O carcinoma hepatocelular (HCC) é a sexta neoplasia mais comum e a terceira causa de mortalidade por câncer no mundo. Está relacionado, principalmente, à exposição a diversos fatores de risco, como a doença hepática gordurosa não alcoólica (NAFLD). O HCC apresenta mau prognóstico; neste sentido, é importante a adoção de medidas de controle, como a quimioprevenção. A quimioprevenção é o método mais apropriado para se evitar o câncer e consiste na prevenção, inibição ou reversão das etapas iniciais da carcinogênese, pela administração de um ou mais compostos químicos sintéticos ou naturais. Diversos compostos presentes nos alimentos podem apresentar atividade quimiopreventiva, dentre esses, a tributirina (TB), pró-fármaco do ácido butírico. Dessa forma, propôs-se avaliar o efeito quimiopreventivo da TB nas etapas de iniciação/promoção inicial, em ratos submetidos ao modelo de hepatocarcinogênese do hepatócito resistente (RH) associado à NAFLD. Ratos Wistar machos foram distribuídos em Grupo dos animais não tratados (NT), e dois outros grupos experimentais, o grupo RH + NAFLD + maltodextrina (grupo controle isocalórico, CI), e o grupo RH + NAFLD + tributirina (grupo TB). Os animais do grupo CI foram tratados diariamente, por gavagem, com emulsão hiperlipídica [1 mL/100 g de peso corpóreo (p. c)], e maltodextrina (300 mg/ 100 g p.c.), enquanto que os animais do Grupo TB foram tratados diariamente, por gavagem, com emulsão hiperlipídica [1 mL/100 g de p. c.], e TB (200 mg/ 100 g de p.c.), durante 13 semanas consecutivas quando foram eutanasiados. Após esse período, a análise por cromatografia a gás revelou que o grupo dos animais tratados com a tributirina apresentou uma concentração de AB 2.118 vezes superior (p < 0.05) às concentrações de AB constatadas no grupo controle. Em relação aos dados de morfometria de LPNs [lesões pré-neoplásicas persistentes (pLPN) ou em remodelação (rLPN)], a atividade quimiopreventiva da tributirina foi observada a partir da redução (p < 0,05) significativa do número de lesões pré-neoplásicas persistentes (pLPN) em comparação ao grupo controle. A área das pLPNs, contudo, foi maior (p < 0,05) no Grupo TB quando comparada à do grupo controle isocalórico. Não foram observadas diferenças estatisticamente significativas (p >= 0,05) no número e na área de rLPN, bem como na % da área do corte do fígado ocupada por LPNs entre os dois grupos experimentais. A quantificação da apoptose por H&E revelou maior (p < 0,05) índice apoptótico em pLPN e em rLPN nos animais tratados com TB, quando comparados aos animais do grupo 6 controle. Nesse sentido, observou-se que os animais tratados com a tributirina apresentaram maior (p < 0,05) ativação de Caspase 3 do que os ratos do grupo controle isocalórico. Em relação à avaliação da proliferação celular, o Grupo TB apresentou redução (p < 0,05) do índice de proliferação tanto em pLPNs como em rLPNs, quando comparado ao do grupo controle isocalórico. Não foi observada diferença estatisticamente significativa (p > 0,05) entre os surroundings dos dois grupos experimentais. A determinação do perfil lipídico sérico revelou que os animais tratados com a tributirina apresentaram menores (p < 0,05) concentrações séricas de LDL-colesterol e maiores (p < 0,05) concentrações séricas de HDL-colesterol, quando comparados às do grupo controle. Além disso, foram observadas menores (p < 0,05) concentrações hepáticas de colesterol no Grupo TB quando comparadas às do Grupo CI. Em relação a análise por PAS, embora, o presente estudo não tenha revelado diferença estatisticamente significativa (p >= 0,05) entre os dois grupos experimentais, o score entre os graus de marcação por PAS apresentou tendência (p = 0,07) a ser maior nos animais tratados com a tributirina quando comparados aos ratos do grupo controle. Em relação à expressão gênica de FGF-21, o Grupo TB apresentou maior (p < 0,05) expressão em comparação a seu grupo controle (Grupo CI). Além disso, esses dados são corroborados por meio da marcação imunoistoquímica de FGF-21, que revelou que os animais tratados com a tributirina apresentaram maior (p < 0,05) score desta marcação quando comparados aos ratos do grupo controle isocalórico. A análise de expressão em nível proteico de PPAR-&#945; revelou que o Grupo TB apresentou maior (p < 0,05) expressão em comparação ao Grupo CI. O presente estudo demonstrou que o tratamento com a tributirina apresentou um efeito quimiopreventivo quando administrada nas etapas de iniciação/promoção inicial em modelo de hepatocarcinogênese associado à NAFLD. Adicionalmente, sugere-se que a atividade como HDACi da TB poderia modular a expressão de genes supressores de tumor, bem como a daqueles relacionados ao metabolismo lipídico, atenuando os fatores de risco relacionados à NAFLD. / Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the third leading cause of cancer mortality in the world. It is mainly related to exposure to several risk factors, such as non-alcoholic fatty liver disease (NAFLD). HCC presents poor prognosis; In this sense, it is important to adopt control measures, such as chemoprevention. Chemoprevention is the most appropriate method to avoid cancer and consists in the prevention, inhibition or reversal of the early stages of carcinogenesis by the administration of one or more synthetic or natural chemical compounds. Several compounds present in foods may present chemopreventive activity, among them, tributyrin (TB), a prodrug of butyric acid. Thus, it was proposed to evaluate the chemopreventive effect of TB in the initiation / initial promotion stages in rats submitted to the hepatocyte-resistant hepatocyte model (HR) associated with NAFLD. Male Wistar rats were divided into two groups: RH + NAFLD + maltodextrin (isocaloric control group, CI), and RH + NAFLD + tributyrin group (TB group). The animals of the CI group were treated daily with gavage with hyperlipid emulsion [1 mL / 100 g body weight (wt.)], and maltodextrin (300 mg / 100 g body wt.), while the animals of the TB Group were treated daily by gavage with hyperlipid emulsion [1 mL / 100 g of body wt.], and TB (200 mg / 100 g body wt.) for 13 consecutive weeks when they were euthanized. After this period, the gas chromatographic analysis revealed that the group of animals treated with tributyrin had a concentration of AB 2184 times higher (p < 0.05) than the concentrations of AB found in the control group. Regarding the morphometry data of PNLs [persistent pre-neoplastic lesions (pPNL) or remodeling (rPNL)], the chemopreventive activity of tributyrin was observed from a significant (p < 0.05) reduction in the number of pPNL compared to the control group. The area of the pPNLs, however, was higher (p < 0.05) in the TB Group when compared to the isocaloric control group. There were no statistically significant differences (p >= 0.05) in the number and area of rPNL, as well as in the area of the cut of the liver occupied by LPNs between the two experimental groups. The quantification of apoptosis by H&E revealed a higher (p < 0.05) apoptotic index in pPNL and rPNL in the TB treated animals, when compared to the animals in the control group. In this sense, animals treated with tributyrin showed greater (p < 0.05) activation of Caspase 3 than the rats of the isocaloric control group. In relation to the evaluation of cell proliferation, the TB group presented a reduction (p < 0.05) in the proliferation index in both pPNLs and rPNLs, when compared to the isocaloric control group. No statistically significant difference (p> 0.05) was observed between the two experimental groups. The determination of serum lipid profile revealed that the animals treated with tributyrin showed lower (p < 0.05) serum LDL-cholesterol concentrations and higher (p < 0.05) serum HDL-cholesterol concentrations when compared to those in the group control. In addition, lower hepatic concentrations (p < 0.05) of cholesterol were observed in the TB Group when compared to those in the CI Group. Regarding the SBP analysis, although the present study did not reveal a statistically significant difference (p >= 0.05) between the two experimental groups, the score between the PAS presented a tendency (p = 0.07) to be greater in animals 8 treated with tributyrin compared to the rats in the control group. In relation to the gene expression of FGF-21, the TB Group presented higher (p <0.05) expression in comparison to its control group (CI Group). In addition, these data are corroborated by FGF-21 immunohistochemical labeling, which revealed that animals treated with tributyrin showed a higher (p <0.05) score for this marker when compared to the isocaloric control rats. Analysis of protein-level expression of PPAR-&#945; revealed that the TB Group had higher (p <0.05) expression compared to the CI Group. The present study demonstrated that treatment with tributyrin had a chemopreventive effect when administered in the initiation / initial promotion steps in a hepatocarcinogenesis model associated with NAFLD. In addition, it is suggested that the activity as HDACi of TB could modulate the expression of tumor suppressor genes, as well as those related to lipid metabolism, attenuating the risk factors related to NAFLD.

Introdução: Já foi mostrado que o óxido nítrico (NO) age como um potente inibidor da peroxidação lipídica, um dos principais contribuintes para a fibrogênese na esteatohepatite não-alcoólica (EHNA). O S-Nitroso-N-acetilcisteína (SNAC), um doador de NO, modula a ativação de células estreladas hepáticas e tem mostrado efeitos benéficos na EHNA experimental. O objetivo deste estudo foi avaliar a eficácia do SNAC como um agente anti-fibrogênico na EHNA experimental. Métodos: Ratos Sprague-Dawley adultos foram alimentados com dieta hiperlipídica deficiente em colina e expostos a dietilnitrosamina (DEN) durante 8 semanas. Dez animais receberam SNAC (1,4 mg/ kg) por gavagem diariamente (grupo SNAC) e 10 receberam veículo (grupo EHNA). Três animais receberam somente dieta padrão e veículo (grupo Controle). Após este período, os animais foram sacrificados e o tecido hepático retirado para avaliação histológica, quantificação do colágeno e análises de expressão gênica. Genes relacionados à fibrose [metaloproteinase de matriz (MMP)- 2, 9 e 13, e, TGF b -1, colágeno-1a, inibidor tecidual de metaloproteinase (TIMP)-1 e 2] e genes relacionados ao estresse oxidativo [proteínas de choque térmico (HSP)-60 e 90] foram avaliados. Resultados: O SNAC levou a atenuação da fibrose hepática verificada pela quantificação de colágeno, e este efeito foi associado com supra-regulação da MMP- 13, MMP-9 e infra-regulação da HSP-60, TIMP-2, TGF b -1 e colágeno-1a. Conclusões: O SNAC apresenta propriedades anti-fibrogênicas no modelo de EHNA empregado, infra-regula moléculas pró-fibrogênicas e supra-regula a MMP-13, um fator determinante da degradação do colágeno intersticial. Nossa hipótese é que o SNAC, pela redução do estresse oxidativo, leva a um padrão de expressão gênica que favorece uma maior remoção de colágeno, com conseqüente atenuação da fibrose hepática na EHNA, sugerindo que SNAC é um potencial agente anti-fibrogênico neste contexto / Introduction: Nitric oxide (NO) has already been shown to act as a potent inhibitor of lipid peroxidation, a major contributor for fibrogenesis in nonalcoholic steatohepatitis (NASH). S-Nitroso-N-acetylcysteine (SNAC), an NO donor, modulates hepatic stellate cells activation and has shown beneficial effects on experimental NASH. The aim of this study was to evaluate the effectiveness of SNAC as an antifibrogenic agent in experimental NASH. Methods: Adult Sprague-Dawley rats were fed choline-deficient high fat diet and exposed to diethylnitrosamine during 8 weeks, during which 10 animals received SNAC (1.4 mg/kg) by daily gavage (SNAC group) and 10 animals received vehicle (NASH group). Another group (Control; n=3) received only standard diet and vehicle. After this period, liver tissue was obtained for histological study, collagen quantification and gene expression analyses. Genes related to fibrosis [matrix metalloproteinase (MMP)-13, 9 and 2, TGFb-1, collagen-1a, tissue inhibitor of metalloproteinase (TIMP)-1 and 2] and genes related to oxidative stress [heat-shock protein (HSP)-60 and 90] were evaluated. Results: SNAC led to attenuation of liver fibrosis, verified through collagen quantification, and this effect was associated with up-regulation of MMP-13, MMP-9 and down-regulation of HSP-60, TIMP-2, TGFb-1 and collagen-1a. Conclusions: SNAC shows antifibrogenic properties in the NASH model employed, down-regulates profibrogenic molecules and up-regulates MMP-13, a key determinant of interstitial collagen degradation. We hypothesize that SNAC, through oxidative stress lowering, leads to a gene expression pattern that favors greater collagen removal, with consequent attenuation of liver fibrosis in NASH. Our findings suggest that SNAC is a potential antifibrogenic agent in this setting

Introdução: O aumento alarmante da obesidade em todo mundo é um fator independente para o aumento da prevalência de doenças crônicas, como diabetes, hipertensão arterial e doença hepática gordurosa não alcoólica (DHGNA). A DHGNA engloba um amplo espectro de doença desde esteatose simples sem sinais inflamatórios, evoluindo para esteatohepatite (ENA) e até cirrose. Por ser uma patologia multifatorial sua patogênese e terapêutica ainda apresentam pontos obscuros. A resistência à insulina é um dos fatores que já foram determinados como importantes na fisiopatogênese da DHGNA e, está diretamente relacionado ao estilo de vida e hábitos alimentares. Objetivo: Avaliar a correlação entre a presença da gordura visceral e subcutânea à resistência insulínica, síndrome metabólica, medidas antropométricas e hábitos alimentares em pacientes portadores DHGNA. Resultados: Sessenta por cento dos pacientes com DHGNA apresentavam Síndrome Plurimetabólica de acordo com os critérios do ATP III. Dentre os critérios mais prevalentes da síndrome metabólica, níveis baixos HDL e circunferência de cintura (CC) aumentada ocorreram em 56%. Ao dividirmos os pacientes em grupos, os critérios se modificaram. Nos pacientes portadores de esteatose simples, o critério mais prevalente foi CC 77%. Já naqueles com ENA o aumento do nível de triglicérides ocorreu em 68% dos pacientes, sendo o de maior prevalência. As correlações entre CT e IMC (p=0,001), CC (p=0,003) e percentual de massa gorda (p=0,031), apresentam significância estatística negativa entre os pacientes com DHGNA. Não houve significância estatística entre CT e HOMA, ingestão total de energia, proteínas, carboidratos e lipídios nestes pacientes mesmo quando foram divididos de acordo com o grau de inflamação, esteatose e ENA. Por outro lado, a CC ao ser correlacionada com IMC (p=0,000), HOMA (p=0,049), ingestão total de energia (p=0,043), lipídios e dos ácidos graxos mono (p=0,020) e poliinsaturados (0,045) apresentaram significância estatística nos pacientes com DHGNA. Quando divididos segundo o grau de esteatose houve correlação positiva entre CC e HOMA (p=0,045) nos pacientes com esteatose simples. Já nos pacientes portadores de ENA houve correlação positiva entre CC e ingestão total de calorias (p=0,031). Houve diferença estatística positiva quanto ao consumo de lipídios totais (p=0,005) entre o grupo de esteatose e ENA, onde os pacientes com ENA, tinham um maior consumo de lipídios. Conclusões: A circunferência de cintura se mostrou melhor parâmetro de diagnóstico complementar ao ser comparado com a tomografia computadorizada em pacientes com DHGNA. Ao associarmos o IMC com a CC conseguimos obter uma avaliação nutricional mais fidedigna dos riscos de doenças decorrentes da síndrome metabólica. O alto consumo de lipídios apresentou correlação positiva com maior grau de inflamação em pacientes com DHGNA. / Introduction: The dramatic increasing of obesity in the world is a predictive factor to increase the prevalence of chronic diseases, as type II diabetes, hypertension and Nonalcoholic fatty liver disease (NAFLD). It is part of large spectrum of liver damage ranging from simple steatosis and cirrhosis. The pathogenesis of NAFLD is multifactorial and some points in the mechanisms and therapeutic still unknown. The insulin resistance is the major risk factors, recognized in the pathophysiology of NAFLD and it is strongly related with life style and diet. Objective: Evaluate the correlation between visceral and subcutaneous fat with insulin resistance, presence of metabolic syndrome, anthropometric measurements and dietary intake in patients with NAFLD. Results: Sixty percent of patients with NAFLD, classified by ATPIII criteria, had the metabolic syndrome. Waist circumference and low HDL -C were the most positive criteria, observed in 56% of total cases. However the criteria modifies when the group were divided. In patients with simple steatosis WC was the most prevalent criteria 77%, where as in NASH patients the high level of TGL 68%, was the most prevalent. Correlations between CT and BMI (p=0,001), WC (p=0,033) and fat mass (p =0,031) had a negative statistic significant correlation in all cases of NAFLD. No significant associations were found between CT (umbilical circumference) and fat mass, WC, HOMA, Intake of calories, carbohydrates, proteins, lipids and fat acids in patients divided in steatosis and NASH patients. The correlation between Waist circumference (WC) with BMI (p=0,000), HOMA (p:0,049), total of energy intake (p=0,043) , monounsaturated (p:0,020) and polyunsaturated (p:0,045) fat acids intake showed a statistic significance in all cases of NAFLD. Significant different correlations were found as the group were divided in steatosis and NASH cases. The values of fat mass correlated with WC was (p: 0,045) in the steatosis group where as in NASH patients the only correlation found was WC and total consumption of daily energy (p=0,031). NASH patients showed a statistic higher intake of lipids (p=0,005), compared with steatosis patients. Conclusions: CT measurement of visceral fat did not contribute to differential diagnosis as much as WC in patients with NAFLD. The use of both measurements BMI and WC showed to be a better predictor of the risks of Metabolic Syndromes. NASH subjects had a higher intake o total fat as well as of lipid fractions.

A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é atualmente uma das formas mais comuns de doença hepática, e está diretamente relacionada com a obesidade. Estudos indicam uma prevalência por volta de 30% na população ocidental e 11% na população oriental. A DGHNA possui um largo espectro abrangendo desde casos de esteatose simples sem inflamação, até casos com esteatohepatite e fibrose, podendo evoluir para cirrose e carcinoma hepatocelular. A fisiopatogênese da DHGNA é baseada em múltiplos eventos: resistência insulínica, aumento da lipólise periférica, aumento da síntese de novo de lipídeos, estresse oxidativo, endotoxemia crônica no desencadeamento da inflamação e fibrose entre outros. A etapa inicial caracteriza-se pelo acúmulo de ácidos graxos no hepatócito, suplantando sua capacidade de metabolização e exportação conseqüente à ação lipogênica da insulina, desta forma o metabolismo de ácidos graxos está intimamente ligado ao desenvolvimento da DHGNA. O Yo Jyo Hen Shi Ko (YHK) é um composto natural usado na medicina japonesa para tratamento de doenças hepáticas e apresenta propriedades antioxidantes, antiinflamatórias e hipolipemiantes. O presente estudo teve como objetivo avaliar o efeito do YHK no metabolismo hepático de lipídeos. Para tanto, foram utilizados camundongos obesos (ob/ob) com esteatohepatite não alcoólica (ENA) induzida por dieta deficiente em colina e metionina (DCM) e foram analisados no tecido hepático, genes relacionados com a síntese de novo de lipídeos (SREBP1c, FASn), genes relacionados com a oxidação e exportação de lipídeos (CPT1a e SCD-1, MTP), assim como genes relacionados com o armazenamento de lipídeos (Perilipina e ADFP). O YHK apresentou um efeito citoprotetor hepático com melhora dos parâmetros histológicos neste modelo experimental de ENA. Associadamente, houve redução na expressão de genes relacionados à síntese de novo como SREBP e da FASn, quando se comparou o grupo tratado com DCM+YHK com o grupo não tratado DCM. Em contrapartida, houve aumento na expressão da MTP e da SCD-1 ocasionando uma maior exportação de triglicerídeos hepáticos nos animais que utilizaram o YHK. Ainda, o YHK modulou as proteínas Perilipina e ADFP. Por outro lado, não houve modificação na oxidação de lipídeos.. Conclui-se que o YHK pode ser uma droga promissora no tratamento da DHGNA, já que age modulando genes envolvidos na síntese e exportação de lipídeos hepáticos, reduzindo o acúmulo de gordura no hepatócito / Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common forms of liver disease, related directly to the increase of obesity in the world. Previous studies indicate a prevalence of around 30% in the western population and 11% in the eastern. This disease covers cases from simple steatosis without inflammation to cases of steato hepatitis (NASH) and fibrosis and it may lead to liver cirrhosis and hepatocellular carcinoma. The pathophysiology of NAFLD is based on multiple events: insulin resistance, increased peripheral lipolysis, increased de novo lipogenesis, oxidative stress, endotoxemia in triggering chronic inflammation and fibrosis and others. The initial stage of the disease is characterized by the accumulation of fatty acids in hepatocytes caused by the lipogenic action of insulin. This condition results in the surpassing of the cells ability to metabolize and export. This way, the metabolism of fatty acids is closely linked to the development of NAFLD. The Yo Jyo Hen Shi Ko (YHK) is a natural compound used in Japanese medicine for the treatment of liver disease and it has antioxidant, anti-inflammatory and lipid lowering properties. This study aimed to evaluate the effect of YHK in the hepatic metabolism of lipids. An experimental model was designed using obese mice (ob/ob) affected by NASH induced by a deficient diet in choline and methionine (MCD). Then, genes from its hepatic tissue related to de novo lipogenesis (SREBP1c, FASN), to lipid oxidation and exportation (CPT1A and SCD-1, MTP) as well as genes related to lipid storage (Perilipin and ADFP) were analyzed. The YHK presented a cytoprotective effect, improving the hepatic histological parameters in this experimental model. Additionally, when comparing the group treated with MCD + YHK (diet + YHK) to the untreated MCD group (diet) there was a reduction in the expression of genes related to de novo synthesis like SREBP1c and FASn. However, an increased expression of MTP and SCD-1 were observed leading to a greater liver exportation of fatty acids in animals that were treated with YHK. Moreover, the YHK modulated the proteins Perilipin and ADFP. On the other hand, there was no changing in lipid oxidation. In summary, the YHK can be a promising drug for the treatment of NAFLD, as it acts by modulating genes involved in the synthesis and exportation of hepatic lipids, reducing the accumulation of fat in hepatocytes

Introdução: A lesão de isquemia/reperfusão do fígado é caracterizada pelo agravamento da lesão isquêmica hepatocelular quando o órgão é revascularizado, podendo originar, nos casos mais graves, uma reação inflamatória sistêmica com lesão de órgãos à distância. O controle desse fenômeno é importante no transplante de fígado, nas cirurgias de ressecção hepática e no choque hemorrágico. A administração de soluções salinas hipertônicas têm se mostrado eficaz no tratamento do choque hemorrágico, pois melhora as alterações hemodinâmicas e, possivelmente, apresenta uma ação antiinflamatória. Neste trabalho foram avaliados os efeitos locais e sistêmicos da administração da solução salina hipertônica na lesão de isquemia/reperfusão hepática em ratos. Métodos: Enquanto 14 ratos Wistar machos, dos 56 utilizados no estudo, compuseram o grupo controle, grupo C; os demais, que foram submetidos à uma hora de isquemia hepática e 4 horas de reperfusão, compuseram outros grupos de 14 animais: o grupo ST, animais que não receberam tratamento; o grupo SSF, animais que receberam 34 mL/kg de NaCl 0,9%, por via endovenosa, 15 minutos antes da reperfusão; o grupo SSH, animais que receberam 4 mL/kg de NaCl 7,5%, 15 minutos antes da reperfusão. Após 4 horas de reperfusão, os materiais foram coletados para análise. Foram realizadas as dosagens das transaminases AST e ALT, a avaliação das funções oxidativas e fosforilativas mitocondriais, a dosagem das interleucinas IL-6 e IL-10, as análises teciduais pulmonares e a análise histológica do fígado isquêmico e não isquêmico. Resultados: Quando comparado aos grupos ST e SSF, o grupo SSH apresentou elevação dos níveis de AST e ALT significantemente menores; preservação da função mitocondrial tanto dos lobos isquêmicos, como dos não isquêmicos, significantemente melhor; elevação dos níveis de IL-6 e IL-10 sem significância estatística; aumento da permeabilidade vascular pulmonar significantemente menor; e elevação da atividade da mieloperoxidase pulmonar sem significância estatística. Em relação à análise histológica da lesão de isquemia/reperfusão hepática, o escore da lesão do grupo SSH foi significantemente menor que o da lesão do grupo C; entretanto, quando comparados os três grupos submetidos à isquemia hepática ST, SSH e SSF não se observaram diferenças significantes estatisticamente. Conclusão: A administração da solução salina hipertônica a 7,5% na isquemia/reperfusão hepática normotérmica melhorou as lesões hepáticas locais e as lesões à distância, principalmente pulmonares / Introduction: During liver ischemia, the drop in mitochondrial energy production leads to cellular damage, which is aggravated during restoration of blood supply. Besides local hepatic injury, the ischemia/reperfusion process can trigger a systemic inflammatory syndrome producing remote organ damage. To control these alterations in clinical conditions like liver transplantation, liver resections and hypovolemic shock, is crucial to achieve proper patient management. Aim: To evaluate the effect of the sodium chloride hypertonic solution on prevention of local and systemic injury during partial liver ischemia/reperfusion. Methods: Animals underwent partial warm liver ischemia/reperfusion. Fity six Wistar male rats were randomly allocated into four groups. Fourteen animals were submitted to sham operation and allocated to C group; 42, submitted to one hour of liver ischemia followed by 4 hours of reperfusion, were allocated in three additional groups: ST group, 14 animals that received no treatment; SSF group, 14 animals that received NaCl 0.9%, 34 mL/kg, intravenously; SSH group, animals that received NaCl 7.5%, 4 mL/kg, intravenously. Blood and tissue samples were collected four hours after reperfusion, when animals were killed. Blood samples were collected to determinate AST, ALT, IL-6 and IL-10 levels. Liver and pulmonary tissues were assembled for liver histology and for liver mitochondrial phosphorylation, pulmonary vascular permeability and myeloperoxidase analyzes. Results: Hypertonic saline solution showed beneficial effects in the treatment of liver ischemia/reperfusion injury. SSH group presented elevation of AST and ALT plasma levels significantly lower than ST and SSF groups. A significant reduction on mitochondrial dysfunction was observed in SSH group compared with ST and SSH groups. Elevation in serum IL-6 and IL-10 was similar among ST, SSF and SSH groups. Pulmonary vascular permeability was significantly lower in group SSH compared with ST and SSF groups. No differences in myeloperoxidase activity were observed among these three groups. Histological score for liver ischemia/reperfusion injury was significantly lower in SSH group compared to ST group, however no differences were observed between SSH and SSF groups. Conclusion: Administration of hypertonic saline solution in an experimental rat model of liver ischemia-reperfusion ameliorated local and systemic injuries

En spectroscopie de résonance magnétique (SRM) in vivo, les principales méthodes utilisées permettent la quantification des concentrations de métabolites en utilisant des signaux à une dimension spectrale. Les travaux réalisés dans le cadre de cette thèse portent sur le développement de méthodes de SRM à deux dimensions spectrales (SRM 2D) de corrélation localisée afin d’accroître le pouvoir de résolution spectrale et la précision de la quantification de la SRM in vivo. Le premier axe de cette thèse concerne le développement d’une méthode fondée sur la spectroscopie 2D de corrélation localisée pour l’exploration des métabolites cérébraux. La séquence L-COSY (spectroscopie de corrélation localisée) est implantée sur imageur petit animal et étudiée. Une procédure de quantification dédiée aux signaux de corrélation acquis est développée. Cette dernière opère dans le domaine d’acquisition du signal, et s’appuie sur : 1) une connaissance a priori forte obtenue par simulation de l’effet quantique des séquences sur les spins des composés présents dans le spectre 2) un modèle de pondération lié aux effets de relaxation agissant sur le signal de SRM 2D. 3) une contrainte sur la relaxation liée aux effets d’inhomogénéités supposés toucher tous les spins de la même manière. Les résultats présentés s’attachent à étudier les performances quantitatives de la SRM 2D de corrélation, en comparaison à la SRM 2D dite J-résolue (avec la séquence JPRESS), de manière expérimentale, sur fantômes de métabolites mais aussi à travers la théorie des bornes de Cramér-Rao (CRBs). La quantification des signaux L-COSY, bien que défavorisée par une perte théorique du rapport signal sur bruit par unité de temps, présente des CRBs théoriques relatives du même ordre de grandeurs voire, pour certains métabolites couplés (e.g la glutamine, le GABA) plus petites que celles correspondantes à la spectroscopie J-résolue pour un même temps d’acquisition. Le second axe de cette thèse porte sur l’adaptation la SRM 2D de corrélation pour l’étude in vivo du métabolisme lipidique du foie et des tissus adipeux sous-cutanés sur un modèle de souris obèse à 7T. Cette application inédite montre la faisabilité de la SRM 2D de corrélation à être acquise sur un tel organe mouvant et sa capacité à être quantitative pour l’étude et la caractérisation des triglycérides hépatiques et sous-cutanées. / In in vivo Magnetic Resonance Spectroscopy (MRS), the main methods used allow metabolite concentration quantification using signals having one spectral dimension. This work focuses on the development of in vivo two dimensional correlated MRS in order to increase spectral resolution and quantification precision. The first axis is about the development of a method based on a 2D localized correlation MRS (L-COSY) for brain metabolite exploration. The L-COSY is implemented and studied on a small animal scanner. A dedicated quantification procedure operating in the acquisition domain is described. This latter is based on 1) a strong prior knowledge obtained by quantum mechanically simulate the effect of sequence on metabolite spin systems 2) a model function taking into account the relaxation weighting 3) constraints on the relaxation term linked to the field inhomogeneity effects which are assumed to act the same way on all the spins. Results are given experimentally using metabolites phantoms and through a comparison to other existing 2D MRS method, namely the J-resolved MRS (with the JPRESS sequence) using the Cramer Rao Lower Bounds (CRBs) theory. Although its inherent loss of signal to noise ratio is a disadvantage compared to J-PRESS, L-COSY quantification shows theoretically competitive relative CRBs, and even smaller CRBs for some coupled metabolites (e.g Glutamine or GABA), for an acquisition time similar to JPRESS. Second axis is about the adaptation of the 2D correlation MRS for the in vivo lipid metabolism study in the liver and subcutaneous adipose tissues of obese mice at 7T. This application shows the feasibility of 2D correlated MRS to be acquired on a moving organ and its quantitative relevance for triglyceride quantification and characterization in fatty liver and subcutaneous tissue.

Abstract Obesity, insulin resistance, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) form a dangerous quartet which threatens human health all over the world. About 25% of adults around the world have NAFLD, which poses risks for cardiovascular and metabolic well-being and may develop into liver cirrhosis and hepatocellular carcinoma. Apart from lifestyle modification, treatment options for NAFLD are scarce. This thesis presents atrial fibrillation (AF) as a new complication of NAFLD among general population of 958 individuals aged 40-60 years participating in the OPERA study. Even after multiple-adjustments for confounding factors, ultrasound-based NAFLD predicted the development of AF during about 16 years of follow-up. Moreover, the association between AF and liver fibrosis in 76 individuals aged 64-82 years in a cross-sectional setting is presented. The thesis also shows that individuals with metabolic syndrome (MetS), with or without NAFLD, are at increased risk of cardiovascular events, T2D and the increase of left ventricular mass index in a study population of 958 individuals aged 40-60 years during a 20-year follow-up. In other words, NAFLD without MetS does not seem to expose to these three cardiometabolic complications. The thesis also shows that rifampicin-activated pregnane X receptor (PXR), a member of the nuclear receptor superfamily of ligand-activated transcription factors with several endobiotic and xenobiotic activators, increases serum levels of cholesterol, phospholipids and certain fatty acids, assessed by nuclear magnetic resonance metabolomics technique, in a randomized, open, placebo-controlled trial among 34 young and healthy individuals. These serum lipids are considered toxic lipids and capable of transforming hepatosteatosis into steatohepatitis and even more severe forms of NAFLD. Moreover, rifampicin-activated PXR has no effect on serum triglycerides, that are non-toxic lipids, or triglyceride accumulation in the liver, assessed by magnetic resonance imaging, in 15 young and healthy individuals. In conclusion, this thesis advances the knowledge in the pathogenesis, lipid metabolism, complications and heterogeneous nature of NAFLD. These may have implications for patient care and follow-up. / Tiivistelmä Maailmanlaajuisesti noin 25% täysi-ikäisistä henkilöistä sairastaa alkoholinkäyttöön liittymätöntä rasvamaksaa. Sen tiedetään altistavan sydän- ja verisuonisairauksille, aineenvaihduntahäiriöille, maksakirroosille ja jopa maksasyövälle, mutta elämäntapahoitoa lukuun ottamatta hoitomahdollisuudet ovat toistaiseksi vähäisiä. Tässä väitöskirjassa osoitetaan ensimmäistä kertaa alkoholinkäyttöön liittymättömän rasvamaksan ennustavan itsenäisesti eteisvärinän ilmaantuvuutta noin 16 vuoden seurannan aikana 958 tavallisen keski-ikäisen ihmisen aineistossa osana OPERA-tutkimusta. Lisäksi väitöskirjassa osoitetaan maksan sidekudosmuodostuksen ja eteisvärinän välillä olevan yhteys poikkileikkausasetelmassa 76 iäkkään ihmisen muodostamassa aineistossa. Väitöstutkimuksessa havaittiin myös, että metabolista oireyhtymää sairastavilla henkilöillä on suurentunut tyypin 2 diabeteksen, sydän- ja verisuonisairauksien sekä vasemman kammion koon suurentumisen riski noin 20 vuoden seurannan aikana 958 tutkittavan henkilön aineistossa riippumatta siitä, onko heillä alkoholinkäyttöön liittymätön rasvamaksa. Toisin sanoen alkoholin käyttöön liittymätön rasvamaksa ilman metabolista oireyhtymää ei lisää edellä mainittujen kolmen sairauden riskiä. Väitöstutkimuksessa esitetään lisäksi, että rifampisiinilla aikaansaatu maksan pregnane X -reseptorin aktivaatio johtaa seerumin fosfolipidien, tiettyjen rasvahappojen sekä usean eri kolesterolityypin lisääntymiseen 34 terveen nuoren henkilön aineistossa. Kirjallisuudessa näiden seerumin rasva-aineiden on esitetty aiheuttavan alkoholin käyttöön liittymätöntä maksatulehdusta ja jopa rasvamaksan vakavimpia muotoja. Toisaalta rifampisiini ei lisännyt seerumin triglyseridipitoisuutta eikä aiheuttanut magneettitutkimuksella mitattuna triglyseridien kertymistä maksaan 15 terveen nuoren henkilön aineistossa. Tämä väitöstutkimus antaa lisätietoa rasvamaksan kehittymisestä, rasva-aineenvaihdunnasta ja komplikaatioista sekä korostaa rasvamaksan monimuotoista luonnetta. Nämä löydökset saattavat parantaa rasvamaksaa sairastavien henkilöiden hoitoa ja seurantaa.

The past two decades have seen an explosive increase of obesity rates worldwide, with more than one billion adults overweight and 300 million of them obese. Obesity and its associated complications have become leading causes of morbidity and mortality in the United States and major contributing factors to the rising costs of national health care. The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation and chronic activation of immune pathways in adipose tissue and liver. The CD40 receptor and its ligand, CD40L, initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. A clear understanding of how CD40 and its ligand communicate to regulate and sustain the inflammatory environment of obesity is lacking. Here we demonstrate that CD40 receptor deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels, but paradoxically exhibit liver steatosis, insulin resistance and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, visceral adipose tissue in CD40 deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8+ effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40-null mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate the chronic inflammation associated with obesity, thereby protecting against hepatic steatosis.

This thesis aims to investigate fluoxetine, a widely prescribed SSRI antidepressant, for its role in the pathogenesis of NAFLD and uncover novel mechanisms by which it may contribute to drug-induced steatosis. We demonstrated that increased hepatic lipid accumulation was mediated, in part, via elevated serotonin production. The inhibition of hepatic serotonin synthesis prevented lipid accumulation in fluoxetine-treated hepatocytes demonstrating a causal role for serotonin in fluoxetine-induced hepatic steatosis. Interestingly, in several studies, serotonin signaling has been shown to impact prostaglandin biosynthesis. As prostaglandins have been implicated in the development of NAFLD, and fluoxetine has previously been shown to alter the production of prostaglandins I assessed the role of prostaglandins in fluoxetine-induced hepatic lipid accumulation. Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes, increased production of prostaglandin 15-deoxy-Δ12,14PGJ2 (PPARG agonist), and elevated PPARG targets involved in fatty acid uptake. Fluoxetine-induced lipid accumulation, 15-deoxy-Δ12,14PGJ2 production, and the expression of PPARG lipogenic genes were attenuated with a PTGS1 specific inhibitor. Taken together these findings suggested that fluoxetine-induced lipid accumulation was mediated via PTGS1 and its downstream product 15-deoxy-Δ12,14PGJ2. Given that Pparg was elevated following fluoxetine treatment, and PPARG regulates microRNA involved in hepatic lipid accumulation, my final project focused on PPARG’s role in altered miRNA expression. Indeed, fluoxetine treatment increased the miRNA expression of miR-122, an effect that was attenuated when fluoxetine treatment was combined with the PPARG antagonist GW9662, suggesting a fluoxetine-PPARG-miR122 axis contributing to hepatic steatosis. While these studies have only been performed in vitro, an understanding of the molecular changes associated with SSRI treatment may lead to the development of strategies to prevent the increased risk of adverse metabolic outcomes associated with the use of SSRI antidepressants. / Dissertation / Doctor of Philosophy (Medical Science) / In adults, major depressive disorder (depression) is one of the most common psychiatric illnesses. Recent data suggests that there are more than 4.1 million Canadians who currently suffer from depression. Depression is commonly treated using selective serotonin reuptake inhibitor (SSRI) antidepressants. While these antidepressants do help manage depressive symptoms, they can also cause unwanted side effects including a build-up of fat in the liver, leading to fatty liver disease. The goal of my research is to understand the link between SSRI use and the development of fatty liver disease. This thesis investigates the effects of fluoxetine (Prozac®), a commonly used SSRI antidepressant, on molecular pathways that can lead to the development of fatty liver disease. An understanding of the molecular changes with SSRI treatment may lead to the development of strategies to prevent the harmful effects of SSRI antidepressants on the liver.

La stéatose hépatique est une affection caractérisée par l'accumulation d'un excès de graisse dans le foie d'un individu sans antécédents d'abus d'alcool. La stéatose hépatique est en train de devenir la maladie chronique du foie la plus répandue dans le monde. En l'absence de thérapies approuvées, les modifications du mode de vie, y compris les interventions diététiques, restent la seule stratégie thérapeutique. Dans cette étude, nous avons cherché à déterminer si un régime riche en graisses (HFD) complété par des triglycérides à chaîne moyenne (TCM) atténue la stéatose hépatique chez les souris. Des souris mâles âgées de 7 semaines C57BL/6 ont été nourries avec un régime riche en graisses puis ont été randomisées pour rester sous régime riche en graisses ou passer à un régime riche en graisses complété par des TCM ou à un régime pauvre en graisses (LFD). Comparées aux souris nourries au HFD, les souris nourries au HFD supplémenté en TCM ont présenté une amélioration significative du degré de stéatose et d'inflammation et une amélioration des tests de tolérance au glucose et à l'insuline avec des changements modestes dans les lipides circulants et les teneurs en triacylglycérol et en cholestérol du foie. Notre étude démontre qu’une diète en gras supplémenté en TCM a des effets bénéfiques sur la stéatose hépatique. Les améliorations observées semblent être liées à la réduction de la teneur hépatique en céramides et en diacylglycérols, améliorant la sensibilité à l’insuline, et faisant des TCM une option non-pharmacologique viable à inclure dans la prise en charge de cette maladie débilitante. / Hepatic steatosis is a condition where excess fat accumulates in an individual's liver without a history of alcohol abuse. Hepatic steatosis is becoming the most common chronic liver disease worldwide. In the absence of approved therapies to specifically treat hepatic steatosis, lifestyle modifications, including dietary interventions, remain the only therapeutic strategy. In this study, we sought to determine whether a high-fat diet (HFD) supplemented with medium-chain triglycerides (MCT) attenuates hepatic steatosis in mice. Seven-week-old C57BL/6 male mice were fed HFD for 12 weeks to induce hepatic steatosis, then mice were randomized to remain on HFD or switch to either HFD supplemented with MCT or low-fat diet (LFD) for 6 weeks. Mice switched to LFD were used as another model of dietary interventions. Compared with mice fed HFD, mice fed HFD supplemented with MCT exhibited significant improvement in the degree of steatosis and inflammation as well as improvement in glucose and insulin tolerance tests with modest changes in circulating lipids and liver triacylglycerol and cholesterol contents, the hallmark of hepatic steatosis. Our study demonstrates that HFD supplement with MCT has beneficial effects on hepatic steatosis. The observed improvements seem to be linked to reductions in hepatic ceramide and diacylglycerol content, which ultimately prevents protein kinase C-ε translocation to the plasma membrane and improves insulin sensitivity, making a dietary supplement like MCTs, a viable non-pharmacological option in the management of this debilitating disease.