Increased susceptibility of ethanol-treated gastric mucosa to naproxen and its inhibition by DA-9601

PO Box 2345, Beijing 100023, ChinaWorld Gastroenterol 2005: 11(47): 7450-7456Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.comELSEVIER9 2005 The w]G Press and Elsevier Inc. All rights reserved.· ASIC ESEARCH·Increased susceptibility of ethanol-treated gastric mucosa tonaproxen and its inhibition by DA-9601 an Artemisia asiaticaextractTae Young Oh, Gook Jun Ahn, Seul Min Choi, Byoung Ok Ahn, Won Bae KimTae Young Oh, Gook Jun Ahn, Seul Min Choi, Byoung Ok Key words: DA-9601; Alcohol; Naproxen; Gastric dam-Ahn, Won Bae Kim, Research Laboratories, Dong-A Pharm Co., age; Malondialdehyde; Prostaglandin Ez; GlutathioneLtd, 47-5, Sanggal, Kiheung, Yongin, Kyunggi,449-905, South Myeloperoxidase; NSAIDsKoreaSupported by the National Ministry of Health and WelfareOh TY, Ahn G, Choi SM, Ahn BO, Kim WB IncreaseCo-first-authors: Tae Young Oh and Gook Jun AhnCorrespondence to: Gook Jun Ahn, Research Laboratories, Ceptibility of ethanol-treated gastric mucosa to naproxenDong-A Pharm. Co, Ltd, 47-5, Sanggal, Kiheung, Yongin, and its inhibition by DA-9601, an Artemisia asiatica extract.Kyunggi, 449-905, South Korea. ahnsnu2@donga. co krWorld Gastroenterol2005;11(47):7450-7456Telephone:+82-31-280-1359Fax:+82-31-282-8564http://www.wignet.com/1007-9327/11/7450.aspReceived: 2005-04-07Accepted: 2005-07-05INTRODUCTIONAbstractThe prevalence of gastrointestinal (Gi) disease isAIM: To examine the effect of DA-9601, a new increasing in subjects aged 65 years and above. Amonggastroprotective agent, on the vulnerability of ethanolthem, gastroesophageal reflux disease(GERD) and peptictreated rats stomach to naproxen(NAp)cer arethe most important and common GI disorders"Peptic ulcer is one of the most common gastrointestinalMETHODS: Male Sprague-Dawley rats were pretreated diseases with 4-5% prevalence in the human societywith 1 mL of 50% ethanol twice a day for 5 d and then Although it was speculated that food or stress was a mainNAP(50 mg/kg)was administered. DA-9601 was admin causative factor in the early 20"century, genetic andstered 1 h before NAP. Four hours after naP the rats environmental factors are considered most relevant and itwere killed to examine gross injury index(mm), histo- is generally accepted that their complicated correlation haslogic change and to determine mucosal levels of malo- a great role in the occurrence of peptic ulcer at presentdialdehyde(MDA), prostaglandin E(PGEz), glutathioneThe number of patients with peptic ulcer is increasing(GSH)and myeloperoxidase( MPO).as a result of complicated social activity and the continuedadvance of civilization. Whereas people over the middleRESULTS: Pretreatment of ethanol significantly in-age were predisposed to have peptic ulcer in the past,creased NAP-induced gastric lesions, as well as aneven teenaged group is also susceptible recently. Majorincrease in MDA and MPO. On the contrary, mucosal etiologic factors of peptic ulcer include Helicobacter pylonPGEz and GSH contents were decreased dramatically by (H pylori)infection, excessive use of drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), irregularDA-9601 significantly reduced NAP-induced gastric injury eating habits, food containing causative materials such asgrossly and microscopically, regardless of pretreatment linoleic acid, smoking, alcohol consumption, psychological,and physiological stress. Chronic medication of NSAIDsmucosal PGEz and GSH in NAP-treated rats(P<0.05), often evokes dyspepsia, ulcer, hemorrhage, and perforationwith reduction in mucosal MDA and mPo levelsof upper gastrointestinal tract-These side effects that arise from NSAIDs arealcohol consumption renders gastric mucosa more sus- augmented by the suppression of prostaglandin(PG)ceptible to NSAIDs though, at least in part, reduction of synthesis and neutrophil-mediated injury secondary toendogenous cytoprotectants including PGE2 and GSHinflammatory mediators such as tumorand increase in MPO activation and that DA-9601, a new necro中国煤化工 leukotrienes(LTs)gastroprotectant, can reduce the increased vulnerability TheCNMHGr the aggravation ofof ethanol consumers to NSAIDs-induced gastric damage gbelieved to be agevia the mechanism in which PGEz and GSH are involvedgender, dyspepsia", clinical history ulcer and complicated2005 The W)G Press and Elsevier Inc. All rights reserved. smoking" and H pylori infectio usage of alcohol andulcer diseasel as well as heavOh TY et al protective effect of DA-9601 on gastritisEspecially, NSAIDs and alcohol consumption increase twice a day(8: 00 a.m. and 4: 00 p.m. for 5 d. Afterthe risk for major upper GI bleeding. Alcohol-induced body weight changes and clinical signs such as diarrheadisorders of the Gi tract are very common. Alcoholic and mortality were carefully examined, DA-9601 andgastritis subsequently leads to the impairment of the naproxen(NAP) were administered orally after the lastintegrity of gastric mucosal barrier, contributing to acid administration of ethanol and fasting animals. Ratsreflux into the subluminal layers of the mucosa and were assigned to one of six experimental groups; groupsubmucosa. The underlying mechanism of mucosal lesionsI: normal age-matched control, group I: alcoholsuch as petechiae, hemorrhage, and erosion elicited by administration, group IlL: NAP 50 mg/kg without alcoholalcohol is similar to various Nsaids 4)administration, group IV: alcohol+NAP, group V: DA-9601In this connection, this study was carried out100 mg/kg pretreatment+NAP without alcohol admiinvestigate the effect of NSAIDs on the susceptibility nistration, group VI: alcohol administration+DA-9601f gastric lesion sensitized by serial administration of 100 mg/kg+NAP. Four hours after NAP administration,alcohol and preventative effect of DA-9601(Stillen M), rats were anesthetized with ether and total extirpation ofArtemisia asiatica extract, on the progression of gastrie stomach was performed. Thirteen milliliters of 4 Clesion and finally to reveal the underlying mechanism of saline was injected into the lumen of isolated stomachaction. DA-9601 is now on the market in South Korea and maintained for 30 min Surface area(mm of gastricand will be on sale in other Asian countries in the near lesion was measured using optical microscope(Olympusfuture. It is reported to be effective to erosive gastritis s x10) following gastrotomy along with greater curvatureand to possess antioxidative and cytoprotective actions Small portion of tissue was fixed in 10%formalin solutionin models of gastric mucosal damage t6 7. The active and mucosal fuid collected from gastric mucosa stored atingredient of DA-9601 is eupatilin, which is reported to -74 C refrigeratorrevent experimental gastric damage induced by a varietyf noxious agentsHomogenization of gastric mucosal fluidFrozen mucosal fluid was thawed at room temperature anddiluted with 1 mL Tris-HCl buffer (pH 7. 4) per 100 mgMATERIALS AND METHODSand homogenized at 3 000 r/min for 10 min SupernatantTest materials and experimental animalswas stored at-20℃.DA-9601 Lot No. DA-9601-L-07) with 0.42% of activeingredient, eupatilin, was extracted from Artemisiae benMeasurement of the level of malondialdehyde in gastricand supplied to this study after HPLC analysis in Dong-A mucosal fluidpharmaceutical institute (S)-6-methoxy-a-methylTBA method was applied to accomplish MDAnapthaleneacetic acid (Naproxen), 10% neutral formalin, measurement. Two millimole per liter of FeCl was addedsodium phosphate monobasic, sodium phosphate to 100 HL supernatant, which was agitated and incubateddibasic, ferric chloride(FeCI), thiobarbituric acid (TBA), in 37C water bath for 30 min. Then 30% TCA,0.75%trichloroacetic acid (TCA), malondialdehyde (MDA), TBa and 5 mol/L HCI were added as well and boiledsulfosalicylic acid, DTNB, B-nicotineamide adenine for reaction in water bath for 15 min. Reacted mixturedinucleotide phosphate reduced form(B-NADPH), was cooled down to room temperature and centrifugedglutathione reductase, hexadecyltrimethylammonium at 3 000 r/min for 10 min Supernatant was assigned tobromide(HTAB), O-dianisidine dihydrochloride, bovine measure absorbance at 550 nm thereby the amount oferum albumin(BSA)were obtained from Sigma ( USA). MDA (nmol/L/mg protein) was generated from standardProstaglandin(PG) Ea[ I] radioimmunoassay kit (NEN) curve of MDawas obtained by DuPont(USA)and p-nitrothiophenol wasobtained from TCI (apan). Absolute ethanol, ether, and Measurement of PGEa in gastric mucosal fluidhydrochloric acid were purchased from Duksan(Korea). The amount of PGEz was measured with[ I RIa kit.DA-9601 was suspended in 5% hydroxypropylmethylcell After sample and tracer were added to the assay buffer,ulose(HPMC)dissolved in sterilized saline(v/). Seven- they were mixed with anti-serum and incubated at 2-8 Cweek-old SPF male Sprague-Dawley rats were obtained for 24 h Precipitating reagent was added to the sample,from Charles River(Kanagawa, Japan) and acclimatized agitated well and then incubated for 30 min at 2-8 C.Theat least 1 wk under standard laboratory conditions amount of PGEz(pg/mg protein) was measured with[temperature: 23#2 C, humidity range: 40-70%, gamma counter using pellet obtained from centrifugationventilation:15-20/h, luminous intensity: 150-300 lux, at 3 000 r/min for 30 min12 h light/ dark cycle (lighting: 7: 00 to 19: 00)]. The ratswere given regular chow and UV-sterilized tap water ad Measurement of the amount of glutathione in gastriclibitum中国煤化工 acid was centrifuged atPreventative effect of DA-9601 on ethanol-induced acute 3 000CNMH G Protein and reactedexperimental gastric lesionRate buffer(pH 7.5)Rats fasted for 24 h were given 50% ETOH orally containing 6 mmol/L DTNB, 0.3 mmol/L NADPH,7452 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2005 Volume 11 Number 47Normal Alcohol NAP Alcohol DA-9601Normal Alcohol NAPDA- 9601 AlcoholNAP + DA-9601gure 2 The effect of DA-9601 on MDA levels in gastric mucosal fluid. There wereno statistical significances among all groupson gastric mucosa with a significantly different area of50 unit glutathione reductase at room temperature. The hemorrhagic lesion(65.3+6.7, P<0. 05), which was moreamount of GSH (nmol/L/mg protein) was generated severe than groups I and I. Group V(DA-9601+NAP)by the absorbance of p-nitrophenol measured at 412 nm significantly inhibited gross gastric lesion in comparisonusing the supernatant from centrifugation at 3 000 r/ minto group Il (4.5+1. 2, P<0.05). The area of gross gastricfor 10 minlesion of group VI(alcohol+DA-9601+NAP)wa20.0+2. 1 which was significantly different from group IvMeasurement of the amount of myeloperoxidase in gastricThe amount of myeloperoxidase(mU/mg protein) was MDA level in gastric mucosal fluidmeasured at gastric mucosa modifying the method of MDa levels(Figure 2)of groups I (normal contro) andprevious reports. After gastric mucosa was homogenized I(alcohol indicating lipid peroxidation were 3.43+0.44in 0.5% HTAB solution containing 50 mmol/L potassium (nmol/L/mg protein) and 3. 66+0.58 with no significantphosphate(pH 6.5), homogenized samples were freezed difference between themfound thatand thawed thrice and homogenized. Supernatant was I(NAP)and IV(alcohol+NAP) had 4.61+0.83 anddded to the reaction buffer containing O-dianisidine 5.30+0.89, respectively. There was a tendency to increasedihydrochloride and H2O2 and the amount of mye- MDA levels of groups Ill and I without statisticalperoxidase was calculated from the absorbance of the significance. Group V(DA-9601+NAP)exhibitedreaction buffer at 460 nm at every 1 minMDA level of 3.16+0. 18 which was reduced by 31.5%compared with group I(NAP), but there was noProtein assaysignificant difference between them. In case of group VIProtein assay was performed by means of measuring (alcohol+DA-9601+NAP), MDA level was 3.92+0.84absorbance at 535 nm compared using standard curve of (nmol/L/mg protein) with 15.0% reduction compared togroup IV, but no significance was foundStatistical analysisProstaglandin E2 level in gastric mucosal fluidData were expressed as the mean+SD and all statistical PGE2 level as a cytoprotection marker in group I(normalanalyses were performed using SigmaStat for Windows 2.0 control) was determined to be 194.5+21. 5(pg/mg proteinsoftware (andel Corporation, USA). Statistical significanc(Figure 3), group I (alcohol) showed 159.5+30.2 whichwas evaluated by one-way analysis of variance(ANOVA) was reduced to the extent of 18.0% compared to group Ifollowed by Bonferroni post boc test or Dunnetts test for a (normal control) but did not produce significance. Groupmultiple comparison. P<0.05 were considered significantIl(NAP) represented 62.0+13.8 indicating significantdifference due to 68. 1% reduction(P<0.05. PG Ez level ofRESULTSgroup Iv (alcohol+NAP)was 47. 1+11.9 which produced70.5% reduction which was significantly different(P<0. 05)Preventative effect of DA-9601 on ethanol-induced acute from group II(alcohol) and 24.0% reduction withoutexperimental gastric lesionmpared to orn而cNAP).3072±273There was an apparent linear lesion on gastric mucosa was中国煤化工 I of group V(Daof group I (alcohol) with area of gross lesion(mm9601CNMHGthan normal(P<0. 05)lesion on gastric mucosa and area of gross lesion was VI (alcohol+ DA-9601+NAP) showed 180.3+18.3 similar27.0+6.0. Group IV(alcohol+NAP)exhibited hemorrhage to group I (normal control) normal control but 282.8%Oh TY et a/ Protective effect of DA-9601 on gastritis7453EB品an2Normal Alcohol NAP Alcohol DA9601 AlcoholNormal Alcohol NAP AlcoholFigure 3 The effect of DA-9601 on PGEa levels in gastric mucosal fluid. Data are Figure 5 The effect of DA-9601 on MPO levels in gastric mucosal fluid. Data aresed as mean*SD, p<0.05 vs normal control, Pc0.05 vs alcohol group, expressed as meantSD. p<0. 05 vs normal control, " P<0.05 vs NAP groupP<0. 05 vs NAP ' p<0.05 vs alcohol+ NAP2Normal Alcohol NAP Alcohol DA-9601ad409601Figure 6 Histopathological changes induced either by alcohol, NAP andnaP administration and its inhibition by DA-9601. A: Minimal inflamchange in gastric mucosa in group I (alcohol); B: Focal epithelial eros4 The effect of DA-9601 on GSH levels in gastric mucosal fluid. Data are inflammatory cell infiltration in gastric mucosa and submucosa of group m(NAP)sed as mean* SD. p<0. 05 vs normal control, "p<0. 05 vs alcohol group,multifocal and diffuse epithelial erosion and severe intvs NAP P<0. 05 vs alcohol+ NAPin gastric mucosa and submucosa of group Iv (alcohol NAP); D: DA.-9601treatment ameliorates the pathological changes of gastric mucosa and submucosainduced by NAP in alcohol pre treated group Vi(alcohol+ DA-9601+NAP)increase with a signifcant difference(P<0.05)compared toup Iv(alcohol+ NAP)0.94+0. 27 of group Iv(alcohol+NAP) generated 21.1%Glutathione levelreduction compared to group I, but was similar to groupThe levels of GSH in gastric mucosa(Figure 4)were IT Group V(DA-9601+ NAP) showed 0.58+0.04 which2.01+0. 14(nmol/L/mg protein) in group I(normalproduced 25.6% reduction in comparison to group Icontrol) and 1. 18*0. 17 in group I(alcohol) and there (normal control) and was significantly decreased comparedwas no significant difference between them. 1.01+0. 2145%5d0 d was significantl NAP, tir. p Iv level of MPO in gre中Ⅵ(D190+NApwignificant difference(P<0.05). GSH level of group Iv 1.08+rly to group I(ale(NAP)from group II (alcohol) and group III (NAP), respectively.Group V(DA-9601+NAP) exhibited 2.68+0.22 withHistopathological examinationsignificant increase compared with group Ill, and that wasstopathological changes are depicted in Figure 6. Inven higher than group I (normal control). The level of group I(alcohol), there was a minimal inflammatoryGSH in group VI was 2.16+0.28 which was significantly change in gastric mucosa; however, it was found that grouphigher than group IV(P<0.05)Ill (NAP) had focal epithelial erosion and inflammatorycell infiltration in gastric mucosa and submucosa. WhereasMyeloperoxidase levelgroup IV(alcohol+ NAP)aggravated the gastric lesion byThe level of MPO in group I (normal control)wasforfocal and diffise enithelial erosion and severe0.78+0. 11(mU/protein)(figure 5). Whereas group infla中国煤化工 mucosa and submucosa,I(alcohol) exhibited significant increase of MPO DA-(1. 19+0. 27)compared to normal (P<. 05), there was anofCNMH Ga induced by nap inincrease of MPO in group III(NAP)without significance. alcohol pre-treated group (group VI)7454 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2005 Volume 11 Number 47DISCUSSIONand free radical formation by extravasated neutrophilConsidering the outcomes from this experiment, itSeveral methods are suggested to alleviate the adversewas demonstrated that co-administration of alcohol effects of NSAIDs until recently. First of all, aspirin isand naproxen, one of the currently available NSAIDs, administered in the form of enteric-coated tablet or directaugmented the gastric mucosal lesion in comparison to the irritation to gastric mucosa is prevented by administrationadministration of alcohol or naproxen alone. Subsequentlytreatment of DA-9601 that is being developed for targepatients being chemically modified with nitric oxide thating a new gastric mucoprotectant was shown to account is known to act as a gastric mucosal protectant similarly tofor the preventative effects on gastric mucosa effectively PG Lastly, NSAIDs with high sensitivity to CoX Iagainst the lesion caused by alcohol and naproxen. Besidesare designed,). Despite these efforts, 62. 2%/ of pattentsthe effects on quantified gastric lesion, the level of MDA treated with NSAIDs on a long-term basis complainedand MPO in DA-9601 treated group exhibited similarof upper gastrointestinal clinical signs of which gastritislevel to that in normal control and it was also revealed (38.5%), gastric ulcer(15.5%), duodenal ulcer (1.9%)werehat DA-9601 contributed to intact maintenance of most prevalent; however, it appears that more patientsthe gastric mucosa at the extent of normal control onuffered from these adverse effects due to complicatedhistopathological examination.drug administration or eating habits. The underlyingThe well-known etiologies for peptic ulcer can be mechanisms that result in gastrointestinal symptoms cancategorized as follows: eating habits, environmental be classified as follows: firstly, it is generally acceptedfactors, stress by social life, drinking behavior, smoking, that NSAIDs inhibit COX responsible for the synthesisand drugs. Among these causative factors, drug-induced of PG. Secondly, NSAIDs have a direct effect on gastricpeptic ulcer is increasing dramatically with respect tomucosa andlastly it is also proposed that overproductionthe overuse of NSAIDs and increased susceptibility of of LTs, especially for LTB4, by 5-lipoxygenase(5-LO)gastric mucosa by single or co-administration of oral resulting from compensation for the inhibition of COXanti-coagulants(warfarin, phenindione, phenprocoumon,is a causative factor 1 32. lts are known to be mediatorsacenocoumarol, anisindione, diphenadione, etc )and that induce inflammatory process and take part in thecorticosteroid hormones(triamcinolone, dexamethasone, formation and infiltration of neutrophils.The mostcortisone, hydrocortisone, prednisolone, prednisone)"). It commonly prescribed formula considering NSAIDis reported that alcohol is responsible for the hemorrhage treatment is the co-administration of extrinsic or intrinsicof gastric mucosa and edema in submucosal muscle layer PG. Whereas Misoprostol, one of the extrinsic P(by means of direct irritation"9) and also leads to acute synthetics, has the advantage in the aspect of potency, andgastritis associated with microcircular stasis 20. Once it exhibits adverse effect such as diarrhea. The intrinsic PGgastric mucosa is injured, thereby ischemia and decreased derivative, ornoprostil, is known to be not effective in thlevel of aTp in gastric mucosa and intervention of patients with gastric ulcer or gastritis because of the factmicrocirculation occurs. Gastric mucosal damage duethat NSAIDs inhibit COX. It was revealed that DA-9601to chronic ingestion of low concentration of alcohol is increased the level of PGE? and GSH dose-dependently ingenerally not discernible on gross examination; however, if normal state as well as alcohol or ammonia-induced gastricgastric mucosa is exposed to an external mucosal irritant, lesion. Moreover, DA-9601 elevated the level of PGE2gastric lesion becomes more extensive and consistent with protecting gastric mucosa without affecting the efficacyincreased susceptibility as well as persistent submucosal of NSAIDs, when DA-9601 was co-administered withmicrocircular stasis It was demonstrated that DA-9601 NSAIDs in arthritis-induced rats. Oxygen free radical and100 mg/kg treatment exerted excellent preventative or lipid peroxidation are regarded as the crucial etiologiestherapeutic effccts on gastric mucosal lesion with action of leading to gastric mucosal lesion by oxidative stress 5)duration being effective for more than 2 h. It was also well Once oxygen free radical triggers and maintains ischemicdocumented that these effects were in accordance with the status in gastric mucosa, hydroxyl radical generated fromfacilitation of the synthesis of PG by DA-9601superoxide anion results in gastric lesion by virtue ofSince aspirin was first synthesized in the year of 1899, decreasing the velocity of blood flow subsequently ola variety of NSAIDs has emerged so far and they areIn this present study, we measured not only the levelbeing used as antipyretic analgesics and anti-inflammatorof MDA and MPO in order to quantify the materialsdrugs. Nowadays, the market size of NSAIDs is more than contributing to gastric mucosal lesion but also the level60 billion/year, in addition, more extended indications are of PG E2 and GSH that are produced as protectivemaking the need for NSAIDs to increase"21. Despite these markers. As a whole, it was demonstrated that the level offacts, NSAIDs were reported to cause gastric lesion since MDA and MPO were decreased similarly to the level of1930s and underlying mechanism was related to the fact norPGF, and GSH were increasedthat NSaiDs inhibit cyclooxygenase(COX) thereby block in D中国煤化工 the previous report,the synthesis of PG. More specifically, NSAIDs elicit Dagastromucosal lesion not only by reducing the physiological preCNMHGability to facilitate theFrom the findings ofrole of PG, but also by directly inducing neutrophilic this study, DA-9601 treatment was proven to protectinfiltration in gastric capillaries causing subsequent stasis effectively against the NSAIDs-induced lesion of gastricOh TY et a/ Protective effect of DA-9601 on gastritis7455mucosa which was more susceptible to NSAIDs by thequantitation of ion fluxes across in vivo human gastric mucosa:administration of alcohol in rats. On closer inspectionopen, ethanol, and hyperosmolarof results, the underlying mechanism of the actionsolutions. Gastroenterology 1984: 86: 60-70of DA-9601 can be centered on the cytoprotective15 Seol sY, Kim MH, Ryu JS, Choi MG, Shin Dw, Ahn BDA-9601 for erosive gastritis: results of a double-blindeffect by normalizing the production of PG, additionallacebo-controlled phase Ill clinical trial. Worid J Gastroeterotimprovement of gastric mucosal function by elevating00410:2379-2382GSH content, alleviation of inflammation by decreasing 16 Oh TY, Ryu Bk, Yang JL, Kim WB, Park JB, Lee SD, Lee EBthe level of MDA and MPO. Bearing all these in mind, itStudies on antiulcer effects of DA-9601, an Artemisia herbais worth making a conclusion that DA-9601 treatment has) Appl Pharmacol 1996; 4: 111-121a protective effect on gastric mucosa of patients who are 17 Oh TY, Ahn BO, Ko JL Ryu BK, Son MW, Kim SH, Kim WB,accustomed to drinking alcohol regularly and will be ofLee wB. 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Turk JRole of oxygen radicals in ischemia-induced lesions in the catGastroenterol 2003: 14: 39-43stomach, Gastroenterology 1986: 90: 362-267Science Editor Wang XL and Guo SY Language Editor Elsevier HK中国煤化工CNMHG