Betulin, a pentacyclic triterpene, possesses antioxidant, anti-inflammatory and hepatoprotective properties. The aim of this study was to evaluate the impact of liver mitochondria in hepatoprotection of betulin using a rat model of alcoholic steatohepatitis induced by ethanol administration (4 g/kg, intragastric) for 8 weeks. The treatment with betulin (50 and 100 mg/kg b.w., intragastric) during this period attenuated the histological signs of steatohepatitis and lowered the serum and liver triglyceride contents, as well as the serum activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Betulin (100 mg/kg) decreased the liver/body weight ratio and inhibited the increase in the serum levels of TNFα, IL-1β, TGFβ, and hyaluronic acid, demonstrating hepatoprotective, anti-inflammatory, and antifibrotic potential. Betulin also inhibited the formation of superoxide anions in mitochondria and the end-products of lipid peroxidation in liver tissue, the amount of which was significantly increased in ethanol-treated rats. The disturbances in mitochondrial respiration, uncoupling of oxidative phosphorylation and decreasing of mitochondrial complex I, II, and IV activities in rats with steatohepatitis, were reverted by betulin administration. The increased susceptibility of mitochondria to Ca2+-induced permeability transition pore formation in the hepatitis group was improved in rats treated with betulin. In conclusion, betulin, having antioxidant properties, exerts a beneficial effect in the rat model of alcoholic steatohepatitis via prevention of liver mitochondria dysfunction, which may be attributed to the inhibition of mitochondrial permeability transition.

The aim of study was to evaluate the hepatoprotective effect of borage oil containing predominantly gamma‐linolenic acid in rats with alcoholic steatohepatitis. Liver of ethanol‐treated animals was characterized by fatty and hydropic dystrophies. Liver triglyceride contents and activitiies of serum marker enzymes were significantly increased. Ethanol increased nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)‐induced chemiluminescence and the contents of liver thiobarbituric acid reactive substances (TBARS). The reduced glutathione content in the liver was decreased. Ethanol enhanced liver microsomal cytochrome P‐450 (CYP450) content, aniline p ‐hydroxylase and amydopyrine‐N‐demethylase activities. The treatment with borage oil improved the liver morphology, decreased triglyceride contents and normalized serum marker enzyme activities. Borage oil developed an antioxidant effect in ethanol‐treated rats. The treatment with this compound decreased NADPH‐induced chemiluminescence and the content of lipid peroxidation products. Borage oil normalized CYP450 content compared with the ethanol‐treated group. CYPI450 2E1 isoform is a main source of free oxygen radicals in the liver of ethanol‐treated rats and we propose that the antioxidant effect of borage oil is realized via the normalization of CYP450 content and activities of CYP450‐related microsomal oxidases, as borage oil can improve the lipid surrounding of CYP450. In our opinion, the hepatoprotection by borage oil in alcoholic steatosis is connected with its antioxidant properties. Copyright © 2012 John Wiley & Sons, Ltd.

Ethanol treatment increases the production of reactive oxygen species (ROS) and lowers antioxidant levels, thereby enhancing oxidative stress in many tissues. ROS are capable of initiating lipid peroxidation and lead to the release of reactive aldehydes with potent pro-inflammatory and pro-fibrotic properties. Various pro-inflammatory cytokines such as TNFα, IL-1, IL-8 and others have been proposed to play a major role in alcoholic steatohepatitis (ASH). Serum concentrations TNF and of various TNF-inducible cytokines such as IL-1 and IL-8 are increased in patients with acute ASH. Several studies in rats, mice and tissue culture focused on the role of cytokines, especially TNF, in experimental models of alcoholic liver disease. Studies neutralizing other pro-inflammatory cytokines such as IL-1 or IL-8 are currently not available. Furthermore, alcohol-associated liver injury is inhibited when the animals are treated with poorly absorbed oral antibiotics or lactobacillus to decrease endotoxaemia, supporting the hypothesis that gut-derived bacterial products such as endotoxin might be important in activation of Kupffer cells and/or other cell types in the liver. This fits with the observation that chronic ethanol feeding causes more severe liver injury in wild-type mice than in CD14 knockouts. These results further support that gut-derived endotoxin acting via its cellular receptor CD14 plays a major role in the development of alcohol-induced liver injury. In experimental models and isolated hepatocytes, alcohol has been shown to induce liver cell apoptosis. Evidence from rat studies indicates that ethanol-induced gut leakiness and endotoxemia precedes liver inflammation, suggesting that indeed ethanol-induced gut damage come firs. Even though cytokine dysregulations in ASH may constitute secondary phenomena, blockade of these highly pro-inflammatory molecules remains a potential treatment concept. # S05.2 HEPATOPROTECTION AND IMMUNE RESPONSE TOWARDS MONOTHERAPY AND COMBINATION THERAPY WITH ANTIBODIES TO TNFα IN EXPERIMENTAL ALCOHOLIC STEATOHEPATITIS {#article-title-2} The activation of TNFα, the key pro-inflammatory cytokine, plays an important role in alcoholic liver disease. TNFα mediates inflammatory process and accelerates lipid accumulation in the liver under alcoholic steatohepatitis (ASH). Therapy with anti-TNFα antibodies is mainly in use in the clinics for acute alcoholic hepatitis but not for ASH. In the study that we carried out, the monoclonal anti-TNFα antibodies remicade (REM; 1 and 10 mg/kg, i.p) administered during 10 days decreased necrotic foci and lymphocytic infiltration but unaffected steatosis in the liver of rats with ASH induced by feeding of the Lieber-DeCarli ethanol-containing diet (LCD). Only the high dose of REM lowered liver triglyceride content and serum alkaline phosphatase (AP) activity. Both REM doses raised phagocytic index and circulating immune complexes and normalized complement activity. As the treatment of ASH with REM rather benefits inflammatory signs, we used REM in combinations with promising hepatoprotectors to improve steatosis. We administered REM simultaneously with i.g. administration of sesamine (500 mg/kg), pioglytasone (10 mg/kg), betaine (100 mg/kg) or metformine (50 mg/kg). The combinations of REM with betaine and sesamine were more effective than REM alone in decreasing serum AP activity, square of sudanophylic staining and triglyceride content in the liver. All combinations did not disturb immunomodulatory properties of REM. These experimental studies suggest that anti-TNFα antibodies combined with the hepatoprotectors (sesamine, betaine) are effective in necrosis, inflammation and steatosis in the experimental model of ASH induced by LCD feeding. # S05.3 IRON METABOLISM AND ALCOHOLIC LIVER DISEASE {#article-title-3} Alcoholic liver disease (ALD) is frequently associated with hepatic iron overload that contributes to liver injury and is considered as a risk factor for the development of liver fibrosis. Dietary iron absorption and body iron traffic are controlled by the iron regulatory hormone hepcidin, a liver-derived peptide that binds to the iron exporter ferroportin and promotes its internalization and degradation. This leads to retention of iron within ferroportin-expressing cells, including duodenal enterocytes, reticuloendothelial macrophages and hepatocytes. The expression of hepcidin is regulated transcriptionally in response to iron, inflammation and stress signals. Experiments in cells and mouse models demonstrated that ethanol suppresses hepcidin mRNA transcription by inhibiting the binding of the constitutive transcription factor C/EBPα within its promoter. Ethanol-dependent downregulation of hepcidin is associated with increased expression of the duodenal iron transporters DMT1 and ferroportin at the apical and basolateral membranes of enterocytes, respectively. These findings provide a framework to understand the molecular mechanisms underlying hepatic iron accumulation in ALD patients. To better characterize the role of iron in chemically induced liver fibrogenesis, hemojuvelin knockout (Hjv − /−) mice, a model of hemochromatosis, and wild-type controls were subjected to intoxication with CCl4. Hjv − /− mice developed earlier and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF. Thus, hepatic iron overload potentiates the effects of chemical intoxication and triggers precocious profibrogenic gene expression. Although the CCl4 model may simulate pathophysiological responses of ALD, further experiments with ethanol intoxication are required to validate these data. # S05.4 H2O2: A LINK BETWEEN INFLAMMATION AND IRON METABOLISM {#article-title-4} Hepatitis of any kind is characterized by increased concentrations of reactive oxygen species. H2O2 is the main reactive oxygen intermediate derived from a multitude of cellular metabolic processes as well as from oxidative burst by granulocytes or macrophages. Due to its relatively long half-life time, there is growing evidence that H2O2 also acts as a signaling molecule. Iron metabolism can be influenced by H2O2 by activating the iron regulatory protein 1, which regulates intracellular iron metabolism but also by increasing transferrin receptor 1 that is responsible for cellular iron uptake. Both mechanisms lead to increased cellular iron uptake and decreased levels of circulating serum iron. Hepcidin, the systemic iron regulator, has not been linked to oxidative stress so far. New experiments, however, point out that H2O2 is able to increase hepcidin via STAT3 signaling, thus decreasing iron recycling by macrophages and iron resorption by the duodenum. This is a third mechanism to lower serum iron during increased oxidative stress and preventing toxic reactions between H2O2 and iron.

In our recently published study we have detected in livers from Sprague-Dawley rats given fructose-enriched diet (FED) and developed NAFLD, increased hepatic triglycerides (+198%), increased hepatic cholesterol (+89%) and hypertriglyceridemia (+223%).The genetic risk factors involved in the pathogenesis of NAFLD are poorly understood.Methods: We investigated the gene expressions of: 1. Microsomal triglyceride transfer protein (MTP) involved in hepatic lipid synthesis, storage and export.2. Manganese-dependent superoxide dismutase 2 (MnSOD2), encoding proteins involved in the response to oxidant stress.3. Peroxisome proliferator-activated receptor (PPAR)-a involved in hepatic fatty acid oxidation, in rats that were administered FED (n = 6) or standard rat chow (n = 3) for five weeks.Intrahepatic messenger RNA (mRNA) levels of MTP, MnSOD2 and PPAR-a were measured by real-time polymerase chain reaction using Taqman gene expression assays (Applied Biosystems, Foster City, CA) in FED rats and in standard rat chow.Results: In the FED rats, liver histology demonstrated diffused macrovesicular and micro-vesicular steatosis.1. MTP mRNA level was statistically significant decreased in FED rats (by 85%) (P < 0.0001) compared with the standard rat chow.2. MnSOD2 mRNA level was statistically significant decreased in FED rats (by 80%) (P < 0.001) compared with the standard rat chow.3. PPAR-a mRNA level was statistically significant decreased in FED rats (by 95%) (P < 0.0001) compared with the standard rat chow.Conclusions: the reduced expression of MTP, MnSOD2 and PPAR-a in the FED rats implies these genes are involved in the pathogenesis of NAFLD.Further studies are needed to confirm our results.

Both obesity and diabetes are risk factors for nonalcoholic steatohepatitis (NASH) and for HCC.Moreover, HCC is a recognized complication of NASH while steatosis might play a role in HCC in non-NASH liver disease.Hepatic stem cell (oval cell) proliferation may play a role in carcinogenesis in this setting.Aim: We sought to develop a rat model which reproduces NASH with cirrhosis and HCC.Methods: Seven adult Sprague-Dawley rats, weighing 250-300g, were fed a choline-deficient diet with high trans-fat content for 16 wk (Rhoster Ltda, Brazil) and simultaneously exposed to diethylnitrosamine (Sigma) 13-15 mg/day in their drinking water (135 mg/l).Afterwards, the animals underwent ultrasound (US) and sacrifice.Liver was assessed grossly and by light microscopy (LM), immunohistochemistry (IHC) for cytokeratin 19 (CK19) and by transmission electron microscopy (TEM).Results: US revealed steatosis in all and focal nodular changes in 6 animals.At sacrifice, all 7 were steatotic and 6 appeared cirrhotic.On LM, all had steatohepatitis defined as inflammation, fibrosis and ballooning with Mallory-Denk bodies (MDB).Areas of more severe injury were associated with CK19 + ductular reaction.Cirrhosis was evident by LM in 6 of 7 while HCC (moderately to poorly differentiated) was evident in all 7. HCC were macro-trabecular or solid with polyhedral cells and occasional steatotic or balloon cells with MDB and hyaline globules.CK19 IHC was positive in solid nests of oval cells and in some neoplastic hepatocytes.One animal had a "biphasic" Hepato-Cholangiocarcinoma. TEM confirmed ballooning change associated with small droplet fat, dilated endoplasmic reticulum and MDB in non-neoplastic hepatocytes and focal small droplet steatosis in some cancer cells which qualitatively had far less mitochondria than non-neoplastic counterpart.Conclusions: This novel model replicates the major stages of NASH including cirrhosis and a form of HCC which appears to be oval cell derived.Our results offer a practical platform on which to further study NASH progression to cirrhosis and HCC and to assess new forms of anti-HCC therapy as well as to investigate alternative forms of imaging of HCC.

The aim of this study was to investigate the antiatherogenic effects of 17 beta-estradiol and 17 alpha-estradiol and its derivative J811 (estra-1,3,5(10),8-tetraene-3, 17 alpha-diol), having a non-feminizing effect and high antioxidant potential, in male rabbits.Male White-Russian rabbits weighing 2.1-2.6 kg were fed either a standard or a high-cholesterol (200 mg/kg) diet, with thyroid function-inhibiting thiouracil (20 mg/kg) combined with cholic acid (40 mg/kg) administered daily in sunflower oil for 3 months. During the last month of the study, estrogens were administered by gavage at a dose of 0.02 or 0.1 mg/kg.All three estrogens exerted remarkable antiatherosclerotic effects. Decreases in serum and aortic-wall lipid parameters and the index of atherogenicity were dependent on estrogen dose. Morphological evaluation of the aortic wall (height of plaques, size of plaque relative to aortic half-circumference) showed only weak therapeutic effects with all three estrogens. It is an open question whether the treatment period was too short to reverse the above changes. On the other hand, the data clearly suggest that 17 alpha-estradiol and J811 offer new perspectives for the prevention of atherosclerosis in men, which is similar to that found with 17 beta-estradiol in women.

The hypolipidemic effects of pantothenic acid derivatives (phosphopantothenate, panthenol and pantethine) were studied in mice with hypothalamic obesity. Hypothalamic obesity in mice was induced by single injection of aurothioglucose (300 mg/kg body wt, i.p.). All the tested substances were administered during the last 10 days before decapitation (i.m., of dosage equivalent to 150 mg/kg body wt of phosphopantothenate). The studied substances inhibited the weight gain of the animals with hypothalamic obesity over the last 10 days of the experiment. The treatment with aurothioglucose increased food intake and mean body weight, blood glucose level; insulin, serum total cholesterol, triglyceride, the sum of LDL + VLDL and LDL-cholesterol concentration; triglyceride and cholesterol fractions in the liver; triglyceride and FFA content as well as lipoprotein lipase activity in adipose tissue of experimental mice. The administration of the assay compounds lowered food intake and mean body weight, insulin and glucose levels and decreased the content of triglycerides, total cholesterol and cholesterol esters in serum and adipose tissue as well as raised the activity of lipoprotein lipase in adipose tissue and serum lipolytic activity in obese mice. Among the compounds studied the reverse effect of panthenol was especially pronounced. The mechanism of hypolipidemic effects of pantothenic acid derivatives can be related to the reduced resistance to insulin and activation of lipolysis in serum and adipose tissue.