S1293 Is There a Role for Adipocytokines On the Effect of Anti-TNF Treatment in An Experimental...
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cells protects these cells from oxidative stress. Increased production of free radicals and/or impaired antioxidant defenses have been demonstrated in both animal models and human inflammatory bowel disease. This study investigated the effect of intestinal epithelial cell- specific PHB overexpression on dextran sodium sulfate (DSS)-induced colitis using PHB transgenic mice. METHODS: The 12.4-kb villin promoter was spliced upstream of the PHB cDNA to generate transgenic villin-PHB mice. Intestinal mucosal expression of PHB mRNA was elevated 800-fold and protein was elevated 2.8-fold in these mice. 6-week-old PHB transgenic mice and their non-transgenic littermates (WT) were administered 3.0% DSS in their drinking water for 7 days to induce colitis. Colitis was determined using established clinical (weight loss, diarrhea and fecal blood; maximal score 11) and histological (crypt damage, ulcerations and inflammatory infiltration; maximal score 12) scoring. RESULTS: Following DSS administration, WT mice developed colitis as evidenced by weight loss, fecal blood and diarrhea. PHB transgenic mice were protected from DSS-induced weight loss and had significantly less fecal blood and diarrhea (clinical score: WT DSS: 9.2 ± 0.7 vs. TG DSS: 6.4 ± 0.5) as well as MPO levels. Clinical data was reflected by histological measurement of intestinal inflammation (WT DSS: 7.8 ± 0.9 vs. TG DSS: 5.3 ± 0.6). CONCLUSIONS: Our results demonstrate that restoration of PHB levels in intestinal epithelial cells ameliorates colitis in mice. PHB may represent a novel therapeutic target in inflammatory bowel disease. S1289 Treatment of Induced Colitis in Mice By the RAS Antagonist Farnesylthiosalicylic Acid (FTS) Shimon Reif Background and aims: Ras proteins have been shown to regulate cell growth, proliferation,dif- ferentiation and apoptosis. Targeting the Ras family of monomeric Guanosine-Tri-Phosphate (GTP)-ases has been suggested as a therapeutic strategy in proliferate and inflammatory diseases. Farnesythiosalicilic acid (FTS) is a synthetic Ras antagonist that inhibits the binding of Ras to discrete membrane sites, thereby down-regulating several Ras-dependent signaling functions and accelerating Ras degradation. This study examines the role of Ras in the inflammatory process of colitis, and examines whether the Ras antagonist FTS can prevent it. Methods: Colitis was induced in 26 Balb/c, 8-10 weeks old, female mice by adding 5% Dextran sodium sulfate (DSS) to their drinking water and allowing them to drink ad libitum for 7 days. Twelve mice were treated with FTS (5mg/kg) 3 times a week, and 14 mice were treated with 0.9% normal saline 3 times a week. After 7 days the mice were sacrificed and the colon was isolated for further evaluation. Colonic damage was assessed clinically by using a disease activity score which combines weight loss and rectal bleeding according to a standard scoring system, and histologically by evaluating colonic segments stained with Haemotoxylin and Eosin. Mucosal Myeloperoxidase activity, Tumor Necrosis Factor-α (TNF- α) and Interleukin- 1β (IL=1β) levels were measured in order to determine the inflammatory response. The expression of Ras and Ras downstream effectors such as P-ERK was determined by immunobloting assays. Results: Mice treated with FTS had a significant lower disease activity score (P=0.0001), and a lower histopathologic score (NS). A significant reduction was found in the inflammatory response in the FTS treated mice expressed by Myeloperoxid- ase activity (P=0.007), The levels of TNF-α (P=0.04) and the levels of interleukin- 1 Beta (P=0.01).The expression of activated Ras was found to be lower in the group treated with FTS (P=0.004), opposing to the expression of P-ERK which was found to be higher in that group (P=0.003). Conclusions: These results indicate that Ras inhibition significantly ameliorates the severity of experimental colitis, and may offer a new therapeutic approach. S1290 NF-κB-Dependent Gene Transcription in Response to Lipopolysaccharide Is Delayed and Prolonged in the Neonatal Intestine Runlan Tian, Shirley X. Liu, Isabelle G. De Plaen Premature infants are prone to necrotizing enterocolitis (NEC), a disease characterized by inflammation and necrosis of the small intestine. We previously showed that the transcription factor nuclear factor-κB (NF-κB) is activated and mediates the bowel injury in a neonatal rat model of NEC. However, the developmental differences in NF-κB dependent gene expression in the immature vs. mature intestine have not been well characterized. In this study, we first examined the gene expression of pro-inflammatory cytokines, CXCL2 (mac- rophage inhibitory protein-2, MIP-2) and TNF, 2 NF-κB target genes, in response to lipopoly- saccharide (LPS), in the intestines of neonatal rats and compare to those of adults. We then compared the time-course of NF-κB dependent protein production in neonatal vs. adult intestine, by determining LPS-induced luciferase production in transgenic mice expressing a GFP-luciferase construct under the control of NF-κB (NGL mice). 24 hour-old neonatal rats were injected i.p. with LPS (10 mg/kg). At 30 min., 1h, 2h, 4h, 8h, they were sacrificed and their intestine removed. Total RNA was extracted and measured by real-time PCR. In a second experiment, 24-hour-old and adult NGL mice were injected i.p. with LPS (10 mg/ kg) for different time periods, then sacrificed and their intestine processed for luciferase assay. In adult intestine CXCL2 and TNF gene expression increased from the baseline at 30 minutes after LPS, peaked at 1 hour (58±23-fold and 427±159-fold at 30 min. and 1 hour respectively for CXCL2 and 7.4±3-fold and 31±14-fold for TNF), and decreased to baseline levels at 2 h. In contrast, in neonatal intestine, CXCL2 and TNF gene expression remained at baseline levels before 1 h (30 min: p<0.05; 1 hour: p<0.01), and peaked at 2 hours (40±14-fold and 17±13-fold respectively). Similarly, in NGL reporter mice, luciferase activity increased within an hour in adult intestine, peaking at 4 hours (4.27±0.38-fold increase), while it remained at baseline levels until 2 hours in the neonatal intestine, and peaked at 12 hours (39.31±10-fold increase). In conclusion, we found developmental differ- ences in the gene expression of 2 NF-κB target genes TNF and CXCL2, as well as NF-κB dependent luciferase. NF-κB-dependent gene transcription was delayed and prolonged in response to LPS in the intestine of newborn mice, compared to those of adults. How these developmental differences might contribute to NEC awaits further investigation. A-219 AGA Abstracts S1291 Roles of Endogenous Prostaglandins/EP4 Receptors in Development and Healing of Indomethacin-Induced Small Intestinal Lesions in Rats: Relation to MUC, iNOS and VEGF Expressions Mayu Tanigami, Michitaka Ogura, Kazuhiko Nagao, Akimu Ochi, Kikuko Amagase, Koji Takeuchi Background/Aim: Endogenous prostaglandins (PGs), especially PGE2, play a role in the mucosal protection against NSAID-induced small intestinal lesions and their healing response. These effects of PGE2 are reportedly mediated by the activation of EP4 receptors, yet the functional mechanisms related to these effects remain unexplored. We investigated using the selective EP4 agonist and antagonist how endogenous PGs affect the development of NSAID-induced intestinal lesions and their healing response, in relation to the functional mechanisms mediated by EP4 receptors. Methods: Male SD without fasting were given indomethacin (10 mg/kg) SC, and killed 3, 6, 24 h and 7 days later. To examine the protective effect the EP4 agonist (0.03-10 µg/kg) was given PO 30 min before and 6 hr after indomethacin, while to examine the healing effect the EP4 agonist was given PO twice daily for 6 days after induction of lesions. Likewise, indomethacin (2 mg/kg, PO) or EP4 antagonist (3 mg/kg, IP) was given twice daily for 6 days after ulceration. Expression of Muc 1~3 and NOS mRNAs or VEGF protein was examined by RT-PCR or Western blot, respectively. Angiogenesis was examined by immunohistochemical staining. Results: Indo- methacin (10 mg/kg) produced lesions in the small intestine, accompanied with the down- regulation of Muc 1 and 2 expressions and the up-regulation of iNOS expression as well as enterobacterial invasion. These lesions were dose-dependently prevented by pretreatment with the EP4 agonist, together with suppression of the changes in Muc and iNOS expressions and bacterial invasion. On the other hand, the healing of these lesions was delayed by the repeated treatment of indomethacin (2 mg/kg). The healing impairment effect of indometh- acin was mimicked by EP4 antagonist given for 6 days and reversed by the co-administration of EP4 agonist. The expression of VEGF was up-regulated in the intestinal mucosa after ulceration. Both indomethacin and EP4 antagonist decreased VEGF expression and the number of microvessels in the mucosa, and these effects were reversed by the co-treatment with EP4 agonist. Conclusion: These results confirmed the importance of endogenous PGs/ EP4 receptors in the mucosal protective and healing responses in the small intestine and further suggested that the protective effect was functionally associated with the increase of both Muc 1 and 2 expressions as well as the decrease of iNOS expression, the events relating to increase of mucus production and suppression of bacterial invasion, respectively, while the healing promoting effect is associated with stimulation of angiogenic response through the up-regulation of VEGF expression. S1292 MMP9 Mediated Tissue Injury Overrides the Protective Effect of MMP2 On the Development of Colitis Pallavi Garg, Matam Vijay-Kumar, Lixin Wang, Anupama Ravi, Andrew T. Gewirtz, Didier Merlin, Shanthi V. Sitaraman Introduction: Matrix metalloproteinase (MMP) gene family plays important role in pathogen- esis of inflammatory bowel disease (IBD). We have demonstrated that two known gelatinases, MMP2 and MMP9, are both upregulated during human, as well as animal models of IBD. We have shown that epithelial-derived MMP9 is an important mediator of colitis and targeted deletion of it attenuates colitis suggesting that pharamacologic inhibition of MMP9 might have therapeutic value in IBD. However, agents designed to inhibit MMP9 also inhibit the structurally-similar gelatinase MMP2, which protects against tissue damage during colonic inflammation and maintains gut barrier function. Thus, therapeutic strategies targeting MMP9 would also likely to inhibit MMP2 activity, which might negate the beneficial effect of MMP9 inhibition. Thus, to gain insight into what might be the outcome of inhibiting both MMP9 and MMP2, we examined the mice lacking both MMP9 and MMP2 in murine models of colitis. Methods: 8 weeks old MMP9-/-/MMP2-/- double knock out mice and age- and sex- matched wild type (WT) mice of C57/B6 background were used for the study. Colitis was induced by oral administration of 3% dextran sodium sulfate (DSS) in drinking water. Inflammation was quantitated by clinical (weight loss, blood in stool and diarrhea-total score 12) score, histological (inflammatory infiltrate, ulceration and crypt damage-total score 11) score and myeloperoxidase (MPO) activity. Another set of mice were administered Salmonella typhimurium SL 3201 by gavage (25 X 104 cfu/mice) with or without pretreatment with streptomycin (n=6 per group). Results: We observed that MMP9 and MMP2 activity was highly upregulated in WT mice treated with DSS or ST compared to WT water using zymography (4+2.4 fold). Interestingly, MMP-9-/-/MMP-2-/- double knockout mice were resistant to the development of colitis induced by DSS or ST compared to WT. This was reflected by rapid weight loss, diarrhea and rectal bleeding in WT mice compared to MMP9-/-/MMP2-/- double knockout mice (Clinical score: MMP9-/-/MMP2-/-: 4.6+1.17, WT: 9.67+0.21). Histological assessment showed massive inflammation and extensive tissue damage among WT mice compared to MMP9-/-/MMP-2-/- double knockout mice (Histolog- ical score: MMP9-/-/MMP2-/- 4+1 WT: 7.34+0.34). MPO indicated decreased infiltration of neutrophils among MMP9-/-/MMP2-/- double knockout mice compared to WT mice. Conclusions: These results suggest an overriding role of MMP9 in mediating tissue injury compared to the protective role of MMP2 in development of colitis. Thus, inhibition of MMP9, even if resulting in inhibition of MMP2, may be beneficial in the treatment of colitis. S1293 Is There a Role for Adipocytokines On the Effect of Anti-TNF Treatment in An Experimental Model of Colitis? Suna Yapali, Mustafa Deniz, Fatih Eren, Nese Imeryuz, Cigdem Celikel, Naziye Ozkan, Veysel Tahan, Hulya O. Hamzaoglu, Nurdan Tozun BACKGROUND:Recently adipocytokines released from mesenteric fat tissue have been found to affect the course and activity of inflammatory bowel disease (IBD). There is paucity of data on adipocytokines and inflammation. AIM: To investigate the changes in serum and AGA Abstracts
Transcript of S1293 Is There a Role for Adipocytokines On the Effect of Anti-TNF Treatment in An Experimental...
cells protects these cells from oxidative stress. Increased production of free radicals and/orimpaired antioxidant defenses have been demonstrated in both animal models and humaninflammatory bowel disease. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on dextran sodium sulfate (DSS)-induced colitis using PHBtransgenic mice. METHODS: The 12.4-kb villin promoter was spliced upstream of the PHBcDNA to generate transgenic villin-PHB mice. Intestinal mucosal expression of PHB mRNAwas elevated 800-fold and protein was elevated 2.8-fold in these mice. 6-week-old PHBtransgenic mice and their non-transgenic littermates (WT) were administered 3.0% DSS intheir drinking water for 7 days to induce colitis. Colitis was determined using establishedclinical (weight loss, diarrhea and fecal blood; maximal score 11) and histological (cryptdamage, ulcerations and inflammatory infiltration; maximal score 12) scoring. RESULTS:Following DSS administration, WT mice developed colitis as evidenced by weight loss, fecalblood and diarrhea. PHB transgenic mice were protected from DSS-induced weight loss andhad significantly less fecal blood and diarrhea (clinical score: WT DSS: 9.2 ± 0.7 vs. TGDSS: 6.4 ± 0.5) as well as MPO levels. Clinical data was reflected by histological measurementof intestinal inflammation (WT DSS: 7.8 ± 0.9 vs. TG DSS: 5.3 ± 0.6). CONCLUSIONS:Our results demonstrate that restoration of PHB levels in intestinal epithelial cells amelioratescolitis in mice. PHB may represent a novel therapeutic target in inflammatory bowel disease.
S1289
Treatment of Induced Colitis in Mice By the RAS AntagonistFarnesylthiosalicylic Acid (FTS)Shimon Reif
Background and aims: Ras proteins have been shown to regulate cell growth, proliferation,dif-ferentiation and apoptosis. Targeting the Ras family of monomeric Guanosine-Tri-Phosphate(GTP)-ases has been suggested as a therapeutic strategy in proliferate and inflammatorydiseases. Farnesythiosalicilic acid (FTS) is a synthetic Ras antagonist that inhibits the bindingof Ras to discrete membrane sites, thereby down-regulating several Ras-dependent signalingfunctions and accelerating Ras degradation. This study examines the role of Ras in theinflammatory process of colitis, and examines whether the Ras antagonist FTS can preventit. Methods: Colitis was induced in 26 Balb/c, 8-10 weeks old, female mice by adding 5%Dextran sodium sulfate (DSS) to their drinking water and allowing them to drink ad libitumfor 7 days. Twelve mice were treated with FTS (5mg/kg) 3 times a week, and 14 mice weretreated with 0.9% normal saline 3 times a week. After 7 days the mice were sacrificed andthe colon was isolated for further evaluation. Colonic damage was assessed clinically byusing a disease activity score which combines weight loss and rectal bleeding according toa standard scoring system, and histologically by evaluating colonic segments stained withHaemotoxylin and Eosin. Mucosal Myeloperoxidase activity, Tumor Necrosis Factor-α (TNF-α) and Interleukin- 1β (IL=1β) levels were measured in order to determine the inflammatoryresponse. The expression of Ras and Ras downstream effectors such as P-ERK was determinedby immunobloting assays. Results: Mice treated with FTS had a significant lower diseaseactivity score (P=0.0001), and a lower histopathologic score (NS). A significant reductionwas found in the inflammatory response in the FTS treated mice expressed by Myeloperoxid-ase activity (P=0.007), The levels of TNF-α (P=0.04) and the levels of interleukin- 1 Beta(P=0.01).The expression of activated Ras was found to be lower in the group treated withFTS (P=0.004), opposing to the expression of P-ERK which was found to be higher inthat group (P=0.003). Conclusions: These results indicate that Ras inhibition significantlyameliorates the severity of experimental colitis, and may offer a new therapeutic approach.
S1290
NF-κB-Dependent Gene Transcription in Response to Lipopolysaccharide IsDelayed and Prolonged in the Neonatal IntestineRunlan Tian, Shirley X. Liu, Isabelle G. De Plaen
Premature infants are prone to necrotizing enterocolitis (NEC), a disease characterized byinflammation and necrosis of the small intestine. We previously showed that the transcriptionfactor nuclear factor-κB (NF-κB) is activated and mediates the bowel injury in a neonatalrat model of NEC. However, the developmental differences in NF-κB dependent geneexpression in the immature vs. mature intestine have not been well characterized. In thisstudy, we first examined the gene expression of pro-inflammatory cytokines, CXCL2 (mac-rophage inhibitory protein-2, MIP-2) and TNF, 2 NF-κB target genes, in response to lipopoly-saccharide (LPS), in the intestines of neonatal rats and compare to those of adults. We thencompared the time-course of NF-κB dependent protein production in neonatal vs. adultintestine, by determining LPS-induced luciferase production in transgenic mice expressinga GFP-luciferase construct under the control of NF-κB (NGL mice). 24 hour-old neonatalrats were injected i.p. with LPS (10 mg/kg). At 30 min., 1h, 2h, 4h, 8h, they were sacrificedand their intestine removed. Total RNA was extracted and measured by real-time PCR. Ina second experiment, 24-hour-old and adult NGL mice were injected i.p. with LPS (10 mg/kg) for different time periods, then sacrificed and their intestine processed for luciferaseassay. In adult intestine CXCL2 and TNF gene expression increased from the baseline at30 minutes after LPS, peaked at 1 hour (58±23-fold and 427±159-fold at 30 min. and 1hour respectively for CXCL2 and 7.4±3-fold and 31±14-fold for TNF), and decreased tobaseline levels at 2 h. In contrast, in neonatal intestine, CXCL2 and TNF gene expressionremained at baseline levels before 1 h (30 min: p<0.05; 1 hour: p<0.01), and peaked at 2hours (40±14-fold and 17±13-fold respectively). Similarly, in NGL reporter mice, luciferaseactivity increased within an hour in adult intestine, peaking at 4 hours (4.27±0.38-foldincrease), while it remained at baseline levels until 2 hours in the neonatal intestine, andpeaked at 12 hours (39.31±10-fold increase). In conclusion, we found developmental differ-ences in the gene expression of 2 NF-κB target genes TNF and CXCL2, as well as NF-κBdependent luciferase. NF-κB-dependent gene transcription was delayed and prolonged inresponse to LPS in the intestine of newborn mice, compared to those of adults. How thesedevelopmental differences might contribute to NEC awaits further investigation.
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S1291
Roles of Endogenous Prostaglandins/EP4 Receptors in Development andHealing of Indomethacin-Induced Small Intestinal Lesions in Rats: Relation toMUC, iNOS and VEGF ExpressionsMayu Tanigami, Michitaka Ogura, Kazuhiko Nagao, Akimu Ochi, Kikuko Amagase, KojiTakeuchi
Background/Aim: Endogenous prostaglandins (PGs), especially PGE2, play a role in themucosal protection against NSAID-induced small intestinal lesions and their healing response.These effects of PGE2 are reportedly mediated by the activation of EP4 receptors, yet thefunctional mechanisms related to these effects remain unexplored. We investigated usingthe selective EP4 agonist and antagonist how endogenous PGs affect the development ofNSAID-induced intestinal lesions and their healing response, in relation to the functionalmechanisms mediated by EP4 receptors. Methods: Male SD without fasting were givenindomethacin (10 mg/kg) SC, and killed 3, 6, 24 h and 7 days later. To examine theprotective effect the EP4 agonist (0.03-10 µg/kg) was given PO 30 min before and 6 hrafter indomethacin, while to examine the healing effect the EP4 agonist was given PO twicedaily for 6 days after induction of lesions. Likewise, indomethacin (2 mg/kg, PO) or EP4antagonist (3 mg/kg, IP) was given twice daily for 6 days after ulceration. Expression ofMuc 1~3 and NOS mRNAs or VEGF protein was examined by RT-PCR or Western blot,respectively. Angiogenesis was examined by immunohistochemical staining. Results: Indo-methacin (10 mg/kg) produced lesions in the small intestine, accompanied with the down-regulation of Muc 1 and 2 expressions and the up-regulation of iNOS expression as wellas enterobacterial invasion. These lesions were dose-dependently prevented by pretreatmentwith the EP4 agonist, together with suppression of the changes in Muc and iNOS expressionsand bacterial invasion. On the other hand, the healing of these lesions was delayed by therepeated treatment of indomethacin (2 mg/kg). The healing impairment effect of indometh-acin was mimicked by EP4 antagonist given for 6 days and reversed by the co-administrationof EP4 agonist. The expression of VEGF was up-regulated in the intestinal mucosa afterulceration. Both indomethacin and EP4 antagonist decreased VEGF expression and thenumber of microvessels in the mucosa, and these effects were reversed by the co-treatmentwith EP4 agonist. Conclusion: These results confirmed the importance of endogenous PGs/EP4 receptors in the mucosal protective and healing responses in the small intestine andfurther suggested that the protective effect was functionally associated with the increase ofboth Muc 1 and 2 expressions as well as the decrease of iNOS expression, the events relatingto increase of mucus production and suppression of bacterial invasion, respectively, whilethe healing promoting effect is associated with stimulation of angiogenic response throughthe up-regulation of VEGF expression.
S1292
MMP9 Mediated Tissue Injury Overrides the Protective Effect of MMP2 Onthe Development of ColitisPallavi Garg, Matam Vijay-Kumar, Lixin Wang, Anupama Ravi, Andrew T. Gewirtz, DidierMerlin, Shanthi V. Sitaraman
Introduction: Matrix metalloproteinase (MMP) gene family plays important role in pathogen-esis of inflammatory bowel disease (IBD). We have demonstrated that two known gelatinases,MMP2 and MMP9, are both upregulated during human, as well as animal models of IBD.We have shown that epithelial-derived MMP9 is an important mediator of colitis and targeteddeletion of it attenuates colitis suggesting that pharamacologic inhibition of MMP9 mighthave therapeutic value in IBD. However, agents designed to inhibit MMP9 also inhibit thestructurally-similar gelatinase MMP2, which protects against tissue damage during colonicinflammation and maintains gut barrier function. Thus, therapeutic strategies targeting MMP9would also likely to inhibit MMP2 activity, which might negate the beneficial effect of MMP9inhibition. Thus, to gain insight into what might be the outcome of inhibiting both MMP9and MMP2, we examined the mice lacking both MMP9 and MMP2 in murine models ofcolitis. Methods: 8 weeks old MMP9-/-/MMP2-/- double knock out mice and age- and sex-matched wild type (WT) mice of C57/B6 background were used for the study. Colitis wasinduced by oral administration of 3% dextran sodium sulfate (DSS) in drinking water.Inflammation was quantitated by clinical (weight loss, blood in stool and diarrhea-totalscore 12) score, histological (inflammatory infiltrate, ulceration and crypt damage-total score11) score and myeloperoxidase (MPO) activity. Another set of mice were administeredSalmonella typhimurium SL 3201 by gavage (25 X 104 cfu/mice) with or without pretreatmentwith streptomycin (n=6 per group). Results: We observed that MMP9 and MMP2 activitywas highly upregulated in WT mice treated with DSS or ST compared to WT water usingzymography (4+2.4 fold). Interestingly, MMP-9-/-/MMP-2-/- double knockout mice wereresistant to the development of colitis induced by DSS or ST compared to WT. This wasreflected by rapid weight loss, diarrhea and rectal bleeding in WT mice compared toMMP9-/-/MMP2-/- double knockout mice (Clinical score: MMP9-/-/MMP2-/-: 4.6+1.17, WT:9.67+0.21). Histological assessment showed massive inflammation and extensive tissuedamage among WT mice compared to MMP9-/-/MMP-2-/- double knockout mice (Histolog-ical score: MMP9-/-/MMP2-/- 4+1 WT: 7.34+0.34). MPO indicated decreased infiltrationof neutrophils among MMP9-/-/MMP2-/- double knockout mice compared to WT mice.Conclusions: These results suggest an overriding role of MMP9 in mediating tissue injurycompared to the protective role of MMP2 in development of colitis. Thus, inhibition ofMMP9, even if resulting in inhibition of MMP2, may be beneficial in the treatment of colitis.
S1293
Is There a Role for Adipocytokines On the Effect of Anti-TNF Treatment in AnExperimental Model of Colitis?Suna Yapali, Mustafa Deniz, Fatih Eren, Nese Imeryuz, Cigdem Celikel, Naziye Ozkan,Veysel Tahan, Hulya O. Hamzaoglu, Nurdan Tozun
BACKGROUND:Recently adipocytokines released from mesenteric fat tissue have been foundto affect the course and activity of inflammatory bowel disease (IBD). There is paucity ofdata on adipocytokines and inflammation. AIM: To investigate the changes in serum and
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stissue concentrations of adipocyte hormones and ghrelin in trinitrobenzene sulphonic acid(TNBS) induced colitis before and after anti TNF treatment in rats. METHODS: Forty SpragueDawley rats were divided into 1. Control, 2. Infliximab (IFX), 3.TNBS, 4.TNBS&IFX groups.Colitis was induced by intrarectal TNBS in groups 3&4 and physiological saline (PS) ingroups 1&2. Infliximab (IFX) 5 mg/kg iv or PS infusion were given in the groups 2&4 andin the groups 1&3 respectively, on day 3 via jugular vein. Rats were decapitated on day 6.Blood, mesenteric fat and colonic samples were obtained. Leptin, adiponectin, resistin,ghrelin, IL-6, TNF-α were determined in serum by ELISA, and in tissue by immunohisto-chemistry semiquantitatively. Macroscopic and histological inflammation was scored. Myelo-peroxidase (MPO), malonyldialdehyde (MDA) and glutathione (GSH) levels were determinedin tissue homogenates. RESULTS: TNBS led to colitis which was ameliorated by IFX macro-scopically and histopathologically. MPO and MDA were increased in the colon and mesentericfat in colitis whereas GSH was decreased. IFX reverted effects of TNBS on MPO and MDA,increased GSH levels. There were no changes in serum levels of adipocytokines exceptadiponectin in the TNBS group. The effects of TNBS and IFX on tissue cytokine immunoex-pressions were expressed as median (range) and depicted in the table. CONCLUSION:Tissue expression of adipocytokines reflects inflammatory activity more accurately thanserum level does. Decreased adiponectin, resistin and ghrelin levels in the mesenteric tissuein the TNBS group may support their anti-inflammatory role. Colonic expressions were inparallel with that of the mesentery except resistin. Paradoxical expression of resistin in thecolon and mesentery may mandate to investigate if resistin has at least dual actions in thosetissues not necessarily related to the inflammation. The mechanisms of action of anti-TNFagent to alleviate the inflammation and to reduce oxidative stress do not involve adipocytok-ines in this experimental setting.
S1294
Role of Corticotropin-Releasing Factors in Pathogenesis of NSAID-InducedSmall Intestinal Lesions in RatsYoshikazu Kubo, Michitaka Ogura, Aiko Kumano, Shinichi Kato, Koji Takeuchi
Background/Aim: Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide, is theprincipal regulator of the hypothalamus-pituitary-adrenal (HPA) axis. Recent studies showedthat CRF and CRF-related peptides affect the mucosal defense of the gastrointestinal tractthrough modulation of various functions under pathophysiological conditions. However,the roles CRF plays in the ulcerogenic response to NSAIDs in the small intestine remainunexplored. We examined using selective CRF receptor (CRFR) agonist and antagonistswhether CRF plays a role in the pathogenesis of indomethacin- induced intestinal lesionsin rats. Methods: Male SD rats were used. Intestinal lesions were induced by a single injectionof indomethacin (10 mg/kg, SC), and the animals were killed 24 h later. Urocortin [CRF1Rand CRF2R agonist: 10-20 µg/kg] was given IV 10 min before indomethacin. Astressin (anonselective CRFR antagonist: 50 µg/kg), NBI-27914 (a CRF1R antagonist: 200 µg/kg) orastressin-2B (a CRF2R antagonist: 60 µg/kg) was given IV 10 min before urocortin orindomethacin. Intestinal motility was measured by a balloon method. The mucosal MPOactivity was determained by o-dianisidine method. Results: Indomethacin provoked multiplehemorrhagic lesions in the small intestine within 24 h, in accompany with a marked increaseof MPO activity. Pretreatment of the animals with astressin, a nonselective CRFR antagonist,aggravated these lesions in a dose-dependent manner. Likewise, a CRF2R antagonist astressin-2B also exacerbated the intestinal ulcerogenic response induced by indomethacin, but aCRF1R antagonist NBI-27914 had no effect. On the other hand, urocortin dose- dependentlyprevented the development of indomethacin-induced intestinal lesions, together with sup-pression of MPO activity, and these effects were significantly reversed by the prior administra-tion of astressin-2B but not NBI-27914. Indomethacin at the ulcerogenic dose markedlyenhanced the intestinal motility. Urocortin suppressed the intestinal hypermotility responseto indomethacin, and this effect was abrogated by astressin-2B but not NBI-27914. Neitherastressin-2B nor NBI-27914 alone had any effect on the intestinal hypermotility response.Conclusion: These results suggest that CRF-related peptides afford a protective influenceon the development of indomethacin- induced small intestinal lesions, and this action ismediated by the activation of CRF2R and functionally associated with suppression of intestinalhypermotility caused by indomethacin. It is assumed that endogenous CRF plays a role inmaintenance of the intestinal mucosal integrity under adverse conditions such as administra-tion of NSAIDs.
S1295
Detrimental Role of Endothelial Nitric Oxide Synthase (eNOS/NNOS3) inAggravation of Indomethacin-Induced Gastric Damage in Adjuvant ArthritisRatsShinichi Kato, Yasuyuki Ito, Fumikazu Ohkawa, Kikuko Amagase, Koji Takeuchi
Background/Aim: We previously reported that indomethacin-induced gastric damage wasmarkedly aggravated in adjuvant arthritic rats. This phenomenon is accounted for by theoverproduction of nitric oxide (NO) derived from inducible NO synthase (iNOS). We furtherobserved that the preventive effect of aminoguanidine, a relatively selective inhibitor ofiNOS, on the aggravation of these lesions in arthritic rats was significantly less than that ofNG-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS. It is therefore
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possible that NO derived from eNOS, in addition to iNOS/NO, also negatively affects theulcerogenic response to indomethacin in arthritic rats. In the present study, we investigatedthe role of eNOS/NO in the pathogenic mechanism for aggravation of these lesions in arthriticrats. Methods: Arthritis was induced in male Dark Agouti rats by injection of Freund'scomplete adjuvant into the right hind paw. Two weeks later, the animals were givenindomethacin (30 mg/kg, PO), and the stomach was examined for lesions 4 h later. Theexpression of NOS isozymes such as neuronal NOS (nNOS), iNOS and eNOS was determinedby RT-PCR and Western blotting. Immunohistochemical study for eNOS, iNOS and CD31,the marker of endothelial cells, were also examined. Results: Oral administration of indometh-acin produced hemorrhagic damage in the gastric mucosa. The severity of damage wasmarkedly aggravated in arthritic rats in comparison with normal rats. Pretreatment with L-NAME (3-30 mg/kg, SC) did not affect the ulcerogenic response to indomethacin in normalrats but dose-dependently prevented the aggravation of these lesions; at 30 mg/kg the damagescore in arthritic rats was almost equivalent to that in normal rats, Likewise, pretreatmentwith aminoguanidine (10-50 mg/kg, SC) or 1400W (10 mg/kg, SC), a selective inhibitor ofiNOS, also significantly prevented the aggravation of damage in arthritic rats, yet the inhibitoryeffects of both these agents were significantly less than that of L-NAME, the lesion scoreobserved when given aminoguanidine or 1400W at maximal doses in arthritic rats beingstill significantly higher than that in normal rats. The gene and protein expressions of eNOSand iNOS but not nNOS clearly enhanced in the gastric mucosa of arthritic rats whencompared with normal rats. Immunohistochemical study further showed the increasedexpression of eNOS in the gastric mucosa, and that was also positive for CD31. Conclusion:These findings suggest that the increased gastric ulcerogenic response to indomethacin inarthritic rats may be mediated by NO derived from eNOS in addition to iNOS.
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A2b Adenosine Receptor Gene Deletion or Antagonism Attenuates MurineColitisVasantha L. Kolachala, Matam Vijay-Kumar, Lixin Wang, Dan Yang, Melissa Marshall,Guoquan Wang, Joel Linden, Andrew T. Gewirtz, Didier Merlin, Katya Ravid, Shanthi V.Sitaraman
Background and significance: The A2b adenosine receptor (A2bR) is the predominant adenos-ine receptor expressed in the colonic epithelia. We have previously shown that A2bR mRNAand protein levels are upregulated during murine and human colitis. In this study weaddressed the role of the A2bR in the development of murine colitis and potential mechanismunderlying its effects. Methods: Dextran sodium sulfate (DSS)- and Salmonella typhimurium(S.T.) were used to induce colitis in A2bR null mice (A2bR-/-) on a C57BL-6 backgroundand their wild type (WT) littermates. Colitis was determined using established clinical(weight loss, diarrhea and blood in stool) and histological (crypt damage, ulcerations andinflammatory infiltration) scoring. In some experiments ATL801 (10 mg/Kg in the diet), ahighly selective inhibitor of A2bR, was used to antagonize the effects of A2bR in WT mice.IL-8 measurements were performed using ELISA. Short circuit current (Isc) was measuredin T84 cells or colonic mucosal explants using Ussing chamber. Results: WT mice givenDSS or S.T. exhibited robust weight loss, diarrhea and rectal bleeding (DSS: clinical score:9.4±0.7, (maximal score 12); histological score: 8.1±0.5 (maximal score 11); S.T. histologicalscore: 10.4±0.4). In contrast, A2bR-/- mice were protected from DSS- and S.T.-inducedcolitis (DSS: clinical score 3.2±1.1; Histological score: 4.0±1.2; S.T. histological score:3.5±1.2). Mice fed with A2bR antagonist, ATL801 also showed significantly reduced extentand severity of colitis in DSS-treated mice (clinical score: 4.4 ± 1.5, histological score: 4.1± 1.8). Flagellin-induced IL-8 levels were attenuated in A2bR-/- mice (WT:153.3±22.4;A2bR-/-: 60.0 ±23.3: ug/ml). Similarly, ATL801 inhibited flagellin-induced IL-8 levels, bothIn Vitro in T84 cells (flagellin alone: 1126±88.1; flagellin+ATL801: 769±13.6 ug/ml), as wellas In Vivo in C57BL-6 mice. Intraperitonial administration of IL-8 rescued neutrophil migra-tion in A2bR-/- mice. Isc in response to adenosine was abolished in A2bR-/- mice andATL801 inhibited adenosine-induced Isc, both in T84 cells as well as in colonic mucosalexplants ex vivo. Conclusions: These data demonstrates for the first time, that A2bR antagon-ism or gene deletion protects against intestinal inflammation. A2bR activation may potentiatesinflammation by stimulating pro-inflammatory chemokine and chloride secretion.
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Role of IL-4 and IL-1β On the Pathogenesis of 5-Fluorouracil (5-FU) InducedIntestinal Inflammation in MicePedro Marcos G. Soares, José Maurício S. Mota, Gerly Anne C. Brito, Fernando Q.Cunha, Ronaldo A. Ribeiro, Marcellus H. Souza
BACKGROUND & AIMS: 5-FU induced- intestinal mucositis is characterized by inflammat-ory epithelial ulcerations in the mucosa. Intestinal mucositis is a frequent side-effect associatedto 5-fluorouracil (5-FU) clinical use. Cytokines participation in the 5-FU- induced intestinalmucositis was not completely elucidated. Our objective was to evaluate the role of IL-4 andIL-1β in 5-FU induced- intestinal mucositis in mice. METHODS: Wild-type C57BL/6 miceor IL-4 deficient mice (-/-) were treated with only one dose of 5-FU (450 mg/kg, i.p.). Othergroup was treated with the soluble receptor IL-1Ra (100 mg/kg, i.p., per day) or saline.Then, received the 5-FU (450 mg/Kg, ip). After three days, mice were sacrificed and piecesof duodenum, jejunum and ileum were harvested for assessment of epithelial damage bymorphometry. Duodenum myeloperoxidase (MPO) activity and cytokines (IL-1β, TNF-αand KC) concentrations were also evaluated by ELISA. RESULTS: 5-FU induced a significant(p<0.01) epithelial intestinal damage (reduction of villus height/crypts depht ratio) in allsegments (duodenum, control= 3.3±0.2, 5- FU= 1.0±0.1; jejunum, control= 2.3±0.2, 5-FU=1.0±0.1; ileum, control= 2.1±0.1, 5-FU= 0.7±0.1), increased MPO activity (number ofneutrophil/mg of tissue) (control= 663.5±76.6, 5-FU= 3579.0±492.0) and cytokines concen-tration (pg/ml) (IL-1β, control= 134.8±31.9, 5-FU= 280.3±41.5; KC, control= 123.9±27.3,5-FU= 177.2±28.2; TNF-α, control= 9.9±0.4, 5-FU= 12.1±1.8). IL4 -/- presented with lessepithelial intestinal damage (duodenum= 2.9±0.2, jejunum= 2.0±0.2, ileum= 1.4±0.1), lessMPO activity (1702±315) and cytokines concentration (IL-1β= 86.5± 29.6; KC= 105.6±21.6,TNF-α= 7.4±0.5). IL-1Ra protected all segments from 5-FU- induced intestinal injury (duo-denum= 1.7±0.1, jejunum= 2.0±0.1, ileum= 1.1±0.1), reduced MPO activity (2061±308) and
