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Skeletal muscle repair can be understood as a balance between fibrosis and regeneration, the result of which may lead to complete recovery or loss of muscle function. To study the involvement of nitric oxide in post-trauma muscle repair, we used an experimental murine model of crush injury muscle. The animals were divided into four groups, (i) control (CO), (ii) sham trauma, (iii) trauma and (iv) trauma+l-NAME. The animals received a single dose of 100mg/kg of the l-NAME, an inhibitor of nitric oxide synthase, 2h after lesion, and the muscle tissue was analyzed in two time-points: 24h and 7 days. Twenty-four hours after injury, the crushed muscle was characterized by an intense inflammatory cell infiltrate and edema demonstrated by histological analysis. These changes were accompanied by increased iNOS, MMP-2 and HGF mRNA transcription and protein expression of the iNOS and MMP-2 in the gastrocnemius muscle. Crushing injury also promoted cell proliferation and increase number satellite cell, responsible for the regeneration of the muscle fiber. Treatment with l-NAME blocking local NO production, greatly attenuated these histological and molecular findings at 24h. On the 7th day the molecular findings of both groups were comparable to the control (sham trauma) group. However, the l-NAME group showed increase deposition of collagen and decrease of SC expression. These findings demonstrate that activation of NO during muscle crush is critical in the early phases of the skeletal muscle repair process and indicate its possible role as a regulator of the balance between fibrosis and muscle regeneration., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background/aims: The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment., Methods: Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry., Results: Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells., Conclusions: NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

The objective of this study was to determine in a rat model of hepatocellular carcinoma (HCC) the effects of the antiangiogenic agent TNP-470 on cell proliferation and effectors of the apoptotic pathway, including p53, p21WAF1/CIP1, cyclin D, and cyclin E. Tumor was induced in male Wistar rats by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, 3 times per week from 20 to 28 weeks. Serum levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor beta (HGFbeta) liver expression were increased by hepatocarcinogenesis (+38% and +183%, respectively), and treatment with TNP-470 was able to prevent the increase in these angiogenic factors induced by HCC. HCC coursed with reduced expression of p21WAF1/CIP1 and p53 (-63% and -60%, respectively). Hepatic expression of cyclin D and cyclin E were significantly increased in rats with HCC (+108% and +115%, respectively). In animals with experimental carcinogenesis, a significant increase in the expression of Cdk4 and CdK2 was also observed (+119% and +187%, respectively). These effects were prevented by TNP-470 administration. In conclusion, cell-cycle inhibition by TNP-470 is mediated at least in part by an activation of p21WAF1/CIP1 because of a p53-dependent mechanism, with reduction of the cyclin D-Cdk4 and cyclin E-Cdk 2 expression. These cytostatic effects should be considered when assessing the efficacy of TNP-470 for anti-angiogenic therapy. These findings may prove useful for the development of therapies for the treatment of human HCC.

In this study we sought to determine whether molecular mechanisms involved in the pathogenesis of fulminant hepatic failure are present in rabbits experimentally infected with rabbit hemorrhagic disease virus (RHDV). The activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, as well as bilirubin concentration, were found to be significantly increased 36 hours after infection. Infected animals also demonstrated significant decreases in factor VII activity, in the Fischer index, and in the deterioration of prothrombin time. The concentration of reduced glutathione was significantly decreased 36 hours after infection, and we noted a marked increase in the ratio of oxidized to reduced glutathione. Infected animals showed progressive decreases in liver activity of the antioxidant enzyme superoxide dismutase. Expression of hepatocyte growth factor and c-met was found to be progressively reduced from 24 hours after infection, during which time we detected no modification in messenger RNA (mRNA) levels of transforming growth factor (TGF)-alpha. TFG-beta 1 was overexpressed 24 and 36 hours after infection, and 36 hours after infection we detected a significant increase in TNF-alpha mRNA levels. Experimental RHDV infection also induced marked activation of nuclear factor-kappaB and a significant increase in inducible nitric oxide synthase mRNA levels from 24 hours after infection. Data obtained from this animal model support its usefulness in the investigation of potential novel therapeutical modalities aimed at neutralizing reactive oxygen species and hepatocyte growth inhibitors or enhancing hepatocyte responsiveness to mitogens.

In this study we sought to characterize a novel model of fulminant liver failure (FLF) by means of experimental infection of rabbits with the rabbit hemorrhagic disease virus (RHDV). Thirty-seven 9-week-old rabbits were injected intramuscularly with 2 x 10(4) hemagglutination units of an RHDV isolate. Eighty-five percent of rabbits died 36 to 54 hours after infection. From 36 hours after infection we noted marked increases in transaminases, lactate dehydrogenase, and total bilirubin. The rabbits exhibited hypoglycemia and coagulation abnormalities, with a significant decrease in factor V, factor VII, and prothrombin. Plasma aromatic amino acids and taurine showed progressive increases, and the Fischer index was significantly reduced. Expression of hepatocyte growth factor messenger RNA was inhibited from 36 hours after infection. Prostration and side recumbency were present at later stages, and neurologic symptoms rapidly progressed to coma. Onset of brain death was associated with a significant increase in intracranial pressure and blood ammonia. RHDV infection reproduces clinical, biochemical, and histologic features of the FLF syndrome and satisfies criteria for a suitable animal model. Rabbit hemorrhagic viral disease could provide a useful tool for the study of FLF and the evaluation of new liver-support technologies in human subjects.

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

This study was designed to investigate the effects of pectin on cholesterol and bile acid metabolism and to elucidate the mechanisms involved in its hypolipidemic effect in rats. The key regulatory enzymes in cholesterol and bile acid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase, were determined. Circulating, hepatic, and biliary lipid concentrations and fecal bile acid excretion were also measured. Male Wistar rats were fed a fiber-free or a pectin-supplemented (7 g/100 g) diet for 4 wk. Bile flow and the biliary secretion of both bile acids and cholesterol were not significantly different than controls in pectin-fed rats. The addition of pectin to the diet resulted in lower serum and liver cholesterol concentrations (-27 and -17%, respectively). Fecal bile acid excretion (+168%) and the hepatic activity of cholesterol 7alpha-hydroxylase (+70%) were significantly higher in pectin-fed animals. HMG-CoA reductase activity was also significantly greater (+11%) in the presence of dietary pectin. Results of our study indicate that pectin, by enhancing fecal bile acid excretion, may cause increased hepatic synthesis of bile acids and liver depletion of cholesterol in rats, which results in a higher rate of cholesterol synthesis and reduced serum cholesterol concentrations.

The purpose of this study was to determine the effects of experimental fasciolosis at various stages of development on the daily food intake of sheep. Five male Churra sheep, 4 months of age, were infected orally with 300 Fasciola hepatica metacercariae over a 30 day period. There was a significant increase in serum glutamate dehydrogenase (GLDH) activity from 40 days post-infection and in aspartate aminotransferase (AST) activity from 60 days post-infection. Both enzyme activities reached maximum levels in the serum of infected animals at 80 days and then progressively decreased. Serum gamma-glutamyltransferase (GGT) activity was significantly increased from 80 to 120 days post-infection. Glycaemia was significantly decreased from 60 days post-infection. The average daily food intake was shown to steadily decrease until approximately 100 days. The coincidence of decreased food intake with the period of significant increase, both in AST and GLDH activities, indicated that damage caused around the time of migration of immature flukes through the liver parenchyma may be involved in appetite depression.