Low dose ethanol potentiates indomethacin induced inhibition of wound re-epithelialization in duodenal monolayers

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce gastroduodenal mucosal injury and ulceration, and delay ulcer healing. In contrast, the effects of low dose ethanol in induction of gastroduodenal mucosal injury, and the subsequent wound repair remains unclear. The aim of this study was to determine, using an in-vitro duodenal epithelial wound model, whether low clinically relevant doses of ethanol or indomethacin interfere with the wound re-epithelialization of duodenal epithelial monolayers. The possible potentiating effect of ethanol on indomethacin modulation of duodenal re-epithelialization was also examined. In-vitro epithelial wounds were created in confluent IEC-6 duodenal epithelial monolayers by a razor blade scrape. Ethanol at low concentrations (0.25, 0.5, 0.75%) did not have significant effect on duodenal wound re-epithelialization. Similarly, low doses of indomethacin (.01, .05, 0.1 mM) also did not have a significant effect on wound re-epithelialization. However, the combination of ethanol (0.5 or 0.75%) and indomethacin (0.1mM) produced a marked inhibition of IEC-6 re-epithelialization. At the low doses used, ethanol and indomethacin (individually or in combination) did not have direct cytotoxic effect on IEC-6 cells. Ethanol or indomethacin (at the studied concentrations) had only minimal effect on the actin stress fibers in the cells at the migration front. However, in combination, they almost completely abolished the actin stress fibers at the migration front. These findings demonstrate that while low clinically relevant doses of ethanol and indomethacin individually do not affect re-epithelialization of wounded duodenal epithelial monolayers, in combination they produce a significant inhibition.     

Nonsteroidal anti-inflammatory drugs inhibit re-epithelialization of wounded gastric monolayers by interfering with actin, Src, FAK, and tensin signaling.

Re-epithelialization is essential for gastrointestinal ulcer and cutaneous wound healing. It requires epithelial cell migration and proliferation, processes that are stimulated by epidermal growth factor (EGF), and dependent on the cell cytoskeleton. Activation of Src and focal adhesion kinase (FAK) has been implicated in EGF-stimulated cell migration. Nonsteroidal anti-inflammatory drugs (NSAIDs) (both nonselective and Cox2-selective) interfere with ulcer healing and re-epithelialization in vitro and in vivo, but the cellular targets and mechanisms remain unexplored forming the basis of this study. Using a wounded gastric epithelial cell monolayer model, we demonstrated that NSAIDs reduce both basal and epidermal growth factor (EGF)-induced re-epithelialization, and that this action involves disruption of actin stress fiber formation, reduced c-Src activity, decreased phosphorylation of focal adhesion kinase (FAK), tensin and their cellular re-distribution. There was a strong correlation between NSAIDs-mediated inhibitory effect on re-epithelialization and loss of stress fibers and reduced tensin signal. Furthermore, NSAIDs significantly reduced EGF-stimulated c-Src association with FAK. These findings suggest that NSAIDs can directly affect the cell cytoskeleton and signaling pathways essential for re-epithelialization.     

Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)-activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP-binding proteins of Rho family, in this study we examined whether VacA affects wound re-epithelialization, cell cytoskeleton-associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re-epithelialization, cell proliferation vs control. VacA-induced cell vacuolation strongly correlated with inhibition of wound re-epithelialization. Furthermore, VacA reduced Rac-1 protein expression and distribution, and C3-mediated ADP-ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re-epithelialization by altering cytoskeleton-dependent cell functions and signaling.     

Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer

Matrix metalloproteinases (MMPs) are suggested to play a critical role in extracellular matrix degradation and remodeling during inflammation and wound healing processes. However, the role of MMPs in indomethacin-induced gastric ulcer and its healing process are not clearly understood. This study is aimed at determining the regulation of MMP-9 and -2 activities in indomethacin-induced acute gastric ulceration and healing. Indomethacin-ulcerated stomach extracts exhibit significant up-regulation of pro-MMP-9 (92 kDa) activity and moderate reduction of MMP-2 activity, which strongly correlate with indomethacin dose and severity of ulcer. The anti-inflammatory and antioxidant properties of curcumin, an active component of turmeric, suggest that curcumin may exert antiulcer activity through scavenging reactive oxygen species, by regulating MMP activity, or both. To test these possibilities, the effect of curcumin in indomethacin-induced gastric ulcer is examined by biochemical and histological methods. The results show that curcumin exhibits potent antiulcer activity in acute ulcer in rat model by preventing glutathione depletion, lipid peroxidation, and protein oxidation. Denudation of epithelial cells during damage of gastric lumen is reversed by curcumin through re-epithelialization. Furthermore, both oral and intraperitoneal administration of curcumin blocks gastric ulceration in a dose-dependent manner. It accelerates the healing process and protects gastric ulcer through attenuation of MMP-9 activity and amelioration of MMP-2 activity. Omeprazole, an established antiulcer drug does not inhibit MMP-9 while protecting indomethacin-induced gastric ulcer. We conclude that antiulcer activity of curcumin is primarily attributed to MMP-9 inhibition, one of the major path-ways of ulcer healing.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

Cyclooxygenase inhibitors not inhibit resting lung cancer A549 cell proliferation

Cyclooxygenase (COX) inhibitors were regarded as anticarcinogenic agents for lung cancer at least partly via PGE2; but these were based on cytokin stimulation experiment on A549 cell. In order to clarify whether COX inhibitors directly inhibit A549 cell, three COX inhibitors, NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), and acetyl salicylic acid (ASA, non-selective COX inhibitor), were studied. NS398, and ASA, can inhibit PGE2 generation via COX-2 inhibition. The viability of A549 cell was assayed by MTT. However, without cytokin stimulation, all the three inhibitors (NS398 0.2-20 microM; SC560 1.0-100 nM; ASA 0.01-1.0 mM) were not able to inhibit A549 cell proliferation, in the other way round, NS398 promoted cell growth. And arachidonic acid (AA) and lipopolysaccharide (LPS) did not disturb the property of its growth. These data suggested that without cytokin stimulation, COX and PGE2 may not be the kernel molecules involved in A549 cell proliferation, and COX inhibitors could not inhibit A549 cell growth directly.     

Indomethacin Inhibits Endothelial Cell Proliferation by Suppressing Cell Cycle Proteins and PRB Phosphorylation: A Key to Its Antiangiogenic Action?

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit angiogenesis in vivo and in vitro, but the mechanism of this action is unclear. Angiogenesis—formation of new capillary vessels—requires endothelial proliferation, migration, and tube formation. It is stimulated by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The cell cycle is regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors (CKI) and the retinoblastoma protein (pRb). Since the effects of NSAIDs on cell cycle-regulatory proteins in endothelial cells remain unknown, we examined the effect of indomethacin on bFGF-stimulated endothelial cell proliferation and on cell cycle regulatory proteins in rat primary aortic endothelial cells (RAEC). Indomethacin significantly inhibited basal and bFGF-stimulated endothelial cell proliferation. This inhibition correlated significantly with reduced cyclin D1 and increased p21 protein expression. Furthermore, indomethacin reduced pRb phosphorylation. These findings suggest that indomethacin arrests endothelial cell proliferation essential for angiogenesis by modulating cell cycle protein levels.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.     

Mechanisms underlying the contraction induced by bradykinin in the guinea pig epithelium-denuded trachea

This study investigates some of the mechanisms by which bradykinin (BK) triggers contraction of epithelium-denuded strips of guinea pig trachea (GPT). Cumulative or single additions of BK, T-BK, L-BK, or ML-BK in the presence of captopril (30 microM) produced graded GPT contractions with the following rank order of potency (EC50 level): T-BK (31.3 nM) > BK (40.0 nM) > L-BK (56.0 nM) > ML-BK (77.0 nM). BK-induced contraction (100 nM) in GPT was completely inhibited by either HOE 140 or NPC 17731 with mean IC50 values of 17 and 217 nM, respectively. Addition of BK (100 nM) at 30 min intervals, induced progressive tachyphylaxis, which was complete after 4 h. The tachyphylaxis induced by BK was unaffected by L-NOARG (nitric oxide synthase inhibitor, 100 microM) or valeryl salicylate (a cyclooxygenase-1 (COX-1) inhibitor, 30 microM), but was prevented by a low concentration of indomethacin, diclofenac (non-selective COX inhibitors, 3 nM each) or by NS 398 (a COX-2 inhibitor, 10 nM). Furthermore, higher concentrations of indomethacin, diclofenac, phenidone (a lypooxygenase (LOX) and COX inhibitor), or NS 398, caused graded inhibition of BK-induced contraction, with mean IC50 values of 0.28, 0.08, 46.37, and 0.15 microM, respectively. Together, these results suggest that BK-induced contraction in GPT involves activation of B2 receptors and release of prostanoids from COX-2 pathway. Furthermore, the tachyphylaxis induced by BK was insensitive to the nitric oxide and COX-1 inhibitors, but was prevented by non-selective and selective COX-2 inhibitors, indicating a mediation via COX-2-derived arachidonic acid metabolites.     

MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.

Limited clinical and experimental studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) may inhibit gastric cancer growth. However, the mechanisms involved are not completely understood and cannot be explained by COX-2 inhibition alone. MAPK signaling pathway is essential for cell proliferation, but the effect of NSAIDs on MAPK activity and phosphorylation in gastric cancer has never been studied. Since increased and unregulated cell proliferation and reduced cell apoptosis are important features of cancer growth, we studied whether NS-398, a selective COX-2 inhibitor and/ or indomethacin (IND), a non-selective NSAID: 1) inhibit gastric cancer cell proliferation, 2) whether this inhibition is mediated via MAPK (ERK2), and 3) whether NSAIDs enhance apoptosis in gastric cancer cells. Human gastric epithelial cells (MKN28) derived from gastric tubular adenocarcinoma were cultured and treated with either vehicle, IND (0.25-0.5mM) or NS-398 (50-100 microM) for 6, 16, 24 and 48h. Studies: 1) Cellular proliferation was determined by 3H-thymidine uptake. 2) MAPK activity was measured by incorporation of radiolabeled phosphate into myelin basic protein. 3) Apoptosis was evaluated using TUNEL assay. IND and NS-398 significantly inhibited the proliferation of MKN28 cells at 24h by 3.5 - 5 fold (p<0.002) and at 48h by 2.5 - 10 fold (p<0.02). Both NSAIDs also significantly inhibited ERK2 activity: IND >53% inhibition, NS-398, 100 microM >72% inhibition; all p<0.05. Both IND and NS-398 significantly increased apoptotic index. In conclusion, IND and NS-398 significantly inhibit proliferation and growth of human gastric cancer cell line MKN28. This effect is mediated by NSAID-induced inhibition of MAPK (ERK2) kinase signaling pathway, essential for cell proliferation. NSAIDs also increase apoptosis in MKN28 cells. In addition to inhibiting cyclooxygenase, NSAIDs inhibit phosphorylating enzymes--kinases essential for signaling cell proliferation.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Effect of local injection with basic fibroblast growth factor (BFGF) and neutralizing antibody to BFGF on gastric ulcer healing, gastric secretion, angiogenesis and gastric blood flow.

Exogenous administration of bFGF was shown to accelerate tissue repair predominantly due to an increase in the formation of new microvessels (angiogenesis) suggesting that bFGF plays an important role in healing of gastric ulcer. This study was designed: 1) to examine the effect of local application of bFGF with or without neutralizing antibody (NA) to bFGF and 2) to determine the role of gastric secretion, gastric blood flow (GBF) at the ulcer margin and angiogenesis during gastric ulcer healing with or without local application of NA, bFGF or the combination of NA and bFGF. Chronic gastric ulcers were induced in Wistar rats by subserosal application of acetic acid (ulcer area 28 mm2) and gastric secretion during ulcer healing was assessed using animals additionally equipped with chronic gastric fistulas. The bFGF without or with NA to bFGF (10 ng/100 microl]), irrelevant antibodies (rabbit IgG; 10 microg/100 microl) or vehicle (saline) were locally injected into the subserosa immediately upon ulcer induction (day 0) and at day 2. Rats with acetic acid ulcers without subserosal injections served as controls. At day 11, all animals were anaesthetized and GBF was determined at the ulcer base, ulcer margin as well as in intact mucosa using the H2-gas clearance technique and the area of gastric ulcers was measured by planimetry. Gastric mucosa with ulcer was excised and the percentage of area covered with blood vessels, the number of fibroblasts and the percentage of connective tissue at the ulcer edge was assessed by histology. The gastric ulcers were healed spontaneously in control vehicle-treated rats at day 11 and this was accompanied by the significant increase in the GBF and number of microvessels in the ulcer area. The gastric secretion was suppressed immediately after ulcer induction and increased significantly at day 2 and day 11 but failed to return to that recorded in intact animals. In contrast, local application of bFGF inhibited gastric acid and pepsin outputs at each study time intervals tested and this effect was reversed by addition of NA to bFGF. Locally applied bFGF accelerated significantly ulcer healing and this was accompanied by the greater rise in the GBF of ulcer margin and more marked increase in number of microvessels as compared to those in vehicle-treated rats. Subserosal application of NA to bFGF prolonged significantly the ulcer healing and this effect was accompanied by a significant fall in the GBF at the ulcer margin and a decrease in number of capillaries in ulcer bed without significant alteration in gastric acid and pepsin outputs. The ulcer healing effect of bFGF and accompanying increase in the GBF at ulcer margin and in thenumber of microvessels as well as inhibition of gastric acid secretion evoked by bFGF were significantly attenuated by the addition of NA to bFGF. The number of fibroblasts and the distribution of connective tissue did not differ between groups studied. We conclude that; 1) depletion of endogenous bFGF at the ulcer area by specific NA to bFGF delays healing of gastric ulcers, reduces angiogenesis of ulcer bed and impairs the microcirculatory effect of this growth factor at the ulcer margin indicating that the availability of bFGF in the ulcer area plays a crucial role in the ulcer healing through induction of angiogenesis; 2) this prominent antiulcer effect of locally applied bFGF depends, at least in part, upon the inhibition of acid secretion by this peptide.     

Growth factors in ulcer healing: lessons from recent studies.

Growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and more recently vascular endothelial growth factor (VEGF) have been used extensively to heal experimental gastric, duodenal and colonic ulcers in animal models. Encouraging results have been reported in clinical trials with EGF and bFGF. Since our laboratory has been involved with the initial ulcer healing studies with bFGF, PDGF and VEGF, we summarize here the major lessons from these studies and from literature data. These conclusions relate to the role of: 1) gastrointestinal (GI) secretion; 2) epithelial versus vascular components of the healing; 3) efficacy in the upper and lower GI tract; 4) quality of ulcer healing; as well as 5) the endogenous origin; and 6) molar potency of growth factors. Namely, among these growth factors only EGF inhibits gastric acid and stimulates duodenal bicarbonate secretion, while chronic administration of bFGF slightly enhances gastric secretion and PDGF has no effect demonstrating that potent ulcer healing can be achieved without influencing acid base and mucus secretion. This might be related to the fact that these growth factors stimulate with varying potency virtually all the cellular elements needed for ulcer healing, e.g., epithelial cell proliferation and migration by EGF > bFGF > PDGF, fibroblast proliferation by bFGF > PDGF and angiogenesis by VEGF > bFGF > PDGF > EGF. Conceptually, the most interesting results were obtained recently with VEGF which is virtually specific for angiogenesis, illustrating that stimulation of vascular factors is sufficient for ulcer healing because epithelial cells apparently spontaneously proliferate and migrate over a dense granulation tissue to complete the healing process. Since these growth factors directly stimulate the cell components of ulcer healing, it is probably not surprising that they are active in both upper and lower GI tract lesions, produce good quality of ulcer healing in comparison with spontaneously healed duodenal ulcers which are hypovascular and muscle regeneration is not part of natural healing. Contrary to other antiulcer drugs, these growth factors are endogenously derived and play a role in the natural history of ulcer healing, and since these relatively large peptides (18-45 kDa) are active in ng quantities, their molar potency is 2-7 million times superior to cimetidine-like drugs. Thus growth factors are endogenously derived very potent antiulcer drugs which act independently of GI secretion, are active in upper and lower GI lesions, and since they stimulate virtually all the cells of the healing process, they produce an excellent quality of ulcer healing.     

Gastroprotective effect of epoxy clerodane diterpene isolated from Tinospora cordifolia Miers (Guduchi) on indomethacin-induced gastric ulcer in rats

The present study evaluated the gastroprotective effect of epoxy clerodane diterpene (ECD), isolated from Tinospora cordifolia on indomethacin-induced gastric ulcer in rats. Administration of indomethacin exhibits extreme levels of ulcer index (UI) and myeloperoxidase (MPO) activity. Indomethacin down regulated PGE₂, anti-inflammatory cytokines (IL-4, IL-10) and pro-angiogenic factors (VEGF and EGF). The ECD pretreatment considerably increased the levels of PGE₂, anti-inflammatory cytokines and pro-angiogenic factors. The ulcer-healing activity of ECD was inhibited by pre-administration of the specific COX-1 inhibitor (SC560) and nonspecific NOS inhibitor (l-NAME), which indicates the involvement of PGE₂ and NOS in ECD induced ulcer healing activity. These findings suggest that ECD exerts its antiulcer activity by reinforcement of defensive elements and diminishing the offensive elements.     

Role of cyclooxygenase-2 in modulating gastric acid secretion in the normal and inflamed rat stomach

Nonsteroidal anti-inflammatory drugs elevate gastric acid secretion, possibly contributing to their ability to interfere with gastric ulcer healing. Inhibitors of cyclooxygenase-2 have been shown to delay experimental gastric ulcer healing. In the present study, we tested the hypothesis that cyclooxygenase-2-derived prostaglandins modulate gastric acid secretion. Studies were performed in normal rats and in rats with iodoacetamide-induced gastritis. Inflammation in the latter group was confirmed histologically and by a threefold increase in tissue levels of the granulocyte marker myeloperoxidase and was also associated with overexpression of cyclooxygenase-2 in the stomach. Basal acid secretion in both groups of rats was not affected by pretreatment with DuP-697, a selective inhibitor of cyclooxygenase-2. A nonselective cyclooxygenase inhibitor, indomethacin, had no effect on acid secretion in normal rats but caused a doubling of acid secretion in the rats with gastritis. DuP-697 had no effect on pentagastrin-induced secretion in either group of rats. Gastritis itself was associated with significantly increased pentagastrin-induced acid secretion, and this was further increased in rats pretreated with indomethacin. These results suggest that in a setting of gastric inflammation, prostaglandins derived from cyclooxygenase-1, not cyclooxygenase-2, exert inhibitory effects on acid secretion.     

Roles of endogenous prostaglandins and nitric oxide in gastroduodenal ulcerogenic responses induced in rats by hypothermic stress.

We examined the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the gastroduodenal ulcerogenic responses to hypothermic stress (28 approximately 30 degrees C) in anesthetized rats. Lowering body temperature provoked damage in the gastroduodenal mucosa, with an increase of gastric acid secretion and motility. These responses were completely abolished by bilateral vagotomy or atropine, while 16,16-dimethyl PGE2 decreased the mucosal ulcerogenic response with no effect on acid secretion. The non-selective COX inhibitors, indomethacin or aspirin, worsened these lesions with enhancement of gastric motility and no effect on acid secretion, while the selective COX-2 inhibitor NS-398 did not affect any of these responses. On the other hand, the non-selective NOS inhibitor L-NAME but not aminoguanidine (a relatively selective inhibitor of iNOS), significantly potentiated the acid secretory and mucosal ulcerogenic responses in the stomach but reduced the duodenal damage in response to hypothermia, the effects being antagonized by co-administration of L-arginine. Hypothermia itself decreased duodenal HCO3- secretion under both basal and mucosal acidification-stimulated conditions. Both indomethacin and aspirin further decreased the HCO3- response to the mucosal acidification, while L-NAME significantly increased the HCO3- secretion even under hypothermic conditions, similar to 16,16-dimethyl PGE2. These results suggest that 1) hypothermic stress caused an increase of acid secretion and motility as well as a decrease of duodenal HCO3-secretion, resulting in damage in both the stomach and duodenum, 2) the COX-1 but not COX-2 inhibition worsened these lesions by enhancing gastric motility and further decreasing duodenal HCO3- response, 3) the cNOS but not iNOS inhibition worsened gastric lesions by increasing acid secretion but decreased duodenal damage by increasing HCO3- secretion. Thus, it is assumed that the gastroduodenal ulcerogenic and functional responses to hypothermic stress are modified by cNOS/NO as well as COX-1/PGs.