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.

Effects of peripherally and centrally applied ghrelin in the pathogenesis of ischemia-reperfusion induced injury of the small intestine.

Ghrelin is an important hormone involved in the control of the human appetite center. Recently, protective properties of this hormone have been recognized in various models of impairment of the gastric mucosa, including stress, ischemia and reperfusion (I/R). Ghrelin is predominantly secreted by the gastric mucosa of stomach, but there are other sources of ghrelin, for example in the hypothalamus and various parts of the central nervous system (CNS) that should be taken into consideration. This hormone exerts biological effects via the activation of growth hormone secretagogue receptor (GHSR), the presence of which was confirmed in different parts of the gastrointestinal (GI) tract and midbrain structures. Although substantial evidence of the divergent biological effects of ghrelin and the mechanism of its action has been emphasized, the precise mechanisms of ghrelin which affords GI protection is still unclear. Particularly, there is a sparse amount of evidence concerning its action on the GI system. The major aim of the present study was to evaluate the importance of peripherally and centrally administered ghrelin at different times of the ischemia and reperfusion (I/R period in the modulation of resistance of the intestinal mucosa to the injury induced by ischemia and subsequent reperfusion. Secondly, we wanted to evaluate the possible mechanism of the action of ghrelin with a particular focus on its influence on the intestinal blood flow. Male Wistar rats were divided into 4 series (A-D) of the experimental groups (n=7). In series A the importance of peripherally administered ghrelin at different time of I/R period was studied. In series B the importance of centrally administered ghrelin at different time of I/R period was evaluated. In series C and D, the mechanisms of peripherally and centrally administered hormone were examined, respectively. Two models of the I/R period were selected: short lasting (30/60 min) and long lasting (60/120 min). The following drugs were used: ghrelin (50 µg/kg i.p. or 1 nmol in 10 µl i.c.v.), 6 hydroxy dopamine (50 mg/kg i.p.), nadolol (0.5 mg/kg i.p.), calcitonin gene related peptide fragment (CGRP(8-37), 100 µg /kg i.p.), capsaicin (5-10 mg/100 ml solution s.c.). The mesenteric blood flow (MBF-ml/min), the intestinal microcirculatory blood flow (LDBF-PU), the arterio-venous oxygen difference (AVO(2)-ml/O(2)/100 ml blood), and the intestinal oxygen uptake (VO(2)) in ml O(2)/min were measured. Mucosal impairment was assessed planimetrically with the use of a digital photo analyzer (LA) and histologically with the use of the six-point Park/Chiu scale. Peripheral administration of ghrelin evoked marked increase of MBF and LDBF by 42% and 48%, respectively, with significant reduction of LA by 38%. When ghrelin was administered at the beginning of the reperfusion period during the short I/R period or prior to the long lasting I/R period, the vascular reactions and protective effects were reduced, but not completely abolished. The central administration of ghrelin before the short I/R period significantly increased the MBF and LDBF by about 32% and 35%, respectively, as well as LA reduction by about 20% in comparison to the control group. However, when ghrelin was administered prior to the long I/R period or after the onset of completed ischemia, neither vascular nor protective effects were noticed. Sensory denervation and the blockade of the CGRP1 receptors totally blocked the protective and hyperemic effects of the peripherally administered ghrelin. Selective blockade of the adrenergic system or blunting of the vagal nerves (vagotomy) significantly but not totally eliminated the effects of centrally applied ghrelin, which were abolished when both adrenergic and parasympathetic pathways were ablated. These results indicate that ghrelin applied centrally or peripherally markedly increases resistance of the intestinal tissue during the I/R period induced mucosal and hyperemic impairment evoked by I/R. Ghrelin is an important mediator of the increase in the intestinal microcirculation and elevation of the intestinal metabolism, which seems to be, at least in part, responsible for the observed protection of the intestine subjected to I/R. Impairment of this microvasculature response due to I/R seems to be responsible for a markedly observed weaker effect of ghrelin when this hormone was administered after the ischemic period. The lack of a protective effect observed after central administration of this peptide against a long lasting I/R period is probably due to damage of neural pathways caused by I/R. Finally, the peripheral activity of ghrelin in the intestine is mediated by the sensory neurons with a prominent role of CGRP released from their endings. However, this peripheral action of ghrelin depends upon the proper functioning of both the sympathetic and parasympathetic system.

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.

Treatment with ghrelin accelerates the healing of acetic acid-induced gastric and duodenal ulcers in rats.

UNLABELLED: Recent studies have shown that ghrelin exhibits gastroprotective effects. The aim of present study was to examine the influence of ghrelin administration on the healing of chronic gastric and duodenal ulcers and to evaluate the role of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in this process. In pituitary-intact or hypophysectomized rats, chronic gastric and duodenal ulcers were induced by acetic acid. After induction of ulcers, rats were treated intraperitoneally twice a day with saline, ghrelin (4, 8 or 16 nmol/kg/dose) or IGF-1 (20 nmol/kg/dose) for six or ten days. In animals with intact pituitary, treatment with ghrelin increased serum level of GH and IGF-1. These effects were accompanied by the increase in mucosal cell proliferation, mucosal blood flow and healing rate of gastric and duodenal ulcers. After hypophysectomy, the significant increase in serum level of endogenous ghrelin was observed, but the healing of gastric and duodenal ulcers was delayed. This effect was accompanied by a significant decrease in serum concentration of endogenous GH and IGF-1, and reduction in mucosal blood flow and DNA synthesis. In hypophysectomized rats, administration of exogenous ghrelin was without any effect on serum level of GH and IGF-1, healing rate of gastroduodenal ulcers or mucosal cell proliferation. In contrast to this effect, administration of IGF-1 increased mucosal cell proliferation, healing rate of gastroduodenal ulcers and mucosal blood flow in hypophysectomized rats. CONCLUSION: Treatment with ghrelin accelerates healing of chronic gastroduodenal ulcers and this effect is mediated by the release of endogenous GH and IGF-1.

Effect of Candida colonization on human ulcerative colitis and the healing of inflammatory changes of the colon in the experimental model of colitis ulcerosa.

The influence of fungal colonization on the course of ulcerative colitis (UC) has not been thoroughly studied. We determined the activity of the disease using clinical, endoscopic and histological index (IACH) criteria in UC patients with fungal colonization and the healing process of UC induced by an intrarectal administration of trinitrobenzene sulfonic acid (TNBS) in rats infected with Candida, without and with antifungal (fluconazole) or probiotic (lacidofil) treatment. The intensity of the healing of the colonic lesions was assessed by macro- and microscopic criteria as well as functional alterations in colonic blood flow (CBF). Myeloperoxidase (MPO) content and plasma proinflammatory cytokines IL-1beta and TNF-alpha levels were evaluated. Candida more frequently colonized patients with a history of UC within a 5-year period, when compared with those of shorter duration of IBS. Among Candida strains colonizing intestinal mucosa, Candida albicans was identified in 91% of cases. Significant inhibition of the UC activity index as reflected by clinical, endoscopical and histological criteria was observed in the Candida group treated with fluconazole, when compared to that without antifungal treatment. In the animal model, Candida infection significantly delayed the healing of TNBS-induced UC, decreased the CBF and raised the plasma IL-1beta and TNF-alpha levels, with these effects reversed by fluconazole or lacidofil treatment. We conclude that 1) Candida delays healing of UC in both humans and that induced by TNBS in rats, and 2) antifungal therapy and probiotic treatment during Candida infection could be beneficial in the restoration and healing of colonic damage in UC.

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.

Mucosal strengthening activity of central and peripheral melatonin in the mechanism of gastric defense.

This review summarizes the involvement of centrally and peripherally applied melatonin, a major hormone of pineal gland, in the mechanism of gastric mucosal integrity, gastroprotection and ulcer healing. Melatonin was originally shown to attenuate gastric mucosal lesions but the controversy exists in the literature as to whether melatonin derived from the pineal gland, considered as the major source of this indole or rather that locally generated from L-tryptophan within gastric mucosa, plays predominant role in the mechanism of gastrointestinal integrity. Both, intragastric (i.g.) and intracerebroventricular (i.c.v.) administration of melatonin and its precursor, L-tryptophan to rats without or with removed pineal gland by pinealectomy attenuates in the dose-dependent manner the formation of on gastric lesions induced by topical irritants and water immersion restraint stress (WRS). Melatonin accelerated the gastric ulcer healing and this was accompanied by the rise in gastric blood flow (GBF), the plasma melatonin and gastrin levels, the mucosal generation of PGE(2) and luminal NO content. Pinealectomy, which suppresses the plasma melatonin levels, markedly aggravated the gastric lesions induced by WRS. Concurrent supplementation of pinealectomized animals with melatonin or L-tryptophan, the melatonin precursor, attenuated the lesions induced by WRS. Treatment with luzindole, an antagonist of Mel(2) receptors, or with L-NNA, the NO-synthase inhibitor, significantly attenuated melatonin- and L-tryptophan-induced protection and the acceleration of ulcer healing and the accompanying increase in the GBF and luminal content of NO. We conclude that 1) exogenous melatonin and that released from the L-tryptophan attenuate lesions induced by topical irritant such as ethanol and WRS via interaction with MT(2) receptors and due to an enhancement of gastric microcirculation, probably mediated by NO and PG derived from cNOS, iNOS and COX-2 overexpression and activity, and 2) the pineal gland plays an important role in the limitation of WRS-induced gastric lesions and acceleration of ulcer healing via releasing melatonin predominately at night time, that exerts gastroprotective and ulcer healing actions.

Effect of neonatal endotoxemia on the pancreas of adult rats.

UNLABELLED: Bacterial endotoxin (lipopolysaccharide, LPS), is the component of the cellular wall of Gram negative bacteria. Endotoxemia (sepsis) could produce multiorgan failure and could be particularly danger in the early period of life. The effects of endotoxemia induced in the neonatal period of life on the pancreatic secretory function and on pancreatic defense of adult organism have not been investigated yet. To induce endotoxemia suckling rats (30 g) have been injected intraperitoneally with LPS from E. coli (5, 10 or 15 mg/kg-day) during 5 consecutive days. Three months later in these animals (300 g) the studies on pancreatic secretion and acute pancreatitis were carried out. In the adult rats, which have been subjected in infancy to endotoxemia, basal pancreatic secretion was unaffected, whereas amylase secretions stimulated by caerulein or by diversion of pancreatic-biliary juice to the exterior were significantly, and dose-dependently reduced as compared to the untreated control. In the rats pretreated with LPS in the suckling period of life caerulein-induced amylase release from isolated pancreatic acini was significantly decreased, and dose-dependent reduction of mRNA signal for CCK1 receptor on pancreatic acini have been observed. Caerulein infusion (25 microg/kg) produced caerulein induced pancreatitis (AP) in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS (10 or 15 mg/kg-day x 5 days) all manifestations of AP have been reduced. In these animals acute inflammatory changes of pancreatic tissue have been significantly diminished. Pancreatic weight and plasma lipase activity, have been markedly decreased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in an antioxidative enzyme; SOD concentration was reversed and accompanied by significant reduction of lipid peroxidation products; MDA+ 4 HNE in the pancreatic tissue. CONCLUSIONS: 1/ neonatal endotoxemia reduces gene expression for CCK1 receptor and could produce impairment of the exocrine pancreatic function at adult age; 2/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age and this effect could be related to the increased concentration of antioxidative enzyme SOD in the pancreatic tissue.

Protective and therapeutic effect of heparin in acute pancreatitis.

UNLABELLED: The initiation and progression of acute pancreatitis is associated with disturbances in pancreatic microcirculatory. Microcirculatory disorders contribute to multiorgan dysfunction syndrome in the course of acute pancreatitis. The aim of this study was to determine the influence of heparin administration on the development and the course of ischemia/reperfusion-induced pancreatitis. METHODS: Acute pancreatitis was induced in rats by pancreatic ischemia followed by reperfusion. In the first series of studies, heparin was administered 0.5 h before induction of acute pancreatitis and the severity of acute pancreatitis was assessed after 6-h reperfusion. In the second series of studies, heparin was administered twice a day, starting 24 h after the initiation of reperfusion. In both series of studies, heparin was administered subcutaneously at the dose of 150 U/kg. RESULTS: Treatment with heparin, before induction of pancreatitis, inhibits the development of morphological signs of acute pancreatitis and reduced the pancreatitis-evoked increase in plasma level of pancreatic enzymes and pro-inflammatory interleukin-1beta. These effects have been accompanied with improvement of pancreatic blood flow, pancreatic DNA synthesis and reduction in plasma concentration of D-dimer. Administration of heparin after induction of acute pancreatitis accelerates normalization of pancreatic histology, and reduces biochemical markers of the severity of acute pancreatitis. These effects have been accompanied with the improvement of pancreatic circulation, increase in APTT and reduction in plasma D-dimer level. CONCLUSIONS: Treatment with heparin inhibits the development of ischemia/reperfusion-induced pancreatitis and accelerates pancreatic regeneration in the course of this disease.

Brain-gut and appetite regulating hormones in the control of gastric secretion and mucosal protection.

The progress in basic and clinical gastrology indicates that gastric mucosal integrity represents a balance between offensive and defensive factors. The main offensive factors appear to be gastric acid and pepsin under health conditions, while the nonsteroidal anti-inflammatory drugs (NSAID) and Helicobacter pylori (H. pylori), infecting this mucosa, are currently considered the most important "aggressive" factors under pathological conditions. To the list of the aggressive factors, also stress, certain cytokines (TNF-alpha, IL-8, IL-11 and IL-18) and oxygen or nitrogen free radicals should be added. The aims of this review is the presentation of the involvement of aggressive and protective factors in the control of gastric acid secretion and appetite regulating hormones in maintaining gastric mucosal integrity and its protection against damaging factors.

Physiological mediators in nonsteroidal anti-inflammatory drugs (NSAIDs)-induced impairment of gastric mucosal defense and adaptation. Focus on nitric oxide and lipoxins.

Prostaglandins mediate various physiological aspects of mucosal defense and the suppression of prostaglandin synthesis in the stomach is a critical event in terms of the development of mucosal injury after NSAID administration. However, it has become clear that other mediators besides prostaglandins can similarly act to protect the stomach from injury. For instance, nitric oxide (NO) released from vascular epithelium, epithelial cells of gastrointestinal tract and sensory nerves can influence many of the same components of mucosal defense as do prostaglandins. Thus, administration of NO in a form of NO-donors exert protective influence on the stomach from the injury that usually occurs when mucosal prostaglandin levels are suppressed. The new class of NO releasing NSAIDs, including NO-aspirin, represent a very promising approach to reducing the toxicity of anti-inflammatory drugs. Lipoxins are another group of lipid mediators that can protect the stomach. Aspirin-triggered lipoxin synthesis, via COX-2, acts to reduce the severity of damage induced by this drug. Lipoxin analogues may prove to be useful for preventing mucosal injury and for modulating mucosal inflammation. Aspirin-triggered lipoxin also seems to play in important role in gastric adaptation during chronic aspirin administration. Suppression of COX-2 activity by selective COX-2 inhibitors abolishes the production of this endogenous gastroprotective substance and diminishes the gastric tolerability of NSAIDS and gastric adaptation to these drugs. This review was designed to give an updated overview on the physiological factors and experimental and clinical attempts that were used or may be used in the future as the therapeutic approach to counteract adverse effects in the stomach associated with NSAID ingestion.

Nitric oxide (NO)-releasing aspirin and (NO) donors in protection of gastric mucosa against stress.

Acute gastric mucosal lesions represent an important clinical problem. The experimental model of acute gastritis such as water immersion restraint (WRS) stress is useful tool in examination of pathomechanism of acute gastric damage. Nitric oxide (NO) plays an important role in the maintenance of gastric barrier, however the role of reactive oxygen species (ROS) in the interaction between NO and gastric mucosa integrity has been little studied. The purpose of our present study was to explain the participation of ROS in healing of WRS-induced gastric lesions accelerated by NO. Experiments were carrying out on 120 male Wistar rats. To assess gastric blood flow (GBF) laser Doppler flowmeter was used. The number of gastric lesions was established by planimetry. The colorimetric assays were used to determine gastric tissue level of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), the products of lipid peroxidation by ROS, as well as superoxide dismutase (SOD) activity, the enzyme scavanger of ROS. We demonstrated that 3.5 h of WRS resulted in appearance of acute gastric mucosal lesions accompanied by a significant decrease of GBF. Biological effects of ROS were estimated by measuring tissue level of MDA and 4-HNE, as well as the SOD activity. It was demonstrated that 3.5 h of WRS led to significant increase of MDA and 4-HNE mucosal level, that was accompanied by a decrease of SOD activity. Pretreatment with NO-donors (SIN-1, SNAP, nitroglycerin, NO-ASA) resulted in reduction of gastric lesions number, increment of GBF, decrease of MDA and 4-HNE tissue level and increase of SOD activity. Suppression of ROS play an important role in NO-donors action in gastroprotection against gastric acute lesions induced by 3.5 h of WRS. NO-donors cause an attenuation of lipid peroxidation as documented by a decrease of MDA and 4-HNE levels and enhancement of antioxidative properties as evidenced by increase of SOD activity.

Molecular mechanism of protection against chemically and gamma-radiation induced apoptosis in human colon cancer cells.

The involvement of peroxisome proliferator-activated receptors (PPARs) in the cancer cell elimination through apoptosis is a generally accepted fact. However, some reports indicate that the activation of PPARgamma is directly responsible for carcinogenesis. Caco-2 cells, a human adenocarcinoma cells, were used as a model of colon cancer. Cell cultures (5 x 10(6) cell per dish) were pretreated for 24 h with PPAR gamma agonists ciglitazone (CI, 1 x 10(-6)M) and retinoic acid (RA, 1 x 10(-6)M) and part of the cultures were subsequently subjected to gamma-radiation (photons) with therapeutic dose of 2,5 Gy. Total cellular RNA and proteins (cytoplasmic and nuclear) were isolated 24h after cultures irradiation or 48 h after stimulation in the non irradiated part of experiment to preserve the equal growth time for all samples. gamma-Irradiation of the cells abolished nuclear translocation of PPARgamma under its agonists treatment and preserved PPARgamma in the cytoplasmic pool. But it did not affect the HSP 70 expression in response to ciglitazone and retinoic acid. Moreover, combined gamma-irradiation and CI/RA treatment of the cells changed the equilibrium between Bax and Bcl-2 mRNA to anti apoptotic state with increased expression of Bcl-2 and almost abolished expression of Bax. In conclusion, this paper provides an evidence for the anti-apoptotic action of PPARgamma agonists used along with the gamma-radiation. Moreover, it shows that the up-regulated HSP70, in response to PPARgamma agonists in gamma-irradiated cultures promotes cell survival.

Ischemic preconditioning of the hindlimb or kidney does not attenuate the severity of acute ischemia/reperfusion-induced pancreatitis in rats.

UNLABELLED: Ischemic preconditioning of several organs, including the pancreas has been shown to protect these organs from injury evoked by subsequent exposure to severe ischemia followed by reperfusion. Moreover, it has been shown that ischemic preconditioning of distant organs such as the kidney, intestine or limb may protect the heart as effectively as cardiac preconditioning itself. This study was designed to determine whether ischemic preconditioning of the kidney or hindlimb protects the pancreas against ischemia/reperfusion-induced pancreatitis. METHODS: In male Wistar rats, remote ischemic preconditioning of the pancreas was performed by clamping of right femoral or renal artery twice for 5 min with 5 min interval. Direct ischemic preconditioning was performed by clamping of celiac artery. Thirty min after ischemic preconditioning or sham-operation, acute pancreatitis was induced by clamping of inferior splenic artery for 30 min followed by reperfusion. After 6, 12 h or 1, 2, 3, 5 or 9 days of reperfusion the experiment was ended. Secretory studies were performed 2 h after exposure to direct or remote ischemic preconditioning of the pancreas in conscious rats with chronic pancreatic fistula. RESULTS: Direct ischemic preconditioning of the pancreas applied alone reduced pancreatic exocrine secretion; whereas ischemic preconditioning of the hindlimb or kidney was without effect on pancreatic secretion. Direct ischemic preconditioning of the pancreas attenuated the severity of acute pancreatitis. It was found as a reduction in the pancreatitis-evoked increase in serum activity of lipase and amylase, a decrease in serum concentration of pro-inflammatory interleukin-1beta, diminution of histological signs of pancreatic damage, as well as, an improvement of pancreatic blood flow and DNA synthesis. Remote ischemic preconditioning of the pancreas evoked by short-lasting ischemia of the hindlimb or kidney was without any protective effect in ischemia/reperfusion-induced pancreatitis. Moreover, this procedure led to a significant increase in serum activity of lipase and amylase, and enhanced the morphological signs of pancreatic damage. CONCLUSION: In contrast to direct ischemic preconditioning, remote ischemic preconditioning of the pancreas is without effect on pancreatic exocrine secretion and does not reduce the severity of ischemia/reperfusion-induced pancreatitis.

Gastric secretion, proinflammatory cytokines and epidermal growth factor (EGF) in the delayed healing of lingual and gastric ulcerations by testosterone.

Hormonal fluctuations are known to predispose ulceration of the upper gastrointestinal tract, but to date no comparative study of their effects on the healing of pre-existing ulcers in the oral cavity and stomach has been made. We studied the effects of depletion of testosterone and of EGF on the healing of acetic acid-induced ulcers using rats having undergone bilateral orchidectomy and/or salivectomy respectively. We measured alterations in gastric acid secretion and blood flow at ulcer margins, as well as plasma levels of testosterone, gastrin and the proinflammatory cytokines IL-1 beta and TNF-alpha. Testosterone (0.01-10 mg/kg/day i. m.) dose-dependently delayed oral and gastric ulcer healing. When applied in an optimal dose of 1 mg/kg/day, this hormone significantly raised gastric acid secretion and plasma IL-1 beta and TNF-alpha levels. Attenuation of plasma testosterone levels via bilateral orchidectomy inhibited gastric acid secretion and accelerated the healing of oral and gastric ulcers, while increasing plasma gastrin levels and these effects were reversed by testosterone. Salivectomy raised plasma testosterone levels, and delayed oral and gastric ulcer healing. Treatment of salivectomised animals with testosterone further inhibited ulcer healing, and this effect was counteracted by EGF. We propose that testosterone delays ulcer healing via a fall in blood flow at the ulcer margin, a rise in plasma levels of IL-1 beta and TNF-alpha and, in the case of gastric ulcers, an increase in gastric acid secretion. EGF released from the salivary glands plays an important role in limitation of the deleterious effects of testosterone on ulcer healing.

Involvement of sensory afferent fibers and lipid peroxidation in the pathogenesis of stress-induced gastric mucosa damage.

Ablation of sensory nerves impairs healing of gastric ulcers, but the role of free radicals in the healing process has been little studied. The aim of our present investigations was to determine the participation of reactive oxygen species (ROS) in sensory nerve activity during WRS. Experiments were carried out on male Wistar rats and the number of gastric lesions was measured by planimetry. Colorimetric assays were used to determine gastric mucosal levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as well as superoxide dismutase (SOD) activity. We found that capsaicin-inactivation of sensory nerves resulted in magnification of gastric mucosal damage induced by the WRS. In this process, oxidative stress occurs, as reflected by an increase of MDA and 4-HNE tissue concentrations (an index of lipid peroxidation), and a decrease of SOD activity, could play an important role. Pentoxyfilline-induced gastroprotection and hyperemia depends upon attenuation of the oxidative stress. This protection and hyperemia were, at least in part, attenuated by ASA. Afferent sensory fibers participate in the pathogenesis of ulcers. Lipid peroxidation plays an important role in this process.

Melatonin stimulates HSP27 phosphorylation in human pancreatic carcinoma cells (PANC-1).

UNLABELLED: Heat shock protein 27 (HSP27) is a cytoprotective chaperone, activated by stressful stimuli. HSP27 modulates aggregation and degradation of many proteins. Recent evidence suggests that HSP27 could be involved in the progression of tumor growth and in the development of resistance of various tumors to chemo- and radiotherapy. It has been reported that melatonin protects pancreatic cells and various tissues against inflammatory damage. Previous experimental studies have shown that melatonin stimulates pancreatic enzyme secretion and improves the outcome of experimental pancreatitis. To investigate whether melatonin could affect HSP27 protein level in human pancreatic carcinoma cells (PANC-1). PANC-1 cells were incubated in the standard medium DMEM supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal conditions or in the presence of decreasing doses of melatonin (10(-6) - 10(-12)M). Control experiments were performed with the vehicle only (0,1% DMSO) without melatonin. After 24 h and 48 h the cells were harvested, the cytoplasmic and nuclear proteins were isolated for western blot and immunoblotting studies. Incubation of the PANC-1 cells with melatonin resulted in the stimulation both cytoplasmic and nuclear nonphosphorylated HSP27 protein levels after 24 h of incubation, however, above pools of nonphosphorylated chaperone protein levels were strongly diminished after subsequent 24 h. These changes were accompanied by marked rise of nuclear phosphorylated HSP27. The significant increase of this nuclear protein was observed after 48h of incubation. CONCLUSION: Melatonin stimulates phosphorylation of HSP27 in human pancreatic carcinoma cells (PANC-1).

Endotoxemia in the infant rats modulates HSP60 protein level in the pancreatic acinar cells.

Lipopolysaccharide (endotoxin, LPS) is responsible for septic shock and multiorgan failure, but pretreatment of the rats with low doses of LPS reduced pancreatic damage produced by caerulein-induced pancreatitis (CIP). In spite of this observations the effects of LPS and caerulein on pro-apoptotic HSP60 and Bax protein expression in the pancreatic acinar cells has not been examined yet. The aim of this study was to assess the effects of endotoxemia induced in the early period of life on the pro-apoptotic nuclear HSP60 and mitochondrial Bax protein expressions detected in the pancreas of adult animals. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days, at the total doses of 25, 50 or 75 mg/kg. Control animals received injections of physiological saline. Two months later the pancreatic acinar cells were isolated from all above groups of rats and subjected to caerulein over stimulation (10(-8)M). Total nuclear HSP60 and mitochondrial Bax protein expression were isolated for Western blot and co-immunoprecipitation studies. High levels of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein has been observed in the pancreatic acinar cells under basal conditions. Pretreatment of newborn rats with LPS failed to affect significantly the HSP60 and Bax protein levels in the pancreatic acini isolated from the same animals 2 months later, as compared to the control group. Caerulein stimulation significantly reduced the level of these proteins. Pretreatment of suckling rats with LPS (at the total doses of 25, 50 or 75 mg/kg) reversed above caerulein-induced suppression of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein levels in the pancreatic acini obtained from adult rats. We conclude that pretreatment of suckling rats with LPS reversed the suppression of pro-apoptotic HSP60 and Bax protein levels produced by caerulein overstimulation in the pancreatic acini. This mechanism could take a part in the LPS-induced protection of the pancreatic tissue against acute damage.

Localization and biological activities of melatonin in intact and diseased gastrointestinal tract (GIT).

Melatonin (MT), an indole formed enzymatically from L-trytophan (Trp), was first discovered in the bovine pineal gland in 1958 by Lerner et al. Melatonin is the most versatile and ubiquitous hormonal molecule produced not only in the pineal gland but also in various other tissues of invertebrates and vertebrates, particularly in the gastrointestinal tract (GIT). This review focuses on the localization, production, metabolism and the functions of MT in GIT and the duodenal unit (liver, biliary routes and pancreas), where multi-step biosynthetic pathways of this indole, similar to those in pinealocytes, have been identified. These biosynthetic steps of MT, including two major rate limiting enzymes; arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), transforming L-tryptophan (Trp), originally identified in pinealocytes, have been also detected in entero-endocrine (EE) cells of GIT, where this indole appears to act in endocrine, paracrine and/or luminal pathway directly or through G-protein coupled MT receptors. Studies of the distribution of MT in GIT mucosa showed that this indole is generated in GIT in much larger amounts than it is produced in the pineal gland. Melatonin acts in GIT, partly locally in paracrine fashion and is partly released into portal circulation, to be taken up by the liver. It is then metabolized and excreted with the bile to small bowel and finally returns to liver through entero-hepatic circulation. The production of MT by the pineal gland shows circadian rhythm with high night-time surge, especially at younger age, followed by the fall during the day-light time. As a highly lipophylic substance, MT reaches all body cells within minutes, thus, serving as a convenient circadian timing signal. Following pinealectomy, the light/dark cycle of plasma MT levels disappears, while its day-time blood concentration is maintained mainly due to its release from the GIT. According to our experience, after oral application of Trp, the plasma MT increases in dose-dependent manner both in intact and pinealectomized animals and humans, indicating that GIT but not the pineal gland is a source of this indole. In GIT MT exhibits a wide spectrum of activities such as circadian entrainment, antioxidant and free radicals scavenging activity, Melatonin (MT), an indole formed enzymatically from L-trytophan (Trp), was first discovered in the bovine pineal gland in 1958 by Lerner et al. Melatonin is the most versatile and ubiquitous hormonal molecule produced not only in the pineal gland but also in various other tissues of invertebrates and vertebrates, particularly in the gastrointestinal tract (GIT). This review focuses on the localization, production, metabolism and the functions of MT in GIT and the duodenal unit (liver, biliary routes and pancreas), where multi-step biosynthetic pathways of this indole, similar to those in pinealocytes, have been identified. These biosynthetic steps of MT, including two major rate limiting enzymes; arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), transforming L-tryptophan (Trp), originally identified in pinealocytes, have been also detected in entero-endocrine (EE) cells of GIT, where this indole appears to act in endocrine, paracrine and/or luminal pathway directly or through G-protein coupled MT receptors. Studies of the distribution of MT in GIT mucosa showed that this indole is generated in GIT in much larger amounts than it is produced in the pineal gland. Melatonin acts in GIT, partly locally in paracrine fashion and is partly released into portal circulation, to be taken up by the liver. It is then metabolized and excreted with the bile to small bowel and finally returns to liver through entero-hepatic circulation. The production of MT by the pineal gland shows circadian rhythm with high night-time surge, especially at younger age, followed by the fall during the day-light time. As a highly lipophylic substance, MT reaches all body cells within minutes, thus, serving as a convenient circadian timing signal. Following pinealectomy, the light/dark cycle of plasma MT levels disappears, while its day-time blood concentration is maintained mainly due to its release from the GIT. According to our experience, after oral application of Trp, the plasma MT increases in dose-dependent manner both in intact and pinealectomized animals and humans, indicating that GIT but not the pineal gland is a source of this indole. In GIT MT exhibits a wide spectrum of activities such as circadian entrainment, antioxidant and free radicals scavenging activity, cytoprotective, anti-inflammatory and healing efficacy of various GIT lesions such as esophagitis, gastritis, peptic ulcer, pancreatitis and colitis. This review concentrates on the generation and pathophysiological implication of MT in GIT and related organs.