Inhibition of endogenous nitric oxide activity impairs the colonic sparing effect of rofecoxib, the cyclooxygenase-2 inhibitor and resveratrol, the preferential cyclooxygenase-1 inhibitor in the course of experimental colitis. Role of oxidative stress biomarkers and proinflammatory cytokines.

The gut mucosal barrier plays a key role in the physiology of gastrointestinal (GI) tract, preventing under homeostatic conditions, the epithelial cells of the gastric mucosa from hydrochloric acid and intestinal mucosa from alkaline secretion, food toxins and pathogenic microbiota. Previous studies have documented that blockade of both isoforms of cyclooxygenase (COX): constitutive (COX-1) and inducible (COX-2), as well NO synthase in the stomach exacerbated the gastric damage induced by various ulcerogens, however, such as effects of non-selective and selective inhibition of COX-1, COX-2 and NOS enzymes on colonic damage have been little studied. The supplementation of NO by intragastric (i.g.) treatment with NO-releasing compound NO-aspirin (NO-ASA) or substrate for NO synthase L-arginine ameliorated the damage of upper GI-tract, but whether similar effect can be observed in colonic mucosa associated with the experimental colitis, and if above mentioned compounds can be effective in aggravation or protection of experimental colitis remains less recognized. In this study rats with experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzosulphonic acid (TNBS) were daily treated for 7 days with: 1) vehicle (i.g.), 2) ASA 40 mg/kg (i.g.), 3) rofecoxib 10 mg/kg (i.g.), 4) resveratrol 10 mg/kg (i.g.), 5) NO-ASA 40 mg/kg (i.g.), 6) L-arginine 200 mg/kg (i.g.) with or without of L-NNA 20 mg/kg (i.p.). The macroscopic and microscopic area of colonic damage was determined planimetrically, the colonic blood flow (CBF) was assessed by Laser flowmetry, and the oxidative stress biomarkers malondialdehyde and 4-hydroxynonenal (MDA+4-HNE), the antioxidative factors superoxide dismutase (SOD) and glutathione (GSH), as well as proinflammatory cytokines in the colonic mucosa (tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1ß)) were measured. We have documented that administration of TNBS produced gross and microscopic colonic damage and significantly decreased CBF (p<0.05). Treatment with ASA significantly increased the area of colonic damage (p<0.05), an effect accompanied by a significant decrease in the CBF, the significant increment of MDA+4-HNE, and the attenuation of the antioxidative properties in colonic mucosa, documented by a significant decrease of SOD activity and GSH concentration, and elevation of the colonic tissue levels of TNF-α and IL-1ß comparing to control Veh-treated TNBS rats. Administration of rofecoxib or resveratrol also significantly increased the colonic damage and significantly decreased the CBF, causing an increase in MDA+4-HNE and mucosal content of TNF-α and IL-1α and a significant decrease of the SOD activity and GSH content (p<0.05), however, these changes were significantly less pronounced as compared with ASA. On the contrary, the treatment with NO-ASA, or L-arginine, significantly diminished the area of colonic lesions, the MDA+4-HNE concentration, attenuated the TNF-α and IL-1ß levels, while increasing the CBF, SOD activity and GSH content (p<0.05). The concomitant treatment of L-NNA with rofecoxib or resveratrol reversed an increase in area of colonic damage and accompanying changes in CBF, colonic mucosa TNF-α and IL-1ß levels, the MDA+4-HNE concentration, and SOD activity and GSH content comparing to those observed in TNBS rats treated with these COX-inhibitors alone (p<0.05). In contrast, co-treatment with L-NNA and NO-ASA or L-arginine failed to significantly affect the decrease of colonic lesions accompanied by the rise in CBF, the attenuation of MDA+4-HNE concentration, TNF-α and IL-1ß levels, SOD activity and GSH content exerted by NO-ASA- or L-arginine treatment of the respective control TNBS-rats without L-NNA administration. These observations suggest that 1) the increase of NO availability either from NO-releasing donors such as NO-ASA or NO precursors such as L-arginine, can inhibit the inflammatory and microvasculature alterations, as well as increase in lipid peroxidation due to the enhanced efficacy of these compounds to increase the antioxidative properties of colonic mucosa, 2) unlike ASA which exacerbated the severity of colitis, the treatment with rofecoxib, the specific 'safer' COX-2 inhibitor or resveratrol, the polyphenolic compound known to act as the dual COX-1 and COX-2 inhibitor, can attenuate the colonic damage during course of TNBS colitis possibly via anti-inflammatory and antioxidative properties, and 3) the blockade of endogenous NO activity by L-NNA which also exacerbated the severity of mucosal damage in colitis, can abolish the sparing effect of rofecoxib and resveratrol indicating the NO bioavailability plays an important role in enhanced efficacy of both specific and dual COX inhibitors to ameliorate the experimental colitis.

The relationship between the presence of Helicobacter pylori and the composition of ionic and organic microelements in drinking water from Cracow.

Although the natural niche for H. pylori (Hp) is the human stomach, for widespread infection to occur this microorganism may need to survive in the external environment. Molecular techniques such as polymerase (PCR) have revealed the presence of Hp DNA in water, indicating that this environment could act as a reservoir for this bacterium. The aim of this study was to analyse the occurrence of Hp in tap water from Cracow and to examine the relationship between 26 parameters and the presence of Hp DNA due to the lack of such information related to this issue in Poland. Additional aim of this study was to determine whether the correlation between Hp DNA detection and seasonal changes of water quality in 379 water samples collected from various water treatment plants (WTPs), could be found. Water samples were subjected to PCR for glmM and cagA genes. Ionic and organic composition of microelements were determined in accordance to Polish and ISO standards. The data obtained from tests show that 212 (55.96%) objects were Hp DNA (glmM) positive and among them 145 (68.40%) waters samples revealed expression of cagA. Linear Discriminant Analysis and Principal Component Analysis were used and provided that the selected variables (p < 0.05): colour, pH, conductivity at 25°C, chlorides, nitrites, nitrates, phosphates, chlorates, chlorites, sulphates, free chlorine, sodium, magnesium, potassium, calcium, organic carbon, trichloromethane, bromodichloromethane, dibromochloroethane, total iron, ammonium ion, and Æ©TMHs distinguished the water samples that contain Hp DNA and do not contain Hp DNA. We conclude that the ionic and organic composition of microelements in water might influence the presence of Hp. Thus, determination of the selected microelements may indirectly indicate or sometimes predict the presence of Hp in drinking water.

Participation of the intestinal microbiota in the mechanism of beneficial effect of treatment with synbiotic Syngut on experimental colitis under stress conditions.

Gut-brain axis plays a central role in the regulation of stress related diseases such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). It is increasingly recognized that stress modulates gut microbiota community structure and activity and represents an important causal factor in dysbiosis. This study was designed to determine the effect of daily treatment with synbiotic (Syngut) containing inulin, Lactobacillus acidophilus, Bifidobacterium lactis W51, Lactobacillus plantarum W21 and Lactococcus lactis applied i.g. at a dose of 50 mg/kg i.g. on the colonic damage and colonic mucosal blood flow in rats with experimentally induced TNBS-colitis that were additionally exposed or not to acute stress (episodes of cold restraint stress every other day before colitis induction). Control rats received daily treatment with vehicle (saline, i.g.) or mesalazine (50 mg/kg-d i.g.), the standard drug recommended in therapy of IBD. At the termination of TNBS colitis, the histologic evaluation of colonic mucosa, mucosal malonyldialdehyde (MDA) level and plasma concentrations of proinflammatory cytokines (TNF-α, IL-1ß) and adipokine adiponectin were assessed. the samples of colonic mucosa not involving colonic lesions and surrounding the flared mucosa were excised for the determination of mRNA expression for proinflammatory biomarkers TNF-α, IL-1ß, IL-10 and COX-2 as well as antioxidazing factors SOD-1 and SOD-2. Finally, the gut microbial profiles were analyzed by 16S rRNA sequencing at phylum, family and genus level. Episodes of cold stress significantly aggravated the course of TNBS colitis, and significantly increased the release of proinflammatory cytokines as well as the significant increase in the MDA concentration has been observed as compared with non-stressed TNBS rats. These changes were followed by the significant fall in the CBF and plasma adiponectin levels and by the overexpression of mRNA of proinflammatory biomarkers. Synbiotic treatment with Syngut significantly reduced the area of colonic lesions observed macroscopically and microscopically in rats with TNBS colitis with or without exposure to cold stress, significantly increased the CBF, normalized plasma adiponectin levels and significantly attenuated the release and colonic expression of proinflammatory cytokines and biomarkers. the analysis of the gut microbiota showed a significant reduction of microbial diversity (Shannon index) in rats with TNBS colitis with or without exposure to stress. The therapy with Syngut failed to significantly affect the alpha diversity. At the phylum level, the significant rise in Proteobacteria has been observed in stressed rats with TNBS colitis and this effects was attenuated by treatment with Syngut. At family level, TNBS colitis alone or in combination with stress led to a significant decrease of SCFA producing bacterial taxa such as Ruminococaceae and Lachnospiraceae and Syngut counteracted this effect. We conclude that: 1) cold stress exacerbates the gastrointestinal inflammation in experimental colitis; 2) the synbiotic therapy with Syngut ameliorates the gut inflammation in rats with TNBS colitis combined with cold stress; 3) the beneficial effect of Syngut is accompanied by increase of anti-inflammatory taxa such as Ruminococaceae and Lachnospiraceae, and 4) the modulation of gut microbiota with Syngut alleviates stress-related intestinal inflammation suggesting a potential usefulness of synbiotic therapy in intestinal disorders accompanied by stress in patients with IBD.

Involvement of epithelial-mesenchymal transition-inducing transcription factors in the mechanism of Helicobacter pylori-induced fibroblasts activation.

Gastric cancer (GC) originating from the lining of the stomach is one of the most frequent malignancies in humans. The most efficient method giving hope of full recovery from GC is gastric resection combined with adjuvant chemotherapy and radiotherapy. However, over 50% of patients after treatment suffer from recurrence and peritoneal metastasis. The bacteria Helicobacter pylori (Hp) is nowadays considered as the major pathogen capable of colonizing gastric mucosa. This bug causes deregulation of multiple signaling pathways including the activation of nuclear factor kappaB (NFκB) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) responsible for inflammation and development of carcinogenic cascade. The pathomechanism of these changes remains little understood, but the Hp virulence factors affecting mainly gastric epithelium have been postulated. Nevertheless, the changes associated with inflammation progressing to cancer are not only limited to epithelial cells. The cells surrounding the tumor, mainly activated fibroblasts (CAFs - cancer-associated fibroblasts) create molecular microenvironment promoting tumorigenesis and cancer invasion. The downstream targets of STAT3, epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) are expressed in activated fibroblasts providing them with additional properties. Thus, our aim was to determine if the Hp strain expressing CagA and VacA cytotoxins may result in the activation/differentiation of rat gastric fibroblasts resulting in NFκB and STAT3 signaling, which could lead to EMT-TFs expression and secretome responsible for inflammatory and EMT inducing microenvironment. In this study, gastric samples were harvested from 8-week-old Spraque-Dowley rats and the primary and secondary fibroblast cultures were established. The 70% confluent secondary fibroblast cultures were infected with 1 x 109 of live Hp expressing cytotoxins CagA VacA per dish and incubated in humidified atmosphere for 3, 24, 48 and 72 hours, before the conditioned media or the cells were used for endpoint experiments. As the control, fibroblast culture in DMEM with 10% FBS and antibiotics, free from Hp infection was used. The expression of mRNA for 18S (control), toll-like receptors: TLR2 and TLR4, STAT3, NFκB p65/Rel A, inhibitor of NF-κB (Iκß), Snail and Twist was determined by RT-PCR. The protein expression of Snail and Twist was assessed by Western blot technique. The fibroblast supernatant was collected at 72 hours from non-infected and Hp (cagA+ vacA+)-infected culture and the concentrations of interleukin 8 (IL-8), hepatocyte growth factor (HGF) and stromal derived factor-1 (SDF-1) were measured by ELISA. In fibroblasts infected with Hp (cagA+ vacA+), the significant increase of mRNA expression for both, TLR2 and TLR4, as well as STAT3, NFκB/RelA subunit was observed already after 3 hours of fibroblasts infection with Hp strain compared with control non-infected fibroblasts. Simultaneously, the significant decrease of Iκß mRNA has been noticed starting from 48 hours after the Hp infection of fibroblasts was carried out. The strong increase in the expression of Snail1 and Twist mRNA was recorded already at 3 hours in Hp-infected fibroblasts comparing to control non-infected fibroblasts and this increase persisted at 24 and 48 hours being the most pronounced at 72 hours post incubation with Hp. The expression of Snail1 protein was observed after 3 hours post Hp infection and this increase persisted throughout entire time periods upon Hp infection. In contrast, no detectable level of Twist protein expression was observed up to 72 hours post-infection neither in control conditions nor in fibroblasts co-infected with Hp. These changes in fibroblasts were accompanied by a significant increase in the release of HGF, SDF-1 and IL-8 determined in cell supernatants collected from Hp-infected fibroblasts. These data indicate that the activation/differentiation of rat gastric fibroblasts can occur directly by Hp releasing CagA and indirectly through TLR2 and TLR4 and these effects can be mediated by transcription factors NFκB and STAT3 signaling leading to rapid Snail1 protein expression. We conclude that NFκB and STAT3 signaling together with Snail1 protein expression may activate the secretome responsible for fibroblasts inflammatory and EMT-inducing microenvironment likely serving as prerequisite for GC development.

Role of curcumin in protection of gastric mucosa against stress-induced gastric mucosal damage. Involvement of hypoacidity, vasoactive mediators and sensory neuropeptides.

The antioxidizing properties of curcumin, a highly pleiotropic substance used for centuries in traditional medicine has been confirmed by numerous experimental and clinical studies. Curcumin exhibits anti-inflammatory, antiproliferative and anti-angiogenic actions inhibiting the development and progression of tumors but the efficacy of this compound to influence gastric acid secretion n in the stomach and to affect the gastric mucosal damage induced by non-topical ulcerogenes such as stress has been little studied. We determined the effect of curcumin on basal and pentagastrin- or histamine-stimulated gastric secretion, in rats with surgically implemented gastric fistulas and we assessed the contribution of gastric secretion, endogenous prostaglandin (PG), endogenous nitric oxide (NO), as well as sensory afferent nerves in the mechanisms underlying the potential gastroprotective effects of curcumin against stress-induced gastric mucosal lesions. Rats exposed to water immersion and restraint stress (WRS) for 3.5 h were pretreated either with: 1) vehicle (saline); 2) curcumin (2.5 - 100 mg/kg i.g.) or 3) curcumin (50 mg/kg i.g.) combined with or without indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.) or rofecoxib (10 mg/kg i.g.); 4) curcumin (50 mg/kg i.g.) co-administered with (L-NNA (20 mg/kg i.p.) with or without L-arginine (200 mg/kg i.g.), a substrate for NO-synthase; 5) curcumin (50 mg/kg i.g.) administered in rats with intact or capsaicin-induced functional ablation of sensory nerve fibers, and 6) curcumin (50 mg/kg i.g.) administered with capsazepine (5 mg/kg i.g.), the antagonist of vanilloid TRPV1 receptor. The number of gastric lesions was determined by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance technique, the plasma gastrin concentrations were measured using the radioimmunoassay (RIA) and the expression of mRNA for tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in gastric mucosa was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Curcumin dose-dependently reduced the WRS-induced gastric lesions, the dose inhibiting these lesions by 50% being about 50 mg/kg. These effects of curcumin were accompanied by an increase in GBF and the reduction in basal and histamine- or pentagastrin-stimulated gastric acid secretion. The protective and hyperemic activities of curcumin (50 mg/kg i.g.) against WRS lesions were significantly attenuated (P < 0.05) in rats pretreated with rofecoxib and SC-560 and completely reversed (P < 0.01) by indomethacin. L-NNA significantly reduced (P < 0.05) the decrease in WRS-induced lesions and the accompanying rise in GBF caused by curcumin and these effects were restored by concurrent treatment with L-arginine (200 mg/kg i.g.). The curcumin-induced decrease in the number of WRS-induced gastric lesions and accompanying increase in the GBF were significantly attenuated (P < 0.05) in capsaicin-denervated rats and in those pretreated with capsazepine. These effects of curcumin in rats with capsaicin denervation were restored by concomitant treatment with exogenous calcitonin gene related pepetide (CGRP) combined with curcumin and subsequently exposed to WRS. The expression of mRNA for TNF-α, COX-2 and iNOS was significantly increased (P < 0.05) in vehicle-pretreated control rats exposed to WRS and significantly attenuated (P < 0.05) by curcumin administered in graded dosages. We conclude that curcumin exerts gastroprotective and hyperemic activities against experimental stress-induced gastric lesions by mechanism involving endogenous prostaglandins, NO, the neuropeptides such as CGRP released from capsaicin-sensitive afferent nerves and the activation of vanilloid TRPV1 receptors located on these sensory nerve terminals.

The renin-angiotensin system and its vasoactive metabolite angiotensin-(1-7) in the mechanism of the healing of preexisting gastric ulcers. The involvement of Mas receptors, nitric oxide, prostaglandins and proinflammatory cytokines.

The inhibition of angiotensin-converting enzyme (ACE) or the blockade of angiotensin (Ang) AT-1 receptors affords protection against acute gastric mucosal injury, but whether the major metabolite of renin-angiotensin system (RAS), Ang-(1-7), accelerates the healing process of preexisting gastric ulcers remains unknown. Previous studies documented that Ang-(1-7) acting via its own Mas receptor exerts vascular responses opposing those of Ang II. We studied the effects of the Ang-(1-7)/Mas receptor axis on the healing rate of acetic-acid-induced gastric ulcers with or without the blockade of Mas receptors by A 779 and compared it with the effects of activation and blockade of the AT-1 receptor by the treatment with Ang II and losartan, respectively, the inhibition of ACE by lisinopril, the NO/cNOS inhibition by L-NAME and inhibition of prostaglandin/COX system by indomethacin in the presence of Ang-(1-7). Additionally, ex vivo metabolism of Ang I in gastric tissue was assessed by LC/MS method. At day 9 after ulcer induction, the area of these ulcers and the accompanying changes in total gastric blood flow (GBF) were determined as were gastric mucosal blood flow (GMBF) at ulcer margin and gastric oxygen uptake (GVO2). The gastric mucosal expression of mRNAs for constitutive nitric oxide synthase (cNOS), superoxide dismutase (SOD), and pro-inflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) and plasma level of both cytokines were determined by RT-PCR and ELISA. The 9 days treatment with Ang II dose-dependently increased the area of gastric ulcers and this effect was accompanied by a significant fall in the GBF, GVO2 and GMBF at ulcer margin. In contrast, treatment with Ang-(1-7) which produced a significant rise in the luminal content of NO significantly reduced the area of gastric ulcer and significantly increased the GBF, GVO2 and the GMBF at ulcer margin. Similar GMBF changes and significant reduction the area of gastric ulcer was observed in rats with gastric ulcers treated with the agonist of Mas receptor, AVE 0991. These effects of Ang-(1-7) and AVE 0991 were eliminated by blockade of the Mas receptor with A779. Similarly to Ang-(1-7), treatment with losartan or lisinopril significantly reduced the area of gastric ulcers and the accompanying increase in the GMBF at ulcer margin and these effects were significantly attenuated by a concomitant administration of L-NAME and indomethacin. The rate of healing of ulcers was associated with a decrease in ex vivo Ang-(1-7) formation and this effect was attenuated by lisinopril. The treatment with Ang-(1- 7) or AVE 0991 increased the expression of mRNA for cNOS and SOD and downregulated that of IL-1ß and TNF-α followed by the decrease in the plasma IL-1ß and TNF-α levels. We conclude that the Ang-(1-7)/Mas receptor system accelerates the healing of preexisting gastric ulcers via an increase in the gastric macro- and microcirculations, and an increase in gastric tissue oxygenation. These effects are mediated by PG and NO derived from overexpression of cNOS, an increase in the expression of antioxidizing enzyme SOD 2 and an anti-inflammatory action involving the inhibition of expression and release of pro-inflammatory cytokines IL-1ß and TNF-α. Our results seem to underlie the importance of the Ang-(1-7), AT-1 and Mas receptors in the regulation of local vascular and metabolic effects associated with mechanism of gastric ulcer healing.

The role of local and systemic cytokines in patients infected with Clostridium difficile.

It is widely accepted that the pathogenesis of Clostridium difficile infection (CDI) is multifactorial, dependent on pathogen virulence factors produced by the organism as well as disorders of the gastrointestinal tract, the alteration in intestinal flora and the immune response of the host. In particular, the immune response in the course of CDI and the involvement of cytokines in the pathogenesis of CDI is not fully understood. The aim of our study was to evaluate the relationship between proinflammatory and anti-inflammatory cytokines and the course of CDI in vivo. We prospectively studied 80 patients. Our study group included 40 patients aged 30-87 years (mean age 66.9 years) with CDI hospitalized at Infectious Diseases Department and Gastroenterology and Hepatology Clinic, University Hospital in Cracow, and 40 healthy volunteers aged 24-62 years (mean age 51.1 years). The serum concentrations of cytokines IL-1ß, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-α), myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were measured using ELISA assays. Additionally, the routine biochemical parameters were assessed including the following: white blood cells with differential leukocyte count, platelets counts, and blood plasma levels of creatinine, alanine transaminase, and C-reactive protein were determined. We noted a significant increase in the concentration of the following cytokines in the CDI group when compared to the control group: IL-1b (4.7 vs. 3.6 pg/ml), IL-6 (21.0 vs. 0.04 pg/ml), IL-10 (8.5 vs. 0.5 pg/ml), TNF-α (7.1 vs. 0.09 pg/ml). In addition the serum concentration of MPO (1056.0 vs. 498.0 pg/ml), and PGE2 (2036.7 vs. 1492.0 pg/ml) showed a significant increase in CDI patients as compared with control subjects. Most CDI patients did not show any increase in the concentration of IL-8. We did observe a direct relationship between TNF-α and creatinine. The course of CDI is characterized by an initial local inflammatory process followed by a systemic inflammatory response, which manifests clinically as fever, and includes leukocytosis, an increase in the level of neutrophils in the blood, and an increase in the serum concentrations of TNF-α, IL-1ß, IL-6, IL-10, MPO and PGE2. Despite the leading role of IL-8 in the local inflammatory process, we postulate TNF-α and IL-6 play a key role in the systemic inflammatory response in CDI, and the plasma TNF-α level seems to act as a major factor of poor prognosis in patients with CDI.

Involvement of cyclooxygenase-1 and cyclooxygenase-2 activity in the therapeutic effect of ghrelin in the course of ethanol-induced gastric ulcers in rats.

Previous studies have shown that treatment with ghrelin exhibits protective and therapeutic effects in the gut. Aim of our present investigation was to examine the influence of ghrelin administration on the healing of ethanol-induced gastric ulcers and determine the role of cyclooxygenase-1 and cyclooxygenase-2 in this effect. Our studies were performed on male Wistar rats. Gastric ulcers were induced by intragastric administration of 75% ethanol. Ghrelin alone or in combination with cyclooxygenase inhibitors was administered twice, 1 and 13 hours after ethanol application. Cyclooxygenase-1 (COX-1) inhibitor (SC-560, 10 mg/kg/dose) or COX-2 inhibitor (celecoxib, 10 mg/kg/dose) were given 30 min prior to ghrelin. Twelve or 24 hours after administration of ethanol, rats were anesthetized and experiments were terminated. The study revealed that administration of ethanol induced gastric ulcers in all animals and this effect was accompanied by the reduction in gastric blood flow and mucosal DNA synthesis. Moreover induction of gastric ulcer by ethanol significantly increased mucosal expression of mRNA for COX-2, IL-1ß and TNF-α. Treatment with ghrelin significantly accelerated gastric ulcer healing. Therapeutic effect of ghrelin was associated with significant reversion of the ulcer-evoked decrease in mucosal blood flow and DNA synthesis. Ghrelin administration also caused the reduction in mucosal expression of mRNA for IL-1ß and TNF-α. Addition of SC-560 slightly reduced the therapeutic effect of ghrelin in the healing of ethanol-induced ulcer and the ulcer area in rats treated SC-560 plus ghrelin was significantly smaller than that observed in rats treated with saline or SC-560 alone. Pretreatment with celecoxib, a COX-2 inhibitor, abolished therapeutic effect of ghrelin. We concluded that treatment with ghrelin increases healing rate of gastric ulcers evoked by ethanol and this effect is related to improvement in mucosal blood flow, an increase in mucosal cell proliferation, and reduction in mucosal expression of proinflammatory cytokines. Ghrelin is able to reverse a deleterious effect of COX-1 inhibitor on healing of ethanol-induced gastric ulcers. Activity of COX-2 is necessary for the therapeutic effect of ghrelin in healing of ethanol-induced gastric ulcers.

Esophagoprotective activity of angiotensin-(1-7) in experimental model of acute reflux esophagitis. Evidence for the role of nitric oxide, sensory nerves, hypoxia-inducible factor-1alpha and proinflammatory cytokines.

Gastroesophageal reflux disease (GERD) is a global disease rapidly increasing among world population. The pathogenesis of reflux esophagitis which is considered as the early stage of GERD is complex, resulting from an imbalance between aggressive factors damaging the esophagus and a number of the natural defense mechanisms. The esophageal mucosa is in a state of continuous exposure to potentially damaging endogenous and exogenous factors. Important aggressive components of gastric refluxate include acid and pepsin and also pancreatic enzymes and bile. Among aggressive factors of exogenous origin, cigarette smoking, non-steroidal anti-inflammatory drugs (NSAID), and steroids are of the utmost importance. The basic level of esophageal defense against acid-pepsin damage consists of the anti-reflux mechanisms such as the luminal acid clearance and removal of the esophageal contents and neutralization of luminal acidity. In addition the esophageal mucosal protection includes the presence of pre-epithelial, epithelial and post-epithelial cellular and functional components. Recently, the progress have been made in the understanding of role of the heptapeptide member of the renin-angiotensin system (RAS), angiotensin-(1-7) (Ang-(1-7)) in the control of gastrointestinal functions. It has been shown that all components of local RAS including Ang-(1-7) are detectable in the gastrointestinal wall including not only the stomach but also the esophagus. Previous studies revealed that Ang-(1-7), which is an important component of the RAS, exerts vasodilatory, anti-inflammatory and antioxidant activities in the stomach. Ang-(1-7) was recently implicated in gastroprotection, but its effects on esophageal mucosa in a rodent model of reflux esophagitis and in human subjects presenting GERD symptoms have not been explored. The present study was aimed to evaluate the possible protective effects of Ang-(1-7) and Mas-receptors upon esophageal mucosal damage in acute reflux esophagitis (RE) induced in anesthetized rats by ligating the pylorus and the limiting ridge (a transitional region between the forestomach and the corpus of stomach). Consequently, the total gastric reservoir to store gastric juice was greatly diminished, resulting in the reflux of this juice into the esophagus. Because Mas receptors are functionally linked to nitric oxide (NO) formation, we also studied involvement of endogenous NO in the mediation of protective and circulatory effects of exogenous Ang-(1-7). Moreover, an attempt was made to assess the possible role of sensory neurons in the modulation of the protective effects exerted by Ang-(1-7)/Mas receptor system. Six series of rats were pretreated 30 min before induction of RE with 1) vehicle (saline), 2) Ang-(1-7) (5-50 µg/kg i.p.), 3) A779 (50 µg/kg i.p.), the selective Mas receptor antagonist applied alone, 4) Ang-(1-7) (50 µg/kg i.p.) combined with A779, 5) L-NNA (20 mg/kg i.p.) administered alone, and 6) Ang-(1-7) (50 µg/kg i.p.) combined with L-NNA. In separate group of rats, capsaicin (total dosage of 125 mg/kg within three days) was administered s.c. 2 weeks before the induction of RE to induce functional ablation of sensory nerves. Rats with intact sensory nerves and those with capsaicin-induced sensory denervation received vehicle (saline) or Ang-(1-7) (50 µg/kg i.p.) to determine whether this vasoactive metabolite of angiotensin I could be also effective in rats with capsaicin-induced impairment of the synthesis and release of sensory neuropeptides such as CGRP. Four hours after induction of RE, the mucosal damage was graded with mucosal lesion index (LI) from 0 to 6, the esophageal microcirculatory blood flow (EBF) was determined by H2-gas clearance technique and plasma level of pro-inflammatory cytokines interleukin-1b (IL-1ß), and tumor necrosis factor-α (TNF-α) was determined by ELISA. The expression of proinflammatory factors including COX-2, cytokine IL-1ß and hypoxia inducible factor 1alpha (Hif1α) was analyzed in the esophageal mucosal biopsies. In rats with RE, the esophageal LI was significantly elevated comparing its value observed in intact rats, and the EBF was significantly decreased as compared with intact mucosa. Pretreatment with Ang-(1-7) of control rats without esophagitis induced increase in EBF by about 25% without any macroscopic changes in the esophageal mucosa or in the plasma level of cytokines. In animals with RE, pretreatment with Ang-(1-7) significantly reduced gross and histological esophageal mucosal injury and significantly increased EBF in comparison to vehicle-pretreated animals. The observed gross and histologic esophagoprotective effect of Ang-(1-7) was totally abolished by A779 so in rats with combined treatment of A779 with Ang-(1-7), the LI was identical with this observed in control RE and the EBF was decreased in these animals by about 39%. Inhibition of NO synthase by L-NNA significantly reduced the LI and the rise in EBF caused by Ang-(1-7). Similarly, the capsaicin denervation also significantly attenuated the vasodilatory and the esophagoprotective effects of Ang-(1-7). The expression of proinflammatory factors COX-2, Hif1α and IL-1ß which was negligible in intact esophageal mucosa, was upregulated in esophageal mucosa of rats with RE. In contrast, the administration of Ang-(1-7) resulted in a downregulation of mRNA for COX-2, Hif1 and IL-1ß in esophageal mucosa an this effect was abolished in A779-dependent manner. The Ang-(1-7) significantly decreased the RE-induced elevation of plasma levels of IL-1ß and TNF-α, and this effect was also reversed by pretreatment with A779, and significantly attenuated by pretreatment with L-NNA and capsaicin-induced sensory denervation. The present study indicates that the protective effect of Ang-(1-7) observed in the esophageal mucosa during early acute stage of gastroesophageal reflux depends upon the enhancement of esophageal microcirculatory blood flow via the activation of Mas receptor possibly due to NO synthase/NO system activation, stimulation of sensory nerves, the inhibition of expression of pro-inflammatory factors including COX-2, Hif1α and IL-1ß and release of proinflammatory cytokines IL-1ß and TNF-α.

Experimental healing of preexisting gastric ulcers induced by hormones controlling food intake ghrelin, orexin-A and nesfatin-1 is impaired under diabetic conditions. A key to understanding the diabetic gastropathy?

Hormonal peptides like ghrelin, orexin A (OXA) or nesfatin-1 not only regulate appetite, which is their basic biological function, but also contribute to mechanisms responsible for maintaining integrity of the gastric mucosa. Previous studies including those from our laboratory have revealed that their gastroprotective effect results from cooperation with other factors responsible for protection of the gastric mucosa, including prostaglandin (PG) synthesis pathway, nitric oxide (NO) and the sensory afferent fibres releasing the vasoactive neurotransmitters. The aim of the present study was to determine whether ghrelin, orexin-A (OX-A) or nesfatin-1 with their protective effect on the gastric mucosa, also can modify the healing of chronic gastric ulcers. Furthermore, an attempt was made to explain participation of these peptides in healing processes of chronic gastric ulcers with comorbid conditions for the human beings resulted from diabetes mellitus. In our study, a model of gastric ulcers caused by concentrated acetic acid to induce the chronic gastric ulcers was used, while the clinical condition corresponding to diabetes was induced by single injection of streptozotocin (STZ). We found that ghrelin, OX-A and nesfatin-1 accelerate dynamics of the acetic acid ulcers healing, confirmed by a reduction in the ulcer area and this effect was accompanied by an increase in gastric blood flow at the ulcer margin. Destruction of sensory afferent fibres with capsaicin or blocking of vanilloid receptors with capsazepine resulted in a significant reduction of ghrelin, OX-A and nesfatin-1-induced acceleration of ulcer healing. Similar results were obtained when an NO-synthase blocker, L-NNA was used in a combination with these peptides. Moreover, it was found that OX-A and nesfatin-1 failed to accelerate the healing process under diabetic condition because both these hormones induced reduction in the ulcer area and the increase in blood flow in normal, non-diabetic rats were completely lost in the group of animals with diabetes. Treatment with OX-A and nesfatin-1 increased superoxide dismutase (SOD) mRNA expression even in acetic acid ulcers concurrent with diabetes. However, the treatment with OX-A and nesfatin-1 failed to alter the increase in gastric mucosal mRNA expression for ghrelin and hypoxia-inducible factor 1-alpha (HIF-1α), this latter effect that had been strongly pronounced in diabetic animals. We conclude that the hormonal peptides involved in the regulation of satiety and hunger such as ghrelin, OX-A and nesfatin-1 contribute to the process of chronic gastric ulcers healing cooperating with NO and sensory afferent nerve endings releasing vasoactive neuropeptide CGRP. Furthermore, OX-A and nesfatin-1, the two relatively unrecognized peptides, play an essential role in healing process of chronic gastric ulcers activating the gastric blood flow at ulcer margin and the mucosal regeneration and both ulcer healing and accompanying hyperemia at ulcer margin are greatly impaired during diabetes. Possibly, loss of the healing effect of these peptides during diabetes results from an interaction with radical generation processes as reflected by an increase of mRNA expression for SOD as well as the failure of their attenuating activity on proinflammatory factors such as HIF-1α.

Molecular alterations in fibroblasts exposed to Helicobacter pylori: a missing link in bacterial inflammation progressing into gastric carcinogenesis?

Major human pathogen Helicobacter pylori (Hp) can colonize the gastric mucosa causing inflammation and being of potential risk for gastric cancer development but the contribution of fibroblasts to the pathogenesis of Hp in the stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts, but their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue. We determined the effect of coincubation of cultured rat gastric fibroblasts with alive Hp on the transdifferentiation of fibroblasts into myofibroblasts associated with Hp-induced inflammation and neoplasia. Gastric mucosal samples were harvested from 8-week-old Spraque-Dowley rats and cultured to obtain the sub-confluent fibroblasts. The isolated fibroblasts were infected with 1 x 10(9) of live Hp (ATCC 700824, cagA+, vacA+) per dish and incubated in humidified atmosphere for 3, 24 and 48 hours. At respective times, fibroblasts were harvested and the expression of mRNA for α-smooth muscle actin (SMA), hypoxia inducible factor (HIF)-1α, collagen I, heat shock protein (HSP)-70, heme oxygenase (HO)-1, Bax and Ki67 transcripts was determined by RT-PCR with specific primers. Hp increased the transdifferentiation of fibroblasts into myofibroblasts as reflected by the time-dependent overexpression of mRNA for α-SMA. The increased expression of HIF-1α and collagen I was observed in fibroblasts co-cultured with Hp. The expression of HSP70 which was negligible in isolated fibroblasts incubated with vehicle (saline) showed time-dependent 2-3 fold increase in those incubated with Hp. The HO-1 mRNA was strongly expressed in rat gastric fibroblasts without or with the co-incubation with Hp. The mRNA for Bax was progressively downregulated within the time of incubation while no significant changes in expression of proliferation marker Ki67 were recorded. We conclude that Hp-induced transdifferentiation of fibroblasts into myofibroblasts involves an increased expression of the early carcinogenic marker HIF-1α, and inhibition of proapoptotic Bax expression, and 2) the overexpression of HSP70 and the unchanged expression HO-1 and Ki67 probably represent the enhanced protective activity of Hp-infected fibroblasts to maintain their own integrity under inflammatory action of this bacteria and its cytotoxins.

Expression and release of leptin and proinflammatory cytokines in patients with ulcerative colitis and infectious diarrhea.

Leptin plays not only an important role in regulation of food intake, but also in the mechanism of inflammation. The universal presence of leptin in the cells of immune system and its secretion by these cells caused increasing interest in the role of this hormone in ulcerative colitis (UC). We determined the role of leptin in 80 patients, aged from 18 to 69 years, including 50 patients with active UC and 30 patients with infectious diarrhea. The tests were performed within 48 hours of the first symptoms, in the period of remission of UC and 8 weeks after resolution of infectious diarrhea. Endoscopy was performed in each patient, and the biopsy samples were taken for the assessments of expression of mRNA for leptin, IL-1ß, IL-6 and TNF-α by RT-PCR and Western blot. Blood tests included concentrations of leptin, IL-1ß, IL-6 and TNF-α. In addition, the plasma levels of leptin, IL-1ß, IL-6 and TNF-α were assessed by ELISA. Serum concentrations of leptin was significantly increased in patients with exacerbation of UC over that in patients with UC in remission. The serum leptin concentration was significantly higher in patients with infectious diarrhea, than the patients that recovered from infectious diarrhea. The leptin protein was overexpressed in the biopsy samples of the mucosa of large intestine compared to those with exacerbation of UC, and in patients after successful recovery from infectious diarrhea. The leptin mRNA was overexpressed in patients with infectious diarrhea compared with that in the group of patients after successful recovery from this condition. Serum concentrations of leptin failed to correlate with severity of exacerbation of UC and with extent of intestinal inflammatory lesions in patients with UC. However, the correlation was observed between serum concentrations of leptin in patients with exacerbation of UC and serum concentrations of proinflammatory cytokines IL-1ß and TNF-α. We conclude that 1) the increased leptin in exacerbated UC is related to the increased serum proinflammatory cytokines IL-1ß, TNF-α and IL-6 levels; 2) In patients with infectious diarrhea, the concentrations of leptin in intestinal mucosa correlates with serum concentrations of cytokines IL-1ß, IL-6 and TNF-α and with an increased expression of leptin mRNA in intestinal mucosa but not with alterations in serum levels of this hormone; 3) leptin may serve as useful predictive marker of inflammation in inflammatory bowel disease (IBD).

Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, interacts with gastric oxidative metabolism and enhances stress-induced gastric lesions.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase known to exert vasoconstriction of vascular bed. The elevation of ADMA has been considered as the cardiovascular risk factor associated with hyperlipidemia, hypercholesterolemia and metabolic syndrome. ADMA is produced by the action of dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Previous studies have shown that endogenous NO plays an important role in the mechanism of gastric mucosal defense, but the role of ADMA in the pathogenesis of serious clinical entity, such as the acute gastric mucosal injury induced by stress has been little studied. In present study, we determined the effect of intragastric (i.g.) pretreatment with ADMA applied in graded doses ranging from 0.1 up to 20 mg/kg on gastric mucosal lesions induced by 3.5 h of water immersion and restraint stress (WRS). The number of gastric lesions was determined by planimetry and the gastric blood flow (GBF) was assessed by laser Doppler technique. The malondialdehyde and 4-hydroxynonenal (MDA+4-HNE) concentration, as an index of oxygen radical-lipid peroxidation was assessed in the gastric mucosa in rats exposed to WRS with or without ADMA administration. Proinflammatory cytokines IL-1ß, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1ß and TNF-α were analyzed by RT-PCR and ELISA, respectively. The exposure of rats to WRS for 3.5 h produced acute gastric lesions accompanied by a significant rise in the plasma ADMA levels and a significant fall in the GBF, an increase in MDA+4-HNE concentrations and the significant increase in the expression and release of IL-1ß and TNF-α. The pretreatment with ADMA, applied i.g. 30 min before WRS dose-dependently, aggravated WRS damage and this effect was accompanied by a further significant fall in the GBF. The ADMA induced exacerbation of WRS lesions and the accompanying rise in the plasma ADMA levels and the fall in GBF were significantly attenuated by concurrent treatment with glyceryl trinitrate (GTN) (10 mg/kg i.g.) in the presence of ADMA. Administration of ADMA resulted in a significant decrease in the expression of SOD and GPx mRNAs and the up-regulation of mRNA for IL-1ß and TNF-α followed by an increase in these plasma cytokine levels as compared to respective values observed in vehicle-pretreated animals. We conclude that 1) ADMA could be implicated in the mechanism of WRS-induced ulcerogenesis, 2) ADMA exacerbates WRS-induced gastric lesions due to enhancement in neutrophil dependent lipid peroxidation and overexpression and release of proinflammatory cytokines IL-1ß and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity.

Novel concept in the mechanism of injury and protection of gastric mucosa: role of renin-angiotensin system and active metabolites of angiotensin.

The term cytoprotection pioneered by Robert and colleagues has been introduced to describe the remarkable ability of endogenous and exogenous prostaglandins (PGs) to prevent acute gastric hemorrhagic lesions induced by noxious stimuli such as ethanol, bile acids, hiperosmolar solutions and nonsteroidal anti-inflammatory agents such as aspirin. Since that time many factors were implicated to possess gastroprotective properties such as growth factors including epidermal growth factor (EGF) and transforming factor alpha (TGFα), vasodilatory mediators such as nitric oxide (NO) and calcitonin gene related peptide (CGRP) as well as appetite gut hormones including gastrin and cholecystokinin (CCK), leptin and recently ghrelin. This protective action of gut peptides has been attributed to the release of PG but question remains whether another peptide angiotensin, the classic component of the systemic and local renin-angiotensin system (RAS) could be involved in the mechanism of gastric integrity and gastroprotection. After renin stimulation, the circulating angiotensin I is converted to angiotensin II (ANG II) by the activity of the Angiotensin Converting Enzyme (ACE). The ANG II acting via its binding to two major receptor subtypes the ANG type 1 (AT1) and type 2 (AT2) has been shown be activated during stress and to contribute to the pathogenesis of cold stress- and ischemia-reperfusion-induced gastric lesions. All bioactive angiotensin peptides can be generated not only in systemic circulation, but also locally in several tissues and organs. Recently the new functional components of RAS, such as Ang-(1-7), Ang IV, Ang-(1-12) and novel pathways ACE2 have been described suggesting the gastroprotective role for the novel ANG II metabolite, Ang-(1-7). The fact that Ang-(1-7) is produced in excessive amounts in the gastric mucosa of rodents and that pretreatment by Ang-(1-7) exhibits a potent gastroprotective activity against the gastric lesions induced by cold-restraint stress suggests that this and possibly other vasoactive metabolites of ANG II pathway could be involved in the mechanism of gastric integrity and gastroprotection. This review summarizes the novel gastroprotective factors and mechanisms associated with metabolic fate of systemic and local RAS activation with major focus to recent advancement in the angiotensin pathways in the gut integrity.

MicroRNAs and chronic inflammation contribution to gastrointestinal integrity.

Recent studies have revealed that chronic inflammation represents a major basis for different forms of human malignancies. Chronic inflammations are involved in the pathogenesis of 15-25% of human malignancies. Gastrointestinal (GI) cancer is one of the most common causes of mortality in the European Union. The mechanisms leading to cancer development and its progression are not completely understood. Advances are required both in early detection and therapy of GI cancers. There are many factors connecting inflammation and cancer. Cytokines that are small protein molecules regulating growth, differentiation, development and immune response mechanisms in cells. Overexpression of cyclooxynenase-2 is associated with decreased apoptosis, cell to cell adhesion, increased proliferation and angiogenesis contributes to the increased immunosuppresion and mediates carcinogenetic effects. MicroRNAs are regarded as a novel class of gene expression regulators. They are gene-silencing RNAs which negatively regulate gene expression. After binding to target mRNAs they lead either to mRNA destruction or inhibition of translation. Hence, they can play an important role in carcinogenesis. Currently, almost all of the miRNA-related studies on cancers based on the different expression profile of miRNAs in cancer cells compared to normal cells. In summary, miRNAs, proinflammatory cytokines and other factors, may be involved in cancer development based on chronic inflammation by controlling cell differentiation and apoptosis. Assessing the role of miRNAs will provide the new insights on their contribution to the link between chronic inflammation and subsequent cancer, and new markers for cancer diagnoses and cancer therapy.

Mechanisms promoting physiological cells progression into tumorigenesis.

Cancer as the most frequent cause of death worldwide requires detailed investigation of its biology. This knowledge may open a new possibilities of generating novel targets, help to overcome issues of drug resistance, improve therapeutic efficacy, and make disease treatment more successful. The major advance in recent years was the discovery of the cancer stem cells (CSCs) population responsible for tumor maintenance. Numerous signalling pathways and genes connected with stem cell biology, such as an alternation in multiple malignancies resulting from the WNT/ß-catenin signalling pathway, have been identified. Crucial is knowledge concerning CSCs dependence and interactions with adjacent stromal cells that comprise a specialized microenvironment or niche. The niche shelters cells from diverse genotoxic factors thereby strengthening antitumor therapy resistance, and supports the growth of primary tumors converting non-tumorigenic cells into CSCs by processes related to the epithelial-to-mesenchymal transition (EMT). Moreover, numerous experiments confirmed that target genes of WNT signalling are implicated in cell-adhesion, which in consequence has an impact on EMT. This suggests a model that integrates a number of fundamental processes that underlie disease development and it should be put forward as an important target for novel therapies. Hence, linking the potency of silanols moiety, as innovative inhibitors of EMT resistant with the anticancergenic properties of selenium agents that targeting genetic basis of cancer stem cells development, requires a need for a design, synthesis and evaluation of novel compounds as a prospective direction of cancer research.

Antibiotic treatment with ampicillin accelerates the healing of colonic damage impaired by aspirin and coxib in the experimental colitis. Importance of intestinal bacteria, colonic microcirculation and proinflammatory cytokines.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory, analgesic and antipyretic effects, however their use is associated with the broad spectrum of side effects observed in human as well as the experimental animals. Despite damaging activity of NSAIDs in upper gastrointestinal (GI) tract, these drugs exert deleterious influence in lower GI tract, including colon. The role of GI microflora in the pathogenesis of NSAIDs-induced experimental colonic damage is not completely understood. The aim of this study was 1) to evaluate the relative importance of the GI microflora on the experimental colonic damage in the presence of caused by NSAID, and 2) to assess the efficacy of antibiotic treatment with ampicillin on the process of healing of colitis. We compared the effect of vehicle, ASA applied 40 mg/kg intragastrically (i.g.) or the selective cyclooxygenase (COX)-2 inhibitor, celecoxib (25 mg/kg i.g.) without or with ampicillin treatment (800 mg/kg i.g.) administered throughout the period of 10 days, on the intensity of TNBS-induced colitis in rats. The severity of colonic damage, the alterations in the colonic blood flow (CBF) and myeloperoxidase (MPO) activity, the mucosal expression of TNF-α, IL-1ß, COX-2, VEGF and iNOS and the plasma concentration of TNF-α and IL-1ß were assessed. In all rats, the faeces samples as well as those from the colonic mucosa, blood, liver and spleen underwent microbiological evaluation for intestinal bacterial species including Escherichia coli and Enterococcus spp. The administration of TNBS resulted in macroscopic and microscopic lesions accompanied by the significant fall in the CBF, an increase in tissue weight and 4-5-fold rise in the MPO activity and a significant increase in the plasma IL-1ß and TNF-α levels. ASA or celecoxib significantly increased the area of colonic lesions, enhanced MPO activity and caused the marked increase in colonic tissue weight and plasma IL-1ß and TNF-α levels, as well as an overexpression of mRNA for IL-1ß and TNF-α, COX-2, VEGF and iNOS in the colonic tissue. ASA and coxib also resulted also in a significant increase of E. coli counts in the stool at day 3 and day 10 day of the observation compared with the intact rats. Moreover, E. coli translocation from the colon to the blood and extraintestinal organs such as liver and spleen in the group of rats treated without or with ASA and coxib. E. coli was the most common bacteria isolated from these organs. Treatment with ampicillin significantly attenuated the ASA- or celecoxib-induced increase in plasma levels of IL-1ß and TNF-α and suppressed the mucosal mRNA expression for IL-1ß and TNF-ß, COX-2, iNOS and VEGF in the colonic mucosa. Ampicillin administration caused a significant fall in the number of E. coli in the faeces at day 3 and day 10 of observation in ASA- and coxib-treated rats with colitis. Antibiotic therapy markedly reduced bacterial translocation to the colonic tissue and the extraintestinal organs such as the liver and spleen. We conclude that administration of ASA and to lesser extent of celecoxib, delays the healing of experimental colitis and enhances the alterations in colonic blood flow, proinflammatory markers such as IL-1ß, TNF-α, COX-2, iNOS and VEGF and increased intestinal mucosal permeability resulting in the intestinal bacterial translocation to the blood, spleen and liver. Antibiotic treatment with ampicillin is effective in the diminishing of the severity of colonic damage, counteracts both the NSAID-induced fall in colonic microcirculation and bacterial E.coli translocation to the extraintestinal organs.

Nitric oxide (NO)-releasing aspirin exhibits a potent esophagoprotection in experimental model of acute reflux esophagitis. Role of nitric oxide and proinflammatory cytokines.

The purpose of this study was to develop an acute animal model of reflux esophagitis, which would be suitable to induce the esophageal damage caused by gastric acid reflux, thus mimicking the esophageal injury of human gastroesophageal reflux disease (GERD). Global research indicates that GERD is rapidly increasing among the world's population. NSAIDs are known to induce gastrointestinal damage and low doses of aspirin (ASA) have been shown to increase the incidences of GERD in humans. Gastric acid and pepsin secretion and enhanced COX-2 expression were implicated in the pathogenesis of reflux esophagitis, but the effect of selective COX-2 inhibitors against lesions induced by the reflux of gastric acid content into esophagus has not been thoroughly studied. Here, we compared the effect of aspirin (ASA) and so called "safe" nitric oxide (NO) derivative of ASA with those of non-selective and selective cyclooxygenase (COX)-1 and COX-2 in rat model of reflux esophagitis. Reflux esophagitis was induced in anesthetized rats by ligating the pylorus and limiting ridge transitional region between the forestomach and the corpus of stomach. Subsequently, the total gastric reservoir to store gastric juice was greatly diminished, resulting in the reflux of this juice into the esophagus. Rats with esophagitis received intragastric (i.g.) pretreatment either with: 1) vehicle (saline), 2) ASA or NO-ASA (100 mg/kg); 3) the non-selective COX inhibitor, indomethacin (5 mg/kg); 4) the selective COX-1 inhibitor, SC-560 (10 mg/kg), and 5) the selective COX-2 inhibitor, celecoxib (5 mg/kg). In a separate series of rats with reflux oesophagitis, the efficacy of ASA combined with a donor of NO, glyceryl trinitrate (GTN; 10 mg/kg i.g.) to prevent esophageal mucosal injury was investigated. Four hours after induction of esophagitis the gross mucosal damage was graded with a macroscopic lesion index (LI) from 0-6. The esophageal blood flow (EBF) was determined by H2-gas clearance technique, the oesophageal mucosal and blood samples were collected for histology and analysis of the RT-PCR expression and release of proinflammatory cytokines IL-1ß, TNF-α and IL-6 using specific ELISA. The exposure of the esophagus to reflux of gastric acid time-dependently increased the esophageal LI and morphologic damage, and decreased EBF with the most significant changes observed at 4 hrs after the ligation procedure. The pretreatment with native ASA in the dose that suppressed the generation of mucosal PGE2, enhanced gross and histologic esophageal damage and produced a significant fall in EBF. NO-ASA or ASA coupled with GTN counteracted the aggravation of the damage and accompanying fall in EBF when compared with native ASA applied alone to rats with esophagitis. The proinflammatory cytokines IL-1ß and TNF-α were overexpressed in rats with esophagitis and those pretreated with ASA but this effect was significantly attenuated by NO-ASA. Plasma IL-1ß, TNF-α and IL-6 were negligible in the intact rats but significantly increased in those with esophagitis, with this effect being further enhanced by non-selective (indomethacin) and selective (SC-560, celecoxib) COX-1 and COX-2 inhibitors. We conclude that conventional NSAID such as aspirin augments esophagitis, while NO-ASA exerts the beneficial protective effect against reflux esophagitis via the enhancement of esophageal microcirculation due to NO release and an inhibitory effect on expression and release of pro-inflammatory cytokines.

Cyclooxygenase-2 (COX-2) is the key event in pathophysiology of Barrett's esophagus. Lesson from experimental animal model and human subjects.

Mixed reflux of the gastroduodenal contents induces the esophageal mucosal damage and inflammation progressing chronic esophagitis and premalignant Barrett's esophagus (BE). The role of cyclooxygenase-2 (COX-2) and chronic inflammation in the progression of BE toward adenocarcinoma of the esophagus has not been extensively studied in experimental models of BE in animals and in human subjects. We evaluated the expression of COX-2 in rat model of BE and examined the usefulness of COX-2 expression in determining the risk of malignant transformation in patients with BE treated with argon plasma coagulation (APC) that allows for effective ablation of metaplastic mucosa (group A) without or with proton pump inhibitors (PPI). In addition, the group B of patients was subjected to laparoscopic Nissen's fundoplication and group K that served as control, received PPI treatment only. Expression of COX-2 was evaluated in fresh-frozen biopsy specimens obtained from the distal esophagus in all 60 patients before and 12 months after treatment. In experimental studies, eighty rats were surgically prepared with esophagogastroduodenal anastomosis (EGDA) resulting in chronic esophagitis. At 4 months, the esophageal damage in EGDA rats was evaluated by macroscopic and histological index score, the plasma IL-1beta and TNF-alpha levels was determined by ELISA and the mucosal expression of COX-2 mRNA and COX-2 protein were assessed by RT-PCR and Western Blot, respectively. Chronic esophagitis was developed in all EGDA animals followed by the rise in the plasma TNF-alpha and IL-1beta levels. Histology revealed extensive esophageal ulcerations with development of columnar epithelium, formation of mucus glands in squamous epithelium, intestinal metaplasia distant to anastomosis consisting of goblet cells, infiltration of inflammatory cells including plasma cells and lymphocytes. COX-2 mRNA was absent in the esophageal mucosa of sham-control animals but strongly upregulated in metaplastic Barrett's epithelium. In BE patients, the overexpression of COX-2 was documented in patients with dysplasia. After APC (group A) or Nissen's fundoplication (group B), the expression of COX-2 mRNA was markedly reduced and these effects were positively correlated with histopathological findings. Controls failed to show significant alterations in COX-2 expression. We conclude that 1) EGDA rats serve as the suitable model of the chronic esophagitis by the gastrointestinal refluxate resembling many features of those observed in human Barrett's esophagus, as confirmed by severe morphology changes, excessive release of proinflammatory cytokines TNF-alpha and IL-1beta and overexpression of COX-2, and 2) the significant correlation of the degree of COX-2 overexpression with histopathological findings indicates the usefulness of this inducible biomarker as a valuable indicator of the risk of malignant transformation in patients with BE.

Monocytes and vascular endothelial cells apoptosis. Role of p-HSP27.

The aim of this study was to find out whether stimulated monocytes could trigger apoptosis of vascular endothelial cells. Human umbilical vein endothelial cells (HUVEC) (EC) were co-cultured for 24 h and 48 h with monocytes isolated from peripheral blood (peripheral blood monocytes) or MonoMac6 cell line activated previously with proinflammatory cytokines. Real-time PCR was conducted to investigate p53 up-regulated modulator of apoptosis (PUMA), heat shock protein HSP70 and HSP27 genes expression. Changes in the level of PUMA, HSP70, HSP27 and phospho-heat shock protein 27 (p-HSP27) proteins were analyzed by means of immunoprecipitation. Apoptosis was determined by TUNEL and poli-(ADP ribose) polymerase ( PARP ) cleavage assay. In HUVEC cells stimulated with monocytes hardly any increase of PUMA mRNA was observed, but the PUMA protein level was significantly up regulated especially after 24 h. Heat shock proteins (HSP70 and HSP27) mRNA expression was elevated after 24 h and 48h and confirmatory up regulation of these proteins was observed in HUVEC cells stimulated with peripheral blood monocytes but not with MonoMac6 cells. Interestingly, in nuclear compartment of HUVECs exposed to the monocytic line and native monocytes, a significant increase of p-HSP27 level has appeared. TUNEL and PARP cleavage assay did not show any apoptotic HUVEC cells after stimulation with monocytes. The main observations of this study indicate that monocytes do not trigger apoptosis of vascular endothelial cells. Proapoptotic activation mediated by PUMA that was observed seemed to be counterbalanced by significant increase of antiapoptotic HSP70, HSP27 and especially phospho-HSP27 proteins level.