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Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt. Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen. Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen. Insgesamt konnten folgende Thesen aufgestellt werden: • Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren. • Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt. • Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden. • Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden. • Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden. • Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.

Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt. Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen. Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen. Insgesamt konnten folgende Thesen aufgestellt werden: • Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren. • Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt. • Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden. • Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden. • Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden. • Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.

O balanço energético corporal é mantido através de alterações na ingestão de calorias e no gasto energético. O prolongado balanço energético positivo, em que a ingestão excede o gasto, promove obesidade. A obesidade resulta do aumento de triacilglicerois no tecido adiposo, que também passa a apresentar inflamação de baixo grau crônica. O resultado disso envolve a redução da capacidade de tamponamento dos ácidos graxos livres circulantes e subsequente deposição de gordura em territórios ectópicos como musculatura esquelética e fígado, além de aumentar o risco de desenvolver o diabetes Mellitus tipo 2. Assim, a obesidade é um fator de risco importante para doenças metabólicas e suas comorbidades, dentre elas a doença gordurosa hepática não alcoólica. Uma proposta terapêutica para intervir na obesidade e/ou nas comorbidades associadas é o uso de substancias com ação anti-inflamatória. Considerando a possibilidade de novo uso para alguns produtos liberados para uso e com potencial tóxico já avaliado, foi escolhido avaliar o efeito do fitoterápico Uncaria tomentosa. Para isso, foram utilizados dois modelos de camundongos: (1) obesidade induzida por dieta hiperlipídica (DH), camundongos machos c57bl/6, alimentado com DH por 10 semanas, e (2) camundongo geneticamente obeso, ob/ob. A dose e tempo de tratamento escolhidos basearam-se no resultado da tolerância à insulina determinada inicialmente. Os animais receberam via oral o extrato da UT na dose de 50 mg/kg, uma vez ao dia, por 5 dias consecutivos. Seguindo-se ao tratamento, os animais foram submetidos a testes de tolerância a glicose e insulina, análise do coeficiente respiratório e depois a eutanásia, para retirada do fígado e sangue. O tecido hepático foi submetido a técnicas histológicas para verificar a morfologia e quantificação das gotículas de gordura, extração das proteínas celulares e RNAm para realização de western blot típico e reação em cadeia da polimerase (PCR), respectivamente. Também foi utilizada imunohistoquímica com anticorpo anti-F4/80 para comprovar infiltrado inflamatório. O tratamento com UT reduziu a glicemia de jejum cerca de 15 e 20% nos modelos DH e ob/ob, respectivamente, e a insulimenia para 54% no grupo DH, enquanto no grupo ob/ob houve um aumento de 2 vezes na insulinemia com o uso da UT. Houve uma redução de 22% no índice de massa corpórea (IMC) acompanhada de maior gasto energético com o tratamento no modelo DH. A esteatose hepática foi reduzida em aproximadamente 30% e a presença de infiltrados inflamatórios em 70% em ambos os modelos. Além disso, o grau de fosforilação do IRS1 no resíduo serina foi reduzido em 25% nos camundongos alimentados com DH após tratamento com UT, acompanhando a maior sensibilidade à insulina desses animais em relação aos obesos não tratados. Diante desses resultados, concluímos que o extrato bruto da Uncaria tomentosa melhora a homeostase da glicose e reverte a esteatohepatite incipiente a estatose benigna. O tratamento com Uncaria tomentosa pode ser uma estratégia terapêutica potencial no combate a alterações metabólicas associadas à obesidade como a doença hepática gordurosa não alcoólica (DHGNA) de início imediato. / The body\'s energy balance is maintained through changes in calorie intake and energy expenditure. The prolonged positive energy balance, in which the intake exceeds the expense, promotes obesity. Obesity results from the increase of triacylglycerols in adipose tissue, which also starts to present chronic low-grade inflammation. The result of this involves the reduction of the buffering capacity of circulating free fatty acids and subsequent fat deposition in ectopic territories such as skeletal muscle and liver, as well as increasing the risk of developing type 2 diabetes mellitus. Thus, obesity is an important risk factor for metabolic diseases and their comorbidities, among them non-alcoholic fatty liver disease. A therapeutic proposal to intervene in obesity and/or associated comorbidities is the use of substances with anti-inflammatory action. Considering the possibility of new use for some products released for use and with toxic potential already evaluated, it was chosen to evaluate the effect of the herbal remedy Uncaria tomentosa (UT), popularly known as cat\'s claw. Two models of mice were used: (1) higt fat diet induced obesity (HFD), male mice c57bl / 6, fed HFD for 12 weeks, and (2) genetically obese mice, ob/ob. The dose and time of treatment chosen were based on the initially determined insulin tolerance result. The animals received orally the UT extract at the dose of 50 mg/kg once daily for 5 consecutive days. Following the treatment, the animals were submitted to glucose and insulin tolerance tests, analysis of the respiratory coefficient and then euthanasia, for removal of the liver and blood. Liver tissue was submitted to histological techniques to verify the morphology and quantification of fat droplets, extraction of cellular proteins and mRNA for typical western blot and polymerase chain reaction (PCR), respectively. Immunohistochemistry with anti-F4 / 80 antibody was also used to prove inflammatory infiltrate. Treatment with UT reduced fasting glucose by 15 and 20% in the HFD and ob/ob models respectively, and the insukinemia to 54% in the HFD group, whereas in the ob/ob group there was a 2-fold increase in insulinemia with the use of UT. There was a 22% reduction in body mass index (BMI) accompanied by greater energy expenditure with HFD treatment. Hepatic steatosis was reduced by approximately 30% and the presence of inflammatory infiltrates by 70% in both models. In addition, the degree of IRS1 phosphorylation at the serine residue was reduced by 25% in the mice fed HFD after treatment with UT, following the higher insulin sensitivity of these animals compared to the untreated obese. In view of these results, we conclude that the crude extract of Uncaria tomentosa improves glucose homeostasis and reverts incipient steatohepatitis to benign steatosis. Treatment with Uncaria tomentosa can be a potential therapeutic strategy in the action against obesity-related metabolic alterations such as non-alcoholic fatty liver disease (NAFLD).

A incidência da obesidade tomou proporções epidêmicas nos últimos anos, atingindo bilhões de indivíduos mundialmente. A DHGNA é uma manifestação hepática das alterações metabólicas causadas pela obesidade e os casos desta doença vêm crescendo cada vez mais. Alternativas capazes de reduzir estas alterações são fundamentais para minimizar o impacto na qualidade de vida da população e na economia do país. Diversos estudos têm mostrado que os compostos bioativos de alimentos possuem efeitos benéficos à saúde. O camu-camu (Myrciaria dubia (H. B. K). Mc Vaugh) é um fruto nativo da região amazônica com potencial agroeconômico ainda inexplorado, que contém um grande número de compostos fitoquímicos que podem atuar sobre o metabolismo corporal. Desta forma, o objetivo deste estudo foi avaliar o efeito dos compostos fenólicos do camu-camu no desenvolvimento da DHGNA em camundongos C57BL/6 que receberam dieta rica em lipídios e sacarose (HFS). O extrato rico em compostos fenólicos da polpa comercial deste fruto foi obtido através de extração em fase sólida e caracterizado por cromatografia líquida de alta eficiência (CLAE/DAD). Os extratos obtidos foram testados em doses de 7 mg e de 14 mg equivalentes de ácido gálico/Kg de peso corporal. Foram investigados os efeitos destes compostos sobre as homeostases glicídica e lipídica através de análises séricas, testes de tolerância à insulina e à glicose e conteúdo de glicogênio e triacilglicerol intra-hepático. O extrato do camu-camu apresentou flavonóis, ácido elágico e elagitaninos em sua composição. A suplementação com extrato fenólico de camu-camu diminuiu a intolerância à glicose, independente da dose administrada, e melhorou a sensibilidade à insulina e regulou o conteúdo de glicogênio intra-hepático na maior dose. Não foi observado efeito sobre os lipídios plasmáticos. Entretanto, nota-se que houve uma melhora na função hepática em decorrência da redução da atividade da alanina aminotransferase (ALT), indicadora de dano celular, independente da dose. Além disso, a suplementação com extratos fenólicos do camu-camu na maior dose reduziu o conteúdo de triacilglicerol intra-hepático (p = 0,0001) e de biomarcadores inflamatórios, como Proteína - C Reativa (PCR) (p = 0,0359) e prostaglandina E2 (PGE2) (p = 0,004). Estes efeitos foram associados, principalmente, à menor ingestão alimentar. Portanto, neste estudo, os compostos fenólicos do camu-camu foram eficientes em prevenir a progressão da DHGNA em camundongos alimentados com dieta HFS. / Obesity has reached epidemic proportions in recent years, affecting billions of people worldwide. Nonalcoholic fatty liver disease (NAFLD) is a hepatic disorder induced by the metabolic changes caused by obesity and its incidence has been growing increasingly. Alternatives designed to reduce such changes are crucial to minimize their impact on the population´s quality of life and countries economy. Several studies have shown that food bioactive compounds have beneficial effects on health. Camu-camu (Myrciaria dubia (H. B. K.) Mc Vaugh) is a native fruit from Amazonan, with unexplored agroeconomic potential, which contains a large number of phytochemical compounds that can act on body metabolism. Therefore, this study was designed to assess the effect of the phenolic compounds of camu-camu in the development of NAFLD in C57BL/6 mice fed with a lipid and saccharose-rich diet (HFS). The phenolic compound-rich extract was obtained from the commercial pulp of this fruit using solid-phase extraction and high-performance liquid chromatography with diode array detector (HPLC/DAD). The resulting extracts were tested at 7 mg and 14 mg gallic acid equivalents/kg body weight. The effects of these compounds on glucose and lipid homeostasis were investigated by serum analyses, insulin and glucose tolerance tests and intrahepatic content of glycogen and triacylglycerol. Camu-camu extract presented flavonols, ellagic acid and ellagitannins in its composition. Supplementation with camu-camu phenolic extract decreased glucose intolerance, regardless the dose, improved insulin sensitivity and normalized the intra-hepatic glycogen content at the highest dose. No effects on plasma lipid were found. However, an improvement in liver function due to the decrease in alanine aminotransferase (ALT) was observed, suggestive of cell damage, regardless the dose. Moreover, the supplementation with phenolic extracts of camu-camu at the highest dose decreased the intrahepatic content of triacylglycerol (p = 0.0001) and inflammatory biomarkers, such as C-reactive protein (CRP) (p = 0.0359) and prostaglandin E2 (PGE2) (p = 0.004). Such effects were primarily associated with lower food intake. Therefore, in this study, the phenolic compounds of camu-camu have shown to be effective in preventing the progression of NAFLD in mice fed with HFS (high-fat/sucrose) diet.

L’obésité est à l’origine de la première cause de maladies hépatiques dans les pays occidentaux. Les lésions hépatiques s’étendent de la stéatose isolée et réversible à la stéatohépatite (NASH), la fibrose, la cirrhose jusqu’au carcinome hépatocellulaire. Aucun traitement pharmacologique n’a montré son efficacité pour éviter cette évolution. La compréhension des mécanismes à l’origine du processus inflammatoire est donc un élément clef pour le développement de nouvelles voies thérapeutiques. Nous avons précédemment montré dans un modèle murin d’obésité que la stéatose induit un recrutement accru de lymphocytes par le foie. Les cellules de Kupffer représentent jusqu’à 20% des cellules immunitaires du foie. Elles peuvent présenter différents phénotypes : pro-inflammatoire ou anti-inflammatoire. Un phénotype pro-inflammatoire engendre la sécrétion de cytokines/chimiokines pro-inflammatoires favorisant une réponse immune de type Th1 et un recrutement de cellules immunitaires. Les cellules de Kupffer pourraient ainsi être des acteurs majeurs dans les étapes précoces du développement de la maladie.Le but de ce travail a été d’étudier le phénotype des cellules de Kupffer au stade de la stéatose et le rôle des lipides dans ce phénotype.Nous avons montré que l'accumulation de lipides dans les cellules de Kupffer est due à un dérèglement du métabolisme des lipides et de leur trafic. Les cellules de Kupffer chargées de lipides ont un phénotype pro-inflammatoire qui induit le recrutement des lymphocytes durant les premiers stades du développement de la NASH et qui est réversible par l'inhibition de la lipogenèse. / We have shown lipid accumulation in fat-laden Kupffer cells is due to a dysregulation of lipid metabolism and trafficking. Fat-laden Kupffer cells are "primed" to recruit lymphocytes and exhibit a pro-inflammatory phenotype at the stage of steatosis, which is reversible with inhibition of lipogenesis

Carcinome hépatocellulaire (CHC) est le sixième cancer et la deuxième cause de mortalité par cancer dans le monde. Dans la majorité des cas, le CHC se développe sur une maladie chronique associée à des étiologies variées telles que l'infection par le virus de l'hépatite B ou l’hépatite C, la consommation excessive d'alcool et des maladies métaboliques. Le développement des maladies chroniques du foie qui conduisent à la cirrhose puis au cancer induisent des modifications de la composition chimique des cellules et des tissus. En effet, la carcinogenèse hépatique est un processus en plusieurs étapes caractérisé par la progression de nodules de régénération, de nodules dysplasiques de bas grade puis de grade et enfin du CHC. Le traitement du CHC reste difficile et la transplantation du foie est la seule option thérapeutique curative à long terme. Le problème est qu'il n'y a pas de marqueur objectifs et quantifiables pour contrôler la qualité d’un greffon. Des biomarqueurs spécifiques marquant la progression du CHC font également défauts.Dans ce travail de thèse, nous avons évalué l’intérêt de la microspectroscopie infrarouge (IR) pour le diagnostic de la stéatose, qui est le facteur le plus important affectant la reprise de la fonction hépatique après greffe de foie. La microspectroscopie infrarouge permet de détecter de façon qualitative et quantitative les caractéristiques biochimiques liées aux différents constituants moléculaires présents dans l'échantillon biologique. Nos travaux ont montré que la progression de la stéatose hépatique correspond non seulement à l'accumulation de lipides, mais également à des changements spectaculaires dans la composition qualitative du tissu. En effet, le bas grade de stéatose présente une diminution de la teneur en glycogène et une augmentation concomitante de lipides par rapport au foie normal. La stéatose intermédiaire montre une augmentation de glycogène et des changements majeurs sont observés en ce qui concerne les lipides, avec une contribution significative des acides gras estérifiés, des chaînes de carbone allongées et des lipides insaturés. Ces caractéristiques sont encore plus prononcées dans les hauts degrés de stéatose. De plus, nous avons mis en évidence que des changements biochimiques majeurs se produisent dans la partie non-stéatosique du tissu malgré son aspect normal sur le plan histologique, ce qui suggère que l’organe dans son ensemble reflète le degré de la stéatose.La deuxième partie de la thèse est focalisée la carcinogenèse hépatique. Il s’agit d’un processus en plusieurs étapes qui se caractérise dans la plupart des foies cirrhotiques par la progression de nodules hyperplasiques de régénération vers des lésions précancéreuses telles que les nodules dysplasiques de bas grade puis de haut grade et enfin le CHC. Le diagnostic différentiel entre nodules dysplasiques en particulier de haut garde et CHC reste extrêmement difficile. Nous avons abordé le potentiel de la microspectroscopie IR pour le diagnostic des nodules cirrhotiques. Nous avons observé de profondes modifications de la composition biochimique du foie pathologique. En effet, des changements importants ont été détectés dans la composition des lipides, des protéines et des sucres mettant en évidence la reprogrammation métabolique dans la carcinogenèse. Les principaux changements ont été observés dans le domaine de fréquence 950-1480 cm-1 dans lequel plusieurs bandes permettaient la discrimination des nodules cirrhotiques, dysplasiques et tumoraux. Enfin, nous avons montré que le diagnostic peut être réalisé à l’aide d’un microscope de laboratoire qui peut être facilement mis en œuvre en milieu hospitalier. / Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the second most common cause of death in the world. Hepatocarcinogenesis is a multistep process characterized in patients with chronic liver diseases by a spectrum of hepatic nodules that mark the progression from regenerative nodules to dysplastic lesions followed by HCC. Liver transplantation remains the curative therapeutic option able to treat both the HCC and the underlying liver disease. The issue is that there is no objective and quantifiable marker for quality control of liver graft. Specific biomarkers of early stages of HCC are also an unmet need.In this study, we have evaluated the potential of infrared (IR) microspectroscopy for the diagnosis of steatosis, one of the most important factors affecting the liver allograft function. Vibrational microspectroscopy, such as Fourier transform infrared microspectroscopy (FTIR), allows detecting spectral characteristics associated with different molecular components present in the biological sample, both qualitatively and quantitatively. Our first working hypothesis was that the progression of liver steatosis corresponds not only to the accumulation of lipids but also to dramatic changes in the qualitative composition of tissue. Indeed, a lower grade of steatosis showed a decrease in glycogen content and concomitant increase in lipids in comparison with normal liver. Intermediate steatosis exhibited an increase in glycogen and major changes in lipids, with a significant contribution of esterified fatty acids with elongated carbon chains and unsaturated lipids, and these features were more pronounced in a high grade of steatosis. Furthermore, we have shown, that FTIR approach allows a systemic discrimination of morphological features, leading to a separate investigation of steatotic vesicles and the non-steatotic counterpart of the tissue. This highlighted the fact that dramatic biochemical changes occur in the non-steatotic part of the tissue also despite its normal histological aspect, suggesting that the whole tissue reflects the grade of steatosis. The second part of the thesis focused on hepatocarcinogenesis; a multistep process that is characterized in most cirrhotic livers by the progression from hyperplastic regenerative nodules to low grade dysplastic nodules (LGDN), high grade dysplastic nodules (HGDN) and finally small HCC which corresponds either to vaguely nodular well differentiated HCC so called early HCC or to distinctly nodular moderately differentiated hepatocellular carcinomas. Since the differential diagnosis between precancerous dysplastic nodules and early HCC remains extremely difficult, we addressed the potential of FTIR microspectroscopy for grading cirrhotic nodules. The study was focused on 39 surgical specimens including normal livers as controls, dysplastic nodules, early HCC and the progressed HCC. Profound alterations of the biochemical composition of the pathological liver were demonstrated by FTIR microspectroscopy. Indeed, dramatic changes were observed in lipids, proteins and sugars highlighting the metabolic reprogramming in carcinogenesis. The major changes were observed in the frequency domain 950-1480 cm-1 in which several bands allowed significant discrimination of cirrhotic nodules, dysplastic lesions and HCC. Finally, a significant discrimination between benign, dysplastic nodules and early HCC remained possible using a FTIR microscope equipped with a laboratory-based infrared source that can be easily implemented in hospital environment. In conclusion, our study positions FTIR microspectroscopy as a versatile and powerful approach for investigating liver diseases, such as steatosis, dysplastic lesions and cancer. Further studies on larger series of patients as well as on biopsies will allow confirming the clinical reliability of such spectral signatures. Therefore, we anticipate that FTIR microspectroscopy will open new avenue in clinical diagnosis.

Introdução: A doença hepática alcóolica (DHA) e a doença hepática gordurosa não alcóolica (DHGNA) estão entre as principais causas de morte por doenças hepáticas no mundo. Apesar das etiologias distintas, sendo a DHA causada pelo consumo excessivo de álcool e a DHGNA por dieta inadequada e sedentarismo, apresentam curso de doença semelhante, que pode evoluir de esteatose, para esteato-hepatite, fibrose, cirrose e carcinoma hepatocelular. Tanto a DHA quanto a DHGNA estão relacionadas à disbiose, aumento de permeabilidade intestinal, inflamação e dano hepático. Entretanto, ainda não está claro se a doença hepática precede as alterações no epitélio intestinal ou se é o aumento da permeabilidade que promove o dano hepático. Objetivo: Avaliar a expressão gênica de Tight Junctions em modelo de DHA e DHGNA em zebrafish (Danio rerio). Métodos: No modelo de DHA, os peixes foram divididos em dois grupos: Etanol (n=30), expostos a 0,5% de etanol por 28 dias e controle (n=30). No modelo de DHGNA, os peixes foram divididos também em dois grupos: Frutose (n=24), expostos à frutose 6% durante 2 horas por 14 dias e controle (n=24). Ao término dos experimentos os animais foram eutanasiados e coletados fígados, para avaliação histológica por coloração hematoxilina-eosina (HE) e de esteatose por Oil Red, e intestinos para avaliação da expressão gênica dos marcadores de permeabilidade intestinal cldnC, cldn15a, cldn15b e f11r por Real Time qPcr. Resultados: Tanto os animais expostos ao álcool quanto à frutose apresentaram esteatose hepática por coloração HE e Oil Red quando comparados aos seus respectivos controles, sem sinais de infiltrados inflamatórios e de fibrose hepática à microscopia óptica. Não houve diferença significativa na expressão gênica das tight junctions intestinais, tanto para a DHA quanto DHGNA (p  0,05). Conclusão: Os resultados sugerem que em estágios iniciais de DHA e DHGNA não ocorre alteração da permeabilidade intestinal, e que possivelmente o dano hepático precede o dano intestinal. / Introduction: Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are among the leading causes of death from liver disease worldwide. Despite the different etiologies, ALD is caused by excessive alcohol consumption and NAFLD is due to inadequate diet and sedentary lifestyle, they have a similar disease course, which can progress from steatosis to steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. Both ALD and NAFLD are related to dysbiosis, increased gut permeability, inflammation, and liver damage. However, it is not yet clear whether liver disease precedes changes in the intestinal epithelium or whether it is the increased permeability that promotes liver damage. Objective: To evaluate the gene expression of tight junctions in ALD and NAFLD models in zebrafish (Danio rerio). Methods: In the ALD model, fish were divided into two groups: Ethanol (n=30), exposed to 0.5% ethanol for 28 days and control (n=30). In the NAFLD model, fish were also divided into two groups: Fructose (n=24), exposed to 6% fructose for 2 hours for 14 days and control (n=24). At the end of the experiments the animals were euthanized and livers were collected for histological evaluation by hematoxylin-eosin (HE) staining and steatosis by oil red staining and intestines for evaluation of the gene expression of gut permeability markers cldnC, cldn15a, cldn15b and f11r by Real Time qPcr. Results: Both animals exposed to alcohol and fructose presented hepatic steatosis by HE and Oil Red staining when compared to their respective controls, without signs of inflammatory infiltrates under optical microscopy. There was no significant difference in the gene expression of the tight junctions for both ALD and NAFLD (p0.05) Conclusion: The results suggest that in the early stages of ALD and NAFLD there are no changes in intestinal tight junctions, and that possibly liver damage precedes intestinal damage.

A doença hepática gordurosa não alcoólica (DHGNA) é definida pelo acúmulo excessivo de gordura nos hepatócitos sem ingestão significativa de álcool. Essa condição é associada à obesidade e a síndrome metabólica (SM) e é considerada um grave problema de saúde pública global. A DHGNA engloba a esteatose pura e a esteatohepatite, esta última caracterizada pela presença de inflamação e balonização dos hepatócitos, com ou sem formação de fibrose com potencial para evolução para formas mais graves como cirrose e carcinoma hepatocelular. Estudos atuais divergem sobre a relação entre a deficiência da vitamina D e a gravidade da DHGNA. O objetivo foi verificar se há associação entre níveis séricos de vitamina D, citocinas relacionadas ao processo inflamatório (IL-6, IL-10 e TNF-?), presença de componentes da SM e a prevalência e a gravidade da DHGNA. Metodologia Estudo transversal que incluiu 40 pacientes do sexo feminino, obesas (IMC>30kg/m²) e com idade superior a 18 anos submetidas e biópsia hepática e obtiveram diagnostico de DHGNA. Foram coletados dados referentes a características clínicas, parâmetros antropométricos e de gordura corporal. Adicionalmente, foram avaliados parâmetros clínicos de SM e dados bioquímicos relacionados à lesão hepática além de dosagem de 25(OH)D e níveis séricos de citocinas. Um grupo controle com 37 indivíduos saudáveis do sexo feminino, não obesas (IMC<30kg/m²), com idade média de 41 anos, foi usado como controle de vitamina D sérica. Resultados Dos 40 pacientes incluídos, 25 tinham esteatose pura e 15 esteatohepatite. A prevalência de insuficiência de vitamina D foi de 70%. Os valores séricos de vitamina D não foram diferentes quando comparados ao grupo controle saudável e comparando-se esteatose e esteatohepatite. Níveis de vitamina D e de citocinas não apresentaram relação com os parâmetros histopatológicos de lesão hepática, exceto a presença de balonização que foi associada a maiores valores de vitamina D. Níveis séricos de vitamina D correlacionaram-se negativamente com o IMC. Níveis séricos de citocinas não foram associados com a gravidade da DHGNA. O escore NAS e o grau de balonização dos hepatócitos foram maiores em pacientes que apresentaram SM. Conclusões Apesar da alta prevalência de insuficiência de vitamina D, níveis séricos de vitamina D e citocinas não foram associados com presença ou gravidade da DHGNA em pacientes obesas do sexo feminino submetidas à cirurgia bariátrica. Balonização dos hepatócitos foi o único parâmetro histopatológico de gravidade de lesão hepática associado com níveis séricos mais altos de vitamina D. SM foi associada a parâmetros de maior gravidade de lesão hepática. / Nonalcoholic fatty liver disease (NAFLD) is defined by excessive fat accumulation in hepatocytes without significant alcohol intake and is associated with obesity and metabolic syndrome. NAFLD is one of the most common causes of chronic liver disease and it is considered a global public health problem. NAFLD includes pure steatosis and steatohepatitis; the last been characterized by the presence of inflammation and hepatocytes ballooning with or without fibrosis and with potential to evolve to more severe forms as cirrhosis and hepatocelular carcinoma. Current studies diverge on the relationship between vitamin D deficiency and the severity of NAFLD. The objective was to assess if there is an association between vitamin D serum levels, cytokines related to inflammation (IL-6, IL-10 and TNF-?) and presence of metabolic syndrome components and the prevalence and severity of NAFLD. Methods Cross-sectional study of 40 obese (BMI > 30kg/m²) female patients above 18 years who underwent hepatic biopsy during bariatric surgery to diagnose NAFLD and assess the degree of liver damage. Clinical data, anthropometric parameters and body fat were collected. In addition, clinical parameters of metabolic syndrome were evaluated and biochemical data relating to liver injury beyond 25(OH)D and serum cytokines. A control group with 37 healthy non obese females, mean age 41 was used as a controls for serum vitamin D. Results Among 40 NAFLD patients 25 had pure steatosis and 15 steatohepatitis. Prevalence of vitamin D insufficiency was 70%. No difference was observed in vitamin D levels comparing healthy control group and NAFLD or comparing steatosis and steatohepatitis. No relationship was observed between vitamin D or cytokine levels with histopathology parameters of liver injury. Higher levels of vitamin D were associated with cellular ballooning .Vitamin D levels negatively correlated with BMI. Serum cytokine levels were not associated with the severity of NAFLD. The NAS score and the ballooning degree were higher in patients with metabolic syndrome. Conclusions Despite high prevalence of vitamin D insufficiency, serum vitamin D and cytokines were not associated with the presence or severity of NAFLD in obese female patients undergoing bariatric surgery. Ballooning was the only histological parameter of liver damage associated with higher serum levels of vitamin D. Metabolic syndrome was associated with parameters of higher severity of NAFLD.

Au cours de l'obésité, l'excès de lipides circulants, est stocké dans les organes périphériques, principalement dans le foie. Ce stockage ectopique de lipide peut avoir, à long terme, des conséquences délétères sur le métabolisme glucidique. Au long cours, l'excès de lipides hépatiques peut conduire au développement de stéatopathies métaboliques pouvant évoluer vers la cirrhose et le cancer du foie. Dans l'hépatocyte, le métabolisme et l'homéostasie lipidique sont finement régulés par la balance entre la synthèse (LDN) et l'utilisation (ß-oxydation) des lipides. Ces deux voies métaboliques sont sous le contrôle de plusieurs facteurs de transcriptions comme ChREBP, SREBP1 PPARƴ ou PPARa. La compaction et l'accessibilité de la chromatine sont des éléments cruciaux pour la régulation indirecte de l'activité de ces facteurs de transcription. Dans le noyau, la compaction de l'ADN est régulée par les histones mais aussi par les protéines High Mobility Group (HMG). Parmi la famille des protéines HMG, la protéine High Mobility Group box 1 (HMGB1), principalement localisée dans le noyau, est capable de réguler de façon indirecte la transcription de gènes dans de nombreux tissus. En plus de son rôle nucléaire, HMGB1 peut être activement sécrétée par les cellules innées au cours de phénomènes inflammatoires aigus. Chez la souris, la délétion totale de cette protéine entraine une hypoglycémie périnatale létale. De plus, chez la souris, les concentrations circulantes d'HMGB1 sont augmentées lors d'un stress métabolique induit par un régime hyper-lipidique (HFD). Tous ces résultats suggèrent un rôle d'HMGB1 dans le métabolisme hépatique et énergétique ainsi que dans les processus inflammatoires de bas-bruits associés au stress métabolique. [...] / During obesity, the excess of circulating lipids, are stored in the peripheral organs, and mainly in the liver. This ectopic storage of lipids may have long-term deleterious consequences on carbohydrate metabolism. Over time, excess of intra-hepatic lipids can lead to the development of steatohepatitis that can evolve to cirrhosis and liver cancer. In the hepatocyte, lipid homeostasis is finely regulated by the balance between synthesis (de novo lipogenesis-DNL) and degradation (ß-oxidation) of lipids. These two metabolic pathways are under the control of several transcription factors like ChREBP, SREBP1, PPARƴ or PPARa. The compaction and accessibility of chromatin are crucial parameters, which regulate the activity of these transcription factors. In the nucleus, the compaction of DNA is regulated by histones but also by High Mobility Group (HMG) proteins. Among the HMG protein family, High Mobility Group box 1 protein (HMGB1), mainly located in the nucleus, is able to indirectly regulate gene transcription in many tissues. In addition to its nuclear role, HMGB1 can be actively secreted by innate immunes cells during acute inflammatory reactions. In mice, the global deletion of Hmgb1 gene leads to perinatal lethality due to a severe hypoglycemia. Moreover, preliminary data from our laboratory show that circulating concentrations of HMGB1 are increased in mice subjected to high fat diet (HFD). All these results support a role of HMGB1 in hepatic and energetic metabolism but also in tissue-low grade inflammation related to metabolic stress. [...]