsin inhibitor, 2 �g/mL aprotinin,nd 50 mmol/L Tris brought to pH 7.0 at 20°C with HClhomogenization buffer), and supernatants after centrif-gation at 12,000g for 20 minutes were used. For p65/elA fraction of NF-�B, cytosolic and nuclear extractsere obtained (see following text).After centrifugation in a microcentrifuge for 15 min-

tes, the proteins present in the supernatant were loaded20 �g) and size separated in 10% sodium dodecyl sul-ate/polyacrylamide gel electrophoresis (90 mV). The gelsere processed against the antigens and after blottingnto a polyvinylidene difluoride membrane (Millipore,edford, MA) were incubated with specific goat poly-lonal anti-rat COX-1 (1:1000), goat polyclonal anti-ratOX-2 (1:1000), rabbit polyclonal anti-rat NOS-2 (1:00), mouse monoclonal anti-rat PPAR� (1:1000), andabbit polyclonal anti-rat p65/RelA (1:1000) antibodiesall from Santa Cruz Biotechnology, Santa Cruz, CA).roteins recognized by the antibody were visualized on
-ray film by chemiluminescence following the manufac- u
urer’s instructions (Amersham lbérica, Madrid, Spain).utoradiographs were quantified by densitometry (Soft-are Total Lab Dynamics Ltd, Phoretix, Newcastle, En-land), and several time expositions were analyzed tonsure the linearity of the band intensities.

Preparation of Cytosolic and NuclearExtractsA modified procedure based on the method of

chreiber et al38 was used. Tissues were homogenizedith 300 �L of buffer (10 mmol/L HEPES, pH 7.9, 1mol/L EDTA, 1 mmol/L ethylene glycol-bis[�-amino-

thyl ether]-N,N,N=,N=-tetraacetic acid, 10 mmol/L KCl, 1mol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfo-

yl fluoride, 0.1 �g/mL aprotinin, 1 �g/mL leupeptin, 1g/mL N�-p-tosyl-L-lysine-chloromethyl ketone, 5 mmol/LaF, 1 mmol/L NaVO4, 0.5 mol/L sucrose, and 10mol/L Na2MoO4). After 15 minutes, Nonidet P40 was

dded to reach a 0.5% concentration. The tubes wereently vortexed for 15 seconds, and nuclei were collectedy centrifugation at 8000g for 5 minutes. Supernatantsere taken as a cytosolic fraction. The pellets were resus-ended in 100 �L of buffer supplemented with 20%lycerol and 0.4 mol/L KCl and gently shaken for 30inutes at 4°C. Nuclear protein extracts were obtained

y centrifugation at 13,000g for 5 minutes, and aliquotsf the supernatant were stored at �80°C. All steps of theractionation were performed at 4°C.

Electron MicroscopyColon segments from 4 rats per group were

xed in 2% glutaraldehyde for 3 hours, cryoprotectedvernight at 4°C in buffer phosphate 0.1 mol/L with0% sucrose, postfixed in 1% osmium tetroxide, stained

n 1% uranyl acetate, and embedded in Epon-Aralditeesin. Ultrathin sections (70 nm) were collected onickel grids. Sections were examined under Zeiss EM-02 (Zeiss, Oberkochen, Germany) transmission elec-ron microscopy. To evaluate changes in tight junc-ions (TJs), the junctional regions of several sections ofongitudinally sectioned villi per animal were exam-ned by a member of the study group blinded to theonditions of the experiment. At least 30 TJs per ani-al were evaluated. Results were expressed as percent-

ge of opened (ie, dilation �20 nm) TJs.

Assessment of Colonic PermeabilityRats were anesthetized with halothane. A catheter

OD, 1 mm) was inserted rectally at 4 cm from the anus.e slowly perfused 1.5 �Ci 51Cr-EDTA (Perkin Elmer

spaña, Madrid, Spain) in 0.5 mL NaCl 0.9% into theolon (0.25 mL/h) to control rats or just after the lastession in stressed animals. After 2 hours, rats were killed
sing sodium pentobarbital and blood was collected by

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ardiac puncture and anticoagulated in the presence ofrisodium citrate (3.15% wt/vol, 1 vol citrate per 9 vollood). The [51Cr]-bound radioactivity was counted usinggamma counter to measure radioactivity of the sam-

les. The permeability was expressed as the ratio betweenlood and total 51Cr instilled and reported as a percent-ge, as previously described.33,34

Bacterial TranslocationRats were anesthetized with sodium pentobarbital

P (320 mg/kg; Vetoquinol), and the abdominal skin washaved and sterilized with an iodine solution. Aftereath, mesenteric lymph nodes, liver, and spleen wereemoved under sterile conditions. After weighing andomogenization, aliquots (2 mL) of serial 10-fold dilu-ions of the suspension were plated onto 5% blood and

acConkey’s agar plates for recovery of aerobic bacteriand Brucella blood agar plates supplemented with vita-in K1 and hemin for anaerobic bacteria. After 24 and 48

ours of incubation at 37°C, for aerobic and anaerobicultures, respectively, colonies were counted. Quantita-ive culture results were expressed as the number ofolony-forming units per gram. Any positive mesentericymph node, liver, or spleen cultures were consideredndicative of bacterial translocation from the intestinalumen. Bacterial strains were identified by Gram stain,iochemical tests, and standard biochemical identifica-ion systems.

Plasma Corticosterone AssayPlasma was obtained within 1 hour of obtaining

lood samples by using a commercially available radio-mmunoassay kit of 131I-labeled rat corticosterone (DPC,os Angeles, CA). The sensitivity of the assay was 10g/mL. The values obtained in control animals (89.3 �.1 ng/mL) matched the kit manufacturer’s expectedalues in adult male Wistar rats at the time of bloodxtraction (�15.00 hours).

Chemicals and Statistical AnalysisChemicals were from Sigma (Barcelona, Spain) or

s indicated. Results are expressed as mean � SE of thendicated number of experiments, and statistical compar-sons were made using the Mann–Whitney test and one-ay analysis of variance followed by the Newman–Keuls

est, as appropriate. P � .05 was considered statisticallyignificant.

ResultsEffect of Immobilization Stress on BodyWeight and Colonic Inflammation and Effectof Pharmacologic PPAR� ActivationRepeated immobilization during 5 days induced

significant weight loss in comparison with non- w

tressed animals (Figure 1A). The macroscopic appear-nce of colonic mucosa was not affected in stressroups compared with control animals (data nothown). Nevertheless, MPO activity in colonic mucosa,hich has been shown to be linearly correlated witheutrophil infiltration,37 resulted in significantlyigher colonic homogenates of rats exposed to acute (6ours) stress and 5 days of immobilization comparedith nonstressed animals (Figure 1B). Intraperitonealdministration of PPAR� agonists rosiglitazone (3 mg/g) and 15d-PGJ2 (120 �g/kg) before each stress ses-ion prevented both weight loss (Figure 1A) and anncrease in MPO (Figure 1B). Administration of ros-glitazone or 15d-PGJ2 to control animals did not alter

igure 1. (A) Effect of immobilization stress during 6 hours, 5 days, and0 days on weight of rats receiving vehicle (saline), rosiglitazone (3 mg/g), or 15d-PGJ2 (120 �g/kg) at the onset of each stress session. Theata represent the mean � SEM of 8 rats. *P � .05 vs control non-tressed animals; #,†P � .05 vs saline. (B) Effect of immobilization stressuring 6 hours, 5 days, and 10 days on colonic MPO activity of ratseceiving vehicle (saline), rosiglitazone (3 mg/kg), or 15d-PGJ2 (120 �g/g) at the onset of each stress session. The data represent the mean �EM of 8 rats. *P � .05 vs control nonstressed animals; #,†P � .05 vsaline.

eight or colonic MPO activity.

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Effect of Immobilization Stress on NOS-2Expression and COX-2 Expression andActivity in the Colon and Effect ofPharmacologic PPAR� ActivationTo clarify the possible molecular pathways in-

olved in stress-induced colonic inflammation, the ex-ression of 2 major inflammatory enzymes, COX-2 andOS-2, in colonic homogenates was assessed. The anal-

sis of COX-2 expression (Figure 2A and B) showed aignificant increase in stressed animals, more evidentfter 6 hours of immobilization, in comparison withontrols. Similarly, NOS-2 expression (Figure 2A and C)as significantly increased after 6 hours and 5 days of

tress exposure, returning to baseline levels after 10 days.he expression of constitutive isoforms, neuronal NOS

NOS-1) and COX-1, remained unchanged during theifferent stress protocols in comparison with control

igure 2. (A) COX-2 and NOS-2 protein expression in colon fromontrol and stressed rats for 6 hours, 5 days, and 10 days. Effect ofosiglitazone (3 mg/kg) and 15d-PGJ2 (120 �g/kg) treatment. Data areepresentative of 3 animals. (B) Densitometric analysis of COX-2 proteinn � 3); mean density was calculated, plotted, and compared for sta-istical significance. (C) Densitometric analysis of NOS-2 protein (n � 3).,#P � .05 vs correspondent saline injected animals exposed to immo-ilization stress. (D) Effect of immobilization stress during 6 hours, 5ays, and 10 days on colonic PGE2 production of rats receiving vehicle

saline), rosiglitazone (3 mg/kg), or 15d-PGJ2 (120 �g/kg) at the onset ofach stress session. The data represent the mean � SEM of 8 rats.P � .05 vs control nonstressed animals; #,†P � .05 vs saline.

nimals (Figure 3). 1

Administration of PPAR� agonists rosiglitazone (3g/kg) and 15d-PGJ2 (120 �g/kg) before each stress

ession abolished the increase in NOS-2 expression ob-erved after stress but had no effect on increased COX-2xpression in stressed animals after 6 hours, 5 days, or 10ays of immobilization exposure (Figure 2A–C). Interest-

ngly, stress induced a significant increase in tissue PGE2

evels, a marker of COX activity, but administration ofPAR� agonists before each stress session markedly de-reased PGE2 production in colon (Figure 2D), indicatingn effect of PPAR� agonists at activity level, not at pro-ein level.

Effect of Immobilization Stress on 15d-PGJ2Levels and PPAR� Expression in the ColonIn parallel to the increase of proinflammatory

olecules, stress induced a significant increase in 15d-GJ2 levels (Figure 4A) and administration of PPAR�gonists before each stress session increased productionf 15d-PGJ2 in the colon (Figure 4A). Similarly, stress also

nduced the expression of PPAR� in the colon (FigureB). The increased expression was more evident after 6ours of immobilization exposure, but it was maintainedfter 5 days and, to a lesser extent, 10 days.

Effect of Immobilization Stress on ColonicPermeability and Bacterial Translocation andEffect of Pharmacologic PPAR� ActivationImmobilization induced a significant increase in co-

onic permeability to 51Cr-EDTA after 6 hours and 5 days,ith a trend to return to baseline values after 10 days of

ustained stress exposure (Figure 5A). In parallel, stressnduced a transient translocation of different bacterial spe-ies to mesenteric lymph nodes (Figure 5B), spleen, and liverdata not shown), as demonstrated by the significant in-rease in the colony-forming units cultured in those tissuesrom rats exposed to stress compared with control animals.solated species included gram-negative (Enterobacter spp,lcaligenes spp, Escherichia coli), gram-positive (Enterococcus

igure 3. Analysis of COX-1 and NOS-1 protein expression by West-rn blot in colon from control and stressed rats for 6 hours, 5 days, and
0 days. Data are representative of 3 animals.

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aecalis, Bacillus spp, coagulase-negative Staphylococcus), andnaerobic bacteria (Propionibacterium acnes, Clostridium aceto-utylicum, Lactobacillus spp, Bacteroides urealyticus, Peptostrepto-occus spp). Both stress-induced increased permeability andacterial translocation were prevented by the administra-ion of PPAR� agonists rosiglitazone and PGJ2 (Figure 5And B, respectively).

Increased colonic paracellular permeability has beenelated previously with TJ disruption.39 – 41 Ultrastruc-ural analysis of TJs by transmission electron microscopyas performed (Figure 6). The presence of electron-denseaterial in the space between cells near the brush border,

ppearing as punctate “kisses” where outer leaflets ofembrane bilayers come into closest contact, reflects theJs. Biopsy specimens from control animals (Figure 6A)isplayed an intact apical intercellular ultrastructure (ie,Js), and intercellular spaces and desmosomes appearedreserved. Conversely, dilated (dilation �20 nm) andisrupted TJs (reduced TJ complex and contained lesslectron-dense material), dilated intercellular spaces, andisrupted desmosomes were observed in those samplesrom animals stressed for 6 hours (Figure 6B) and 5 daysFigure 6C). TJs from animals stressed for 10 days (Figure

igure 4. (A) Effect of immobilization stress during 6 hours, 5 days, and0 days on colonic 15d-PGJ2 production of rats receiving vehicle (sa-

ine), rosiglitazone (3 mg/kg), or 15d-PGJ2 (120 �g/kg) at the onset ofach stress session. The data represent the mean � SEM of 8 rats.P � .05 vs control nonstressed animals; #,†P � .05 vs saline. (B)nalysis of PPAR� expression by Western blot in colon from control andtressed rats for 6 hours, 5 days, and 10 days. Data are representativef 3 animals.

D) were morphologically indistinguishable from con- .

rol. Quantitative analysis showed a higher proportion ofisrupted and dilated TJs in those animals stressed for 6ours and, to a lesser extent, for 5 consecutive days

control, 8.8%; stress 6 hours, 67.5%; stress 5 days, 32.5%;tress 10 days, 12.2%).

Effect of Immobilization Stress on ColonicImmunoglobulin A Production and Effect ofPharmacologic PPAR� ActivationTo clarify possible local homeostatic mechanisms

nvolved in stress-induced colonic dysfunction and bac-erial crossing to abdominal organs, levels of one of the

ajor local defense mechanisms, immunoglobulin (Ig) A,ere assessed in colon. Interestingly, acute stress induced

igure 5. Colonic permeability to 51Cr-EDTA (expressed as the ratioetween blood and total 51Cr instilled and reported as a percentage) inats exposed to immobilization stress during 6 hours, 5 days, and 10ays. Effect of rosiglitazone (3 mg/kg) and 15d-PGJ2 (120 �g/kg) treat-ent. The data represent the mean � SEM of 10 rats. *P � .05 vs

ontrol nonstressed animals; #,†P � .05 vs saline. (B) Bacterial translo-ation to mesenteric lymph nodes in rats exposed to immobilizationtress during 6 hours, 5 days, and 10 days. Effect of rosiglitazone3 mg/kg) and 15d-PGJ2 (120 �g/kg) treatment. The data represent the

ean � SEM of 8 rats. *P � .05 vs control nonstressed animals; #P �
05 vs saline.

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significant decrease in colonic IgA production thateturned to baseline values after 5 days of repeated stressxposure (Figure 7). Both PPAR� agonists evaluated pre-ented the decrease in IgA after acute stress.

igure 7. Effect of immobilization stress during 6 hours, 5 days, and 10ays on colonic IgA synthesis of rats receiving vehicle (saline), rosiglita-one (3 mg/kg), or 15d-PGJ2 (120 �g/kg) at the onset of each stressession. The data represent the mean � SEM of 8 rats. *P � .05 vs

igure 6. Transmission elec-ron micrographs of the TJ re-ion of colonic epithelial cells

rom (A) control rats or animalsxposed to immobilization stressuring (B) 6 hours, (C) 5 days, and

D) 10 days. Immobilization stressor 6 hours and 5 days inducedn increased proportion of TJ di-

atation and disruption (white ar-ows) versus control animals (seeumerical data in the text).

aontrol nonstressed animals; #,†P � .05 vs saline.

Effect of Immobilization Stress onCorticosterone Release and Effect ofPharmacologic PPAR� ActivationTo test the possibility that the effects of selective

PAR� activation were related to interference on theeneral response to stress, we determined plasma corti-osterone levels in all of the experimental groups studied.he effects elicited by the PPAR� ligands after stress in

he colon are independent of the systemic stress response,ecause plasma corticosterone levels after 6 hours, 5 days,r 10 days were not different between the saline, rosigli-azone, or 15d-PGJ2 groups. Similarly, nonstressed ani-

als receiving rosiglitazone or 15d-PGJ2 did not haveodified plasma corticosterone levels when comparedith control (Figure 8).

Effect of Pharmacologic PPAR� Inhibition onAcute Stress-Induced Colonic Inflammationand DysfunctionTo verify the anti-inflammatory role of PPAR� in

he colon in vivo, the specific PPAR� antagonist0070907 was used in different sets of experiments. Ad-inistration of T0070907 (2 mg/kg IP) 1 hour before the

nset of each stress session exacerbated colonic inflam-ation (increased MPO activity [Figure 9A], PGE2 syn-

hesis [Figure 9B], and NOS-2 expression [Figure 9C]) inhose animals treated with PPAR� agonists rosiglitazone
nd 15d-PGJ2. Furthermore, T0070907 blocked the pre-

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entive effect of PPAR� agonists on the stress-inducedolonic dysfunction as seen by 51Cr-EDTA permeabilitytudies (Figure 9D) and bacterial translocation to mesen-eric lymph nodes (Figure 9E). Moreover, T0070907locked the effects of PPAR� agonists on stress-inducedecreased IgA synthesis in colonic homogenates (Fig-re 9F).

DiscussionThe reported results showed that (1) exposure to

cute (6 hours) or subacute (5 days) immobilizationtress induces weight loss and an increase in MPO activ-ty reflecting neutrophil infiltration of the colonic mu-osa, as well as an increase in the expression of enzymaticnflammatory sources NOS-2 and COX-2; (2) colonicpithelial barrier results morphologically and function-lly altered after stress exposure and IgA productionecrease after stress; (3) stress leads to an early inductionf PPAR� expression and 15d-PGJ2 synthesis in the colon;nd (4) this stress-induced inflammation and dysfunc-ion was prevented with both natural and syntheticPAR� ligands (15d-PGJ2 and rosiglitazone), whereas ad-inistration of an specific PPAR� antagonist reverted

hose effects. On the other hand, subchronic (10 days)tress exposure leads to the normalization of MPO activ-ty, proinflammatory enzyme expression, colonic perme-bility, and colonic IgA production.

Growing evidence from experimental studies supportshat psychological and physical stress can lead to acutentestinal inflammatory response and barrier dysfunctionesulting in bacterial translocation and enhanced uptakef luminal antigens, which, consequently, are associated

igure 8. Effect of immobilization stress during 6 hours, 5 days, and 10ays on corticosterone release of rats receiving vehicle (saline), rosigli-azone (3 mg/kg), or 15d-PGJ2 (120 �g/kg) at the onset of each stressession. The data represent the mean � SEM of 8 rats. *P � .05 vsontrol nonstressed animals; #,†P � NS vs saline.

ith maintained stimulation of lamina propria and sub- P

ucosa immune cells and finally with chronic inflamma-ion.42,43 Interestingly, our results indicate a normaliza-ion of the colonic inflammatory response andonsequent barrier dysfunction after repeated predictabletressful stimuli. The trend to the normalization of MPO,roinflammatory enzymes, and colonic barrier functionbserved in our experiments after 10 days of repeatedtress could be related to an adaptation to the stressorith lack of corticotropin-releasing factor response.owever, taking into account that corticosterone levels

re still significantly higher after 10 days of immobiliza-ion stress compared with control animals, the activationf additional compensatory mechanisms that counteracthort exposure to stress-induced colonic inflammationnd dysfunction is suggested. In this vein, our experi-ents have focused on PPAR� because of the increas-

ngly recognized anti-inflammatory activity of this recep-or44 and because the colon is the organ, together withhe adipose tissue, with the greatest concentration ofPAR� messenger RNA in the organism,20 suggesting aivotal role of this receptor in the regulation of colonicomeostasis.Numerous experimental and clinical studies demon-

trate anti-inflammatory effects of PPAR� agonists in theut; treatment with PPAR� agonists such as thiazo-idinediones, 15d-PGJ2, and other members of the cyclo-entenone family of prostaglandins, or conjugated lino-

eic acid, has been shown to protect against inflammatoryrocesses in the gut.22,23,45– 48 PPAR� heterozygous micehow a significantly enhanced susceptibility to 2,4,6-tri-itrobenzene sulfonic acid–induced colitis,23 PPAR� ex-ression has been found to be impaired in the colonicucosa of patients with ulcerative colitis,49 and a pilot

tudy assessing the efficacy of the PPAR� agonist rosigli-azone in patients with ulcerative colitis has shownromising results that could be the starting point for theesign of future randomized controlled trials.50

PPAR� exerts its anti-inflammatory effects throughifferent mechanisms: inhibition of proinflammatory cy-okines,51 down-regulation of the expression of adhesion

olecules,52 and inhibition of the NOS-2 expression andctivity.53 All these effects seem to be mediated by thenterference with NF-�B transcriptional activity.22,54 In-eed, we have found that the expression of the RelAubunit of NF-�B in nuclear extracts from colonic mu-osa increased after 6 hours and 5 days of stress exposuren comparison with control, but not after 10 days oftress (data not shown).

Among the identified regulatory mechanisms ofPAR� expression, glucocorticoids could play a centralole in the stress-induced up-regulation observed in our

odel. Glucocorticoids induce PPAR� and PPAR� ex-ression in various cell types,55–58 and immobilizationtress has been demonstrated as a potent stimulator ofPAR� expression in the liver.56 On the other hand,
PAR� is considered to be a ligand-activated transcrip-

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igure 9. Effect of immobilization stress during 6 hours on (A) colonic MPO activity, (B) PGE2 production, (C) NOS-2 expression (representativeestern blot [inset] and densitometry plot, n � 3), (D) colonic permeability to 51Cr-EDTA, (E) bacterial translocation to mesenteric lymph nodes, and

F) colonic IgA production of rats receiving vehicle (saline), T0070907 (2 mg/kg), rosiglitazone (3 mg/kg), 15d-PGJ2 (120 �g/kg), T0070907 plusosiglitazone, or T0070907 plus 15d-PGJ . The data represent the mean � SEM of 8 rats. *P � .05 vs rosiglitazone-treated animals; #P � .05 vs
2
5d-PGJ2–treated animals.

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ion factor, and its regulation, apart from the gene ex-ression level, can occur depending on ligand availability;hus, taking into account the possibility of ligand-stim-lated displacement of the transcriptional corepressorhat is constitutively associated with the PPAR/RXR het-rodimer,59 the stress-induced increase in 15d-PGJ2 ob-erved may explain the increase in PPAR� activation.

Furthermore, our results show that PPAR� activationlso inhibits the stress-induced increased colonic perme-bility, decreased IgA production, and bacterial translo-ation. The results obtained with the PPAR� antagonisturther support the role of this receptor. Activation ofPAR� by butyrate has been shown to reduce colonicermeability, probably through the promotion of intes-inal epithelial cell differentiation and the consequenteinforcement of the TJs.60 Additionally, the effects ofPAR� on the regulation of colonic permeability coulde also related to the ability, shown in this report, ofecreasing NOS-2 expression and PGE2 synthesis. NOS-– derived NO has been shown to increase intestinalpithelial permeability both in vitro61,62 and in vivo63 inn effect that seems to be related to NO-induced cy-oskeleton rearrangement and subsequent TJ dysfunc-ion.61,63– 65 Finally, the release of PGs after lamina pro-ria mast cell degranulation induced by acute stress haseen proposed as a mechanism of stress-induced in-reased colonic permeability.66

The stress-induced increase of 15d-PGJ2 in the coloneems to run parallel with the expression of COX-2, asreviously shown in the brain.36 It has been suggestedhat the enhanced production of 15d-PGJ2 in the acti-ated macrophages during the inflammatory process mayepresent a response that contributes to negative regula-ion of inflammation,67 probably through its ability toctivate PPAR�.28 Apart from the anti-inflammatory ef-ects mediated by 15d-PGJ2 as a high-affinity PPAR�igand, it has shown important PPAR�-independent anti-nflammatory activity by inhibiting transcriptional acti-ation via NF-�B.68,69 In this vein, we have shown aown-regulation of NOS-2 by 15d-PGJ2 in our model, ingreement with previous findings in neurons and mac-ophages.36,70,71 Furthermore, the findings of an in-reased severity of dextran sodium sulfate–induced colitisn rats by pretreatment with selective COX-2 inhibitors72

nd of the induction of a colonic secretory dysfunctiony pre-exposure of normal colonic tissue to PGD2, butot to 15d-PGJ2,73 supports the hypothesis that COX-2–erived 15d-PGJ2 could exert a protective and anti-in-ammatory role in the gut. Interestingly, it has beenhown that PGE2 synthesis predominates during thearly inflammatory step in carrageenin-induced pleurisyn rats, whereas 15d-PGJ2 substitutes PGE2 formation athe end of the process, coincident with the accumulationf macrophages.74 Interestingly, 15d-PGJ2 has recently
een shown to decrease PGE2 synthesis by means of
icrosomal PGE synthase down-regulation in chondro-ytes75 and intestinal epithelial cells.76

Although others have found a decrease in salivary andaginal secretion IgA levels after stress exposure,77,78 thiss the first report, to our knowledge, of stress-inducedecrease of IgA production in the colonic mucosa.mong the mechanisms responsible for the stress-in-uced decrease in IgA, endogenous glucocorticoids couldlay a central role. Previous studies have shown that IgA

evels in serum were elevated when increasing doses ofexamethasone were administered to rats; in contrast,gA levels significantly decreased in saliva and vaginalecretions over the same dose range.77 If the stress-in-uced diminished IgA levels in the colonic mucosa resultrom a true decrease of IgA synthesis by lamina proprialasma cells or if there is a lack of transportation to theolonic lumen deserves further investigation. Neverthe-ess, there is evidence supporting the first hypothesis: (1)lucocorticoids significantly reduced estradiol-inducedevels of IgA in uterine secretions with no effect onstradiol-induced levels of the secretory component ofhe polymeric Ig receptor79 and (2) glucocorticoids stim-late secretory component synthesis by hepatocytes inulture.80 In our model, the effect of PPAR� agonists onhe restoration of colonic IgA levels seems to have anndependent component on glucocorticoid secretion, be-ause treatment of stressed rats with rosiglitazone or5d-PGJ2 does not affect plasma corticosterone levels inhose animals.

In summary, the present work shows that acute immo-ilization stress leads to colonic barrier dysfunction thateems to be mediated by the increased expression ofnflammatory enzymes COX-2 and NOS-2. In parallel,arly PPAR� up-regulation and activation by its endoge-ous agonist 15d-PGJ2 as well as direct anti-inflamma-ory effects by 15d-PGJ2 are responsible, at least in part,or the return to baseline values of biochemical inflam-

atory markers and mucosal permeability observed after0 days of stress exposure, representing a physiologic,nti-inflammatory pathway that counteracts initial co-onic inflammation and dysfunction. The pharmacologic

odulation of this pathway represents a promising ther-peutic approach in stress-related intestinal disorders.

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Received May 12, 2006. Accepted January 18, 2007.Address requests for reprints to: Luis Menchén, MD, PhD, Sección de

astroenterología, Servicio de Aparato Digestivo, Hospital Generalniversitario “Gregorio Marañón,” C/ Dr Esquerdo 46, 28007adrid, Spain. e-mail: [email protected]; fax: (34)14265024.Á.P. and J.R.C. contributed equally to this work.Supported by grants from Spanish Ministry of Education and Sci-

nce (DGI SAF 04/27) and Fundación Santander/UCM (PR27/05) (to.C.L.), Asociación Castellana de Aparato Digestivo (ACAD/2004), andundación para la Investigación en Gastroenterología y Hepatología
to L.M.)
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