Produção Nacional
Revisado por pares
Unfractionated Heparin and New Heparin Analogues from Ascidians (Chordate-Tunicate) Ameliorate Colitis in Rats
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m807211200
ISSN1083-351X
AutoresCelso L.R. Belmiro, Morgana T. Castelo-Branco, Leandra M.C. Melim, Alberto Schanaider, Celeste C. Elia, Kalil Madi, Mauro S.G. Pavão, Heitor Siffert Pereira de Souza,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoThe anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-α, TGF-β, and VEGF was observed, as well as a reduction of NF-κB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin. The anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-α, TGF-β, and VEGF was observed, as well as a reduction of NF-κB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin. Inflammatory bowel diseases (IBD) 3The abbreviations used are: IBD, inflammatory bowel diseases; CD, Crohn disease; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; GAG, glycosaminoglycan; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; VEGF, vascular endothelial growth factor; LPS, lipopolysaccharide; ANOVA, analysis of variance; TNBS, trinitrobenzene sulfonic acid; aPTT, activated partial thromboplastin time; DS, dermatan sulfate; MAP, mitogen-activated protein; HS, heparan sulfate; CS, chondroitin sulfate. comprise basically Crohn disease (CD) and ulcerative colitis and are characterized by chronic inflammation of the gastrointestinal tract. The etiology of IBD is complex and multifactorial, involving genetic predisposition and environmental triggers, as well as microbial and immune factors (1.Xavier R.J. Podolsky D.K. Nature. 2007; 448: 427-434Crossref PubMed Scopus (3303) Google Scholar). In CD, the chronic inflammatory process is a consequence of an imbalance in the production of proinflammatory and immunoregulatory cytokines, which results in a T helper cell type 1 (Th1) phenotype (2.Papadakis K. Targan S. Inflamm. Bowel Dis. 2000; 6: 303-313Crossref PubMed Scopus (105) Google Scholar, 3.Reinecker H. Steffen M. Witthoeft T. Pflueger I. Schreiber S. MacDermott R. Raedler A. Clin. Exp. Immunol. 1993; 94: 174-181Crossref PubMed Scopus (788) Google Scholar). Th1-type response is characterized by the production of tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-18, interferon (IFN)-γ, among other proinflammatory cytokines, and also involves the production of growth factors such as transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) (4.Fiocchi C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1870) Google Scholar, 5.Podolsky D. N. Engl. J. Med. 2002; 347: 417-429Crossref PubMed Scopus (3184) Google Scholar, 6.Macdonald T.T. Monteleone G. Science. 2005; 307: 1920-1925Crossref PubMed Scopus (816) Google Scholar). In this context, the nuclear transcription factor κB (NF-κB) was identified as a key regulator of the expression of proinflammatory genes, determining the course of mucosal inflammation in IBD (7.Atreya I. Atreya R. Neurath M.F. J. Intern. Med. 2008; 263: 591-596Crossref PubMed Scopus (598) Google Scholar). The increased secretion of proinflammatory cytokines in IBD intestinal mucosa is accompanied by the over expression of adhesion molecules, including the selectins (8.Schurmann G.M. Bishop A.E. Facer P. Vecchio M. Lee J.C. Rampton D.S. Polak J.M. Gut. 1995; 36: 411-418Crossref PubMed Scopus (87) Google Scholar, 9.Andoh A. Tsujikawa T. Hata K. Araki Y. Kitoh K. Sasaki M. Yoshida T. Fujiyama Y. Am. J. Gastroenterol. 2005; 100: 2042-2048Crossref PubMed Scopus (68) Google Scholar). Animal models of mucosal inflammation have been utilized to investigate the pathogenesis of IBD and to evaluate possible new therapies. The trinitrobenzene sulfonic acid (TNBS)-induced colitis constitutes an established experimental model, which exhibits clinical and histological similarities to CD, and the course of colonic damage has been well characterized as a Th1-type immune response, with the resulting production of proinflammatory cytokines (10.Strober W. Fuss I.J. Blumberg R.S. Annu. Rev. Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1154) Google Scholar). Heparin is a sulfated glycosaminoglycan (GAG) largely utilized in the clinical practice for anticoagulation and prevention and treatment of vascular thromboembolism (11.Roden L. Ananth S. Campbell P. Curenton T. Ekborg G. Manzella S. Pillion D. Meezan E. Adv. Exp. Med. Biol. 1992; 313: 1-20Crossref PubMed Scopus (27) Google Scholar, 12.Hirsh J. Warkentin T.E. Shaughnessy S.G. Anand S.S. Halperin J.L. Raschke R. Granger C. Ohman E.M. Dalen J.E. Chest. 2001; 119: 64S-94SAbstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar). Apart from its well-established anticoagulant and antithrombotic effects, heparin has anti-inflammatory properties such as inhibition of leukocyte adhesion and migration (13.Papa A. Danese S. Gasbarrini A. Gasbarrini G. Aliment Pharmacol. Ther. 2000; 14: 1403-1409Crossref PubMed Scopus (71) Google Scholar), and modulation of cytokine production (14.Elsayed E. Becker R.C. J. Thromb. Thrombolysis. 2003; 15: 11-18Crossref PubMed Scopus (59) Google Scholar). Based on these activities, and the suggested efficacy observed in several open clinical studies (15.Gaffney P.R. Doyle C.T. Gaffney A. Hogan J. Hayes D.P. Annis P. Am. J. Gastroenterol. 1995; 90: 220-223PubMed Google Scholar, 16.Folwaczny C. Wiebecke B. Loeschke K. Am. J. Gastroenterol. 1999; 94: 1551-1555Crossref PubMed Scopus (123) Google Scholar, 17.Ang Y.S. Mahmud N. White B. Byrne M. Kelly A. Lawler M. McDonald G.S. Smith O.P. Keeling P.W. Aliment Pharmacol. Ther. 2000; 14: 1015-1022Crossref PubMed Scopus (93) Google Scholar), heparin has been proposed as an alternative for the treatment of IBD. However, heparin therapy may cause hemorrhage and other adverse side effects (18.Schulman S. Beyth R. Kearon C. Levine M. Chest. 2008; 133: 257S-298SAbstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). Our laboratory has isolated and characterized several heparin analogues from marine invertebrates (19.Santos J. Mesquita J. Belmiro C. da Silveira C. Viskov C. Mourier P. Pavao M. Thromb. Res. 2007; 121: 213-223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20.Cardilo-Reis L.C.M. Silveira C.B. Pavao M.S. Braz J. Med. Biol. Res. 2006; 39: 1409-1415Crossref PubMed Scopus (12) Google Scholar, 21.Pavao M. An. Acad. Bras. Cienc. 2002; 74: 105-112Crossref PubMed Scopus (26) Google Scholar, 22.Vicente C. Zancan P. Peixoto L. Alves-Sa R. Araujo F. Mourao P. Pavão M.S. Thromb. Haemost. 2001; 86: 1215-1220Crossref PubMed Scopus (37) Google Scholar, 23.Pavao M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 24.Pavao M. Mourao P. Mulloy B. Tollefsen D. J. Biol. Chem. 1995; 270: 31027-31036Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The study of the anticoagulant properties of the marine invertebrate glycans indicated that although they are capable of inhibiting venous and arterial thrombosis, they are less anticoagulant and have no bleeding effect after intravenous administration to experimental animals. Therefore, in the present work, we investigate the anti-inflammatory effect of heparin analogues obtained from the ascidian Styela plicata in a rat model of colon inflammation. Animals—Male Wistar rats (each weighing between 250 and 300 g) obtained from a local supplier were maintained on a 12-h/12-h light and dark cycle in a temperature-controlled room (24 °C). Animals were housed in rack-mounted, wire cages with 3 animals per cage. Standard laboratory pelleted formula and tap water were provided ad libitum. The care and use of animals, as well as procedures reported in this study were approved by the institutional care committee of the Federal University of Rio de Janeiro and are in accordance with the guidelines of the International Care and Use Committee of the National Institutes of Health, and Guide for the Care and Use of Laboratory Animals (25.Institute of Laboratory Animal Resources, Commission on Live Sciences, National Research Council (1996), National Academic Press, Washington, D.C.Google Scholar). Induction of Colitis—On day (d) 0, rats were anesthetized subcutaneously with ketamine (35 mg/kg) and xylazine (5 mg/kg), and colitis was induced by intracolonic instillation of 0.8 ml of a solution containing 20 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Sigma) in 20% ethanol (Merck, Damstadt, Germany) using a rubber cannula (8-cm long) inserted through the rectum. Thereafter, animals were allowed access to standard chow and water ad libitum. Clinical manifestations such as diarrhea, bleeding, and weight loss were observed during this period. Experimental Design—After an initial acclimation period of 1 week, animals were assigned randomly to one of five groups of 10 animals each, and followed during 1 week. The colitis group (TNBS) was submitted to colitis induction but did not receive any treatment, and animals were sacrificed on experimental day 7. There were 4 GAG-treated groups: mam Hep, mammalian heparin (Hep); mam DS, mammalian dermatan sulfate (DS); S. plicata Hep and S. plicata DS. These groups were submitted to colitis induction followed by treatment with the indicated GAG (4 or 8 mg/kg per day) by a subcutaneous route over 7 days. In control experiments, the ascidian GAGs were either treated with chondroitin ABC-lyase or nitrous acid. Normal rats not submitted to any intervention constituted the control group, which was sacrificed after the acclimation period. For the surgical procedure, animals were anesthetized as described in the previous paragraph and submitted to a mild laparotomy under sterile technique. The distal colon was removed, opened longitudinally, rinsed with sterile saline, and scored. After scoring, three tissue samples were excised from the colon for histological assessment. A quick death procedure by cervical dislocation was uniformly performed in all animals. Histological Inflammatory Scores of the Colon—Specimens were fixed in 40 g/liter formaldehyde saline, embedded in paraffin, cut into 5-μm sections, stained with hematoxylin-eosin stain, and examined microscopically by two independent observers. The following histological parameters were studied: ulceration, hyperplasia, and inflammatory infiltrate. For both inflammatory infiltrate and hyperplasia, grading was considered: 3, severe; 2, moderate; 1, mild; 0, absent. For ulcers, grading was: 4, diffuse glandular disruption or extensive deep ulceration; 3, glandular disruption or focal deep ulceration; 2, diffuse superficial ulceration; 1, focal superficial ulceration; and 0, absent (26.Hahm K. Im Y. Parks T. Park S. Markowitz S. Jung H. Green J. Kim S. Gut. 2001; 49: 190-198Crossref PubMed Scopus (170) Google Scholar). Immunohistochemical Analysis of the Colon—Tissue samples were immediately embedded in Tissue-Tek O.C.T. compound (Miles Scientific Laboratories Ltd, Naperville, IL) and snap-frozen in isopentane in a liquid nitrogen bath. Samples were then stored at -80 °C until processing, and cut into 6-μm section in a cryostat maintained at -20 °C. Tissue sections were air-dried and fixed for 10 min in a 1:1 solution of chloroformacetone. Immunologic assessment of the intestinal mucosa was made using indirect immunoperoxidase technique using the following antibodies: mouse monoclonal anti-rat ED1 (Serotec Ltd., Oxford, UK) to macrophages; mouse monoclonal anti-rat CD3 (PC3/188) (Santa Cruz Biotechnology) to lymphocytes; mouse monoclonal anti-rat p65 (F-6) (Santa Cruz Biotechnology) to NF-κB. Briefly, frozen sections were immersed in 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. After rinsing in phosphate-buffered saline (PBS) containing 0.5% Tween 20 for 10 min, tissue sections were incubated with nonimmune horse serum for 30 min and, subsequently, with the respective monoclonal antibody in a humidified chamber overnight, at room temperature. Two sections from each sample were incubated with either PBS alone or mouse monoclonal IgG1 (concentration-matched) (Dako A/S) and served as negative controls. After rinsing in PBS for 10 min, all tissue sections were incubated for 30 min with a goat anti-mouse peroxidase conjugate (Zymed Laboratories Inc., Inc., San Francisco, CA). Additional rinsing was followed by development with a solution containing hydrogen peroxide and diaminobenzidine, dehydrated, and mounted in histological mounting medium. Immunofluorescence and Confocal Microscopy—For the indirect immunofluorescence study, frozen sections were incubated at room temperature with 2.5% bovine serum albumin + 2.0% nonfat milk + 8.0% fetal calf serum blocking buffer under shaking for 30 min. The sections were then incubated with appropriately diluted primary antibodies in PBS solution + 1.0% fetal calf serum for 1 h in wet atmosphere at 37 °C. The primary antibody used was the monoclonal mouse anti-rat P-ERK1/2 (1:50) (Santa Cruz Biotechnology). After rinsing three times in PBS for 5 min each, tissue sections were incubated for 1 h with a FITC-conjugated Fab fraction of goat anti-mouse IgG antibody (Dako A/S). Slides were counterstained with Evan blue-diluted 0.01% in phosphate-buffered saline (PBS) and incubated at 37 °C for 15 min, mounted in an anti-fading medium containing buffered glycerol and p-phenylenediamine (Sigma), and then observed with a Zeiss LSM 510 confocal laser scanning microscope. At least four representative images from each slide were captured. Two sections from each sample were incubated with either PBS alone or FITC-conjugated anti-mouse IgG antibody and served as negative controls. Assessment of Collagen Deposition in the Colon—Specimens were fixed in 40 g/liter formaldehyde saline, embedded in paraffin, and cut into 5-μm sections. The phosphomolibidic acid-picro-sirius red dye was used to stain collagen fibers in tissue (27.Dolber P. Spach M. Stain Technol. 1987; 62: 23-26Crossref PubMed Scopus (87) Google Scholar, 28.Dolber P. Spach M. J. Histochem. Cytochem. 1993; 41: 465-469Crossref PubMed Scopus (145) Google Scholar). At least 15 different areas per tissue section were analyzed under light microscopy. Detection of Apoptosis using TUNEL Assay—Paraffin-embedded colon samples were de-waxed in xylene twice for 5 min each time and then rehydrated in graded ethanol (100-70%) three times, followed by rehydration in PBS for 30 min. Apoptotic cells were detected by the terminal deoxynucleotidyltransferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) assay, using the in situ apoptosis detection kit ApopTag Fluorescein (Chemicon International, Inc. Temecula, CA), according to the instructions of the manufacturer. Sections were analyzed in a confocal microscope. Quantitative Assessment of Colon Sections—Quantitative analysis of tissue sections (under light microscopy) and captured immunofluorescence images (under confocal laser-scanning microscope) was carried out using a computer-assisted image analyser (Image-Pro Plus Version 4.1 for Windows, Media Cybernetics, LP, Silver Spring, MD). Any epithelial and lamina propria cells exhibiting identifiable reactivity distinct from background were regarded as positive. In the immunoperoxidase and immunofluorescence studies, the densities of the different cell subsets were defined by the number of immunoreactive cells in the lamina propria per millimeter-squared (counted in at least 10 different areas). In the epithelium, the density of apoptotic cells was defined as the percentage of immunoreactive cells within at least 500 epithelial cells in the crypts and in the surface epithelium of longitudinally sectioned colonic pits. The density of collagen fibers was defined by the area positively stained for collagen in relation to total intestinal tissue per millimeter-squared using an imaging analysis system. Two independent observers who were unaware of the experimental animal data examined all tissue sections and captured images. Organ Culture and Cytokine Measurements—Colonic mucosal explants were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen), 2 mm l-glutamine (Sigma), 50 μm 2-mercaptoethanol (Sigma), 10 mm HEPES (Promega), penicillin (100 killiunits/liter) and streptomycin (100 mg/liter) (Sigma) for 24 h at 37 °C in a 5% CO2 humidified incubator. After incubation for 24 h, the supernatant was collected and stocked at -20 °C. Samples were centrifuged, and the supernatants used for measurement of the concentration of cytokines TNF-α, TGF-β, and VEGF by a commercial sensitive enzyme-linked immunosorbent assay (ELISA) method (R&D System, MN). The total protein content of the biopsy specimens was estimated by the Lowry method. In our data, biopsy specimen wet weight was shown to correlate closely with protein content of tissue homogenates. The minimum detectable concentration of rat TNF-α, TGF-β, and VEGF was typically less than 5.0 ng/liter. Isolation and Quantification of GAGs—The dried intestinal samples (∼0.5 g) were individually suspended in 10 ml of 0.1 m sodium acetate buffer (pH 5.5), containing 50 mg of papain, 5 mm EDTA, and 5 mm cysteine, and incubated at 60 °C for 24 h. The mixtures were centrifuged (2000 × g for 10 min at room temperature). Another 50 mg of papain in 10 ml of the same buffer, containing 5 mm EDTA and 5 mm cysteine was added to the precipitate. The mixture was then incubated for another 24 h. The clear supernatants from the two extractions were combined, and the GAGs precipitated with a solution of cetylpyridinum chloride (0.5% final concentration), followed by 2 vol. of 95% ethanol and maintained at 4 °C for 24 h. The precipitate formed was collected by centrifugation (2000 × g for 10 min at room temperature), freeze-dried, and dissolved in 2 ml of distilled water. The amount of GAGs in the renal samples was estimated by the content of hexuronic acid, using the carbazole reaction (29.Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5218) Google Scholar). Agarose Gel Electrophoresis—The intact or enzyme-degraded GAGs from the different intestinal samples were analyzed by agarose gel electrophoresis, as described previously (23.Pavao M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Briefly, about 1.5 μg (as uronic acid) of the glycans, and a mixture of standard GAGs, containing chondroitin sulfate, dermatan sulfate, and heparan sulfate (1.5 μg as uronic acid of each) were applied to a 0.5% agarose gel in 0.05 m 1,3-diaminopropane/acetate (pH 9.0), and run for 1 h at 110 mV. After electrophoresis, the GAGs were fixed with aqueous 0.1% cetylmethylammonium bromide solution and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v/v). The relative proportions of the GAGs were estimated by densitometry of the metachromatic bands on a Bio-Rad densitometer, following agarose gel electrophoresis. The identity of GAGs was determined by agarose gel electrophoresis before and after incubation with specific GAG-lyases (Chondroitin AC-lyase, Chondroitin ABC-lyase) or deaminative cleavage with nitrous acid as described previously (23.Pavao M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Macrophage Activation Assay—Rat peritoneal macrophages were obtained by washing out the peritoneal cavity with 5 ml of ice-cold sterile RPMI 1640 serum-free medium and cultured at 37 °C for 4 h in a humidified 5% CO2 incubator. After incubation, non-adherent cells were removed by washing with serum-free medium. Adherent cells were incubated in 24-well tissue culture plates at a density of 106 cells per well with RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (Invitrogen) for 24 h in the presence of a range of heparin concentrations (5.0, 10, 20, and 40 μg/ml) plus LPS (1 μg/ml) (Sigma), added either concomitantly or 2 h after heparin under 5% CO2 atmosphere. In some experiments LPS was omitted to check the effect of heparin on macrophages. Determination of TNF-α Production—The cell culture supernatant was collected from the cultures of macrophages cells stimulated with heparin or LPS or heparin plus LPS. The concentration of TNF-α was determined by enzyme-linked immunosorbent assay kit, according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN). Ex Vivo Anticoagulant Action Measured by aPTT (Activated Partial Thromboplastin Time)—At experimental day 7, animals from the different groups were anesthetized with an intramuscular injection of 100 mg/kg of ketamine (Cristália, São Paulo, Brazil) and 16 mg/kg of xylazine (Bayer AS, São Paulo, Brazil), supplemented as needed. The right carotid artery was isolated and cannulated with a 22-gauge catheter (Jelco, Johnson & Johnson Medical Inc.) for blood collection. Blood samples (∼500 μl) were collected into 2.8% sodium citrate (9:1, v/v) for analysis of aPTT. At least 5 animals were used per group. aPTT was carried out as following: rat plasma (100 μl) was incubated with 100 μl of aPTT reagent (Celite-Biolab) at 37 °C. After 2 min of incubation 100 μl of 0.25 m CaCl2 were added to the mixtures and the clotting time recorded in a coagulometer (Amelung KC4A). Platelet Counts—Blood samples were carefully drawn on heparin from the portal vein during surgery just prior to colon removal. The platelet count in the whole blood was measured on an automatic hematology analyzer. Results are expressed as number of cells per cubic millimeter. Statistical Analysis—Statistical analyses were performed using the statistical software SPSS for Windows (Version 10.1, SPSS Inc., 1989–1999). Statistical differences among the experimental groups were evaluated with the one-way ANOVA test in which pairwise multiple comparisons were carried out using the Dunnett T3 test. Correlations between inflammatory scores, the densities of positive cells measured by immunohistochemistry, and the cytokine levels were assessed using the Spearman rank correlation coefficient. The level of significance was set at p < 0.05. Histomorphological Changes in the Colonic Tissue—We found that animals subjected to TNBS treatment developed colitis accompanied by a significant weight loss. Evident morphological changes were observed predominantly in the distal colon of the animals. Inflammatory lesions in the colon included mucosal edema, ulceration, and evidence of transmural inflammation. Therapeutic approach consisted of subcutaneous administration of ascidian or mammalian heparin and dermatan sulfate (DS), initiated concomitantly with TNBS treatment and continued for 7 days. A drastic attenuation of the inflammatory lesions was observed macroscopically in the animals after subcutaneous administration of mammalian or ascidian GAGs at the dose of 8 mg/kg. No significant changes were observed at the dose of 4 mg/kg (not shown). Histological analysis of formalin-fixed HE-stained intestinal sections revealed an increase in the microscopic damage score in all TNBS-treated animals, when compared with the normal mucosa of control animals (Fig. 1, A and B). Administration of mammalian or ascidian GAGs (8 mg/kg) significantly reduced the inflammatory scores in the TNBS-treated group. However, the scores obtained by ascidian heparin were significantly (p < 0.001) lower than that obtained by the mammalian counterpart. Administration of nitrous acid-treated S. plicata heparin or chondroitin ABC-lyase-treated S. plicata DS to the TNBS group abolished the beneficial effect of the GAGs (Fig. 1C). Cellular Infiltration in the Colonic Tissue—TNBS colitis is characterized by a Th1-mediated immune response with intense infiltration of lymphocytes and macrophages (10.Strober W. Fuss I.J. Blumberg R.S. Annu. Rev. Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1154) Google Scholar). Therefore, we evaluated the infiltrating cell profile in inflamed colonic tissue. The number of macrophages and T-cells significantly increased in the colonic lamina propria of all TNBS-treated animals, when compared with that of the control group (Fig. 2). Subcutaneous administration of mammalian or ascidian GAGs reduced the number of both macrophages (Fig. 2) and T-cells (Fig. 3) in TNBS-treated animals. Heparin administration, regardless of its source, was more effective in reducing infiltrating cells, when compared with DS (Figs. 2 and 3). However, ascidian heparin was more efficient in reducing macrophages than the mammalian counterpart (Fig. 2). Mammalian or ascidian DS reduced macrophages and CD4+ T-cells at the same extent (Figs. 2 and 3).FIGURE 3Effect of heparin analogues on lymphocyte infiltration into the inflamed colon. Colonic samples from Wistar rats after TNBS-induced colitis without or with heparin or ascidian heparin analogues administration were immediately embedded in Tissue-Tek O.C.T. compound, snap-frozen in isopentane in a liquid nitrogen bath, and submitted to immunohistochemical analysis using mouse monoclonal anti-rat CD3. Heparin and heparin analogues were administered to the animals as described in the legend of Fig. 1. The number of immunoreactive cells per millimeter-squared was counted in at least 10 different areas. Quantitative analysis of tissue sections were carried out under light microscopy at ×400 magnification. Values are mean ± S.E. of 10 animals/group. Statistical differences among the experimental groups were evaluated with the one-way ANOVA test. The level of significance was set at p < 0.05. Mam, mammalian. Scale bar, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cytokine Production and Intracellular Signaling in the Colonic Tissue—In IBD, increasing TNF-α production is associated with immunologically mediated tissue damage (30.Schreiber S. Nikolaus S. Hampe J. Hamling J. Koop I. Groessner B. Lochs H. Raedler A. Lancet. 1999; 353: 459-461Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and induces the activation of the NF-κB pathway in a variety of cell types. Therefore, we investigated whether the administration of mammalian or ascidian GAG could reduce TNF-α production and NF-κB activation in the inflamed colon. TNBS treatment induced a ∼4-fold increase in the levels of TNF-α in the colonic tissue (Fig. 4A). The increase of the cytokine was accompanied by a clear increase in NF-κB activation (Fig. 5). Subcutaneous administration of GAGs, regardless of its source, drastically reduced TNF-α production to values observed in the basal level (p < 0.001) (Fig. 4A), and no significant difference was observed on the effect of DS or heparin. Similarly, NF-κB activation drastically reduced in inflamed colon after GAG administration (Fig. 5). No significant difference was observed in the effect of mammalian or ascidian DS and heparin. However, ascidian heparin induced the highest reduction in NF-κB activation.FIGURE 5Effect of heparin analogues on NF-κB activation in the inflamed colon. Colonic samples from Wistar rats after TNBS-induced colitis without or with heparin or ascidian heparin analogues administration were immediately embedded in Tissue-Tek O.C.T. compound, snap-frozen, and submitted to immunohistochemical analysis using mouse monoclonal anti-rat p65. Heparin and heparin analogues were administered to the animals as described in the legend of Fig. 1. Quantitative analysis of tissue sections were carried out under light microscopy at ×800 magnification. The number of cells with nuclear NF-κB-staining (NF-κB-positive cells) per millimeter-squared was counted in at least 10 different areas. Values are mean ± S.E. of 10 animals/group. Statistical differences among the experimental groups were evaluated with the one-way ANOVA test. The level of significance was set at p < 0.05. Mam, mammalian. Scale bar, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) T
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