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Background: There is accumulating evidence for the role of Helicobacter pylori in the development of gastric cancer as well as of lymphomas that arise in mucosa-associated lymphoid tissue (MALT). We reported recently that gastric cancer patients show high prevalence of cagA-positive H. pylori and express gastrin and gastrin receptors enabling them to stimulate tumour growth in autocrine fashion. Aims: Since the H. pylori infection is considered to be more strongly associated with MALT lymphoma than with gastric cancer, we decided to determine the gastrin and its receptors’ mRNA expression and gastrin content in this tumour as well as the release of this hormone both into plasma and gastric lumen. Twenty MALT lymphoma patients were compared with 100 age- and gender-matched controls with similar dyspeptic symptoms. Results: The overall H. pylori seropositivity in MALT lymphoma was about 90% and CagA positivity was 70%, compared to 56% and 33%, respectively, in controls. The serum gastrin in MALT lymphoma was about sixfold higher than in controls while gastric luminal gastrin in these patients was over 70 times higher than in controls. Gastrin content in tumour was about 10-fold higher than in antral mucosa. Gastrin and gastrin-receptor (CCKB-receptor) mRNA were detected by reverse transcriptase–polymerase chain reaction in cancer tissue whilst in the fundic and antral mucosa, only enhanced expression of CCKB-receptor mRNA and gastrin mRNA was detected, respectively. Histamine stimulation in MALT lymphoma induced acid secretion that was only about 30% of control value due to atrophic gastritis. This study confirms an important role of CagA-positive H. pylori in the pathogenesis of MALT lymphoma and shows that this lymphoma is capable of synthesizing and releasing potent growth promoting gastrin, possibly due to the action on G-cells of H. pylori-originated Nα-methyl histamine and cytokines (tumour necrosis factor alpha and interleukin-8). Conclusions: Gastric MALT lymphoma is closely linked to CagA-positive H. pylori infection. Gastrin and its receptors may be implicated in the pathogenesis of gastric lymphoma.

Ischemia followed by reperfusion is known to produce gastric lesions due to oxidative stress, but the role of gastric H(+) secretion in the formation of this mucosal injury remains unknown. We studied alterations in gastric acid secretion and gastric histamine content, as well as the expression of histidine-decarboxylase and interleukin-1beta during the mucosal recovery from ischemia-reperfusion erosions. Gastric secretion was studied in rats (series A) with gastric fistula before, during and after the ischemia induced by clamping of celiac artery for 0.5 h followed by reperfusion in animals pretreated with vehicle (saline), omeprazole, a proton pump inhibitor, or ranitidine, a histamine (H(2)) receptor antagonist. In series B, the animals were submitted to 0.5 h of ischemia followed by 1 h of reperfusion and then anesthetized at 0, 3, 12 and 24 h or 3, 5, 10 or 15 days after the end of ischemia-reperfusion to determine gastric blood flow by H(2)-gas clearance technique, area of gastric lesions, plasma gastrin and interleukin-1beta levels, histamine content by radioimmunoassay (RIA) and expression of histidine-decarboxylase and interleukin-1beta mRNA by reverse transcription polymerase chain reaction. Clamping of celiac artery caused cessation of gastric blood flow and almost complete suppression of basal gastric acid secretion (series A) that returned gradually to the control value at day 3 after ischemia-reperfusion, accompanied by the rise in plasma gastrin levels, pronounced expression of histidine-decarboxylase mRNA and increased mucosal histamine content. Ischemia, followed by 1 h of reperfusion, produced gastric erosions (series B) that reached maximum at 12 h, but then declined at 24 h. These erosions progressed at day 3 into deeper ulcers whose area declined progressively within the next 5-15 days. The gastric blood ceased to flow (series B) during 30 min of clamping and was reduced throughout the period of healing of acute erosions, being accompanied by a gradual rise in mucosal interleukin-1beta mRNA content and in plasma interleukin-1beta levels. Treatment with omeprazole or ranitidine, which completely suppressed gastric acid secretion and significantly raised plasma gastrin level, greatly reduced the formation of erosive lesions preventing the progression of these lesions to chronic gastric ulcers, and this was accompanied by the rise in gastric blood flow and plasma gastrin levels and the significant attenuation of plasma interleukin-1beta levels. The ranitidine and omeprazole-induced suppression of ischemia-reperfusion erosions were abolished by the instillation of exogenous 0.2 N HCl into the stomach of these rats. The histidine-decarboxylase was faintly expressed in the intact gastric mucosa, but strongly upregulated during mucosal recovery from the damage induced by ischemia-reperfusion. We conclude that following ischemia-reperfusion: (1) gastric acid secretion, gastric microcirculation and histamine production markedly decline, while interleukin-1beta release significantly increases, probably playing an important role in the progression of acute lesions into chronic gastric ulcerations; (2) the suppression of gastric acid secretion by omeprazole and ranitidine, that induces hypergastrinemia, prevents the progression of gastric erosions into ulcers; and (3) the addition of exogenous acid restores the progression of the acute lesions into gastric ulcers, indicating that gastric acid plays a key role in ulcerogenesis induced by ischemia-reperfusion.

Lipopolysaccharides (LPS), the component of the cell wall of gram-negative bacteria, have been implicated in the pathogenesis of acute pancreatitis, but the mechanism of their action on the pancreas has not been fully explored. The aim of this study was to investigate the effects of various doses of LPS on the integrity of intact pancreas and that involved in acute caerulein-induced pancreatitis (CIP) in the rat and to compare these effects with those of nitric oxide (NO) donor, S-nitrose-acetylpenicillamine (SNAP). The expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA was also examined in the isolated pancreatic acini obtained from the inflamed pancreas of rats treated with LPS. CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 μg/kg·h for 5 h) to conscious rats. Bolus injections of various doses of LPS (0.1, 1, 10, 20 or 40 mg/kg) or SNAP (1.5, 3 or 6 mg/kg) were made intraperitoneally (i.p.) either alone or 30 min prior to s.c. infusion of caerulein to induce CIP. Infusion of caerulein produced acute pancreatitis confirmed by histological examination and manifested by an increase of pancreatic mass (by about 200%). Blood levels of amylase and lipase were augmented by 400 and 800% respectively, whereas the pancreatic blood flow (PBF) was decreased by 50% in rats with CIP. Injection of low doses of LPS (0.1–1 mg/kg i.p.) or SNAP (1.5–3 mg/kg i.p.) 30 min prior to caerulein infusion reversed the harmful effects of pancreatic overstimulation with caerulein and reduced significantly the histological manifestations of CIP such as edema, neutrophil infiltration and vacuolization of the acinar cells. These protective effects of low doses of LPS pretreatment on the pancreas were completely antagonized by the suppression of the activity of NO synthase (NOS) with NG-nitro-L-arginine (L-NNA) applied (20 mg/kg i.p.) 15 min prior to the LPS injection. Combination of L-arginine (100 mg/kg i.p.), a substrate for NOS, with L-NNA given prior to low doses of LPS, restored the LPS-induced protection of the pancreas in rats with CIP. In contrast, higher doses of LPS (20–40 mg/kg i.p.) or SNAP (6 mg/kg i.p.), which produced a significant fall of the PBF, did not protect the pancreas against CIP. Administration of various doses of LPS to rats with CIP resulted in significant and dose-dependent stimulation of NO biosynthesis in the isolated acini obtained from the pancreas of these animals. LPS enhanced the expression of both cNOS and iNOS in the pancreatic acini obtained from rats subjected to CIP. The signal for cNOS mRNA was detected in all samples, reaching peak at the protective dose of LPS (1 mg/kg i.p.), while iNOS was overexpressed only at the highest doses of LPS that failed to exhibit the protective activity. We conclude that the pretreatment with low doses of LPS protects the pancreas against the damage provoked by CIP and this effect could be attributed, at least in part, to the activation of L-arginine-NO system in the pancreas.

Leptin, detected recently in the stomach, is a product of the ob gene released by cholecystokinin (CCK) and plays an important role in the control of food intake but its influence on gastroprotection against the damage caused by noxious agents has not been studied. This study was designed to compare the effects of leptin and cholecystokinin-8 (CCK-8) on gastric mucosal lesions induced by topical application of 75% ethanol or acidified aspirin. Four series of Wistar rats (A, B, C and D) were used to determine the effects of: (A) suppression of prostaglandin biosynthesis by indomethacin (5 mg/kg i.p.); (B) inhibition of nitric oxide (NO)-synthase by nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v.); (C) blockade of sensory nerves by capsaicin (125 mg/kg s.c.) and (D) bilateral vagotomy, on the gastric lesions induced by intragastric (i.g.) application of ethanol with or without pretreatment with CCK-8, a known gastroprotective substance or leptin. CCK-8 (1-100 microg/kg i.p.) and leptin (0.1-50 microg/kg i.p.) dose dependently attenuated gastric lesions induced by 75% ethanol; the dose reducing these lesions by 50% being about 10 microg/kg and 8 microg/kg, respectively. The protective effects of CCK-8 and leptin were accompanied by a significant rise in gastric blood flow (GBF) and luminal NO concentration. Leptin was also effective to attenuate aspirin-induced damage and the accompanying fall in the GBF, whereas CCK-8 dose dependently worsened aspirin damage and failed to influence GBF. CCK (1-100 microg/kg i.p.), given in graded doses, produced a dose-dependent increase in the plasma leptin level and a rise of the expression of ob messenger RNA (mRNA) in gastric mucosa, the maximum being reached at a dose of 100 microg/kg. Pretreatment with CCK-8 (10 microg/kg i.p.) or with 8% peptone, that is known to stimulate CCK release, also produced a significant rise in plasma leptin levels and up-regulation of ob mRNA while reducing significantly the gastric lesions induced by 75% ethanol to the same extent as that induced by exogenous leptin (10 microg/kg i.p.). Indomethacin, which suppressed prostaglandin generation by approximately 90%, failed to influence leptin- or CCK-8-induced protection against ethanol, whereas L-NAME attenuated significantly CCK-8- and leptin-induced protection and hyperemia but addition to L-NAME of L-arginine, but not D-arginine, restored the protective and hyperemic effects of both hormones. The ob mRNA was detected as a weak signal in the intact gastric mucosa and in that exposed to ethanol alone but this was further enhanced after treatment with graded doses of CCK-8 or peptone meal applied prior to ethanol. We conclude that: (1) exogenous leptin or that released endogenously by CCK or meal exerts a potent gastroprotective action depending upon vagal activity, and involving hyperemia probably mediated by NO and sensory nerves but unrelated to endogenous prostaglandins; (2) leptin mimics the gastroprotective effect of CCK and probably mediates the protective and hyperemic actions of CCK in the rat stomach.

Background : Helicobacter pylori infection in humans is a major risk factor for peptic ulcer, but studies on the relation between H. pylori infection and gastric pathology are limited due to a deficiency of convenient animal models resembling this infection in humans. Methods : We studied the effects of inoculation of conventional BALB/c mice with CagA and VacA positive (type I) H. pylori or CagA and VacA negative H. pylori (type II) strains on gastric secretion and healing of chronic acetic acid-induced ulcers in mouse stomachs. The ulcer area, gastric blood flow, plasma interleukin (IL)-1β and IL-12, as well as plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy samples were also taken for assessment of the presence of viable H. pylori using a rapid urease test, H. pylori-culture and the RT-PCR analysis of the signal for H. pylori CagA. Results : Gastric acid and pepsin secretion was reduced by over 50% immediately after H. pylori inoculation and accompanied by a significant increment in plasma gastrin and fall in gastric luminal somatostatin content observed over all test days, particularly in mice infected with type I H. pylori. The area of ulcers in vehicle-treated controls decreased significantly starting from day 2 after ulcer induction and then continued to decline for a further 14 days to heal almost completely after 28 days. In contrast, the ulcers were present until day 28 in all mice infected with type I or type II H. pylori strains, being significantly larger, especially with type I H. pylori infection. The gastric blood flow at the ulcer margin and ulcer crater in vehicle-treated mice gradually increased with decreasing ulcer size, after 14 and 28 days reaching a value which was not significantly different from that in vehicle-administered mice. In contrast, the gastric blood flow in type I H. pylori and, to a lesser extent, in type II H. pylori infected mice was significantly lower than in vehicle controls, both at the margin and at the crater of ulcers at all tested days. Histological changes such as oedema or congestion of surface epithelium were found after 7 days whereas mucosal inflammatory infiltration appeared after 14 days with a further increase after 28 days, especially in type I H. pylori and to a lesser extent in type II H. pylori infected mice. Plasma IL-1β and IL-12 were significantly elevated at all tested days of ulcer healing and their increments were significantly higher in type I than in type II H. pylori infection. Conclusions : Conventional mice with gastric ulcers can be successfully infected by both toxigenic and nontoxigenic H. pylori strains, and this infection causes an immediate suppression of gastric secretion and markedly delays the healing of ulcers due to the fall in mucosal microcirculation in the ulcer region, cytokine release and an impairment in the gastrin–somatostatin link that appears to be independent of gastritis and more pronounced with infection of toxigenic than nontoxigenic strains.

Pancreatic secretory trypsin inhibitor (PSTI) is an inhibitor of serine-proteinases including pancreatic trypsin that prevents excessive digestion of the gastrointestinal mucus, but its role in the mechanism of mucosal defense has been little studied. This study was designed to determine the effect of base variant of human PSTI (R44S-PSTI) on gastric secretion, healing of gastric lesions induced by stress and the expression of PSTI during mucosal recovery from stress lesions. Recombinant R44S-PSTI was obtained using by site-directed mutagenesis due to replacement of arginine by serine that led to longer half life of this peptide than its natural form. Stress ulcerations were induced by exposure of rats to a standard 3.5 h of water immersion and restraint stress with or without pretreatment with vehicle or R44S-PSTI (0.1 mg/kg) applied s.c. 30 min before and immediately after the end of stress. Rats were then sacrificed immediately (time 0) and at 6 h or 12 h after the termination of stress. The gastric blood flow (GBF) was measured by H 2 -gas clearance technique at each time period and gastric mucosal samples were excised for assessment of PSTI immunohistochemical expression and PSTI messenger RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Southern hybridization. Stress produced numerous gastric lesions and decreased the GBF by about 30% as compared to the respective value in vehicle-treated non-stressed gastric mucosa. R44S-PSTI given s.c. in graded doses (0.01–1 mg/kg) inhibited dose-dependently gastric acid and pepsin outputs, in rats with gastric fistula and accelerated the healing of stress-induced gastric lesions significantly. The healing effects of R44S-PSTI (0.1 mg/kg s.c.) recorded at 6 h and 12 h after the end of stress were accompanied by a significant rise in the GBF. The expression of PSTI mRNA in the intact mucosa was weak, but following exposure to stress it was significantly augmented to reach the highest observed value at 6 h after the stress. We conclude that (1) base variant of human PSTI accelerates healing of stress-induced gastric lesions probably due to its antisecretory activity and enhancement of mucosal blood flow and (2) the expression of genes for PSTI plays an important role in the mechanism of mucosal recovery from gastric lesions induced by stress.

Background: NSAIDs, such as aspirin (ASA), cause widespread mucosal damage, but repeated ASA insults appear to induce mucosal tolerance (adaptation) to this␣injury. The mechanism of the gastric adaptation to the damage induced by ASA has not been fully explained. Aim: To determine the role of the mucosal gene expression for spasmolitic peptide (SP) (a member of trefoil peptides) and transforming growth factor alpha (TGFα) as well as for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to ASA in rats. Methods: Gastric lesions were produced by ASA (100 mg/kg in 1.5 mL of 0.2 M HCl) applied intragastrically (i.g.) as a single dose, every day for 5 days. Control rats were given 1.5 mL of vehicle (0.2 M HCl i.g.) as a single dose, during 5 consecutive days. Gastric blood flow (GBF) was measured by H2-gas clearance technique and gastric mucosal specimens were taken for the assessment of cell␣proliferation rate in gastric mucosa by bromodeoxyuridine (BrdU) uptake, mucosal generation of prostaglandin E2 measured by radioimmunoassay, and for expression of SP, TGFα COX-1 and COX-2 mRNA as determined by RT-PCR. To quantify the relative amounts of mRNA for SP and TGFα, southern blotting analysis of the PCR products was performed and the intensity of PCR products was compared with that of β-actin used as a standard. Results: ASA applied once produced numerous gastric erosions, but with repeated ASA doses the adaptation to this NSAID developed, the area of gastric lesions being reduced by 86% after six consecutive ASA insults. This adaptation to ASA was accompanied by approximately a 90% reduction in prostaglandin E2 biosynthesis, by a significant rise in BrdU uptake by glandular cells predominantly in the neck region of gastric glands and by expression of SP (SP/β-actin ratio; 0.96 ± 0.08 in ASA-adapted mucosa vs. 0.38 ± 0.05 in the control mucosa) and TGFα (TGFα/β-actin ratio: 0.97 ± 0.07 in ASA-adapted mucosa vs. 0.77 ± 0.06 in the control mucosa). COX-1 expression was detected in vehicle-control gastric mucosa and after single exposure to ASA or after six consecutive ASA insults, while COX-2 mRNA was not detected in vehicle-control gastric mucosa, but appeared after single ASA insult and was sustained after subsequent ASA doses. Conclusions: (i) Gastric adaptation to aspirin injury involves enhanced cell proliferation which appears to be mediated by increased expression of SP and TGFα, and (ii) rapid upregulation of COX-2 expression following single and repeated ASA insults may represent a compensatory response to suppression of prostaglandin generation by this NSAID.

Background Epidermal growth (EGF) and transforming growth factor alpha (TGFalpha) are potent gastric secretory inhibitors, mitogens, and mucosal protectors, but the impact of Helicobacter pylori infection on their mucosal expression and luminal release has not been clarified. Methods In this study, gene and immunoreactive and immunohistochemical expressions of EGF and TGFalpha were assessed in the gastric mucosa of 15 H. pylori-negative healthy normals, in 22 H. pylori-positive duodenal ulcer patients (DU) and in 24 H. pylori-positive non-ulcer dyspepsia patients (NUD). All studies in DU and NUD patients were repeated after 2 weeks of triple therapy (amoxicillin + clarithromycin + omeprazole) and 4 weeks and 2 years later. Results Immunohistochemical expression of EGF and TGFalpha in H. pylori-positive DU and NUD was significantly higher than in H. pylori-negative normals, and this increase persisted at 2 and 4 weeks after therapy but normalized 2 years later. EGF mRNA was detected in the gastric mucosa of H. pylori-positive DU before and at 2 and 4 weeks after H. pylori eradication, but it was not found 2 years after the eradication of H. pylori or in gastric mucosa of H. pylori-negative control subjects. TGFalpha mRNA was detected in the gastric mucosa independently of H. pylori status, with the stronger expression observed in the gastric mucosa of H. pylori-positive DU and NUD before eradication than after this procedure. Plasma gastrin, which was significantly increased in H. pylori-positive DU, normalized already after 2 weeks of triple therapy. The eradication rate as determined by histology after triple therapy reached 86.3% in DU patients and 90.5% in NUD patients. Two years after the eradication the H. pylori reinfection rate was 4.5% among DU patients and 4.2% among NUD. Treatment of DU patients with triple therapy resulted in complete ulcer healing. Conclusions 1) Chronic H. pylori infection and resulting antral gastritis are associated with increased plasma gastrin and increased mucosal cell proliferation, probably due to enhanced expression of EGF and TGFalpha, and 2) the H. pylori eradication results in a decrease in plasma gastrin, but the increase in gastric TGFalpha and EGF content is sustained, suggesting that they may be involved in ulcer healing.

Lipopolysaccharide (LPS) has been proposed to act as one of the pathogens in endotoxemia-induced gastric lesions, but its action on mucosal integrity has not been fully clarified.We compared the effects of LPS originating from Escherichia coli and the chemical donor of nitric oxide (NO), S-nitroso-acetylpenicillamine (SNAP), on acute gastric lesions induced by 100% ethanol, mucosal blood flow (GBF), and mucosal generation of prostaglandin E2 (PGE2) and examined the expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA in the gastric mucosa of rats treated with LPS, by using reverse transcription polymerase chain reaction (RT-PCR).LPS (0.01-1.0 mg/kg) or SNAP (0.37-3.0 mg/kg) given intraperitoneally, dose-dependently prevented ethanol-induced mucosal lesions, and these protective effects were accompanied by a significant increase in the GBF and excessive mucosal release of NO. Suppression of NOS activity by NG-nitro-L-arginine methyl ester (L-NAME) (20mg/kg intravenously) or L-NG-(1-iminoethyl)-lysine (L-NIL) (30mg/kg intraperitoneally) and NOS induction by treatment with dexamethasone (2 mg/kg intraperitoneally) reversed the protective and hyperemic effects of LPS, and this reversal by L-NAME was significantly antagonized by addition of the substrate for NOS, L-arginine, but not D-arginine. Both LPS and SNAP increased PGE2 generation significantly, and this effect was reduced by pretreatment with L-NAME, L-NIL, or dexamethasone. Expression of cNOS was detected by RT-PCR in the intact mucosa, but intense signals for expression of both cNOS and iNOS were detected in the mucosa of LPS-treated rats.Parenteral LPS, similarly to the chemical NO donor, SNAP, protects the gastric mucosa against ethanol-induced damage via an increase in GBF mediated by NO due to the activation of arginine-NO system and possibly also enhanced generation of PGE2.

Overexpression of transforming growth factors (TGF) in acute pancreatitis (AP) suggested that these substances play an important role in pancreatic repair and remodeling but the contribution of epidermal growth factor (EGF), that is well known to promote cell growth and regeneration, has not been investigated. The aim of this study was to compare the gene and immunohistochemical expression of EGF and TGF-β1, cell proliferation, and biochemical parameters in AP induced by infusion of a supramaximal dose of caerulein in rats. The rats were sacrificed at 0, 12, 24, 48, 72 h, 5 and 10 days after the termination of caerulein infusion. Pancreatic tissue DNA synthesis, cell proliferation, histological and immunohistochemical assessments and plasma amylase were estimated following induction of AP. The mRNA expression for EGF and TGF-β1 was evaluated by reverse transcription-polymerase chain reaction. During 10 days of the study after induction of AP a gradual normalization of biochemical and histological parameters was observed. DNA synthesis and cell proliferation which were significantly decreased at 0 and 24 h, increased significantly at 48 and 72 h, and then gradually decreased reaching at day 10 the values similar to those of vehicle-treated control rats. In these control rats the EGF mRNA or immunohistochemical expression was not detected, while the TGF-β1 expression was weak. After induction of AP, the mRNA and immunohistochemical expression of EGF showed an increase during the initial 5 days, while those of TGF-β1 showed a marked increase between 0 and 48 h and then again at day 10. We confirm that: (1) the expression of TGF-β1 during AP is biphasic with an initial increase probably related to pancreatic damage and inhibition of cell proliferation and with the later phase of increase accompanied by the stimulation of the synthesis of extracellular matrix components and (2) AP is accompanied by an induction of synthesis of EGF that occurs in the initial phase of AP, probably limiting the extent of AP, and enhancing the stimulation of the pancreatic repair and regeneration.

Background: Autoantibodies against epitopes located at the canaliculi of human parietal cells occur in about 30% of Helicobacter pylori-infected patients. This has led to the hypothesis that gastric secretory function could be inhibited by anticanalicular autoantibodies in H. pylori gastritis. Methods: Forty-four H. pylori-infected patients with and without duodenal ulcers were screened for anticanalicular autoantibodies by means of immunohistochemistry. Plasma gastrin levels and basal and maximal gastric acid output were determined. Results: Fasting gastrin levels were significantly increased in the group with anticanalicular autoantibodies. In the group of patients with non-ulcer dyspepsia the presence of anticanalicular autoantibodies was significantly correlated with an impaired basal acid secretion. Conclusions: Antigastric autoimmunity in H. pylori gastritis seems to be relevant for gastric hyposecretion either directly by inhibiting the proton pump or indirectly through the development of gastric m...

Melatonin, a pineal hormone that is biosynthesized from L-tryptophan, is known to scavenge oxygen free radicals and to be present in the gut, but little is known about the role of this hormone and its precursor, L-tryptophan, in protecting the gastric mucosa from damage accompanied by increase in the generation of oxygen radicals.This study was designed to determine the effects of melatonin and L-tryptophan on the formation of acute gastric lesions induced by stress and ischaemia reperfusion and, for comparison, by topical irritants such as 100% ethanol or acidified acetylsalicylic acid.It was found that pretreatment with melatonin in doses ranging from 1.2 to 10 mg/kg dose-dependently reduced the stress-induced gastric lesions and was accompanied by a reduction in blood-free radicals and by attenuation of the fall in gastric blood flow. L-tryptophan applied intragastrically in doses ranging from 1 to 100 mg/kg also reduced dose-dependently the lesions induced by stress; this effect too was accompanied by a rise in gastric blood flow. Pretreatment with indomethacin, to block the biosynthesis of prostaglandins, significantly augmented the lesions produced by stress and completely abolished the protective effects of melatonin or L-tryptophan. Both melatonin and tryptophan reduced the formation of acute gastric lesions provoked by ischaemia reperfusion; this was accompanied by an increase in gastric blood flow. In contrast, melatonin and L-tryptophan failed to influence acute gastric lesions induced by topical irritants such as 100% ethanol or acidified aspirin.Melatonin and L-tryptophan protect the gastric mucosa from damage by stress and ischaemia reperfusion, and this action is mediated, at least in part, by the limitation in the free radicals, the stimulation of mucosal generation of PG and by the increase in gastric blood flow.

The gastric hormone ghrelin is known as an important factor for energy homeostasis, appetite regulation and control of body weight. So far, ghrelin has mainly been examined as a serological marker for gastrointestinal diseases, and only a few publications have highlighted its role in local effects like mucus secretion. Ghrelin can be regarded as a gastroprotective factor, but little is known about the distribution and activity of ghrelin cells in pathologically modified tissues. We aimed to examine the morphological changes in ghrelin expression under several inflammatory, metaplastic and carcinogenic conditions of the upper gastrointestinal tract. In particular, autoimmune gastritis showed interesting remodeling effects in terms of ghrelin expression within neuroendocrine cell hyperplasia by immunohistochemistry. Using confocal laser microscopy, the gastrin/cholecystokinin receptor (CCKB) could be detected on normal ghrelin cells as well as in autoimmune gastritis. Functionally, we found evidence for a physiological interaction between gastrin and ghrelin in a primary rodent cell culture model. Additionally, we gathered serological data from patients with different basic gastrin levels due to long-term autoimmune gastritis or short-term proton pump inhibitor treatment with slightly reactive plasma gastrin elevations. Total ghrelin plasma levels showed a significantly inverse correlation with gastrin under long-term conditions. Autoimmune gastritis as a relevant condition within gastric carcinogenesis therefore has two effects on ghrelin-positive cells due to hypergastrinemia. On the one hand, gastrin stimulates the proliferation of ghrelinpositive cells as integral part of neuroendocrine cell hyperplasia, while on the other hand, plasma ghrelin is reduced by gastrin and lost in pseudopyloric and intestinal metaplastic areas. Ghrelin is necessary for the maintenance of the mucosal barrier and might play a role in gastric carcinogenesis, if altered under these pre neoplastic conditions.

BACKGROUND: Application of neutralising antibodies (NAs) to transforming growth factor beta 1 (TGF beta 1) improves wound healing in experimental glomerulonephritis and dermal incision wounds. TGF beta 1 has been detected in the stomach, but despite the fact that this cytokine plays a central part in wound healing no information is available to determine if modulation of the TGF beta 1 profile influences the healing of gastric ulcers. This study examines gastric ulcer healing in the rat after local injection of NAs to TGF beta 1. METHOD: Chronic gastric ulcers were induced in Wistar rats by the application of 100% acetic acid to the serosal surface of the stomach. Immediately after ulcer induction and on day 2, NAs to TGF beta 1 (50 micrograms), TGF beta 1 (50 ng), saline or control antibodies (IgG; 50 micrograms) were locally injected into the subserosa. Controls received no subserosal injections. Animals were killed on day 5 or 11, the ulcer area was measured planimetrically, sections were embedded in paraffin wax, and stained with trichrome or haematoxylin and eosin. Depth of residual ulcer was assessed on day 11 by a scale of 0-3, the percentage of connective tissue was determined by a semiquantitative matrix score and granulocytes and macrophages in the ulcer bed were also assessed. RESULTS: The application of NAs to TGF beta 1 led to a significant acceleration of gastric ulcer healing on day 11 (0.6 (SD 0.8) v 3.7 (SD 2.6) mm2), a reduction in macrophages (23.7 (SD 22.6) v 38 (26) per 40 x power field) and granulocytes (8.5 (SD 5.6) v 20 (10) per 40 x power field), fewer histological residual ulcers (mean 1 (SD 0.9) v 2 (1.1)), a reduced matrix score, and a regenerative healing pattern. Excessive scarring was seen in the TGF beta 1 treated group. CONCLUSION: Further treatment of gastric ulcers may induce a new treatment modality by local injection of NA to TGF beta 1 in an attempt to accelerate and improve ulcer healing.

The urease-ammonia (NH4OH) system has been proposed to play a major role in the pathogenesis of the Helicobacter pylori-associated gastritis, but the mechanism of the mucosal damage has not been fully explained. This study was designed to examine possible adaptive cytoprotection and the adaptation of rat gastric mucosa to the irritant action of NH4OH and urease.Single application of NH4OH alone in various concentrations (15-500 mM) caused concentration-dependent mucosal damage starting with 30 mM and reaching a maximum at 250 mM NH4OH, similar to that obtained with 100% ethanol; it was accompanied by a decrease in gastric blood flow (GBF) to approximately 30% of the normal value. When the mucosa was first exposed to the low, non-damaging concentration (15 mM) of NH4OH and then insulted with 100% ethanol, the extent of ethanol damage was greatly attenuated as compared with that caused by ethanol alone. This adaptive cytoprotection was accompanied by the rise in GBF and reversed, in part, by the pretreatment with indomethacin, an inhibitor of prostaglandin (PG)-cyclooxygenase; with L-NAME, a blocker of NO-synthase; or with capsaicin deactivating the sensory nerves. Damaging concentrations of NH4OH (125 mM) caused widespread mucosal damage after the first application, but with repeated insults with 125 mM NH4OH a gradual reduction in the mucosal lesions, accompanied by enhanced mucosal cell proliferation and over-expression of epidermal growth factor (EGF) (using immunocytochemistry) and mRNA of EGF (using trans-reverse polymerase chain reaction), were observed.NH4OH alone damages gastric mucosa only at the concentration exceeding that found in H. pylori-infected stomachs, whereas at lower concentrations it acts as 'mild' irritant to induce adaptive cytoprotection. This adaptive cytoprotection appears to be mediated, in part, by endogenous PG, sensory nerves, and an arginine-NO-dependent pathway, and repeated applications of NH4OH induce gastric adaptation, probably mediated by enhanced expression of EGF and its receptors and by an increased cell proliferation.

Non-alcoholic fatty liver disease (NAFLD), most common chronic hepatic pathology, that occurs in the developed countries is estimated at 1/3 of the population. Amongst the numerous pathogenetic factors, oxidative stress and apoptosis of hepatocytes initiate many inflammatory processes and are involved in the progression of disease, particularly in transformation of non-alcoholic steatohepatitis (NASH) to cirrhosis. The aim of our study was to determine the effects of tryptophan and melatonin on the selected biochemical parameters in patients with NAFLD, and additionally, to evaluate the effects of tryptophan and melatonin in improvement of liver tissue in selected NAFLD patients. Seventy four patients with NAFLD confirmed by histopathological examination of liver biopsy samples, were admitted to the study. They were randomly assigned to three groups. Group I received the preparation Essentiale forte in the dose of 3 x 1 tablet per day and tryptophan 2 x 500 mg/day over the period of 14 months, group II received Essentiale forte and melatonin 2 x 5 mg/day over 14 months and group III received only Essentiale over the period of 14 months. In nine patients of groups I, II, and III, the liver biopsy was performed after 14-months of treatment period. Out of nine patients whom biopsy was performed, three of them were from group I, four from group II and two of them were from group III, respectively. After the 14-month treatment period, gamma-glutamyl transferase (GGPT) activity and levels of triglycerides and LDL-cholesterol were found to be significantly reduced in group I and II. The level of melatonin after the therapy was significantly elevated in group I and II and did not change in group III. Statistically significantly lower levels of IL-1, IL-6 and TNF-α were observed in patients receiving melatonin and tryptophan, comparing with group III treated with Essentiale forte only. These study findings demonstrate that melatonin and tryptophan substantially reduce the levels of pro-inflammatory cytokines and improve some parameters of fat metabolism in patients with NAFLD. In few patients with NASH melatonin and tryptophan reduced the inflammation in liver. We conclude that melatonin is worth considering for the therapy of NAFLD, particularly in patients with impaired fat metabolism accompanied by hypertriglyceridemia and hyper-LDL cholesterolemia.

The incidence of the obstructive sleep apnoea syndrome (OSAS) is rising as it is often associated with obesity. Actually, the adipose tissue is working as an endocrine organ with complex interactions. Recently, many adipokines such as omentin-1 were discovered. The role of omentin-1 in the pathogenesis of OSAS has not been clearly determined. Melatonin has a known influence on the sleep and wake rhythm. The data on the involvement of melatonin in OSAS are rare. Therefore we evaluated the changes in plasma levels of omentin-1 and melatonin before and after continuous positive airway pressure (CPAP) therapy in OSA patients. 10 patients with newly diagnosed OSAS were included in the study. They underwent diagnostic polysomnography with blood drawings in a 2 hour interval for 24 hours. In the second night sufficient CPAP therapy was established. After three months of CPAP therapy the measurements were repeated. As controls 10 healthy volunteers were recruited. The same blood analysis and a polygraphic measurement were made and compared with the patients. OSA patients showed significantly higher omentin-1 plasma levels (17.22±13.94 versus 9.24±4.85 ng/ml, p0.05). After three months of therapy the plasma levels of omentin-1 decreased toward the values observed in the controls at 8.00 a.m. Melatonin showed the usual peak at 2.00 a.m. in the volunteer group. OSA patients showed a later peak of melatonin at 6.00 a.m. which returned to 2.00 a.m. after CPAP therapy. We conclude that omentin as well as melatonin seem to be involved in pathogenesis of OSAS. To what exent, further studies will have to face that question.

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-1β, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1β 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-1β 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-1β 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-1β and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity.

Nitric oxide (NO) is a local mediator in inflammation and allergy. The aim of this study was to investigate whether live incubated colorectal mucosal tissue shows a direct NO response ex vivo to nonspecific and specific immunological stimuli and whether there are disease-specific differences between allergic and chronic inflammatory bowel disease (IBD). We took biopsies (n=188) from 17 patients with confirmed gastrointestinally mediated food allergy, six patients with inflammatory bowel disease, and six control patients. To detect NO we employed an NO probe (WPI GmbH, Berlin, Germany) that upon stimulation with nonspecific toxins (ethanol, acetic acid, lipopolysaccharides), histamine (10(-8)-10(-4)M), and immune-specific stimuli (anti-IgE, anti-IgG, known food allergens) directly determined NO production during mucosal oxygenation. Non-immune stimulation of the colorectal mucosa with calcium ionophore (A23187), acetic acid, and ethanol induced a significant NO release in all groups and all biopsies. Whereas, immune-specific stimulation with allergens or anti-human IgE or -IgG antibodies did not produce significant release of NO in controls or IBD. Incubation with anti-human IgE antibodies or allergens produced a ninefold increase in histamine release in gastrointestinally mediated allergy (p0.001), but anti-human IgE antibodies induced NO release in only 18% of the allergy patients. Histamine release in response to allergens or anti-human IgE antibodies did not correlate with NO release (r(2)=0.11, p=0.28). These data show that nonspecific calcium-dependent and toxic mechanisms induce NO release in response to a nonspecific inflammatory signal. In contrast, mechanisms underlying immune-specific stimuli do not induce NO production immediately.

Gastric microcirculation plays an important role in the maintenance of the mucosal gastric integrity and the mechanism of injury as well as providing protection to the gastric mucosa. Disturbances in the blood perfusion, through the microcapillaries within the gastric mucosa may result in the formation of mucosal damage. Acute gastric mucosal lesions constitute an important clinical problem. Originally, one of the essential component of maintaining the gastric mucosal integrity was the biosynthesis of prostaglandins (PGs), an issue that has captured the attention of numerous investigations. PGs form due to the activity of cyclooxygenase (COX), an enzyme which is divided into 2 isoforms: constitutive (COX-1) and inducible (COX-2) ones. The inhibition of COX-1 by SC-560, or COX-2 by rofecoxib, reduces gastric blood flow (GBF) and impairs gastric mucosal integrity. Another detrimental effect on the gastric mucosal barrier results from the ablation of sensory afferent nerves by neurotoxic doses of capsaicin. Functional ablation of the sensory afferent nerves by capsaicin attenuates GBF and also renders the gastric mucosa more susceptible to gastric mucosal damage induced by ethanol, aspirin and stress. However, the role of reactive oxygen species (ROS) in the interaction between COX specific inhibitors and afferent sensory nerves has not been extensively studied. The aim of our present study was to determine the participation of ROS in pathogenesis of stress-induced gastric lesions in rats administered with SC-560 or rofecoxib, with or without ablation of the sensory afferent nerves. ROS were estimated by measuring the gastric mucosal tissue level of MDA and 4-HNE, the products of lipid peroxidation by ROS as well as the SOD activity and reduced glutathione (GSH) levels, both considered to be scavengers of ROS. It was demonstrated that exposure to 3.5 h of WRS resulted in gastric lesions, causing a significant increase of MDA and 4-HNE in the gastric mucosa, accompanied by a decrease of SOD activity and mucosal GSH level. Pretreatment with COX-1 and COX-2 inhibitors (SC-560 and rofecoxib, respectively) aggravated the number of gastric lesions, decreased GBF, attenuated GSH level without further significant changes in MDA and 4-HNE tissue levels and SOD activity. Furthermore, the capsaicin--nactivation of sensory nerves resulted in exaggeration of gastric mucosal damage induced by WRS and this was further augmented by rofecoxib. We conclude that oxidative stress, as reflected by an increase of MDA and 4-HNE tissue concentrations (an index of lipid peroxidation), as well as decrease of SOD activity and the fall in GSH tissue level, may play an important role in the mechanism of interaction between the inhibition of COX activity and afferent sensory nerves releasing vasoactive neuropeptides. This is supported by the fact that the addition of specific COX-1 or COX-2 inhibitors to animals with capsaicin denervation led to exacerbation of gastric lesions, and further fall in the antioxidizing status of gastric mucosa exposed to stress.

Nitric oxide (NO) was shown to mediate gastric hyperemia following secretory stimulation but its role in the control of gastric secretion has not been clarified. Secretory studies were carried out on conscious dogs with chronic gastric fistula, Heidenhain pouch and esophageal fistula, while changes in gastric blood flow were measured in the mucosa of Heidenhain pouuch by laser Doppler flowmetry. Plasma gastrin was determined by radioimmunoassay. Infusion of N G - nitro- l -arginine ( l -NNA) (bolus i.v. injection of 2.5 mg/kg followed by infusion of 0.5 mg/kg/h), a potent inhibitor of nitric oxide synthase, failed to affect basal gastric secretion or plasma gastrin level but suppressed an increase of this secretion induced by sham-feeding, ordinary meat feeding or i.v. infusion of bombesin (0.5 μg/kg/h), pentagastrin (4 μg/kg/h) or histamine (40 μg/kg/h). In tests with feeding and bombesin infusion, l -NNA caused a significant and dose-dependent reduction in plasma gastrin levels. The inhibition by l -NNA of gastric acid secretory response to pentagastrin, histamine or feeding was accompanied by a decline in blood flow. Addition of l -arginine (bolus i.v. dose of 50 mg/kg followed by infusion of 5 mg/kg/h) significantly attenuated the l -NNA induced inhibition of gastric secretion and the reduction in plasma gastrin response as well as in the fall of gastric blood flow. We conclude that endogenous nitric oxide affects the gastric secretion and that this effect is mediated, at least in part, by the changes in the gastrin release and gastric blood flow.

The present study represents the follow-up of our initial observations designed to investigate whether in patients with nonalcoholic steatohepatitis (NASH) the beneficial effect of 12-week course of melatonin (MT) on liver enzymes could be maintained with prolonged period of treatment and to analyze whether biochemical treatment responses could be sustainable after melatonin discontinuation. Forty two patients with histologically proven NASH (30 treated with melatonin 2x5 mg daily, 12 controls receiving placebo) enrolled to our previous 3-month study agreed to take part of subsequent 12 weeks treatment followed by 12-week follow-up period. Enrolled patients had biochemical determinations every six weeks during the melatonin treatment period and again after 12 weeks of follow-up. Significant reduction in median alanine aminotransferase (ALT) levels between baseline and week 18, week 24 and follow-up was observed in both MT-treated and control group: 43% and 31%, 42% and 33%, 32% and 31%. Aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT) levels decrease significantly only in MT-treated group. In MT-treated group mean percentage change in AST level below baseline at week 18, at week 24 and at follow-up was 45%, 33% (p0.05) and 8% (ns), respectively. The evolution of GGT levels was as follows: the mean percentage reduction in GGT below baseline level at week 18, 24 and follow-up was: 48%, 52% and 38% (p0.05), respectively. In both MT-treated and control group plasma cholesterol, triglicerydes and glucose concentrations as well as plasma alkaline phosphatase persisted within normal values during the prolonged study period. Plasma concentration of melatonin (pg/ml) in MT-treated group averaged 7.5±3.5 at baseline and increased to 52.5±17.5 at 24th week. The results of our study demonstrating beneficial effect of melatonin on liver enzymes in patients with NASH would seem to encourage further controlled trials of melatonin given over a longer period of time with liver histology as end point.

Melatonin (MT) and its precursor L-tryptophan (TRP) are implicated in the protection of gastric mucosa against aspirin-induced lesions and in the acceleration of healing of idiopathic gastro-duodenal ulcers, but no information is available whether these agents are also effective in healing of gastroduodenal ulcers accompanied by Helicobacter pylori (H. pylori) infection. In this study three groups A, B and C, each including 7 H. pylori-positive patients with gastric ulcers and 7 H. pylori-positive patients with duodenal ulcers, aging 28-50 years, were randomly assigned for the treatment with omeprazole 20 mg twice daily combined with placebo (group A), MT administered in a dose of 5 mg twice daily (group B) or TRP applied in a dose of 250 mg twice daily (group C). All patients underwent routine endoscopy at day 0 during which the gastric mucosa was evaluated and gastric biopsies were taken for the presence of H. pylori and histopathological evaluation. The rate of ulcer healing was determined by gastroduodenoscopy at day 0, 7, 14 and 21 after the initiation of the therapy. Plasma MT, gastrin, ghrelin and leptin were measured by specific RIA. At day 21, all ulcers were healed in patients of groups B and C but only 3 out of 7 in group A of gastric ulcers and 3 out of 7 in duodenal ulcers. Initial plasma MT showed similar low levels in all three groups but it increased several folds above initial values in ulcer patients at day 7, 14 and 21. Plasma gastrin and leptin levels showed a significant rise over initial values in patients treated with omeprazole and placebo, MT or TRP while plasma ghrelin levels were not significantly affected by these treatments. We conclude that MT or TRP added to omeprazole treatment, significantly accelerates healing rate of H. pylori infected chronic gastroduodenal ulcers over that obtained with omeprazole alone and this likely depends upon the significant rise in plasma MT and possibly also in leptin levels, both hormones involved in the mechanism of gastroprotection and ulcer healing.

The current scientific literature is replete with investigations providing information on the molecular mechanisms governing the regulation of circadian rhythms by neurons in the suprachiasmatic nucleus (SCN), the master circadian generator. Virtually every function in an organism changes in a highly regular manner during every 24-hour period. These rhythms are believed to be a consequence of the SCN, via neural and humoral means, regulating the intrinsic clocks that perhaps all cells in organisms possess. These rhythms optimize the functions of cells and thereby prevent or lower the incidence of pathologies. Since these cyclic events are essential for improved cellular physiology, it is imperative that the SCN provide the peripheral cellular oscillators with the appropriate time cues. Inasmuch as the 24-hour light:dark cycle is a primary input to the central circadian clock, it is obvious that disturbances in the photoperiodic environment, e.g., light exposure at night, would cause disruption in the function of the SCN which would then pass this inappropriate information to cells in the periphery. One circadian rhythm that transfers time of day information to the organism is the melatonin cycle which is always at low levels in the blood during the day and at high levels during darkness. With light exposure at night the amount of melatonin produced is compromised and this important rhythm is disturbed. Another important source of melatonin is the gastrointestinal tract (GIT) that also influences the circulating melatonin is the generation of this hormone by the entero-endocrine (EE) cells in the gut following ingestion of tryptophan-containing meal. The consequences of the altered melatonin cycle with the chronodisruption as well as the alterations of GIT melatonin that have been linked to a variety of pathologies, including those of the gastrointestinal tract.

Circadian and seasonal rhythms are a fundamental feature of all living organisms and their organelles. Biological rhythms are responsible for daily food intake; the period of hunger and satiety is controlled by the central pacemaker, which resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, and communicates with tissues via bidirectional neuronal and humoral pathways. The molecular basis for circadian timing in the gastrointestinal tract (GIT) involves interlocking transcriptional/translational feedback loops which culminate in the rhythmic expression and activity of a set of clock genes and related hormones. Interestingly, it has been found that clocks in the GIT are responsible for the periodic activity (PA) of its various segments and transit along the GIT; they are localized in special interstitial cells, with unstable membrane potentials located between the longitudinal and circular muscle layers. The rhythm of slow waves is controlled in various segments of the GIT: in the stomach (about 3 cycles per min), in the duodenum (12 cycle per min), in the jejunum and ileum (from 7 to 10 cycles per min), and in the colon (12 cycles per min). The migrating motor complex (MMC) starts in the stomach and moves along the gut causing peristaltic contractions when the electrical activity spikes are superimposed on the slow waves. GIT hormones, such as motilin and ghrelin, are involved in the generation of MMCs, while others (gastrin, ghrelin, cholecystokinin, serotonin) are involved in the generation of spikes upon the slow waves, resulting in peristaltic or segmental contractions in the small (duodenum, jejunum ileum) and large bowel (colon). Additionally, melatonin, produced by neuro-endocrine cells of the GIT mucosa, plays an important role in the internal biological clock, related to food intake (hunger and satiety) and the myoelectric rhythm (produced primarily by the pineal gland during the dark period of the light-dark cycle). This appears to be an endocrine encoding of the environmental light-dark cycle, conveying photic information which is used by organisms for both circadian and seasonal organization. Motor and secretory activity, as well as the rhythm of cell proliferation in the GIT and liver, are subject to many circadian rhythms, mediated by autonomic cells and some enterohormones (gastrin, ghrelin and somatostatin). Disruption of circadian physiology, due to sleep disturbance or shift work, may result in various gastrointestinal diseases, such as irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD) or peptic ulcer disease. In addition, circadian disruption accelerates aging, and promotes tumorigenesis in the liver and GIT. Identification of the molecular basis and role of melatonin in the regulation of circadian rhythm allows researchers and clinicians to approach gastrointestinal diseases from a chronobiological perspective. Clinical studies have demonstrated that the administration of melatonin improves symptoms in patients with IBS and GERD. Moreover, our own studies indicate that melatonin significantly protects gastrointestinal mucosa, and has strong protective effects on the liver in patients with non-alcoholic steatohepatitis (NASH). Recently, it has been postulated that disruption of circadian regulation may lead to obesity by shifting food intake schedules. Future research should focus on the role of clock genes in the pathophysiology of the GIT and liver.

The mechanism by which nonalcoholic fatty liver disease (NAFLD) progresses into nonalcoholic steatohepatitis (NASH) is unknown, however, the major process is oxidative stress with increased production of reactive oxygen species and excessive inflammatory cytokine generation. To date, there are no effective treatments for NASH and the published data with treatment using antioxidants are not satisfactory. Melatonin (MT), the potent endogenous antioxidant secreted in circadian rhythm by pinealocytes and in large amounts in the digestive system, was reported to improve oxidative status and to exert beneficial effects in NASH pathology in experimental animals, but no study attempted to determine the possible effectiveness of MT in humans with NASH. In this study, 42 patients (12 placebo controls and 30 MT-treated) with histological evidence (liver biopsy) of NASH and no history of alcohol abuse, were included. The treatment group took melatonin (2x5 mg/daily orally), while controls were treated with placebo. At baseline no significant differences between the groups were found for age, body mass index (BMI) as well as for plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and concentrations of cholesterol, triglycerides (TG), glucose and MT. During the study period plasma ALT level and cholesterol concentration decreased significantly in both MT-treated and control groups, however AST and GGT levels decreased significantly only in MT-treated groups. Median value of AST level at baseline was 76.5 (64.2-114.2) IU/L and its percentage decrease at 4, 8 and 12 week was 20, 36 and 38%, resp. Baseline GGT median level was 113 (75.7-210.7) IU/L and its mean percentage decrease at week 4, 8 and 12 was 46, 48 and 47%, resp. Plasma ALP levels did not change significantly during MT treatment. Median value of plasma concentrations of MT (pg/mL) in MT-treated group rose from 7.5 (5.0-14.25) at baseline to 35.5(18.8-110.0), 43.5(17.0-102.5) and 49.5(18.0-99.5) at the end of 4, 8 and 12 week of treatment, respectively. Plasma levels of TG and glucose as well as BMI in controls and MT-treated patients were not significantly different from baseline. This study demonstrates for the first time in humans that three months treatment with MT significantly improves plasma liver enzymes in patients with NASH without causing any side-effect. Plasma MT levels during the whole period of MT treatment persisted above that at baseline. Our findings show that treatment with MT significantly improves plasma liver enzymes in NASH patients, but larger cohort trials and longer treatment with MT are required before this indole could be included into the spectrum of the NASH treatment.

Previous studies have shown that stimulation of cannabinoid 1 (CB1) receptor protects the gastric mucosa against stress-induced lesion. Aim of the present study was to examine the influence of anandamide on lipid peroxidation and antioxidant defense system in gastric mucosa and the role of sensory nerves in gastroprotective effects of cannabinoids. Studies were performed on rats with intact or ablated sensory nerves (by neurotoxic doses of capsaicin). Gastric lesions were induced by water immersion and restrain stress (WRS). Anandamide was administered at the dose of 0.3, 1.5 or 3.0 μmol/kg, 30 min before exposure to WRS. CB1 receptor antagonist, AM251 (4.0 μmol/kg) was administered 40 min before WRS. WRS induced gastric lesions associated with the decrease in gastric blood flow, mucosal DNA synthesis and mucosal activity of superoxide dismutase (SOD). Serum level of interleukin-1β (IL-1β) and mucosal level of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were increased. Administration of anandamide reduced the ulcers area, generation of MDA+4-HNE and serum level of IL-1β, and this effect was associated with the reduction in the WRS-induced decrease in gastric mucosal blood flow, mucosal DNA synthesis and SOD activity. Ablation of sensory nerves increased the area of ulcers, serum level of IL-1β and mucosal content of MDA+4-HNE, whereas mucosal DNA synthesis, SOD activity and blood flow were additionally decreased. In rats with ablation of sensory nerves, administration of anandamide at the high doses (1.5 and 3.0 μmol/kg) partly reduced deleterious effect of WRS on gastric mucosa, but this effect was weaker than in animals with intact sensory nerves. Low dose of anandamide (0.3 μmol/kg) was ineffective in the protection of gastric mucosa against the WRS-induced lesions in rats with ablation of sensory nerves. In rats with intact sensory nerves and exposed to WRS, administration of AM251 exhibited deleterious effect. In rats with ablation of sensory nerves and exposed to WRS, AM251 failed to affect mucosal injury in the stomach. We conclude that anandamide reduces the mucosal oxidative stress and exhibits gastroprotective effect against WRS-induced ulcers. These effects are partly mediated by sensory nerves.

Nonalcoholic fatty liver disease is the most common chronic liver disease and nonalcocholic steatohepatitis (NASH) is its advanced form. Oxidative stress and hepatocyte apoptosis may be involved in pathogenesis of NASH and particularly in progress of NASH to liver fibrosis and cirrhosis, which are initiated by the inflammation and which promote the progress of the disease. The aim of this study was to evaluate the effects of melatonin and L-tryptophan on selected biochemical parameters of blood in patients with NASH. Forty five patients with NASH, confirmed by histopathological examination of liver biopsy samples, were admitted to the study. They were divided into three groups (I, II and III). The first group (group I, n=15) received preparation Essentiale forte 3 times a day and tryptophan 500 mg twice daily for 4 weeks. In the second group (group II, n=15), Essentiale forte three times a day was administered with melatonin 5 mg applied twice a day for 4 weeks. The third group (group III, n=15) received only Essentiale forte with placebo three times a day for 4 weeks. After four-week treatment we found statistically significant reduction in GGTP, triglycerides and proinflammatory cytokine levels in the melatonin-treated (group I) and the L-tryptophan-treated patients (group II). Plasma level of melatonin was significantly elevated in groups treated with tryptophan (group I) and melatonin (group II), but remained unchanged in placebo-treated group (group III). Among patients from the third group (treated with placebo) no statistically significant differences in the measured biochemical parameters were observed. The present study suggests that melatonin and tryptophan have the significant impact on the reduction in plasma levels of proinflammatory cytokines and may be useful in the treatment of patients with NASH.

Melatonin and its precursor, l-tryptophan, have been shown to exert gastroprotective effects in animals, but their influence on the gastric damage by aspirin (ASA) in humans has been sparingly investigated. In this study, we designed to determine the effects of melatonin and l-tryptophan on ASA-induced gastric mucosal damage, gastric microbleeding, mucosal generation of prostaglandin E(2), and plasma melatonin, and gastrin levels. Three groups of healthy male volunteers (n = 30) with intact gastric mucosa received daily for 11 days either ASA alone or that combined with melatonin or tryptophan. Gastric blood loss and mucosal damage were evaluated at 3rd, 7th, and 11th days of ASA administration by endoscopy using Lanza score. ASA alone caused a marked rise of gastric damage and gastric blood loss, mainly at day 3rd and 7th, but they were significantly reduced at 11th day. Pretreatment with melatonin or tryptophan remarkably reduced ASA induced gastric lesions and microbleeding. Gastric mucosal generation of PGE(2) was suppressed by about 90% in all subjects treated with ASA alone without or with addition of melatonin or tryptophan. Plasma melatonin was markedly increased after treatment with melatonin or tryptophan plus ASA, but it was also raised significantly after application of ASA alone. Plasma gastrin levels were raised in subjects given melatonin or tryptophan plus ASA, but not in those with ASA alone. We conclude that melatonin and its precursor tryptophan given orally significantly reduce gastric lesions induced by ASA possibly due to (a) direct gastroprotective action of exogenous melatonin or that generated from tryptophan and (b) gastrin released from the gastric mucosa by melatonin or tryptophan.

Histamine is not only a potent stimulator of gastric acid secretion, but it also plays a central role in gastroduodenal ulcerogenesis. In the present study we tested the effect of pre-treatment with exogenous prostaglandin E(2) (PGE(2)) in a new rat model of experimental gastric ulcers induced by combination of histamine and gastric ischemia.In male Wistar rats, a chronic ischemia of gastric mucosa was induced via the clamping of the left gastric artery and vein (L-AV) in combination with pylorus ligation. The following treatment groups of rats (6 rats/group) were investigated: 1) histamine alone (40 mg/kg twice s.c.); 2) vehicle (saline) followed 30 min later by gastric mucosal L-AV ischemia and pylorus ligation combined with histamine (40 mg/kg twice s.c.) and 3) PGE(2) (5 microg/kg i.g.) followed 30 min later by gastric mucosal L-AV ischemia combined with histamine (40 mg/kg twice s.c.) and pylorus ligation. At 4 hr after the clamping of L-AV and pylorus ligation, the area of gastric lesions and gastric acid secretion was determined.Histamine treatment failed to produce gastric lesions, but when it was combined with ischemia, the widespread gastric lesions in the corpus mucosa, but not in the antrum, were observed. This damaging effect and decrease in the GBF were significantly attenuated by pretreatment with PGE(2).The present study demonstrates that gastric hypersecretion induced by histamine in combination with gastric mucosal ischemia results in gastric lesions which progress into chronic gastric ulcers.

Diabetes mellitus increases susceptibility to acute gastric injury and impairs ulcer healing. Pioglitazone as an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma) is used as anti-diabetic drug and has additionally gastroprotective activities. However, the effect of pioglitazone on the protection and healing of gastric mucosa under diabetic conditions is poorly understood. The aim of the present study was: 1) to compare the effects of treatment with PPARg ligand (pioglitazone) on healing of acetic acid-induced gastric ulcers and prevention of acute water immersion and restraint stress (WRS)-induced gastric lesions in normal rats and those with streptozotocin (STZ)-induced diabetes mellitus; 2) to assess the effects of pioglitazone on the mRNA expression of cyclooxygenase-2 (COX-2), c-NOS, interleukin-1beta and hypoxia inducible factor-1 alpha (HIF-1alpha) in the gastric mucosa of rats with or without STZ-induced diabetes mellitus; 3) to investigate the involvement of endogenous NO and proinflammatory cytokines (IL-1beta, TNF-alpha) in healing of chronic gastric ulcers and in prevention of acute stress lesions by pioglitazone in rats with or without STZ-induced diabetes mellitus. Diabetes was induced in rats by single injection of STZ (70 mg/kg i.p.) four weeks prior to production of gastric ulcers by acetic acid method or induction of stress lesions by 3.5 hours of WRS. Non-diabetic rats were used as controls. Two major animal groups (A and B) were tested; A) diabetic and non-diabetic rats with chronic gastric ulcers treated with 1) pioglitazone (40 mg/kg-d i.g.), 2) pioglitazone in combination of blocker of NO synthase (L-NNA 20 mg/kg-d i.p.), and 3) saline (vehicle-control); and B) diabetic and non-diabetic rats exposed to 3.5 hours of WRS and pretreated with 1) pioglitazone (40 mg/kg i.g.), 2) pioglitazone in combination of blocker of NO synthase (L-NNA 20 mg/kg i.p.), and 3) saline (vehicle-control). The gastric mucosal blood flow was assessed by H(2)-gas clearance method. The area of chronic acetic acid ulcers and number of acute WRS-induced gastric lesions were assessed by planimetry or by counting of number of lesions, respectively. In rats with chronic ulcers, the mRNA expression of HIF-1alpha, IL-1beta and COX-2 was assessed by RT-PCR and protein expression of platelet endothelial cell adhesion molecule-1 (PECAM-1), COX-2 and cNOS was examined by Western blot. In rats with stress lesions, the protein expression of COX-2, cNOS, catalase, PPAR and heat shock protein 70 (HSP70) was examined by Western blot. In diabetic rats, a marked delay in ulcer healing and increased susceptibility to WRS lesions were observed and these effects were accompanied by a significant decrease in GBF. Pioglitazone significantly increased healing of chronic gastric ulcers and exerted a strong protective effect against WRS-induced lesions, but these effects were attenuated by NO-inhibition with L-NNA. Interestingly, the ulcer healing and gastroprotective effects of pioglitazone were weak under diabetic conditions, and this effect on ulcer healing was accompanied by impaired angiogenesis due to decreased PECAM-1 expression, attenuated expression of COX-2 and the increased expression of proinflammatory cytokines compared to those in diabetic rats treated with vehicle. We conclude that: 1) experimental diabetes in rats impairs healing of chronic ulcers and enhances acute stress lesions due to an increase in the expression and release of proinflammatory cytokines such as TNF-alpha and IL-1beta; 2) the ulcer healing effect of pioglitazone, which is, at least in part, mediated by endogenous NO, is significantly attenuated by L-NNA in diabetic rats despite increased COX-2 expression at the ulcer edge; 3) the formation of acute gastric lesions induced by WRS is also attenuated by pretreatment with pioglitazone due to increased GBF probably mediated by NO, as the administration of L-NNA reversed, in part, the preventive action induced by this PPARgamma ligand, and 4) pioglitazone is effective both in healing of chronic ulcers and protection against WRS lesions though its action under diabetic conditions seems to be attenuated, possibly due to reduction in NOS-NO system, angiogenesis and increased expression and release of proinflammatory cytokines.

Probiotic bacteria Escherichia coli Nissle (EcN) was shown to prevent or heal acute murine colitis, but gastroprotective effects of EcN against mucosal injury have been little studied. In this study, the effects of EcN on formation of stress-induced gastric erosions were assessed in rats. Rats were divided in following treatment groups: 1) vehicle (control); 2) EcN 10(1) CFU/ml; 3) EcN 10(4) CFU/ml and 4) EcN 10(8) CFU/ml. One hour after treatment, the rats were exposed to 3.5 h of water immersion and restraint stress (WRS) and then sacrificed. Involvement of prostaglandins was tested using indomethacin given one hour before EcN, whereas that of sensory nerves was assessed using neurotoxic dose of capsaicin in rats pretreated with EcN or vehicle. The expression of proinflammatory cytokine (IL-1beta), ghrelin, peroxisome proliferator receptor gamma (PPARgamma) and heat-shock protein (HSP70) was assessed by RT-PCR and Western blot. Exposure to WRS in vehicle-pretreated rats induced acute erosions. Pretreatment with EcN significantly reduced WRS lesions and increased gastric blood flow. This protective effect was completely abolished by indomethacin and significantly attenuated by capsaicin-denervation. The exposure to WRS was accompanied by an increase in gastric mucosal expression of IL-1beta, ghrelin, PPARgamma, HSP70 and COX-2. In rats pretreated with EcN, a significant downregulation of mRNA and protein expression for IL-1beta, COX-2 and PPARgamma and increased expression of HSP70 without major change in activation of NFkappaB were observed. We conclude that EcN protects gastric mucosa against WRS erosions due to antiinflammatory and vasodilatory actions involving HSP70, prostaglandins and sensory afferent neurons.

Bile salts play an important pathogenic role in the development of Barrett adenocarcinoma (BA). However, the precise role of different bile salts in this process is still unknown. The aim of the present study was to compare the effects of two different bile salts, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on the expression of COX-2, CDX-2 and DNA repair enzymes (MUTYH, OGG-1) in the Barrett epithelial cancer cells (OE-19). OE-19 cells were incubated with DCAor UDCA(100 microM or 300 microM at pH=7.0) over 24 h. To investigate the involvement of NF kappaB, in separate experiments the cells were incubated with DCA in the presence of proteosome inhibitor (MG-132). Cells cycle and apoptosis were analyzed by FACS analysis. After incubation of OE-19 cells with bile salts, the expression of mRNA of COX-2, DNA repair enzymes (MUTYH, OGG-1) and caudal-related homebox transcription factor CDX-2 were measured by quantitative RT-PCR. OE-19 cell were also transfected with siRNA-RelA (p65) to asses effect of NF kappaB inactivation on COX-2 and CDX2 expression. DCA caused a stronger reduction in cell survival of OE-19 cells than UDCA. In addition, DCA stimulated directly the translocation of NF kappaB p65 (active form) in the nuclei of OE-19 cells. DCA caused stronger than UDCA stimulation of the COX-2 mRNA expression in these cells and this effect was significantly attenuated by the addition of inhibitor of NF kappaB activity (proteosome inhibitor MG-132). siRNA-RelA reduced expression not only of NF kappaB but also expression of COX-2 as well as CDX-2 mRNA. DCA caused stronger downregulation of mRNA for DNA repair enzymes MUTYH and OGG-1 than UDCA. In contrast, UDCA induced stronger CDX-2 mRNA expression than DCA in OE-19 cells. We conclude that bile salts are involved in the carcinogenesis of Barrett adenocarcinoma via inhibition of DNA repair enzymes and induction of COX-2 and this last effect is, at least partly, mediated by NF kappaB. DCA shows carcinogenic potential due to high upregulation of COX-2, CDX-2 and downregulation of DNA repair enzymes.

This study was performed to assess whether mice lacking the cannabinoid receptor CB1, CB2 or both receptors show increased susceptibility to TNBS colitis in comparison to wildtype mice. Previously, activation of CB1 and CB2 receptors showed attenuation of TNBS colitis in mice. The aim of the study was to investigate the susceptibility of three mouse strains CB1-, CB2- and CB1+2 double knockout mice in the model of TNBS colitis. The different knockout mice were given each a single enema with TNBS 7 mg, volume 150 microl (in 50% ethanol solution) on day 1. Control group (C57BL/6 mice) received the same concentration of TNBS enema and each strain received vehicle application of 150 microl 50% ethanol solution. After a 3-day period, the animals were sacrificed and their colon excised. A scoring system was used to describe macroscopical and histological changes. Messenger RNA-expression of TNF-alpha and IL-1beta as pro-inflammatory markers was measured by RT-PCR. All three knockout strains showed increased susceptibility to TNBS colitis quantified by macroscopical and histological scoring systems and pro-inflammatory cytokine expression in comparison to the TNBS control group (wild type C57BL/6 animals). Mice lacking the CB1-, CB2-receptor or both receptors showed aggravation of inflammation in the model of TNBS colitis. Lacking of both cannabinoid receptors did not result in potentiation of colitis severity compared to lacking of each CB1 or CB2, respectively. These results suggest that the endocannabinoid system may have tonic inhibitory effects on inflammatory responses in the colon.

Gastric cancer (GS) remains one of the most common cancers worldwide. It is considered as the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Many studies before linked Helicobacter pylori (Hp) which is now considered as an important pathogen, to the risk of developing noncardia GS. This overview attempts to summarize the recent basic and clinical evidence on the link between H. pylori and gastric cancer, after the award of the Nobel Prize for Physiology or Medicine to Drs. J.R. Warren and B.J. Marshall for the first culture and isolation of Hp and the investigation of their relevance to peptic ulcer disease. It become evident that Hp eradication by antibiotic treatment combined with proton pump inhibitor (PPI) serves as the primary chemoprevention strategy to reduce gastric cancer incidence. Moreover, the eradication therapy reduces gastric cancer incidence in patients without any precancerous lesions at the baseline and is most effective before the development of atrophic gastritis. Due to understanding the molecular nature of GC which has been nowadays under intense investigation, our review attempts to highlight recent progress in the field of research on Hp-induced GS. We discuss the geographical diversity in Hp infection and cancer incidence and the mechanistic role of gastrin, cyclooxygenase-2 (COX-2), growth factor, nitric oxide (NO)/NO synthase and E-cadherin/beta-cathenin systems, apoptosis and angiogenesis in Hp-induced gastric carcinogenesis. In addition host-related genetic susceptibility and the role of overexpression of a proinflammatory cytokines and their polymorphism is discussed in the relation to the cascade of events such as gastric atrophy, intestinal metaplasia and dysplasia that finally lead to adenocarcinoma.

Ghrelin is a novel growth hormone (GH)-releasing and orexigenic peptide with anti-inflammatory activities. However, the role of ghrelin in the colonic inflammation is still controversial. The aim of the present study was: 1) to examine the expression of ghrelin and TNF-alpha mRNA in the inflamed colonic mucosa of patients with ulcerative colitis (UC), 2) to analyze the effect of treatment with exogenous ghrelin on the healing of trinitrobenze sulphonic acid (TNBS)-induced colitis in rats, and 3) to assess the effects of ghrelin treatment on mRNA expression for iNOS and protein expression for COX-2 and PPARalpha in intact colonic mucosa and in that with TNBS-induced colitis. Fifteen patients with UC and fifteen healthy controls were enrolled in this study. Expression of ghrelin and TNF-alpha was assessed by semi-quantitative RT-PCR in the colonic mucosal biopsies from UC patients and healthy controls. In addition, the effect of exogenous ghrelin on healing of TNBS colitis was tested in rats without or with capsaicin-induced functional ablation of sensory nerves. Patients with UC showed a significant upregulation of mRNA for ghrelin and TNF-alpha in colonic mucosa as compared to that observed in healthy controls. The expression of ghrelin correlated with the grade of inflammation and expression of TNF-alpha. In rats the exogenous ghrelin administered daily at a dose of 20 microg/kg i.p. significantly accelerated the healing of TNBS colitis and this effect was accompanied by an increase in mRNA expression for iNOS and protein expression for COX-2 in the colonic mucosa. The protein expression for PPARgamma, which was down-regulated in rat colonic mucosa after exposure to TNBS as compared to that in intact colonic mucosa, was not significantly influenced by ghrelin treatment. We conclude that 1) patients with UC show an increased mucosal expression of mRNA for ghrelin in the colonic mucosa which could trigger protective response in inflamed colon; and 2) exogenous ghrelin accelerates healing of colonic lesions in animal model of ulcerative colitis via increased release of NO and PGE(2) due to an increase in iNOS and COX-2 expression and stimulation of sensory neuropeptides such as CGRP released from sensory afferent endings.

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.