In der vorliegenden Arbeit wurde der Einfluss von Prä- und Probiotika auf bakterielle Translokation (BT) und postoperative Infektionen nach großen viszeralchirurgischen Eingriffen untersucht. Dabei wurde aus methodischen Gründen zunächst BT nach Leber- (LR) und Colonresektion (CR) allein oder in Kombination experimentell im Rattenmodell quantifiziert und deren potentielle Pathomechanismen untersucht. Anschließend wurde der Einfluss von einzelnen Probiotika und einer Kombination verschiedener Probiotika und Präbiotika auf die Inzidenz bakterieller Infektionen nach Lebertransplantation (LTX) und pyloruserhaltender Pankreaskopfresektion (PPPD) in zwei prospektiv randomisierten klinischen Studien analysiert. Im experimentellen Teil der Untersuchungen wurde BT nach LR vor allem in Leber und Milz, nach CR hauptsächlich in mesenterialen Lymphknoten (MLK) und Milz nachgewiesen. Kombination von LR und CR führte zu einer Potenzierung der BT, parallel zum Ausmaß der LR. Durch Gabe von Probiotika wurde die Konzentration von Bakterien in den MLK signifikant gesenkt. Tiere mit einer hohen coecalen Laktobazillenkonzentration hatten eine signifikant niedrigere bakterielle Konzentration in allen untersuchten Organen als Tiere mit weniger Laktobazillen. CR führten zu einer Zunahme der coecalen gramnegativen Bakterienkonzentration und zu einer Abnahme der Laktobazillen. Histologische Veränderungen der Darmmukosa wurden nicht beobachtet. Die parazelluläre Permeabilität für Ionen, nicht aber für die höhermolekulare Laktulose war im Colon in allen Gruppen im Vergleich zur Kontrollgruppe erhöht. Probiotika beeinflussten die Zusammensetzung der coecalen Flora und damit auch die BT. In der ersten klinischen Studie verminderte postoperative orale Gabe von Laktobazillus plantarum und einer ballaststoffhaltigen Ernährungslösung die Inzidenz von bakteriellen Infektionen nach LTX im Vergleich zu selektiver Darmdekontamination und ballaststofffreier Ernährung signifikant. Die Gabe von Ballaststoffen und hitzeinaktivierten Laktobazillen führte zwar auch zu einer geringen Reduktion der Infektionen; diese war jedoch nicht signifikant. In der Mehrzahl wurden enteropathogene Bakterien isoliert. Die zweite klinische Studie untersuchte den Einfluss einer Kombination von vier verschiedenen Milchsäurebakterien und vier Präbiotika auf die Inzidenz bakterieller Infektionen nach LTX und PPPD. Im Vergleich zu Präbiotika und Placebo kam es zu einer deutlichen Verminderung der Infekte, die nach LTX auch signifikant war. In beiden Studien wurde die enterale Ernährung gut vertragen mit relativ wenig Nebenwirkungen. BT tritt somit sehr häufig auch nach kleineren viszeralchirurgischen Eingriffen auf und hat organspezifisch verschiedene Ursachen. Probiotika konnten sowohl tierexperimentell die BT vermindern als auch klinisch die Inzidenz bakterieller Infektionen nach großen viszeralchirurgischen Eingriffen senken. Da sie leicht zu verabreichen sind und wenig Nebenwirkungen verursachen, könnten sie breit eingesetzt werden. / In the present study, the impact of pre- and probiotics on bacterial translocation (BT) and postoperative bacterial infection rates was assessed. Due to methodological reasons, we first quantified BT following single liver (LR) and colon resection (CR) or a combination of both and analysed potential pathogenic mechanisms for BT. Then, we performed two prospective randomised clinical studies to analyse the influence of a single probiotic strain and a combination of different pre- and probiotics on the incidence of bacterial infections in patients with liver transplantation (OLT) or pylorus preserving partial pancreatoduodenectomy (PPPD). In the rat model, BT after LR mainly occurred in the liver and spleen, after CR mainly in the mesenteric lymph nodes (MLN) and spleen. BT was increased in the animals with combined operation, in parallel to the extent of liver resection. Probiotics significantly decreased the bacterial concentration in the MLN. Animals with a high cecal concentration of lactobacilli had significantly less BT than the others. CR led to an increase of cecal gramnegative bacterial concentrations and to a decrease of lactobacilli. No histological changes were observed in the intestine. Paracellular permeability for ions, but not for the larger molecule lactulose, was increased in the colon in all groups compared to the sham group. Probiotics had an influence on cecal bacterial concentration. In the first clinical study, postoperative oral administration of Lactobacillus plantarum and a fibre-enriched enteral diet significantly decreased bacterial infection rates after OLT compared to selective bowel decontamination and a fibre-free diet. Fibre and heat-inactivated Lactobacillus also led to a slight, but not significant decrease of infections. Mainly gut-derived bacteria were isolated. The second clinical study analysed the influence of a combination of four different lactic acid bacteria and fibres on bacterial infection rates after OLT and PPPD. Compared to fibres and placebo, infection rates were significantly lower after OLT and markedly lower after PPPD. In both studies, the study substances were well tolerated without serious side effects. BT even occurs following minor abdominal surgery and is caused by different mechanisms related to the kind of operation. Probiotics were able to diminish BT in the rat model as well as to decrease bacterial infection rates following major abdominal surgery in the clinical studies. As they are easy to administer and do not cause severe side effects, they could be useful in clinical practice.

INTRODUÇÃO: O transplante hepático se consolidou como terapêutica para crianças com doença hepática em estágio terminal. A principal indicação nessa faixa etária é a atresia das vias biliares, doença colestática que leva à cirrose hepática na maioria dos casos nos primeiros anos de vida mesmo nas crianças submetidas a portoenterostomia. Essas crianças evoluem entre outras complicações com desnutrição grave e necessitam do transplante precocemente. A relação ideal do peso do enxerto hepático e o peso do receptor varia de 1 a 3%. No entanto, quando o transplante é realizado em crianças em idade inferior a 2 anos, essa relação na maioria das vezes é maior do que 5%. Essa situação é conhecida como large-for-size, em que a perfusão sanguínea do enxerto pode ser insuficiente, o que leva a disfunção do órgão. Os mecanismos de lesão neste cenário ainda não estão bem esclarecidos. A lesão de isquemia-reperfusão (IR) é apontada como um dos fatores para o mal funcionamento do enxerto bem como de outras complicações. Nessa lesão estão envolvidos elementos da cascata inflamatória culminando em morte celular por apoptose bem como mecanismos de regeneração celular. Um fenômeno conhecido que atenua a lesão de IR é o pré-condicionamento isquêmico (PCI). Nenhum trabalho experimental foi realizado com o objetivo de avaliar a lesão hepática na situação large-for-size e o efeito do PCI nessa situação. MÉTODOS: Para a padronização do modelo foram realizados 8 transplantes, tendo sido testados os pesos dos animais, relação entre o peso do enxerto hepático e peso do receptor e técnica cirúrgica. Na fase experimental, foram realizados 21 transplantes e os animais foram divididos em 3 grupos. No primeiro grupo (controle) o transplante era realizado com doadores e receptores com pesos similares. Para caracterização da situação large-for-size, no segundo grupo os doadores tinham aproximadamente o dobro do peso dos receptores (LFS). No terceiro grupo a distribuição de peso foi semelhante a do segundo, porém era realizado o PCI no doador (LFS+PCI). Os procedimentos foram realizados sob anestesia geral e técnica asséptica e os animais foram mantidos vivos por 3 horas após a reperfusão. Foram obtidas amostras de sangue em 3 períodos (logo após abertura da cavidade abdominal, 1 e 3 horas após reperfusão) e fragmentos de fígado 1 e 3 horas após reperfusão. As amostras de sangue foram submetidas a gasometria arterial, dosagem de sódio, potássio e enzimas hepatocelulares. Os fragmentos de tecido hepático foram submetidos a análise histológica pela coloração da hematoxilina-eosina (HE) e a análise de quantificação da expressão dos genes pró-apoptótico (Bax), anti-apoptótico (Bcl- XL), da óxido nítrico-sintase endotelial (eNOS) e da interleucina-6 (IL-6). RESULTADOS: A mortalidade observada foi de 28%. A relação peso do enxerto e peso do paciente foi de 2,9% no grupo controle, 6,3% no grupo LFS e 6,4% no grupo LFS+PCI. A natremia 1 hora após a reperfusão foi mais elevada no grupo controle do que nos demais, já com relação ao potássio, a dosagem foi mais baixa nesse grupo no mesmo intervalo. A dosagem da aspartato aminotransferase (AST) foi mais elevada nos grupos LFS e LFS+PCI do que no controle. A análise histológica não Resumo demonstrou diferença entre os grupos. A expressão do gene Bax foi mais elevada no grupo LFS do que nos demais, enquanto que a do gene eNOS foi mais acentuada no grupo LFS+PCI do que nos demais nos dois intervalos estudados. Após 3 horas de reperfusão a expressão do gene IL-6 foi maior no grupo LFS+PCI. CONCLUSÕES: O modelo de transplante hepático large-for-size em suínos foi exequível e adequado para a pesquisa. A lesão IR foi mais acentuada no grupo LFS do que no grupo controle e apesar de não ter alterado a elevação de enzimas hepatocelulares, o PCI teve papel de aumentar a expressão dos genes IL-6 e eNOS e diminuir a do gene Bax / INTRODUCTION: Liver transplantation has been established as therapy for children with end-stage liver disease. The main indication is biliary atresia, cholestatic disease that leads to cirrhosis in most cases even in children undergoing portoenterostomy. These children develop other complications as severely malnutrition and need early transplantation. The ideal graft to body weight ratio (GBWR) for transplantation varies from 1 to 3%. However, when transplantation is performed in children younger than 2 years, this ratio in most cases is higher than 5%. This situation, known as large-for-size, may be related to insufficient graft perfusion and liver disfunction. The mechanisms of injury involved in this scenario are not well established. Ischemia-reperfusion injury (IRI) has been related as a factor for poor graft function and other complications. Inflammatory cascade culminating in cell death by apoptosis and cellular mechanisms of regeneration are the elements involved in IRI. Ischemic preconditioning (IPC) is a phenomenon that attenuates IR injury. There are no experimental studies conducted to evaluate the hepatic injury in large-for-size situation and the effect of IPC in this situation. METHODS: 8 procedures were required for standardize the model. Weight of the animals, GBWR and surgical technique were evaluated. In the experimental phase, 21 transplantations were performed. The animals were divided in three groups. In the first group (control), liver transplantation was performed with donors and recipients with similar weights. To characterize large-for-size situation, donors of the second group had approximately twice the weight receptor (LFS). In the third group the weight distribution was similar to the second but IPC was performed in donor (LFS+IPC). Procedures were performed under general anesthesia and aseptic technique and the animals were kept alive for 3 hours after reperfusion. Blood samples were obtained at three times (immediately after opening the abdominal cavity, 1 and 3 hours after reperfusion) and fragments of liver 1 and 3 hours after reperfusion. Blood samples were analyzed for arterial blood gases, sodium, potassium and hepatocellular enzymes. The fragments of liver tissues were subjected to histological analysis by hematoxylin-eosin staining (HE). Molecular biology involved quantification of the expression of Bax, Bcl-XL, endothelial nitric oxide synthase (eNOS) and interleukin- 6 (IL-6) genes. RESULTS: The mortality rate was 28%. The GBWR was 2.9% in the control group, 6.3% in the LFS group and 6.4% in the LFS+ICP group. The natremia 1 hour after reperfusion was higher in the control group and potassium dosage was lower in this group in the same period. The dosage of aspartate aminotransferase (AST) was higher in LFS and LFS+IPC groups than control. Histological analysis showed no difference between groups. The Bax gene expression was higher in the LFS, while the eNOS gene was more expressed in LFS + PCI group. After 3 hours of reperfusion the expression of IL-6 gene was higher in the PCI + LFS. CONCLUSIONS: The model of large-for-size liver transplantation in swine was feasible and appropriate for the research. IR injury was more pronounced in the Summary group LFS than in the control group and despite not having changed the elevation of hepatocellular enzymes, the PCI was responsible for increasing the expression of IL- 6 and eNOS and decreasing the Bax gene expression

O presente trabalho avaliou o papel da solução parenteral de fatores hepatotróficos nutricionais em animais com cirrose submetidos à hepatectomia parcial. Este procedimento é temido nestes animais devido à possibilidade de ocorrência de falência hepática aguda, já que a remoção de um fragmento do fígado reduz ainda mais a capacidade funcional de um órgão já comprometido. Além disso, é conhecido que o fígado cirrótico diminui sua capacidade regenerativa, fato que atrasa a recuperação do animal, bem como também regenera cirroticamente. Esses fatores, aliados, contribuem para uma considerável taxa de mortalidade pós-operatória. Porém, há algumas situações em que estes pacientes precisam ser submetidos a ressecções hepáticas, tais como traumas, infecções e neoplasias. De fato, a presença de hepatocarcinomas representa a maior indicação deste procedimento em fígados cirróticos. Por outro lado, tem-se mostrado que a administração parenteral de solução de fatores hepatotróficos nutricionais (FHN), uma mistura de aminoácidos, vitaminas, sais minerais e hormônios, aumenta consideravelmente a proliferação celular e o tamanho do fígado em animais sadios, com fibrose e com cirrose. Nestes dois últimos, além do crescimento hepático, ocorre também uma importante redução na quantidade de colágeno, significando uma melhora morfológica que, por muitas vezes, resulta em uma melhora funcional. Sendo assim, o objetivo do presente trabalho foi o verificar se o uso de fatores hepatotróficos nutricionais traria também uma melhora morfológica e funcional em animais com cirrose induzida por tiocetamida após uma ressecção hepática de 40%. Utilizou-se 40 ratos (Rattus norvegicus) Wistar fêmeas cuja indução da cirrose foi pela administração intraperitoneal de tiocetamida. Ao final deste período, e após 10 dias de descanso, todos os animais foram submetidos a uma hepatectomia parcial (HP) de 40%. Foram então divididos em dois grupos: um que recebeu intraperitonealmente a solução de fatores hepatotróficos nutricionais durante 12 dias, designado grupo HP+FHN, e outro que recebeu solução fisiológica nas mesmas condições, formando o grupo HP+S. Os seguintes parâmetros foram avaliados no término do período experimental: dados biométricos (peso do fígado, IHS: índice hepassomático e IHC: índice hepatocarcaça), bioquímica hepática plasmática (AST, ALT, fosfatase alcalina, bilirrubina total e albumina), quantificação da densidade volumétrica de colágeno hepático por morfometria, quantificação do índice de proliferação celular por imunohistoquímica para PCNA e expressão de genes pró-fibróticos (MMP2, TIMP1, Cola1 e TGFb1) por PCR em tempo real. De fato, os fígados dos animais do grupo HP+FHN estavam maiores do que os animais do grupo HP+S (aumentos de 8,4%, 5,6% e 8,4% no peso do fígado, IHS e IHC respectivamente), e também apresentaram maior índice de proliferação de hepatócitos (44,9%). Ocorreu também redução de 27,9% na densidade volumétrica do colágeno hepático no grupo que recebeu FHN comparandose com o grupo que recebeu solução salina. Esta redução também foi observada na expressão do gene de colágeno a1, que foi de 53%. Porém, não houve diferença nos demais genes avaliados. Dentre os parâmetros bioquímicos, apenas a fosfatase alcalina mostrou redução. Os resultados obtidos permitem concluir que o uso de FHN acarreta em um aumento da regeneração hepática acompanhado de uma redução da quantidade de colágeno e, esses achados, em conjunto, podem representar uma condição benéfica na recuperação de pacientes com cirrose submetidos à ressecção hepática / The current study evaluated the role of parenteral solution of nutritional hepatotrophic factors in animals with cirrhosis undergoing partial hepatectomy. This procedure is fearful in these animals due to the possibility of acute liver failure, since removal of a liver fragment further reduces the functional capacity of an already compromised organ. Moreover, it is known that cirrhotic liver decreases its regenerative capacity, which impairs the recovery of the animal, and also regenerates cirrhotic. These factors, together, contribute to a considerable rate of postoperative mortality. However, there are some situations when these patients need to be submitted to liver resection, such as trauma, infections and neoplasm. In fact, the presence of hepatocellular carcinoma represents the most important indication of this procedure in patients with cirrhosis. On the other hand, it has been shown that parenteral administration of a solution of the nutritional hepatotrophic factors (NHF), a mixture of amino acids, vitamins, minerals and hormones, significantly increases cell proliferation and liver size in healthy, fibrotic and cirrhotic animals. In the two latter, beyond the liver growth, there is also a significant reduction in the amount of collagen, meaning a morphological enhancement, resulting in a functional improvement. Therefore, the objective of this study was to determine whether the use of nutritional hepatotrophic factors would also lead a morphological and functional improvement in animals with thioacetamide-induced cirrhosis after 40% liver resection. We used 40 rats (Rattus norvegicus) female Wistar whose cirrhosis was induced by intraperitoneal administration thioacetamide. At the end of this period, and after 10 days of rest, all animals were underwent a partial hepatectomy (PH) of 40%. They were then divided into two groups: one that received intraperitoneally a solution of nutritional hepatotrophic factors for 12 days, designated PH+NHF group, and another that received saline under the same conditions, forming the PH+S group. The following parameters were evaluated at the end of the trial period: biometrics (liver weight, HSI hepatossomatic index, and HCI hepatocarcass index), plasmatic liver biochemistry (AST, ALT, alkaline phosphatase, total bilirubin and albumin), quantification of volume density of collagen in liver morphology, quantification of cell proliferation by immunohistochemistry for PCNA and expression of pro-fibrotic genes (MMP2, TIMP1, TGF1 and Cola1) by real-time PCR. In fact, the livers of animals in group PH+NHF were larger than the animals in PH+S group (increases of 8.4%, 5.6% and 8.4% in liver weight, HSI and HCI, respectively), and also had higher rates of proliferation of hepatocytes (44.9%). There was also a 27.9% reduction in liver volume density of collagen in the group receiving NHF compared with the group that received saline. This reduction was also observed in gene expression of collagen a1, which was 53%. However, there were no differences in other genes evaluated. Among biochemical parameters, only the alkaline phosphatase showed a reduction. The results indicate that the use of NHF leads to an increase in liver regeneration accompanied by a reduction in the amount of collagen, and these findings, together, can represent a beneficial condition in the recovery of patients with cirrhosis undergoing liver resection

Evolutionär konservierte, kleine offene Leserahmen (upstream open reading frames, uORFs) sind translational aktive Kontrollelemente, die bevorzugt in Boten-Ribonukleinsäuren von Schlüsselgenen zur Regulation von Zellwachstum, Proliferation und Differenzierung vorkommen. In dieser Arbeit wurden Mäuse analysiert, die defizient für das uORF Initiationscodon des Transkriptionsfaktors CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF) sind. Proteinanalysen verschiedener Gewebe zeigten, dass C/EBPbeta-Delta-uORF Mäuse im Gegensatz zu Wildtyptieren nicht in der Lage sind, die kurze, auto-antagonistische C/EBPbeta LIP Isoform zu induzieren. Die verminderte LIP Expression verursachte eine gestörte Differenzierung knochenabbauender Osteoklasten und ging mit einer Zunahme von mineralisiertem Knochengewebe in C/EBPbeta-Delta-uORF Mäusen einher. Nach partieller Hepatektomie führte der Verlust der uORF-vermittelten Induktion von LIP in regenerierenden C/EBPbeta-Delta-uORF Lebern zu einer Überaktivierung C/EBPbeta-regulierter Akute Phase Gene. Im Vergleich zum Wildtyp wiesen Hepatozyten von C/EBPbeta-Delta-uORF Tieren einen verzögerten und abgeschwächten Wiedereintritt in die S-Phase des Zellzyklus auf. Genomweite Genexpressionsanalysen zeigten, dass die verminderte S-Phase Aktivität in regenerierenden C/EBPbeta-Delta-uORF Lebern mit einer persistierenden Repression von Zellzyklusgenen korrelierte, wobei insbesondere die verminderte Expression zahlreicher E2F-regulierter Gene auffällig wurde. Chromatinimmunpräzipitations- und Reportergenexperimente führten zur Entwicklung eines mechanistischen Modells, das eine isoformspezifische C/EBPbeta-Koregulation E2F-kontrollierter Zellzyklusgene vorschlägt. Die Analyse der C/EBPbeta-Delta-uORF Mäuse belegt erstmals die Funktionalität der uORF-gesteuerten translationalen Kontrolle im Säugetier und weist auf eine entscheidende Bedeutung dieses Kontrollmechanismus bei zahlreichen physiologischen und pathopysiologischen Prozessen hin. / Evolutionary conserved small upstream open reading frames (uORFs) are translational control elements predominantly prevalent in the 5'' mRNA regions of key regulatory genes of growth, proliferation, and differentiation. This thesis comprises the evaluation of mice deficient for the uORF initiation codon of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF). Protein analysis of various tissues demonstrated that C/EBPbeta-Delta-uORF mice, in contrast to wildtype control animals (C/EBPbeta-WT), fail to induce translation of the truncated, auto-antagonistic C/EBPbeta LIP isoform. The reduced expression of LIP was associated with impaired differentiation of bone resorbing osteclasts and resulted in an increased bone volume of C/EBPbeta-Delta-uORF mice. After partial hepatectomy the loss of uORF-mediated LIP induction resulted in super activation of acute phase response genes in regenerating livers. Furthermore, C/EBPbeta-Delta-uORF hepatocytes showed a delayed and blunted re-entry into the cell cycle after partial hepatectomy as compared to C/EBPbeta-WT animals. Genome-wide transcript expression analyses revealed that the reduced S-phase activity in regenerating C/EBPbeta-Delta-uORF livers correlated with a persistent repression of cell cycle regulatory genes and showed a remarkable underrepresentation of genes regulated by the E2F family of transcription factors. Chromatinimmunoprecipitations and luciferase reporter gene assays allowed the development of a mechanistic model that suggests C/EBPbeta isoform-specific co-regulation of E2F-controled cell cycle genes. The analysis of C/EBPbeta-Delta-uORF mice validates the functionality of uORF-mediated translational control in vertebrates and suggests a comprehensive role of uORF regulation in physiology and the etiology of disease.

No description available.

O presente trabalho avaliou o papel da solução parenteral de fatores hepatotróficos nutricionais em animais com cirrose submetidos à hepatectomia parcial. Este procedimento é temido nestes animais devido à possibilidade de ocorrência de falência hepática aguda, já que a remoção de um fragmento do fígado reduz ainda mais a capacidade funcional de um órgão já comprometido. Além disso, é conhecido que o fígado cirrótico diminui sua capacidade regenerativa, fato que atrasa a recuperação do animal, bem como também regenera cirroticamente. Esses fatores, aliados, contribuem para uma considerável taxa de mortalidade pós-operatória. Porém, há algumas situações em que estes pacientes precisam ser submetidos a ressecções hepáticas, tais como traumas, infecções e neoplasias. De fato, a presença de hepatocarcinomas representa a maior indicação deste procedimento em fígados cirróticos. Por outro lado, tem-se mostrado que a administração parenteral de solução de fatores hepatotróficos nutricionais (FHN), uma mistura de aminoácidos, vitaminas, sais minerais e hormônios, aumenta consideravelmente a proliferação celular e o tamanho do fígado em animais sadios, com fibrose e com cirrose. Nestes dois últimos, além do crescimento hepático, ocorre também uma importante redução na quantidade de colágeno, significando uma melhora morfológica que, por muitas vezes, resulta em uma melhora funcional. Sendo assim, o objetivo do presente trabalho foi o verificar se o uso de fatores hepatotróficos nutricionais traria também uma melhora morfológica e funcional em animais com cirrose induzida por tiocetamida após uma ressecção hepática de 40%. Utilizou-se 40 ratos (Rattus norvegicus) Wistar fêmeas cuja indução da cirrose foi pela administração intraperitoneal de tiocetamida. Ao final deste período, e após 10 dias de descanso, todos os animais foram submetidos a uma hepatectomia parcial (HP) de 40%. Foram então divididos em dois grupos: um que recebeu intraperitonealmente a solução de fatores hepatotróficos nutricionais durante 12 dias, designado grupo HP+FHN, e outro que recebeu solução fisiológica nas mesmas condições, formando o grupo HP+S. Os seguintes parâmetros foram avaliados no término do período experimental: dados biométricos (peso do fígado, IHS: índice hepassomático e IHC: índice hepatocarcaça), bioquímica hepática plasmática (AST, ALT, fosfatase alcalina, bilirrubina total e albumina), quantificação da densidade volumétrica de colágeno hepático por morfometria, quantificação do índice de proliferação celular por imunohistoquímica para PCNA e expressão de genes pró-fibróticos (MMP2, TIMP1, Cola1 e TGFb1) por PCR em tempo real. De fato, os fígados dos animais do grupo HP+FHN estavam maiores do que os animais do grupo HP+S (aumentos de 8,4%, 5,6% e 8,4% no peso do fígado, IHS e IHC respectivamente), e também apresentaram maior índice de proliferação de hepatócitos (44,9%). Ocorreu também redução de 27,9% na densidade volumétrica do colágeno hepático no grupo que recebeu FHN comparandose com o grupo que recebeu solução salina. Esta redução também foi observada na expressão do gene de colágeno a1, que foi de 53%. Porém, não houve diferença nos demais genes avaliados. Dentre os parâmetros bioquímicos, apenas a fosfatase alcalina mostrou redução. Os resultados obtidos permitem concluir que o uso de FHN acarreta em um aumento da regeneração hepática acompanhado de uma redução da quantidade de colágeno e, esses achados, em conjunto, podem representar uma condição benéfica na recuperação de pacientes com cirrose submetidos à ressecção hepática / The current study evaluated the role of parenteral solution of nutritional hepatotrophic factors in animals with cirrhosis undergoing partial hepatectomy. This procedure is fearful in these animals due to the possibility of acute liver failure, since removal of a liver fragment further reduces the functional capacity of an already compromised organ. Moreover, it is known that cirrhotic liver decreases its regenerative capacity, which impairs the recovery of the animal, and also regenerates cirrhotic. These factors, together, contribute to a considerable rate of postoperative mortality. However, there are some situations when these patients need to be submitted to liver resection, such as trauma, infections and neoplasm. In fact, the presence of hepatocellular carcinoma represents the most important indication of this procedure in patients with cirrhosis. On the other hand, it has been shown that parenteral administration of a solution of the nutritional hepatotrophic factors (NHF), a mixture of amino acids, vitamins, minerals and hormones, significantly increases cell proliferation and liver size in healthy, fibrotic and cirrhotic animals. In the two latter, beyond the liver growth, there is also a significant reduction in the amount of collagen, meaning a morphological enhancement, resulting in a functional improvement. Therefore, the objective of this study was to determine whether the use of nutritional hepatotrophic factors would also lead a morphological and functional improvement in animals with thioacetamide-induced cirrhosis after 40% liver resection. We used 40 rats (Rattus norvegicus) female Wistar whose cirrhosis was induced by intraperitoneal administration thioacetamide. At the end of this period, and after 10 days of rest, all animals were underwent a partial hepatectomy (PH) of 40%. They were then divided into two groups: one that received intraperitoneally a solution of nutritional hepatotrophic factors for 12 days, designated PH+NHF group, and another that received saline under the same conditions, forming the PH+S group. The following parameters were evaluated at the end of the trial period: biometrics (liver weight, HSI hepatossomatic index, and HCI hepatocarcass index), plasmatic liver biochemistry (AST, ALT, alkaline phosphatase, total bilirubin and albumin), quantification of volume density of collagen in liver morphology, quantification of cell proliferation by immunohistochemistry for PCNA and expression of pro-fibrotic genes (MMP2, TIMP1, TGF1 and Cola1) by real-time PCR. In fact, the livers of animals in group PH+NHF were larger than the animals in PH+S group (increases of 8.4%, 5.6% and 8.4% in liver weight, HSI and HCI, respectively), and also had higher rates of proliferation of hepatocytes (44.9%). There was also a 27.9% reduction in liver volume density of collagen in the group receiving NHF compared with the group that received saline. This reduction was also observed in gene expression of collagen a1, which was 53%. However, there were no differences in other genes evaluated. Among biochemical parameters, only the alkaline phosphatase showed a reduction. The results indicate that the use of NHF leads to an increase in liver regeneration accompanied by a reduction in the amount of collagen, and these findings, together, can represent a beneficial condition in the recovery of patients with cirrhosis undergoing liver resection

A Glutamina (Gln) melhora a regeneração hepática em ratos nutridos. Para avaliar o efeito molecular da Gln endovenosa no remanescente hepático, 52 ratos foram classificados em: 1. Nutridos com hepatectomia parcial (HP) e Gln (N-Gln=10) ou prolina (N-Pro=10); 2. Desnutridos com HP: (D-Gln=10) ou (D-Pro=10); 3. Nutridos e Desnutridos sem HP (N-Controle=6) e (D-Controle=6). Após 96 horas da HP os resultados foram: DNA = (D-Gln, D-Pro e D-Controle < N-Gln, N-Pro e N-controle), (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln); RNA= (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln e N-Pro). Não houve diferença nos genes transcritos (GT) para HGF, TGF-a e timidina kinase. Concluímos que: A desnutrição e HP reduzem DNA e RNA; A Gln não altera os GT estudados em ratos desnutridos 96 horas após HP / Glutamine dipeptide (Gln) improve hepatic regeneration of nourished rats. To evaluate molecular effect of Gln on liver remnant 52 rats was classified into: 1. Nourished with partial hepatectomy (PH) and Gln (N-Gln=10) or proline (N-Pro=10); 2. Malnourished (MN) with PH: (MN-Gln=10) or (MN-Pro=10); 3. Nourished and Malnourished rat without PH (N-Control=6) and (MN-Control=6). Results: DNA = (MN-Gln, MN-Pro and MN-Control < N-Gln, N-Pro and N-control), (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln); RNA= (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln and N-Pro). There was no difference on transcript genes (TG) for HGF, TGF-a and thymidine kinase. Conclusions: Malnutrition and PH decrease hepatic DNA and RNA; Gln does not modify TG studied 96 hours after PH in MN rats

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / The maternal liver shows marked growth during pregnancy to accommodate the development and metabolic needs of the placenta and fetus. Previous study has shown that the maternal liver grows proportionally to the increase in body weight during gestation by hyperplasia and hypertrophy of hepatocytes. As the maternal liver is enlarged, the transcript level of Ascl1, a transcription factor essential to progenitor cells of the central nervous system and peripheral nervous system, is highly upregulated. The aims of the study were to (1) identify hepatic Ascl1-expressing cells, and (2) study the functions of Ascl1 in maternal liver during pregnancy. In situ hybridization shows that most cell types (parenchymal, nonparenchymal, and mesothelial cells) express Ascl1 mRNA in maternal livers during gestation and in male regenerating livers. Notably, hepatic mesothelial cells abundantly express Ascl1 during pregnancy and liver regeneration. Inducible ablation of Ascl1 gene during pregnancy results in maternal liver enlargement, litter size reduction, and fetal growth retardation. In addition, maternal hepatocytes deficient in Ascl1 gene lack majority of their cytosols and exhibit β-catenin nuclear translocation, while maintaining their cellular boundary and identity. In summary, in both maternal liver during pregnancy and regenerating liver, the expression of Ascl1 is induced in most cell types. Mesothelial cells are potential origin of Ascl1-expressing cells. Ascl1 gene is essential for the progression of normal pregnancy

Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis.