<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

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No. of bitstreams: 1 paulohenriquediasdecarvalho.pdf: 3125617 bytes, checksum: 3e951b5c5e73d3862e1ed9b6e9e28451 (MD5) Previous issue date: 2013-07-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A descoberta de protótipos naturais associada às metodologias de isolamento e identificação química de constituintes a partir de novas fontes botânicas, bem como da avaliação das atividades farmacológicas e toxicológicas dessas moléculas demonstram grandes perspectivas para o desenvolvimento racional de novos fármacos. Tendo em vista a alta incidência de doenças do fígado, no Brasil e no mundo, e que existem poucos medicamentos eficazes e capazes de reverter ou reduzir a progressão destas, o isolamento e a identificação de substâncias com potencial hepatoprotetor é, hoje, uma área promissora na busca de novas substâncias bioativas. Tradicionalmente, as raízes de Glycyrrhiza sp., conhecidas como licorice, são usadas na medicina alternativa com inúmeras finalidades, dentre elas hepatoprotetora. Entretanto, até o momento, não existem relatos desta atividade vinculada à licochalcona A, uma das substâncias majoritárias nas raízes de Glycyrrhiza inflata. No presente trabalho avaliaram-se as atividades da licochalcona A em ensaios de viabilidade celular das linhagens de fibroblásto (NIH/3T3) e carcinoma hepatocelular humano (HepG-2), adesão celular de HepG-2 e em modelo de lesão hepática induzida por ligação do ducto biliar (BDL) em ratos Wistar. Além disso, desenvolveu-se uma metodologia para determinação da licochalcona A em CLAE-UV, utilizando coluna C18, fase móvel em gradiente de água acidificada (0,1% H3PO4) e metanol (50:50 – 20:80 v/v), fluxo de 1,1 mL/min e comprimento de onda para detecção em 372 nm. A licochalcona A isolada a partir do extrato seco das raízes de G. inflata foi identificada por RMN 1H e 13C. O isolamento apresentou-se satisfatório, bem como a metodologia proposta para quantificação desta substância por CLAE-UV, que apresentou excelente linearidade, precisão e exatidão. Nos experimentos in vitro, a licochalcona A não demonstrou redução significativa na viabilidade das células da linhagem HepG-2 (IC50 > 200 μM) e da NIH/3T3 (IC50 > 100 μM), bem como no experimento de adesão das células HepG-2 (IC50 > 200 μM) (p>0,05). Estes dados corroboram com aqueles encontrados no experimento in vivo, no qual a licochalcona A (50 mg/Kg) também não apresentou toxicidade ao fígado, já que os resultados encontrados não foram significativamente diferentes aos do grupo controle (p>0,05). Contudo, ela também não demonstrou capacidade de promover ou reduzir os danos hepáticos causado pelo BDL, no tempo de tratamento do estudo realizado (48 h). / The natural prototypes discovery associated with methods of chemical constituents isolation and identification from new botanical sources, as well as the evaluation of pharmacological and toxicological activities of these molecules show great prospects for the new drugs rational development. In view of the high incidence of liver disease in Brazil and the world, and there are few effective drugs and able to reverse or slow the progression of these disease, the substances isolation and identification with potential hepatoprotective today is a promising area in search for new bioactive substances. Traditionally, the roots of Glycyrrhiza sp., known as licorice, are used in alternative medicine with numerous purposes, among them hepatoprotective. However, to date, there are no reports of this activity linked to licochalcona A, one majority of the substances in the roots of Glycyrrhiza inflata. In the present study evaluated the activities of licochalcone A in cell viability assays of strains fibroblast (NIH/3T3) and human hepatocellular carcinoma (HepG-2), cell adhesion HepG-2 and model of liver injury induced by bile duct ligation (BDL) in Wistar rats. In addition, we developed a methodology for determining the licochalcone A quantitative HPLC-UV, using C18 column and a mobile phase gradient of acidified water (0.1% H3PO4) and methanol (50:50 - 20:80 v/v), flow rate of 1.1 mL/min and detection wavelength at 372 nm. The licochalcone A isolated from the dried extract of the roots of G. inflata was identified by 1H and 13C NMR. The isolation had to be satisfactory, as well as the proposed methodology for quantification of this substance by HPLC-UV, which showed excellent linearity, reproducibility and accuracy. In in vitro experiments, licochalcone A showed no significant reduction in the viability of the cell line HepG-2 (IC50 > 200 μM) and NIH/3T3 (IC50 > 100 μM), as well as in cell adhesion HepG-2 experiments (IC50 > 200 μM) (p> 0.05). These data corroborate those found in the in vivo experiment, in which the licochalcone A (50 mg/kg) also showed no toxicity to the liver, since the results were not significantly different to the control group (p>0.05). Nevertheless, it has not shown the ability to promote or reduce liver damage caused by BDL, at the treatment time of the study (48 h).

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17α− ethinylestradiol induced cholestasis, but the increase of b-series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatis induced bile duct ligation (HO-1 activator- hemine, HO-1 inhibitor- Sn-mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals, but a decrease of some a- and b- series gangliosides was observed. In group with activated HO-1 total sialic acid increased, but the composition of gangliosides...

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17ethinylestradiol induced cholestasis, but the increase of b- series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatic rats (HO-1 activator- hemine, HO- 1 inhibitor- Sn- mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals but without significant changes in gangliosides composition. Lipid sialic acid and gangliosides were not changed in animals with hemine activated HO-1. Expression of mRNA of key...

Η ηπατική εγκεφαλοπάθεια είναι ένα πολύπλοκο νευροψυχιατρικό σύνδρομο που έχει συσχετισθεί με οξείες και χρόνιες ηπατοπάθειες. Τα τελευταία χρόνια συσσωρεύονται πληροφορίες που εμπλέκουν το οξειδωτικό στρες (φορτίο) ως παράγοντα-κλειδί στην παθογένεση της ηπατικής εγκεφαλοπάθειας σε μελέτες που χρησιμοποιούν ως μετρούμενους δείκτες την υπεροξείδωση λιπιδίων και το οξειδοαναγωγικό ζεύγος της γλουταθειόνης (GSH/GSSG). Στην παρούσα μελέτη χρησιμοποιήθηκε ένα μοντέλο πειραματικού αποφρακτικού ίκτερου με απολίνωση του χοληδόχου πόρου. Αρσενικοί αρουραίοι χωρίστηκαν σε ομάδες ελέγχου, ψευδώς χειρουργημένων, και σε ομάδες απολίνωσης χοληδόχου πόρου που είτε θυσιάστηκαν την 5η ημέρα, είτε τη 10η ημέρα, ή τους χορηγήθηκαν αντιοξειδωτικοί παράγοντες (NAC, ALP, Vit-E). Στη συνέχεια, μελετήθηκε η θειολική οξειδοαναγωγική κατάσταση στον εγκέφαλο των αρουραίων, και μάλιστα ανά περιοχές (εγκεφαλικός φλοιός, στέλεχος, παρεγκεφαλίδα), καθώς και η επίδραση των επιλεγμένων αντιοξειδωτικών παραγόντων σε αυτήν. Χρησιμοποιήθηκε για πρώτη φορά μια πιο αντιπροσωπευτική εκτίμηση του οξειδωτικού στρες, καθώς ποσοτικοποιήθηκαν συγκεκριμένοι δείκτες υψηλού (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, υπεροξείδια λιπιδίων) και χαμηλού (GSH, CSH, PSH) οξειδωτικού στρες. Τα αποτελέσματά μας δείχνουν αύξηση των πρώτων και μείωση των τελευταίων σε όλες τις εγκεφαλικές περιοχές καταδεικνύοντας έτσι την παρουσία αυξημένου οξειδωτικού φορτίου στον αποφρακτικό ίκτερο. Το σημαντικότερο αποτέλεσμα αυτής της μελέτης είναι ότι κατέδειξε πρώιμα σημεία οξειδωτικού στρες στον εγκέφαλο ήδη από την 5η ημέρα μετά την απολίνωση του χοληδόχου πόρου. Οι μεταβολές των βιοχημικών δεικτών, και αυτό αφορά σε όλους τους δείκτες και σε όλες τις εγκεφαλικές περιοχές, αρχίζουν να φαίνονται από την 5η ημέρα και γίνονται στατιστικά σημαντικές τη 10η ημέρα από την απολίνωση του χοληδόχου πόρου. Διαπιστώσαμε επιπλέον ότι η GSH είχε περίπου διπλάσιες τιμές στον εγκεφαλικό φλοιό από ό, τι στο στέλεχος και την παρεγκεφαλίδα, και ότι στο στέλεχος παρατηρήθηκε μια δραματική αύξηση των επιπέδων των NPSSR τη 10η ημέρα μετά την απολίνωση του χοληδόχου πόρου, τα οποία όμως παρέμειναν χαμηλά στις άλλες δύο περιοχές. Καθώς είναι γνωστό πως το οξειδωτικό στρες έχει ενοχοποιηθεί στην παθογένεση διαφόρων νευροεκφυλιστικών παθήσεων στον άνθρωπο, τα ευρήματα αυτά θα μπορούσαν να συσχετισθούν με διαφορές στη φυσιολογία και τη βιοχημεία των περιοχών αυτών και ενδεχομένως να σχετίζονται με τον τρόπο που το οξειδωτικό στρες εκφράζεται στην παθοφυσιολογία και άλλων νοσολογικών καταστάσεων όπως η πλάγια αμυοτροφική σκλήρυνση, η νόσος Parkinson, και η νόσος Alzheimer. Έχει ενδιαφέρον το γεγονός ότι τα βασικά γάγγλια και οι πυρήνες του στελέχους είναι θέσεις εκλεκτικής βλάβης στην εγκεφαλοπάθεια από χολερυθρίνη στο νεογνικό ίκτερο. Στη δεύτερη πειραματική φάση, στους ικτερικούς αρουραίους χορηγήσαμε αντιοξειδωτικούς παράγοντες, που έχουν επανειλημμένα μελετηθεί τόσο in vitro όσο και σε ζωικά μοντέλα, σε μια προσπάθεια να αναστρέψουμε τις διαταραχές που είχαν παρατηρηθεί. Ένα πρώτο εύρημα ήταν η ευεργετική δράση στην υπεροξείδωση των λιπιδίων, που ποσοτικοποιήθηκε με τον υπολογισμό της MDA, στις ομάδες και των τριών αντιοξειδωτών αλλά κυρίως στις ALP και Vit-E. Και στις τρεις ομάδες που έλαβαν αντιοξειδωτικά, αντίθετα με την ομάδα απολίνωσης χοληδόχου πόρου, δεν παρατηρήθηκε εξάντληση του συνολικού κυτταρικού περιεχόμενου γλουταθειόνης. Επιπλέον, και στις τρεις ομάδες αντιοξειδωτικών δεν παρατηρήθηκε αύξηση των NPSSR στο εγκεφαλικό στέλεχος, γεγονός που υποδηλώνει ότι η συσσώρευση των μη-πρωτεϊνικών δισουλφιδίων στο στέλεχος εμποδίστηκε από τους αντιοξειδωτικούς παράγοντες. Η ανισορροπία των πρωτεϊνικών θειολών, όπως αυτή φάνηκε από τη μείωση της PSH και την αύξηση του PSSP, αναστράφηκε σημαντικά μόνο στην ομάδα NAC στην οποία η PSH αυξήθηκε στα φυσιολογικά επίπεδα. Εν συντομία, η παρούσα μελέτη είναι η πρώτη που καταδεικνύει με σαφήνεια το οξειδωτικό στρες στον εγκέφαλο στο μοντέλο του αποφρακτικού ίκτερου και μάλιστα αρκετά πρώιμα. Χρησιμοποιεί μια συστοιχία βιοχημικών δεικτών που περιγράφουν την θειολική αναγωγική κατάσταση και την υπεροξείδωση των λιπιδίων και μελετά τις ευεργετικές επιδράσεις γνωστών αντιοξειδωτικών παραγόντων στον πειραματικό αποφρακτικό ίκτερο. Τα αποτελέσματά της θα μπορούσαν να προσφέρουν κάποια βοήθεια στην κατανόηση ορισμένων μηχανισμών της ηπατικής εγκεφαλοπάθειας στους ανθρώπους. Μελλοντικές έρευνες θα απαντήσουν ερωτήματα σχετικά με τα γενεσιουργά αίτια του οξειδωτικού στρες, την ίδια την παρουσία των ελευθέρων ριζών και την παθοφυσιολογία του φαινομένου. / Hepatic encephalopathy is a complex neuropsychiatric syndrome that has been associated with acute and chronic liver diseases. Accumulated evidence over the last several years has implicated oxidative stress a key factor in the pathogenesis of hepatic encephalopathy. These studies utilize measurements of lipid peroxidation products and glutathione (GSH) and its oxidized disulfide GSSG. A model of experimental obstructive jaundice after ligation of the biliary duct has been used in the present study. Male Wistar rats were divided into control, sham operated and bile duct ligated groups that were sacrificed either on the 5th or the 10th day, or they have been treated with antioxidant agents (NAC, ALP, Vit-E). Subsequently, the thiol redox state of various areas (cortex, midbrain and cerebellum) of the rat brain and the effect of selected antioxidants were studied. For the first time specific markers of high oxidative stress (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, lipid peroxides) and low oxidative stress (GSH, CSH, PSH) were quantified providing a more detailed assessment of the phenomenon. Our results show increase in the first and decrease in the latter group of markers in all studied brain areas, therefore demonstrating high oxidative stress in the obstructive jaundice. The major impact of the present study is the demonstration of early signs of oxidative stress in the brain. Using this battery of biochemical markers, deviations from control and sham animals occurred as early as 5 days following bile duct ligation; by the 10th day the majority of these changes became statistically significant. It was also observed that GSH values in cerebral cortex were twice as high as those in midbrain and cerebellum and a dramatic increase in the levels of NPSSR on the 10th day after bile duct ligation in midbrain that was not observed in the other brain areas. These findings could be attributed to specificities of metabolic or biochemical status of neurons and astrocytes and alterations of blood-brain barrier permeability in different brain areas and probably should be taken into account in further studies, since, as we know, oxidative stress has been implicated in the pathogenesis of many human diseases like Parkinson’s , Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS). It is of interest that basal ganglia and brainstem nuclei are, as well, the sites of selective damage in bilirubin encephalopathy in jaundiced neonates. Jaundiced rats were treated with agents that have frequently been used in vitro and in vivo for their antioxidant effects, in an effort to reverse the observed alterations in redox state. In the treated groups, there was no decrease in the total cell glutathione content unlike the bile duct ligated rats. There was also no significant difference in the levels of lipid peroxidation as compared with control and sham groups. The imbalance of protein thiols demonstrated by the decrease of PSH and the increase of PSSP was considerably reversed only in the NAC group. In all treated groups, no NPSSR increase was found suggesting that the antioxidant agents suppressed the accumulation of non-protein disulfides in the midbrain. In brief, this experimental study demonstrates the oxidative profile of the brain associated with obstructive jaundice at an early and later stage. A battery of biochemical markers that define the thiol redox state is utilized and the beneficial effects of known antioxidants are examined. The evidence presented supports the concept that oxidative stress is neither a uniform matter affecting brain in a general way nor that any antioxidant agent could prevent damage by enhancing equally well different defence system. It is also likely that oxidative stress is one of the important mechanisms of jaundice-induced encephalopathy. Further studies could provide with more evidence on the pathogenetic mechanisms and generative causes of the oxidative stress in obstructive jaundice.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-12T13:37:04Z No. of bitstreams: 1 pedroluizrodriguesguedes.pdf: 3448334 bytes, checksum: 3bac263ee478fd60c02c5d6036a0e81e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:44:09Z (GMT) No. of bitstreams: 1 pedroluizrodriguesguedes.pdf: 3448334 bytes, checksum: 3bac263ee478fd60c02c5d6036a0e81e (MD5) / Made available in DSpace on 2017-05-12T15:44:09Z (GMT). No. of bitstreams: 1 pedroluizrodriguesguedes.pdf: 3448334 bytes, checksum: 3bac263ee478fd60c02c5d6036a0e81e (MD5) Previous issue date: 2013-07-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Condroitim sulfato (CS) é um glicosaminoglicano (GAG), presente na matriz extracelular (MEC) de vários tecidos de mamíferos, utilizado para o tratamento da osteoartrite e, recentemente, tem despertado grande interesse devido ao seu potencial anti-inflamatório. Vários modelos experimentais in vivo de inflamação são empregados para o estudo da atividade anti-inflamatória, entre eles o modelo de fibrose induzida por colestase extra-hepática. A colestase produz lesão hepatocelular com edema do trato portal, infiltrado leucocitário, proliferação de células epiteliais biliares e fibrose do trato portal. O objetivo deste trabalho foi analisar os efeitos do CS no modelo de colestase extra-hepática emperimental induzido por laqueadura do ducto biliar (BDL) em ratos Wistar. Para isso foram utilizados animais (n = 82) de 6 a 8 semanas de idade eutanasiados 2, 7 ou 14 dias após o procedimento cirúrgico divididos nos grupos: BDL, BDL tratado com CS, Sham e Sham tratado com CS. Foram avaliados peso corporal e do fígado dos animais, concentrações séricas de bilirrubina direta (BD), globulinas, atividades de gama glutamil transpeptidase (Gama GT), fosfatase alcalina (FA), alanina transaminase (ALT) e aspartato transaminase (AST), alterações morfológicas no tecido, atividade de mieloperoxidase (MPO), atividade de metaloproteases (MMP-9, MMP-2 e pró MMP-2) e conteúdo de GAGs no fígado dos animais, além da análise histopatológica do tecido hepático. O CS obtido para a realização do trabalho apresentou teores superiores a 92%, com peso molecular de aproximadamente 40 kDa e um conteúdo dissacarídico com predominância de Δdi4S (65%). BDL gerou vários sintomas relacionados à lesão celular e ao processo inflamatório como aumento dos níveis séricos de BD e globulinas, aumento das atividades de Gama GT, FA, ALT e AST, infiltrado inflamatório e modificação morfométrica, com proliferação ductular, e na MEC do fígado dos animais induzidos. CS levou a redução do aumento inicial das transaminases indicando proteção dos tecidos lesados no procedimento cirúrgico. O tratamento levou à redução do infiltrado inflamatório no tecido, expresso pela diminuição significativa da atividade de MPO no homogenato. A remodelação tecidual também foi reduzida, havendo diminuição da atividade de MMP-9, pró MMP-2 e MMP-2 e ainda dos níveis dos GAGs dermatam sulfato e heparam sulfato presentes, produzidos por células estreladas em resposta ao dano no tecido. Estes resultados mostram que o CS reduziu os efeitos da lesão hepática do modelo e foi capaz de retardar a fibrogênese hepática. / Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) present in the extracellular matrix (ECM) of many mammalian tissue, used for osteoarthritis treatment and, recently, has aroused great interest due to its anti-inflammatory potential. Several in vivo inflammation experimental models are employed to study anti-inflammatory activity, including extra-hepatic cholestasis induced fibrosis. Cholestasis produces hepatocellular injury with portal tract edema, leukocyte infiltration, biliary epithelial cells proliferation and portal tract fibrosis. The aim of this work was to analyze CS effects on an extra-hepatic cholestasis experimental model induced by bile duct ligation (BDL) on Wistar rats. For this purpose 6 to 8 weeks old animals (n = 82) were euthanized 2, 7 or 14 days after surgical procedure, previously divided into groups: BDL, CS treated BDL, Sham, CS treated Sham. To analyze disease evolution body and liver weight, serum concentrations of direct bilirubin (BD), globulins, activities of gamma glutamyl transferase (Gama GT), alkaline phosphatase (FA), alanine and aspartate aminotransferases (ALT and AST), morphological changes on tissue, mieloperoxidase (MPO) activity, matrix metalloproteinases (MMP-9, pró MMP-2 and MMP-2) activities and liver GAGs content, besides histopathological analysis of the tissue. CS acquired presented over 92% tenor, molecular weight of approximately 40 kDa and disaccharide content of Δdi4S predominantly (65%).BDL caused many symptoms related to cellular damage and inflammatory process such as increasing BD and globulins, elevation of Gama GT, FA, ALT and AST activities, inflammatory infiltrate and changes on liver morphometry, with ductular proliferation, and on the ECM. CS reduced initial burst on aminotransferases, indicating protection of tissues injured on surgery procedure. Treatment led to reduction of inflammatory infiltrate, showed by significant decreasing on MPO activity. Tissue remodeling was also reduced, with decrease of MMP-9, pro MMP-2 and MMP-2 activities and also of GAGs dermatam sulfate and heparam sulfate levels, produced by hepatic stellate cells in response of tissue damage. These results show that CS reduced cholestasis hepatic injury effects, being capable to slow down liver fibrogenesis.

L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique dû à une dysfonction hépatique où l'ammoniaque est un facteur central. Il a déjà été rapporté que l’intoxication aiguë d'ammoniaque induise le stress oxydatif/nitrosatif. La présente étude cible à évaluer le rôle du stress oxydatif/nitrosatif dans 2 modèles de l’EH chronique : (1) l’anastomose portocave (PCA) et (2) la ligation de la voie biliaire (BDL). Ces 2 modèles sont caractérisés par une hyperammoniémie et une augmentation d’ammoniaque centrale, cependant l’œdème cérébral est trouvé seulement chez les rats BDL. Des marqueurs du stress oxydatif/nitrosatif ont été évaluées dans le plasma et cortex frontal. Un stress nitrosatif central a été observé chez les rats PCA; tandis qu’un stress oxydatif/nitrosatif systémique a été démontré seulement chez les rats BDL. Ces résultats suggèrent (1) que l’hyperammoniémie chronique n’induise pas le stress oxydatif/nitrosatif systémique et (2) qu’un synergisme existe entre l’ammoniaque et le stress oxydatif/nitrosatif, en association avec l’œdème cérébral. / Hepatic encephalopathy (HE) is a neuropsychiatric complication due to liver failure where ammonia is believed to be central in the pathogenesis. Acute ammonia intoxication has demonstrated to induce oxidative/nitrosative stress in both in vivo and in vitro models. The present study was aimed to assess the role of oxidative/nitrosative stress in 2 models of chronic liver failure/HE; 1. portacaval anastomosis (PCA) and 2. bile duct ligation (BDL). Both models are characterised with hyperammonemia and increased brain ammonia however cerebral edema is only found in BDL rats. Oxidative/nitrosative stress markers were evaluated in plasma and frontal cortex of both animal models. Central nitrosative stress was observed in PCA rats, but systemic oxidative/ntrosative stress was demonstrated only in BDL rats. The results of our study suggest i) chronic hyperammonemia does not induce oxidative stress and ii) a synergistic effect between ammonia and systemic oxidative/nitrosative stress is associated with cerebral edema.