The Role of PPARγ on Restoration of Colonic Homeostasis After Experimental Stress-Induced Inflammation and Dysfunction
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.02.032
ISSN1528-0012
AutoresÁngel Ponferrada, Javier R. Caso, Luís Alou, Arturo Colón Rodríguez, David Sevillano, Marı́a A. Moro, Ignacio Lizasoaín, Pedro Menchén, Marı́a Luisa Gómez-Lus, Pedro Lorenzo, Enrique Cos, Juan C. Leza, Luís Menchén,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoBackground & Aims: Psychological stress has been implicated in the clinical course of several gastrointestinal diseases, but the mechanisms implicated and the effects of stress on the normal colon are not yet fully understood. Methods: Male Wistar rats were exposed to various immobilization periods as a stress paradigm. Colon was processed to assess myeloperoxidase activity, nitric oxide synthase 2, cyclooxygenase 2, and peroxisome proliferator-activated receptor gamma (PPARγ) expression and production of prostaglandins. Colonic permeability, bacterial translocation, tight junctions ultrastructure, and immunoglobulin (Ig) A levels were also evaluated. Results: Exposure to acute (6 hours) immobilization stress produced an increase in myeloperoxidase activity and nitric oxide synthase 2 and cyclooxygenase 2 expression. All these parameters remained increased after 5 days of repeated stress exposure, showing a trend to normalize after 10 days. Levels of the anti-inflammatory eicosanoid 15-deoxy-Δ12Collins S.M. McHugh K. Jacobson K. Khan I. Riddell R. Murase K. Weingarten H.P. Previous inflammation alters the response of the rat colon to stress.Gastroenterology. 1996; 111: 1509-1515Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 14Ferrier L. Mazelin L. Cenac N. Desreumaux P. Janin A. Emilie D. Colombel J.F. Garcia-Villar R. Fioramonti J. Bueno L. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice.Gastroenterology. 2003; 125: 795-804Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar-prostaglandin J2 (15d-PGJ2) and expression of PPARγ run parallel with these changes. Colonic epithelial barrier was altered after stress exposure, and a significant decrease in colonic IgA levels after acute stress exposure was observed. Pretreatment with PPARγ agonists 15d-PGJ2 and rosiglitazone prevented colonic inflammation and barrier dysfunction as well as the decrease of IgA production induced after acute stress; PPARγ specific antagonist T0070907 reverted these effects. Conclusions: Activation of PPARγ in rat colon in vivo seems to counteract colonic inflammation and dysfunction induced by stress. On the other hand, PPARγ ligands may be therapeutically useful in conditions in which inflammation and barrier dysfunction takes place in colon after exposure to stress. Background & Aims: Psychological stress has been implicated in the clinical course of several gastrointestinal diseases, but the mechanisms implicated and the effects of stress on the normal colon are not yet fully understood. Methods: Male Wistar rats were exposed to various immobilization periods as a stress paradigm. Colon was processed to assess myeloperoxidase activity, nitric oxide synthase 2, cyclooxygenase 2, and peroxisome proliferator-activated receptor gamma (PPARγ) expression and production of prostaglandins. Colonic permeability, bacterial translocation, tight junctions ultrastructure, and immunoglobulin (Ig) A levels were also evaluated. Results: Exposure to acute (6 hours) immobilization stress produced an increase in myeloperoxidase activity and nitric oxide synthase 2 and cyclooxygenase 2 expression. All these parameters remained increased after 5 days of repeated stress exposure, showing a trend to normalize after 10 days. Levels of the anti-inflammatory eicosanoid 15-deoxy-Δ12Collins S.M. McHugh K. Jacobson K. Khan I. Riddell R. Murase K. Weingarten H.P. Previous inflammation alters the response of the rat colon to stress.Gastroenterology. 1996; 111: 1509-1515Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 14Ferrier L. Mazelin L. Cenac N. Desreumaux P. Janin A. Emilie D. Colombel J.F. Garcia-Villar R. Fioramonti J. Bueno L. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice.Gastroenterology. 2003; 125: 795-804Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar-prostaglandin J2 (15d-PGJ2) and expression of PPARγ run parallel with these changes. Colonic epithelial barrier was altered after stress exposure, and a significant decrease in colonic IgA levels after acute stress exposure was observed. Pretreatment with PPARγ agonists 15d-PGJ2 and rosiglitazone prevented colonic inflammation and barrier dysfunction as well as the decrease of IgA production induced after acute stress; PPARγ specific antagonist T0070907 reverted these effects. Conclusions: Activation of PPARγ in rat colon in vivo seems to counteract colonic inflammation and dysfunction induced by stress. On the other hand, PPARγ ligands may be therapeutically useful in conditions in which inflammation and barrier dysfunction takes place in colon after exposure to stress. The relationship between psychosocial stress and several gastrointestinal disorders is widely accepted by both patients and physicians, although the rational demonstration of a casual nexus remains difficult because of methodological aspects and interpretation of clinical studies and the shortage of experimental approaches. Stress influences subjective perception of gastrointestinal symptoms by patients1Dickhaus B. Mayer E.A. Firooz N. Stains J. Conde F. Olivas T.I. Fass R. Chang L. Mayer M. Naliboff B.D. Irritable bowel syndrome patients show enhanced modulation of visceral perception by auditory stress.Am J Gastroenterol. 2003; 98: 135-143Crossref PubMed Scopus (182) Google Scholar and has been shown to trigger the onset and modify the clinical course of certain gastrointestinal diseases.2Wilhelmsen I. The role of psychosocial factors in gastrointestinal disorders.Gut. 2000; 47: iv73-iv75PubMed Google Scholar Irritable bowel syndrome and functional dyspepsia are the most prevalent among the gastrointestinal disorders that have been related with psychological factors.3Mayer E.A. Naliboff B.D. Chang L. Coutinho S.V.V. Stress and irritable bowel syndrome.Am J Physiol Gastrointest Liver Physiol. 2001; 280: G519-G524PubMed Google Scholar It has been reported that patients with irritable bowel syndrome and functional dyspepsia had been exposed to stressful events more frequently than control individuals and that life stress was highly predictive of symptom intensity.4Bennett E.J. Tennant C.C. Piesse C. Badcock C.A. Kellow J.E. Level of chronic life stress predicts clinical outcome in irritable bowel syndrome.Gut. 1998; 43: 256-261Crossref PubMed Scopus (348) Google Scholar Stress has been associated also with inflammatory activity of ulcerative colitis5Duffy L.C. Zielezny M.A. Marshall J.R. Byers T.E. Weiser M.M. Phillips J.F. Calkins B.M. Ogra P.L. Graham S. Relevance of major stress events as an indicator of disease activity prevalence in inflammatory bowel disease.Behav Med. 1991; 17: 101-110Crossref PubMed Scopus (156) Google Scholar, 6Levenstein S. Prantera C. Varvo V. Scribano M.L. Berto E. Andreoli A. Luzi C. Psychological stress and disease activity in ulcerative colitis: a multidimensional cross-sectional study.Am J Gastroenterol. 1994; 89: 1219-1225PubMed Google Scholar and with an increased risk of disease exacerbation.5Duffy L.C. Zielezny M.A. Marshall J.R. Byers T.E. Weiser M.M. Phillips J.F. Calkins B.M. Ogra P.L. Graham S. Relevance of major stress events as an indicator of disease activity prevalence in inflammatory bowel disease.Behav Med. 1991; 17: 101-110Crossref PubMed Scopus (156) Google Scholar, 7Levenstein S. Prantera C. Varvo V. Scribano M.L. Andreoli A. Luzi C. Arca M. Berto E. Milite G. Marcheggiano A. Stress and exacerbation in ulcerative colitis: a prospective study of patients enrolled in remission.Am J Gastroenterol. 2000; 95: 1213-1220Crossref PubMed Google Scholar, 8Bitton A. Sewitch M.J. Peppercorn M.A. de Edwardess M.D. Shah S. Ransil B. Locke S.E. Psychosocial determinants of relapse in ulcerative colitis: a longitudinal study.Am J Gastroenterol. 2003; 98: 2203-2208Crossref PubMed Scopus (174) Google Scholar The high response to placebo frequently observed in inflammatory bowel disease clinical trials9Ilnyckyj A. Shanahan F. Anton P. Cheang M. Bernstein C. Quantification of the placebo response in ulcerative colitis.Gastroenterology. 1997; 112: 1854-1858Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Su C. Lichtenstein G. Krok K. Brensinger C. Lewis J. 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Previous inflammation alters the response of the rat colon to stress.Gastroenterology. 1996; 111: 1509-1515Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 13Saunders P.R. Hanssen N.P. Perdue M.H. Cholinergic nerves mediate stress-induced intestinal transport abnormalities in Wistar-Kyoto rats.Am J Physiol. 1997; 273: G486-G490PubMed Google Scholar, 14Ferrier L. Mazelin L. Cenac N. Desreumaux P. Janin A. Emilie D. Colombel J.F. Garcia-Villar R. Fioramonti J. Bueno L. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice.Gastroenterology. 2003; 125: 795-804Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar (2) increase the severity of experimental colitis,15Gue M. Bonbonne C. Fioramonti J. More J. Del Rio-Lacheze C. Comera C. Bueno L. Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved.Am J Physiol. 1997; 272: G84-G91PubMed Google Scholar and (3) even reactivate a quiescent colitis.16Qiu B.S. Vallance B.A. Blennerhassett P.A. Collins S.M. The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis.Nat Med. 1999; 5: 1178-1182Crossref PubMed Scopus (24) Google Scholar Previous findings of our group showed that stress induces macroscopic and histologic colonic inflammation, increasing inducible nitric oxide synthase (NOS-2) activity and membrane lipid peroxidation, after a low dose of trinitrobenzene sulfonic acid unable to induce such changes in nonstressed animals.17Colon A.L. Madrigal J.L. Menchen L.A. Moro M.A. Lizasoain I. Lorenzo P. Leza J.C. Stress increases susceptibility to oxidative/nitrosative mucosal damage in an experimental model of colitis in rats.Dig Dis Sci. 2004; 49: 1713-1721Crossref PubMed Scopus (26) Google Scholar Also, it has been shown that stress increases colonic permeability, facilitating the entry of luminal contents that activate previously sensitized CD4+ T cells in the colon,16Qiu B.S. Vallance B.A. Blennerhassett P.A. Collins S.M. The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis.Nat Med. 1999; 5: 1178-1182Crossref PubMed Scopus (24) Google Scholar initiating or perpetuating an inflammatory response, but the exact mechanisms of the association are still poorly understood. A question of special relevance is the possibility that some of the changes observed in stress would be a feature not of the deleterious effects of stress but of some potentially reversible type of adaptive plasticity, as proposed by McEwen.18McEwen B. Protective and damaging effects of stress mediators.N Engl J Med. 1998; 338: 171-179Crossref PubMed Scopus (4759) Google Scholar Most species of higher multicellular organisms have evolved complex homeostatic and defense mechanisms that allow cells to overcome stressful stimuli. One such mechanism that has received considerable experimental attention as a possible anti-inflammatory pathway is peroxisome proliferator-activated receptors (PPARs), a group of 3 ligand-regulated transcription factors originally implicated in adipocyte differentiation and glucose homeostasis. One of its isoforms, PPARγ, exerts an anti-inflammatory effect by interfering with the activity of inflammatory transcription factors, such as Activator Protein-1 (AP-1), Signal Transducers and Activators of Transcription (STAT), and nuclear factor κB (NF-κB).19Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. 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Previous inflammation alters the response of the rat colon to stress.Gastroenterology. 1996; 111: 1509-1515Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 14Ferrier L. Mazelin L. Cenac N. Desreumaux P. Janin A. Emilie D. Colombel J.F. Garcia-Villar R. Fioramonti J. Bueno L. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice.Gastroenterology. 2003; 125: 795-804Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar-prostaglandin J2 (15d-PGJ2),21Kliewer S.A. Xu H.E. Lambert M.H. Willson T.M. Peroxisome proliferator-activated receptors: from genes to physiology.Recent Prog Horm Res. 2001; 56: 239-263Crossref PubMed Scopus (369) Google Scholar or synthetic agonists can be considered as a pathway to halt the inflammatory response. Indeed, PPARγ ligands have been shown to reduce markedly colonic inflammation in both dextran sodium sulfate and trinitrobenzene sulfonic acid colitis models in mice,22Su C.G. Wen X. Bailey S.T. Jiang W. Rangwala S.M. Keilbaugh S.A. Flanigan A. Murthy S. Lazar M.A. Wu G.D. A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response.J Clin Invest. 1999; 104: 383-389Crossref PubMed Scopus (737) Google Scholar, 23Desreumaux P. Dubuquoy L. Nutten S. Peuchmaur M. Englaro W. Schoonjans K. Derijard B. Desvergne B. Wahli W. Chambon P. Leibowitz M.D. Colombel J.F. Auwerx J. Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer A basis for new therapeutic strategies.J Exp Med. 2001; 193: 827-838Crossref PubMed Scopus (410) Google Scholar probably through inhibition of NF-κB activity and decrease of proinflammatory cytokine production. To further investigate the possible mechanisms involved in stress-induced inflammation and dysfunction in the normal colon and the role of PPARγ as a possible homeostatic mechanism in the colon of stressed animals, we decided to explore whether this may serve as a new target for protective pharmacologic maneuvers. We used a stress paradigm that has been reported to cause accumulation of oxidative mediators in the gut.17Colon A.L. Madrigal J.L. Menchen L.A. Moro M.A. Lizasoain I. Lorenzo P. Leza J.C. Stress increases susceptibility to oxidative/nitrosative mucosal damage in an experimental model of colitis in rats.Dig Dis Sci. 2004; 49: 1713-1721Crossref PubMed Scopus (26) Google Scholar Acute or repeated immobilization stress is an easy, reproducible, and widely used model to study pathophysiologic aspects of the stress response and to study possible pharmacologic manipulations to decrease the negative effects of stress exposure on gastrointestinal, cardiovascular, or central nervous systems.24Pare W.P. Glavin G.B. Restraint stress in biomedical research: a review.Neurosci Biobehav Rev. 1986; 10: 339-370Crossref PubMed Scopus (332) Google Scholar We used a pharmacologic approach using natural and synthetic PPARγ agonists (the deoxy-prostaglandin 15d-PGJ2 and the thiazolidinedione drug rosiglitazone, respectively) as well as the specific PPARγ antagonist T0070907. Rosiglitazone is a well-documented high-affinity and selective ligand for PPARγ25Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma).J Biol Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3489) Google Scholar and has been shown to activate PPARγ in intestinal epithelial cells both in vitro26Gupta R.A. Brockman J.A. Sarraf P. Willson T.M. DuBois R.N. Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells.J Biol Chem. 2001; 276: 29681-29687Crossref PubMed Scopus (180) Google Scholar and in vivo.27Adachi M. Kurotani R. Morimura K. Shah Y. Sanford M. Madison B.B. Gumucio D.L. Marin H.E. Peters J.M. Young H.A. Gonzalez F.J. Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.Gut. 2006; 55: 1104-1113Crossref PubMed Scopus (168) Google Scholar 15d-PGJ2 has also been shown to be a high-affinity ligand for PPARγ.28Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma.Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2754) Google Scholar, 29Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation.Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1881) Google Scholar On the other hand, T0070907 has previously been shown to inhibit PPARγ activity both in vitro30Lee G. Elwood F. McNally J. Weiszmann J. Lindstrom M. Amaral K. Nakamura M. Miao S. Cao P. Learned R.M. Chen J.L. Li Y. T0070907, a selective ligand for peroxisome proliferator-activated receptor gamma, functions as an antagonist of biochemical and cellular activities.J Biol Chem. 2002; 277: 19649-19657Crossref PubMed Scopus (250) Google Scholar and in vivo.31Collin M. Murch O. Thiemermann C. Peroxisome proliferator-activated receptor-gamma antagonists GW9662 and T0070907 reduce the protective effects of lipopolysaccharide preconditioning against organ failure caused by endotoxemia.Crit Care Med. 2006; 34: 1131-1138Crossref PubMed Scopus (25) Google Scholar Young adult male Wistar rats (Animal Care Facility of the Complutense University, Madrid, Spain) weighing 210–230 g were used in this study. All experimental protocols adhered to the guidelines of the Animal Welfare Committee of the Universidad Complutense following European legislation (2003/65/EC). The rats were housed individually under standard conditions of temperature and humidity and a 12-hour light/dark cycle (lights on at 8 am) with free access to standard pelleted formula and tap water. All animals were maintained under constant conditions for 4 days before study. The restraint was performed using a plastic rodent restrainer that allowed for a close fit to rats in their home cages.32Leza J.C. Salas E. Sawicki G. Russell J.C. Radomski M.W. The effects of stress on homeostasis in JCR-LA-cp rats: the role of nitric oxide.J Pharmacol Exp Ther. 1998; 286: 1397-1403PubMed Google Scholar Stress consisted of 6 hours of immobilization starting at 9 am for 1, 5, or 10 consecutive days. Control animals were not subjected to stress but were handled at 9 am for a few seconds. Immediately after restraint (still in the restrainer), animals were weighed and killed using intraperitoneally (IP) administered sodium pentobarbital (320 mg/kg; Vetoquinol, Madrid, Spain). Blood for plasma determinations was collected by cardiac puncture and anticoagulated in the presence of trisodium citrate (3.15% wt/vol, 1 vol citrate per 9 vol blood). The colon was removed, and after assessment of macroscopic damage, the distal 8-cm portion of the colon was removed and cut longitudinally for the determinations described in the following text. For biochemical determinations, tissue was frozen at −80°C until assayed. For image studies, tissue was processed as described in the following text. A separate group of animals was used for colonic paracellular permeability assay immediately after the stress sessions for the in vivo lumen-to-blood ratio after 51Cr-EDTA instillation.33Aiko S. Fuseler J. Grisham M.B. Effects of nitric oxide synthase inhibition or sulfasalazine on the spontaneous colitis observed in HLA-B27 transgenic rats.J Pharmacol Exp Ther. 1998; 284: 722-727PubMed Google Scholar, 34Stein J. Ries J. Barrett K.E. Disruption of intestinal barrier function associated with experimental colitis: possible role of mast cells.Am J Physiol. 1998; 274: G203-G209PubMed Google Scholar Various groups of animals were IP injected with rosiglitazone maleate (3 mg/kg, dissolved in saline; Alexis Corp, San Diego, CA) or 15d-PGJ2 (120 μg/kg, dissolved in dimethyl sulfoxide 10%; Cayman Chemical, Ann Arbor, MI). The doses of both drugs were chosen based on previous findings showing maximum inhibitory activity on NOS-2 expression and tumor necrosis factor α release in a model of stress-induced brain damage.35Garcia-Bueno B. Madrigal J.L. Lizasoain I. Moro M.A. Lorenzo P. Leza J.C. Peroxisome proliferator-activated receptor gamma activation decreases neuroinflammation in brain after stress in rats.Biol Psychiatry. 2005; 57: 885-894Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 36Garcia-Bueno B. Madrigal J.L. Lizasoain I. Moro M.A. Lorenzo P. Leza J.C. The anti-inflammatory prostaglandin 15d-PGJ2 decreases oxidative/nitrosative mediators in brain after acute stress in rats.Psychopharmacology. 2005; 180: 513-522Crossref PubMed Scopus (34) Google Scholar None of the parameters studied were modified in saline-treated rats when compared with noninjected animals. The drugs were injected at the onset of the daily stress session. Animals receiving saline at the onset of stress (either in acute or in repeated exposure) were used as the control group in Results and the figures. Finally, 2 groups of nonstressed animals received 3 mg/kg rosiglitazone IP or 120 μg/kg 15d-PGJ2 IP at 9 am. On the other hand, various groups of rats were IP injected with T0070907 (2-chloro-5-nitro-N-4-pyridinyl-benzamide; Cayman Chemical), a specific PPARγ antagonist. The drug was injected IP 1 hour before the onset of the stress and the injection of PPARγ agonists. Each experimental group contained at least 8 animals. Immediately after the animals were killed and dissected, colonic samples were minced on ice and homogenized (glass/glass) in 0.5% hexadecyltrimethylammonium bromide, 0.5% Nonidet P40 (Boehringer, Mannheim, Germany) in 20 mmol/L phosphate buffer, pH 6.0. The homogenates were then centrifuged for 20 minutes at 12,000g. Tissue levels of myeloperoxidase (MPO) activity were determined on supernatants using hydrogen peroxide as substrate for the enzyme. A unit of MPO activity was defined as that converting 1 μmol of hydrogen peroxide to water in 1 minute at 40°C.37Bradley P.P. Priebat D.A. Christensen R.D. Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.J Invest Dermatol. 1982; 78: 206-209Crossref PubMed Scopus (3129) Google Scholar Colonic PGE2 and PGJ2 levels were determined using enzyme immunoassay kits (Biolink, Barcelona, Spain, and Amersham, Buckinghamshire, England, respectively). Samples were purified using Amprep minicolumns (Amersham). After homogenizing colons by sonication (Branson Sonifier 250; American Lab Trading, Niantic, CT) in the same buffer used for Western blot analysis (see following text), the fraction needed for enzyme immunoassay isolation was obtained and PG quantification was performed following the manufacturer’s instructions. To determine the levels of NOS-2, cyclooxygenase (COX)-1, COX-2, and PPARγ, tissues were homogenized at 4°C in 5 vol of buffer containing 320 mmol/L sucrose, 1 mmol/L dl-dithiothreitol, 10 μg/mL leupeptin, 10 μg/mL soybean trypsin inhibitor, 2 μg/mL aprotinin, and 50 mmol/L Tris brought to pH 7.0 at 20°C with HCl (homogenization buffer), and supernatants after centrifugation at 12,000g for 20 minutes were used. For p65/RelA fraction of NF-κB, cytosolic and nuclear extracts were obtained (see following text). After centrifugation in a microcentrifuge for 15 minutes, the proteins present in the supernatant were loaded (20 μg) and size separated in 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (90 mV). The gels were processed against the antigens and after blotting onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) were incubated with specific goat polyclonal anti-rat COX-1 (1:1000), goat polyclonal anti-rat COX-2 (1:1000), rabbit polyclonal anti-rat NOS-2 (1:500), mouse monoclonal anti-rat PPARγ (1:1000), and rabbit polyclonal anti-rat p65/RelA (1:1000) antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA). Proteins recognized by the antibody were visualized on X-ray film by chemiluminescence following the manufacturer’s instructions (Amersham lbérica, Madrid, Spain). Autoradiographs were quantified by densitometry (Software Total Lab Dynamics Ltd, Phoretix, Newcastle, England), and several time expositions were analyzed to ensure the linearity of the band intensities. A modified procedure based on the method of Schreiber et al38Schreiber E. Matthias P. Muller M.M. Schaffner W. Rapid detection of octamer binding proteins with “mini-extracts,” prepared from a small number of cells.Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (4058) Google Scholar was used. Tissues were homogenized with 300 μL of buffer (10 mmol/L HEPES, pH 7.9, 1 mmol/L EDTA, 1 mmol/L ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid, 10 mmol/L KCl, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL Nα-p-tosyl-l-lysine-chloromethyl ketone, 5 mmol/L NaF, 1 mmol/L NaVO4, 0.5 mol/L sucrose, and 10 mmol/L Na2MoO4). After 15 minutes, Nonidet P40 was added to reach a 0.5% concentration. The tubes were gently vortexed for 15 seconds, and nuclei were collected by centrifugation at 8000g for 5 minutes. Supernatants were taken as a cytosolic fraction. The pellets were resuspended in 100 μL of buffer supplemented with 20% glycerol and 0.4 mol/L KCl and gently shaken for 30 minutes at 4°C. Nuclear protein extracts were obtained by centrifugation at 13,000g for 5 minutes, and aliquots of the supernatant were stored at −80°C. All steps of the fractionation were performed at 4°C. Colon segments from 4 rats per group were fixed in 2% glutaraldehyde for 3 hours, cryoprotected overnight at 4°C in buffer phosphate 0.1 mol/L with 30% sucrose, postfixed in 1% osmium tetroxide, stained in 1% uranyl acetate, and embedded in Epon-Araldite resin. Ultrathin sections (70 nm) were collected on nickel grids. Sections were examined under Zeiss EM-902 (Zeiss, Oberkochen, Germany) transmission electron microscopy. To evaluate changes in tight junctions (TJs), the junctional regions of several sections of longitudinally sectioned villi per animal were examined by a member of the study group blinded to the conditions of the experiment. At least 30 TJs per animal were evaluated. Results were expressed as percentage of opened (ie, dilation >20 nm) TJs. Rats were anesthetized with halothane. A catheter (OD, 1 mm) was inserted rectally at 4 cm from the anus. We slowly perfused 1.5 μCi 51Cr-EDTA (Perkin Elmer España, Madrid, Spain) in 0.5 mL NaCl 0.9% into the colon (0.25 mL/h) to control rats or just after the last session in stressed animals. After 2 hours, rats were killed using sodium pentobarbital and blood was collected by cardiac puncture and anticoagulated in the presence of trisodium citrate (3.15% wt/vol, 1 vol citrate per 9 vol blood). The [51Cr]-bound radioactivity was counted using a gamma counter to measure radioactivity of the samples. The permeability was expressed as the ratio between blood and total 51Cr instilled and reported as a percentage, as previously described.33Aiko S. Fuseler J. Grisham M.B. Effects of nitric oxide synthase inhibition or sulfasalazine on the spontaneous colitis observed in HLA-B27 transgenic rats.J Pharmacol Exp Ther. 1998; 284: 722-727PubMed Google Scholar, 34Stein J. Ries J. Barrett K.E. Disruption of intestinal barrier function associated with experimental colitis: possible role of mast cells.Am J Physiol. 1998; 274: G203-G209PubMed Google Scholar Rats were anesthetized with sodium pentobarbital IP (320 mg/kg; Vetoquinol), and the abdominal skin was shaved and sterilized with an iodine solution. After death, mesenteric lymph nodes, liver, and spleen were removed under sterile conditions. After weighing and homogenization, aliquots (2 mL) of serial 10-fold dilutions of the suspension were plated onto 5% blood and MacConkey’s agar plates for recovery of aerobic bacteria and Brucella blood agar plates supplemented with vitamin K1 and hemin for anaerobic bacteria. After 24 and
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