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Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose. / This study was designed to evaluate the effect of long-term pre-treatment with celecoxib, a cyclooxygenase-2 inhibitor, on myocardial infarct size. Since celecoxib is an anti-inflammatory and that myocardial damages can be present in the occurrence of inflammatory processes, inhibition of inflammation should hypothetically reduce the size of an eventual infarct. Celecoxib (3 mg/kg/day i.p.) or vehicle (DMSO 50%; EtOH 15%; distilled water) was administered chronically to male Sprague-Dawley rats (n=18 per group) through ALZET osmotic pumps for 28 days. Under anaesthesia, the animals were then subjected to left anterior descending coronary artery occlusion for 40 minutes, followed by 24-hour reperfusion. The results show that myocardial infarct size in celecoxib-treated rats was significantly reduced compared to the control group (37.5±2.5% versus 48.0±2.6% of the area at risk, p < 0.05). Accumulation of neutrophils, estimated by myeloperoxidase levels, indicated an increase in the ischemic area without any significant difference between groups. No significant difference was observed between the treated and vehicle groups in terms of plasma prostaglandin E2 and tumour necrosis factor-alpha. Apoptosis, evaluated by Bax/Bcl-2 and terminal dUTP nick-end labelled-positive cells, was significantly decreased in the subendocardial layer of the ischemic area in celecoxib-treated rats. Adenosine diphosphate-induced platelet aggregation in whole blood suggested that celecoxib diminished platelet aggregation. This study indicates that pre-treatment with celecoxib can reduce infarct size by a mechanism which may involve apoptosis.

Chu Wai Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-96). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / Publication --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Table of Contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of gastric cancer --- p.1 / Chapter 1.2 --- Risk factors associated with gastric cancer --- p.7 / Chapter 1.3 --- Prevention of Gastric Cancer --- p.9 / Chapter 1.4 --- H. pylori eradication and gastric cancer development --- p.11 / Chapter 1.5 --- Non-steroidal anti-inflammatory drugs and gastric cancer prevention --- p.13 / Chapter 1.6 --- COX-2 independent pathway --- p.14 / Chapter 1.7 --- Animal model of gastric cancer --- p.15 / Chapter 1.8 --- Microarray system --- p.16 / Chapter 1.9 --- Hypothesis --- p.18 / Chapter 1.10 --- Aim of study --- p.19 / Chapter Chapter 2 --- Chemoprevention of gastric cancer by celecoxib --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Material and Methods --- p.22 / Chapter 2.2.1 --- Animals --- p.22 / Chapter 2.2.2 --- Chemicals --- p.22 / Chapter 2.2.3 --- Study design --- p.23 / Chapter 2.2.4 --- Cell Culture --- p.24 / Chapter 2.2.5 --- Celecoxib treatment --- p.24 / Chapter 2.2.6 --- Cell proliferation assay --- p.25 / Chapter 2.3 --- Results --- p.26 / Chapter 2.3.1 --- Chemoprevention of gastric cancer by celecoxib in rats --- p.26 / Chapter 2.3.2 --- Effects of celecoxib on growth of human gastric cancer cells --- p.29 / Chapter 2.4 --- Discussion --- p.30 / Chapter 2.4.1 --- MNNG induced gastric cancer effectively --- p.30 / Chapter 2.4.2 --- Celecoxib significantly suppressed gastric carcinogenesis in rats --- p.31 / Chapter 2.4.3 --- Celecoxib inhibited the growth of MKN 45 in a concentration-dependent manner --- p.31 / Chapter 2.4.4 --- Celecoxib may exert its anti-tumor property through COX independent pathway --- p.32 / Chapter Chapter 3 --- Gene expression profiles of celecoxib treated rat gastric tumor and human gastric cells --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Material and Methods --- p.34 / Chapter 3.2.1 --- RNA extraction --- p.34 / Chapter 3.2.2 --- Target preparation and Array hybridization --- p.35 / Chapter 3.2.3 --- Post-hybridization processing and Scanning --- p.36 / Chapter 3.2.4 --- Microarray data analysis --- p.36 / Chapter 3.2.5 --- Quantitative RT-PCR --- p.37 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- Gene expression profiles of rat gastric tumors --- p.39 / Chapter 3.3.1.1 --- Genes differentially expressed in MNNG induced gastric tumors --- p.39 / Chapter 3.3.1.2 --- Genes differentially expressed in celecoxib treated group --- p.42 / Chapter 3.3.1.3 --- Mechanisms underlying chemoprevention of celecoxib --- p.43 / Chapter 3.3.2 --- Verification of gene expression by quantitative RT-PCR --- p.55 / Chapter 3.3.3 --- Confirmation of the gene expression profiles in human by quantitative RT-PCR --- p.59 / Chapter 3.4 --- Discussions --- p.63 / Chapter Chapter 4 --- Effects of celecoxib on Akt pathway in gastric cancer cells --- p.68 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Material and methods --- p.72 / Chapter 4.2.1 --- Protein extraction --- p.72 / Chapter 4.2.2 --- Western blotting --- p.72 / Chapter 4.3 --- Results --- p.74 / Chapter 4.3.1 --- Expression of the Akt pathway after treatment with celecoxib --- p.74 / Chapter 4.4 --- Discussions --- p.78 / Chapter Chapter 5 --- Conclusion --- p.82 / References --- p.87

Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose. / This study was designed to evaluate the effect of long-term pre-treatment with celecoxib, a cyclooxygenase-2 inhibitor, on myocardial infarct size. Since celecoxib is an anti-inflammatory and that myocardial damages can be present in the occurrence of inflammatory processes, inhibition of inflammation should hypothetically reduce the size of an eventual infarct. Celecoxib (3 mg/kg/day i.p.) or vehicle (DMSO 50%; EtOH 15%; distilled water) was administered chronically to male Sprague-Dawley rats (n=18 per group) through ALZET osmotic pumps for 28 days. Under anaesthesia, the animals were then subjected to left anterior descending coronary artery occlusion for 40 minutes, followed by 24-hour reperfusion. The results show that myocardial infarct size in celecoxib-treated rats was significantly reduced compared to the control group (37.5±2.5% versus 48.0±2.6% of the area at risk, p < 0.05). Accumulation of neutrophils, estimated by myeloperoxidase levels, indicated an increase in the ischemic area without any significant difference between groups. No significant difference was observed between the treated and vehicle groups in terms of plasma prostaglandin E2 and tumour necrosis factor-alpha. Apoptosis, evaluated by Bax/Bcl-2 and terminal dUTP nick-end labelled-positive cells, was significantly decreased in the subendocardial layer of the ischemic area in celecoxib-treated rats. Adenosine diphosphate-induced platelet aggregation in whole blood suggested that celecoxib diminished platelet aggregation. This study indicates that pre-treatment with celecoxib can reduce infarct size by a mechanism which may involve apoptosis.

Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease with no available pharmacological treatment. AAA formation reduces the structural integrity of the vessel and increases the susceptibility to rupture. The inflammatory response within human aneurysmal tissue is characterized by increased expression of cyclooxygenase-2 (COX-2). Similarly, in a mouse model of the disease induced by chronic Angiotensin II (AngII) infusion, we have shown that COX-2 expression in the abdominal aortic smooth muscle layer increases early in the development of the disease. Furthermore, genetic or pharmacological inactivation of COX-2 prior to disease initiation reduces AAA incidence. The current study utilized nonhyperlipidemic mice to determine the effectiveness of COX-2 inhibition initiated after AAA formation. COX-2 inhibitor treatment was initiated 5 days after beginning the AngII infusion, a time-point where significant aneurysmal pathology is observed. COX-2 inhibition with celecoxib significantly reduced the incidence as well as severity of AAAs as compared to the control group. Celecoxib treatment also protected the mice from aortic rupture and death. AAA development is characterized by degradation of the aortic smooth muscle layer with loss of the contractile phenotype. We found that the effectiveness of celecoxib was associated with significantly increased mRNA expression of alpha-actin, SM22alpha and desmin, all of which are markers of a differentiated smooth muscle cell phenotype. Celecoxib treatment also decreased mRNA expression of a marker of dedifferentiated smooth muscle (hyaluronic acid synthase 2). We also examined the role of altered expression of COX-2 in the increased susceptibility of the abdominal segment to AAA formation. We found a prolonged and greater induction of COX-2 in the abdominal aortic smooth muscle layer in contrast to a transient induction of COX-2 in the other regions of the aorta throughout disease progression. Overall, these findings suggest that COX-2 plays an important role in AAA development in mice, and COX-2 inhibition with celecoxib attenuates progression of aneurysm development by maintaining a differentiated phenotype in abdominal aortic smooth muscle cells.

The inflammatory arthritis Ankylosing Spondylitis (AS) is linked to the human leucocyte antigen HLA-B27. HLA-B27 is thought to drive AS because it misfolds during assembly in the endoplasmic reticulum (ER), inducing ER cell stress. Modulating HLA-B27 folding in the ER is therefore a therapeutic target pathway. The recent discovery of polymorphisms in the ER-resident peptidase ERAP1 that can impact on HLA-B27 and AS, makes ERAP1 one such target. Exosomes are small, typically 50-200 nm sized particles, formed in the endosomal recycling pathway, which can be released into the extracellular environment. Exosomes have a wide range of biological activities depending on the cell type of origin, and on the delivered cargo, which can include bio-active proteins, lipids, mRNA and miRNA. There is interest in the use of exosomes as drug delivery agents. Here, exosomes were studied as a delivery agent to modulate ERAP1, as a potential therapeutic tool for the treatment of AS. Exosomes, isolated from cell lines including CEM and Jurkat (T cell lineage), Jesthom (B cell lineage), U937 (monocyte lineage) and the epithelial HeLa cell line, were characterized by nanoparticle tracking analysis, flow cytometry and immunoblotting using markers including CD9, CD63, CD81 and TSG101. Differential expression of these markers in the immune cell lines indicated the complexity of defining exosomes. EVs were then tested using cell penetrating peptides, electroporation, lipid transfection and sonication for their ability to load FITC-siRNA or FITC-antibody as cargo. Significantly, post-loading RNase A or trypsin incubation demonstrated that many techniques do not lead to efficient cargo loading of exosomes. Sonication proved the most effective technique, with up to 30% efficiency. Loading of exosomes with ERAP1-targetted siRNA did not however lead to notable ERAP1 inhibition. The data indicates that external loading of exosomes with cargo remains a significant challenge in developing exosomes as therapeutic tools.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O sobrepeso induzido por uma dieta rica em gordura atrasa a cicatrização através do prolongamento da fase inflamatória, entretanto, quando recebem uma dieta obesogênica, alguns ratos são suscetíveis a desenvolver sobrepeso, enquanto outros são resistentes. Drogas anti-inflamatórias não-esteróides são frequentemente utilizadas para reduzir a inflamação. Este estudo investigou a cicatrização cutânea em ratos propensos a obesidade induzida por dieta (DIO) e em ratos resistentes a dieta (DR) e avaliou a participação da administração do celecoxibe na cicatrização cutânea destes animais. Ratos machos foram alimentados com uma dieta padrão (Controle, C) ou com uma dieta rica em gordura saturada (30%). Após 19 semanas, o grupo experimental foi subdividido nos grupos DIO e DR. Uma lesão excisional foi feita e os animais foram mortos 7 ou 14 dias depois. Os grupos tratados receberam uma dose diária de 5 ou 10 mg/kg/dia de celecoxibe a partir de dois dias antes da lesão até 7 dias após a lesão, quando foram mortos. O peso corporal foi maior no grupo DIO comparado aos grupos C e DR. A gordura retroperitoneal foi maior no grupo DIO do que nos grupos C e DR e foi maior no grupo DR do que no grupo C. O tratamento com o celecoxibe não alterou o maior peso corporal apresentado pelo grupo DIO ou a maior porcentagem de gordura retroperitoneal apresentada pelos grupos DIO e DR. Todos os grupos tratados com celecoxibe 10 mg apresentaram atraso na cicatrização e não foram mais analisados. O grupo DIO apresentou intolerância a glicose, e ambos os grupos DIO e DR apresentaram atraso na contração e na reepitelização da lesão. O tratamento com celecoxibe 5 mg reverteu a intolerância a glicose no grupo DIO e a contração atrasada nos grupos DIO e DR. Comparado ao grupo DR, o grupo DIO apresentou maior quantidade de células inflamatórias, assim como maiores níveis de peroxidação lipídica. O tratamento com celecoxib (5 mg) não reduziu o número de PMN, mas reduziu o número de mastócitos no grupo DIO, o número de macrófagos e a peroxidação lipídica em ambos os grupos. A diferenciação miofibroblástica e o remodelamento dos vasos foram atrasados em ambos os grupos DIO e DR. O tratamento com celecoxibe 5 mg aumentou a diferenciação miofibroblástica, mas não alterou os vasos sanguíneos. A quantidade de hidroxiprolina foi semelhante nos grupos DIO e DR. O tratamento com celecoxibe 5 mg aumentou a quantidade de hidroxiprolina em todos os grupos. A quantidade de nitrito foi menor no grupo do que no grupo DR. A expressão de TNF-&#945; foi aumentada no grupo DIO comparada ao grupo DR. Nossos resultados mostraram que os ratos DIO assim como os ratos DR apresentam retardo na cicatrização cutânea devido principalmente a intensa inflamação, e a baixa dose de celecoxibe acelerou o reparo cutâneo nestas condições. / Overweight induced by high-fat diet delays wound healing through elongation of inflammatory phase, however, when receiving an obesogenic diet, some rats are susceptible to eveloping the overweight phenotype, whereas others are resistant. Nonsteroidal antiinflammatory drugs are frequently used to reduce the inflammation. This study investigated cutaneous wound healing in diet-induced obesity (DIO)-prone and diet-resistant (DR) rats and evaluated the contribution of celecoxib administration on cutaneous wound healing of these animals. Male rats were fed with a standard (Control, C) or high-saturated fat (30%) diet. After 19 weeks, experimental group was subdivided into DIO and DR groups. An excisional lesion was made and the animals were killed 7 or 14 days later. Treated groups received a daily dose of celecoxib 5 or 10 mg/kg/day from two days before wounding until 7 days after wounding when were killer. The body weight was higher in the DIO group compared to the C and DR groups. Retroperitoneal fat was higher in the DIO group than in the C and DR groups and was higher in the DR group than in the C group. Celecoxib-treatment did not alter the higher body weight presented by DIO group or higher retroperitoneal fat percentage displayed by DIO and DR groups. All groups treated with celecoxib 10 mg showed delayed wound healing, and werent further analysed. The DIO group presented glucose intolerance, and both the DIO and DR groups presented delayed wound contraction and re-epithelialisation. The celecoxib 5 mg-treatment reversed the glucose intolerance in the DIO group and the delayed contraction in the DIO and DR groups. Compared to the DR group, the DIO group displayed higher amounts of inflammatory cells as well as higher levels of lipid peroxidation. Celecoxib-treatment (5 mg) did not reduce the number of PMN, but reduce mast cells number in DIO group and macrophages number and lipid peroxidation in both groups. Myofibroblastic differentiation and vessel remodeling were delayed in both the DIO and DR groups. The celecoxib 5 mg-treatment increased the myofibroblastic differentiation, but did not alter the blood vessels. Hydroxyproline levels were similar in the DIO and DR groups. The celecoxib 5 mg-treatment increased the hydroxyproline levels in all groups. Nitrite levels were lower in the DIO group than in the DR group. TNF-&#945; expression was increased in the DIO group compared to the DR group. Our results showed that DIO as well as DR rats present delays in cutaneous wound healing due mainly to intense inflammation, and a lowdose of celecoxib accelerates cutaneous repair in these conditions.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O sobrepeso induzido por uma dieta rica em gordura atrasa a cicatrização através do prolongamento da fase inflamatória, entretanto, quando recebem uma dieta obesogênica, alguns ratos são suscetíveis a desenvolver sobrepeso, enquanto outros são resistentes. Drogas anti-inflamatórias não-esteróides são frequentemente utilizadas para reduzir a inflamação. Este estudo investigou a cicatrização cutânea em ratos propensos a obesidade induzida por dieta (DIO) e em ratos resistentes a dieta (DR) e avaliou a participação da administração do celecoxibe na cicatrização cutânea destes animais. Ratos machos foram alimentados com uma dieta padrão (Controle, C) ou com uma dieta rica em gordura saturada (30%). Após 19 semanas, o grupo experimental foi subdividido nos grupos DIO e DR. Uma lesão excisional foi feita e os animais foram mortos 7 ou 14 dias depois. Os grupos tratados receberam uma dose diária de 5 ou 10 mg/kg/dia de celecoxibe a partir de dois dias antes da lesão até 7 dias após a lesão, quando foram mortos. O peso corporal foi maior no grupo DIO comparado aos grupos C e DR. A gordura retroperitoneal foi maior no grupo DIO do que nos grupos C e DR e foi maior no grupo DR do que no grupo C. O tratamento com o celecoxibe não alterou o maior peso corporal apresentado pelo grupo DIO ou a maior porcentagem de gordura retroperitoneal apresentada pelos grupos DIO e DR. Todos os grupos tratados com celecoxibe 10 mg apresentaram atraso na cicatrização e não foram mais analisados. O grupo DIO apresentou intolerância a glicose, e ambos os grupos DIO e DR apresentaram atraso na contração e na reepitelização da lesão. O tratamento com celecoxibe 5 mg reverteu a intolerância a glicose no grupo DIO e a contração atrasada nos grupos DIO e DR. Comparado ao grupo DR, o grupo DIO apresentou maior quantidade de células inflamatórias, assim como maiores níveis de peroxidação lipídica. O tratamento com celecoxib (5 mg) não reduziu o número de PMN, mas reduziu o número de mastócitos no grupo DIO, o número de macrófagos e a peroxidação lipídica em ambos os grupos. A diferenciação miofibroblástica e o remodelamento dos vasos foram atrasados em ambos os grupos DIO e DR. O tratamento com celecoxibe 5 mg aumentou a diferenciação miofibroblástica, mas não alterou os vasos sanguíneos. A quantidade de hidroxiprolina foi semelhante nos grupos DIO e DR. O tratamento com celecoxibe 5 mg aumentou a quantidade de hidroxiprolina em todos os grupos. A quantidade de nitrito foi menor no grupo do que no grupo DR. A expressão de TNF-&#945; foi aumentada no grupo DIO comparada ao grupo DR. Nossos resultados mostraram que os ratos DIO assim como os ratos DR apresentam retardo na cicatrização cutânea devido principalmente a intensa inflamação, e a baixa dose de celecoxibe acelerou o reparo cutâneo nestas condições. / Overweight induced by high-fat diet delays wound healing through elongation of inflammatory phase, however, when receiving an obesogenic diet, some rats are susceptible to eveloping the overweight phenotype, whereas others are resistant. Nonsteroidal antiinflammatory drugs are frequently used to reduce the inflammation. This study investigated cutaneous wound healing in diet-induced obesity (DIO)-prone and diet-resistant (DR) rats and evaluated the contribution of celecoxib administration on cutaneous wound healing of these animals. Male rats were fed with a standard (Control, C) or high-saturated fat (30%) diet. After 19 weeks, experimental group was subdivided into DIO and DR groups. An excisional lesion was made and the animals were killed 7 or 14 days later. Treated groups received a daily dose of celecoxib 5 or 10 mg/kg/day from two days before wounding until 7 days after wounding when were killer. The body weight was higher in the DIO group compared to the C and DR groups. Retroperitoneal fat was higher in the DIO group than in the C and DR groups and was higher in the DR group than in the C group. Celecoxib-treatment did not alter the higher body weight presented by DIO group or higher retroperitoneal fat percentage displayed by DIO and DR groups. All groups treated with celecoxib 10 mg showed delayed wound healing, and werent further analysed. The DIO group presented glucose intolerance, and both the DIO and DR groups presented delayed wound contraction and re-epithelialisation. The celecoxib 5 mg-treatment reversed the glucose intolerance in the DIO group and the delayed contraction in the DIO and DR groups. Compared to the DR group, the DIO group displayed higher amounts of inflammatory cells as well as higher levels of lipid peroxidation. Celecoxib-treatment (5 mg) did not reduce the number of PMN, but reduce mast cells number in DIO group and macrophages number and lipid peroxidation in both groups. Myofibroblastic differentiation and vessel remodeling were delayed in both the DIO and DR groups. The celecoxib 5 mg-treatment increased the myofibroblastic differentiation, but did not alter the blood vessels. Hydroxyproline levels were similar in the DIO and DR groups. The celecoxib 5 mg-treatment increased the hydroxyproline levels in all groups. Nitrite levels were lower in the DIO group than in the DR group. TNF-&#945; expression was increased in the DIO group compared to the DR group. Our results showed that DIO as well as DR rats present delays in cutaneous wound healing due mainly to intense inflammation, and a lowdose of celecoxib accelerates cutaneous repair in these conditions.

nÃo hà / Esse estudo tem por finalidade avaliar os efeitos de inibidores seletivos da COX (cetorolaco e celecoxibe) e de inibidores nÃo seletivos (indometacina) da COX sobre o transporte ileal de Ãgua e eletrÃlitos em ratos anestesiados. TambÃm se propÃe a avaliar as alteraÃÃes vilo/cripta do Ãleo de ratos tratados com inibidores seletivos e nÃo seletivos da COX-1 e 2 (cetorolaco-inibidor especÃfico da COX-1 na dose de 3mg/Kg, celecoxibe-inibidor especÃfico da COX-2 e indometacina-inibidor nÃo seletivo da COX). Os animais foram tratados per os durante trÃs dias consecutivos com uma das seguintes substÃncias: cetorolaco (3mg/Kg), celecoxibe (10mg/Kg), indometacina (5mg/Kg), cetorolaco + celecoxibe, salina 0,9% ou tampÃo fosfato. Todos os AINES aqui utilizados foram diluÃdos em soluÃÃo salina (Nacl a 0,9%) exceto a indometacina que foi diluÃda em soluÃÃo tampÃo fosfato. ApÃs jejum de 24 horas com livre acesso à Ãgua, os animais foram anestesiados com Uretana (1,2mg /kg corporal, i.p). A seguir realizou-se laparotomia mediana para isolamento do segmento a ser perfundido. CÃnulas foram introduzidas nas extremidades proximal e distal do segmento mediante criaÃÃo de fÃstulas. O segmento isolado e as cÃnulas formaram o circuito que foi perfundido. Para a perfusÃo foi utilizada soluÃÃo modificada de Ringer e FenolsulftaleÃna 50mg/L como marcador nÃo absorvÃvel. O perfusato foi coletado em tubos de ensaio apÃs 40min (03 amostras). TambÃm foram obtidas 3 (trÃs) amostras no inÃcio e no final (03 amostras) do experimento, para determinaÃÃo dos parÃmetros controle. Foram determinadas as diferenÃas entre as amostras controle e as coletas do perfusato quanto aos valores das concentraÃÃes de sÃdio, potÃssio e cloreto (mmol/L). Ao final do experimento, os animais foram sacrificados e o segmento ileal perfundido retirado, sendo imediatamente pesado, depois retirados anÃis distais de aproximadamente 0,5 cm para o histopatolÃgico. Nova mediÃÃo de peso desse segmento foi realizada apÃs o mesmo ser mantido em estufa a 100oC por 48h, de modo a permitir a correÃÃo dos parÃmetros funcionais. A administraÃÃo de cetorolaco aos animais promoveu secreÃÃo ileal de Ãgua, de sÃdio, cloreto e potÃssio. Por outro lado, o tratamento com celecoxib, sozinho ou em associaÃÃo com cetorolaco, bem como o tratamento com indometacina nÃo desencadearam alteraÃÃes significativas na secreÃÃo de Ãgua, sÃdio, cloro e potÃssio, quando comparado com os grupos controles. Em relaÃÃo as alteraÃÃes morfomÃtricas, os tratamentos com celecoxibe sozinho ou em associaÃÃo com cetorolaco, promoveram um aumento da relaÃÃo vilo/cripta. Por outro lado, a administraÃÃo de cetorolaco aos animais nÃo modificou a relaÃÃo vilo/cripta quando comparado com o controle. Por outro lado, no tratamento com indometacina, a administraÃÃo de indometacina apresentou uma diminuiÃÃo na relaÃÃo vilo/cripta, quando comparado com o controle. Baseado nestes dados podemos concluir, que a inibiÃÃo da COX-1 pelo cetorolaco, mas nÃo a inibiÃÃo da COX-2 pelo celecoxib, desencadeia uma alteraÃÃo funcional na secreÃÃo de Ãgua e eletrÃlitos no Ãleo, pois nÃo houve lesÃo do epitÃlio na anÃlise histolÃgica do intestino de ratos tratados com a cetorolaco. / The aim of this study was to evaluate the effects of selective and non-selective COX inhibitors on water and electrolyte transport and the villous/crypt ratio in the ileum of anesthetized rats. Thirty-six animals distributed in 6 groups were treated with 3mg/Kg ketorolac (a selective COX-1 inhibitor), 10mg/Kg celecoxib (a selective COX-2 inhibitor), 5mg/Kg indometacin (a non-selective COX inhibitor),ketorolac+celecoxib, 0.9% saline solution or phosphate buffer for 3 consecutive days. Ketorolac and celecoxib were diluted in 0.9% saline solution, while indometacin was diluted in phosphate buffer. Following 24 hours of fasting with access to water ad libitum, the animals were anesthetized with 1.2mg/Kg urethane i.p. and submitted to median laparotomy to isolate an ileal segment for perfusion. Cannulae were introduced through surgically created fistulas in the proximal and distal extremities of the segment. Perfusion was performed with modified Ringer solution containing 50mg/L phenolsulfonphthalein (a non-absorbable marker). After 40 minutes, 3 samples of perfusate were collected. In addition, 3 control samples were collected at baseline and by the end of the experiment for comparison of sodium, potassium and chloride concentrations (mmol/L). Finally the animals were euthanized, the extremities of the perfused segment were cut off (5-mm rings) for histopathological examination and the segment was weighed. After 48 hours of storage at 100oC, the segment was weighed again in order to correct the functional parameters. The administration of ketorolac promoted secretion of ileal water, sodium, potassium and chlorine. However, treatment with celecoxib alone or with ketorolac, and indomethacin treatment did not induce significant changes in the secretion of water, sodium, potassium and chlorine, when compared with control groups. In the histological evaluation, treatment with celecoxib alone or in combination with ketorolac, induced an increase in villous / crypt ration. On the other hand, ketorolac administration did not change the villous / crypt ration when we compared with the control. Indomethacin treatment induced a decrease in villous / crypt ration compared with the control. Based on these data we can conclude that inhibition of COX-1 by ketorolac, but not COX-2 inhibition by celecoxib, induced a functional change in the ileal water and electrolytes secretion, since there was not epithelial damage in the histological analysis of intestine of rats treated with ketorolac.

Einleitung: Die Mucositis enoralis stellt die bedeutendste und schwerwiegendste frühe Nebenwirkung bei der Strahlentherapie fortgeschrittener Kopf-Hals-Tumoren dar. Sie führt häufig zu einer Unterbrechung der Behandlung, mit der Folge einer reduzierten Tumorheilungschance. Des Weiteren wirkt sie sich nachteilig auf die Lebensqualität aus, erhöht das Risiko später Nebenwirkungen und Infektionen und stellt einen erheblichen Kostenfaktor dar. Trotz umfangreicher experimenteller und klinischer Untersuchungen wurde bisher keine allgemein anerkannte Strategie zur Prophylaxe oder Therapie der Mucositis enoralis klinisch etabliert. Die Blockade der Cyclooxygenase-2 (COX-2), welche in einer Reihe von Tumoren überexprimiert wird, wird in der Kombination mit einer Strahlentherapie diskutiert. Außerdem kommen COX-2-Inhibitoren als entzündungshemmende Medikamente therapiebegleitend zum Einsatz. C-Jun und NF-kB p50 sind Transkriptionsfaktoren, die möglicherweise in der Pathogenese der radiogenen Mukositis eine wichtige Rolle spielen. Ziel der Untersuchungen: Ziel der vorliegenden Arbeit ist es, die Wirkung von Celecoxib, einem selektiven COX-2-Inhibitor, auf die Strahlenreaktion der oralen Mukosa (Zellzahl, Epitheldicke) und die epitheliale Expression von c-Jun und NF-kB p50 im etablierten Tiermodell der Schleimhaut der Zungenunterseite der Maus zu untersuchen. So soll festgestellt werden, ob eine Interaktion zwischen den begleitenden Entzündungsreaktionen und der eigentlichen epithelialen Strahlenreaktion besteht. Material und Methoden: In vorangegangenen Untersuchungen wurden die Schnauzen von Mäusen des Stammes C3H/Neu mit 5x3 Gy/Woche über 2 Wochen (Tag 0-4, 7-14) bestrahlt (200 kV Röntgenstrahlung). In einer weiteren Versuchsgruppe erhielten die Tiere täglich an den Tagen 0 bis 13 25 mg/kg/Tag Celecoxib oral per Schlundsonde. Im vierzehntägigen Untersuchungszeitraum wurden täglich jeweils 5 Mäuse je Versuchsgruppe getötet, deren Zungen entnommen und fixiert. In diesem Zustand wurde das Material von der Autorin dieser Arbeit übernommen. Mit Hilfe eines Zählrasters wurden in den vorliegenden Untersuchungen die Zellzahl, die Epitheldicke und die Expression von c-Jun und NF-kB p50 im Epithel der Zungenunterseite der unbehandelten Kontrolle (n = 5), während alleiniger Bestrahlung, sowie nach Bestrahlung und Gabe von Celecoxib manuell erfasst. Aufgrund der geringen Tierzahlen pro Untersuchungszeitpunkt (n = 5) und Versuchsgruppe wurde auf eingehende statistische Analysen verzichtet. Die vorliegende Arbeit beschränkt sich auf eine beschreibende Darstellung des Verlaufs der Einzelparameter über den Gesamtzeitraum. Ergebnisse: Das unbehandelte Epithel besteht aus 402 ± 11 Zellen, wobei 281 ± 8 Zellen der Germinativ- und 121 ± 4 Zellen der funktionellen Schicht angehören. Die Dicke des Gesamtepithels beträgt 68 ± 3 μm. Davon nehmen Germinativ- und Keratinschicht je 15 ± 1 μm und die funktionelle Schicht 38 ± 4 μm ein. Im Kontrollepithel wird c-Jun von 81 % der Zellen der funktionellen und von 80 % der Zellen der Germinativschicht exprimiert, wobei die funktionelle Schicht scheinbar eine größere Färbeintensität aufweist. NF-kB p50 wird von 79 % der Epithelzellen exprimiert, wobei eine stärkere Expression in der Germinativschicht zu beobachten ist. Bei alleiniger fraktionierter Bestrahlung verringert sich die Zellzahl innerhalb der ersten Woche auf 68 % des Kontrollwertes und bleibt anschließend trotz weiterer Bestrahlung weitestgehend konstant. Der größere Anteil an Zellen geht dabei in der funktionellen Schicht verloren. Die Epitheldicke nimmt in der ersten Bestrahlungswoche bis auf 113 % zu und liegt in der folgenden Woche im normalen bis subnormalen Bereich. Die epitheliale c-Jun-Expression nimmt unmittelbar nach Bestrahlungsbeginn zu und liegt anschließend während des gesamten Beobachtungszeitraums über dem Kontrollbereich. Während der gesamten Bestrahlung liegt die Färbeintensität der funktionellen Schicht weiterhin über derer der Germinativschicht. Auch bei NF-kB p50 nimmt die Färbeintensität in der ersten Bestrahlungswoche zu und bleibt anschließend erhöht. Der Anteil NF-kB p50 exprimierender Zellen ist in der Germinativschicht scheinbar größer als in der funktionellen Schicht. Unter zusätzlicher Celecoxibgabe liegen die Zellzahlen vom 6.-12. Tag des Beobachtungszeitraums vermutlich unterhalb derjenigen bei alleiniger Bestrahlung. Dieser Effekt ist besonders innerhalb der Germinativschicht sichtbar. Der Anstieg der Epitheldicke in der ersten Bestrahlungswoche fällt bei zusätzlicher Celecoxibtherapie stärker aus. Im weiteren Verlauf sind kaum Differenzen zu verzeichnen. Die epitheliale Färbeintensität von c-Jun unterscheidet sich kaum von den alleinig bestrahlten Tieren. Bei NF-kB p50 ist dies mit Ausnahme einer scheinbar geringeren Expressionsintensität am 8. und 13. Beobachtungstag ebenso. Schlussfolgerungen: Bei der frühen Strahlenreaktion der Mundschleimhaut werden c-Jun und NF-kB p50 verändert exprimiert. Celecoxib reduziert im Vergleich zur alleinigen Bestrahlung die Zellzahl und verstärkt die Zunahme der Epitheldicke. Es hat keinen Einfluss auf die Expression von c-Jun und NF-kB p50. Somit ist die Regulation der Aktivität von c-Jun und NF-kB p50 unabhängig von der Aktivität von COX-2 erfolgt.