Effects of selective and unselective cyclooxygenase inhibitors on prostanoid release from various rat organs

It has been assumed that cyclooxygenase-2 (COX-2) is solely responsible for inflammatory processes. Recently, this view has been challenged because COX-2-selective agents caused a delay of gastric ulcer healing and exacerbation of inflammation in rats. To further characterize organ-specific toxic effects of selective and nonselective COX inhibitors, we assessed the eicosanoid release from different rat organs ex vivo after oral administration of the COX-2-selective inhibitor NS-398 and the unselective COX inhibitors diclofenac, meloxicam, and ketorolac. Prostanoid and leukotriene release from tissue fragments of the stomach, kidney, lung, and brain were determined after ex vivo incubation of tissue fragments in Tyrode's solution for 10 min at 37 degrees C. Ketorolac (0.1, 0.3, and 0.9 mg/kg) inhibited prostanoid release from all organs most potently and led to a significant increase of leukotriene release from the lung. Effects of diclofenac and meloxicam (1, 3, and 9 mg/kg each) were similar for all organs tested. At 9 mg/kg, 6keto-prostaglandin F (PGF)(1alpha) release from gastric mucosa was reduced by 79.1 +/- 11.4 and 87.6 +/- 7.7% and PGE(2) release from rat kidney was inhibited by 60.4 +/- 6.8 and 78.6 +/- 16.6% by diclofenac and meloxicam, respectively. NS-398 did not reduce prostanoid release from the lung. Consistent with the reported constitutive expression of COX-2, prostanoid release from kidney and brain was reduced by 20 to 30%. The release of 6keto-PGF(1alpha) from gastric mucosa was reduced by 34.7 +/- 22.2% at 3 mg/kg and by 86.9 +/- 12.7% at 9 mg/kg. At these doses, NS-398 has been previously shown to be COX-2 selective. Because PGF(1alpha) is the stable breakdown product of PGI(2), these results suggest that COX-2 contributes to PGI(2) synthesis in the rat stomach.     

Interaction of cyclooxygenase isoenzymes, nitric oxide, and afferent neurons in gastric mucosal defense in rats

The cyclooxygenase (COX)-2 inhibitors 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5II)-furanone (DFU) (0.02-2 mg/kg) and N-[2-(cyclohexyloxy)-4-nitrofenyl]-methanesulfonamide (NS-398) (0.01-1 mg/kg), the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (0.05-5 mg/kg), and dexamethasone (1 mg/kg) were studied in rats challenged with intragastric acid (300 mM HCl). All compounds induced severe gastric damage when rats were treated concurrently with the inhibitor of constitutive and inducible nitric-oxide (NO) synthase N(G)-monomethyl-L-arginine methyl ester (L-NAME) (3 or 40 mg/kg). DFU and NS-398 caused significantly less damage in rats receiving the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl)acetamidine (1400W) (0.3 mg/kg). The COX-1 inhibitor SC-560 induced moderate damage in the acid-challenged stomach even without suppression of NO, but damage was aggravated by L-NAME. The COX-3 inhibitor phenacetin (400 mg/kg) did not injure the gastric mucosa despite suppression of NO. Furthermore, DFU, NS-398, SC-560, and dexamethasone caused severe injury in the acid-challenged stomach of rats pretreated with capsaicin to ablate afferent neurons. The mucosal damage induced by the COX-1 inhibitor, the COX-2 inhibitors, and dexamethasone in L-NAME- or capsaicin-treated rats was reversed by coadministration of 16,16-dimethyl-prostaglandin E2 (2 x 8 ng/kg). Gross mucosal damage was paralleled by histology. Our results support the concept that endogenous NO, prostaglandins, and afferent neurons act in concert in the regulation of gastric mucosal integrity. The prostaglandins necessary for mucosal defense in the face of NO suppression, and afferent nerve ablation can be derived either from COX-1 or COX-2. The data do not propose a protective role for a phenacetin-sensitive COX-3. Our findings suggest that not only COX-1 but also COX-2 has important functions in the maintenance of gastric integrity.     

Cyclooxygenase-2-mediated angiogenesis in carrageenin-induced granulation tissue in rats

The possible participation of cyclooxygenase (COX)-2 in angiogenesis in granulation tissue was analyzed using an air pouch-type carrageenin-induced inflammation model in rats. Injection of carrageenin solution into an air pouch induced gradual increases in the pouch fluid volume and granulation tissue weight as well as angiogenesis in granulation tissue. NS-398 (10-100 microg) inhibited all of these parameters in a dose-dependent manner. NS-398 (100 microg), indomethacin (100 microg), and dexamethasone (10 microg) markedly reduced prostaglandin (PG) E(2) levels in the pouch fluid at day 6. NS-398 and indomethacin did not affect protein levels of COX-1 and COX-2 but dexamethasone significantly reduced the level of COX-2 in granulation tissue at day 6. Protein levels of vascular endothelial growth factor (VEGF) in granulation tissue and in the pouch fluid were higher at day 6 than at day 3, and the levels were decreased by treatment with NS-398 (10-100 microg) in a dose-dependent manner. The inhibitory effects of NS-398 (100 microg) were almost the same as those of indomethacin (100 microg). Dexamethasone (10 microg) also reduced VEGF protein levels in granulation tissue at day 6. To clarify the role of PGE(2) in VEGF production, minced granulation tissue obtained 3 days after carrageenin injection from the indomethacin-treated rats was incubated in the presence of various concentrations of PGE(2). It was shown that VEGF mRNA and protein levels in the minced granulation tissue were increased by PGE(2) in a concentration-dependent manner. These findings suggest that COX-2-derived PGE(2) plays a significant role in angiogenesis in the carrageenin-induced granulation tissue through VEGF formation.     

Blockade of central cyclooxygenase (COX) pathways enhances the cannabinoid-induced antinociceptive effects on inflammatory temporomandibular joint (TMJ) nociception

The present study is the first to investigate the participation of central cyclooxygenase (COX) pathways in modulating the antinociceptive effects of intracisternally administered cannabinoid on nociception induced by inflammation of the temporomandibular joint (TMJ) in freely moving rats. Following intra-articular injection of 5% formalin in the TMJ, nociceptive scratching behavior was recorded for nine successive 5-min intervals in Sprague-Dawley rats. Intracisternal injection of 30 microg of WIN 55,212-2, a synthetic non-subtype-selective CB1/2 agonist, administered 20 min prior to formalin injection significantly reduced the number of scratches and duration of scratching induced by formalin compared with the vehicle-treated group. Antinociceptive effect of WIN 55,212-2 was blocked by intracisternal injection of 10 microg of AM251, a CB1 receptor-selective antagonist, but not by AM630, a CB2 receptor-selective antagonist. A 10 microg dose of WIN 55,212-2 that was ineffective in producing antinociception became effective following intracisternal administration of NS-398, a selective COX-2 inhibitor; indomethacin, a non-selective COX 1/2 inhibitor; acetaminophen, a putative COX-3 inhibitor, but not following pretreatment with the selective COX-1 inhibitor, SC-560. The ED(50) value of WIN 55,212-2 in the NS-398-treated group was significantly lower than that in the vehicle-treated group. Importantly, administration of low doses of COX inhibitors alone did not attenuate nociception. These results indicate that inhibition of central COX pathways, presumably via COX-2 inhibition, reduces inflammatory pain by enhancing the cannabinoid-induced antinociceptive effect. Based on our observations, combined administration of cannabinoids with COX inhibitors may hold a therapeutic promise in the treatment of inflammatory TMJ pain.     

Nonsteroidal anti-inflammatory drug-activated gene-1 plays a role in the impairing effects of cyclooxygenase inhibitors on gastric ulcer healing

Nonsteroidal anti-inflammatory drugs (NSAIDs) can impair gastric ulcer healing. This study investigates the involvement of NSAID-activated gene-1 (NAG-1) in ulcer repair impairment by cyclooxygenase (COX) inhibitors. Gastric ulcers were induced in rats by acetic acid. Four days later, animals received daily intragastric indomethacin (nonselective COX-1/COX-2 inhibitor; 1 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (selective COX-1 inhibitor; 2.5 mg/kg), (5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) (selective COX-2 inhibitor; 5 mg/kg), celecoxib (selective COX-2 inhibitor; 1 mg/kg), and valdecoxib (selective COX-2 inhibitor; 1 mg/kg), for 1, 3, or 7 days. Ulcerated tissues were processed to assess: 1) COX-1, COX-2, NAG-1, proliferating cell nuclear antigen (PCNA), and activated caspase-3 expression; 2) ulcer area; and 3) prostaglandin E(2) (PGE(2)) levels. COX-1 expression in ulcerated tissues was decreased, whereas COX-2 expression was enhanced. Ulcer healing was delayed by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. Ulcer PGE(2) levels were decreased by SC-560, DFU, celecoxib, valdecoxib, and indomethacin. NAG-1 was overexpressed in ulcerated tissues and further enhanced by indomethacin, DFU, and SC-560, but not by celecoxib or valdecoxib. PCNA expression in ulcerated areas was reduced by indomethacin, but not by the other test drugs. The expression of activated caspase-3 in ulcers was increased and enhanced further by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. These findings indicate that: 1) COX inhibitors exert differential impairing effects on gastric ulcer healing, through mechanisms unrelated to the inhibition of COX isoforms and prostaglandin production; and 2) NAG-1 induction, followed by activation of proapoptotic pathways, can contribute to the impairing effects of COX inhibitors on ulcer healing.     

Adaptive gastric cytoprotection is mediated by prostaglandin EP1 receptors: a study using rats and knockout mice

Endogenous prostaglandins (PGs) play a central role in adaptive cytoprotection induced in the stomach by mild irritants. In the present study, we used taurocholate (TC) as a mild irritant in both rats and EP-receptor knockout mice, and examined which EP receptor is responsible for the adaptive gastric cytoprotection. Gastric lesions were induced by p.o. administration of HCl/ethanol (60% ethanol in 150 mM HCl). TC (5-20 mM) or PGE2 was administered p.o. 30 min before HCl/ethanol. HCl/ethanol-induced gastric lesions were dose dependently prevented by TC, and the effect at 20 mM was equivalent to that induced by PGE2 at 0.3 mg/kg. The protective effect of TC was significantly attenuated by indomethacin as well as ONO-AE-829, the EP1 antagonist, but not by either NS-398, the selective cyclooxygenase (COX)-2 inhibitor, or chemical ablation of capsaicin-sensitive sensory neurons. Likewise, the protective action of PGE2 was also antagonized by ONO-AE-829 but not chemical deafferentation. TC significantly increased PGE2 contents in the stomach, with or without chemical deafferentation, and this effect was blocked in the presence of indomethacin but not NS-398 or ONO-AE-829. TC increased the mucosal PGE2 contents similarly in both wild-type and knockout mice lacking EP1 or EP3 receptors, yet the protective action of TC against HCl/ethanol was observed in both wild-type and EP3 receptor knockout mice, but not in mice lacking EP1 receptors. The present findings confirmed a role for endogenous PGE2 produced by COX-1 in adaptive gastric cytoprotection and suggested that this action is mediated by activation of EP1-receptors but not associated with capsaicin-sensitive afferent neurons.     

Peroxisome proliferator-activated receptor gamma mediates protection against cyclooxygenase-2-induced gut dysfunction in a rodent model of mesenteric ischemia/reperfusion

Cyclooxygenase (COX)-2 has been identified as an important mediator elaborated during ischemia/reperfusion, with pro- and anti-inflammatory properties having been reported. As the role of COX-2 in the small intestine remains unclear, we hypothesized that COX-2 expression would mediate mesenteric ischemia/reperfusion-induced gut injury, inflammation, and impaired transit and that these deleterious effects could be reversed by the selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphanamide (NS-398). Additionally, we sought to determine the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in mediating protection by NS-398 in this model. Rats underwent sham surgery or were pretreated with NS-398 (3, 10, or 30 mg/kg) intraperitoneally 1 h before 60 min of superior mesenteric artery occlusion and 30 min to 6 h of reperfusion. In some experiments, NS-398 (30 mg/kg) was administered postischemia. Ileum was harvested for COX-2 mRNA and protein, PGE2, myeloperoxidase (inflammation), histology (injury), intestinal transit and PPARgamma protein expression, and DNA-binding activity. COX-2 expression and PGE2 production increased after mesenteric ischemia/reperfusion and were associated with gut inflammation, injury, and impaired transit. Inhibition of COX-2 by NS-398 (30 mg/kg, but not 3 or 10 mg/kg) not only reversed the deleterious effects of COX-2, but additionally induced expression and nuclear translocation of PPARgamma. NS-398 given postischemia was equally protective. In conclusion, COX-2 may function as a proinflammatory mediator in a rodent model of mesenteric ischemia/reperfusion. Reversal of gut inflammation, injury, and impaired transit by high-dose NS-398 is associated with PPAR activation, suggesting a potential role for PPAR-gamma in shock-induced gut protection.     

Roles of endogenous prostaglandins and cyclooxygenase isozymes in healing of indomethacin-induced small intestinal lesions in rats

The role of prostaglandins (PGs)/cyclooxygenase (COX) in the healing of indomethacin-induced small intestinal ulcers was examined in rats. Animals were given indomethacin (10 mg/kg s.c.) and killed 1, 2, 3, 5, and 7 days later. Indomethacin (2 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560; COX-1 inhibitor; 3 mg/kg), and rofecoxib (COX-2 inhibitor; 3 mg/kg) were given p.o. once daily for 6 days, during the first 3 days or last 3 days of the experimental period. All COX inhibitors given for 6 days significantly impaired the healing of these ulcers. Healing was also impaired by rofecoxib given for the first 3 days or by SC560 given for the last 3 days. The expression of COX-2 mRNA in the intestine was up-regulated after ulceration, persisting for 3 days and dissipating thereafter. Mucosal PGE2 contents decreased within 3 h after ulceration, recovered 24 h later, and increased above normal 1 approximately 3 days later. The PGE2 content at 4 days after ulceration was decreased by rofecoxib but not SC560, whereas that at 7 days was suppressed by SC560 but not rofecoxib. Vascular content in the ulcerated mucosa decreased when the healing was impaired by COX inhibitors. The deleterious effect of indomethacin on healing was mimicked by a prostacyclin E receptor (EP) 4 antagonist and reversed by coadministration of PGE2 as well as an EP4 agonist. In conclusion, endogenous PGs play a role in the healing of intestinal ulcers through EP4 receptors, yet the COX isozyme involved differs depending on the stage of healing; COX-2 in the early stage and COX-1 in the late stage.     

Cyclooxygenase-2-mediated regulation of Kupffer cell interleukin-6 production following trauma-hemorrhage and subsequent sepsis

Studies indicate that trauma-hemorrhage results in activation of Kupffer cells to release inflammatory mediators and it leads to immunosuppression and increased susceptibility to subsequent sepsis. The cyclooxygenase (COX) product prostaglandin (PG) E2 appears to be central to this process, however, non-selective inhibition of COX activity with non-steroidal anti-inflammatory agents that block both the constitutive (COX-1) and inducible (COX-2) isoforms of cyclooxygenase has not yielded promising results in trauma patients. Nonetheless, it remains unknown whether selective inhibition of COX-2 activity has any salutary effect following trauma-hemorrhage and subsequent induction of sepsis. To study this, male C3H/HeN mice were subjected to laparotomy (i.e., soft-tissue trauma) and hemorrhagic shock (35 +/- 5 mmHg for 90 min, then resuscitated) or to sham operation. Twenty-four hours later, the mice were subjected to sepsis by cecal ligation and puncture (CLP) or to sham CLP. The mice were treated with the COX-2 inhibitor NS-398 (10 mg/kg body weight, intraperitoneally) or vehicle immediately after trauma-hemorrhage or sham operation, 12 h thereafter, and following CLP or sham CLP. At 5 h after CLP, plasma PGE2, Interleukin-(IL) 6, and TNF-alpha levels were determined along with Kupffer cell IL-6 and TNF-alpha production in vitro. NS-398 treatment markedly suppressed the elevation in plasma PGE2 levels following CLP. The increase in plasma IL-6 levels after CLP were also significantly attenuated by NS-398 treatment. In vitro Kupffer cell IL-6 production after CLP was significantly reduced by in vivo NS-398 treatment. However, NS-398 had no effect on TNF-alpha levels, in vivo and in vitro. These findings indicate that activation of COX-2 following trauma-hemorrhage and subsequent sepsis up-regulates Kupffer cell IL-6 production. Thus, selective inhibition of COX-2 activity may reduce the deleterious consequences of sepsis under such conditions.     

Role of cyclooxygenase (COX)-1 and COX-2 inhibition in nonsteroidal anti-inflammatory drug-induced intestinal damage in rats: relation to various pathogenic events

We recently reported that cyclooxygenase (COX)-2 expression was up-regulated in the rat small intestine after administration of indomethacin, and this may be a key to nonsteroidal anti-inflammatory drug (NSAID)-induced intestinal damage. In the present study, we investigated the effect of inhibiting COX-1 or COX-2 on various intestinal events occurring in association with NSAID-induced intestinal damage. Rats without fasting were treated with indomethacin, SC-560 (a selective COX-1 inhibitor), rofecoxib (a selective COX-2 inhibitor), or SC-560 plus rofecoxib, and the following parameters were examined in the small intestine: the lesion score, the enterobacterial number, myeloperoxidase (MPO) and inducible nitric-oxide synthase (iNOS) activity, and intestinal motility. Indomethacin decreased mucosal prostaglandin (PG)E2 content and caused damage in the intestine within 24 h, accompanied by an increase in intestinal contractility, bacterial numbers, and MPO as well as iNOS activity, together with the up-regulation of COX-2 and iNOS mRNA expression. Neither SC-560 nor rofecoxib alone caused intestinal damage, but their combined administration produced lesions. SC-560, but not rofecoxib, caused intestinal hypermotility, bacterial invasion, and COX-2 as well as iNOS mRNA expression, yet the iNOS and MPO activity was increased only when rofecoxib was also administered. Although SC-560 inhibited the PG production, the level of PGE2 was restored 6 h later, in a rofecoxib-dependent manner. We conclude that inhibition of COX-1, despite causing intestinal hypermotility, bacterial invasion, and iNOS expression, up-regulates the expression of COX-2, and the PGE2 produced by COX-2 counteracts deleterious events, and maintains the mucosal integrity. This sequence of events explains why intestinal damage occurs only when both COX-1 and COX-2 are inhibited.     

Inhibition of cyclooxygenase-2 decreases DNA synthesis induced by platelet-derived growth factor in Swiss 3T3 fibroblasts

NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide], a selective inhibitor of cyclooxygenase-2 (COX-2), inhibited proliferation induced by platelet-derived growth factor (PDGF) in Swiss 3T3 fibroblasts. The effect of NS-398 was found to be concentration-dependent. The half-maximal effect occurred at approximately 0.1 microM. NS-398 decreased mitogenesis at subsaturating PDGF concentrations and the inhibitory effect of NS-398 was overcome by increasing PDGF concentration. SC-236, another COX-2 selective inhibitor, also inhibited PDGF-induced proliferation. In contrast, two selective COX-1 inhibitors, valeryl salicylate and ketorolac, had no significant inhibitory effect on PDGF-stimulated DNA synthesis. The inhibition was obtained when NS-398 was added during the first hour after PDGF addition. At 1 h, PDGF induced COX-2 protein and prostaglandin (PG)E(2) synthesis, and NS-398 blocked the synthesis of PGE(2). The inhibitory effect of NS-398 on PDGF-stimulated DNA synthesis was counteracted by 280 nM PGE(2). The antimitogenic action of NS-398 and SC-236 suggests that selective inhibition of COX-2 may produce antiproliferative effects with substantial safety advantages over nonselective COX inhibitors.     

Endogenous nitric oxide enhances prostaglandin production in a model of renal inflammation.

The interaction between nitric oxide (NO) and cyclooxygenase (COX) was studied in a rabbit model of renal inflammation, the ureteral obstructed hydronephrotic kidney (HNK). Ex vivo perfusion of the HNK but not the control kidney (e.g., unobstructed contralateral kidney, CLK), led to a time-dependent release of nitrite (NO2-), a breakdown product of NO. Stimulation of the HNK with bradykinin (BK) evoked a time-dependent increase in prostaglandin E2 (PGE2) production. NG-monomethyl-L-arginine (L-NMMA), which blocks the activity of both constitutive and inducible nitric oxide synthase (cNOS and iNOS), aminoguanidine, a recently described selective iNOS inhibitor, dexamethasone, or cycloheximide abolished the release of NO2- and attenuated the exaggerated BK-induced PGE2 production. This supports the existence of iNOS and COX-2 in the HNK. In the CLK, BK elicited release of both NO2- and PGE2 but this did not augment with time. L-NMMA but not aminoguanidine, dexamethasone, or cycloheximide attenuated NO2- and PGE2 release indicative of the presence of constitutive but not inducible NOS or COX. The current study suggests that the endogenous release of NO from cNOS in the CLK activates a constitutive COX resulting in optimal PGE2 release by BK. In addition, in the HNK, NO release from iNOS activates the induced COX resulting in markedly increased release of proinflammatory prostaglandin. The broader implication of this study is that the cyclooxygenase isozymes are potential receptor targets for nitric oxide.     

Significance of cyclooxygenase-2 induced via p38 mitogen-activated protein kinase in mechanical stimulus-induced peritoneal adhesion in mice

Postoperative peritoneal adhesion represents a major complication of surgery, but the molecular mechanism underlying pathogenesis of adhesion is not fully understood. The present study investigated the roles of cyclooxygenase (COX)-1 and COX-2 in peritoneal adhesion induced by scraping the surface of the cecum and abdominal wall in mice. Slight, but macroscopically observable, peritoneal adhesion was induced even on day 1, and the extent of adhesion reached a maximum on day 7 and beyond. COX-1 mRNA was constitutively expressed in the intact cecum, and its expression level was not altered after the mechanical stimulus. In contrast, expression of the COX-2 gene was markedly increased after the stimulus, and maximum expression was observed on days 3 to 7. Mofezolac, a specific COX-1 inhibitor, had no effect on peritoneal adhesion at 30 mg/kg and had only marginal effects on prostaglandin (PG)E2 levels in the cecum or peritoneal fluid. On the other hand, two highly selective inhibitors for COX-2, NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide) and CAY10404 [3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole], dose-dependently inhibited both adhesion formation and the increase in PGE2 levels (3-30 mg/kg). The effects of NS-398 were eliminated when PGE2 or (R)-butaprost was administered exogenously. A COX-2 antisense oligonucleotide also attenuated adhesion formation. Activation of p38 mitogen-activated protein (MAP) kinase was observed in the traumatized cecum, and an MAP kinase inhibitor, SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole], inhibited adhesion formation (54% inhibition at 15 microM) and also reduced the COX-2 mRNA level and PGE2 levels. In conclusion, COX-2, but not COX-1, plays a significant role in mechanical stimulus-induced peritoneal formation in the mouse cecum.     

环氧酶2介导鞘内注射血小板活化因子诱发的触觉痛敏和热痛敏

目的：研究环氧酶（COX）及前列腺素（PGs）在鞘内注射血小板活化因子（PAF）诱导大鼠痛敏机制中的作用。方法：鞘内置管的雄性SD大鼠60只,随机分为脊液组（注射人工脑脊液）,PAF组（鞘内注射PAF）,盐水组（注射盐水）,SC-560组（注射PAF及SC-560）,NS-398组（注射PAF及NS-398）,吲哚美辛组（注射PAF及吲哚美辛）各10只。各组注射前及鞘内给药后均检测大鼠机械缩爪阈值和热缩爪潜伏期,每15min测1次,5h后RT-PCR和放射免疫分析检测腰段脊髓COX-1,COX-2及COX-3的表达和PGE2的含量。结果：鞘内注射PAF后的PAF组迅速诱发大鼠触觉痛敏和热痛敏,并诱导脊髓COX-2表达增强和PGE2含量升高;与PAF组比较,NS-398组和吲哚美辛组能明显抑制PAF诱发的痛敏和脊髓PGE2含量升高。结论：鞘内注射PAF可诱发大鼠触觉异常痛敏和热痛敏;脊髓COX-2的激活和表达增强以及PGE2的产生可能参与其机制。     

Role of prostaglandins in maintaining gastric mucus-cell permeability against acid exposure

Regulation of gastric epithelial permeability is important in the protection of the gastric mucosa from secreted acid. However, the mechanism(s) for this regulation in gastric mucus cells remains unknown. In this study, we evaluated gastric epithelial-cell permeability in response to acid exposure by monitoring trans-epithelial electrical resistance (TEER) and paracellular permeability with carbon 14-labeled mannitol. We also examined the role of prostaglandins on gastric epithelial permeability. Rat gastric epithelial cells (RGM-1) were plated on 8-microm-pore tissue-culture inserts. Cells were exposed to solutions of differing pH (3-7.4), with and without the nonsteroidal antiinflammatory drug (NSAID) indomethacin (10(-7) mol/L), for 60 to 120 minutes. Transepithelial permeability was measured on the basis of TEER and the diffusion rate of [(14)C]mannitol. Prostaglandin E(2) (PGE(2)) was administered in some experiments with NSAIDs. After acid exposure (pH 3.0-5.0), TEER rapidly and significantly increased, peaking in 5 minutes. Diffusion of [(14)C]mannitol was blocked during the period when TEER increased. Pretreatment with the cyclooxygenase (COX) inhibitor indomethacin blocked the rapid acid-induced increase in TEER. A specific COX-2 inhibitor had no effect on this rapid increase in TEER. The blockade by indomethacin was eliminated by the addition of PGE(2). These findings suggest that when gastric-surface mucus cells are exposed to acid, gastric epithelial permeability decreases rapidly to inhibit acid back-diffusion. Prostaglandins play an important role in this protective response to acid exposure. COX inhibitors such as indomethacin may inhibit the regulation of epithelial permeability by reducing the concentration of PGE(2).     

Blocking prostaglandin E2 after trauma attenuates pro-inflammatory cytokines and improves survival

Major injury leads to impaired immune responses and increases the risk of infectious complications. Following trauma, increased prostaglandin E2 (PGE2) levels may be important in immunodysregulation. We hypothesized that blocking PGE2 with NS-398, a selective COX-2 inhibitor, during the first 24 h after injury may modify the immune response and protect the host from a subsequent septic challenge. BALB/c mice were given NS-398 (10 mg/kg) immediately after injury, at 12, and at 24 h after sham injury or trauma (femur fracture and 40% hemorrhage). On day 7 after injury, splenic macrophages were evaluated for cytokine production and COX-2 mRNA. In a separate study mice were injured, then given 3 doses of NS-398. After 7 days, cecal ligation and puncture was performed and mice were followed for survival. Traumatized mice given NS-398 had a significant survival advantage compared with trauma mice alone (P < 0.001). Macrophages from traumatized mice showed increased COX-2 mRNA and proinflammatory cytokines compared with controls (P < 0.05), whereas treatment of injured mice with NS-398 significantly decreased proinflammatory cytokine production (P < 0.05) and COX-2 mRNA. Therefore NS-398 given within 24 h of injury suppressed PGE2 through inhibition of cyclooxygenase, in addition to decreasing proinflammatory cytokines, and providing a survival advantage to the host.     

Neuroinflammatory role of prostaglandins during experimental meningitis: evidence suggestive of an in vivo relationship between nitric oxide and prostaglandins

Nitric oxide (NO) and prostaglandins are inflammatory mediators produced during meningitis. The purpose of the present study was to pharmacologically inhibit cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) to 1) explore the prostaglandin contribution to blood-cerebrospinal fluid barrier permeability alterations and 2) elucidate the in vivo concentration relationship between prostaglandin E2 (PGE2) and NO during experimental meningitis. Intracisternal injection of lipopolysaccharides (LPSs, 200 microg) induced neuroinflammation. Rats were dosed with nimesulide (COX-2 inhibitor), aminoguanidine (iNOS inhibitor), or vehicle. Evans blue was used to assess blood-cerebrospinal fluid barrier permeability. Meningeal NO and cerebrospinal fluid PGE2 were assayed using conventional methods. (Results are expressed as mean +/- S.E.M. of 5-9 rats/group.) Nimesulide failed to prevent blood-cerebrospinal fluid barrier disruption [cerebrospinal fluid Evans blue (micrograms per milliliter): control, 0.22 +/- 0.22*; LPS, 11.58 +/- 0.66; LPS + nimesulide, 10.58 +/- 0.86; *p < 0.05; ANOVA]. Although nimesulide decreased PGE2 (picograms per microliter; p < 0.01) in LPS + nimesulide rats (13.9 +/- 1.96) versus LPS + vehicle (73.8 +/- 12.4), meningeal NO production (picomoles/30 min/10(6) cells; p < 0.01) increased unexpectedly in LPS + nimesulide rats (439 +/- 47) versus LPS + vehicle rats (211 +/- 31). In contrast, aminoguanidine inhibited meningeal NO (picomoles/30 min/10(6) cells; p < 0.005) in LPS + aminoguanidine (111 +/- 20) versus LPS (337 +/- 48) but had no effects (p > 0.05) on PGE2. The in vivo relationship between PGE2 and NO was mathematically described by a biphasic, bell-shaped curve (r2 = 0.42; n = 27 rats; p < 0.0001). Based on these results, inhibition of prostaglandin synthesis not only fails to prevent blood-cerebrospinal fluid barrier disruption during neuroinflammation and but also promotes increased meningeal NO production. The in vivo concentration relationship between PGE2 and NO is biphasic, suggesting that inhibition of COX-2 alone may promote NO toxicity through enhanced NO synthesis.     

选择性COX-2抑制剂NS-398对人胃腺癌细胞增殖和凋亡的影响

目的通过COX-2抑制剂NS-398对胃癌培养细胞系SGC7901增殖及凋亡影响的研究证实非甾体类抗炎药（NSAID。）可在体外抑制胃癌细胞的生长。方法应用MTT法检测NS-398对胃癌细胞SGC7901细胞增殖的影响；应用流式细胞仪、透射电镜检测NS-398对胃癌细胞SGC7901细胞凋亡的影响。结果MTT结果显示NS-398对胃癌细胞均抑制作用，并随剂量的增加及作用时间延长，其抑制作用要明显增加；透射电镜观察发现未经NS-398作用的胃癌培养细胞的细胞核和细胞器亚微结构清晰，核模完整，而经不同浓度的舒林酸和尼美舒利作用后细胞呈现典型的凋亡形态学变化，并可见凋亡小体形成；流式细胞仪结果显示不同浓度的NS-398作用SGC7901细胞72h后，均可出现亚G1峰，呈剂量依赖关系诱导胃癌细胞凋亡，并呈浓度依赖性改变的细胞周期分布，一方面增高G0／G期细胞比例，另一方面降低S期和G2／M期细胞比例。结论NS-398可抑制胃癌SGC7901细胞的增殖；NS-398促进胃癌SGC7901细胞的凋亡；NS-398抑制胃癌的机制可能是通过抑制胃癌细胞COX-2的活性，从而抑制前列腺素E2的释放而抑制胃癌细胞的生长。