O objetivo deste estudo foi avaliar o efeito da sinvastatina, isolada e associada ao α-tocoferol e à L-arginina, sobre os inibidores endógenos da óxido nítrico sintase, os metabólitos do óxido nítrico e tióis, em pacientes hipercolesterolêmicos. Analisou-se um grupo de 16 pacientes hipercolesterolêmicos que seguiram o seguinte protocolo: período de washout (sem medicação), 1 mês; sinvastatina (20mg/dia), 2 meses; sinvastatina (20mg/dia) + α-tocoferol (400U/dia), 2 meses; sinvastatina (20mg/dia, washout), 1 mês; sinvastatina (20mg/dia) + L-arginina (7g/dia), 2 meses. A sinvastatina reduziu significativamente as concentrações do colesterol total e LDL-colesterol e a razão LDL-colesterol/HDL-colesterol. O tratamento com sinvastatina, isolada e associada ao α-tocoferol, promoveu diminuição nas concentrações de S-nitrosotióis. A L-arginina associada à sinvastatina, aumentou os níveis de colesterol total quando comparada com a sinvastatina isoladamente. As concentrações plasmáticas de α-tocoferol e L-arginina não aumentaram em decorrência da suplementação, devido à grande dispersão dos dados obtidos, embora as medianas das concentrações plasmáticas de Larginina e α-tocoferol tenham sido mais elevadas após as suplementações. O tratamento com sinvastatina, isolada ou associada à L-arginina e ao α-tocoferol, não alterou as concentrações dos inibidores endógenos da óxido nítrico sintase (ADMA e SDMA), dos metabólitos do óxido nítrico, da nitrotirosina total e dos tióis analisados. / The aim of this study was to evaluate the effect of sinvastatin, isolated and associated to α-tocopherol and to L-arginine, on the endogenous inhibitors of nitric oxide synthase, on nitric oxide metabolytes and thiols, in hypercholesterolemic patients. A group of 16 hypercholesterolemic patients were analysed, acconting to the protocol: a washout period (without medication) of 1 month, sinvastatin (20 mg/day) for 2 months; sinvastatin (20 mg/day) + α-tocopherol (400U/day) for 2 months; sinvastatin (20 mg/day) for 1 months (washout period), sinvastatin (20 mg/day) + L-arginine (7g/day) for 2 months. Sinvastatin significantly reduced the concentrations of total cholesterol and LDL-cholesterol, as well as the LDL-cholesterol/HDLcholesterol ratio. The treatment with sinvastatina, alone and associate to α-tocoferol, resulted in a reduction of RSNO concentration. The L-arginine associated with sinvastatin, increase the level of total cholesterol as compared with simvastatin alone. The plasma concentrations of a-tocopherol and Larginine did not increase following supplementation due to the large dispersion of the data obtained, even though the median plasma concentrations of L-arginine and a-tocopherol were elevated after supplementation. Treatment with simvastatin, alone or associated to L-arginine and a-tocopherol did not alter the concentrations of the endogenous inhibitors of nitric oxide synthase (ADMA and SDMA), or that of nitric oxide metabolytes, total nitrotyrosine or the thiols analysed.

INTRODUÇÂO: A morte encefálica (ME) induz instabilidade hemodinâmica com hipoperfusão microcirculatória, desencadeando inflamação e disfunção de órgãos. OBJETIVO: Este estudo teve como objetivo investigar os efeitos da solução salina hipertônica (SH) 7,5% na evolução da resposta inflamatória no tecido mesentérico de ratos submetidos à ME. MÉTODOS: Ratos Wistar machos foram anestesiados e ventilados mecanicamente. A ME foi induzida pela insuflação rápida de um balão posicionado na cavidade intracraniana (Fogart 4F). Os ratos foram divididos aleatoriamente em: 1) Falso-operado, ratos submetidos aos procedimentos cirúrgicos e trepanação (FO, n=17); 2) Controle, ratos tratados com solução salina isotônica (NaCl 0,9%, 4 mL/kg) imediatamente após ME (CO, n=17); 3) Solução hipertônica 1, ratos tratados com solução hipertônica (NaCl 7,5%, 4 mL/kg) imediatamente após ME (SH1, n=17); 4) Solução hipertônica 60, ratos tratados com solução hipertônica 60 min após ME (SH60, n=17). Três horas após a indução da ME ou o término do procedimento cirúrgico para os animais do grupo FO, foram coletados os seguintes dados: (a) perfusão mesentérica, fluxo sanguíneo e interações leucócito-endotélio no mesentério, pela técnica de microscopia intravital; (b) expressão de proteínas de óxido nítrico sintase endotelial (eNOS), endotelina-1, P-selectina e molécula de adesão intercelular (ICAM)-1, por imunohistoquímica; (c) expressão gênica de eNOS e endotelina-1, por reação em cadeia da polimerase em tempo real (PCR); (d) concentrações séricas de citocinas, quimiocinas e corticosterona por meio de enzimaimunoensaio (ELISA). RESULTADOS: Todos os grupos submetidos a ME apresentaram um comportamento semelhante da pressão arterial, sendo observado um pico hipertensivo, seguido de período de hipotensão, logo após a insuflação do cateter intra-craniano. A proporção de pequenos vasos perfundidos foi diminuída no grupo CO (46%) em comparação com FO (74%, p=0,0039). A SH foi capaz de restaurar a proporção de vasos perfundidos (SH1=71%, p=0,0018). Não houve diferenças no fluxo sanguíneo mesentérico entre os grupos. A expressão proteica de eNOS aumentou significativamente em ratos com SH (SH1 e SH60, p=0,0002) em comparação ao grupo CO. Resultados semelhantes foram observados em relação à endotelina-1 (p < 0,0001). Não houve diferenças na expressão gênica de eNOS e endotelina-1. O aumento no número de leucócitos \"rollers\" (p=0,0015) e migrados (p=0,0063) foi observado no grupo CO em comparação com FO. Ratos com SH demonstraram redução significativa em todos os parâmetros da interação leucócito-endotélio. Com relação às moléculas de adesão, a expressão de ICAM-1 estava elevada no grupo CO em comparação com o FO, enquanto que o tratamento com SH diminuiu a expressão de ICAM-1 (SH1 e SH60, p=0,0002). CONCLUSÕES: O emprego da solução salina hipertônica melhorou a perfusão mesentérica, influenciou positivamente o metabolismo do óxido nítrico e reduziu a inflamação no mesentério, com diminuição da adesão e migração leucocitária, em ratos submetidos a ME / BACKGROUND: Brain death (BD) induces hemodynamic instability with microcirculatory hypoperfusion leading to increased organ inflammation and dysfunction. OBJETIVE: To investigate the effects of 7.5% hypertonic saline solution (HS) on the course of the inflammatory response in rats submitted to BD. METHODS: Male Wistar rats were anesthetized and mechanically ventilated. BD was induced by rapid inflation of intracranial balloon catheter (Fogart 4F). Rats were randomly divided in: 1) Sham-operated, rats submitted only to trepanation (SH, n=17); 2) Control, rats treated with normal saline solution (NaCl 0.9%, 4 mL/kg) immediately after BD (CO, n=17); 3) Hypertonic solution 1, rats treated with hypertonic solution (NaCl 7.5%, 4 mL/kg) immediately after BD (HS1, n=17); 4) Hypertonic solution 60, rats treated with hypertonic solution 60 min after BD (HS60, n=17). Hundred eighty minutes thereafter the following experiments were performed: (a) mesenteric perfusion, blood flow, and leukocyte-endothelial interactions, by intravital microscopy; (b) protein expression of endothelial nitric oxide synthase (eNOS), endothelin-1, P-selectin, and intercellular cell adhesion molecule (ICAM)-1, by immunohistochemistry; (c) gene expression of eNOS, and endothelin-1, by real-time polymerase chain reaction (PCR); (d) serum concentrations of cytokines, chemokines and corticosterone by enzyme-linked immunosorbent assay (ELISA). RESULTS: All BD groups presented similar hypertensive peak followed by hypotension. The proportion of perfused small vessels was decreased in CO group (46%) compared to SH (74%, p=0.0039). HS was able to restore the proportion of perfused vessels (HS1=71%, p=0.0018). There were no differences in mesenteric blood flow between groups. eNOS protein expression significantly increased in rats given HS (HS1, and HS60, p=0.0002). Similar results were observed regarding endothelin-1 (p < 0.0001). There were no differences in eNOS and endothelin-1 gene expression. Increased numbers of rolling (p=0.0015) and migrated (p=0.0063) leukocytes were observed in CO group compared to SH. Rats given HS demonstrated an overall reduction in leukocyte-endothelial interactions. Levels of ICAM-1 increased in CO group compared to SH, and decreased in HS-treated groups (p=0.0002). CONCLUSIONS: Hypertonic saline improves mesenteric perfusion, increased eNOS and endothelin-1 protein expression, and reduced inflammation by decreasing leukocyte adhesion and migration in BD rats

A úlcera gástrica é uma doença crônica, de alta prevalência, e a eficácia dos tratamentos farmacológicos disponíveis é limitada pela alta incidência de efeitos adversos. Neste trabalho é mostrado o mecanismo de ação terapêutica e os efeitos toxicológicos da molécula indol-tiazolidínica LYSO-7 em diferentes modelos experimentais de úlcera gástrica. Camundongos Swiss machos foram tratados com veículo, LYSO-7 (5, 25 ou 50 mg/kg, v.o.) ou bezafibrato (25 ou 50 mg/kg, v.o.) 1 hora antes da administração oral de Et/HCl (60%/0,03 M) ou indometacina (100 mg/kg). Em outro conjunto de ensaios, animais foram pré-tratados com GW9962, um antagonista PPAR&#947; (2 mg/kg, i.p.); anticorpo anti-granulócito (50 &#181;L, i.p.), ou L-NAME (70 mg/kg, i.p) 1 hora antes dos tratamentos com veículo ou LYSO-7. Uma hora após administração da solução de Et/HCl, os neutrófilos foram quantificados no sangue e medula óssea, a rede microcirculatória gástrica foi estudada em in situ, utilizando a técnica de microscopia intravital; o tecido gástrico foi utilizado para quantificar a percentagem de área lesada, atividade da MPO, a expressão gênica e proteica de PPAR&#947;, expressão proteica de iNOS e eNOS, e a atividade das enzimas catalase, SOD, GPx, GR e GST. Uma hora após a administração de indometacina, o tecido gástrico foi removido para avaliar a eficácia do tratamento e a secreção de mediadores inflamatórios. Ensaio de úlcera crônica, induzida por ácido acético, foi realizado em camundongos Balb/c WT ou ANXA1-/-, aplicando-se 20&#181;L de ácido acético na camada subserosa do estômago e 24 horas após a indução, os animais foram tratados, uma vez ao dia, durante sete dias com LYSO-7 (50 mg/kg), bezafibrato (50 mg/kg) ou veículo. Foram realizados ensaios com macrófagos recrutados para o peritônio pela ação do tioglicolato de sódio (3%, i.p.) e com neutrófilos recrutados pela ação do glicogênio de ostra (1%, i.p.). Ensaios de toxicologia aguda, crônica e mutagenicidade também foram realizados. Os resultados obtidos mostram que o tratamento com LYSO-7 reduz a área lesada, o influxo de neutrófilos e a estase da rede microcirculatória provocada pela administração de Et/HCl. Os efeitos protetores foram revertidos em animais pré-tratados com GW9962, indicando a participação do PPAR&#947; no efeito. O influxo de neutrófilos é determinante para a lesão, uma vez que a depleção destas células reduziu a ulceração gástrica, e indica que o bloqueio da mobilização de neutrófilos da medula óssea para o sangue e destes para o tecido lesado pela LYSO-7 pode ser um mecanismo de ação gastroprotetora desta molécula. A reversão da estase vascular na microcirculação, mas não o influxo de neutrófilos, é mediado pelo NO, pois o pré-tratamento com L-NAME aboliu os efeitos da LYSO-7 no restabelecimento do fluxo sanguíneo da microcirculação. Este efeito pode ser dependente da maior e menor expressão proteica de eNOS e iNOS, respectivamente. A LYSO-7 foi capaz de alterar favoravelmente a atividade das enzimas antioxidantes no tecido gástrico. Ainda, a LYSO-7 diminuiu a área lesada e reduziu a concentração de TNF&#945; e aumentou a de IL-10 no tecido gástrico lesado pela indometacina. Na resolução do processo inflamatório, o tratamento com LYSO-7 diminuiu a percentagem de área lesada, aumentou a apoptose de neutrófilos e a eferocitose de neutrófilos por macrófagos peritoneais, inibiu a secreção de TNF&#945; e aumentou a secreção de IL-10, TFG-1&#946; e VEGF para o sobrenadante de macrófagos em fagocitose. A resolução de lesão gástrica, bem como a indução da fagocitose pela LYSO-7 foi reduzida em animais ANXA1-/-. As investigações destes últimos dados mostraram a relação da ANXA1 e PPAR&#947;, já que a expressão do receptor é reduzida em macrófagos obtidos de animais depletados de ANXA1. Os estudos toxicológicos mostraram que a LYSO-7 apresenta baixa toxicidade aguda e crônica in vivo, além de não ocasionar mutagenicidade em eritrócitos da medula óssea. Os dados obtidos mostram que a molécula LYSO-7 atua como agonista PPAR&#947; na modulação da úlcera gástrica e modula a migração de neutrófilos e o fluxo sanguíneo na microcirculação. A transativação e transrepressão de eNOS e iNOS, respectivamente, o bloqueio da migração de neutrófilos para a lesão e a inibição da atividade de enzimas oxidativa, ativação de enzimas antioxidantes no epitélio gástrico e a inibição da secreção de mediadores inflamatórios parecem ser os mecanismos de ação da LYSO-7 na citoproteção gástrica. Adicionalmente, a LYSO-7 atua na resolução do processo inflamatório promovendo downregulation na secreção de mediadores inflamatórios, aumento na apoptose de neutrófilos e eferocitose de neutrófilos apoptóticos. / Gastric ulcer is a chronic disease that presents high prevalence, and effectiveness of pharmacological treatments available is limited by several adverse effects. In this study is shown the mechanism of action and toxicological effects of the molecule indole-thiazolidine LYSO-7 in different models of gastric ulcer. Male Swiss mice were treated with vehicle LYSO-7 (5, 25, or 50 mg/kg, p.o.) or bezafibrate (25 or 50 mg/kg, p.o.) 1 hour before the oral administration of Et/HCl (60%/0.03 M) or indomethacin (100 mg/kg). In another set of assays, animals were pre-treated with GW9962, a PPAR&#947; antagonist (2 mg/kg, i.p.), anti-granulocyte antibody (50 &#181;L, i.p.) or L-NAME (70 mg/kg, i.p.) 1 hour before the treatment with vehicle or LYSO-7. One hour after administration of the Et/HCl solution, neutrophils were quantified in the blood and bone marrow, the gastric microcirculatory network was studied in situ by intravital microscopy, in the gastric tissue were quantified the percentage of injured area, MPO activity, PPAR&#947; gene and protein expression, iNOS and eNOS protein expression, and catalase, SOD, GPx, GR and GST activity. One hour after indomethacin administration, gastric tissue was removed to verify the efficacy of LYSO-7 on inflammatory mediator secretion. Chronic ulcer assay induced by acetic acid was carried out in Balb/c WT or ANXA1-/-, applying 20&#181;L of acetic acid in the subserosal layer of the stomach and 24 hours after induction, animals were treated during seven days, once a day, with LYSO-7 (50 mg/kg), bezafibrate (50 mg/kg) or vehicle. Assays were performed with macrophages recruited to the peritoneum by sodium thioglycollate (3%, i.p.) and neutrophils by oyster glycogen (1%, i.p.). Acute and chronic toxicological and mutagenicity assays were also conducted. The results obtained show that LYSO-7 treatment decrease the injured area, neutrophil influx and microcirculatory stasis evoked by Et/HCl administration. Protective effects were reversed in animals pretreated with GW9962, indicating the involvement of PPAR&#947;. Neutrophil influx is a determinant of the gastric lesion, once the depletion of these cells decreased the gastric damage, indicating that in the neutrophil mobilization blockade from the bone marrow to blood and to injured tissue may be a gastroprotective mechanism of LYSO-7. The vascular stasis reversion in the microcirculation is mediated by NO, but not the neutrophil influx, since the pretreatment with L-NAME abolished the effects of LYSO-7 on blood flow. This effect was dependent on increase and decrease of eNOS and iNOS protein expression, respectively. LYSO-7 positively altered the activity of antioxidant enzymes in the gastric tissue. Furthermore, LYSO-7 reduced the injured area and the concentration of TNF&#945; and increased IL-10 in the gastric tissue in the indomethacin-induced ulcer model. In the resolution of inflammation, LYSO-7 treatment decreased the percentage of the injured area, increased the neutrophils apoptosis and the efferocytosis of apoptotic neutrophils by peritoneal macrophages, inhibited the TNF&#945; release and increased the secretion of IL-10, IL-1&#946; and VEGF in the supernatant of phagocytosis assay. The resolution of gastric lesions, as well as, the induction of phagocytosis by LYSO-7 was reduced in animals ANXA1-/-. This data shown the relation of PPAR&#947; and ANXA1, as PPAR&#947; expression is reduced in macrophages obtained from ANXA1-/- animals. Toxicological studies showed that LYSO-7 has low acute and chronic toxicity in vivo, and did not cause mutagenicity in bone marrow erythrocytes. The data obtained show that LYSO-7 acts as PPAR&#947; in the modulation of gastric ulcer and modulate neutrophil migration and blood flow in the microcirculation. The transactivation and transrepression of eNOS and iNOS, respectively, blocking the neutrophil influx into the injury, antioxidant enzymes activation in the gastric epithelium and inhibition of inflammatory mediators release seem to be the mechanisms action of LYSO-7 in gastric cytoprotection. Additionally, LYSO-7 operates in the resolution of inflammation promoting downregulation in the secretion of inflammatory mediators and increases the neutrophil apoptosis and efferocytosis of apoptotic neutrophils.

A úlcera gástrica é uma doença crônica, de alta prevalência, e a eficácia dos tratamentos farmacológicos disponíveis é limitada pela alta incidência de efeitos adversos. Neste trabalho é mostrado o mecanismo de ação terapêutica e os efeitos toxicológicos da molécula indol-tiazolidínica LYSO-7 em diferentes modelos experimentais de úlcera gástrica. Camundongos Swiss machos foram tratados com veículo, LYSO-7 (5, 25 ou 50 mg/kg, v.o.) ou bezafibrato (25 ou 50 mg/kg, v.o.) 1 hora antes da administração oral de Et/HCl (60%/0,03 M) ou indometacina (100 mg/kg). Em outro conjunto de ensaios, animais foram pré-tratados com GW9962, um antagonista PPAR&#947; (2 mg/kg, i.p.); anticorpo anti-granulócito (50 &#181;L, i.p.), ou L-NAME (70 mg/kg, i.p) 1 hora antes dos tratamentos com veículo ou LYSO-7. Uma hora após administração da solução de Et/HCl, os neutrófilos foram quantificados no sangue e medula óssea, a rede microcirculatória gástrica foi estudada em in situ, utilizando a técnica de microscopia intravital; o tecido gástrico foi utilizado para quantificar a percentagem de área lesada, atividade da MPO, a expressão gênica e proteica de PPAR&#947;, expressão proteica de iNOS e eNOS, e a atividade das enzimas catalase, SOD, GPx, GR e GST. Uma hora após a administração de indometacina, o tecido gástrico foi removido para avaliar a eficácia do tratamento e a secreção de mediadores inflamatórios. Ensaio de úlcera crônica, induzida por ácido acético, foi realizado em camundongos Balb/c WT ou ANXA1-/-, aplicando-se 20&#181;L de ácido acético na camada subserosa do estômago e 24 horas após a indução, os animais foram tratados, uma vez ao dia, durante sete dias com LYSO-7 (50 mg/kg), bezafibrato (50 mg/kg) ou veículo. Foram realizados ensaios com macrófagos recrutados para o peritônio pela ação do tioglicolato de sódio (3%, i.p.) e com neutrófilos recrutados pela ação do glicogênio de ostra (1%, i.p.). Ensaios de toxicologia aguda, crônica e mutagenicidade também foram realizados. Os resultados obtidos mostram que o tratamento com LYSO-7 reduz a área lesada, o influxo de neutrófilos e a estase da rede microcirculatória provocada pela administração de Et/HCl. Os efeitos protetores foram revertidos em animais pré-tratados com GW9962, indicando a participação do PPAR&#947; no efeito. O influxo de neutrófilos é determinante para a lesão, uma vez que a depleção destas células reduziu a ulceração gástrica, e indica que o bloqueio da mobilização de neutrófilos da medula óssea para o sangue e destes para o tecido lesado pela LYSO-7 pode ser um mecanismo de ação gastroprotetora desta molécula. A reversão da estase vascular na microcirculação, mas não o influxo de neutrófilos, é mediado pelo NO, pois o pré-tratamento com L-NAME aboliu os efeitos da LYSO-7 no restabelecimento do fluxo sanguíneo da microcirculação. Este efeito pode ser dependente da maior e menor expressão proteica de eNOS e iNOS, respectivamente. A LYSO-7 foi capaz de alterar favoravelmente a atividade das enzimas antioxidantes no tecido gástrico. Ainda, a LYSO-7 diminuiu a área lesada e reduziu a concentração de TNF&#945; e aumentou a de IL-10 no tecido gástrico lesado pela indometacina. Na resolução do processo inflamatório, o tratamento com LYSO-7 diminuiu a percentagem de área lesada, aumentou a apoptose de neutrófilos e a eferocitose de neutrófilos por macrófagos peritoneais, inibiu a secreção de TNF&#945; e aumentou a secreção de IL-10, TFG-1&#946; e VEGF para o sobrenadante de macrófagos em fagocitose. A resolução de lesão gástrica, bem como a indução da fagocitose pela LYSO-7 foi reduzida em animais ANXA1-/-. As investigações destes últimos dados mostraram a relação da ANXA1 e PPAR&#947;, já que a expressão do receptor é reduzida em macrófagos obtidos de animais depletados de ANXA1. Os estudos toxicológicos mostraram que a LYSO-7 apresenta baixa toxicidade aguda e crônica in vivo, além de não ocasionar mutagenicidade em eritrócitos da medula óssea. Os dados obtidos mostram que a molécula LYSO-7 atua como agonista PPAR&#947; na modulação da úlcera gástrica e modula a migração de neutrófilos e o fluxo sanguíneo na microcirculação. A transativação e transrepressão de eNOS e iNOS, respectivamente, o bloqueio da migração de neutrófilos para a lesão e a inibição da atividade de enzimas oxidativa, ativação de enzimas antioxidantes no epitélio gástrico e a inibição da secreção de mediadores inflamatórios parecem ser os mecanismos de ação da LYSO-7 na citoproteção gástrica. Adicionalmente, a LYSO-7 atua na resolução do processo inflamatório promovendo downregulation na secreção de mediadores inflamatórios, aumento na apoptose de neutrófilos e eferocitose de neutrófilos apoptóticos. / Gastric ulcer is a chronic disease that presents high prevalence, and effectiveness of pharmacological treatments available is limited by several adverse effects. In this study is shown the mechanism of action and toxicological effects of the molecule indole-thiazolidine LYSO-7 in different models of gastric ulcer. Male Swiss mice were treated with vehicle LYSO-7 (5, 25, or 50 mg/kg, p.o.) or bezafibrate (25 or 50 mg/kg, p.o.) 1 hour before the oral administration of Et/HCl (60%/0.03 M) or indomethacin (100 mg/kg). In another set of assays, animals were pre-treated with GW9962, a PPAR&#947; antagonist (2 mg/kg, i.p.), anti-granulocyte antibody (50 &#181;L, i.p.) or L-NAME (70 mg/kg, i.p.) 1 hour before the treatment with vehicle or LYSO-7. One hour after administration of the Et/HCl solution, neutrophils were quantified in the blood and bone marrow, the gastric microcirculatory network was studied in situ by intravital microscopy, in the gastric tissue were quantified the percentage of injured area, MPO activity, PPAR&#947; gene and protein expression, iNOS and eNOS protein expression, and catalase, SOD, GPx, GR and GST activity. One hour after indomethacin administration, gastric tissue was removed to verify the efficacy of LYSO-7 on inflammatory mediator secretion. Chronic ulcer assay induced by acetic acid was carried out in Balb/c WT or ANXA1-/-, applying 20&#181;L of acetic acid in the subserosal layer of the stomach and 24 hours after induction, animals were treated during seven days, once a day, with LYSO-7 (50 mg/kg), bezafibrate (50 mg/kg) or vehicle. Assays were performed with macrophages recruited to the peritoneum by sodium thioglycollate (3%, i.p.) and neutrophils by oyster glycogen (1%, i.p.). Acute and chronic toxicological and mutagenicity assays were also conducted. The results obtained show that LYSO-7 treatment decrease the injured area, neutrophil influx and microcirculatory stasis evoked by Et/HCl administration. Protective effects were reversed in animals pretreated with GW9962, indicating the involvement of PPAR&#947;. Neutrophil influx is a determinant of the gastric lesion, once the depletion of these cells decreased the gastric damage, indicating that in the neutrophil mobilization blockade from the bone marrow to blood and to injured tissue may be a gastroprotective mechanism of LYSO-7. The vascular stasis reversion in the microcirculation is mediated by NO, but not the neutrophil influx, since the pretreatment with L-NAME abolished the effects of LYSO-7 on blood flow. This effect was dependent on increase and decrease of eNOS and iNOS protein expression, respectively. LYSO-7 positively altered the activity of antioxidant enzymes in the gastric tissue. Furthermore, LYSO-7 reduced the injured area and the concentration of TNF&#945; and increased IL-10 in the gastric tissue in the indomethacin-induced ulcer model. In the resolution of inflammation, LYSO-7 treatment decreased the percentage of the injured area, increased the neutrophils apoptosis and the efferocytosis of apoptotic neutrophils by peritoneal macrophages, inhibited the TNF&#945; release and increased the secretion of IL-10, IL-1&#946; and VEGF in the supernatant of phagocytosis assay. The resolution of gastric lesions, as well as, the induction of phagocytosis by LYSO-7 was reduced in animals ANXA1-/-. This data shown the relation of PPAR&#947; and ANXA1, as PPAR&#947; expression is reduced in macrophages obtained from ANXA1-/- animals. Toxicological studies showed that LYSO-7 has low acute and chronic toxicity in vivo, and did not cause mutagenicity in bone marrow erythrocytes. The data obtained show that LYSO-7 acts as PPAR&#947; in the modulation of gastric ulcer and modulate neutrophil migration and blood flow in the microcirculation. The transactivation and transrepression of eNOS and iNOS, respectively, blocking the neutrophil influx into the injury, antioxidant enzymes activation in the gastric epithelium and inhibition of inflammatory mediators release seem to be the mechanisms action of LYSO-7 in gastric cytoprotection. Additionally, LYSO-7 operates in the resolution of inflammation promoting downregulation in the secretion of inflammatory mediators and increases the neutrophil apoptosis and efferocytosis of apoptotic neutrophils.

La palourde japonaise, Ruditapes philippinarum, a été introduite en France en 1972 suite à une volonté de diversification de la production aquacole des bivalves. A la fin des années 1980, des épisodes de mortalité massive ont été observés dans les parcs vénéricoles du pays des Abers (Finistère, France). La mortalité massive des palourdes a été associée à la maladie de l’anneau brun (MAB), une vibriose dont l’agent étiologique est Vibrio tapetis. Le développement de la MAB en milieu naturel a été associé à la modulation de certains facteurs environnementaux tels que la température ou la présence de ressource trophique. Dans le cadre de ce travail et dans un premier temps, des infections expérimentales de palourdes avec différentes souches de V. tapetis ont été effectuées conjointement à une acclimatation des animaux à deux températures contrastées. L’augmentation de la température de 15 à 22 °C a été associée à de plus fortes activités enzymatiques de la superoxyde dismutase (SOD), impliquée dans le système antioxydant, et de la phénoloxydase (PO), impliquée dans le système immunitaire inné. L’augmentation de la température a également conduit à une diminution de la virulence de Vibrio tapetis. Ensemble, ces résultats permettent d’expliquer la baisse de la prévalence et de l’intensité de la MAB à 22 °C. Dans un second temps, des palourdes sexuellement matures nourries avec deux algues contrastées en termes de composition lipidique ont été infectées par V. tapetis. Si la qualité des microalgues n’a pas conduit à des différences de prévalence et d’intensité de la MAB, le statut de reproduction des animaux a eu une influence sur l’intensité de la maladie. Les réponses métaboliques de R. philippinarum exposée à V. tapetis n’ont pas été influencées par la qualité de la nourriture mais par le développement de la MAB. Ces réponses consistaient en une modulation de l’activité d’enzymes antioxydantes (SOD, catalase, glutathion peroxydase, glutathion réductase,glutathion-S-transférase) et d’enzymes reliées au système immunitaire inné (PO et oxyde nitrique synthase inductible) en fonction de la présence de signes cliniques de la MAB. Finalement, l’utilisation de ces indicateurs biochimiques pourrait permettre d’établir des critères de sélection d’individus résistants à la MAB. / The Manila clam, Ruditapes philippinarum, was introduced in France in 1972 following the willingness of bivalve aquaculture diversification. In the late 1980s, episodic mass mortality events were observed in ponds of the “Pays des Abers” region (Finistère, France). The massive mortality of clams was associated brown ring disease (BRD), a vibriosis which causative agent is Vibrio tapetis. BRD development in field has been associated with the modulation of environmental factors such as temperature or the presence of trophic resource. Firstly in the frame of the present work, experimental infections of clams with different strains of V. tapetis were performed together with animal acclimation at two contrasted temperatures. The increase of temperature from 15 to 22 ° C was associated with higher enzymatic activities of superoxide dismutase (SOD), involved in the antioxidant system, and the phenoloxidase (PO), involved in the innate immune system. Temperature increase also led to a decrease in virulence of V. tapetis. Together, these results might explain the decline in BRD prevalence and intensity observed at 22 ° C. Secondly, sexually mature clams fed with two microalgal diets contrasted in terms of lipid composition were infected with V. tapetis. Although microalgae quality did not lead to any difference in BRD prevalence and intensity, the reproductive status of clams influenced BRD intensity. Metabolic responses of R. philippinarum exposed to V. tapetis were not influenced by the food quality but mainly by BRD development. These responses consisted in a modulation of the activity of antioxidant enzymes (SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase) and enzymes related to innate immune system (PO and inducible nitric oxide synthase) according to the presence of BRD clinical signs. Finally, the use of these biochemical indicators could allow for new criteria for selection of BRD resistant clams.

The citrulline-nitric oxide (NO) cycle, comprised of the enzymes argininosuccinate synthase (AS), argininosuccinate lyase (AL) and endothelial nitric oxide synthase (eNOS), is responsible for the regulated production of endothelial NO. Although most studies have focused on eNOS to uncover important regulatory mechanisms, we and others have determined that AS is an essential and regulated step in endothelial NO production. AS is rate limiting for endothelial NO production and is the primary source of arginine, the substrate for eNOS-mediated NO production, despite saturating intracellular levels of arginine and available arginine transport systems. AS is essential for endothelial cell viability and its expression is regulated coordinately with eNOS by TNF and thiazolidenediones with concomitant effects on NO production. Given the importance of AS for endothelial health, we explored three independent regulatory mechanisms. In Chapter One, the functional consequences of altered AS expression due to overexpression, insulin, VEGF and ceramide were studied. We demonstrated that overexpression of AS leads to enhanced NO production and that insulin, VEGF and ceramide coordinately regulate the expression of AS and eNOS. In Chapter Two, the first post-translational modifications of AS in the endothelium were characterized. We determined that AS is an endogenous phosphoprotein in the endothelium, described several levels of biological significance of AS phosphorylation, identified 7 sites of AS phosphorylation and began to uncover the direct impact of phosphorylation on AS function. Finally, in Chapter Three, endothelial AS subcellular localization was defined and important protein interactions were identified including caveolin-1 and HSP90. The work presented in this dissertation demonstrates that multiple mechanisms regulate the function of AS, often coordinately with eNOS, and have a direct impact on nitric oxide production. Our findings suggest that the global understanding of the citrulline-NO cycle as a metabolic unit will unravel new paradigms that will re-define our understanding of the regulation of vascular function by NO.

<p>The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. <i>Helicobacter pylori</i> might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of <i>H. pylori</i> on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat.</p><p>A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (<20μm) and discontinuous in the small intestine.</p><p>Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from <i>H. pylori</i> (HPE). In animals with a chronic <i>H. pylori</i> infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. </p><p>HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.</p>

<p>Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.</p><p>Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.</p><p>Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. <i>Trans</i>-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.</p><p>In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.</p>

The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. Helicobacter pylori might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of H. pylori on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat. A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (&lt;20μm) and discontinuous in the small intestine. Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from H. pylori (HPE). In animals with a chronic H. pylori infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented. Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA. Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs. In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.