Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats.

Angiogenesis is an important event for gastric ulcer healing. Vascular endothelial growth factor (VEGF) is known to be a potent stimulator of angiogenesis. This study consequently examined VEGF production, VEGF mRNA expression and angiogenesis during the spontaneous and indomethacin-delayed healing of acetic acid-induced ulcers in rats. The production of VEGF, taking place in the normal mucosa, was significantly elevated by ulceration. The mRNA expression of three isoforms of VEGF (VEGF188, VEGF164 and VEGF120) was also detected. Following the increase in VEGF production, angiogenesis was significantly promoted in the ulcer base. VEGF-immunoreactivity was observed in granulocytes, fibroblasts and regenerated epithelial cells. Indomethacin markedly inhibited prostaglandin E2 synthesis in the ulcer base, resulting in the prevention of ulcer healing. Angiogenesis was also significantly inhibited by indomethacin, but neither VEGF production nor VEGF mRNA expression was reduced. Such results suggest that VEGF might play a role in angiogenesis in the spontaneous healing of gastric ulcers in rats. However, the inhibition of angiogenesis in indomethacin-delayed ulcer healing is not explainable on VEGF expression.     

The healing effects of Centella extract and asiaticoside on acetic acid induced gastric ulcers in rats

In this study, the healing effects of Centella asiatica water extract (CE) and asiaticoside (AC), an active constituent of CE, on acetic acid induced gastric ulcers (kissing ulcers) in rats were examined. CE was prepared from Centella asiatica dry plant and the concentration of AC in CE was quantitatively determined with the use of high performance liquid chromatography analysis. Different concentrations of CE and AC were orally administered to rats with kissing ulcers. They were found to reduce the size of the ulcers at day 3 and 7 in a dose-dependent manner, with a concomitant attenuation of myeloperoxidase activity at the ulcer tissues. Epithelial cell proliferation and angiogenesis were on the other hand promoted. The expression of basic fibroblast growth factor, an important angiogenic factor, was also upregulated in the ulcer tissues in rats treated with CE or AC. These results further suggest the potential use of Centella asiatica and its active ingredient as anti-gastric ulcers drugs.     

Mechanism of the antiulcerogenic effect of IL-11 on acetic acid-induced gastric ulcer in rats

The purpose of this study was to evaluate the healing effect of interleukin-11 (IL-11) on acetic acid-induced gastric ulcer in rats. Gastric ulcers were induced in male Wistar rats by applying acetic acid to the fundus of the stomach. Recombinant human interleukin-11 (rhIL-11 100 microg/kg/twice daily, subcutaneously) was administered starting on the 2nd day before ulcer induction up through the 7th day after ulcer induction. Control rats were injected with bovine serum albumin. At 12 hours and 7 days after ulcer induction, the animals were sacrificed, and the ulcer index, proliferating cell nuclear antigen (PCNA) expression, and IL-11alpha receptor expression in the gastric tissues were studied. The ulcer index of the rhIL-11-treated rats was significantly lower than that of the control rats at the 7th day. The expression of PCNA as evaluated by Western blotting and immunohistochemistry, was enhanced in both the mucosal proliferative zone and proper muscle layer of the rhIL-11-treated rats in comparison with that in the control rats. IL-11alpha receptor expression was observed in the mucosal neck cells of the rhIL-11-treated rats and control rats. These findings suggest that IL-11 accelerates ulcer healing by inducing the proliferation of mucosal and muscular cells.     

Role of p38 mitogen-activated protein kinase in the healing of gastric ulcers in rats.

p38 belongs to the mitogen-activated protein kinase family and plays a crucial role in cellular responses to both cytokines and various stresses. We investigated the role of p38 in the healing of experimental gastric ulcers. Gastric ulcers were induced by submucosal injection of acetic acid solution into male rats. Western blotting and a kinase assay examined the p38 activity and phosphorylation state in ulcerated tisue. After orally administering FR167653 (p38 kinase inhibitor) for 3 to 14 days, the production level of cytokines and the protein-level expression of cyclooxygenase and inducible nitric oxide synthase were examined by enzyme-linked immunosorbent assay and Western blotting. Only in fibroblasts and macrophages/monocytes in ulcerated tissue, p38 was found to be phosphorylated with an elevated kinase activity level. FR 167653 inhibited the activity of p38, yet had no effect on its phosphorylation state. The drug significantly impaired ulcer healing (without affecting acid secretion) and angiogenesis in the ulcer base. The production of interleukin-1beta and tumor necrosis factor-alpha were significantly reduced after FR167653 treatment. In addition the expression of cyclooxygenase-2 and inducible nitric oxide synthase proteins increased PGE2 generation and NOx secretion in the ulcerated stomach were suppressed by FR167653. From these findings, we conclude that p38, activated by gastric ulceration, might play some role in the healing of gastic ulcers in rats.     

Characterization of "unhealed gastric ulcers" produced with chronic exposure of acetic acid ulcers to indomethacin in rats.

We previously discovered that a 4-wk course of indomethacin delivered to rats with acetic acid ulcers resulted in production of "unhealed gastric ulcers" that persisted for up to 12 wks after treatment cessation. The present study examined the mechanism underlying such "unhealed gastric ulcers" with biochemical and histological procedures. "Unhealed gastric ulcers" were induced with a 4-wk indomethacin treatment (1 mg/kg, twice daily) in rats with acetic acid ulcers. Two and 4 wks after treatment cessation, ulcer size was significantly larger in rats receiving indomethacin compared with control animals. Ulcerated tissue prostaglandin E2 levels were significantly lower during indomethacin treatment, but the levels tended to increase after treatment cessation compared with levels measure in the group receiving vehicle. Myeloperoxidase activity levels were significantly higher during indomethacin treatment; such levels persisted after treatment cessation. Histologically, greater degrees of fibrosis and neutrophil accumulation, as well as a lesser degree of angiogenesis were observed in the "unhealed gastric ulcers" compared to ulcers that healed in a normal fashion. It was concluded that severe fibrosis, persistent neutrophil infiltration, and poor angiogenesis in the ulcer base might represent factors involved in the mechanism underlying production of "unhealed gastric ulcers".     

Naloxone enhancement of acetic acid-induced writhing in rats

Long-lasting visceral pain was produced in rats by the intraperitoneal injection of dilute acetic acid, and the response to this stimulus measured by observing the number of writhes over a subsequent 50 min. period. Prior treatment of the animals with naloxone or saline showed that naloxone increased the frequency of writhing. The enhancement by naloxone of the response to this nociceptive stimulus suggests that the binding of an endogenous opioid to the opiate receptors increases during prolonged nociceptive stimulation.     

Chaetognaths: a useful model for studying heat shock proteins. Effect of wound healing

The pattern of expression of Heat Shock Protein 70 (Hsp70), a highly conserved cellular protein chaperone, was investigated in Chaetognatha, a very important phylum of marine worms, which play a major role in marine food webs. The in toto distribution of Hsp70-like immunoreactivity was assessed in both intact and experimentally wounded specimens of Spadella cephaloptera Busch, 1851 that have been cut transversally just above the seminal vesicles. In intact animals, the ciliary organs, the corona ciliata, the coronal nerve and the spermatocytes express Hsp70 proteins. The kinetic of the expression pattern has been followed during the wound healing from the lesion to 5 days after. The Hsp70 immunoreactivity was observed according to time in the cerebral and suboesophageal ganglia and in the plexuses of the hood, the mouth, the neck and the tail region. At the wound level, the time-dependent Hsp70 expression was detected in the epidermal cells and along numerous muscle fibres of the tail region. Five days after the tail section, two Hsp70 immunoreactive areas were observed on both sides of the healed wound that correspond to the sites of formation of two new seminal vesicles. It is suggested that, in Chaetognaths, Hsp70 may be implicated in the regulation of several cellular processes especially at the level of the spermatocytes in intact and wounded specimens, and of the nervous system and muscular apparatus during the wound healing. It appears that chaetognaths are a good model as indicator of stress responses for experimental studies at the level of a whole organism.     

Role of leukotrienes in the genesis and healing of water immersion stress-induced gastric ulcers in rats

We investigated the role of leukotrienes (LTs) in the genesis of water immersion stress-induced gastric ulcers. Peptide LTs were detected after 4 h stress (3.7 +/- 0.5 ng/g tissue), although they were not detected after 2 h stress, and considerable amounts (20.3 +/- 2.3 ng/g tissue) were detected after 6 h stress. In contrast, AA-861 (100 mg/kg, p.o.), a 5-lipoxygenase inhibitor, reduced ulcer indices significantly after 6 h stress, although no significant changes were observed after 2 or 4 h stress compared with the control group. Peptide LTs were not detected after 4 h and those detected after 6 h stress were remarkably reduced by AA-861 treatment. The role of LTs in the healing of water immersion stress-induced gastric ulcers was also investigated. Significant ulcer healing was not observed within 24 h after stress but was significantly recovered after 48 h. Peptide LTs decreased time-dependently and 48 h after treatment they were not detected. In the rats treated with AA-861, ulcer indices and peptide LTs levels were remarkably reduced after 12 h, concomitantly. These results suggest that the increase in mucosal peptide LTs might be an inhibitory factor to ulcer healing.     

Effect of adrenomedullin administration on acetic acid-induced colitis in rats

Adrenomedullin (AM) administered intracolonically ameliorated the severity of acetic acid-induced colonic ulceration in rats. Ulcers were induced by subserosal injection of acetic acid into the colon. AM-treated group was administered 0.25–1.0 μg of AM in 0.5 ml of saline intracolonically once a day; the control group received only saline. AM administration dose-dependently and significantly reduced the size of the ulcerative lesions, the associated edema, and the infiltration of the affected area by inflammatory cells. AM also reduced tissue levels of interleukin-6, but not interferon-γ. AM reduces the severity of acetic acid-induced colitis in rats, probably by inhibiting the production and/or release of Th-2 cell-derived factors such as interleukin-6.     

A new ulcer model, "unhealed gastric ulcers", induced by chronic treatment with indomethacin in rats with acetic acid ulcers.

The healing of experimental gastric ulcers induced in rats is consistently delayed upon chronic treatment with indomethacin. This study was designed to examine both the fate of such delayed ulcers and the effects of various antiulcer drugs on the delayed ulcers. Four-week treatment with indomethacin significantly delayed the healing of acetic acid ulcers. Such ulcers remained unhealed for up to 12 weeks after cessation of indomethacin treatment, and were thus designated as "unhealed ulcers". Two-week administration of sucralfate, cimetidine or omeprazole significantly reduced the ulcerated area, yet aluminum hydroxide had little or no effect. Four-week administration of sucralfate also extensively reduced the size of the "unhealed ulcers", yet aluminum hydroxide, cimetidine and omeprazole had an insignificant effect on "unhealed ulcers". In the 2 and 4 week sucralfate-treated group, the pH of the gastric contents was 4.0 vs. 1.7 and 4.5 vs. 2.1 in the control groups, respectively. Gastric acid secretion was extensively inhibited by cimetidine and omeprazole. It is concluded that prolonged indomethacin treatment results in the development of "unhealed ulcers" and that only sucralfate has a beneficial effect on such ulcers, irrespective of the length of the treatment.     

Fish oil-enriched diet is mucosal protective against acetic acid-induced colitis in rats.

Products of arachidonic acid metabolism are elevated in patients with inflammatory bowel disease and this elevation is correlated with disease activity. Eicosapentaenoic acid competes with arachidonic acid and alters eicosanoid biosynthesis. In this experiment, the possibility that eicosapentaenoic acid could be used in the treatment of inflammatory bowel disease was investigated by determining the effect of 6 weeks of a fish oil-supplemented diet, enriched in eicosapentaenoic acid, on colonic and ileal morphology, histology, and in vivo fluid absorption in rats with 4% acetic acid-induced colitis. The results of an eicosapentaenoic acid-enriched diet were compared with results of saturated and polyunsaturated fatty acid-enriched diets. In rats with misoprostol pretreated acetic acid-induced colitis, an eicosapentaenoic acid-enriched diet reversed net colonic fluid secretion to absorption and prevented macroscopic and histologic injury, compared with saturated and poly-unsaturated fatty acid-enriched diets, which did not. The fish oil mucosal protective effect occurred in the presence of a 30-fold enhancement of PGE2 synthesis. In rats with non-misoprostol pretreated acetic acid-induced colitis, an eicosapentaenoic acid-enriched diet returned ileal fluid absorption to control levels, as compared with saturated and polyunsaturated fatty acid-enriched diets, which did not. In conclusion, a fish oil (eicosapentaenoic acid)-enriched diet, but not a saturated- or a polyunsaturated-enriched diet, protected colonic and ileal net fluid absorption in an experimental model of inflammatory bowel disease.     

Effects of histamine H1 antagonist dithiaden on acetic acid-induced colitis in rats.

To assess the possible involvement of mast cells and/or their mediators in inflammatory bowel diseases, the effect of the histamine H1 antagonist Dithiaden was studied on a model of acetic acid-induced colitis in rats. Dithiaden pretreatment by intracolonic administration was found to reduce the extent of acute inflammatory colonic injury. This was manifested by a decrease in the score of gross mucosal injury, by lowered colonic wet weight and by diminished myeloperoxidase activity reflecting reduced leukocyte infiltration. Vascular permeability and gamma-glutamyl transpeptidase activity, elevated by acetic acid exposure, were decreased after Dithiaden pretreatment. The results indicate that locally administered Dithiaden may protect the colonic mucosa against an acute inflammatory attack by interfering with the action of the major mast cell mediator histamine.     

Deficiency of glucocorticoid production delays the healing of acute gastric mucosal erosion and chronic ulcers in rats

Effects of glucocorticoid deficiency followed by corticosterone replacement on the healing of gastric erosions and chronic gastric ulcers have been investigated in rats. Glucocorticoid deficiency was induced by adrenalectomy performed after the formation of gastric erosions or ulcers. Gastric erosions were produced by indomethacin (35 mg/kg, i.p.) or by 6 h immobilization at temperature 8 degrees C, chronic gastric ulcers were induced by 60% acetic acid. All ulcerogenic stimuli caused an increase in corticosterone production. Adrenalectomy created corticosterone deficiency and delayed the healing of gastric erosions and chronic gastric ulcers. The effect of adrenalectomy was more evident in the indomethacin ulcerogenic model. Replacement by corticosterone prompted the healing of gastric erosions and ulcers in adrenalectomized animals. These data suggest a participation of endogenous glucocorticoids in a restoration of gastric mucosal integrity.     

Protective and therapeutic effects of resveratrol on acetic acid-induced gastric ulcer

Sprague Dawley rats of both sexes were injected with either saline or RVT (10 mg/kg) either before or after acetic acid ulcer induction and decapitated 3, 5 or 10 days after ulcer. In the saline-treated ulcer groups, macroscopically evident ulcers were observed, while RVT-pretreated or RVT-treated groups had lower macroscopic ulcer scores. Likewise, the microscopic damage scores were lower for the RVT-administered groups. Gastric myeloperoxidase activity, malondialdehyde, collagen and tumour necrosis factor-alpha levels, as well as luminol- and lucigenin-enhanced chemiluminescence levels that were elevated in the saline-administered ulcer groups, were depressed with both RVT-pretreatment and RVT-treatment. Moreover, depleted glutathione levels in the ulcer groups were increased back to control levels by both pre- and post-treatments of RVT. Results demonstrate that resveratrol has both protective and therapeutic effects on oxidative gastric damage by suppressing pro-inflammatory cascades, including the activation of pro-inflammatory cytokines, accumulation of neutrophils and release of oxygen-derived free radicals.     

Autophosphorylation of 70 kDa heat shock proteins.

Several members of the 70 kDa heat shock protein group are known to be phosphorylated in vivo and have recently been found to undergo a Ca(2+)-stimulated autophosphorylation. The characteristics of the autophosphorylation reaction with Escherichia coli DnaK the mitochondrial and chloroplast homologs, and the endoplasmic reticulum Bip/Grp78 are discussed. Some common features are a requirement for Ca2+, inhibition by Mg2+ and phosphorylation solely on a threonine residue. Although the role of autophosphorylation of these proteins is not clear, it is known that the level of phosphorylation of some Hsp70 proteins in vivo is responsive to stress and other cellular conditions.     

Involvement of GDNF and LIMK1 and heat shock proteins in drosophila learning and memory formation

Molecular mechanisms of the synapse and dendrite integrity maintenance and their disruption in psychiatric and neurodegenerative diseases (NDD) are being studied intensively to identify target genes for therapeutic activities. It is suggested that synapse is a tripartite system in which glia, alongside with well-studied pre- and postsynaptic neurons, represents a third, poorly studied partner in synaptic transmission involved in a positive feedback loop between the other two partners. It is the glia cell-derived neurotrophic factor (GDNF) and the transmembrane proteins, neuregulins, that mediate bidirectional coupling between presynaptic neurons and their postsynaptic targets. Neuregulins are structurally related to the epidermal growth factor and have a cytoplasmic domain that interacts with intracellular LIM kinase 1 (LIMK1), the key enzyme of actin remodeling. Since neurons and axons that do not receive a sufficient GDNF supply are at risk of degeneration and synapse elimination, GDNF became a central target factor in human NDD therapy. The delivery of GDNF-producing stem cells to the nidus of neurodegeneration by transplantation surgery is an efficient tool for NDD treatment. A new approach is proposed based on the use of the Drosophila heat shock (hs) promoter that responds to the mammalian body temperature and ensures constant expression of the human GDNF gene. The Drosophila models facilitate studying the role of each component of the bidirectional signaling between pre- and postsynaptic neurons in the development of the key diagnostic NDD symptom—a defective memory formation resulted from synaptic atrophy. To assess the efficiency of memory formation depending on the level of GDNF and LIMK1 brain expression, we used the Drosophila strains simulating different nervous system disorders: GDNF, the transgenic flies that carry the human GDNF gene under hs-promoter, l(1)ts403, the mutants with disruption of heat shock proteins (HSPs) mRNA nuclear trafficking, and agn ^ts3 carrying a mutation in LIMK1 gene. We investigated at the behavioral (learning/memory) level the functional connections between GDNF, LIMK1 and HSP signaling transductions that might offer promising targets for complex approaches to NDD treatment.