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Su1734 Impact of Probiotic Strain E.coli Nissle (EcN) on Gastric Mucosal Protection
mg/kg), WT mice were administered two TGR5 agonists, ciprofloxacin [Cipriani S., et al. Plos One 2011] and betulinic acid, a naturally occurring pentacyclic tripernoid found in the bark of several plants, and the gastric and intestinal mucosal injury assessed. Effects of TGR5 deletion were also assessed in chronic models of gastric and intestinal injury caused by naproxen, ASA + celecoxib, and in response to L-NAME. Results. TGR5 mRNA was detected in the human (intact margins of resected tumors) and mice stomachs by RT-PCR and immune-histochemistry in epithelial cells and submucosa structtures including vessel's endothelium. TGR5 deficiency exacerbated mucosal injury induced by ASA: not only fasting per se caused significant gastric injury in TGR5-/- but not in WT mice, but the mucosal injury scores measured in TGR-/- mice was higher than that observed in WT mice at each dose. TGR5 deficiency increased neutrophils infiltration (MPO activity) in the gastric and intetsinal mucosa. Expression of CSE and CBS (two H2S generating enzymes)and eNOS was reduced in the stomach of TGR-/- while no changes were detected in the expression of COX1 and COX-2. By In Vitro investigations we found that eNOS is regulated in a TGR5dependent manner. TGR5 activation by ciprofloxacin and betulinic acid rescued against development of mucosal damage induced by ASA in WT but not in TGR5-/- mice. TGR5 agonism restored CSE and eNOS expression. An abnormal intestinal architecture associated with altered molecular morphology of intestinal thight junctions proteins (Zonulin 1, occluding and E cadherin) and increased intestinal permeability was detected in TGR-/- mice and intestinal injury caused by naproxen was exacerbated by TGR5 deficiency. Conclusion. TGR5 deficiency predisposes to gastric and intestinal damage induced by ASA and NSAIDs while its activation protects against injury. Gastric protection exerted by TGR5 involves, but is not limited to, regulation of NO and H2S production.
Su1731 Novel Nongenomic Bacterial Component Sensing in Duodenum via Pattern Recognition Receptors Yasutada Akiba, Masaaki Higashiyama, Sergiv Rudenkyy, Paul H. Guth, Eli Engel, Jonathan D. Kaunitz Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3- secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunner's glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS + MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.
Su1734 Impact of Probiotic Strain E.coli Nissle (EcN) on Gastric Mucosal Protection Peter C. Konturek, Stanislaw Konturek, Tomasz Brzozowski Background and Aims: Despite the fact that gastric mucosa is exposed continuously to different injurious factors, it maintains the structural integrity and function. This phenomenon is attributed to the mucosal defence. However, the effect of probiotics on gastric mucosal defence is still poorly understood. The aim of the present study was to assess the effect of pretreatment with probiotic strain E.coli Nissle (EcN) on the development of acute gastric lesions in different models of acute injury (ethanol-induced lesions, stress-induced lesions) in rats. Material and Methods: Rats were divided in the following treatment groups: 1) vehicle; 2) EcN 101 CFU, 3) EcN 104 CFU and 4) EcN 108 CFU given 1 hr before exposure to acute mucosal injury by water immersion stress (WRS) or ethanol (75% EtOH). Involvement of prostaglandins was tested using indomethacin given one hour before EcN, whereas that of sensory nerves was assessed using neurotoxic dose of capsaicin in rats pretreated with EcN or vehicle. The expression of proinflammatory cytokines (IL1β, TNFα), toll-like receptor 4 (TLR4) and the plasma levels of ILβ, TNFα was assessed by RT-PCR and ELISA, respectively. Lesion area and gastric mucosal blood flow was assessed by planimetry and H2 gas clearance method, respectively. In additional In Vitro experiments the effect of EcN on the apoptosis and the expression of apoptosis related genes bax and bcl-2 was assessed by FACS and RT-PCR, respectively. Results: Pretreatment with EcN significantly reduced acute gastric lesions, increased gastric blood flow and decreased plasma levels of IL1β and TNFα. This protective effect was completely abolished by indomethacin and significantly attenuated by capsaicin-denervation. TLR4mRNA was detected in the gastric mucosa. EcN attenuated apoptosis induced by ethanol as shown by FACS and RT-PCR. Conclusions: Probiotic EcN protects gastric mucosa via TLR4 against noxious factors such as 75% ethanol or stress. The gastroprotective mechanisms of EcN involve the vasodilatatory and anti-inflammatory actions via prostaglandins and sensory afferent neurons. EcN attenuates the apoptosis induced by ethanol.
Su1732 Estrogen Upregulates Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Duodenal Mucosa Biguang Tuo, Wen Guorong, Jingyu Xu, Xue Wang, Juan Li, Hui Dong Background & purpose: Duodenal mucosal HCO3- secretion is currently accepted as an important protective factor in protecting duodenal mucosa from acid-inducing injury. Our recent study showed that estrogen is involved in the regulation of duodenal mucosal HCO3secretion in human, but the mechanisms whereby estrogen regulates duodenal HCO3secretion are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel, mediating duodenal mucosal epithelial HCO3- transport, and plays important role in duodenal mucosal HCO3- secretion. In the present study, therefore, we investigated the effect of estrogen on CFTR in duodenal mucosal epithelium. Methods: The study was performed in adult females (20-30 years) and age-matched males, and female mice (4-5weeks). The mice were divided into three groups: ovariectomy, ovariectomy plus estradiol replacement, and control. The expressions of CFTR mRNA and protein in duodenal mucosa were analyzed by real-time RT-PCR and western blot. Serum estradiol concentration in human was measured by chemiluminescent enzyme immunoassay. Duodenal mucosal HCO3- secretion in mice was measured by Ussing chamber. Results: The expression levels of CFTR mRNA and protein of duodenal mucosae in preovulatory phases of females were markedly higher than those in premenstrual phases of these females and in aged-matched males, whereas there was no significant difference in CFTR mRNA and protein expression between premenstrual females and age-matched males. Consistent with this change, the serum estradiol concentrations in preovulatory phases of females were markedly higher than those in premenstrual phases of these females and in aged-matched males, and there was no significant difference in estradiol levels between premenstrual females and age-matched males. Compared with control, the expression levels of CFTR mRNA and protein of duodenal mucosae in mice after ovariectomy were markedly decreased. The administration of estradiol rescued the change of CFTR in ovariectomized mice. Functional experiments showed that forskolin-stimulated duodenal mucosal HCO3- secretion in ovariectomized mice was markedly lower than that in control mice. Likewise, the administration of estradiol rescued duodenal mucosal HCO3- secretory capacity in ovariectomized mice. Conclusion: Estrogen upregulates the expression of CFTR in duodenal mucosa and its functional role, which contribute to explain the cellular mechanism whereby estrogen regulate duodenal mucosal HCO3- secretion.
Su1735 Effects of Melatonin and Tryptophan on Healing of Gastric and Duodenal Ulcers With Helicobacter pylori Infection in Humans Krzysztof Celinski, Stanislaw Konturek, Maria Slomka, Halina Cichoz-Lach, Tomasz Brzozowski, Wladyslaw Bielanski, Peter C. Konturek Melatonin (MT) and its precursor L-tryptophan (Trp) are implicated in the protection of gastric mucosa against aspirin-induced lesions and in the acceleration of healing of experimentally-induced gastric ulcers in rats. However, no information is available whether these agents are also effective in healing of gastroduodenal ulcers accompanied by Helicobacter pylori (H. pylori) infection. In this study three groups (A, B and C) of H. pylori-positive chronic gastric or duodenal ulcer patients, aging 28-50 years, were randomly assigned for the treatment with omeprazole (OME) 20 mg twice daily combined with placebo (group A, N=14) or melatonin 5 mg twice daily (group B, N=14), or Trp (group C, N=14). All patients underwent routine endoscopy at day 0 during which the gastric mucosa was evaluated and gastric biopsies were taken for the presence of H. pylori and histopathological evaluation. The rate of ulcer healing was determined by gastroduodenoscopy at day 0, 7, 14 and 21 after the initiation of the therapy. Plasma melatonin, gastrin, ghrelin and leptin were measured by RIA. At day 21, all ulcers were healed in patients treated with OME + MT or OME + Trp but only in 7 patients (50%) in group A. Initial plasma melatonin showed similar low values levels in all three groups but it increased several folds above initial value in ulcer patients treated with MT or Trp at day 7, 14 and 21. Plasma gastrin and leptin levels also rose significantly over initial value in patients treated in group A, B, and C but showed significantly higher levels in group B and C than in group A. In contrast, plasma ghrelin levels were not significantly affected by these treatment. We conclude that MT or Trp added to omeprazole treatment, significantly accelerates healing of H. pylori infected chronic gastroduodenal ulcers and this likely depends upon the significant rise in plasma MT and leptin levels.
Su1733 The Bile Acid Receptor TGR5 Maintains Gastrointestinal Homeostasis and Its Activation Rescues From Gastrointestinal Injury Caused by ASA and NSAIDs Sabrina Cipriani, Andrea Mencarelli, Barbara Renga, Claudio D'Amore, Eleonora Distrutti, Stefano Fiorucci Background. TGR5, a member of the rhodopsin-like superfamily of GPCRs, was identified as bile acid-activated receptor. Expression of TGR5 has been detected in the ileum, colon and stomach. In the intestine TGR5 ablations predisposes to development of inflammation. ASA and non selective NSAIDs are widely prescribed drugs despite their use associate with gastrointestinal complications, including bleeding and perforation. Aims. To evaluate the role of TGR5 and TGR5 ligands in protecting against gastrointestinal injury caused by ASA and NSAIDs. Methods. TGR5 deficient (TGR5 -/-) and congenic wild type (WT) mice were treated orally with ASA,10,30 or 50 mg/kg and 3 hours later gastric mucosal damage assessed. To investigate whether TGR5 activation protects ag ainst gastric injury caused by ASA(50
S-491
AGA Abstracts
AGA Abstracts
significance of Candida in the disease has not yet been established, it is considered to play an etiological role in ulcer formation, since it was detected in both the original and recurrent lesions in an HP-negative patient who had not taken NSAIDs.
Su1731 Novel Nongenomic Bacterial Component Sensing in Duodenum via Pattern Recognition Receptors Yasutada Akiba, Masaaki Higashiyama, Sergiv Rudenkyy, Paul H. Guth, Eli Engel, Jonathan D. Kaunitz Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3- secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunner's glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS + MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.
Su1734 Impact of Probiotic Strain E.coli Nissle (EcN) on Gastric Mucosal Protection Peter C. Konturek, Stanislaw Konturek, Tomasz Brzozowski Background and Aims: Despite the fact that gastric mucosa is exposed continuously to different injurious factors, it maintains the structural integrity and function. This phenomenon is attributed to the mucosal defence. However, the effect of probiotics on gastric mucosal defence is still poorly understood. The aim of the present study was to assess the effect of pretreatment with probiotic strain E.coli Nissle (EcN) on the development of acute gastric lesions in different models of acute injury (ethanol-induced lesions, stress-induced lesions) in rats. Material and Methods: Rats were divided in the following treatment groups: 1) vehicle; 2) EcN 101 CFU, 3) EcN 104 CFU and 4) EcN 108 CFU given 1 hr before exposure to acute mucosal injury by water immersion stress (WRS) or ethanol (75% EtOH). Involvement of prostaglandins was tested using indomethacin given one hour before EcN, whereas that of sensory nerves was assessed using neurotoxic dose of capsaicin in rats pretreated with EcN or vehicle. The expression of proinflammatory cytokines (IL1β, TNFα), toll-like receptor 4 (TLR4) and the plasma levels of ILβ, TNFα was assessed by RT-PCR and ELISA, respectively. Lesion area and gastric mucosal blood flow was assessed by planimetry and H2 gas clearance method, respectively. In additional In Vitro experiments the effect of EcN on the apoptosis and the expression of apoptosis related genes bax and bcl-2 was assessed by FACS and RT-PCR, respectively. Results: Pretreatment with EcN significantly reduced acute gastric lesions, increased gastric blood flow and decreased plasma levels of IL1β and TNFα. This protective effect was completely abolished by indomethacin and significantly attenuated by capsaicin-denervation. TLR4mRNA was detected in the gastric mucosa. EcN attenuated apoptosis induced by ethanol as shown by FACS and RT-PCR. Conclusions: Probiotic EcN protects gastric mucosa via TLR4 against noxious factors such as 75% ethanol or stress. The gastroprotective mechanisms of EcN involve the vasodilatatory and anti-inflammatory actions via prostaglandins and sensory afferent neurons. EcN attenuates the apoptosis induced by ethanol.
Su1732 Estrogen Upregulates Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Duodenal Mucosa Biguang Tuo, Wen Guorong, Jingyu Xu, Xue Wang, Juan Li, Hui Dong Background & purpose: Duodenal mucosal HCO3- secretion is currently accepted as an important protective factor in protecting duodenal mucosa from acid-inducing injury. Our recent study showed that estrogen is involved in the regulation of duodenal mucosal HCO3secretion in human, but the mechanisms whereby estrogen regulates duodenal HCO3secretion are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel, mediating duodenal mucosal epithelial HCO3- transport, and plays important role in duodenal mucosal HCO3- secretion. In the present study, therefore, we investigated the effect of estrogen on CFTR in duodenal mucosal epithelium. Methods: The study was performed in adult females (20-30 years) and age-matched males, and female mice (4-5weeks). The mice were divided into three groups: ovariectomy, ovariectomy plus estradiol replacement, and control. The expressions of CFTR mRNA and protein in duodenal mucosa were analyzed by real-time RT-PCR and western blot. Serum estradiol concentration in human was measured by chemiluminescent enzyme immunoassay. Duodenal mucosal HCO3- secretion in mice was measured by Ussing chamber. Results: The expression levels of CFTR mRNA and protein of duodenal mucosae in preovulatory phases of females were markedly higher than those in premenstrual phases of these females and in aged-matched males, whereas there was no significant difference in CFTR mRNA and protein expression between premenstrual females and age-matched males. Consistent with this change, the serum estradiol concentrations in preovulatory phases of females were markedly higher than those in premenstrual phases of these females and in aged-matched males, and there was no significant difference in estradiol levels between premenstrual females and age-matched males. Compared with control, the expression levels of CFTR mRNA and protein of duodenal mucosae in mice after ovariectomy were markedly decreased. The administration of estradiol rescued the change of CFTR in ovariectomized mice. Functional experiments showed that forskolin-stimulated duodenal mucosal HCO3- secretion in ovariectomized mice was markedly lower than that in control mice. Likewise, the administration of estradiol rescued duodenal mucosal HCO3- secretory capacity in ovariectomized mice. Conclusion: Estrogen upregulates the expression of CFTR in duodenal mucosa and its functional role, which contribute to explain the cellular mechanism whereby estrogen regulate duodenal mucosal HCO3- secretion.
Su1735 Effects of Melatonin and Tryptophan on Healing of Gastric and Duodenal Ulcers With Helicobacter pylori Infection in Humans Krzysztof Celinski, Stanislaw Konturek, Maria Slomka, Halina Cichoz-Lach, Tomasz Brzozowski, Wladyslaw Bielanski, Peter C. Konturek Melatonin (MT) and its precursor L-tryptophan (Trp) are implicated in the protection of gastric mucosa against aspirin-induced lesions and in the acceleration of healing of experimentally-induced gastric ulcers in rats. However, no information is available whether these agents are also effective in healing of gastroduodenal ulcers accompanied by Helicobacter pylori (H. pylori) infection. In this study three groups (A, B and C) of H. pylori-positive chronic gastric or duodenal ulcer patients, aging 28-50 years, were randomly assigned for the treatment with omeprazole (OME) 20 mg twice daily combined with placebo (group A, N=14) or melatonin 5 mg twice daily (group B, N=14), or Trp (group C, N=14). All patients underwent routine endoscopy at day 0 during which the gastric mucosa was evaluated and gastric biopsies were taken for the presence of H. pylori and histopathological evaluation. The rate of ulcer healing was determined by gastroduodenoscopy at day 0, 7, 14 and 21 after the initiation of the therapy. Plasma melatonin, gastrin, ghrelin and leptin were measured by RIA. At day 21, all ulcers were healed in patients treated with OME + MT or OME + Trp but only in 7 patients (50%) in group A. Initial plasma melatonin showed similar low values levels in all three groups but it increased several folds above initial value in ulcer patients treated with MT or Trp at day 7, 14 and 21. Plasma gastrin and leptin levels also rose significantly over initial value in patients treated in group A, B, and C but showed significantly higher levels in group B and C than in group A. In contrast, plasma ghrelin levels were not significantly affected by these treatment. We conclude that MT or Trp added to omeprazole treatment, significantly accelerates healing of H. pylori infected chronic gastroduodenal ulcers and this likely depends upon the significant rise in plasma MT and leptin levels.
Su1733 The Bile Acid Receptor TGR5 Maintains Gastrointestinal Homeostasis and Its Activation Rescues From Gastrointestinal Injury Caused by ASA and NSAIDs Sabrina Cipriani, Andrea Mencarelli, Barbara Renga, Claudio D'Amore, Eleonora Distrutti, Stefano Fiorucci Background. TGR5, a member of the rhodopsin-like superfamily of GPCRs, was identified as bile acid-activated receptor. Expression of TGR5 has been detected in the ileum, colon and stomach. In the intestine TGR5 ablations predisposes to development of inflammation. ASA and non selective NSAIDs are widely prescribed drugs despite their use associate with gastrointestinal complications, including bleeding and perforation. Aims. To evaluate the role of TGR5 and TGR5 ligands in protecting against gastrointestinal injury caused by ASA and NSAIDs. Methods. TGR5 deficient (TGR5 -/-) and congenic wild type (WT) mice were treated orally with ASA,10,30 or 50 mg/kg and 3 hours later gastric mucosal damage assessed. To investigate whether TGR5 activation protects ag ainst gastric injury caused by ASA(50
S-491
AGA Abstracts
AGA Abstracts
significance of Candida in the disease has not yet been established, it is considered to play an etiological role in ulcer formation, since it was detected in both the original and recurrent lesions in an HP-negative patient who had not taken NSAIDs.