Evaluation of the acute toxicity, analgesic and CNS activities of different species ofTeucrium genus

Methanol and dichloromethanol extracts of the leaves and stems of four Teucrium species (T. cartaginenses, T. flavum, T. pumillum and T. buxifolium) have been tested for their toxicity, analgesic and central depressor effects. The intraperitoneal administration of the different extracts showed a CNS depressant activity in mice, but they lacked anticonvulsive effects. When tested for analgesic activity none of the extracts increased the threshold of pain thermal stimulus. However, the methanol and dichloromethanol extracts of T. cartaginenses and T. buxifolium species showed a significant analgesic effect in models of pain induced by chemical or mechanical stimulation.

The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD

The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6(-/-) mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also ex…

Synthesis of nitric oxide in the dorsal motor nucleus of the vagus mediates the inhibition of gastric acid secretion by central bombesin

1. Central administration of bombesin inhibits gastric acid production independently of the centrally or peripherally-acting stimuli employed. This study evaluates the role and location of the cerebral nitric oxide (NO) implicated in the inhibitory effect of central bombesin on in vivo rat gastric acid secretion, as induced by distension with 15 cm H2O, insulin (0.75 u.i. kg-1 i.p.) TRH (1.2 microg kg-1, i.c.) or pentagastrin (100 microg kg-1, i.p.). 2. The acid-inhibitory effect of i.c. bombesin (40 ng kg-1) was prevented by prior administration of L-NAME (80 microg kg-1) in the dorsal motor nucleus of the vagus (DMN). This dose of L-NAME when administered into the nucleus of the tractus s…

P045 Local inflammation modulates vitamin D receptor protein levels in fibroblasts

DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn′s Disease

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtai…

Antiinflammatory activity and effects on isolated smooth muscle of extracts from differentTeucrium species

The present study analyses the antiinflammatory effects and the action on in vitro motility of methanol and dichloromethanol extracts and stems of four Teucrium species (T. flavum, T. cartaginenses, T. buxifolium and T. pumillum). The antiinflammatory activity was tested in the carrageenan-induced paw oedema in rats. T. flavum methanol (200 mg/kg, i.p.) and dichloromethanol (138 mg/kg, i.p.) extracts showed a significant anti-inflammatory effect through the 24 h experimental period and reduced the Emax induced by histamine and serotonin in vitro on guinea-pig ileum and rat uterus respectively. These extracts did not modify the contractile effects induced by acetylcholine on rat duodenum and…

P017 C86/CD16 macrophages accumulate in the mucosa of B3 patients and could mediate EMT in Crohn’s disease

Abstract Background Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. Methods The aim of the present study is to analyse the pattern of expression of macrophages, of EMT-related genes and cytokines in surgical resections from Crohn’s disease (CD, n = 43) patients which were categorised according to Montreal classification (B2 or B3); unaffected mucosa of patients with ileocecal cancer was used as control (n = 20). mRNA was isolated from intestinal samples and the expression of macrophage, EMT markers and cytokines were analysed by RT-PCR. PBMCS were isolated from healthy donors and treated during…

P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of …

Pharmacological evaluation of the dichloromethanol extract fromInula crithmoides L.

The pharmacological effect of the dichloromethanol extract of Inula crithmoides L. was analysed in in vitro and in vivo models. The extract dose-dependently decreased arterial blood pressure and furthermore it showed low acute toxicity, CNS depressor activity and analgesic and antiinflammatory effects. Preincubation of the guinea-pig ileum and rat duodenum (100 μg/mL) produced a significant reduction in the contractile effects of histamine and acetylcholine and a concentration-related inhibition of the effects of serotonin. Following further fractionation the methylene chloride/acetone (50/50) fraction caused a significant decrease in motor activity and significantly reduced the threshold o…

OP03 Inhibition of autophagy exacerbates intestinal fibrosis and EMT

P041 Differences in NOTCH signalling between stricturing and penetrating behaviour in Crohn’s disease

DOP87 SUCNR1 a novel key protagonist in fistula development

Abstract Background Intestinal fistula is a common complication in CD patients whose aetiology is still not well-characterised. It is associated with an exacerbated inflammation and epithelial-to-mesenchymal transition (EMT), a process which allows a switch from epithelial towards a fibrotic behaviour. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis1 but its role in fistula has not yet been analysed. Therefore, we aim to analyse the role of SUCNR1 in EMT and in fistula formation. Methods Intestinal resections were obtained from CD and non-IBD patients. Fistula specimens were identified by the surgeons and collected from B3-CD patients. The expression of S…

Nitric oxide and sensory afferent neurones modulate the protective effects of low-dose endotoxin on rat gastric mucosal damage

Pretreatment (1 h) with low doses (5-40 micrograms/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats. Treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 micrograms/kg i.p.). The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.p.). The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg s.c.). However, ablation of sensory affe…

Effects on Arterial Blood Pressure of the Methanol and Dichloromethanol Extracts from Schinus molle L. in Rats

The effects on arterial blood pressure of the methanol and dichloromethanol extracts from Schinus molle L. were analysed in urethane anaesthetized rats. In normotensive rats, the mean arterial blood pressure was significantly reduced by the i.v. administration of both extracts. The dichloromethanol extract inhibited the effects of noradrenaline on arterial blood pressure in the anaesthetized rat and it reduced the maximal contractile effect (Emax) induced by noradrenaline on rat vas deferens in the organ bath. However, the methanol extract did not modify the effects of noradrenaline in the evaluated tests.

Inhibition of gastric acid secretion by stress: A protective reflex mediated by cerebral nitric oxide

Moderate somatic stress inhibits gastric acid secretion. We have investigated the role of endogenously released NO in this phenomenon. Elevation of body temperature by 3°C or a reduction of 35 mmHg (1 mmHg = 133 Pa) in blood pressure for 10 min produced a rapid and long-lasting reduction of distension-stimulated acid secretion in the rat perfused stomach in vivo . A similar inhibitory effect on acid secretion was produced by the intracisternal (i.c.) administration of oxytocin, a peptide known to be released during stress. Intracisternal administration of the NO-synthase inhibitor, N G -nitro- l -arginine methyl ester ( l -NAME) reversed the antisecretory effect induced by all these stimul…

Analgesic Activity and Effects on Isolated Smooth Muscle of Different Fractions of Hexane Extract fromAraujia sericifera Brot

The present study analyses the analgesic activity and action on in vitro motility of methanol soluble and methanol insoluble fractions obtained from the hexane extract of fruits from Araujia sericifera. The methanol fraction did not show any pharmacological activity on the different tests evaluated. However, the insoluble methanol fraction exhibited an interesting analgesic effect in models of chemical and thermal stimulus and it reduced the E max induced by histamine in vitro on guinea-pig ileum. This extract lacked any central depressor activity since it did not modify the number of mouse movements in the activity cage.

Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system

1. The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. The i.c.v. administration of E. coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3. By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v. administr…

P061 Succinate promotes EMT in intestinal epithelial cells through SUCNR1: Relevance in fistula development

M1 Macrophages Activate Notch Signalling in Epithelial Cells: Relevance in Crohn's Disease

Background: The Notch signalling pathway plays an essential role in mucosal regeneration, which constitutes a key goal of Crohn's disease (CD) treatment. Macrophages coordinate tissue repair and several phenotypes have been reported which differ in the expression of surface proteins, cytokines and hypoxia-inducible factors (HIFs). We analysed the role of HIFs in the expression of Notch ligands in macrophages and the relevance of this pathway in mucosal regeneration. Methods: Human monocytes and U937-derived macrophages were polarized towards the M1 and M2 phenotypes and the expression levels of HIF-1α, HIF-2α, Jagged 1 (Jag1) and delta-like 4 (Dll4) were evaluated. The effects of macrophage…

Involvement of prostaglandins and 5-hydroxytryptamine in the contractile effect of platelet-activating factor in rat isolated gastric corpus

Abstract The present study characterizes the nature of the response to the platelet-activating factor (PAF) in isolated gastric corpus with and without mucosa. PAF (10-8 M) induced contraction of rat isolated gastric corpus strips followed by desensitization of this tissue. Incubation of strips with the specific PAF-receptor antagonist WEB 2086 (5 × 10-8 − 5 × 10-5 M), the prostaglandin blocker indomethacin (10-6 M) and the 5-hydroxytryptamine antagonist methysergide (10-5 M) reduced significantly the contraction induced by PAF. Neither of the histamine H1/H2 antagonists diphenhydramine (10-6 M) or cimetidine (10-5 M) affected the contraction induced by PAF. In contrast with the whole gastr…

Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD.

A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1 alpha and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of beta-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial …

Protection by Almagate of Ethanol-induced Gastric Mucosal Damage in Rats

Abstract The study was designed to analyse the protective effects of almagate on a model of gastric injury, ethanol-induced mucosal damage, in which acid plays little, if any, role. Pretreatment with almagate dose-dependently reduced the level of gastric damage induced by oral administration of 1mL 100% ethanol. Administration of 12 μmol kg−1 almagate 30 min before ethanol significantly reduced the area of mucosal damage by 65 ± 10%, and the maximum level of inhibition (74 ± 11%) was obtained with 150 μmol kg−1 almagate. Administration of higher doses of almagate (200–250 μmol kg−1) did not result in any further increase in the level of protection against ethanol-induced gastric damage. Adm…

P051 Macrophages as a source of Notch Ligands in Crohn’s disease: implications in fibrosis

Abstract Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cyt…

P073 An increased autophagy and decreased apoptosis is detected in intestinal fibroblasts from Crohn’s Disease patients

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) phenotype, with no effective treatment. This process is characterized by a disequilibrium between the production and degradation of the extracellular matrix (ECM), mainly regulated by myofibroblasts. We aim to analyse here, the expression of markers of autophagy, apoptosis and proliferation in intestinal fibroblasts from CD patients. Methods Fibroblasts were isolated from the damaged intestinal mucosa of CD patients with a penetrating and stenotic behaviour. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. …

P028 The Apa I polymorphism in the Vitamin D receptor gene is associated with a stricturing behaviour in Crohn′s disease patients

Abstract Background Vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors that plays an immunomodulatory role in the gastrointestinal tract through binding Vitamin D. Single-nucleotide polymorphisms (SNPs) in the VDR gene have been related to inflammatory bowel disease. Indeed, Crohn′s disease (CD) patients carrying the Taq I polymorphism in VDR gene run a higher risk of developing a penetrating behaviour. We analyse here the association between the VDR SNPs Taq I, Bsm I, Apa I and Fok I and the clinical characteristics of CD. Methods DNA was extracted from blood samples from 80 patients diagnosed with CD from the Hospital of Manises (Valencia). Four p…

Involvement of endogenous nitric oxide in the inhibition by endotoxin and interleukin-1 beta of gastric acid secretion.

Administration of Escherichia coli endotoxin abolished the acid secretory response induced by a bolus injection of pentagastrin in the continuously perfused stomach of the anaesthetized rat. Likewise, acid secretion stimulated by the continuous intravenous perfusion of pentagastrin was inhibited by administration of interleukin-1 beta (IL-1 beta). In both cases pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) but not dexamethasone or indomethacin substantially restored the secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine but not by its enantiomer D-arginine. Even though L-NAME increased blood pressure, this does no…

Nitric Oxide Modulates the Acute Increase of Gastrointestinal Transit Induced by Endotoxin in Rats: a Possible Role for Tachykinins

Abstract Because of the evidence that endogenous nitric oxide (NO) plays an essential role in the physiological regulation of gastrointestinal motility we have investigated, by use of the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), the role of endogenous NO in the acute endotoxin-induced changes of gastrointestinal transit. Pre-treatment with E. coli endotoxin (100 μg kg−, i.v.) induced a significant increase in the gastrointestinal transit of a charcoal suspension in anaesthetized rats. Previous administration of the NO synthase inhibitor, l-NAME (10 mg kg−, i.v.) significantly prevented the effects of endotoxin. l-arginine (200 mg kg−, i.v.) and the substance P antag…

Macrophages as an Emerging Source of Wnt Ligands: Relevance in Mucosal Integrity

The Wnt signaling pathway is a conserved pathway involved in important cellular processes such as the control of embryonic development, cellular polarity, cellular migration, and cell proliferation. In addition to playing a central role during embryogenesis, this pathway is also an essential part of adult homeostasis. Indeed, it controls the proliferation of epithelial cells in different organs such as intestine, lung, and kidney, and guarantees the maintenance of the mucosa in physiological conditions. The origin of this molecular pathway is the binding between Wnt ligands (belonging to a family of 19 different homologous secreted glycoproteins) and their specific membrane receptors, from …

P003 C86/CD16 macrophages may act as a source of WNT2b in intestinal tissue from B3 Crohn’s disease patients

P089 IFNγ-macrophages could mediate EMT in Crohn’s disease

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT). Methods The mRNA and protein expression of IFN in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (2 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. IFNγ-U937 were coculture with HT29…

Modulation by peripheral opioids of basal and distension-stimulated gastric acid secretion in the rat.

1. The influence of opioids in modulating gastric acid secretory responses has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. Intravenous administration of morphine (0.75-3 mg kg-1) or the peripherally acting enkephalin analogue, BW443C (0.75-3 mg kg-1), substantially augmented acid secretion in basal conditions. These effects were significantly inhibited by the opioid antagonists naloxone (1 mg kg-1) and the peripherally acting N-methylnalorphine (2 mg kg-1). When administered alone, neither opioid antagonist influenced basal acid output. 3. Acid secretory responses to different levels of gastric distension (5-20 cmH2O) were significantly and dose-depen…

Pharmacological screening of the methanol and dichloromethanol extracts ofGenista patens

The pharmacological effects of the dichloromethanol and methanol extracts obtained from leaves and stems of Genista patens DC were analysed in in vitro and in vivo models. Both extracts showed low acute toxicity (LD 50 >3 g/kg), CNS depressor and antiinflammatory activity, and similar analgesic effect in models of chemical and thermal stimulation. Furthermore, the dichloromethanol extract (1-20 mg/kg) induced a pronounced dose-dependent decrease on blood pressure. On isolated organs, the dichloromethanol extract (1, 10, 100 μg/mL) shifted the concentration-effect curve to the right for ACh and reduced the E max induced by histamine without modifying responses induced by noradrenaline and se…

Analgesic and central depressor effects of the dichloromethanol extract fromSchinus molle L.

The analgesic and central depressor effects of the dichloromethanol extract of Schinus molle L. were analysed in in vivo models. This extract showed low acute toxicity, CNS depressor activity and analgesic effect. Following further fractionation, the hexane/dichloromethane (75/25) fraction showed the most interesting results. Thus, this fraction caused a total inhibition of motor activity and significantly reduced the threshold of pain to chemical stimulus. © 1997 John Wiley & Sons, Ltd.

P030 SUCNR1 mediates inflammasome activation: Relevance in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon1. Inflammasome complex is involved in the intestinal homeostasis regulation, but its role in UC has not been established yet. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis2. We aim to analyze the role of SUCNR1 in inflammasome activation and UC. Methods Intestinal resections from UC and non-IBD patients were obtained. HT29 cells were treated with succinate 1mM and an inflammasome activator cocktail (TNF-α 25ng/ml, IFN-γ 20 ng/ml and LPS 1µg/ml) for 24 hours and transfected with SUCNR1 siRNA. Chronic …