Células madre adiposas humanas disminuyen el daño de la fibrosis hepática con baja persistencia de células trasplantadas en ratas / Human adipose stem cells decrease liver fibrosis damage with low persistence of transplanted cells in rats

RESUMEN: En la enfermedad hepática crónica el trasplante ortotópico es la única alternativa terapéutica actual pero es limitada por falta de donantes. Ensayos con células madre adultas en daño hepático agudo evidencian promisorios resultados. El objetivo de este trabajo fue evaluar en ratas con daño hepático crónico la efectividad de la infusión de células madre adiposas humanas (CMAd-h). Ratas con fibrosis hepática inducida por tioacetamida fueron agrupadas en: grupo I control que no recibió tioacetamida ni células madre, grupo II recibió tioacetamida y suero fisiológico i.v., grupo III recibió tioacetamida y células madre adiposas 1 x 106/kg i.v. vía vena de la cola. La regeneración hepática histológica se evaluó por el index METAVIR, mientras las Macrophagocytus stellatus, células estrelladas a- SMA+ y células colágeno I+ por inmunohistoquímica; el daño funcional se evaluó por los niveles sanguíneos de los analitos Aspartato Aminotransferasa (AST), Alanina Aminotransferasa (ALT), Fosfatasa Alcalina (ALP), úrea y nitrógeno ureico (BUN) y hemograma. Los resultados muestran atenuación del daño estructural hepático evidenciado por disminución de los nódulos, del grado de lesión histológica en el score Metavir, y disminución de Macrophagocytus stellatus, células a-SMA+ y células colágeno tipo I+; funcionalmente hay reducción moderada de AST, ALT, urea, BUN y disminución moderada de células blancas pero efecto favorable sobre el volumen corpuscular media y la hemoglobina corpuscular media. Ocho semanas después de la infusión hay escasa población de CMAd-h en el hígado. En conclusión la infusión intravenosa de CMAd-h en ratas disminuye el daño funcional y estructural de la fibrosis hepática con escasa persistencia de CMAd-h en el parénquima hepático. A nuestro conocimiento este es el primer trabajo que evalúa el efecto de las CMAd-h en el modelo daño hepático crónico murino y la persistencia de las células trasplantadas.

SUMMARY: In chronic liver disease, orthotopic transplantation is the only current therapeutic alternative but it is limited due to lack of donors. Trials with adult stem cells in acute liver damage show promising results. The aim of this work was to evaluate the effectiveness of human adipose stem cell (h-ASC) infusion in rats with chronic liver damage. Rats with thioacetamide- induced liver fibrosis were grouped into: group I control that did not receive thioacetamide and h-ASC, group II received thioacetamide and saline i.v., group III received thioacetamide and h-ASC 1 x 106/ kg i.v. via tail vein. Histological liver regeneration was evaluated by METAVIR index, while Macrophagocytus stellatus (Kupffer cells), stellate cells a-SMA+ and collagen I+ cells by immunohistochemistry; functional damage was evaluated by blood levels of the analytes Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Urea and Blood Urea Nitrogen (BUN) and hemogram. The results show attenuation of structural liver damage evidenced by decreased nodules, degree of histologic injury on Metavir score, and decreased Macrophagocytus stellatus, a-SMA+ cells and type I+ collagen cells; functionally there is moderate reduction of AST, ALT, urea, BUN and moderate decrease of white cells but favorable effect on mean corpuscular volume and mean corpuscular hemoglobin. Eight weeks after infusion there is a small population of h-ASC in the liver. In conclusion, intravenous infusion of h-ASC in rats reduces functional and structural damage of hepatic fibrosis with low persistence of h- ASC in the liver parenchyma. To our knowledge this is the first work that evaluates the effect of h-SC in the model of chronic murine liver damage and the persistence of transplanted cells.

Observation of the effect of bone marrow mesenchymal stem cell transplantation by different interventions on cirrhotic rats

Bone marrow mesenchymal stem cells (BMSCs) transplantation has attracted attention for the treatment of liver cirrhosis and end-stage liver diseases. Therefore, in this study, we evaluated the effect of different methods of BMSCs transplantation in the treatment of liver cirrhosis in rats. Seventy-two male Sprague-Dawley rats were divided into 7 groups: 10 were used to extract BMSCs, 10 were used as normal group, and the remaining 52 rats were randomly divided into five groups for testing: control group, BMSCs group, BMSCs+granulocyte colony-stimulating factor (G-CSF) group, and BMSCs+Jisheng Shenqi decoction (JSSQ) group. After the end of the intervention course, liver tissue sections of rats were subjected to hematoxylin and eosin (H&E) and Masson staining, and pathological grades were scored. Liver function [aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB)] and hepatic fibrosis markers [hyaluronidase (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (CIV)] were measured. BMSCs+JSSQ group had the best effect of reducing ALT and increasing ALB after intervention therapy (P<0.05). The reducing pathological scores and LN, PCIII, CIV of BMSCs+G-CSF group and BMSCs+JSSQ group after intervention therapy were significant, but there was no significant difference between the two groups (P>0.05). The effect of JSSQ on improving stem cell transplantation in rats with liver cirrhosis was confirmed. JSSQ combined with BMSCs could significantly improve liver function and liver pathology scores of rats with liver cirrhosis.

Cirrhosis inducss apoptosis in renal tissue through intracellular oxidative stress / Cirrose induz apoptose em tecido renal através de estresse oxidativo intracelular

Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis. .

Contexto A falência renal é uma complicação grave e frequente em pacientes com cirrose descompensada. Objetivo Avaliar o estresse oxidativo, o dano ao DNA e alterações na função celular no rim em um modelo animal de cirrose. Métodos A cirrose biliar secundária foi induzida em ratos através da ligadura do duto biliar comum. Foi medido no rim o TBARS (substâncias que reagem ao ácido tiobarbitúrico), ERO (espécies reativas de oxigênio), o potencial de membrana mitocondrial e a atividade das enzimas antioxidantes. A viabilidade celular foi determinada utilizando o ensaio de exclusão do trypan-blue. Para distinguir células em apoptose ou necrose foram usados os marcadores: Anexina V-PE e 7-AAD e o ensaio cometa foi utilizado para determinar dano ao DNA. Resultados Em animais cirróticos houve um aumento significativo da lipoperoxidação no rim e na quantidade de ERO intracelular. Foi observado um aumento na atividade de todas as enzimas antioxidantes. A porcentagem de viabilidade celular foi superior a 90% no grupo controle e de 64,66% no grupo da ligadura do duto biliar. O padrão de morte celular predominante foi apoptose e houve dano ao DNA no grupo da ligadura do duto biliar. Observou-se uma redução no potencial de membrana mitocondrial no grupo da ligadura do duto biliar (34,48% ± 11,40%) em comparação aos controles (71,40% ± 6,35%). Conclusão Esses resultados parecem indicar que nos animais cirróticos ocorre um aumento no dano oxidativo e ao DNA levando as células renais à apoptose, o que contribui para a falência renal na cirrose. .

Lung and liver hhanges due to the induction of cirrhosis in two experimental models / Alteracoes hepaticas e pulmonares decorrentes da inducao de cirrose em dois modelos experimentais.

Context To evaluate lung and liver changes in two experimental models using intraperitoneal carbon tetrachloride (CCl4) and bile duct ligation (BDL). Methods Twenty-four male Wistar rats were divided into a control group (CO) and an experimental group (EX). We evaluated the liver transaminases (AST, ALT, AP), arterial blood gases (PaO2, PCO2 and SpO2) and lipid peroxidation by TBARS (substances that react to thiobarbituric acid) and chemiluminescence. We also evaluated the antioxidant enzyme superoxide dismutase (SOD) and histology of lung tissue and liver. Results There were significant differences in AST, ALT, ALP and PaO2 between CO group and EX group (P<0.05). The levels of TBARS, chemiluminescence and activity of enzyme superoxide dismutase were increased to different degrees in the CCl4 groups: CO and in the BDL -EX (P<0.05, respectively). In the lung histology, an increase in the wall thickness of the pulmonary artery and a diameter reduction in the CCl4 animal model were observed: comparing CO group with EX group, we observed a reduction in thickness and an increase in the diameter of the artery wall lung. Conclusion Both experimental models have caused liver damage and alterations in the artery wall that are associated with major changes in pulmonary gas exchange. .

Objetivo Avaliar as alterações pulmonares e hepáticas em dois modelos experimentais de cirrose hepática pelo uso de tetracloreto de carbono intraperitoneal (CCl4) e ligadura de ducto biliar. Métodos Vinte e quatro ratos machos Wistar foram divididos em grupo controle (CO) e experimental (EX). Foram avaliadas as transaminases hepáticas (AST, ALT, FA), gasometria arterial (PaO2, PCO2 e SatO2) e a lipoperoxidação através de TBARS (substâncias que reagem ao ácido tiobarbitúrico) e por quimiluminescência. Também foi avaliada a atividade antioxidante da enzima superóxido dismutase e a histologia do tecido pulmonar e hepático. Resultados Nas enzimas hepáticas (AST, ALT e FA), bem como na PaO2 foram observadas diferenças significativas (P≤0,05) entre os grupos CO vs EX em ambos modelos. Os níveis de TBARS, quimiluminescência e a atividade da enzima superóxido dismutase encontram-se aumentados nos grupos CCl4 e ligadura de ducto biliar: CO vs EX (P≤0,05). Na análise histológica do pulmão observamos um aumento na espessura da parede da artéria pulmonar e uma redução no diâmetro no modelo CCl4: CO vs EX, e no modelo de ligadura de ducto biliar podemos observar uma redução da espessura e aumento no diâmetro da parede da artéria pulmonar. Conclusão Ambos os modelos experimentais provocaram dano hepático, além de causar alterações na parede da artéria pulmonar contribuindo na redução das trocas gasosas. .

Relationship between serum transforming growth factor ß1 and liver collagen content in rats treated with carbon tetrachloride / Relação entre fator transformador de crescimento β1 e conteúdo hepático de colágeno em ratos tratados com tetracloreto de carbono

CONTEXT: Transforming Growth Factor ß1 (TGFß1) plays a fundamental role in fibrogenesis, although its importance as a biomarker of liver disease is still matter of debate. OBJECTIVE: Quantify serum TGFß1 and its association to liver collagen content in rats exposed to Carbon Tetrachloride (CCl4). METHODS: Rats were submitted to a fibrosis model using CCl4 and sacrificed after 6, 10, 12 and 16 weeks of treatment. Serum levels of TGFß1 were measured by ELISA and collagen content was defined by morphometric analysis. RESULTS: Serum levels of TGFß1 increased between 6 and 10 weeks, whereas collagen density increased between 12 and 16 weeks. A negative correlation was observed between liver collagen deposition and serum levels of TGFß1 (r = -0. 48; P<0. 05). CONCLUSION: Serum levels of TGFß1 were inversely proportional to collagen intensity in cirrhotic livers of rats exposed to CCl4, thus suggesting a limited use as biomarker in advanced liver disease.

CONTEXTO: A citocina TGFß1 (Fator Transformador de Crescimento, TGFß1) desempenha um papel fundamental na fibrogênese, mas sua importância como biomarcador da doença hepática ainda tem sido debatida. OBJETIVO: Quantificar o TGFß1 sérico e estudar a sua associação com o conteúdo de colágeno no tecido hepático em ratos expostos ao Tetracloreto de Carbono (CCl4). MÉTODOS: Ratos foram submetidos ao modelo de fibrose por CCl4 e sacrificados após 6, 10, 12 e 16 semanas de tratamento. Os níveis séricos de TGFß1 foram quantificados por ELISA e a densidade de colágeno foi definida por morfometria. RESULTADOS: Os níveis séricos de TGFß1 aumentaram entre 6 e 10 semanas, enquanto a densidade de colágeno aumentou entre 12 e 16 semanas. Foi detectada uma correlação negativa entre a deposição hepática de colágeno e a concentração sérica de TGFß1 (r = -0,48; P<0,05). CONCLUSÃO: O nível sérico de TGFß1 foi inversamente proporcional à intensidade do colágeno no fígado de ratos com cirrose por CCl4, o que indica que seu uso como biomarcador em estágios avançados da doença pode ter utilidade limitada.

The effect of down-regulation of Smad3 by RNAi on hepatic stellate cells and a carbon tetrachloride-induced rat model of hepatic fibrosis

Searching for effective Smad3 gene-based gene therapies for hepatic fibrosis, we constructed siRNA expression plasmids targeting the rat Smad3 gene and then delivered these plasmids into hepatic stellate cells (HSCs). The effect of siRNAs on the mRNA levels of Smad2, Smad3, Smad4, and collagens I-α1, III-α1 and IV-α1 (Colα1, Col3α1, Col4α1, respectively) was determined by RT-PCR. Eighty adult male Sprague-Dawley rats were randomly divided into three groups. Twice a week for 8 weeks, the untreated hepatic fibrosis model (N = 30) and the treated group (N = 20) were injected subcutaneously with 40 percent (v/v) carbon tetrachloride (CCl4)-olive oil (3 mL/kg), and the normal control group (N = 30) was injected with olive oil (3 mL/kg). In the 4th week, the treated rats were injected subcutaneously with liposome-encapsulated plasmids (150 µg/kg) into the right liver lobe under general anesthesia once every 2 weeks, and the untreated rats were injected with the same volume of buffer. At the end of the 6th and 8th weeks, liver tissue and sera were collected. Pathological changes were assessed by a semi-quantitative scoring system (SSS), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin, and hyaluronic acid). The mRNA expression levels of the above cited genes were reduced in the HSCs transfected with the siRNA expression plasmids. Moreover, in the treated group, fibrosis evaluated by the SSS was significantly reduced (P < 0.05) and the serum indices were greatly improved (P < 0.01). These results suggest that Smad3 siRNA expression plasmids have an anti-fibrotic effect.

Dynamics of Capillaria-hepatica-induced hepatic septal fibrosis in rats / Dinâmica da fibrose septal hepática induzida por Capillaria hepatica em ratos

INTRODUCTION: The pathogenesis of septal hepatic fibrosis, induced in rats by Capillaria hepatica infection, was studied with the aid of a large collection of stored paraffin blocks, representative of the different evolutive phases of fibrosis which appeared in 100 percent of infected rats. METHODS: Studies were conducted involving histology, immunohistochemistry, immunofluorescence and morphometric methods, in order to observe the dynamic behavior of the cellular and matrix components of fibrosis, over a one year period of evolution. RESULTS: Observation verified that septal fibrosis originates from several portal spaces simultaneously. Its origin and progression involve blood vessel proliferation (angiogenesis), multiplication of actin-positive cells (pericytes and myofibroblasts) and progressive collagen deposition. By the end of 4-5 months, a progressive decrease in all these components was observed, when signs of regression of septal fibrosis became more evident over time. CONCLUSIONS: Besides indicating the fundamental role played by angiogenesis in the pathogenesis of fibrosis, these morphological data concerning the dynamics of this C. hepatica experimental model proved to be adequate for future investigations regarding the functional aspects of fibrosis induction, progression and regression.

INTRODUÇÃO: Um extenso material de patologia experimental arquivado em blocos de parafina, ilustrativo das diferentes fases da fibrose hepática septal, que 100 por cento dos ratos desenvolvem em seguida uma infecção com o nematódeo Capillaria hepatica. MÉTODOS: O material foi sistematicamente estudado com métodos morfológicos e morfométricos, no sentido de se verificar o comportamento dos elementos celulares e matriciais durante a evolução da fibrose hepática septal ao longo de um período de um ano. RESULTADOS: Foi constatado que a fibrose septal se origina de vários espaços porta ao mesmo tempo, com proliferação vascular (angiogênese), multiplicação de células actino-positivas (pericitos, miofibroblastas) e progressivo depósito de colágeno. Ao fim dos 4-5 meses há uma involução regressiva de todos estes indícios morfológicos, mas com alguns septos persistindo bem evidentes até o fim de um ano. CONCLUSÕES: Além de ilustrar o papel fundamental desempenhado pela angiogênese, o modelo se mostrou adequado para futuros estudos funcionais relacionados com a indução, progressão e regressão da fibrose hepática.

Capillaria hepatica-induced septal fibrosis in rats: a contribution to the study of liver fibrogenesis / Contribuição para o estudo da fibrogenesis hepática em ratos infectados com Capillaria hepatica

INTRODUCTION: Septal fibrosis of the liver regularly develops in rats infected with the nematode Capillaria hepatica. Curative treatment of the infection prevents the development of septal fibrosis when intervention occurs up to postinfection day (PID) 15, but not later. The present investigation aimed to demonstrate which parasitic factors are present when the process of septal fibrosis can no longer be prevented by curative treatment. METHODS: Wistar rats were infected with 600 embryonated eggs of C. hepatica administered by gavage and treated with ivermectin and mebendazole in separate groups at PIDs 10, 12, 15, 17 or 20. Rats from each group and their nontreated controls, were killed and examined 40 days after the end of treatment. RESULTS: Findings by PID 15 were compatible with the stage of complete maturation of infection, when worms and eggs were fully developed and a complex host-parasite multifocal necroinflammatory reaction showed greater intensity, but with no signs of septal fibrosis, which appeared from PID 17 onward. CONCLUSIONS: Since the worms spontaneously died by PID 15, not only septal fibrosis production, but also its maintenance and further development appeared dependent on the presence of eggs, which were the only parasitic factor remaining thereafter.

INTRODUÇÃO: A fibrose septal do fígado se desenvolve regularmente em ratos infectados pelo nematódeo Capillaria hepatica. O tratamento curativo da infecção, feito antes do 15º dia da infecção, mas não mais tarde, impediu o aparecimento da fibrose septal. O presente trabalho procura verificar qual o estado do parasitismo aos 15 dias da infecção, crucial para patogenia da fibrose septal. MÉTODOS: Ratos foram infectados por via digestiva com 600 ovos embrionados de C. hepatica e tratados com Ivermectina e mebendazol, em grupos separados, aos 10, 12, 15, 17 ou 20 dias após a infecção. O animal de cada grupo e seus respectivos controles foram mortos e examinados aos 40 dias após o fim do tratamento. RESULTADOS: Os achados aos 15 dias da infecção mostraram a maturação completa da parasitose, com presença de ovos e vermes, circundados por reação necro-inflamatória, mas ainda sem fibrose septal. Daí por diante, a fibrose septal se fez presente. CONCLUSÕES: Como os vermes morrem espontaneamente após o 15º dia da infecção, não apenas a origem, mas o posterior crescimento e a manutenção da fibrose septal dependem da presença dos ovos acumulados no fígado, os quais são os únicos elementos parasitários presentes após o 15º dia da infecção por C. hepatica no rato.

Angiogenesis and experimental hepatic fibrosis

Angiogenesis is a basic change occurring during repair by granulation tissue. This process seems to precede fibrosis formation in most types of chronic liver disease. To examine its presence and significance in different types of hepatic insults, this paper sought to identify the presence, evolution and peculiarities of angiogenesis in the most common experimental models of hepatic fibrosis. The characterization of cells, vessels and extracellular matrix and the identification of factors associated with endothelium (factor VIII RA), vascular basement membrane, other components of the vascular walls (actin, elastin) and the presence of the vascular-endothelial growth factor were investigated. The models examined included Capillaria hepatica septal fibrosis, whole pig serum injections, carbon tetrachloride administration, main bile duct ligation and Schistosoma mansoni infection. The first four models were performed in rats, while the last used mice. All models studied exhibited prominent angiogenesis. The most evident relationship between angiogenesis and fibrosis occurred with the C. hepatica model due to circumstances to be discussed. Special attention was paid to the presence of pericytes and to their tendency to become detached from the vascular wall and be transformed into myofibroblasts, which is a sequence of events that explains the decisive role angiogenesis plays in fibrosis.

Manson's schistosomiasis in the undernourished mouse: some recent findings

This paper deals with current knowledge of the interrelationships between Schistosoma infection and malnutrition. It emphasizes the relevance of these investigations in the face of dynamic and evolving changes occurring in population diets and changes in the epidemiological patterns of schistosomiasis in endemic countries. The paper further discusses the basis for continuing the studies on this subject and the reasons why it represents a misunderstood association. This review also focuses on the cellular and humoral immune responses in the undernourished mouse model infected with Schistosoma mansoni, with updated information on the immune response in wild-type and iNOS knockout mice concerning soluble egg antigen specific antibodies and kinetics of IFN-ã, IL-4, IL-10 and IL-13 cytokines, in the chronic phase of Manson's schistosomiasis. There is indication that schistosome-infected undernourished mice are able to develop a humoral immune response, but antibody titres are much lower than in the control animals. Cytokine production (IFN-ã, IL-4, IL-10) is lower in the undernourished mice, but as infection progresses to the chronic phase its kinetics run an antagonistic course when compared to that of well-nourished animals. Marked variation in the secretion of IL-13 (a fibrogenic cytokine) could explain why undernourished mice do not develop liver "pipe-stem" fibrosis described in previous papers on well-nourished animals.

Melatonin protects the liver and erythrocytes against oxidative stress in cirrhotic rats / Melatonina protege o fígado e eritrócitos do estresse oxidativo em ratos cirróticos

CONTEXT: Cirrhosis is a progressive chronic hepatopathy which constitutes an irreversible stage of liver dysfunction. OBJECTIVES: To evaluate the oxidative stress in the blood of cirrhotic rats treated with the antioxidant melatonin. METHODS: Cirrhosis was induced through inhalation of carbon tetrachloride. Liver integrity was evaluated by measuring serum enzymes, oxidative damage measured by lipoperoxidation, and antioxidant enzyme activity in erythrocytes. Lipoperoxidation, total nitrates, collagen, and histology by picrosirius staining were evaluated in the livers of these animals (n = 15), which were divided in three groups: control, carbon tetrachloride, and carbon tetrachloride + melatonin. Melatonin (20 mg/kg) was administered intraperitoneal from week 10 of carbon tetrachloride inhalation. In order to shorten the cirrhosis induction time, phenobarbital (0.3 g/L) was added to the animals' drinking water. RESULTS: A significant impairment in the liver integrity of melatonin-treated animals as compared to cirrhotic animals was observed. In rat erythrocytes and liver, lipoperoxidation was significantly increased in the cirrhotic rats as compared to controls, as measured through thiobarbituric acid reactive substances, and significantly decreased in melatonin-treated animals as compared to cirrhotic ones. In blood, a decrease in superoxide dismutase and glutathione peroxidase enzymes was detected in the cirrhotic group as compared to the control group, with increased superoxide dismutase activity when melatonin was administered. A reduction in the levels of total nitrates was detected in the hepatic tissue of the animals in the carbon tetrachloride group as compared to the control group and an increase of these levels in the carbon tetrachloride + melatonin group. As for hepatic collagen, we found a significant increase in the carbon tetrachloride group as compared to the controls and a regression of these values in the treated group. ...

CONTEXTO: A cirrose é uma hepatopatia crônica e progressiva que constitui estágio irreversível de disfunção hepática. É associada a alterações na circulação sistêmica. OBJETIVOS: Avaliar o estresse oxidativo no sangue de ratos cirróticos e tratados com antioxidante melatonina. MÉTODOS: A cirrose foi induzida através da inalação de tetracloreto de carbono. Foram avaliadas as provas de integridade hepática através das medidas das enzimas séricas, o dano oxidativo medido pela lipoperoxidação e a atividade das enzimas antioxidantes no eritrócito. No fígado desses animais, foram avaliados a lipoperoxidação, os nitratos totais, colágeno e histologia através de picrosíruis. Os animais (n = 15) foram divididos em três grupos experimentais: controle, tetracloreto de carbono e tetracloreto de carbono + melatonina. A melatonina foi administrada por via intraperitonial após a 10ª semana de inalação na concentração de 20 mg/kg. Com o objetivo de abreviar o tempo de indução, foi administrado para todos animais, fenobarbital na água de beber na concentração de 0,3 g/L. RESULTADOS: Observou-se redução significativa nas provas de integridade hepática nos animais tratados com melatonina, em relação aos animais cirróticos. Nos eritrócitos e fígados dos ratos, foi observado aumento significativo da lipoperoxidação nos ratos cirróticos em comparação com os controles, através da medida das substâncias que reagem ao ácido tiobarbitúrico, e redução nos animais tratados com melatonina. No sangue, observou-se diminuição dos valores das enzimas superóxido dismutase e glutationa peroxidase do grupo cirrótico em comparação ao grupo controle, elevando a atividade da superóxido dismutase quando administrada melatonina. Na avaliação dos nitratos totais, no tecido hepático, observou-se redução dos valores nos animais do grupo tetracloreto de carbono em comparação ao grupo CO e um aumento desses valores nos ratos do grupo tratado com melatonina. Na medida do colágeno ...

Hepatotrophic factors reduce hepatic fibrosis in rats / Fatores hepatotróficos reduzem a fibrose hepática em ratos

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2 percent). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6 percent) and perisinusoidal spaces (42.3 percent), as well as around the hepatic terminal vein (57.7 percent). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.

CONTEXTO: A fibrose hepática ocorre em resposta a diversos agentes agressores e é um fator predisponente da cirrose. Fatores hepatotróficos são conhecidos por estimular o crescimento hepático e restaurar a arquitetura histológica do fígado. Promovem, também, melhora na função hepática e aceleram a reversão da fibrose antes de sua progressão para cirrose. OBJETIVO: Testar os efeitos de uma solução de fatores hepatotróficos, composta por aminoácidos, vitaminas, glicose, insulina, glugacon e triiodotironina na fibrose hepática em ratos. MéTODOS: No presente estudo, a fibrose foi induzida em ratos pela administração de dimetilnitrosamina (10 mg/kg) durante 5 semanas. Após a biopsia do fígado, os ratos receberam a solução de fatores hepatotróficos (40 mg/kg/dia) ou solução salina por injeção intraperitonial, durante 10 dias. Amostras sanguíneas e fragmentos do fígado foram coletados para análise da função hepática, avaliação do critério histopatológico e quantificação morfométrica do colágeno. RESULTADOS: Os ratos do grupo fatores hepatotróficos demonstraram diminuição dos componentes histopatológicos da fibrose e aumento de massa hepática (12,2 por cento). Não houve o desenvolvimento de lesões neoplásicas em ambos os grupos. Comparado com o grupo de salina, no grupo fatores hepatotróficos também houve diminuição nos níveis dos marcadores sanguíneos de lesão hepática (AST e ALT), e diminuição da quantidade de colágeno nos espaços porta (31,6 por cento) e espaços perissinusoidais (42,3 por cento), assim como ao redor das veias terminais hepáticas (57,7 por cento). Assim, a administração de fatores hepatotróficos no sangue portal promoveu resposta regenerativa hepática, com redução da densidade volumétrica de colágeno, melhora na função hepática e melhora geral nos aspectos histopatológicos da fibrose. CONCLUSÃO: Juntos, estes resultados sugerem o potencial uso terapêutico desta solução de fatores hepatotróficos para tratar doenças hepáticas crônicas.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53 percent), TIMP-1 (-31.7 percent), TGF-β1 (-57.7 percent), and MMP-2 (-41.6 percent), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1 percent), ALT (-17.6 percent), and AST (-12.2 percent) serum levels; c) increased liver weight (11.3 percent), and reduced liver collagen (-37.1 percent), regenerative nodules size (-22.1 percent), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5 percent, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64 percent, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36 percent, P < 0.05) vs control (2.2 ± 1.21 percent). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3 percent, P < 0.01; collagen III: 1.3 ± 0.2 percent, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06 percent, P < 0.05; collagen III: 1.5 ± 0.8 percent, P < 0.01) vs control (collagen I: 0.38 ± 0.11 percent; collagen III: 0.25 ± 0.06 percent). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8 percent, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21 percent, P < 0.01) vs control (7.9 ± 0.8 percent). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Modelos experimentais para avaliação das alterações pulmonares na síndrome hepatopulmonar / Experimental models for assessment of pulmonary alterations in hepatopulmonary syndrome

OBJETIVO: O objetivo deste trabalho foi avaliar o melhor modelo experimental para observar alterações pulmonares que caracterizam a síndrome hepatopulmonar (SHP). MÉTODOS: Ratos machos Wistar, com peso médio de 250 g foram usados em quatro modelos experimentais: tetracloreto de carbono inalatório; tetracloreto de carbono intraperitoneal; ligadura parcial de veia porta; e ligadura de ducto biliar (LDB). Em todos os grupos os animais foram divididos em controle e experimental. Foram avaliadas as seguintes variáveis: transaminases; gasometria; lipoperoxidação por substâncias que reagem ao ácido tiobarbitúrico (TBARS) e por quimiluminescência; e atividade antioxidante da enzima superóxido dismutase (SOD). Foi feito também o exame anatomopatológico do pulmão. RESULTADOS: Observou-se diferenças significativas entre os grupos LDB controle e experimental: aspartato amino transferase (105,3 ± 43 vs. 500,5 ± 90,3 UI/L); alanino aminotransferase (78,75 ± 37,7 vs. 162,75 ± 35,4 UI/L); fosfatase alcalina (160 ± 20,45 vs. 373,25 ± 45,44 UI/L); pressão parcial de oxigênio (85,25 ± 8,1 vs. 49,9 ± 22,5 mmHg); e saturação de hemoglobina (95 ± 0,7 vs. 73,3 ± 12,07 por cento). A lipoperoxidação e a atividade antioxidante também demonstrou diferenças entre os dois grupos LDB (controle vs. experimental): TBARS (0,87 ± 0,3 vs. 2,01 ± 0,9 nmol/mg proteína); quimiluminescência (16008,41 ± 1171,45 vs. 20250,36 ± 827,82 cps/mg proteína); e SOD (6,66 ± 1,34 vs. 16,06 ± 2,67 UI/mg proteína). No exame anatomopatológico observou-se vasodilatação pulmonar no modelo de LDB. CONCLUSÕES: Os dados sugerem que o modelo de LDB pode ser usado para outros estudos envolvendo alterações hepáticas e suas relações com o estresse oxidativo e a SHP.

OBJECTIVE: The aim of this study was to identify the best experimental model in which to observe the pulmonary alterations characterizing hepatopulmonary syndrome (HPS). METHODS: Male Wistar rats, with mean weight of 250 g, were used in four experimental models: inhaled carbon tetrachloride; intraperitoneal carbon tetrachloride; partial portal vein ligation; and bile duct ligation (BDL). The animals in all groups were divided into control and experimental subgroups. The following variables were measured: transaminase levels; blood gases; lipoperoxidation, using thiobarbituric acid reactive substances (TBARS) and chemiluminescence; and levels of superoxide dismutase (SOD) anti-oxidant activity. Anatomopathological examination of the lung was also performed. RESULTS: There were statistically significant differences between the BDL control and BDL experimental groups: aspartate aminotransferase (105.3 ± 43 vs. 500.5 ± 90.3 IU/L); alanine aminotransferase (78.75 ± 37.7 vs. 162.75 ± 35.4 IU/L); alkaline phosphatase (160 ± 20.45 vs. 373.25 ± 45.44 IU/L); arterial oxygen tension (85.25 ± 8.1 vs. 49.9 ± 22.5 mmHg); and oxygen saturation (95 ± 0.7 vs. 73.3 ± 12.07 percent). Lipoperoxidation and antioxidant activity also differed significantly between the two BDL groups (control vs. experimental): TBARS (0.87 ± 0.3 vs. 2.01 ± 0.9 nmol/mg protein); chemiluminescence (16008.41 ± 1171.45 vs. 20250.36 ± 827.82 cps/mg protein); and SOD (6.66 ± 1.34 vs. 16.06 ± 2.67 IU/mg protein). The anatomopathological examination confirmed pulmonary vasodilatation in the BDL model. In the other models, there were no alterations that were characteristic of HPS. CONCLUSIONS: The data obtained suggest that the BDL model can be used in future studies involving hepatic alterations related to oxidative stress and HPS.

Role of N-acetylcysteine on fibrosis and oxidative stress in cirrhotic rats / Papel da N-acetilcisteína na fibrose e estresse oxidativo em ratos cirróticos

BACKGROUND: Hepatic cirrhosis is the final stage of liver dysfunction, characterized by diffuse fibrosis which is the main response to the liver injury. The inhalatory carbon tetrachloride is an effective experimental model that triggers cirrhosis and allows to obtain histological and physiological modifications similar to the one seen in humans. AIM: To investigate the effects of N-acetylcysteine (NAC) on the fibrosis and oxidative stress in the liver of cirrhotic rats, analyzing liver function tests, lipoperoxidation, activity of glutathione peroxidase enzyme, collagen quantification, histopathology, as well as the nitric oxide role. METHODS: The animals were randomly in three experimentals groups: control (CO); cirrhotic (CCl4) and CCl4 + NAC. Evaluate the lipid peroxidation, the glutathione peroxidase enzyme, the collagen and the expression of inducible nitric oxide synthase (iNOS). RESULTS: The cirrhotic group treated with N-acetylcysteine showed trough the histological analysis and collagen quantification lower degrees of fibrosis. This group has also shown less damage to the cellular membranes, less decrease on the glutathione peroxidase levels and less expression of inducible nitric oxide synthase when matched with the cirrhotic group without treatment. CONCLUSION: N-acetylcysteine seams to offer protection against hepatic fibrosis and oxidative stress in cirrhotic rat livers.

RACIONAL: A cirrose é o estágio final da disfunção hepática, sendo caracterizada por fibrose difusa, que compõe a resposta principal do organismo ao dano hepático. O tetracloreto de carbono inalatório é um modelo experimental efetivo, que desencadeia a cirrose e permite obter modificações histológicas e fisiológicas similares às vistas em humanos. OBJETIVO: Investigar os efeitos da N-acetilcisteina (NAC) sobre a fibrose e o estresse oxidativo no fígado de ratos cirróticos, analisando as provas hepáticas, a lipoperoxidação, a atividade da enzima glutationa peroxidase, a quantificação do colágeno, a histopatologia, bem como o papel do óxido nítrico. MÉTODOS: Os animais foram divididos em três grupos experimentais: controle (CO); cirrótico (CCl4) e CCl4 + NAC. Foram avaliados a lipoperoxidação, a enzima glutationa peroxidase, a histologia hepática, a quantificação de colágeno e a expressão da óxido nítrico síntase induzível (iNOS). RESULTADOS: O grupo cirrótico tratado com a NAC demonstrou, através da análise histológica e da quantificação de colágeno, menores graus de fibrose. Este grupo demonstrou, ainda, menos dano às membranas celulares, menor decréscimo nos níveis de glutationa peroxidase e menor expressão da iNOS quando comparado com o grupo cirrótico sem tratamento. CONCLUSÃO: A NAC parece oferecer proteção contra a fibrose hepática e o estresse oxidativo no fígado de ratos cirróticos.

Role of partial hepatectomy on Capillaria hepatica-induced hepatic fibrosis in rats

It is known that hepatic fibrosis may regress following partial hepatectomy, since the hepatic parenchyma regenerates very rapidly, but not the excess of fibrous tissue. The present study evaluated this hypothesis by observing the behavior of systematized septal fibrosis induced by either 30 or 90-day-old Capillaria hepatica infection, in rats subjected to partial hepatectomy. The results revealed that the morphology of the fibrosis was unaffected, but its relative quantity within the microscope field appeared significantly decreased, as a consequence of the increased liver tissue mass following regeneration.

Sabe-se que a fibrose hepática pode sofrer uma redução em seqüência uma hepatectomia parcial, uma vez que o parênquima hepático se regenera muito rápido, mas não o excesso de tecido fibroso. O presente trabalho avalia esta hipótese ao observar como se comporta a fibrose septal sistematizada induzida pela Capillaria hepática no rato, após infecção de 30 ou 90 dias de duração, em animais submetidos à hepatectomia parcial. Os resultados revelaram que a fibrose em si mesma não foi afetada na sua morfologia, mas a sua quantidade relativa apareceu diminuída significativamente no campo microscópico como conseqüência do aumento da massa de tecido hepático pós-regeneração.

Seminiferous epithelium of rats with food restriction and carbon tetrachloride-induced cirrhosis

OBJECTIVE: Analyze the changes in the seminiferous epithelium in rats with carbon tetrachloride-induced cirrhosis (CCl4). MATERIALS AND METHODS: Forty-eight male Wistar rats aged 45-50 days, weighing 150-180 grams were used. Twenty-two rats underwent CCl4-induced cirrhosis with CCl4 0.25 mL/Kg weekly intragastrically once a week, during 10 weeks. Additionally, they had a 44 percent food restriction diet (Group 1). The control group was divided in two subgroups: 13 rats had a 44 percent food restriction diet and no CCl4 (Group 2) and 10 rats were not submitted to CCl4 or food restriction (Group 3). After 10 weeks, the rats were sacrificed and liver sections were collected for histological analysis. The testicular analysis was carried out to evaluate the frequency of tubules in stages VIII and XIV. RESULTS: The mean rates of stage VIII in animals with food restriction plus CCl4-induced cirrhosis and food restriction without CCl4 were significantly different from animals without either food restriction or CCl4 (18.1 ± 5.5 percent, 20.5 ± 2.5 percent and 13.4 ± 3.5 percent, respectively, p = 0.002). The mean rate of stage VIII in rats with cirrhosis was not significantly different from rats without cirrhosis (18.1 ± 5.5 percent and 17.4 ± 4.6 percent respectively). The mean frequency of stage XIV in rats with cirrhosis was significantly greater than rats without cirrhosis (4.7 ± 2.3 percent and 6.8 ± 1.9 percent respectively, p = 0.027). CONCLUSION: Animals with CCl4-induced cirrhosis and food restriction have shown alterations in spermatogenic cycle that were not seen in rats without CCl4-induced cirrhosis and food restriction.

Changes in Serum Cytokine Concentration: A Morphological Study of Liver Cirrhosis Induced by Common Bile Duct Ligation in Rats

BACKGROUND: Liver cirrhosis is a diffuse hepatic fibrosis and nodule formation. The transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) are very important cytokines in hepatic fibrogenesis. The aim of this study was to examine the relationship between the changes of the serum cytokines and morphological changes following common bile duct ligation in experimental rats. METHODS: Common bile ducts of fifty male Sprague-Dawley rats were ligated and seven male rats were set aside as controls. Five rats each were sacrificed in 1, 2, 4, 6, 8, and 10 experimental weeks. Light microscopic studies and liver function tests were performed during the above experimental weeks. The levels of serum TGF-beta1 and IL-10 were analyzed by ELISA. Also, alpha smooth muscle actin (alpha-SMA) immunohistochemical stains were performed. RESULTS: On the eighth week after common bile duct ligation, most hepatic lobular areas had been replaced by proliferated bile ducts and fibrous tissue (typical biliary cirrhosis). Serum TGF-beta1 levels between the control group and the common bile duct ligation group showed statistically significant changes. The alpha-SMA was stained at proliferated bile ducts. These findings were correlated with each other. CONCLUSION: Thus, this experiment may clarify our understanding of the mechanism in liver fibrogenesis. Also, indicated is a need to explore the therapeutic potential of these cytokines as anti-fibrotic agents.

Worm load and septal fibrosis of the liver in Capillaria hepatica-infected rats

Inocula, varying from 15 to 1,000 embryonated Capillaria hepatica eggs, were administered to young adult rats by gastric tube, in an attempt to investigate the influence of worm load in the production of septal fibrosis of the liver. Low doses of 15, 30 or 50 eggs were sufficient to produce septal fibrosis, but it appeared with variable degrees of intensity and always with focal distribution. Septal fibrosis became diffuse, progressive with time, and already well developed 40 days after infection, when 100 eggs or more were administered. However, higher inocula (200, 500 and 1,000 eggs) did not intensify septal fibrosis, although the number of parasitic focal lesions proportionally augmented