Konturek Sj

Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage

Ghrelin, identified in the gastric mucosa has been involved in control of food intake and growth hormone (GH) release but little is known about its influence on gastric secretion and mucosal integrity. The effects of ghrelin on gastric secretion, plasma gastrin and gastric lesions induced in rats by 75% ethanol or 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous ghrelin (5, 10, 20, 40 and 80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by ethanol and WRS and this was accompanied by the significant rise in plasma ghrelin level, gastric mucosal blood flow (GBF) and luminal NO concentrations. Ghrelin-induced protection was abolished by vagotomy and attenuated by suppression of COX, deactivation of afferent nerves with neurotoxic dose of capsaicin or CGRP(8-37) and by inhibition of NOS with L-NNA but not influenced by medullectomy and administration of 6-hydroxydopamine. We conclude that ghrelin exerts a potent protective action on the stomach of rats exposed to ethanol and WRS, and these effects depend upon vagal activity, sensory nerves and hyperemia mediated by NOS-NO and COX-PG systems.

Activation of Genes for Superoxide Dismutase, Interleukin-1ß, Tumor Necrosis Factor-a, and Intercellular Adhesion Molecule-1 during Healing of Ischemia-Reperfusion-Induced Gastric Injury

Background: Ischemia followed by reperfusion (I/R) induces gastric lesions, probably due to excessive formation of free radicals, but the role of the scavenger of these radicals, proinflammatory cytokines such as interleukin-1b (IL-1b) and tumor necrosis factora (TNF-a), in the healing of these lesions has not been extensively studied. It is also unknown whether expression of intercellular adhesion molecule-1 (ICAM1), which mediates neutrophil-induced injury and neutrophil infiltration, is involved in the recovery from I/ R lesions.Methods: I/R lesions were induced in Wistar rats by applying a small clamp to the celiac artery for 30 min (ischemia phase), followed by the removal of the clamp for 60 min (reperfusion phase). The influence of I/R on gastric secretion was also tested in rats equipped with a gastric fistula (GF) without or with the exposure to a standard period of I/R. Two series of rats (A and B) were used to determine the effects of exogenous and endogenous superoxide dismutase SOD (series A) and allopurinol, a xanthine oxidase inhibitor (series B), on the mucosal recovery from the gastric lesions induced by I/R. The animals were killed immediately after the exposure to I/R (0 h) and at 3 h, 24 h, or 3, 5, or 10 days after this I/R, the area of gastric lesions being measured by planimetry, and the gastric blood flow (GBF) determined by the H2 gas clearance method. Blood was withdrawn for measurement of plasma IL-1 b and TNF-a levels with enzyme-linked immunosorbent assay, and plasma gastrin with radioimmunoassay. Biopsy samples of oxyntic mucosa were taken for the assessment of SOD, IL-1 b, TNF-a, and ICAM-1 mRNAs by reversetranscription polymerase chain reaction and Southern blot. Results: Exposure to I/R resulted in acute gastric erosions, with the maximal increase of the area of these lesions observed 3 h after the end of I/R. This effect was accompanied by a decrease in the GBF, a significant increase in blood free radicals and plasma gastrin increments, and almost complete suppression of gastric secretion. Starting 24 h after I/R, the gastric superficial lesions progressed into deeper ulcers that healed progressively within 10 days, and this was accompanied by gradual restoration of the gastric secretion and the GBF. Treatment with SOD and allopurinol accelerated significantly the healing of I/R erosions, and this effect was accompanied by a significant increase in the GBF and the attenuation of blood free radicals. At 0, 3, and 12 h after I/R a significant decrease in SOD mRNA was observed, whereas expression of TNFa, IL-1b, and ICAM-1 showed a progressive increase starting immediately after I/R, reaching a maximum on day 3. The plasma level of TNF-a and IL-1b started to increase on day 3 and peaked on day 5 after I/R, being still significantly higher at day 10 than that measured in the vehicle-treated control gastric mucosa. On day 10 the gastric ulcers were almost completely healed, and a decrease in the expression for TNFa, IL-1b, and ICAM-1 mRNA and an increase in the expression of SOD mRNA were observed. Conclusions:1) exposure to I/R produces gastric lesions mediated by the excessive formation of free radicals, resulting in suppression of both gastric microcirculation and secretory activity of the stomach; 2) SOD and allopurinol accelerate the healing of I/R lesions, probably due to suppression of oxygen free radicals and improvement of gastric microcirculation; and 3) the upregulation of SOD mRNA, with subsequent increase in the SOD production and local release of IL-1 b and TNF-a, may activate ICAM-1 expression and neutrophil infiltration, which appear to play an important role in the progression of I/R-induced acute gastric erosions into chronic ulcers.BACKGROUND Ischemia followed by reperfusion (I/R) induces gastric lesions, probably due to excessive formation of free radicals, but the role of the scavenger of these radicals, proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in the healing of these lesions has not been extensively studied. It is also unknown whether expression of intercellular adhesion molecule-1 (ICAM-1), which mediates neutrophil-induced injury and neutrophil infiltration, is involved in the recovery from I/R lesions. METHODS I/R lesions were induced in Wistar rats by applying a small clamp to the celiac artery for 30 min (ischemia phase), followed by the removal of the clamp for 60 min (reperfusion phase). The influence of I/R on gastric secretion was also tested in rats equipped with a gastric fistula (GF) without or with the exposure to a standard period of I/R. Two series of rats (A and B) were used to determine the effects of exogenous and endogenous superoxide dismutase SOD (series A) and allopurinol, a xanthine oxidase inhibitor (series B), on the mucosal recovery from the gastric lesions induced by I/R. The animals were killed immediately after the exposure to I/R (0 h) and at 3 h, 24 h, or 3, 5, or 10 days after this I/R, the area of gastric lesions being measured by planimetry, and the gastric blood flow (GBF) determined by the H2 gas clearance method. Blood was withdrawn for measurement of plasma IL-1beta and TNF-alpha levels with enzyme-linked immunosorbent assay, and plasma gastrin with radioimmunoassay. Biopsy samples of oxyntic mucosa were taken for the assessment of SOD, IL-1beta, TNF-alpha, and ICAM-1 mRNAs by reverse-transcription polymerase chain reaction and Southern blot. RESULTS Exposure to I/R resulted in acute gastric erosions, with the maximal increase of the area of these lesions observed 3 h after the end of I/R. This effect was accompanied by a decrease in the GBF, a significant increase in blood free radicals and plasma gastrin increments, and almost complete suppression of gastric secretion. Starting 24 h after I/R, the gastric superficial lesions progressed into deeper ulcers that healed progressively within 10 days, and this was accompanied by gradual restoration of the gastric secretion and the GBF. Treatment with SOD and allopurinol accelerated significantly the healing of I/R erosions, and this effect was accompanied by a significant increase in the GBF and the attenuation of blood free radicals. At 0, 3, and 12 h after I/R a significant decrease in SOD mRNA was observed, whereas expression of TNF-alpha, IL-1beta, and ICAM-1 showed a progressive increase starting immediately after I/R, reaching a maximum on day 3. The plasma level of TNF-alpha and IL-1beta started to increase on day 3 and peaked on day 5 after I/R, being still significantly higher at day 10 than that measured in the vehicle-treated control gastric mucosa. On day 10 the gastric ulcers were almost completely healed, and a decrease in the expression for TNF-alpha, IL-1beta, and ICAM-1 mRNA and an increase in the expression of SOD mRNA were observed. CONCLUSIONS 1) exposure to I/R produces gastric lesions mediated by the excessive formation of free radicals, resulting in suppression of both gastric microcirculation and secretory activity of the stomach; 2) SOD and allopurinol accelerate the healing of I/R lesions, probably due to suppression of oxygen free radicals and improvement of gastric microcirculation; and 3) the upregulation of SOD mRNA, with subsequent increase in the SOD production and local release of IL-1beta and TNF-alpha, may activate ICAM-1 expression and neutrophil infiltration, which appear to play an important role in the progression of I/R-induced acute gastric erosions into chronic ulcers.

Expression of survivin and caspase-3 in gastric cancer.

Gastric cancer is one of the most common malignant tumors of the gastrointestinal tract. However, the molecular pathways involved in the regulation of gastric carcinogenesis are not completely elucidated. In the last decade, basic cancer research has been focused on the deregulation of apoptosis as a central event in the process of carcinogenesis. Caspase-3 and survivin are regulators of apoptosis and have been implicated in the development of gastric cancer. The aim of the present study was to compare the expression of mRNA and protein for survivin and caspase-3 in the gastric cancer and in the cancer margin with that in normal human gastric mucosa. Fifteen patients with advanced gastric cancer (all H. pylori-positive) and 15 matched control subjects with normal gastric mucosa were included in this study. The biospy specimens for histology and for molecular analyses were taken from gastric tumor, tumor surrounding gastric mucosa and in normal patients from the mucosa of antrum and corpus. Survivin mRNA expression was very weak, but detectable, in the normal gastric mucosa. However, at the protein level, no expression for survivin was detected in the normal gastric mucosa. In the biopsy specimens from tumor and surrounding gastric mucosa, a significant increase in survivin mRNA and protein expression was observed. The expression of survivin was higher in the tumor than in the tumor margin. The mRNA and protein expression of caspase-3 was detected in the gastric mucosa of normal subjects. In gastric cancer only the expression of procaspase-3 was observed, while the expression of active caspase-3 was completely undetectable. In the gastric mucosa surrounding gastric cancer, no gene and protein expression for caspase-3 was detected. We conclude that the changes in the level of caspase-3 and survivin play an important role in the transformation from normal gastric mucosa to gastric career.

Pioglitazone, a Specific Ligand of the Peroxisome Proliferator-Activated Receptor Gamma Reduces Gastric Mucosal Injury Induced by Ischaemia/Reperfusion in Rat

Background: The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor that has been implicated in the control of metabolism and numerous cellular processes, including cell cycle control, carcinogenesis, and inflammation. The present study was designed to investigate the effect of the specific PPARγ ligand, pioglitazone, on the mucosal lesions induced by ischaemia and reperfusion (I/R) in rats. Methods: I/R lesions were induced in Wistar rats by applying a small clamp to the coeliac artery for 30 min (ischaemic phase), followed by the removal of the clamp for 3 h (reperfusion phase). Vehicle (saline) or increasing doses of pioglitazone (2.5, 10, and 30 mg/kg i.g.) were given 30 min before exposure to I/R. The animals were killed immediately after the end of the reperfusion phase (time 0) and at 12 and 24 h after I/R. The area of gastric lesions was measured by planimetry, and the gastric blood flow was determined by the H[Formula: See Text] gas clearance method. The gastric mucosal gene expressions of PPARγ, interleukin-1beta (IL-1β), tumour necrosis factor alpha (TNF-α), leptin, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were examined by RT-PCR. In addition, protein expression of COX-2 and leptin was assessed by Western blot. Results: The pretreatment with pioglitazone reduced in a dose-dependent manner the mean lesion area induced by I/R, and this effect was accompanied by a significant increase in the gastric blood flow. The decrease in gastric ulcerations by pioglitazone was also observed 12 and 24 h after the I/R. The PPARγ mRNA was weakly expressed in the intact gastric mucosa, but significantly up-regulated after exposure to I/R at each time interval studied. The expression of IL-1β was not changed significantly after pioglitazone applied i.g. at doses 2.5 and 10 mg/kg, but it was down-regulated at the dose 30 mg/kg. TNFα mRNA was strongly increased after the exposure to I/R, but it was down-regulated after pioglitazone pretreatment. In contrast, both leptin and COX-2 mRNA and protein expression were increased in the gastric mucosa after exposure to I/R. The pretreatment with pioglitazone caused a significant up-regulation of mRNA and protein expression of leptin, reaching its peak at the dose 30 mg/kg i.g. In contrast, COX-2 expression did not change significantly after the 2.5 and 10 mg/kg of pioglitazone, but it significantly decreased after pioglitazone at dose 30 mg/kg given to rats before exposure to I/R. Conclusions: Pioglitazone reduces the acute erosions and deeper gastric lesions induced by I/R. The beneficial effect of this PPARγ ligand on I/R-induced gastric damage may be due to its anti-inflammatory properties, especially to the reduction in TNF-α expression and to up-regulation of leptin mRNA in the gastric mucosa. The inhibition of COX-2 expression by pioglitazone may reflect the anti-inflammatory properties of this compound.

Pioglitazone, a specific ligand of peroxisome proliferator-activated receptor-gamma, accelerates gastric ulcer healing in rat

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation and carcinogenesis. We determined the effect of the specific PPAR-gamma ligand, pioglitazone (5-40 mg/kg intragastrically), on the healing of acetic-acid gastric ulcers in rats. At day 8 after ulcer induction, the ulcer area, the gastric blood flow and mucosal expression of proinflammatory cytokines such as interleukin-1beta, tumour necrosis factor alpha (TNF-alpha) and cyclooxygenase-1, cyclooxygenase-2, constitutive nitric oxide synthase (cNOS), inducible nitric oxide synthase (iNOS) and heat shock protein 70 (HSP70) was determined. Pioglitazone reduced the area of gastric ulcers and raised significantly the gastric blood flow at the ulcer margin and downregulated the mRNA for interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS while cyclooxygenase-1 mRNA was not affected. The expression of PPAR-gamma mRNA was increased in the ulcerated gastric mucosa. We conclude that pioglitazone accelerates the healing of preexisting gastric ulcers due to the hyperemia at ulcer margin and the anti-inflammatory action including suppression of interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS and by an overexpression of HSP70.

Expression of hepatocyte growth factor, transforming growth factor alpha, apoptosis related proteins Bax and Bcl-2, and gastrin in human gastric cancer

Gastric cancer is one of the most frequent neoplasms and a leading cause of the death world‐wide. In recent years, epidemiological and animal studies demonstrated a link between gastric cancer and chronic infection with H. pylori. The exact mechanism responsible for the development of gastric cancer in H. pylori‐infected patients still remains unclear. There is evidence that the up‐regulation of certain growth factors could play an important role in the promotion of the gastric carcinogenesis.

Epidermal growth factor, polyamines, and prostaglandins in healing of stress-induced gastric lesions in rats

Previous studies demonstrated that epidermal growth factor (EGF) and polyamines (PA) are capable of protecting gastric mucosa against topical irritants. This study was designed to examine whether EGF, PA, and PG affect the healing of acute gastric lesions induced by water immersion and restraint stress. It was found that the healing process of stress lesions in sham-operated rats was significant after 6 hr after stress, and after 24 hr the number of stress lesions was reduced by about 75%. In sham-operated rats, the healing of ulcerations observed at 6, 12, and 24 hr after the stress was accompanied by gradual restoration of DNA synthesis, and both these processes were significantly reduced by administration of DFMO (an inhibitor of ornithine decarboxylase activity) or indomethacin (an inhibitor of cyclooxygenase). In salivectomized rats, the healing was significantly delayed and the DNA was lowered at all time intervals after the stress. Administration of EGF, spermine, aminoguanidine (an inhibitor of degradation of PA), or 16,16-dmPGE2 after stress promoted significantly the healing and DNA synthesis, but pretreatment with DFMO abolished the effect of EGF but not that of spermine. We conclude that EGF, PA, and PG are implicated in healing of stress lesions and that EGF acts, at least in part, by the stimulation of PA formation in the gastric mucosa.

Helicobacter pylori and CagA status, serum gastrin, interleukin-8 and gastric acid secretion in gastric cancer.

Background: Despite numerous epidemiological studies, the association between Helicobacter pylori infection and gastric cancer (GC) remains unexplained. This study was designed to determine the seropositivity of H. pylori and cytotoxin-associated gene A (CagA), serum gastrin and interleukin-8 (IL-8) levels as well as basal intragastric pH and maximal histamine-induced gastric acid outputs (MAO) in a large series of GC patients and controls. Methods: 337 GC patients (118 men and 219 women; median age 59.4; range 21-87) and 337 controls randomized for sex and age entered the study. Serum IgG antibodies to H. pylori and CagA and serum levels of IL-8 were measured by enzyme-linked immunosorbent assay, while serum-amidated gastrin was determined by specific radioimmunoassay and correlated with gastric luminal pH. Results: The numbers of GC patients and controls involved in the study in various age groups, ranging from 20 to >70 years, were similar, but overall H. pylori IgG seropositivity in GC patients was significantly higher (90.8%) than in controls (79.2%). The overall CagA seropositivity in GC patients was about double (58.2%) that in controls (25.2%). Serum gastrin levels over the calculated cut-off value (38.88 pM/L) were found in several-fold larger number in GC patients (48%) than in controls (8.3%) and, similarly, serum IL-8 values over the cut-off point (1.77 pg/mL) occurred in almost all (99.7%) GC patients but in only a few controls (0.3%). Basal intragastric pH above the cut-off point (pH = 4.50) was observed in about 58.2% of GC patients compared to 15.1% in controls, and strong correlation between the serum gastrin and gastric pH was found in GC but weak in controls. The cut-off value for MAO was 12.3 mml/h; MAO below this cut-off value occurred in 89.9% of GC patients and in only 4.7% of controls. A summary odds ratio (SOR) in GC for H. pylori IgG was 2.59 (95% Cl; 1.61-4.22) for CagA - 4.12 (95% Cl; 2.93-5.8), for serum gastrin - 10.25 (95%; 6.47-16.47) and for MAO - 15.2 (95% Cl; 9.45-39.82). Multivariable analysis of serum gastrin, IgG and CagA, and luminal pH and MAO values revealed that only gastrin and CagA have significant influence on GC formation (OR >1 in logistic regression). Conclusions: 1. CG patients show significantly higher H. pylori IgG and CagA seropositivity than dyspeptic age- and gender-matched controls, confirming that gastric infection with CagA expressing H. pylori greatly increases the risk of GC. 2. Serum gastrin levels in GC but not in controls are correlated with the rise in intragastric pH, indicating that excessive gastrin release in GC is affected by lower intragastric pH. 3. Serum gastrin level and CagA seropositivity are significantly increased in the majority of GC patients, and are the only variables in multivariable analysis to have a predominant influence on GC formation, which suggests that both these parameters may be implicated in H. pylori -related gastric carcinogenesis. 4. H. pylori -infected GC patients produce significantly more IL-8 than do non-GC controls, probably reflecting CagA-positive H. pylori -associated gastritis.

Leptin Protects the Pancreas from Damage Induced by Caerulein Overstimulation by Modulating Cytokine Production

Background: Recent identification of specific leptin receptors in the pancreas suggests that this peptide may also play some role in this gland. Aim: To examine the effect of intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of leptin in rats on caerulein-induced pancreatitis (CIP), pancreatic gene expression of leptin and inflammatory cytokine production. Methods: caerulein (25 µg/kg) was infused subcutaneously into conscious rats over 5 h to produce CIP. Leptin (1, 5, or 10 µg/kg) was injected i.p. or i.c.v. 30 min prior to the CIP induction. The plasma level of TNFα and IL-4 was determined by ELISA, while plasma leptin was measured by RIA and leptin gene expression in pancreas by RT-PCR. Results: CIP was characterized by the usual pancreatic edema, reduction in pancreatic blood flow (PBF) and an increase in serum levels of amylase, TNFα and IL-4. Pretreatment with i.p. or i.c.v. leptin of the CIP rats partially reversed the harmful effects of CIP on the pancreas, and reduced pancreatic inflammation and the fall in PBF. This was accompanied by a dose-dependent reduction in serum levels of amylase and TNFα, while serum IL-4 in the CIP rats pretreated with leptin rose dose-dependently as compared to control rats with CIP alone. Pretreatment with leptin resulted in the dose-dependent rise in plasma leptin level over that observed in vehicle-treated controls. Leptin mRNA expression in the pancreas was dose-dependently increased after infusion of caerulein. Leptin content in isolated pancreatic acini was also increased dose-dependently by caerulein added to the incubation medium bathing these acini. Conclusions: (1) Exogenous leptin protects the pancreas against damage by CIP; (2) endogenous leptin seems to limit the extend of pancreatic damage, and (3) these protective effects of leptin could be attributed to the reduction in TNFα and to the increase in IL-4 production.

Nitric oxide in gastroprotection by sucralfate, mild irritant, and nocloprost : role of mucosal blood flow

Pretreatment with sucralfate is known to protect gastric mucosa against the damaging effect of strong irritants, and this protection is accompanied by an increase in mucosal blood flow but the mechanisms underlying these effects have not been elucidated. Similar gastroprotective and hyperemic effects can be obtained with exogenous prostaglandins (PG), mild irritants such as dilute ethanol, and by capsaicin. In this study we investigated the role of nitric oxide (NO) in the prevention of ethanol-induced gastric damage and gastric blood flow by sucralfate, mild irritant such as 20% ethanol, capsaicin, and nocloprost, a stable PGE2 analog. Pretreatment withNG-nitro-l-arginine (l-NNA), an inhibitor of NO synthase, enhanced ethanol-induced mucosal damage and reduced dose-dependently the gastroprotective and hyperemic effects of sucralfate, dilute ethanol, and capsaicin. The doses ofl-NNA attenuating significantly the protective effects of sucralfate or 20% ethanol were 25–50 mg/kg, while those reducing the protection by capsaicin were 6.2–12.5 mg/kg. The attenuating effect ofl-NNA on gastroprotection was reversed byl-arginine but notd-arginine. For comparison, the gastroprotective (but not hyperemic) effect of nocloprost was not affected by the pretreatment withl-NNA and/or arginine. We conclude that sucralfate, mild irritant, and capsaicin activate the NO system that may contribute to their gastroprotective effect through enhancing mucosal circulation but that NO is not essential for the mucosal protection by PGE2 analog.