Potential therapeutic role of melatonin in hepatobiliary diseases: current evidence and clinical observations.

Melatonin (MEL) is produced and secreted by the pineal gland as well as the small intestine, liver, retina, lymphocytes, and melanocytes in the skin in both experimental animals as well as in humans. While pineal and retinas MEL is closely related to the light/dark cycle, the production of MEL by other so called extrapineal tissues is independent of such circadian rhythm. Among the primary mechanisms of action of MEL in humans, the most important are interaction of MEL with specific receptors (M1, M2, M3) and the MEL 'scavenging' activity against the formation of free oxygen metabolites as a result of MEL's ability to transfer free electrons and stimulation of the expression of redox reaction enzymes. In addition, MEL binds to intracellular proteins such as calmodulin, thereby affecting the course of cell cycle, and it has been shown to activate of nuclear receptors belonging to the retinoid orphan receptors/retinoid Z receptors (ROR/RZR) subfamily. MEL exerts regulatory effects on the master clock regulating diurnal rhythms. This updated review presents current view on the synthesis and metabolism of MEL and the growing body of experimental evidence transferable to the practical medicine supporting a pleiotropic molecule beneficial effects on the health including protection against various organ abnormalities, including internal organs such as the liver. Although the beneficial effects of MEL in various types of liver damage have been well documented in experimental studies, there are relatively few studies on liver dysfunction in humans. Considering the worldwide obesity pandemic often associated with the occurrence of steatohepatitis and cirrhosis, the beneficial effects of MEL in liver pathology should be proven in randomized trials involving patients presenting with hepatic disorders.

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

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

Role of sensory afferent nerves, lipid peroxidation and antioxidative enzymes in the carbon monoxide-induced gastroprotection against stress ulcerogenesis.

Carbon monoxide (CO) is a physiological gaseous mediator recently implicated in the mechanism of gastric mucosal defense due to its vasodilatory and antioxidative properties. Small quantities of endogenous CO are produced during heme degradation by heme oxygenase (HO-1), however, the involvement of the capsaicin-sensitive afferent neurons releasing calcitonin gene related peptide (CGRP) and anti-oxidative factors and mechanisms in the CO-induced gastroprotection against stress ulcerogenesis has been little studied. We investigated the possible role of CO released from the CO donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) in the protection against water immersion and restraint stress (WRS)-induced lesions in rats with intact sensory nerves and those with capsaicin denervation and the accompanying changes in malondialdehyde (MDA) content considered as an index of lipid peroxidation, the activity of GSH and SOD-2 and gastric mucosal expression of antioxidative enzymes glutathione peroxidase (GPx) and SOD-2. Wistar rats with intact sensory nerves or those with capsaicin administered in total dose of 125 mg/kg s.c. within 3 days (capsaicin denervation) were pretreated either with 1) vehicle (saline) or 2) CORM-2 (0.1 - 0 mg/kg i.g.) with or without exogenous CGRP (10 µg/kg i.p.) and 30 min later exposed to 3.5 h of WRS. At the termination of WRS, the number of gastric lesions was counted and gastric blood flow (GBF) was assessed by H2-gas clearance technique. The mucosal content of MDA and reduced glutathione (GSH) and the activity of SOD-2 were determined and the expression of GPx-1 and SOD-2 mRNA in the gastric mucosa was analyzed by real-time PCR. The exposure of rats to 3.5 h of WRS resulted in numerous hemorrhagic gastric lesions and significantly decreased the GBF, raised MDA content and significantly decreased the mucosal SOD and GSH contents compared with intact gastric mucosa and these changes were exacerbated in rats with capsaicin denervation. Pretreatment with CORM-2 (1 mg/kg i.g.) which in our previous studies significantly reduced the ethanol and aspirin-induced gastric damage, significantly decreased the number of WRS-induced gastric lesions while raising the GBF and significantly increasing the activity of SOD and GSH (P < 0.05). The pretreatment with CORM-2 significantly decreased MDA content as compared with vehicle-pretreated rats exposed to WRS (P < 0.05). The reduction of WRS damage and the accompanying increase in the GBF as well as the significant decrease in MDA content and the increase in GSH content and SOD activity induced by CORM-2 (1 µg/kg i.g.) were all significantly altered in rats with capsaicin denervation (P < 0.05). The concurrent treatment of CORM-2 with exogenous CGRP in rats with or without sensory nerves tended to decrease the number of WRS lesions as compared with CORM-2 alone pretreated animals and significantly increased the GBF over the values measured in gastric mucosa of CORM-2 alone pretreated rats with or without capsaicin denervation. Such combined administration of CORM-2 and CGRP in rats with capsaicin denervation significantly inhibited an increase in MDA and 4-HNE content and evoked a significant increase in the GSH concentration (P < 0.05) remaining without significant effect on the increase in SOD activity observed with CORM-2 alone. The gastric mucosal expression of SOD-2- and GPx-1 mRNA was significantly increased as compared with those in intact gastric mucosa (P < 0.05). The pretreatment with CORM-2 applied with or without CGRP failed to significantly alter the mRNA expression for SOD-2 and GPx in the gastric mucosa of rats exposed to WRS. Both, the expression of SOD-2- and GPx-1 mRNA was significantly increased in capsaicin denervated rats exposed to WRS rats (P < 0.05) and this effect was abolished by the pretreatment with CORM-2. The expression of SOD-2 tended to decrease, though insignificantly, in rats pretreated with the combination of CORM-2 and CGRP as compared with that detected in CORM-2 alone in rats with capsaicin denervation. In contrast, the mRNA expression of GPx-1 was significantly decreased in gastric mucosa of capsaicin-denervated rats treated with the combination of CORM-2 and CGRP as compared with CORM-2 alone pretreated animals. We conclude that 1) CORM-2 releasing CO exerts gastroprotective activity against stress ulcerogenesis and this effect depends upon an increase in the gastric microcirculation and the vasodilatory activity of this gaseous mediator, and 2) the sensory nerve endings releasing CGRP can contribute, at least in part, to the CO-induced gastric hyperemia, the attenuation of gastric mucosal lipid peroxidation and prevention of oxidative stress as indicated by the CORM-2-induced normalization of the antioxidative enzyme expression enhanced in gastric mucosa of capsaicin-denervated rats.

Activation of genes for spasmolytic peptide, transforming growth factor alpha and for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to aspirin damage in rats.

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 (TGF alpha) 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, TGF alpha COX-1 and COX-2 mRNA as determined by RT-PCR. To quantify the relative amounts of mRNA for SP and TGF alpha, southern blotting analysis of the PCR products was performed and the intensity of PCR products was compared with that of beta-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/beta-actin ratio; 0.96 +/- 0.08 in ASA-adapted mucosa vs. 0.38 +/- 0.05 in the control mucosa) and TGF alpha (TGF alpha/beta-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 TGF alpha, 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.

Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.

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)-1beta 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-1beta 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.

Classic NSAID and selective cyclooxygenase (COX)-1 and COX-2 inhibitors in healing of chronic gastric ulcers.

Prostaglandins (PG) derived from COX-1 are essential for the maintenance of mucosal integrity but COX-2 isoform synthesizes PG at a site of inflammation. Recently, COX-2 mRNA expression was demonstrated at the ulcer edge during healing of chronic gastric ulcers but the role for expression of COX-2 and its products such as PGE(2) and cytokines including interleukin (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in ulcer healing remains unknown. In this study, Wistar rats with gastric ulcers produced by serosal application of acetic acid (ulcer area 28 mm(2)) received daily treatment either with: (1) vehicle (saline); (2) NS-398 (10 mg/kg-d i.g.) and Vioxx (5 mg/kg-d i.g.), both, highly specific COX-2 inhibitors; (3) meloxicam (5 mg/kg-d i.g.), a preferential inhibitor of COX-2; (4) resveratrol (10 mg/kg-d i.g.), a specific COX-1 inhibitor; (5) indomethacin (5 mg/kg-d i.g); and (6) aspirin (ASA; 50 mg/kg-d i.g.), non-selective inhibitors of both COX-1 and COX-2. At day 3, 7, and 14 after ulcer induction, the animals were sacrificed and the area of gastric ulcers was determined by planimetry and histology, gastric blood flow (GBF) at ulcer base and margin was measured by H(2) clearance technique, and blood was withdrawn for measurement of plasma IL-1beta and TNFalpha levels. The mucosal biopsy samples were taken for the determination of PGE(2) generation by RIA and expression of COX-1, COX-2, IL-1beta, and TNFalpha mRNA by RT-PCR. In vehicle-treated rats, gastric ulcers healed progressively and at day 14 the healing was completed, accompanied by a significant rise in the GBF at ulcer margin. The IL-1beta, TNFalpha, and COX-1 mRNA were detected in intact and ulcerated gastric mucosa, whereas COX-2 mRNA were upregulated only in ulcerated mucosa with peak observed at day 3 after ulcer induction. The plasma IL-1beta level was significantly increased at day 3 and 7 but then declined at day 14 to that measured in vehicle-controls. Indomethacin and ASA, which suppressed PGE(2) generation both in the non-ulcerated and ulcerated gastric mucosa, significantly delayed the rate of ulcer healing and this was accompanied by the fall in GBF at ulcer margin and further elevation of plasma IL-1beta and TNFalpha levels, which was sustained up to the end of the study. Treatment with NS-398 and Vioxx, which caused only a moderate decrease in the PGE(2) generation in the non-ulcerated gastric mucosa, delayed ulcer healing and attenuated significantly the GBF at ulcer margin and PGE(2) generation in the ulcerated tissue, while raising the plasma IL-1beta and TNFalpha similarly to those observed in indomethacin- and ASA-treated rats. Resveratrol, which suppressed the PGE(2) generation in both non-ulcerated and ulcerated gastric mucosa, prolonged ulcer healing and this was accompanied by the fall in the GBF at the ulcer margin and a significant increase in plasma IL-1beta and TNFalpha levels. We conclude that (1) classic NSAID delay ulcer healing due to suppression of endogenous PG, impairment in GBF at ulcer area, and excessive cytokine expression and release, and (2) this deleterious effect of classic NSAID on the healing of pre-existing ulcers can be reproduced by selective COX-1 and COX-2 inhibitors, suggesting that both COX isoforms are important sources of PG that appear to contribute to ulcer healing.

Involvement of gastrin in gastric secretory and protective actions of N-alpha-methyl histamine.

N alpha-methylhistamine (N alpha-MH) is one of an unusual metabolite of histamine that was found in Helicobacter pylori-infected stomachs and is believed to interact with specific histamine H(1), H(2) and H(3)-receptors to stimulate gastric acid secretion and gastrin release from isolated G-cells but the effects of N alpha-MH on gastric mucosal integrity have been little studied. This study was designed; (1) to compare the effect of exogenous N alpha-MH with that of standard histamine on gastric secretion and plasma gastrin levels in rats equipped with gastric fistula (series A); and (2) to assess the action of N alpha-MH on gastric lesions induced by 100% ethanol (series B) in rats with or without removal of antral portion of the stomach (antrectomy). Rats of series B were pretreated intragastrically (i.g.) or intraperitoneally (i.p.) with N alpha-MH or histamine (0.1-2 mg/kg) 30 min prior to 100% ethanol (1.5 ml, i.g.) with or without: (1) vehicle (saline); (2) RPR 102681 (30 mg/kg i.p.), to block CCK-B/gastrin receptors; and (3) ranitidine (40 mg/kg s.c.) to inhibit histamine H(2)-receptors. The area of gastric lesions was determined planimetrically, gastric blood flow (GBF) was assessed by H(2)-gas clearance method and venous blood was collected for determination of plasma gastrin levels by radioimmunoassay (RIA). N alpha-MH and histamine dose-dependently increased gastric acid output (series A); the dose increasing this secretion by 50% (ED(50)) being 2 and 5 mg/kg i.g or i.p., respectively, and this effect was accompanied by a significant rise in plasma gastrin levels. Both, N alpha-MH and histamine attenuated dose-dependently the area of gastric lesions induced by 100% ethanol (series B) while producing significant rise in the GBF and plasma immunoreactive gastrin increments. These secretory, protective, hipergastrinemic and hyperemic effects of N alpha-MH and histamine were completely abolished by antrectomy, whereas pretreatment with RPR 102681 attenuated significantly the N alpha-MH and histamine-induced protection against ethanol damage and accompanying hyperemia. Ranitidine, that produced achlorhydria and a further increase in plasma gastrin levels, failed to influence the N alpha-MH- and histamine-induced protection and accompanying rise in the GBF. We conclude that (1) N alpha-MH stimulates gastric acid secretion and exhibit gastroprotective activity against acid-independent noxious agents in the manner similar to that afforded by histamine; and (2) this protection involves an enhancement in the gastric microcirculation and release of gastrin acting via specific CCK-B/gastrin receptors but unexpectedly, appears to be unrelated to histamine H(2)-receptors.

Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress.

Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg x kg(-1) i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg x kg(-1) i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg x kg(-1) i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg x kg(-1) i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5-30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg x kg(-1) i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 microg x kg(-1) i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors.

Base variant of human pancreatic secretory trypsin inhibitor in healing of stress-induced gastric lesions in rats.

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 H2-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.

Acceleration of ulcer healing by cholecystokinin (CCK): role of CCK-A receptors, somatostatin, nitric oxide and sensory nerves.

CCK exhibits a potent cytoprotective activity against acute gastric lesions, but its role in ulcer healing has been little examined. In this study we determined whether exogenous CCK or endogenously released CCK by camostate, an inhibitor of luminal proteases, or by the diversion of pancreatico-biliary secretion from the duodenum, could affect ulcer healing. In addition, the effects of antagonism of CCK-A receptors (by loxiglumide, LOX) or CCK-B receptors (by L-365,260), an inhibition of NO-synthase by N(G)-nitro-L-arginine (L-NNA), or sensory denervation by large neurotoxic dose of capsaicin on CCK-induced ulcer healing were examined. Gastric ulcers were produced by serosal application of acetic acid and animals were sacrificed 9 days after ulcer induction. The area of ulcers and blood flow at the ulcer area were determined. Plasma levels of gastrin and CCK and luminal somatostatin were measured by RIA and mucosal biopsy samples were taken for histological evaluation and measurement of DNA synthesis. CCK given s.c. reduced dose dependently the ulcer area; the threshold dose of CCK being 1 nmol/kg and the dose inhibiting this area by 50% being 5 nmol/kg. This healing effect of CCK was accompanied by a significant increase in the GBF at ulcer margin and the rise in luminal NO production, plasma gastrin level and DNA synthesis. Concurrent treatment with LOX, completely abolished the CCK-8-induced acceleration of the ulcer healing and the rise in the GBF at the ulcer margin, whereas L-365,260 remained without any influence. Treatment with camostate or diversion of pancreatic juice that raised plasma CCK level to that observed with administration of CCK-8, also accelerated ulcer healing and this effect was also attenuated by LOX but not by L-365,260. Inhibition of NO-synthase by L-NNA significantly delayed ulcer healing and reversed the CCK-8 induced acceleration of ulcer healing, hyperemia at the ulcer margin and luminal NO release, and these effects were restored by the addition to L-NNA of L-arginine but not D-arginine. Capsaicin denervation attenuated CCK-induced ulcer healing, and the accompanying rise in the GBF at the ulcer margin and decreased plasma gastrin and luminal release of somatostatin when compared to those in rats with intact sensory nerves. Detectable signals for CCK-A and B receptor mRNAs as well as for cNOS mRNA expression were recorded by RT-PCR in the vehicle control gastric mucosa. The expression of CCK-A receptor mRNA and cNOS mRNA was significantly increased in rats treated with CCK-8 and camostate, whereas CCK-B receptor mRNA remained unaffected. We conclude that CCK accelerates ulcer healing by the mechanism involving upregulation of specific CCK-A receptors, enhancement of somatostatin release, stimulation of sensory nerves and hyperemia in the ulcer area, possibly mediated by NO.

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.

Ischemic preconditioning, the most effective gastroprotective intervention: involvement of prostaglandins, nitric oxide, adenosine and sensory nerves.

Various organs, including heart, kidneys, liver or brain, respond to brief exposures to ischemia with an increased resistance to severe ischemia/reperfusion and this phenomenon is called "preconditioning". No study so far has been undertaken to check whether such short, repeated gastric ischemic episodes protect gastric mucosa against severe damage caused by subsequent prolonged ischemia/reperfusion and, if so, what could be the mechanism of this phenomenon. The ischemic preconditioning was induced by short episodes of gastric ischemia (occlusion of celiac artery from one to five times, for 5 min each) applied 30 min before prolonged (30 min) ischemia followed by 3 h of reperfusion or 30 min before topical application of strong mucosal irritants, such as 100% ethanol, 25% NaCl or 80 mM taurocholate. Exposure to regular 30-min ischemia, followed by 3-h reperfusion, produced numerous severe gastric lesions and significant fall in the gastric blood flow and prostaglandin E(2) generation. Short (5-min) ischemic episodes (1-5 times) by itself failed to cause any gastric lesions, but significantly attenuated those produced by ischemia/reperfusion. This protection was accompanied by a reversal of the fall in the gastric blood flow and prostaglandin E(2) generation and resembled that induced by classic gastric mild irritants. These protective and hyperemic effects of standard preconditioning were significantly attenuated by pretreatment with cyclooxygenase-2 and cyclooxygenase-1 inhibitors, such as indomethacin, Vioxx, resveratrol and nitric oxide (NO)-synthase inhibitor, N(G)-nitro-L-arginine (L-NNA). The protective and hyperemic effects of standard preconditioning were restored by addition of 16,16 dm prostaglandin E(2) or L-arginine, a substrate for NO synthase, respectively. Gastroprotective and hyperemic actions of standard ischemic preconditioning were abolished by pretreatment with capsaicin-inactivating sensory nerves, but restored by the administration of exogenous CGRP to capsaicin-treated animals. Gene and protein expression of cyclooxygenase-1, but not cyclooxygenase-2, were detected in intact gastric mucosa and in that exposed to ischemia/reperfusion with or without ischemic preconditioning, whereas cyclooxygenase-2 was overexpressed only in preconditioned mucosa. We conclude that: (1) gastric ischemic preconditioning represents one of the most powerful protective interventions against the mucosal damage induced by severe ischemia/reperfusion as well as by topical mucosal irritants in the stomach; (2) gastric ischemic preconditioning resembles the protective effect of "mild irritants" against the damage by necrotizing substances in the stomach acting via "adaptive cytoprotection" and involves several mediators, such as prostaglandin derived from cyclooxygenase-1 and cyclooxygenase-2, NO originating from NO synthase and sensory nerves that appear to play a key mechanism of gastric ischemic preconditioning.

Role of gastric acid secretion in progression of acute gastric erosions induced by ischemia-reperfusion into gastric ulcers.

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.

Leptin in gastroprotection induced by cholecystokinin or by a meal. Role of vagal and sensory nerves and nitric oxide.

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.

Role of prostaglandins generated by cyclooxygenase-1 and cyclooxygenase-2 in healing of ischemia-reperfusion-induced gastric lesions.

In this study, ischemia-reperfusion produced in rats by clamping the celiac artery for 0.5 h followed by 1 h of reperfusion was used to develop a new model of superficial gastric erosions progressing to deeper ulcers. Ischemia alone resulted in an immediate fall in gastric blood flow but no gross mucosal lesions were observed. When ischemia was followed by reperfusion, gastric erosive lesions occurred, reached a maximum at 12 h and then declined after 24 h. These acute erosions progressed into deeper lesions 24 h after ischemia-reperfusion and reached a peak after 3 days. Gastric blood flow and the mucosal generation of prostaglandin E(2) were significantly suppressed immediately following ischemia-reperfusion, but with the healing of deeper gastric ulcers, both gastric blood flow and prostaglandin E(2) generation were gradually restored. Cyclooxygenase-1 mRNA was detected by reverse transcription-polymerase chain reaction in intact gastric mucosa and throughout the recovery of the mucosa from acute ischemia-reperfusion lesions, whereas cyclooxygenase-2 mRNA, was recorded only after ischemia-reperfusion. NS-398 and rofecoxib, selective inhibitors of cyclooxyganase-2, failed to affect prostaglandin E(2) generation in the non-ulcerated gastric mucosa but inhibited it significantly in the ulcer area. The two cyclooxygenase-2 inhibitors as well as resveratrol, a specific cyclooxygenase-1 inhibitor and indomethacin and meloxicam, non-specific inhibitors of cyclooxygenase, augmented acute gastric erosions induced by ischemia-reperfusion and delayed significantly the progression of these lesions into deeper ulcers at each time interval after ischemia-reperfusion. We conclude that prostaglandins generated by both cyclooxygenase-1 and cyclooxygenase-2 contribute to the healing of gastric lesions induced by ischemia-reperfusion.

Gastroprotective and ulcer healing effects of nitric oxide-releasing non-steroidal anti-inflammatory drugs.

BACKGROUND & AIM: New class of nitric oxide-releasing non-steroidal anti-inflammatory drugs was shown to inhibit cyclooxygenase and prostaglandin generation without causing mucosal damage but whether these agents are capable of affecting gastric mucosal damage induced by strong irritants and healing of chronic gastric ulcers remains to be studied. In this investigation, effects of nitric oxide-releasing aspirin and nitric oxide-releasing naproxen were compared with those of native agents on gastric lesions provoked by 100% ethanol and on healing of chronic acetic acid ulcers. RESULTS: Both, nitric oxide-releasing aspirin and naproxen dose-dependently attenuated ethanol-induced damage and produced a significant rise in gastric blood flow but did not delay healing of gastric ulcers while native aspirin and naproxen had no influence on ethanol-induced gastric damage but significantly prolonged ulcer healing, reduced gastric blood flow and suppressed mucosal generation of prostaglandin E2. The gastroprotective and hyperaemic effects of both nitric oxide-non-steroidal anti-inflammatory drugs were completely abolished by ODQ, an inhibitor of guanylyl cyclase-cGMP system but not influenced by suppression of nitric oxide-synthase with L-NNA. The damaging effects of native acetyl salicylate acid or naproxen were aggravated by acidification of these non-steroidal anti-inflammatory drugs but the exogenous acid added to nitric oxide-acetyl salicylate acid or nitric oxide-naproxen failed to influence their effect. Despite inhibiting of PGE2 generation, both nitric oxide-releasing derivatives and native aspirin and naproxen failed to affect expression of cyclooxygenase-1 mRNA but upregulated the cyclooxygenase-2 mRNA. Concurrent inhibition of cyclooxygenase-2 by selective inhibitor NS-398 which by itself delayed ulcer healing and attenuated the gastric blood flow at ulcer margin, significantly worsened the effects of these nitric oxide-non-steroidal anti-inflammatory drugs and their parent drugs on ulcer healing and the gastric blood flow at the ulcer margin. CONCLUSIONS: 1) Coupling of nitric oxide to aspirin or naproxen attenuates ethanol-induced damage, possibly due to an increase in gastric microcirculation mediated by excessive release and action of nitric oxide that probably compensates for PG deficiency induced by non-steroidal anti-inflammatory drugs; and 2) nitric oxide-non-steroidal anti-inflammatory drug, unlike classic non-steroidal anti-inflammatory drugs, does not affect intact gastric mucosa and fails to delay the healing of pre-existing ulcers.

Sensory nerves and calcitonin gene related peptide in the effect of ischemic preconditioning on acute and chronic gastric lesions induced by ischemia-reperfusion.

Ischemic preconditioning is considered as the most powerful gastroprotective intervention against mucosal lesions and ulcerations but the mechanism of this phenomenon has been little examined. In this study we tested the effects of inactivation of sensory nerves in new rat model combining acute gastric erosions with subsequent ulcers induced by ischemia-reperfusion (I/R). I/R lesions were produced in rats by clamping the celiac artery for 0.5 h followed by 3 h of reperfusion in rats with intact or inactivated sensory nerves by pretreatment with capsaicin for two weeks before the I/R. The animals were killed at 0 and 3 h and 3 days after I/R and the area of gastric lesions was determined planimetrically, the gastric blood flow (GBF) by H2-gas clearance technique and the plasma levels of gastrin by RIA. Gastric mucosal content of calcitonin gene related peptide (CGRP) was assessed by RIA. Following I/R, gastric erosive lesions occurred after 3 h and these erosive lesions then progressed into gastric ulcers within 3 days in all rats. Sensory-inactivation with capsaicin caused several fold increase in the area of early (at 3 h) acute lesions and later (at 3 d) gastric ulcers induced by I/R. This enhancement of acute and then chronic gastric lesions was accompanied by a significant fall in GBF, an elevation of plasma gastrin and a decrease in mucosal expression of CGRP. Ischemic preconditioning markedly reduced acute lesions and chronic ulcerations induced by I/R and attenuated the changes in plasma gastrin and mucosal CGRP contents but these effects were significantly more pronounced in rats with intact sensory nerves but less in capsaicin-inactivated animals. We conclude that: 1) The I/R resulted in the formation of early acute gastric lesions followed 3 days later by chronic gastric ulcers and this gastric injury was accompanied by an impairment of gastric microcirculation, hypergastrinemia and suppression the gastric mucosal CGRP; 2) Gastric ischemic-preconditioning significantly attenuated both acute mucosal damage and chronic ulcers induced by I/R and this was accompanied by a rise in gastric blood flow; 3) The inactivation of sensory nerves with capsaicin enhanced the formation of I/R-induced acute and chronic gastric lesions and strongly attenuated the gastroprotection afforded by I/R possibly due to the decline in mucosal blood flow and the fall in expression of integrity peptides such as CGRP and 4) The excessive release of gastrin may limit the extent of mucosal lesions observed during progression of gastric erosions into ulcers induced by I/R.

SU-840, a novel synthetic flavonoid derivative of sophoradin, with potent gastroprotective and ulcer healing activity.

Flavonois derived from sophoradine are known to exhibit gastroprotective and ulcer healing properties but the mechanism of these actions are not fully explained. In this study we determined the effect of novel flavonoid derivative of sophoradin, SU-840, on gastric secretion, acute gastric lesions induced by acid-independent (100% ethanol) or acid-dependent ulcerogens (acidified aspirin (ASA) and stress) and on the healing of chronic gastric ulcers in rats. The number and area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured using H2-gas clearance technique and the mucosal samples were excised for the measurement of PGE2 generation by radioimmunoassay. Exposure of rats to 100% ethanol or acidified ASA (100 mg/kg dissolved in 0.2 N HCl) or to water immersion and restraint stress (WRS) resulted in hemorrhagic gastric lesions accompanied by drastic fall in the GBF as compared to the values recorded in vehicle treated gastric mucosa. SU-840 (6.25-100 mg/kg i.g.) reduced dose-dependently gastric acid and pepsin secretion and gastric lesions induced by ethanol, acidified ASA and WRS, the dose inhibiting by 50% of these lesions (ID50) being 28, 17 and 95 mg/kg, respectively. This protection required much lower doses as compared to original sofalcone or sucralfate and was obtained when this sofalcone-like drug was administered via parenteral route. The protective effect of SU-840 given i.g. or i.p. was accompanied by a marked rise in the GBF and mucosal generation of PGE2. The protective activity of SU-840 showed longer duration of the action than that of sofalcone and occurred in the doses that failed to affect gastric secretion. Pretreatment with indomethacin to suppress endogenous PG reversed completely the protective and hyperemic effects of SU-840 against ethanol and stress induced damage whereas L-NNA, a potent inhibitor of NO-synthase, failed to affect protection but completely abolished the hyperemia evoked by this agent. NEM, an sulfhydryl alkylator, significantly attenuated the protective and hyperemic effects of SU-840 suggesting that endogenous sulfhydryls are involved in these effects. Seven day treatment with SU-840 accelerated significantly healing rate of chronic gastric ulcers and increased the GBF at the ulcer crater and ulcer margin. These effects were reversed by L-NNA and further restored by the addition to L-NNA of L-arginine, a substrate for NO-synthase. We conclude that SU-840 exhibits gastroprotective and hyperemic activity against acid-independent and acid-dependent irritants involving endogenous PG, sulfhydryls and hyperemia mediated by NO and 2) enhancement in gastric blood flow in the ulcer area mediated by NO appears to be essential for the acceleration of the ulcer healing by SU-840.

Involvement of cyclooxygenase (COX)-2 products in acceleration of ulcer healing by gastrin and hepatocyte growth factor.

Ulcer healing involves expression of various growth factors including hepatocyte growth factor (HGF) at the ulcer margin and the rise in plasma gastrin but the effects of locally applied HGF and gastrin, which are known to act as trophic factors for the gastric mucosa, with or without neutralizing antibodies against HGF and gastrin or COX-1 and COX-2 inhibitors on ulcer healing and the expression of cyclooxygenase (COX)-1 and COX-2 during this healing have been little studied. Rats with gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm2) received a submucosal injection of either: 1)vehicle (saline), 2) HGF and 3) gastrin with or without neutralizing antibodies against HGF and gastrin or treatment with indomethacin (2 mg/kg-d i.p.), a non-specific inhibitor of COX, or NS-398 (5 mg/kg-d i.g.) and Vioxx (10 mg/kg-d i.g.), both highly specific COX-2 inhibitors. Each growth factor and specific antibodies against HGF and gastrin (100 ng/100 microl each) were injected just around the ulcer immediately after ulcer induction and this local application was repeated at day 2 following anesthesia and laparotomy. At day 13 and 21, the area of ulcers was determined by planimetry, the gastric blood flow (GBF) at ulcer margin was examined by H2-gas clearance technique and mucosal generation of PGE2 and the expression of COX-1 and COX-2 mRNA in the non-ulcerated and ulcerated gastric mucosa was analyzed using RT-PCR. The gastric ulcers healed progressively within 21 days and this effect was accompanied by significant increase in the GBF at the ulcer margin and expression of COX-2 mRNA and COX-2 protein at the ulcer area. Treatment with HGF and gastrin significantly accelerated the rate of ulcer healing and raised GBF at ulcer margin causing further significant upregulation of COX-2 mRNA and COX-2 protein (but not of COX-1 mRNA ) in the ulcerated mucosa. The upregulation of COX-2 mRNA induced by HGF was significantly attenuated by the concurrent local treatment with antibody against this growth peptide. Indomethacin and both COX-2 inhibitors significantly prolonged the ulcer healing, while suppressing the generation of PGE2 in non-ulcerated and ulcerated gastric mucosa and the GBF at ulcer margin. The acceleration of ulcer healing by HGF and gastrin and accompanying rise in the GBF at ulcer margin were significantly attenuated by the concurrent treatment with indomethacin or NS-398 and Vioxx. HGF injections produced a significant rise in the plasma gastrin levels and this was significantly attenuated by the cotreatment with NS-398. We conclude that 1) neutralization of HGF and gastrin by their specificantibodies delays ulcer healing due fall in the microcirculation around the ulcer and a decrease in the COX-2 expression, 2) COX-2 derived prostaglandins may play an important role in acceleration of the ulcer healing by various growth factors including HGF and gastrin, 3) enhancement of the local pool for growth factors such as HGF and gastrin at the ulcer site could offer a new modality for treatment of gastric ulcer.

Brain-gut axis in gastroprotection by leptin and cholecystokinin against ischemia-reperfusion induced gastric lesions.

Leptin, a product of ob gene controlling food intake, has recently been detected in the stomach and shown to be released by CCK and implicated in gastroprotection against various noxious agents but it is unknown whether centrally applied leptin influences ischemia-reperfusion (I/R)-induced gastric erosions that progress into deeper gastric ulcerations. In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) on gastric mucosal lesions induced by I/R and topical application of 75% ethanol. Several major series of Wistar rats were used to examine the effects of leptin and CCK applied centrally on gastroprotection against I/R and ethanol in rats with A) vagotomy by cutting of vagal nerves, B) suppression of NO-synthase with L-NNA (20 mg/kg i.p.), C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.) and D) inhibition of CGRP receptors with CGR(8-37) (100 microg/kg i.p.) applied with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration or at 3 h and 3 days upon the end of ischemia to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay (RIA). Leptin (0.1-20 microg/kg i.p.) dose-dependently attenuated gastric lesions induced by 75% ethanol and I/R; the dose reducing these lesions by 50% (ED50) was 8 microg/kg and 6 microg/kg, respectively and this protective effect was similar to that obtained with CCK-8 applied in a standard dose of 10 microg/kg i.p. This protective effect of leptin was accompanied by a significant increase in GBF and plasma gastrin levels whereas CCK-8 increased plasma leptin levels but failed to affect plasma gastrin levels. Leptin and CCK-8 applied i.c.v. in a dose of 625 ng/rat reduced significantly the area of I/R induced gastric lesions and raised the GBF and plasma leptin levels with the extent similar to those achieved with peripheral administration of leptin or CCK-8 (10 microg/kg i.p.). The protective and hyperemic effects of centrally administered leptin or CCK-8 (625 ng/rat i.c.v.) were completely abolished by vagotomy and significantly attenuated by sensory denervation with capsaicin or by CGRP antagonist, CGRP(8-37). The pretreatment with L-NNA to inhibit NO-synthase activity attenuated significantly the protective and hyperemic effects of CCK but not those of leptin while capsaicin denervation counteracted leptin-induced protection and rise in the GBF but attenuated significantly those of CCK. We conclude that: 1) central leptin exerts a potent gastroprotective activity against I/R-induced gastric erosions that progress into deeper gastric lesions and this protection depends upon vagal activity and sensory nerves and involves hyperemia probably mediated by NO and 2) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide against mucosal damage evoked by I/R.