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The effect of tachykinin NK2 receptor blockade on proto-oncogene expression following colorectal distension or experimental colitis
A580 AGA ABSTRACTS
3018 PEROXYNITRITE INDUCED MUCOSAL CELL APOPTOSIS IN HAPTEN·INDUCED COLITIS IN THE RAT. Gang Vue, Pi-Shiang Lai, CongLi Wang, Robert G. Nagele, Frank F. Sun, Patrick Y-K Wong, UMDNJ Sch Osteopath Med, Stratford, NJ. We have demonstrated previously that peroxynitrite is a potent agent inducing apoptosis of human promyelocyte HL-60 cells in vitro. In this work, we investigated the role of peroxynitrite as an important mediator of cell apoptosis in inflamed colon tissue of rat colitis. Rat colon inflammation was induced by intracolonic administration of TNBS. The expression of nitric oxide synthases (cNOS and iNOS) in inflamed lesions was located by immunohistochemistry and measured by enzyme activities. The presence of peroxynitrite attack was detected by immunostaining of its nitrotyrosine product. DNA fragmentation in the apoptotic cells was examined by in situ terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay and gel electrophoresis. A selective iNOS inhibitor L-N 6-[I-iminoethyl] lysine (NIL)was given to colitic animals to suppress the iNOS activity in inflamed lesions. Morphological evidence as well as TUNEL assay showed massive cell apoptosis with intense DNA fragmentation occurred in mucosal epithelial cells and granulocytes of the lamina propria and submucosa. These apoptotic cells displayed strong iNOS and nitrotyrosine immunoreactivities suggesting peroxynitrite assault. The administration of NIL reduced iNOS activity in 24 hour lesions by 84% also significantly attenuated both nitrotyrosine staining and apoptotic cell counts. The data strongly suggest that the elevated iNOS generated peroxynitrite is a major contributor of mucosal cell apoptosis during colon inflammation. 3019 THE EFFECT OF BERBERINE AND GENIPOSIDE ON EXPERI· MENTAL COLITIS. Haiyan Zhou, Satoru Mineshita, Tokyo Med and Dental Univ, Tokyo. Japan. Objectives: We have reported that Oren-gedoku-to (OGT, a complex mixure of ingredients derived from plants) is effective on trinitrobenzene sulfonic acid (TNB) induced colitis in rats. Berberine and geniposide are the main components of OGT which have several pharmacological and biological activities. The aims of this study are to evaluate the effect of berberine and geniposide on the TNB-induced colitis in rats. Methods: Sixty SD male rats were used. Ten of them were used as a blank control. Colitis was induced by intracolonic administration ofTNB/ethanol (Morris et all) in 50 rats. The rats treated with TNB were randomly divided into five groups. Four groups were treated with berberine (7.5, or 15 mg kg") or geniposide (10, or 20 mg kg') orally via a stomach tube once a day for one week. One group was used as a control, and received an equal volume of vehicle. Observation were made on days 3, and 7 after the administration of TNB. A 7-cm segment of the distal colon was removed for functional and morphological studies. The measurements of colonic levels of myeloperoxygenase (MPO) were made by the method of Krawisz et a1 2 • Result: The body weight of rats treated with TNB decreased throughout the 5-day post-treatment period. The groups administrated berberine (7.5, or 15 mg kg') for one week showed quick recovery of their body weight. The macroscopic and histological damage score, colon weight, colonic wall thickness, ulceration diameter were lower or smaller in rats treated with berberine for I week than in the TNB control group. The MPO activity in TNB control group was increased on day 3, and 7 (9.63:t2.53, and 10.50:t6.38 U/100mg of wet tissue) compared with blank control (1.53:t0.45, and 1.90:t0.52 U/lOOmg of wet tissue) (P
GASTROENTEROLOGY Vol. 118, No.4
clinical trials. Preliminary results show that Thalidomide may be an effective adjunctive therapy in these patients. Aim: The aim of our study was to investigate the effects of thalidomide on cytokine profiles in patients with steroid-resistant Crohn's disease. Methods: Secretion of TNFa, IL-1{3 and IL-6 was investigated in short-time cultures of periperal blood monocytes (PBMC) and colonic Lamina propria mononuclear cells (LpMNC) stimulated with LPS or PWM, respectively. Seven patients with steroid resistant chronic active Crohn's disease receiving an average daily dose of 300 mg thalidomide were investigated before and after 12 weeks treatment. Results: Clinically, patients improved with a decrease in COAl from 305 :t 27 to 232 :t 19. This decrease was accompanied with a decrease of TNFa-secretion in short-time cultures of PBMC and LpMNC in 6 of 7 patients, although in one patient secretion of TNFa slightly increased after 12 weeks treatment with thalidomide. TNFa secretion by PBMC: 62.8 :t 14.6 (pg/ml; :tSEM) before treatment, 22.5 :t 9.2 after 12 weeks treatment. TNFa secretion by LpMNC: 42.3 :t 9.8 before treatment, 15.9 :t 7.3 after 12 weeks treatment. Overall, TNFa secretion did change significantly. Secretion ofIL-1{3 by MNC: 387 :t 77 before treatment, 406 :t 63 after 12 weeks. Secretion ofIL-1{3 by LpMNC: 465 :t 74 vs. 417 z; 49. Secretion of IL-6 by PMN: 2193 :t 236 vs. 1961 :t 270, by LpMNC: 2498 :t 286 vs. 2702 :t 376. Secretion of IL-I{3 and IL-6 was not altered significantly. Conclusion: Our data support the hypothesis that the clinical effects of Thalidomide in Crohn's disease are mediated by its inhibitory effects on TNFa. 3021 THE EFFECT OF TACHYKININ NK2 RECEPTOR BLOCKADE ON PROTO·ONCOGENE EXPRESSION FOLLOWING COLO· RECTAL DISTENSION OR EXPERIMENTAL COLITIS. Lori A. Birder, Susanna Kiss, Alessandro Lecci, William C. de Groat, Michelle VanBibber, Carlo A. Maggi, Univ of Pittsburgh, Pittsburgh, PA. Tachykinin NK2 receptors have been implicated in inflammatory responses such as those occurring in inflammatory bowel disease. We investigated the effect of the nonpeptide NK2 antagonist, MEN 11420, on induction of proto-oncogene expression in spinal cord (SC) and dorsal root ganglion (DRG) neurons following noxious and non-noxious stimulation of the colon in anesthetized Sprague Dawley rats. Physiological distension and noxious stimulation were used to activate colon afferents. Colitis was induced with trinitrobenzene sulfonic acid (TNBS)(Wallace et al., 1998). Either I m1 of saline, TNBS or ethanol vehicle was instilled in the colon via a cannula inserted 6 em proximal to the anus. A small number of normal untreated rats received repetitive (5 min on/IO min off) constant pressure inflation (10 mmHg) of a balloon catheter to produce non-noxious colorectal distension (CRD). Half of TNBS-treated rats received repetitive constant high pressure inflation (60 mmHg) to produce noxious CRD. Rats were sacrificed (2-72 hr later) via intracardiac perfusion of fixative and sections of SC and DRG were processed for either Fos or Jun (markers for cellular activation) using specific antisera. In normal untreated rats, following saline instillation or non-noxious CRD, low levels of Fos-immunoreactivity (IR) «15 cells/SC section) or Jun-IR (5% of DRG neurons) were detected. Instillation of ethanol vehicle slightly increased (ave. 10%) the number of Fos/Jun cells. TNBS instillation significantly increased Fos-IR (ave 105 :t II cells/section) in four L6-S I SC regions: medial and lateral dorsal horn, dorsal commissure and the region including the sacral parasympathetic nucleus (SPN). In DRG, TNBS produced a 3 fold increase in Jun-IR whereas distension of sensitized colon produced a 5 fold increase in the number of Fos/Jun-IR cells. MEN 11420 (150 nmol/kg i.v., 30 min prior) had no affect on the number of Fos/Jun neurons induced by nonnoxious CRD. In contrast, MEN 11420 significantly decreased Fos-IR in the dorsal horn (45-75%) and SPN (35-55%) and decreased Jun-IR in DRG by 50-60% after TNBS or distension of the TNBS-sensitized colon. These findings indicate that MEN 11420 does not affect responses of colonic afferent neurons to physiological stimuli, but suppresses the responses of these neurons to nociceptive stimuli. These results suggest that NK2 receptor antagonists may be beneficial in the treatment of colitis. 3022 SHORT·CHAIN FATTY ACID IRRIGATION IN THE TREATMENT OF DIVERSION COLITIS. A PROSPECTIVE DOUBLEBLIND RANDOMIZED STUDY. John W. Briel, David D. Zimmerman, Luuk M. de Boer, Theo H. van der Kwast, Willem R. Schouten, Univ Hosp Digest, Rotterdam, Netherlands. Introduction:Diversion Colitis is found after surgical diversion of the fecal stream. In literature, major randomized studies with prolonged treatment protocols investigating the role of Short-Chain Fatty Acids (SCFA) in the treatment of Diversion Colitis are lacking. Therefore, the current study was conducted. Methods:All patients with persistent or recurrent proctitis (> 3 months) after subtotal colectomy for inflammatory bowel disease were included. Patients were randomized and irrigated their rectum twice daily with 60 ml SCFA or Placebo-solution (P), containing normal saline only. Clinical Signs (CS) were noted. Furthermore, Endoscopic and Histologic
3018 PEROXYNITRITE INDUCED MUCOSAL CELL APOPTOSIS IN HAPTEN·INDUCED COLITIS IN THE RAT. Gang Vue, Pi-Shiang Lai, CongLi Wang, Robert G. Nagele, Frank F. Sun, Patrick Y-K Wong, UMDNJ Sch Osteopath Med, Stratford, NJ. We have demonstrated previously that peroxynitrite is a potent agent inducing apoptosis of human promyelocyte HL-60 cells in vitro. In this work, we investigated the role of peroxynitrite as an important mediator of cell apoptosis in inflamed colon tissue of rat colitis. Rat colon inflammation was induced by intracolonic administration of TNBS. The expression of nitric oxide synthases (cNOS and iNOS) in inflamed lesions was located by immunohistochemistry and measured by enzyme activities. The presence of peroxynitrite attack was detected by immunostaining of its nitrotyrosine product. DNA fragmentation in the apoptotic cells was examined by in situ terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay and gel electrophoresis. A selective iNOS inhibitor L-N 6-[I-iminoethyl] lysine (NIL)was given to colitic animals to suppress the iNOS activity in inflamed lesions. Morphological evidence as well as TUNEL assay showed massive cell apoptosis with intense DNA fragmentation occurred in mucosal epithelial cells and granulocytes of the lamina propria and submucosa. These apoptotic cells displayed strong iNOS and nitrotyrosine immunoreactivities suggesting peroxynitrite assault. The administration of NIL reduced iNOS activity in 24 hour lesions by 84% also significantly attenuated both nitrotyrosine staining and apoptotic cell counts. The data strongly suggest that the elevated iNOS generated peroxynitrite is a major contributor of mucosal cell apoptosis during colon inflammation. 3019 THE EFFECT OF BERBERINE AND GENIPOSIDE ON EXPERI· MENTAL COLITIS. Haiyan Zhou, Satoru Mineshita, Tokyo Med and Dental Univ, Tokyo. Japan. Objectives: We have reported that Oren-gedoku-to (OGT, a complex mixure of ingredients derived from plants) is effective on trinitrobenzene sulfonic acid (TNB) induced colitis in rats. Berberine and geniposide are the main components of OGT which have several pharmacological and biological activities. The aims of this study are to evaluate the effect of berberine and geniposide on the TNB-induced colitis in rats. Methods: Sixty SD male rats were used. Ten of them were used as a blank control. Colitis was induced by intracolonic administration ofTNB/ethanol (Morris et all) in 50 rats. The rats treated with TNB were randomly divided into five groups. Four groups were treated with berberine (7.5, or 15 mg kg") or geniposide (10, or 20 mg kg') orally via a stomach tube once a day for one week. One group was used as a control, and received an equal volume of vehicle. Observation were made on days 3, and 7 after the administration of TNB. A 7-cm segment of the distal colon was removed for functional and morphological studies. The measurements of colonic levels of myeloperoxygenase (MPO) were made by the method of Krawisz et a1 2 • Result: The body weight of rats treated with TNB decreased throughout the 5-day post-treatment period. The groups administrated berberine (7.5, or 15 mg kg') for one week showed quick recovery of their body weight. The macroscopic and histological damage score, colon weight, colonic wall thickness, ulceration diameter were lower or smaller in rats treated with berberine for I week than in the TNB control group. The MPO activity in TNB control group was increased on day 3, and 7 (9.63:t2.53, and 10.50:t6.38 U/100mg of wet tissue) compared with blank control (1.53:t0.45, and 1.90:t0.52 U/lOOmg of wet tissue) (P
GASTROENTEROLOGY Vol. 118, No.4
clinical trials. Preliminary results show that Thalidomide may be an effective adjunctive therapy in these patients. Aim: The aim of our study was to investigate the effects of thalidomide on cytokine profiles in patients with steroid-resistant Crohn's disease. Methods: Secretion of TNFa, IL-1{3 and IL-6 was investigated in short-time cultures of periperal blood monocytes (PBMC) and colonic Lamina propria mononuclear cells (LpMNC) stimulated with LPS or PWM, respectively. Seven patients with steroid resistant chronic active Crohn's disease receiving an average daily dose of 300 mg thalidomide were investigated before and after 12 weeks treatment. Results: Clinically, patients improved with a decrease in COAl from 305 :t 27 to 232 :t 19. This decrease was accompanied with a decrease of TNFa-secretion in short-time cultures of PBMC and LpMNC in 6 of 7 patients, although in one patient secretion of TNFa slightly increased after 12 weeks treatment with thalidomide. TNFa secretion by PBMC: 62.8 :t 14.6 (pg/ml; :tSEM) before treatment, 22.5 :t 9.2 after 12 weeks treatment. TNFa secretion by LpMNC: 42.3 :t 9.8 before treatment, 15.9 :t 7.3 after 12 weeks treatment. Overall, TNFa secretion did change significantly. Secretion ofIL-1{3 by MNC: 387 :t 77 before treatment, 406 :t 63 after 12 weeks. Secretion ofIL-1{3 by LpMNC: 465 :t 74 vs. 417 z; 49. Secretion of IL-6 by PMN: 2193 :t 236 vs. 1961 :t 270, by LpMNC: 2498 :t 286 vs. 2702 :t 376. Secretion of IL-I{3 and IL-6 was not altered significantly. Conclusion: Our data support the hypothesis that the clinical effects of Thalidomide in Crohn's disease are mediated by its inhibitory effects on TNFa. 3021 THE EFFECT OF TACHYKININ NK2 RECEPTOR BLOCKADE ON PROTO·ONCOGENE EXPRESSION FOLLOWING COLO· RECTAL DISTENSION OR EXPERIMENTAL COLITIS. Lori A. Birder, Susanna Kiss, Alessandro Lecci, William C. de Groat, Michelle VanBibber, Carlo A. Maggi, Univ of Pittsburgh, Pittsburgh, PA. Tachykinin NK2 receptors have been implicated in inflammatory responses such as those occurring in inflammatory bowel disease. We investigated the effect of the nonpeptide NK2 antagonist, MEN 11420, on induction of proto-oncogene expression in spinal cord (SC) and dorsal root ganglion (DRG) neurons following noxious and non-noxious stimulation of the colon in anesthetized Sprague Dawley rats. Physiological distension and noxious stimulation were used to activate colon afferents. Colitis was induced with trinitrobenzene sulfonic acid (TNBS)(Wallace et al., 1998). Either I m1 of saline, TNBS or ethanol vehicle was instilled in the colon via a cannula inserted 6 em proximal to the anus. A small number of normal untreated rats received repetitive (5 min on/IO min off) constant pressure inflation (10 mmHg) of a balloon catheter to produce non-noxious colorectal distension (CRD). Half of TNBS-treated rats received repetitive constant high pressure inflation (60 mmHg) to produce noxious CRD. Rats were sacrificed (2-72 hr later) via intracardiac perfusion of fixative and sections of SC and DRG were processed for either Fos or Jun (markers for cellular activation) using specific antisera. In normal untreated rats, following saline instillation or non-noxious CRD, low levels of Fos-immunoreactivity (IR) «15 cells/SC section) or Jun-IR (5% of DRG neurons) were detected. Instillation of ethanol vehicle slightly increased (ave. 10%) the number of Fos/Jun cells. TNBS instillation significantly increased Fos-IR (ave 105 :t II cells/section) in four L6-S I SC regions: medial and lateral dorsal horn, dorsal commissure and the region including the sacral parasympathetic nucleus (SPN). In DRG, TNBS produced a 3 fold increase in Jun-IR whereas distension of sensitized colon produced a 5 fold increase in the number of Fos/Jun-IR cells. MEN 11420 (150 nmol/kg i.v., 30 min prior) had no affect on the number of Fos/Jun neurons induced by nonnoxious CRD. In contrast, MEN 11420 significantly decreased Fos-IR in the dorsal horn (45-75%) and SPN (35-55%) and decreased Jun-IR in DRG by 50-60% after TNBS or distension of the TNBS-sensitized colon. These findings indicate that MEN 11420 does not affect responses of colonic afferent neurons to physiological stimuli, but suppresses the responses of these neurons to nociceptive stimuli. These results suggest that NK2 receptor antagonists may be beneficial in the treatment of colitis. 3022 SHORT·CHAIN FATTY ACID IRRIGATION IN THE TREATMENT OF DIVERSION COLITIS. A PROSPECTIVE DOUBLEBLIND RANDOMIZED STUDY. John W. Briel, David D. Zimmerman, Luuk M. de Boer, Theo H. van der Kwast, Willem R. Schouten, Univ Hosp Digest, Rotterdam, Netherlands. Introduction:Diversion Colitis is found after surgical diversion of the fecal stream. In literature, major randomized studies with prolonged treatment protocols investigating the role of Short-Chain Fatty Acids (SCFA) in the treatment of Diversion Colitis are lacking. Therefore, the current study was conducted. Methods:All patients with persistent or recurrent proctitis (> 3 months) after subtotal colectomy for inflammatory bowel disease were included. Patients were randomized and irrigated their rectum twice daily with 60 ml SCFA or Placebo-solution (P), containing normal saline only. Clinical Signs (CS) were noted. Furthermore, Endoscopic and Histologic