Loss of estrogen-mediated immunoprotection underlies female gender bias in experimental Crohn's-like ileitis.

The incidence and severity of Crohn's disease (CD) are increased in female patients. Using SAMP1/YitFc (SAMP) mice, a spontaneous model of chronic intestinal inflammation that displays histologic and pathogenic similarities to human CD, we investigated the potential mechanism(s) contributing to sex differences observed in CD. Similar to gender differences observed in CD patients, SAMP female (SAMP-F) mice displayed an earlier onset and more severe ileitis compared with SAMP male (SAMP-M) mice. Furthermore, T-regulatory cells (Tregs) from gut-associated lymphoid tissue (GALT) of SAMP-F mice were reduced in frequency and impaired in their in vitro and in vivo suppressive functions compared with that of SAMP-M mice. Given the interaction between sex hormones and Treg function, we investigated the possible role of estrogen (E2) in SAMP ileitis. SAMP-M mice responded to exogenous E2 administration by expanding Treg frequency and reducing ileal inflammation, whereas SAMP-F mice were resistant. Conventional T cells and Tregs responded differentially to estrogen signaling, leading to distinct immunoprotective effects mediated by distinct estrogen receptor (ER) isoforms. These mechanisms were impaired in T cells from SAMP-F mice. Thus, hormone signaling influences the expansion and function of GALT Tregs in an ER-dependent manner and contributes to gender-based differences in experimental CD.

Dietary antioxidants protect gut epithelial cells from oxidant-induced apoptosis.

BACKGROUND: The potential of ascorbic acid and two botanical decoctions, green tea and cat's claw, to limit cell death in response to oxidants were evaluated in vitro. METHODS: Cultured human gastric epithelial cells (AGS) or murine small intestinal epithelial cells (IEC-18) were exposed to oxidants - DPPH (3 microM), H2O2 (50 microM), peroxynitrite (300 microM) - followed by incubation for 24 hours, with antioxidants (10 microg/ml) administered as a 1 hour pretreatment. Cell number (MTT assay) and death via apoptosis or necrosis (ELISA, LDH release) was determined. The direct interactions between antioxidants and DPPH (100 microM) or H2O2 (50 microM) were evaluated by spectroscopy. RESULTS: The decoctions did not interact with H2O2, but quenched DPPH although less effectively than vitamin C. In contrast, vitamin C was significantly less effective in protecting human gastric epithelial cells (AGS) from apoptosis induced by DPPH, peroxynitrite and H2O2 (P < 0.001). Green tea and cat's claw were equally protective against peroxynitrite and H2O2, but green tea was more effective than cat's claw in reducing DPPH-induced apoptosis (P < 0.01). Necrotic cell death was marginally evident at these low concentrations of peroxynitrite and H2O2, and was attenuated both by cat's claw and green tea (P < 0.01). In IEC-18 cells, all antioxidants were equally effective as anti-apoptotic agents. CONCLUSIONS: These results indicate that dietary antioxidants can limit epithelial cell death in response to oxidant stress. In the case of green tea and cat's claw, the cytoprotective response exceed their inherent ability to interact with the injurious oxidant, suggestive of actions on intracellular pathways regulating cell death.

NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2.

BACKGROUND & AIMS: Selective cyclooxygenase (COX)-2 inhibitors produce less gastric damage than conventional nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that NSAIDs cause damage by inhibiting COX-1. We tested this hypothesis in rats by using a selective COX-1 inhibitor (SC-560). METHODS: The effects of SC-560, celecoxib (selective COX-2 inhibitor), or a combination of both inhibitors on gastric damage and prostaglandin synthesis were determined. Selectivity of the drugs for COX-1 vs. COX-2 was assessed in the carrageenan-airpouch model. A COX-1-preferential inhibitor, ketorolac, was also evaluated. The effects of these inhibitors on leukocyte adherence to vascular endothelium and on gastric blood flow were assessed. RESULTS: SC-560 markedly reduced gastric prostaglandin synthesis and platelet COX-1 activity, but spared COX-2 and did not cause gastric damage. Celecoxib did not affect gastric prostaglandin E(2) synthesis and did not cause gastric damage. However, the combination of SC-560 and celecoxib invariably caused hemorrhagic erosion formation, comparable to that seen with indomethacin. Ketorolac caused damage only at doses that inhibited both COX isoforms, or when given with a COX-2 inhibitor. Celecoxib, but not SC-560, significantly increased leukocyte adherence, whereas SC-560, but not celecoxib, reduced gastric blood flow. CONCLUSIONS: Inhibition of both COX-1 and COX-2 is required for NSAID-induced gastric injury in the rat.

Phosphodiesterase inhibitors prevent NSAID enteropathy independently of effects on TNF-alpha release.

Although the ability of nonsteriodal anti-inflammatory drugs (NSAIDs) to injure the small intestine has been well established in humans and animals, the mechanism involved in this type of injury has yet to be elucidated. The cytokine tumor necrosis factor-alpha (TNF-alpha) has recently been demonstrated to play a critical role in the pathogenesis of NSAID-induced gastric damage. We therefore assessed the possibility that TNF-alpha is similarly involved in the pathogenesis of NSAID-induced small intestinal injury. Administration of multiple doses (n = 4) of diclofenac, but not a single dose, resulted in profound macroscopic damage in the intestine and significantly increased levels of TNF-alpha in intestinal tissue and bile. Pretreatment of rats with a phosphodiesterase inhibitor, pentoxifylline, theophylline, or rolipram, significantly attenuated the macroscopic intestinal ulceration produced by diclofenac administration. However, inhibition of TNF-alpha release with thalidomide or immunoneutralization with a polyclonal antibody directed against TNF-alpha failed to afford any protection. These results suggest that the cytokine TNF-alpha does not play a critical role in NSAID-induced small intestinal injury. Therefore, phosphodiesterase inhibitors mediate their protective effect through a mechanism independent of TNF-alpha synthesis inhibition.

Selective inhibitors of cyclooxygenase-2: are they really effective, selective, and GI-safe?

Selective inhibitors of the "inducible" isoform of cyclooxygenase (COX-2) have been suggested to be effective analgesic and anti-inflammatory drugs while sparing the gastrointestinal (GI) tract of injury. There is some experimental and early clinical evidence to support this hypothesis. However, some important questions remain regarding the utility of selective COX-2 inhibitors. For example, estimates of the selectivity of COX-2 inhibitors based on in vitro studies are likely to be poor predictors of selectivity in vivo. Efficacy with selective blockade of COX-2 may be inferior to that achieved with combined inhibition of COX-1 and COX-2. Furthermore, in situations in which there is inflammation or ulceration in the GI tract, COX-2 produces prostaglandins that are essential for repair. In these circumstances, inhibition of COX-2 leads to delay of ulcer healing and exacerbation of inflammation. Some caution should therefore be exercised before the theory is fully accepted that selective COX-2 inhibitors are effective anti-inflammatory drugs that spare the GI tract of injury.

Induction of cyclooxygenase 1 and 2 in the rat stomach during endotoxemia: role in resistance to damage.

BACKGROUND & AIMS: Prostaglandins and nitric oxide are key mediators of gastric mucosal defense. Endotoxemia alters gastric resistance to damage, but little is known of the effects of chronic endotoxemia on the expression of prostaglandin and nitric oxide synthases (NOSs). The effects of short- vs. long-term administration of endotoxin on gastric resistance to damage and on expression of NOS and prostaglandin synthesis were compared. METHODS: Rats were treated with short- or long-term bacterial endotoxin, after which susceptibility to ethanol-induced damage was assessed. The effects of various inhibitors of prostaglandin and NOS were examined. Expression of gastric NOS and cyclooxygenase (COX) messenger RNA (mRNA) were examined. RESULTS: Repeated administration of endotoxin increased gastric resistance to ethanol- but not indomethacin-induced injury. Indomethacin, but not a highly selective COX-2 inhibitor or an inducible NOS inhibitor, abolished long-term endotoxin-induced gastric resistance to injury. Expression of mRNA for both COX-1 and -2, but not for endothelial or inducible NOS, were significantly increased after long-term endotoxin administration. CONCLUSIONS: Repeated exposure to endotoxin resulted in increased resistance of the gastric mucosa to injury through a prostaglandin-dependent pathway. These prostaglandins were produced via COX-1, which like COX-2, is induced by endotoxin administration.

Nonsteroidal anti-inflammatory drug enteropathy in rats: role of permeability, bacteria, and enterohepatic circulation.

BACKGROUND & AIMS: The pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage remains poorly understood. The aim of this study was to examine the relative importance of the three suggested components of the pathogenesis of NSAID enteropathy, namely, epithelial permeability, enteric bacterial numbers, and enterohepatic recirculation, using an NSAID derivative (nitrofenac) that does not cause small intestinal damage. METHODS: Rats were given diclofenac or nitrofenac at 12-hour intervals. Epithelial permeability to [51Cr]-ethylenediaminetetraacetic acid and enteric bacterial numbers were determined after 1-4 doses of the drugs. Serum levels and biliary excretion of the two drugs were determined by high-performance liquid chromatography. RESULTS: Diclofenac caused a progressive increase in epithelial permeability, marked increases in enteric gram-negative bacterial numbers, and frank intestinal ulceration. Nitrofenac caused similar changes in intestinal permeability after a single dose but no further increase with repeated administration. Moreover, nitrofenac had no effect on enteric bacterial numbers and did not cause frank ulceration. Unlike diclofenac, nitrofenac did not undergo extensive enterohepatic recirculation. Two other NSAIDs that do not undergo enterohepatic recirculation (nabumetone and aspirin) also did not modify enteric bacterial numbers or cause intestinal ulceration. CONCLUSIONS: Enterohepatic recirculation of NSAIDs is of paramount importance in the pathogenesis of enteropathy caused by these drugs, whereas suppression of prostaglandin synthesis is relatively unimportant.

Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2.

Cyclooxygenase type 1 is constitutively expressed and accounts for synthesis of prostaglandins in the normal gastrointestinal tract. Cyclooxygenase-2 is expressed at sites of inflammation. Selective inhibitors of cyclooxygenase-2 have been suggested to spare gastrointestinal prostaglandin synthesis, and therefore lack the ulcerogenic effects associated with standard nonsteroidal antiinflammatory drugs. However, the effects of cyclooxygenase-2 inhibitors on inflamed gastrointestinal mucosa have not been examined. We examined cyclooxygenase-2 mRNA and protein expression before and after induction of colitis in the rat, the contribution of cyclooxygenase-2 to colonic prostaglandin synthesis during colitis and the effects of selective inhibitors of cyclooxygenase-2 on colonic injury in this model. Cyclooxygenase-2 mRNA expression increased three to sixfold during the period 24 h to 1 wk after induction of colitis, with marked increases in cyclooxygenase-2 protein expression in the lamina propria and muscularis of the colon during colitis. Cyclooxygenase-1 expression (mRNA and protein) was not affected by the induction of colitis. The prostaglandins produced during colitis were largely derived from cyclooxygenase-2. Treatment with selective cyclooxygenase-2 inhibitors resulted in exacerbation of colitis, with perforation occurring when the compounds were administered for a week. These studies demonstrate that suppression of cyclooxygenase-2 can result in exacerbation of inflammation-associated colonic injury.

Markedly reduced intestinal toxicity of a diclofenac derivative.

Addition of a nitroxybutyl moiety to diclofenac greatly reduces its damaging effects on the gastric mucosa without altering its ability to suppress prostaglandin synthesis and exert anti-inflammatory actions. The present study was performed in order to determine if this derivative of diclofenac, called nitrofenac, would also have less toxicity in the small and large intestine when administered repeatedly over a 1-2 week period. Healthy rats were given equimolar doses of diclofenac (10 mg/kg) or nitrofenac (15 mg/kg) twice daily for up to two weeks. All 10 rats receiving diclofenac died prior to completion of the study, exhibiting massive small intestinal ulceration and perforation. No deaths were observed in the rats treated with nitrofenac, and the only small intestinal abnormality observed was diffuse hyperemia. As nonsteroidal anti-inflammatory drugs have been shown to exacerbate colitis, we compared the effects of twice daily treatment with diclofenac (1-10 mg/kg) or nitrofenac (1.5-15 mg/kg) for 1 week in rats in which colitis had been induced with trinitrobenzene sulfonic acid. Diclofenac administration resulted in mortality which increased dose-dependently (e.g. 86% at 5 mg/kg) and was associated with perforation of the colon. Mortality was not observed with nitrofenac at doses of 1.5 or 7.5 mg/kg, while at 15 mg/kg the mortality rate was 33%. None of the doses of nitrofenac significantly augmented colonic injury or granulocyte infiltration (measured by myeloperoxidase activity). Suppression of colonic prostaglandin E2 synthesis was comparable with equimolar doses of diclofenac and nitrofenace. These studies demonstrate that nitrofenac has markedly reduced intestinal toxicity in healthy and colitic rats when compared to diclofenac.

Nitric oxide-releasing non-steroidal anti-inflammatory drugs: a novel approach for reducing gastrointestinal toxicity.

The use of non-steroidal anti-inflammatory drugs (NSAID) for treatment of inflammatory conditions is significantly limited by the untoward effects of these compounds on the gastrointestinal tract. While the pathogenesis of 'NSAID-gastropathy' is not completely understood, there is good evidence that the process is directly linked to suppression of prostaglandin synthesis and possibly to neutrophil adherence to the vascular endothelium. Pretreatment of rats with a nitric oxide (NO) donor (sodium nitroprusside) was found to significantly reduce the extent of gastric injury induced by flurbiprofen. We therefore tested the effects of a novel derivative of flurbiprofen. This compound contains a moiety similar to the NO-releasing moieties found in many organic nitrates. This compound suppressed gastric prostaglandin synthesis as effectively as flurbiprofen, but caused significantly less haemorrhagic damage. The compound was also found not to induce small intestinal injury. Since the compound was found to exert anti-inflammatory effects comparable with flurbiprofen, NO-releasing NSAID may represent a novel class of drugs with markedly reduced gastrointestinal toxicity.