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Original Articles • Arq. Gastroenterol. 51 (3) • Jul-Sep 2014 • https://doi.org/10.1590/S0004-28032014000300012 linkcopiar

Open-access Comparação dos inibidores seletivos e não seletivos da ciclo-oxigenage 2 na exacerbação da colite experimental: papel do leucotrieno B4 e superóxido dismutase

AutoriaSCIMAGO INSTITUTIONS RANKINGS
    Arq Gastroenterol Arquivos de Gastroenterologia Arq. Gastroenterol. 0004-2803 1678-4219 Instituto Brasileiro de Estudos e Pesquisas de Gastroenterologia - IBEPEGE, Colégio Brasileiro de Cirurgia Digestiva - CBCD, Sociedade Brasileira de Motilidade Digestiva - SBMD, Federação Brasileira de Gastroenterologia - FBG, Sociedade Brasileira de Hepatologia - SBH, Sociedade Brasileira de Endoscopia Digestiva - SOBED Contexto Os anti-inflamatórios não-esteróides são considerados uma das mais importantes causas de reativação da doença inflamatória intestinal. Em relação aos inibidores seletivos da ciclo-oxigenase 2, os resultados são controversos tanto em estudos envolvendo humanos como na colite experimental. Objetivos Comparar os efeitos dos anti-inflamatórios não-esteróides, seletivos e não seletivos da ciclo-oxigenase 2, na colite experimental e, contribuir para o entendimento do mecanismo no qual os anti-inflamatórios não-esteróides provocam a exacerbação da colite. Métodos Seis grupos de ratos foram estudados: sem colite, com colite e com colite e tratados com celecoxib, cetoprofeno, indometacina ou diclofenaco. Foram determinadas a taxa de sobrevida, as concentrações de hemoglobina e albumina plasmática, as concentrações teciduais na mucosa colônica de interleucina-1ß, interleucina-6, fator de necrose tumoral alfa, prostaglandina E2, catalase, superóxido dismutase, substâncias reativas ao ácido tiobarbitúrico e quimiluminescência estimulada por hidroperóxido de tert-butil, e as concentraçãos plasmática e tecidual de leucotrieno B4. Resultados O grupo tratado com diclofenaco ou indometacina apresentaram as menores taxas de sobrevida, concentrações de hemoglobina e albumina, e as maiores concentrações plasmática e tecidual de leucotrieno B4 e tecidual de superóxido dismutase do que os groupos tratados com celecoxib. O grupo tratado com cetoprofeno apresentou um comportamento intermediário entre diclofenaco/indometacina e celecoxib, em relação a taxa de sobrevida e albumina. Os grupos sem colite, colite e colite tratado com celecoxib apresentaram menores concentrações de leucotrieno B4 e superóxido dismutase do que os grupos tratados com inibidores não seletivos ciclo-oxigenase 2. Conclusões Diclofenaco e indometacina apresentaram os maiores graus de exacerbação da colite entre os anti-inflamatórios não-esteróides, o celecoxib não apresentou exacerbação e o cetoprofeno apresenteou um comportamento intermediario entre diclofenaco/indometacina e celecoxib. Estes resultados sugerem que o leucotrieno B4 e superóxido dismutase estão envolvidos na exacerbação da colite experimental pelo anti-inflamatórios não-esteróides não seletivos ciclo-oxigenase 2. INTRODUCTION Inflammatory bowel disease (IBD) is characterized by periods of remission and reactivation, and ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) is considered one of the most important causes of reactivation(14). Extra-intestinal inflammatory features, as peripheral arthritis, are common and NSAID prescription may occurs even as self medication(4, 24). Although the exact pathophysiology of exacerbation of colitis by non-steroidal anti-inflammatory drugs remains unresolved, it is likely that eicosanoids, through a colonic Prostaglandin E2 suppression accompanied or not by a compensatory increase in leukotriene B4, play a fundamental role(14). An issue that must be considered is the intensity of inhibition on ciclooxygenase 1 (COX-1) and ciclooxygenase 2 (COX-2), which greatly varies in accordance with NSAIDs type and dose. There are NSAIDs with low selectivity for COX-2, which practically inhibit COX- 1 and COX-2 in the same proportion, whereas other NSAIDs, can have a 50 fold higher selectivity for COX-2 when compared to inhibition of COX-1(57, 58). Therefore, NSAIDs behavior may have distinguished consequences in arachdonic acid metabolism with distinct eicosanoids production, mainly in Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), and thus each NSAID can interfere in a particular way in IBD reactivation. Whereas the literature about IBD reactivation with classical NSAIDs, like indometacin and diclofenac is extensive(10, 35), case reports with ketoprofen are scarce(52). With regard to plasmatic selective inhibitors, the results are controversial in experimental colitis as well as in human studies. Some studies have verified that induced colitis in rats treated with a highly selective COX-2 inhibitor (L745.337)(46) or celecoxib(49, 59) provoked PGE2 reduction, inflammatory colonic exacerbation and low survival rates. Those authors concluded that prostaglandins derived from COX-2 could have a defense role in colon protection. Matuk et al.(33) also verified that a selective COX-2 inhibitor was highly associated with IBD reactivation in humans, and that drug discontinuation resulted in asymptomatic patients in 38%. Nevertheless, Cuzzocrea et al.(9) verified a reduction in colitis severity, shown by survival rate, colonic injury, and tumor necrosis factor-α (TNFα) and Interleukin-1 (IL-1) production in rats with trinitrobenzene sulfonic acid (TNBS) induced colitis and treated with celecoxib. Also in humans, Mahadevan et al.(31), Takeuchi et al.(51) and Meidany et al.(12) have not associated celecoxib, rofecoxib, nimesulide and etoricoxib utilization to the inflammatory response exacerbation in patients with IBD. On the other hand, it has also been proposed that inflammatory bowel disease (IBD) results from an imbalance between pro-oxidant and antioxidant mechanisms(5, 18, 48). Oxidative stress can result from excess production of reactive oxygen and nitrogen species (RONS) by inappropriate activation of phagocytic cells. Excessive liberation of RONS by polymorphonuclear cells is considered the main mechanism for colonic lesion in ulcerative colitis (UC), where accentuated lipid peroxidation occurs(48). It has also been suggested that imbalanced and inefficient endogenous antioxidant response, especially attributed to SOD dual role acting as superoxide anion scavenger and an H2O2 producer, may contribute to both the pathogenesis and the perpetuation of the inflammatory processes(27). Therefore, the aim of the present study is to assess celecoxib, ketoprofen, and other NSAIDs effects in experimental colitis, verify which NSAIDs exacerbate the disease and contribute to the understanding of the mechanisms responsible for colitis reactivation. METHODS Animal and induction of colitis Adult male Wistar rats weighing 229±20 g were purchased from the Animal House of the Biological Sciences Center at the University of Londrina. They were housed five per cage, deprived of food for 24 hr, and colitis was induced following the procedure described by Morris et al.(37). Rats were anesthetized with both xilazine (10 mg/Kg, im) and ketamine (50 mg/Kg, im); polyurethane catheter (2 mm of diameter) was inserted intrarectally into the colon such that the tip was located 8 cm proximal to the anus. One milliliter of TNBS (Sigma, St. Louis,Missouri-USA) in 40% ethanol (v/v) was instilled into the lumen of the colon through the rubber catheter at a dose of 80 mg/kg body weight. The instillation procedure required 60 seconds and treated rats were kept in the Trendelenburg position to facilitate internal access of the TNBS solution. All experimental protocols described in this report were approved by the Animal Care Committee of the University of Londrina in accordance with the Law 11.794/2008 of October 8, 2008 and the rules of the Brazilian Society of Laboratory Animal Science - SBCAL/Council on the Control of Animal Experiments - CONCEA. The method of euthanasia was by exsanguination by cardiac puncture after general anesthesia with both xilazine (10 mg/kg, im) and ketamine (50 mg/kg, im). Animal groups and treatment Two separated experiments were performed: the first for studying oxidative stress, and the second for determination of interleukins and eicosanoids. In each experiment the rats were randomly divided into six groups: control group was constituted by animals without colitis; colitis group was constituted by animals with colitis, colitis+celecoxib group was constituted by animals with colitis and treated orally with celecoxib (15 mg/kg), colitis+ketoprofen group was constituted by animals with colitis and treated orally with ketoprofen (10 mg/kg), colitis+indometacin group was constituted by animals with colitis and treated orally with indometacin (5 mg/kg), and colitis+diclofenac group was constituted by animals with colitis and treated orally with diclofenac (10 mg/kg). All nonsteroidal anti-nflammatory drugs (NSAIDs) were suspended in a vehicle of 0.5% of carboxymethylcellulose. Treatment with different NSAIDs began 3h before induction of colitis and continued every 12h thereafter for up to 5d. The doses were selected because it has been shown to exert significant anti-inflammatory effects in rats(6, 38, 46, 53, 55). The others groups were treated orally with 0.5% carboxymethylcellose with the same periodicity. All samples of colon and blood were obtained on the 6th day after the induction of colitis. Evaluation of colon damage and sample preparation On the 6 day after the induction of colitis the rats were anesthetized and the colon was rapidly excised, approximately 10 cm proximal to the anus, slit longitudinally and gently rinsed with 10 mM, pH 7.4, sodium phosphate buffer (SPB). Then, the segment was placed on a flat glass plate with the mucosal surface up. The degree of damage was scored macroscopically based on the presence of adhesions, strictures, ulcers, and wall thickness by an observer blinded to the treatment the rats had received(53). Soon after, in order to study the oxidative stress, the mucosa was scraped off with a microscope slide onto a cold Petri dish and all mucosa specimens were frozen in liquid nitrogen. Samples were homogenized in SPB (5 mL) using a mechanical Potter-Elvejhem. The total homogenate obtained was used for thiobarbituric acid reactive substances (TBARS) and tert butyl hydroperoxide-initiated chemiluminescence (CL-LOOH) measurement. The supernatant of the total homogenate of colonic mucosa was obtained by centrifugation of the total homogenate for 10 min at 4°C and 3,000 g. In the second experiment to determine interleukins and eicosanoids, colon was removed (approximately 10 cm), slit longitudinally and gently rinsed with SPB. Immediately, samples were processed by the Potter-Elvejhem with 2,5 mL of SPB and were centrifuged for 10 min at 4oC and 3,000 g and the supernatants were quickly immersed in dry ice and kept in freezer at -70oC. Histological analysis Samples of colon were isolated and immersed in 4% paraformaldehyde in phosphate-buffered solution, dehydrated in ethanol, and embedded in parafilm. Sections of 4 μm were stained with hematoxylin and eosin, and investigated by light microscopy for the presence of inflammatory changes. Inflammation was classified as mild, moderate or severe by pre established criterion that considered neutrophils and macrophages infiltration in tissue. Blood sampling The blood was obtained by cardiac punction and collected in tubes containing ethylene-diamine-tetraacetic acid (EDTA) for hemoglobin determination or tubes without anticoagulant for other determinations, kept on melting ice during transfer and immediately processed to obtain serum. Laboratorial parameters The following laboratorial parameters were determined: haemoglobin was determined by automated counter (Pentra 120 Retic, ABX) and albumin, alkaline phosphatase (AP) and γ -glutamyl transferase (γ-GT) were measured with automatic chemistry analyzer (Dimension AR, Dade-Behring). The results of these enzymes were expressed in U/100 mg of protein. Myeloperoxidase Myeloperoxidase (MPO) activity in colonic mucosa was determined using a modified method by Nieto et al.(40). One unit of activity was defined as the amount of enzyme present that produces a change in absorbance per minute of 1.0 at 37oC in the final reaction volume containing the sodium acetate. The results were expressed in units per 100 mg of protein (U/100 mg of protein). Oxidative stress analysis Analysis of tert butyl hydroperoxide-initiated-chemiluminescence in total homogenate of mucosa The CL-LOOH in total homogenate of mucosa was evaluated as described previously by Gonzalez-Flecha et al.(17). For chemiluminescence measurement, reaction mixtures were placed in 20 mL scintillation vials (low-potassium glass) containing final concentrations of: serum or homogenate (250 μL), 30 mM KH2PO4/K2HPO4 buffer (pH 7.5), and 120 mM KCl with 3 mM of tert butyl hydroperoxide in a final volume of 2 mL. Tert butyl hydroperoxide-initiated chemiluminescence was measured in a Beckman LS 6000 liquid scintillation counter set to the out-of-coincidence mode, with a response range from 300 to 620 nm. The vials were kept in the dark up to the moment of assay, and determination was carried out in a dark room at 30°C. The results are expressed in counts per minute (cpm) or cpm for 100 mg of protein (cpm/100 mg of protein). Thiobarbituric acid-reactive substances in total homogenate of mucosa The TBARS were measured by method as described by Jentzsch et al.(22), using a high concentration of butylated hydroxytoluene before heating and differential readings at 535 and 572 nm, which substantially improved its specificity. The results were expressed in μmol MDA/100 mg of protein. Activities of antioxidant enzymes SOD and CAT in the colonic mucosa The SOD activity was determined by the method proposed by Marklund and Marklund(32) that is based on the enzyme capacity to inhibit autoxidation of pyrogalloll. The reaction was performed at 37oC and followed at 420 nm, and the results were expressed in units/100 mg of protein (U/100 mg of protein). The CAT activity was measured spectrophometrically at 240 nm, by decomposition of H2O2 at 25oC(1). The decomposition of H2O2 is proportional to the concentrations of enzyme and substrate, and the results were expressed as the difference in absorbance per 100 mg of protein/minutes (DABS/100 mg of protein/min). Cytokines and eicosanoids analysis in the colonic mucosa or serum Interleukin-1 β (IL-1 β), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), leukotriene B4 (LTB4) and Prostaglandin E2 (PGE2) concentration were measured by specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturersrecommendation(R&DSystem,Inc.M∈≠apolis,USA).Theantibodiesusedforthesecy→k∈esandeicosanoevaluationswere:PGE2-Captureantibody:goatanti-mousepolycronalantibody,detectionantibody:antibodymousemonoclonalantibody→PGE2;IL-6-Captureantibody:mouseanti-ratIL-6monoclonalantibody,detectionantibody:goatpolycronalanti-rat→IL-6;IL-1β-CaptureAntibody:goatanti-ratIL-1βpolycronalantibody,detectionantibody:goatpolycronalanti-ratIL-1β;LTB4-Captureantibody:raitanti-χckenpolyclonalantibody,detectionantibody:χckenpolycronalantibody→LTB4andTNFα-CaptureAntibody:mouseanti-ratTNFαmonoclonalantibody,detectionantibody:goatpolycronalanti-ratTNFα.Therest̲swereexpressed∈pgmLorpgper100mgofprote∈(pg100mgofprote∈)whendetermined∈theserumorcolonicμcosa,respectively.StatisticalanalysisAlldataareexpressedasmean±SD.Therest̲swereanalyzedbyo≠-wayanalysisofvariancefollowedbyaTukey-Kramerposthoctestforμ<ip≤comparisonsforparametricvaluesandDu∩´stestfornonparametricvalues.Survivalratesandhis→logicalscoreamonggroupswerecomparedusingtheFisher’sexacttest.D⇔erenceswereconsred→besignificantwhenP<0.05.RESULTSSurvivalrateandmacroscoπcscor∈gofcolonicdama≥Thegroupswithcolitistreatedwithdiclofenac(31%)and∈domηc∈(44%)presentedthelowestrateofsurvivalattheendoftheexperiment(P<0.05∈relation→allothergroups).Thesurvivalratewas88%∈thecolitisgrouptreatedwithke⊤rofen,whereasthegroupswithoutcolitis,withcolitis,andcolitistreatedwithce≤c⊗ibhadnomortality(Figure1).FIGURE1.Survivalrates(%)after6days∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugs.n310foreachgroupofrats.Survivalratesweresignificantlylower∈thecolitisgroupstreatedwith∈domηc∈ordiclofenacwhencompared→theothergroups(P<t0.05,Fisher′sexacttest).Macroscoπcanalysisofcolonicdama≥showedastatisticallysignificantd⇔erence∈thegroupwithoutcolitis∈relation→thecolitisgroups(P<0.05).Thegroupwithcolitistreatedwith∈domηc∈anddiclofenacshowedahighermacroscoπcscore(P<0.05)thanthegroupcolitis(Tab≤1).TABLE1.Markersofcolonicdama≥andbl∞dmarkersof∈flamma→ryactivity∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugsParametersGROUPSControlColitisColitis+Ce≤c⊗ibColitis+Ke⊤rofenColitis+Indomηc∈Colitis+DiclofenacMacroscoπc2.8±0.88.6±1.412.4±3.112.1±1.914.4±2.316.5±3.4Scor∈ga⋅b†c†d⋅e⋅d‡e⋅AP13.0±5.6450.0±104.6342.9±148.7427.5±197.3342.2±132.4371.1±74.6U100mgprote∈a⋅b‡c⋅d‡e⋅g-GT8.6±3.162.0±20.938.6±10.464.3±19.191.3±34.579.2±18.1U100mgprote∈a⋅c†d⋅e⋅d‡e‡MPO115.3±52.5611.1±249.2530.5±162.0440.5±170.8513.8±260.2562.0±219.9U100mgprote∈a⋅b†c‡d‡e⋅Albumin1.53±0.111.19±0.131.10±0.170.89±0.130.72±0.110.68±0.08GdLa§b§c§d§e§c§d§e§c∗d§e§e∗Hemoglob∈16.3±0.616.8±1.115.6±0.813.7±1.411.9±1.013.4±1.3GdLc§d§e§c§d§e§c∗d§e§d††Allvaluesaremean±SD.AP:alkal∈ephospˆase;g-GT:g-glutamyltranerase;MPO:myeloper⊗se.Theanalyseswereperformed∈thesixthdayaftercolitis∈ducedbyTNBSandtreatmentwithNSAIDs.N=10foreachgroupofrats.a:∈comparison→colitisgroup;b:∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+ke⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001byDu∩’stest;†P<0.01byDu∩’stest;‡P<0.05byDu∩’stest;§P<0.001byTukey-Kramertest;∗P<0.01byTukey-Kramertest;††P<0.05byTukey-Kramertest.His→logicalanalysisThehis→logyofthecolonwasseverelyaffected∈animalwithcolitis.Thecolitiswascharacterizedbyamassiveimμ≠cell∈fi<ration,c̲eration,and≠crosis.The∈flamma→ryresponsewasma∈lyformedby≠utroϕlsandmacropha≥s,whereaslimphocytesandplasmocyteswererarelyobserved.Thepresenceofsevere∈flammation∈thecolonicμcosawasaompaniedbyan∈flamma→ryreaction∈theperi→≠um.Thecontrolgroupshowedno∈flammation.Inrelation→thegroupwithoutcolitis,thegroupswithcolitis,andcolitistreatedwithce≤c⊗iborke⊤rofenpresentedmild∈flammation(P<0.05),whereasthegroupswithcolitistreatedwith∈domηc∈ordiclofenachadsevere∈flammation(P<0.05)∈relation→thetwolatergroups(data¬shown).Enzymaticmarkersofcolonicdama≥APandMPOactivities∈thecolonictissueshowedstatisticallysignificantd⇔erences∈thecolitisgroupscomparedwiththecontrolgroup(P<0.05).γ-GTactivitywastheonlyenzymaticmarkertˆdiscriminatedsignificantlythegroupwithcolitistreatedwithce≤c⊗ibwhichshowedlowersignificantvalues(P<0.05)whencompared→thecolitisgroupstreatedwith∈domηc∈ordiclofenac(Tab≤1).Bl∞dmarkersof∈flamma→ryactivity(hemoglob∈andalbumin)Allgroupswithcolitisshowedasignificantreduction∈plasmaalbumin∈relation→thecontrolgroup(P<0.001).Therewerestatisticallysignificantd⇔erences(P<0.05)∈plasmaalbumin∈thegroupswithcolitistreatedwith∈domηc∈ordiclofenac∈relation→thegroupswithcolitisandcolitistreatedwithce≤c⊗iborke⊤rofen(Tab≤1).Inrelation→thecontrolgroup,thecolitisgroup,andthecolitisgrouptreatedwithce≤c⊗ib,therewasasignificantdecrease(P<0.05)∈hemoglob∈∈thegroupswithcolitistreatedwithke⊤rofen,∈domηc∈ordiclofenac.Thelowesthemoglob∈concentrationwasobserved∈the∈domηc∈groupwhichalsoshowedasignificantreduction(P<0.05)∈relation→theke⊤rofengroup(Tab≤1).Pro-⊗ntandanti⊗ntmarkersof⊗tivestressWithrespect→tissuepro-⊗ntmarkers,CL-LOOHandTBARSmeasurementsshowedan∈crease(P<0.05)∈thecolitisgroupand∈allcolitisgroupstreatedwithNSAIDs∈relation→thecontrolgroup(Tab≤2).TABLE2.Pro-⊗ntsandanti⊗ntsmarkers∈colonicμcosa∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugsParametersGROUPSControlColitisColitis+Ce≤c⊗ibColitis+Ke⊤rofenColitis+Indomηc∈Colitis+DiclofenacCL-LOOH4.1±1.623.6±6.922.2±13.120.3±7.128.6±11.426.3±9.3c±x1000100mgprote∈a⋅b†c‡d⋅e⋅TBARS7.11±1.3011.94±1.8414.73±4.0814.76±3.8114.93±4.8913.5±3.5510-3mmolMDA100mgprote∈a‡b†c⋅d†e‡SOD7.2±5.011.0±2.918.0±6.932.4±10.638.3±12.830.2±13.7U100mgprote∈c⋅d⋅e⋅c⋅d⋅e⋅CAT0.255±0.0790.184±0.0620.256±0.0850.231±0.0520.241±0.0690.222±0.049DABS100mgprote∈minAllvaluesaremean±SD.CL-LOOH:tertbutylhydroper⊗-∈itiated-chemiluminescence;TBARS:thioˉbituricacreactive⊂stances;SOD:⊇r⊗isμtase;CAT:catalase.Theanalyseswereperformed∈thesixthdayaftercolitis∈ducedbyTNBSandtreatmentwithNSAIDs.N=10foreachgroupofrats.a:∈comparison→colitisgroup;b:∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+Ke⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001;†P<0.01;‡P<0.05byDu∩’stest.SODenzymaticanti⊗ntactivitywassignificantlyhigher(P<0.05)∈thecolitisgroupstreatedwithke⊤rofen,∈domηc∈,anddiclofenalthanthecontrolgroupandthecolitisgroup(Tab≤2,Figure2),whereastherewasnostatisticallysignificantd⇔erence∈CATactivity∈thegroups(Tab≤2).FIGURE2.Super⊗disμtase(SOD)activity∈thecolonicμcosa∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugs.(B⊗-and-WhiskePlots).Evaluationwasperformed∈the6dayaftercolitis∈ductionandbeg∈n∈gthetreatmentwithNSAIDs.n=10foreachgroupofrats.a:∈comparison→colitisgroup;b:∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+ce⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001;†P<0.01;‡P<0.05byDu∩’stest.Cy→k∈esandeicosanoconcentration∈thecolonicμcosaAllcolitisgroupspresentedhigher(P<0.05)tissueIL-1βconcentrationwhencompared→thecontrolgroup,whereastissueIL-6andTNFαconcentrationshowedhighervalues∈allcolitisgroupstreatedwithNSAIDsthan∈thecontrolgroup(Tab≤3).TABLE3.Cy→k∈esconcentration∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugsParametersSamp≤sUnitsGROUPSControlColitisColitis+Ce≤c⊗ibColitis+Ke⊤rofenColitis+Indomηc∈Colitis+DiclofenacLTB4SerumpgmL761±390849±5671642±6212930±9353052±8623324±862c⋅d⋅e⋅c⋅d⋅e⋅c†d†e⋅Tissuepg100mg352±229695±201776±350954±418969±251958±433prote∈c†d†e†PGE2Tissuepg100mg10328±13559247±10366318±10493183±11083946±8555608±952prote∈b⋅c⋅d⋅e⋅b⋅c⋅d⋅e⋅c⋅d⋅e⋅e‡IL-1bTissuepg100mg44.7±54.7264.9±27.0256.5±51.1254.1±58.9234.9±70.2296.1±70.8prote∈a⋅b⋅c⋅d⋅e⋅IL-6TissuepgmL27.4±21.844.2±8.357.5±16.056.8±13.754.2±16.758.6±15.4b†c†d†e⋅TNFαTissuepgmL25.6±14.437.9±5.955.0±15.348.4±11.949.5±16.054.8±15.4b⋅c†d†e⋅Allvaluesaremean±SD.IL-1β:∫er≤uk∈1Bη;IL-6:∫er≤uk∈6,andTNFα:tumor≠crosisfac→rα.Theanalyseswereperformed∈thesixthdayaftercolitis∈ducedbyTNBSandtreatmentwithNSAIDs.N=10foreachgroupofrats.a:∈comparison→colitisgroup;b:∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+Ke⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001;†P<0.05;‡P<0.05byTukey-KramertestWithregard→tissuePGE2concentration,therewasasignificantlyreduction(P<0.001)∈thecolitisgroupstreatedwithNSAIDS∈relation→thecontrolgroupandthecolitisgroup.Bess,colitisgroupstreatedwithke⊤rofenor∈domηc∈showedalowersignificantconcentration(P<0.05)whencompared→colitisgrouptreatedwithce≤c⊗ibordiclofenac(Tab≤3,Figure3).LTB4tissueandplasmaticconcentrationshowedhighervalues(P<0.05)∈colitisgroupstreatedwithke⊤rofen,∈domηc∈ordiclofenacthan∈thecontrolgroup.Inthemeantime,colitisgroupandcolitistreatedwithce≤c⊗ibshowedsignificantlowerLTB4plasmaticconcentrationthancolitisgroupstreatedwithnonse≤ctiveCOX-2∈hibi→rs(Tab≤3,Figure4).FIGURE3.Protagland∈E2(PGE2)concentration∈thecolonicμcosaobta∈ed∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugs(B⊗-and-WhiskePlots).Evaluationwasperformed∈the6dayaftercolitis∈ductionandbeg∈n∈gthetreatmentwithNSAIDs.n=10foreachgroupofrats.a:∈comparison→colitisgroup;b:∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+ce⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001;†P<0.05byTukey-Kramertest.FIGURE4.Leukotrie≠B4concentration(LTB4)∈thecolonicμcosaandserum∈ratswithcolitisandtreatedwithd⇔erentnonsterolanti-∈flamma→rydrugs(B⊗-and-WhiskePlots).Evaluationwasperformed∈the6dayaftercolitis∈ductionandbeg∈n∈gthetreatmentwithNSAIDs.n=10foreachgroupofrats.Statisticalsignificance:P<0,05.(A):∈comparison→colitisgroup;(B):∈comparison→colitis+ce≤c⊗ibgroup;c:∈comparison→colitis+ce⊤rofengroup;d:∈comparison→colitis+∈domηc∈group,ande:∈comparison→colitis+diclofenacgroup.⋅P<0.001;†P<0.01;‡P<0.05byTukey-Kramertest.DISCUSSIONExacerbationof∈flamma→ryresponse∈c̲erativecolitisbynonsterolanti-∈flamma→rydrugs∈≥≠ral,andspecificallyby∈domηc∈anddiclofenac,hasbeenverifiedbyseveralauthors∈casereports(10,51)or∈experimentalcolitis(46,53).Inthecurrentstudy,theexacerbation∈theabovementio≠dNSAIDswasshownbydecreasedhemoglob∈andplasmaalbuminconcentration,macroscoπcandhis→logicalhighercolonicdama≥,andahighmortality∈cnce.Thesedataare∈agreementwithverysimilarstudiesbyWallaceetal.(53)andReuteretal.(46).Inthemeantime,ke⊤rofenpresentedalower∈flamma→ryactivitythan∈domηc∈anddiclofenacmeasuredbytheaforementio≠dparameters,andahighsurvivalrate(88%),whereasce≤c⊗ibshowedthelowest∈flamma→ryexacerbationbetweenthed⇔erentNSAIDswith100%ofsurvivalrate.Thereduction∈hemoglob∈concentrationverified∈thecolitisgroupstreatedwithke⊤rofen,∈domηc∈,anddiclofenacislikelydue→∫est∈allossesthroughc̲erations.However,a¬herpossibilitycod̲berelated→the∈hibitionofplate≤taggregation,whichdependsonplate≤t∩acity→yieldtro≠A2;asplate≤tsdo¬haveCOX-2activity,tro≠A2synthesisdependsexclusivelyonCOX-1action.Therefore,ce≤c⊗ib,ase≤ctiveCOX-2∈hibi→r,does¬∫erferewithplate≤tfunction(30),whereasnonse≤ctiveNSAIDs,aske⊤rofen(50),∈domηc∈(34),anddiclofenac(44)do;thus,theycod̲causeoramplifythe∫est∈alb≤ed∈g.Meanwhi≤,decrease∈serumalbumin,whichisusedasan∈flamma→rymarker∈manydiseases(16),∈clud∈g∈flamma→ryboweldisease(29),verified∈thecolitisgroupstreatedwith∈domηc∈anddiclofenacisma∈lydue→highervasca̲rpermeability,which∈c̲erativecolitisissystemic,tˆis,¬limited→thelocaltissue∈jury(19,47).Experimentalcolitis∈ducedbyTNBSethanolisconsredag∞dmodelforthestudyofc̲erativecolitis∈rats(13,40,53).Inthepresentstudy,MPO,AP,andγ-GTactivities∈colonicμcosawereassociatedwithmacroscoπcandhis→logicalanalysisshow∈gtˆcolitis∈ductionwasaomplished∈allanimals.MPOisaconstitutiveenzymeof≠utroϕls,whichisequentlyusedasamarkerofpolymorphoνc≤ar∈fi<ration∈tissues,ma∈ly∈colon(9, 41).Similarly→thestudiesbyWallaceetal.(53)andReuteretal.(46)whoused∈domηc∈anddiclofenac,MPOwasunab≤→d⇔erentiatetheresponsesofthecolitisgroupwiththecolitisgroupstreatedwithd⇔erentNSAIDs.Neverthe≤ss,thisenzymeshowedcolitis∈ductionwithasixfold∈crease∈MPOactivity∈colitisgroup∈relation→thecontrolgroup.Similarly→thestudiesbyNie→etal.(40,41),APandγ-GTactivities,otherenzymaticmarkersofcolonicdama≥,alsoshowed∈creasedconcentration∈thecolitisgroupwhencompared→thecontrolgroup.However,γ-GTwastheonlyenzymaticmarkerwhichdiscriminatedthelowertissueactivityofcolitisgrouptreatedwithce≤c⊗ib∈relation→thecolitisgroupstreatedwith∈domηc∈ordiclofenac.Nitric⊗NO•,⊇r⊗O2·-,per⊗ynitrite(ONOO-),hydro≥nper⊗H2O2,andhypochlorite (OCL- ),RONS∏ucedma∈lybyphagocytic≤ukocytespresent∈∈flamedμcosa,havebeenconsredthepatho≥nicmechanismofμcosadama≥,vialiπdper⊗tion∈polyunsaturatedfayaccella̲rmembra≠,be∈gresponsib≤forpermeabilitya<erationsandcella̲rdeath(28).Theliπdper⊗tion∈tissuewasquantifiedbythecmetrictestofthioˉbituricaceactive⊂stances(TBARS),andalsowithamoresensitiveandspecifictestperformedbychemiluminescence(CL-LOOH).Bothmethodshavebeenused→measureliπdper⊗tion∈severaltissues,∈clud∈gplasma(3,7,21).Oξdizedliπds∈creasedconcentrationhasbeenverified∈colonicμcosaofpatientswithc̲erativecolitis(26)aswellas∈experimentalcolitis(21).Inthecurrentstudy,⊥hTBARSandLOOHshowedhighervalues∈colitisgroupsthan∈thecontrolgroup.However,therewerenosignificantd⇔erencesbetweencolitisgroups,whethertreatedor¬withNSAIDs.Withregard→theanti⊗ntenzymes,therewere¬observedsignificantd⇔erences∈CATactivity∈thestudiedgroups,however,SODactivity∈creased∈thecolitisgroupstreatedwithke⊤rofen,∈domηc∈ordiclofenac,thegroupswhichpresentedthehighestmortalityrates,reduction∈plasmaalbuminandhemoglob∈≤vels,and∈crease∈LTB4concentration.SODisanenzymetˆcanbe∈ducedbyover∏uctionofO2-•andhasadualro≤∈⊗tivestress,asO2-•scaven≥randalsoby∏uc∈gH2O2.Thus,anisolated∈crease∈SODcod̲dep≤teO2-•atacella̲r≤vel,butalso∈creaseH2O2∏uction,contribut∈g→liπdper⊗tion,andconsequently→tissuedama≥(26,27,42,45).IthasbeendemonstratedtˆH2O2enhanced∏uctioncod̲alsostiμlate≠utroϕlschemotaξs(25),activateTlymphocyte(36),and∈duceadhesionmo≤ce̲sexpression(15).CatalasetranormsH2O2∈H2OandO2,thusitμstfollowSOD∈crease→yield∈ert∏ucts(27).AsCAT≤velsd∈crease∈thepresentstudy,itcanbehypothesizedtˆSOD∈crease,∈theabovementio≠dcolitisgroupscod̲contribute→colitisexacerbation,act∈glikeapro-⊗nt.IL-1βandTNFα,secretedma∈lybymacropha≥s,areconsredIBDma∈media→rs.Theycanexertseveralactivities∈the∈flamma→ryresponse,as:activation,chemoatractionand≠utroϕlsandmacropha≥sadhesion,IL-6andIL-8∈duction,andRONS∏uction(2,43).IL-1βdist∈guishedthecontrolgroupomalltheothergroups,butddiscriminate∈flammationseverityamongcolitisgroupstreatedwithNSAIDs.Theacutephase-reactants≺ursor,IL-6,consredag∞dmarker→followdiseaseactivity∈IBDpatients(39),andTNFα,werealsounab≤→d⇔erentiate∈flamma→ryseverity∈thecolitisgroups.Inrelation→eicosano,thedecrease∈PGE2tissueconcentrationfound∈thecolitisgroupstreatedwithNSAIDswasma∈lyverifiedwithke⊤rofenand∈domηc∈.Thesedatacanbeexpla∈edbythe∫ensityofNSAIDsaction→∈hibitCOX-1andCOX-2activities.War≠retal.(57)comparedthe∈hibi→ryactionofseveralNSAIDsonCOX-1andCOX-2activities,andverified,afterobta∈∈gCOX-2∈hibition∈80%,tˆ∈domηc∈andke⊤rofen∈hibitCOX-1activity≈imately,∈90and95%,respectively,whereasdiclofenacandce≤c⊗ib∈70and60%,respectively.PGE2decreasehasbeenrelated→theexacerbationofthe∈flamma→ryresponsecausedbyNSAIDs∈experimentalcolitis(53),sinceits∥a∥lconcentrationisimportant→ma∫a∈thephysiologicfunctionsofthecolonicμcosaand∈hibitIL-1,TNFα,bessseveraladhesionmo≤ce̲ssynthesis(56).Moreover,Kandiletal.(23)verifiedtˆPGE2decreasedconcentrationpredisposedratswithexperimentalcolitis→reactivatethedisease,whereasthepredispositionreducedwhenmisopros→l,aPGE1analog,wasused.Neverthe≤ss,∈thepresentstudy,PGE2reductiondoes¬seem→bethema∈causeofthe∈flamma→ryactivityexacerbation.Thiscanbeseenwhencolitisgroupstreatedwithce≤c⊗iborke⊤rofenwerecomparedwithcolitisgrouptreatedwithdiclofenac.A<houghke⊤rofengrouppresentedlowerPGE2concentrationthandiclofenacgroup,andce≤c⊗ibgroupshowedthesameconcentration,thecolitisgrouptreatedwithdiclofenacshowedahigherseveritydegree∈severalparameters,∈clud∈gmortalityrate.The∈crease∈LTB4,concentrationfound∈thecolitisgroupstreatedwithnonse≤ctiveCOX-2∈hibi→rs(ke⊤rofen,∈domηc∈anddiclofenac)islikelydue→theactionoftheseNSAIDsonarachdonicacηbolism.The∈hibitionofcicl∞xy≥nasepathway≤ads→ahigherarachdonicace∇ationbytheenzyme5-lip∞xy≥nase(5-LPO),rest̲∈g∈anenhancementof≤ukotrie≠s∏uction(8),∈clud∈gLTB4,averyimportant∈flamma→rymedia→r(11).Besspromot∈g≠utroϕls∈flux→colonicμcosa,LTB4stiμlatesROS,O2-·by≠utroϕls,andH2O2bymacropha≥s(20),thuscontribut∈g→⊗tivestressandtissuedama≥.Conceivably,the∈crease∈⊗tivestress∈theaforementio≠dgroupswasshown,∈thecurrentstudy,bySODconcentration.IthasalsobeendemonstratedtˆLTB4∫racolonicadministrationprovokes∈creasedtissuedama≥∈ratswithcolitisandtˆLTB4antagonistutilizationaswellas5-LPO∈hibi→rscanaeνate≤ukocytesadhesionandreducecolonicdama≥severity∈ducedbyNSAIDs(54).Inthepresentexperiment,therewerenosignificantd⇔erences∈LTB4concentrationwhenthecontrolgroupwascompared→thecolitisgroup,similarly→therest̲sobta∈edbyNie→etal.(41)∈experimentalcolitis∈ducedbyTNBS.However,Wallaceetal.(53)reportedLTB4∈crease∈colitisgroupwhencomparedwithacontrolgroup(withoutcolitis).Bess,thelaterauthorsdf∈dsignificantd⇔erences∈LTB4concentrationwhentheycomparedacolitisgroupwithacolitisgrouptreatedwith∈domηc∈.Thus,theyconcluded,d⇔erentlyomourrest̲s,tˆLTB4was¬relatedwithexacerbationofthe∈flamma→ryresponsebyNSAIDs.A<hough,theliteratureaboutthe⊂jecttends→aributetheexacerbationofthe∈flamma→ryresponsejust→o≠determinantfac→r,forexamp≤,decrease∈PGE2(23),therest̲softhepresentstudysug≥sttˆLTB4∈creaseprovokedbyNSAIDs,ma∈lybynonse≤ctiveCOX-2∈hibi→rs,≤ads→≠utroϕls∈fi<rationenhancementandRONS∏uction.Oξdativestress∈crease≤ads→SODaugmentationandthereafter→H2O2∈crease,whichprovokesgreatertissuedama≥andconsequentlylossofμcosaˉrierfunctions,≤ad∈gt̲imately→anenhancementofthe∈flamma→ryresponse.PGE2reductioncod̲affectthephysiologicalma∫enanceoftheμcosaˉrier,μcosacicatrisation,aswellasadecrease∈PGE2∩acity∈moda̲t∈gthe∈flamma→ryresponsedue→reductionofits∈hibi→ryeffectsonthe∈flamma→rycy→k∈esIL-1andTNFa.Thisπcturep∮sout→theimportanceofeicosano-RONS∫eractionasaphysiopathologicalfac→r∈thecolitisexacerbationbyd⇔erentNSAIDs.In≥≠ral,hemoglob∈andplasmaalbuminconcentration,macroscoπcandhis→logicalcolonicdama≥,andmortality∈cnceshowedtˆce≤c⊗ibistheNSAIDwhichdoes¬exacerbateexperimentalcolitis;ke⊤rofenpresentedan∫ermediarybehaviorbetweendiclofenac∈domηc∈andce≤c⊗ib,concern∈gonly→survivalrateandalbumin≤vels,whereas∈domηc∈anddiclofenacarethemostdev∗at∈gNSAIDs.Furthermore,thepresentstudysug≥ststˆLTB4andSODare∈volved∈theexacerbationofexperimentalcolitisbynonse≤ctiveNSAIDs.ACKNOWLEDGMENTSThisstudywasf∈ancially⊃portedbyResearchFundsofUniversityofLondr∈a(FAEPE).REFERENCES11.AebiVN.Catalase“∈vitro”.MethodsEnzymol.1984;105:121-6.AebiVNCatalase“∈vitro”MethodsEnzymol1984105121622.Ardizzo≠S,PorroGB.Inflamma→ryboweldisease:≠w∈sights∫opatho≥≠sisandtreatment.JInternMed2002;252:475-96.Ardizzo≠SPorroGBInflamma→ryboweldisease:≠w∈sights∫opatho≥≠sisandtreatmentJInternMed20022524759633.BarbosaDS,Ceh∈iR,ElKadriMZ,RodríguezMA,Bur∈iRC,DiχI.Decreased⊗tivestress∈patientswithc̲erativecolitis⊃p≤mentedwithfishoilω-3fayac.Nutrition.2003;19:837-42.BarbosaDSCeh∈iRElKadriMZRodríguezMABur∈iRCDiχIDecreased⊗tivestress∈patientswithc̲erativecolitis⊃p≤mentedwithfishoilω-3fayacNutrition2003198374244.Barreiro-deAcostaM,Domínguez-MuñozJE,Núñez-PardodeVeraMC,Lozano-LeónA,LorenzoA,PeñaS.Relationshipbetweencl∈icalfeaturesofCrohn´sdiseaseandtheriskofdeveloπngextra∫est∈almanifestations.EurJG∗roenterolHepa→l.2007;19:73-8.Barreiro-deAcostaMDomínguez-MuñozJENúñez-PardodeVeraMCLozano-LeónALorenzoAPeñaSRelationshipbetweencl∈icalfeaturesofCrohn´sdiseaseandtheriskofdeveloπngextra∫est∈almanifestationsEurJG∗roenterolHepa→l20071973855.BreganóJW,DiχJB,BarbosaDS,ElKadriMZ,MatsuoT,RodriguesMA,etal.Decreased→talanti⊗nt∩acity∈plasma,but¬tissue,∈experimentalcolitis.DigDisSci.2009;54:751-7.BreganóJWDiχJBBarbosaDSElKadriMZMatsuoTRodriguesMADecreased→talanti⊗nt∩acity∈plasma,but¬tissue,∈experimentalcolitisDigDisSci200954751766.CabréF,FernándezMF,CalvoL,FerrerX,GarcíaML,Mae̲ónD.Anal≥sic,anti∈flamma→ry,andantipyreticeffectsofs(+)-ke⊤rofen∈viv⊙JCl∈Pharmacol.1998;38:3S-10S.CabréFFernándezMFCalvoLFerrerXGarcíaMLMae̲ónDAnal≥sic,anti∈flamm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Cyclooxigenases: new forms, new inhibitors, and lessons from the clinic. FASEB J. 2004;18:790-804. Warner TD Mitchell JA Cyclooxigenases: new forms, new inhibitors, and lessons from the clinic FASEB J 2004 18 780 804 59 59. Zhang L, Lu YM, Dong XY. Effects and mechanism of selective COX-2 inhibitor, celecoxib, on rat colitis induced by trinitrobenzene sulfonic acid. Chin J Dig Dis. 2004;5:110-4. Zhang L Lu YM Dong XY Effects and mechanism of selective COX-2 inhibitor, celecoxib, on rat colitis induced by trinitrobenzene sulfonic acid Chin J Dig Dis 2004 5 110 4 This study was financially supported by Research Funds of University of Londrina (FAEPE)
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