Dual action of nitric oxide in pathogenesis of indomethacin-induced small intestinal ulceration in rats.

We investigated the pathogenic role of nitric oxide (NO) in indomethacin-induced intestinal ulceration in rats. Nonfasting animals responded to a single administration of indomethacin (10 mg/kg, s.c.), resulting in multiple hemorrhagic lesions in the small intestine, mostly the jejunum and ileum. The damage was first observed 6 hr after indomethacin, the severity increasing progressively with time up to 24 hr later, accompanied with the gene expression of inducible NO synthase (iNOS) and the increase of nitrite and nitrate (NOx) contents in the mucosa. The ocurrence of damage was significantly prevented when iNOS induction was inhibited by dexamethasone given either once 0.5 hr before or twice 0.5 hr before and 6 hr after indomethacin. Likewise, aminoguanidine (a relatively selective iNOS inhibitor) reduced the severity of damage, irrespective whether given twice or as a single injection 6 hr after indomethacin. By contrast, the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) exhibited a biphasic effect, depending on the time of administration; the pre-administration worsened the damage, while the later administration reduced the severity of these lesions, yet both responses occureed in a L-arginine-sensitive manner. Pre-administration of L-NAME, but not aminoguanidine, significantly decreased NOx production in the intestinal mucosa of normal rats, while the increase of NOx production following indomethacin was significantly suppressed by the later administration of aminoguanidine as well as L-NAME. These results suggest that NO exerts a dual action in the pathogenesis of indomethacin-induced intestinal ulceration; NO generated by cNOS is protective against indomethacin, by maintaining the integrity of intestinal mucosa, while NO derived by iNOS plays a key pathogenic role in the ulcerogenic process.     

Role of endogenous nitric oxide (NO) and NO synthases in healing of indomethacin-induced intestinal ulcers in rats

We examined the roles of nitric oxide (NO) and NO synthase (NOS) isozymes in the healing of indomethacin-induced small intestinal ulcers in rats. Animals were given indomethacin (10 mg/kg, s.c.) and killed 1, 4 and 7 days after the administration. Indomethacin (2 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor: 10 mg/kg) and aminoguanine (a relatively selective iNOS inhibitor: 20 mg/kg) were given s.c. once daily for 6 days, the first 3 days or the last 3 days during a 7-day experimental period. Both indomethacin and L-NAME significantly impaired healing of these lesions, irrespective of whether they were given for 6 days, first 3 days or last 3 days. The healing was also impaired by aminoguanine given for the first 3 days but not for the last 3 days. Expression of iNOS mRNA in the intestine was up-regulated after ulceration, persisting for 2 days thereafter, and the Ca(2+)-independent iNOS activity also markedly increased with a peak response during 1-2 days after ulceration. Vascular content in the ulcerated mucosa as measured by carmine incorporation was decreased when the healing was impaired by indomethacin and L-NAME given for either the first or last 3 days as well as aminoguanidine given for the first 3 days. These results suggest that endogenous NO plays a role in healing of intestinal lesions, in addition to prostaglandins, yet the NOS isozyme mainly responsible for NO production differs depending on the stage of healing: iNOS in the early stage and cNOS in the late stage.     

Protection by constitutively formed nitric oxide of intestinal damage induced by indomethacin in rats.

In the present study, we investigated a protective role of constitutively occurred nitric oxide (NO) against indomethacin-induced intestinal lesions in rats. Indomethacin (10 mg/kg) was given s.c. to animals without fasting, and the intestinal mucosa was examined for lesions 24 h later. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) was given s.c. 0.5 h before or 6 hr after indomethacin, while the NO donor (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexnamine (NOR-3) was given s.c. 0.5 h before indomethacin. Indomethacin caused hemorrhagic lesions in the small intestine, accompanied with an increase in intestinal motility and bacterial translocation. These lesions were markedly prevented or worsened, respectively, by later or prior administration of L-NAME (20 mg/kg), in a L-arginine-sensitive manner. The worsening effect of L-NAME (5-20 mg/kg) on these lesions was dose-dependently observed in association with further enhancement of the bacterial translocation and intestinal hypermotility following indomethacin. By contrast, prior administration of NOR-3 (1-6 mg/kg) dose-dependently prevented the development of intestinal lesions, together with suppression of the bacterial translocation and intestinal hypermotility in response to indomethacin. On the other hand, both indomethacin and L-NAME decreased intestinal mucus and fluid (water) secretion in the small intestine, while NOR-3 increased these secretions. These results suggest that (1) NO occurred constitutively exerts a protective action against indomethacin-induced intestinal ulceration, and (2) this effect is related with prevention of bacterial translocation, the process functionally associated with increase of mucus and fluid secretions as well as inhibition of intestinal hypermotility.     

Gastroprotective effect of leptin in indomethacin-induced gastric injury

This study investigated the involvement of neutrophil infiltration, disturbances in nitric oxide (NO) generation and oxidative stress in indomethacin-induced gastric ulcer, and the possible gastroprotective potentials of leptin, known for its angiogenic effect. Male Wistar albino rats (180–220 g) were allocated into a normal control group, ulcer control group (received a single dose of indomethacin 40 mg/kg p.o.) and an ulcer group pretreated with leptin (10 μg/kg i.p. 30 min before ulcer induction). The animals were killed 6 h after indomethacin administration and their gastric juice, serum and mucosal tissue were used for gastric injury evaluation. Indomethacin produced multiple lesions in glandular mucosa, evidenced by marked increase in gastric ulcer index (GUI) accompanied by significant increases in gastric juice acidity, tissue myeloperoxidase (MPO) activity, serum NO and tissue conjugated diene (CD), and marked decreases in tissue NO and glutathione (GSH) as well as glutathione reductase (GR) and superoxide dismutase (SOD) activities, while gastric juice mucin and tissue glutathione peroxidase (GPx) were not affected. Leptin exerted significant gastroprotection as evidenced by significantly decreased GUI and attenuated neutrophil infiltration. Leptin significantly increased mucin and tissue NO, restored GR and SOD activities and up-regulated GPx activity. It failed to affect acidity, serum NO, GSH and CD. These results suggest that leptin confers significant gastroprotection against indomethacin-induced injury through interfering with neutrophil infiltration, NO production and oxidative stress.     

The selective inhibition of inducible nitric oxide synthase prevents intestinal ischemia-reperfusion injury in mice.

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.     

Increased susceptibility of small intestine to NSAID-provoked ulceration in rats with adjuvant-induced arthritis: involvement of enhanced expression of TLR4

NSAIDs damage the small intestine as well as the stomach as adverse effects. We previously reported that the gastric ulcerogenic response to NSAIDs was markedly increased in arthritic rats. The present study was designed to examine the intestinal ulcerogenic property of indomethacin in adjuvant-induced arthritic rats in comparison with normal animals. Arthritis was induced in male Dark Agouti rats by injection of Freund's complete adjuvant into the right hindfoot. Two weeks later, indomethacin was given orally and the intestine was examined for lesions at several time points after indomethacin. Indomethacin produced intestinal lesions in both normal and arthritic rats, but in the latter, the ulcerogenic response occurred much earlier and the severity was markedly enhanced. Aminoguanidine, an inhibitor of iNOS, significantly suppressed the damage, yet the efficacy differed in normal and arthritic rats, depending on the dose schedule; the effect of post-administration (6 h after) was greater than that of pre-administration (0.5 h before) in normal rats, whereas that of post-administration was less than that of pre-administration in arthritic rats. The expression of iNOS and TLR4 in the intestine was enhanced in arthritic rats as compared with normal rats. These results suggest that the intestinal ulcerogenic response to indomethacin is markedly aggravated in arthritic rats. Notably, the onset of the ulceration was much earlier in arthritic rats than normal rats. These phenomena may be accounted for by the upregulation of iNOS/NO through the increased expression of TLR4 in the small intestine of arthritic rats.     

Effects of JBP485 on the expression and function of PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells

To investigate the effect of JBP485 (an anti-inflammatory dipeptide) on PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells, the activity and expression of PEPT1 were examined. The effects of treatment with indomethacin and co-treatment with JBP485 were examined in terms of intestinal histological changes, MDA and MPO levels in rats; as well as LDH-release and oxidative stress in Caco-2 cells. Uptake of glycylsarcosine (Gly-Sar) by PEPT1 was determined by in vivo, in vitro and in situ studies. RT-PCR and Western blot were used to assess the expression of PEPT1 in rat intestine and Caco-2 cells. JBP485 caused a significant decrease in MDA and MPO levels, and improved the pathological condition of rat intestine, while attenuating Caco-2 cells damage induced by indomethacin. Uptake of Gly-Sar by PEPT1 was decreased by indomethacin treatment, whereas the Gly-Sar plasma concentration was markedly increased in JBP485 co-treated rats. Indomethacin down-regulated the expression of PEPT1 mRNA and protein in rat intestine and Caco-2 cells, and the effects were reversed after administration of JBP485. These results indicated that JBP485 not only improved intestinal injury and cell damage but also partially blocked the down-regulation of PEPT1 expression and function induced by indomethacin.     

Hypertension alters the participation of contractile prostanoids and superoxide anions in lipopolysaccharide effects on small mesenteric arteries

The involvement of cyclooxygenase-2 (COX-2)-derived products and superoxide anion in the effect of lipopolysaccharide in noradrenaline (NA)-induced contraction was investigated in small mesenteric arteries (SMA) from normotensive, Wistar Kyoto (WKY), and spontaneously hypertensive (SHR) rats. In WKY, lipopolysaccharide (10 microg/ml, 1 and 5 h) only inhibited the NA response (0.1-30 microM) in the presence of dexamethasone (1 microM), indomethacin (10 microM), the selective COX-2 inhibitor, NS 398 (10 microM), and the TXA(2)/PGH(2) receptor antagonist, SQ 29,548 (10 microM) but not of superoxide dismutase (SOD, 100 U/ml). In SHR, lipopolysaccharide inhibited the NA response by itself; this inhibition was potentiated by dexamethasone, indomethacin, NS 398, SQ 29,548 and SOD. The effect of lipopolysaccharide plus indomethacin, NS 398 or SQ 29,548 was higher in SMA from WKY than SHR only after 1 h lipopolysaccharide incubation. N(G)-nitro-L-arginine methyl ester (100 microM) and endothelium removal abolished the indomethacin-induced potentiatory effect of lipopolysaccharide in both strains. Endothelium removal also abolished the SOD potentiatory effect in SMA from SHR. Lipopolysaccharide increases COX-2 expression to a similar level in both strains and iNOS expression in a greater extent in SHR; these increases were reduced by dexamethasone. These results indicate: 1) lipopolysaccharide induces the endothelial production of contractile prostanoids from COX-2 in SMA, probably to compensate the increase in NO from iNOS; 2) the production of prostanoids in the presence of lipopolysaccharide seems to be greater in normotensive than hypertensive rats only after lipopolysaccharide short incubation times; 3) endothelial production of O(2)(.-) contributes to counteract depression of NA contraction caused by lipopolysaccharide only in SHR.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Protection by aspirin of indomethacin-induced small intestinal damage in rats: mediation by salicylic acid.

Most of non-steroidal anti-inflammatory drugs (NSAIDs) except aspirin (ASA) produce intestinal damage in rats. In the present study, we re-examined the intestinal toxic effect of ASA in rats, in comparison with various NSAIDs, and investigated why ASA does not cause damage in the small intestine, in relation to its metabolite salicylic acid (SA). Various NSAIDs (indomethacin; 10 mg/kg; flurbiprofen; 20 mg/kg; naproxen; 40 mg/kg; dicrofenac; 40 mg/kg; ASA; 20-200 mg/kg) were administered s.c., and the small intestinal mucosa was examined macroscopically 24 h later. All NSAIDs tested, except ASA, caused hemorrhagic lesions in the small intestine, with a decrease of mucosal PGE(2) contents. ASA did not provoke any damage, despite inhibiting (prostaglandin) PG production, and prevented the occurrence of intestinal lesions induced by indomethacin, in a dose-related manner. This protective action of ASA was mimicked by the equimolar doses of SA (17.8-178 mg/kg). Indomethacin caused intestinal hypermotility, in preceding to the occurrence of lesion, and this event was followed by increases of enterobacterial translocation in the mucosa. Both ASA and SA prevented both the intestinal hypermotility and the bacterial translocation seen after indomethacin treatment. In addition, the protective effect of SA was not significantly influenced by either the adenosine deaminase or the adenosine receptor antagonists. Following administration of ASA, the blood SA levels reached a peak within 30 min and remained elevated for more than 7 h. These results suggest that SA has a cytoprotective action against indomethacin-induced small intestinal lesions, and this action may be associated with inhibition of the intestinal hypermotility and the bacterial translocation, but not mediated by endogenous adenosine. Failure of ASA to induce intestinal damage may be explained, at least partly, by a protective action of SA, the metabolite of ASA.     

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-κB and AP-1 in INT-407 cells

Multiple mucosal immune factors, such as TNF-α and IL-1β, are thought to be key mediators involved in inflammatory bowel disease. We evaluated the role of the pro-inflammatory cytokine TNF-α on nitric oxide synthase (NOS) expression in indomethacin-induced jejunoileitis in rats. Jejunoileitis was induced in rats with subcutaneous injections of indomethacin (7.5 mg/kg) 24 h apart for two consecutive days, and animals were randomized into four groups. Group 1 received only indomethacin. Group 2 was treated with a daily dose of phosphodiesterase (PDE) inhibitor (theophylline or pentoxifylline) by oral gavage for 2 days before and 4 days after indomethacin. Group 3 received a single dose of anti-TNF-α monoclonal antibody (TNF-Ab, IP) 30 min before indomethacin. Group 4 was treated with 1 h hyperbaric oxygenation (HBO₂) for 5 days after indomethacin. Rats were sacrificed at 12 h or 4 days after final indomethacin injection. PDE inhibitor, TNF-Ab, or HBO₂ treatment significantly decreased indomethacin-induced ulceration, myeloperoxidase activity, and disease activity index. Although indomethacin significantly increased serum TNF-α and nitrate/nitrite (NOx) concentrations above control values at 12 h, inducible NOS (iNOS) expression was detected only at day 4. Serum IL-1β levels did not change at 12 h but increased 4-fold after 4 days. Indomethacin had no effect on constitutive NOS. Treatment with PDE inhibitor, TNF-Ab, or HBO₂ significantly reduced serum/tissue TNF-α, IL-1β, NOx, and iNOS expression. Our data show TNF-α plays an early pro-inflammatory role in indomethacin-induced jejunoileitis. Additionally, down-regulation of NOx by PDE inhibitors, TNF-Ab, or HBO₂ suggests that TNF-α modulates iNOS expression.     

Stimulation by nitric oxide of gastric acid secretion in bullfrog fundic mucosa in vitro.

We examined the effect of NO on acid secretion in vitro using isolated preparations of Bullfrog stomach. The bullfrog fundic mucosa was bathed in unbuffered Ringer solution gassed with 100% O2 on the mucosal side and HCO3- Ringer's solution gassed with 95% O2/5% CO2 on the serosal side, and the acid secretion was measured at pH 5.0 using the pH-stat method and by adding 5 mM NaOH. Serosal addition of a NO donor NOR-3 (10(-5) approximately 10(-3) M: (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexnamine) caused an increase of acid secretion in a dose-dependent manner, the effect lasting about 1 hr and reaching a maximal level of 2-fold the basal values. The acid stimulatory effect of NOR-3 was mimicked by another NO donor SNAP (10(-3) mol/L: S-nitroso-O-N-acetyl-penicillamine) and markedly and markedly inhibited by prior administration of cimetidine (10(-5) mol/L) as well as compound 48/80 (the mast cell degranulator). Likewise, the increased acid response to NOR-3 was significantly mitigatd by pretreatment with carboxy-PTIO (a NO scavenger) or superoxide dismutase (SOD), but not by indomethacin or methylene blue (a guanylyl cyclase inhibitor). Neoither L-NAME, L-arginine nor dibutyryl guanosine-3',5'-cyclic monophosphate (dbcGMP) has any effect on the basal acid secretion. Serosal addition of NOR-3 caused a significant increase in the luminal release of histamine, and this response was inhibited by pretreatment with either compound 48/80, carboxy-PTIO or SOD. These results suggest that the NO donor increases gastric acid secretion in the isolated frog stomach in vitro, and this action is mediated by endogenous histamine released from mast cells, the process being cGMP-independent but requiring the presence of superoxide radicals. In addition, it was speculated that the histamine releasing action of NO may be due to peroxynitrite produced by NO and superoxide radicals.     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Modulation of indomethacin-induced gastric injury by spermine and taurine in rats

This study investigated the involvement of neutrophil infiltration, nitric oxide (NO) generation, and oxidative stress in indomethacin-induced ulcer and the possible gastroprotective potentials of spermine and taurine, known for their tissue regenerating and antioxidant effects, respectively. Male Wistar albino rats (180-220 g) were allocated into a normal control group, ulcer control group (received a single dose of indomethacin 40 mg-kg p.o.), and two ulcer groups pretreated with spermine (150 mg-kg p.o. 1 h before ulcer induction) and taurine (250 mg-kg i.p. for three consecutive days before ulcer induction). The animals were killed 6 h after indomethacin administration, and the gastric juice, serum, and mucosal tissue were used for gastric injury evaluation. Both modulators significantly ameliorated the indomethacin-induced gastric lesions in glandular mucosa. Notably, spermine exhibited the most pronounced effect as manifested by great reduction in the gastric ulcer index, normalization of the elevated gastric acidity, and triggering of mucin production. Spermine and taurine were able to decrease the elevated levels of gastric myeloperoxidase, conjugated diene, and serum NO. However, the lowered tissue NO content was markedly elevated only by taurine. The antioxidant action of taurine was illustrated by restoration of the depressed content of glutathione, normalization of the inhibited activities of glutathione reductase, and superoxide dismutase. These results suggest that spermine and taurine confer significant gastroprotection against indomethacin-induced gastric injury with the priority of spermine.     

Role of prostaglandins and nitric oxide in the lipopolysaccharide-induced ACTH and corticosterone response.

This study was designed to determine the role of endogenous prostaglandins (PG) and nitric oxide (NO) in the lipopolysaccharide (LPS)-induced ACTH and corticosterone secretion in conscious rats. LPS (0.5 and 1 mg/kg) given i.p. stimulated the hypothalamic-pituitary-adrenocortical (HPA) activity measured 2 h later. A non-selective cyclooxygenase inhibitor indomethacin (10 mg/kg i.p.), piroxicam (2 mg/kg i.p.), a more potent antagonist of constitutive cyclooxygenase (COX-1) and compound NS-398 (2 mg/kg i.p.), a selective inhibitor of inducible cyclooxygenase (COX-2) given 30 min before LPS (1 mg/kg i.p.) significantly diminished both the LPS-induced ACTH and corticosterone secretion. COX-2 blocker was the most potent inhibitor of ACTH secretion (72.3%). Nomega-nitro-L-arginine methyl ester (L-NAME 2 and 10 mg/kg i.p.), a non-selective nitric oxide synthase (NOS) blocker given 15 min before LPS did not substantially alter plasma ACTH and corticosterone levels 2 h later. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, considerably enhanced ACTH and corticosterone secretion induced by a lower dose (0.5 mg/kg) of LPS and did not significantly alter this secretion after a larger dose (1 mg/kg) of LPS. L-NAME did not markedly affect the indomethacin-induced inhibition of ACTH and corticosterone response. By contrast, aminoguanidine abolished the indomethacin-induced reduction of ACTH and corticosterone secretion after LPS. These results indicate an opposite action of PG generated by cyclooxygenase and NO synthesized by iNOS in the LPS-induced HPA-response.     

Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation

The mechanism of indomethacin-induced gastric ulcer healing by ellagic acid (EA) in experimental mice model is described in our study. Ulcer index (UI) and myeloperoxidase (MPO) activity of the stomach tissues showed maximum ulceration on the third day after indomethacin (18 mg/kg, single dose) administration. Preliminary observation of UI and MPO activity suggests that EA possesses ulcer-healing activity. Other anti-ulcer parameters such as the levels of prostaglandin E(2), cyclooxygenase (COX) 1 and 2 enzymes, anti-inflammatory cytokines [interleukin (IL)-4 and -5], pro-angiogenic factors, e.g. vascular endothelial growth factor, hepatocyte growth factor (HGF), and endothelial growth factor (EGF) were down-regulated by indomethacin. EA (7 mg/kg/day) treatment for 3 days shifted the indomethacin-induced pro-inflammatory biochemical parameters to the healing side. These activities were correlated with the ability of EA to alter the COX-2-dependent healing pathways. The ulcer-healing activity of EA was, however, compromised by pre-administration of the specific COX-2 inhibitor, celecoxib, and NS-398. Taken together, these results suggested that the EA treatment accelerates ulcer healing by inducing IL-4, EGF/HGF levels and enhances COX-2 expression.     

Prostaglandin E prevents indomethacin-induced gastric and intestinal damage through different EP receptor subtypes

Gastrointestinal ulcerogenic effect of indomethacin is causally related with an endogenous prostaglandin (PG) deficiency, yet the detailed mechanism remains unknown. We examined the effect of various PGE analogues specific to EP receptor subtypes on these lesions in rats and mice, and investigated which EP receptor subtype is involved in the protective action of PGE(2). Fasted or non-fasted animals were given indomethacin s.c. at 35 mg/kg for induction of gastric lesions or 10-30 mg/kg for intestinal lesions, and they were killed 4 or 24 h later, respectively. Various EP agonists were given i.v. 10 min before indomethacin. Indomethacin caused hemorrhagic lesions in both the stomach and intestine. Prior administration of 16,16-dimethyl PGE(2) (dmPGE(2)) prevented the development of damage in both tissues, and the effect in the stomach was mimicked by 17-phenyl PGE2 (EP1), while that in the small intestine was reproduced by ONO-NT-012 (EP3) and ONO-AE-329 (EP4). Butaprost (EP2) did not have any effect on either gastric or intestinal lesions induced by indomethacin. Similar to the findings in rats, indomethacin caused gastric and intestinal lesions in both wild-type and knockout mice lacking EP1 or EP3 receptors. However, the protective action of dmPGE(2) in the stomach was observed in wild-type and EP3 receptor knockout mice but not in mice lacking EP1 receptors, while that in the intestine was observed in EP1 knockout as well as wild-type mice but not in the animals lacking EP3 receptors. These results suggest that indomethacin produced damage in the stomach and intestine in a PGE(2)-sensitive manner, and exogenous PGE(2) prevents gastric and intestinal ulcerogenic response to indomethacin through different EP receptor subtypes; the protection in the stomach is mediated by EP1 receptors, while that in the intestine mediated by EP3/EP4 receptors.