Activation Of Hepatic Cells Research Articles

Impact of Oxygenation of Bioartificial Liver Using Perfluorocarbon Emulsion Perftoran on Metabolism of Human Hepatoma C3A Cells

One of the most important challenges in bioartificial liver designed for patients suffering from acute liver failure is oxygenation of cells within the bioreactor. The aim of this study was to evaluate the impact of oxygenation of bioartificial liver using perfluorocarbon (PFC) emulsion on the metabolic activity of hepatic cells in vitro. Mineral fibers coated with collagen type I were used as scaffolds for hepatic cells. Significantly higher total protein synthesis by hepatic C3A cells cultured in the bioreactor for 24 hours, in the group treated with medium supplemented with PFC emulsion, was observed in comparison with medium without PFC. Albumin production increased in the group treated with PFC after 1 hour of perfusion and was continuously, statistically, significantly higher during perfusion then the control group. In conclusion, the use of oxygen carriers, such as the PFC emulsion, can significantly improve synthetic performance of the bioreactor. Mineral fibers coated with extracellular proteins may serve as support for hepatic cells in the bioreactor.

Hepatitis B Virus X Protein Induces Hepatic Steatosis Via Transcriptional Activation of SREBP1 and PPARγ

Hepatic steatosis occurs frequently in patients with chronic hepatitis B virus (HBV) or chronic hepatitis C virus (HCV) infection. Recently, several studies suggested that steatosis plays an important role as a cofactor in other liver diseases such as hepatic fibrosis, hepatitis, and liver cancer. In contrast to HCV, however, the molecular mechanism by which HBV mediates hepatic steatosis has not been clearly studied. Here, we show the molecular mechanism by which hepatitis B virus X protein (HBx) induces hepatic steatosis. Lipid accumulation and the expression of various lipid metabolic genes were investigated in HBx-transfected Chang liver cells, HepG2-HBx stable cells, and HBx-transgenic mice. Overexpression of HBx induced hepatic lipid accumulation in HepG2-HBx stable cells and HBx-transgenic mice. It also up-regulated the messenger RNA and protein levels of sterol regulatory element binding protein 1, but not peroxisome proliferator-activated receptor alpha (PPARalpha). Moreover, we also determined that the expression of HBx increases PPARgamma gene expression as well as its transcriptional activity in hepatic cells, mediated by CCAAT enhancer binding protein alpha activation. Finally, we showed that HBx expression is able to up-regulate the gene expressions of various lipogenic and adipogenic enzymes in hepatic cells. We showed that the increased HBx expression causes lipid accumulation in hepatic cells mediated by sterol regulatory element binding protein 1 and PPARgamma, which could be a putative molecular mechanism mediating the pathophysiology of HBV infection.

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE −/− mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE −/− mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE −/− mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE −/− mice on WD.

IL-10 attenuates IFN-α-activated STAT1 in the liver: involvement of SOCS2 and SOCS3

Interleukin-10 (IL-10) has been used in the treatment of viral hepatitis in interferon-α (IFN-α) non-responders while patients who have high levels of IL-10 are poorly responsive to IFN-α. The mechanism underlying such controversial functions of IL-10 remains unknown. Here we demonstrated that injection of IL-10 into mice attenuated IFN-α-induced signal transducer and activator transcription factor (STAT)1 tyrosine phosphorylation in the liver. Reverse transcriptase-polymerase chain reaction assay demonstrated that mouse liver expressed high levels of IL-10 receptor 2 (IL-10R2) but low levels of IL-10R1. Injection of IL-10 into mice activated STAT3 but not STAT1 tyrosine phosphorylation and induced suppressor of cytokine signal 2 (SOCS2), SOCS3, and cytokine-inducible SH2 protein (CIS) mRNA expression in the liver. Furthermore, overexpression of SOCS2 or SOCS3 inhibited IFN-α-induced reporter activity in hepatic cells. These findings suggest that IL-10 inhibits IFN-α-activated STAT1 in the liver, at least in part, by inducing SOCS2, SOCS3, and CIS expression, which may be responsible for the resistance of IFN-α therapy in patients who have high levels of IL-10 and recommends that IL-10 treatment for viral hepatitis should be cautious.

Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells

Background/Aims: The human serum albumin (HSA) conjugate Dexa 10-HSA was designed to specifically deliver the anti-inflammatory drug dexamethasone (Dexa) to nonparenchymal cells (NPC) in the rat liver. NPC play an important role in the pathogenesis of acute and chronic inflammatory liver diseases like fibrosis. Targeting Dexa to these cells might reduce its adverse effects and increase the efficacy. Methods: Tissue and intrahepatic distributions of Dexa 10-HSA were assessed in normal and fibrotic rats with 125I-labelled conjugate and by immunohistochemistry. The effect of the conjugate on lipopolysaccharide (LPS)-induced inflammation and cell activation was studied in vitro with precision-cut liver slices and in vivo. Results: Ten minutes after i.v. injection 72±13% and 65±12% of a tracer dose of Dexa 10-HSA had been taken up in normal and fibrotic livers, respectively. Unconjugated Dexa also accumulated in livers, but cellular distribution studies revealed an accumulation in parenchymal cells (NPC vs. parenchymal cell (PC) ratio=0.29±11, p<0.005) whereas Dexa 10-HSA accumulated in nonparenchymal cells (NPC/PC ratio=7.9±3.1). Both coupled and uncoupled Dexa showed effective inhibition of LPS-induced NO x and TNFα production in precision-cut liver slices. At low concentrations (0.02 μM), however, Dexa 10-HSA was more efficient at inhibiting TNFα production than uncoupled Dexa. In fibrotic rats Dexa 10-HSA (3 mg/kg) and an equimolar amount of uncoupled Dexa (0.22 mg/kg) both significantly promoted survival after LPS-induced acute inflammation. Conclusion: Dexa 10-HSA was at least as effective as uncoupled Dexa at inhibiting LPS-induced effects, which indicates that HSA-bound Dexa is pharmacologically active. Coupling Dexa to HSA shifted the accumulation of Dexa from the PC to the NPC of livers. Since mediator release from NPC is crucial in the initiation and propagation of the fibrotic process, selective delivery of Dexa in NPC may improve the efficacy and safety of corticosteroid treatment of liver fibrosis.

Effect of chemical sympathectomy on serum levels of thyroid hormones and the biochemical profile of domestic pigeons

Recent studies have stressed the importance of the cholinergic system on avian metabolism. However, the role of the sympathetic nervous system (SNS) remains unclear. The present study was, therefore, aimed to probe the mechanisms for modulation of avian metabolism by the sympathetic nervous system after inhibition of the adrenergic responses. Activities of serum thyroid hormones (tri-iodothyronine, T 3, and thyroxine, T 4), body weight, hepatic weight, as well as total lipid and water content in the liver and body temperature were some of the parameters examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA) and reserpine treatment in 24-h starved pigeons. In addition, glucose was administered to the pigeons to identify the regulatory role played by glucose after disruption of the SNS. A reduction in body weight of the pigeons and an enhancement in the lipogenic machinery along with a corresponding increase in water content were some of the obvious effects in 6-OHDA + reserpine treated, as well as glucose-loaded sympathectomized birds. The cloacal temperature ( T c ) and both the thyroid hormones showed a drastic decrease while the T 3/T 4 ratio was augmented as a result of sympathectomy. However, serum T 3 and T 4 levels were restored to control values when glucose load was given, indicating that glucose might be reversing some of the detrimental effects of 6-OHDA treatment by activating intrinsic autoregulatory mechanisms of thyroid gland, thereby reviving the levels of thyroid hormones. Thus, the influence of SNS appears to be crucial in the maintenance of serum thyroid hormones and body temperature, as well as metabolic activities of hepatic cells.

Functional characterization of a unique liver gene promoter.

Human phenylalanine hydroxylase (PAH) is specifically expressed in the liver to convert L-phenylalanine to L-tyrosine. Deficiency of the PAH enzyme causes classic phenylketonuria, a common genetic disorder. The human PAH gene has a TATA-less promoter with multiple transcriptional initiation sites. A 9-kilobase DNA fragment 5'-flanking to the human PAH gene is sufficient to confer tissue- and developmental stage-specific expression of a reporter gene in transgenic mice. Deletion studies showed that the -121-base pair proximal promoter still retained a significant level of activity in hepatic cells. At least two protein binding sites, PAH-A and PAH-B, were identified in the proximal region of the human PAH promoter using rat liver nuclear extract. The PAH-A site covers a unique palindromic sequence, and the PAH-B site contains CCCTCCC repeats. Both elements are ubiquitous and essential regulatory elements for transcriptional activity. Nuclear protein factors that bind to the PAH-A and -B sites are detected in different cell types and are distinct from previously characterized transcription factors. No tissue-specific transcription factor binding sites have been detected within the proximal promoter region of the human PAH gene. These results suggest that the PAH gene promoter has a unique organization of regulatory elements for its tissue-specific expression in comparison with other liver gene promoters.

Nuclear factors AF-1 and C/EBP bind to the human ApoB gene promoter and modulate its transcriptional activity in hepatic cells.

ApoB-100, the major protein of low density lipoprotein, is produced primarily in the liver. The apoB promoter region from -86 to -52 binds two hepatic nuclear proteins. The protein that binds to the sequence between -86 to -61, called AF-1, was characterized and also shown to interact with the apoCIII, apoA-I, and apoCII promoters. We now show that the second protein, which by DNase I footprinting binds to the sequence between -69 and -52, is the heat stable hepatic factor C/EBP. The binding of this protein is competed by a known C/EBP-binding sequence in the transthyretin gene enhancer (TRR oligo 3). In addition, the protein protects the same region in DNase I footprinting as purified C/EBP. Mutants that affect AF-1 or C/EBP binding to the apoB promoter were created. These mutants were analyzed by the gel mobility shift assay and transient transfections into HepG2 cells. We have demonstrated that the two factors bind to overlapping sites and that they modulate apoB transcriptional activity.

A Regulatory Element in the ApoCIII Promoter That Directs Hepatic Specific Transcription Binds to Proteins in Expressing and Nonexpressing Cell Types

To better understand the mechanisms that determine cell type-specific gene expression, we have examined the transcriptional activity of a 13-nucleotide long sequence element, designated C3P, located in the promoter of the apoCIII gene. We demonstrate that this element is required for high levels of apoCIII gene expression in hepatic cells and is sufficient to determine hepatic specific expression when introduced into a heterologous promoter. A protein was identified in hepatic cell nuclear extracts, designated AF-1, that binds to this sequence and is presumably responsible for its transcriptional activity in hepatic cells. Even though the C3P element is not active in HeLa cells, a protein with C3P binding specificity was identified in HeLa cell nuclear extracts. While the HeLa protein is similar to the hepatic AF-1 in its binding specificity and relative abundance, it has approximately twice the molecular weight of the hepatic protein, indicating that they are different proteins or different forms of the same protein. A variety of murine tissue types, including those that do not express the apoCIII gene, were found to contain C3P binding proteins. We conclude that the cell type-specific activity of the C3P element is not due to the absence of C3P binding proteins in nonexpressing cells but is the result of qualitative differences in C3P binding proteins in different cell types.

Biochemical evaluation of naphthalene intoxication in the tropical acrid blood clam Anadara granosa

Exposure of the tropical arcid blood clam Anadara granosa L., collected from Bombay waters in 1986, to sublethal concentrations of naphthalene (Nap) (5, 10 and 15μg Nap ml-1), induced various changes in vital biochemical systems. The condition index (CI) remained largely within the control range, although tissue water content after 96h exposure increased significantly (p<0.001). Glycogen levels appreciably decreased, both in muscles (p<0.001) and digestive glands (p<0.01). AMP activity remained unaltered irrespective of the ambient Nap concentration. ADP levels decreased by more than 20% and ATP levels also decreased significantly (p≪0.001) by more than 60% with increases in ambient Nap levels from 5 to 15 μg ml-1. Lysosomal marker enzymes-arylsulphatase and acid phosphatase-showed various changes. Levels of the former were 10 to 17 times higher than the latter. Total and free arylsulphatase activity in hepatic cells decreased on exposure of clams to 5 and 10μg Nap ml-1, but not significantly. However, it was significantly inhibited (p<0.001) towards the upper limits of exposure concentration. The same was also true of acid phosphatase activity. Nicotinamide adenine dinucleotide (NADH2) oxidation of lipochrome showed a dramatic decrease in activity in the range of 37 to 88% over controls with increasing Nap from 5 to 15 μg ml-1. Fatty acids also exhibited significant changes in composition with a decrease in polyunsaturated acids and an increase in certain saturated fatty acids. Protein and carbohydrate levels increased significantly (p<0.001) towards upper limits of exposure concentration. Pyruvic acid levels increased significantly by 2 to 6 times over controls on exposure to 5 to 15 μg Nap ml-1. Explanation of these changes is attempted in terms of the glycolysis pathway involving pyruvic acid oxidation and oxidative phosphorylation in the electron transport system.

Effect of six Chinese drugs on serum transaminase activity and liver tissue in mice

The methanol extracts ofPatriniae radix,Dianthii herba,Melandrii herba,Echinopii radix,Siegesbeckiae herba andMagnoliae cortex showed a significant elevation of serum transaminase activity accompanied by fatty degeneration and Kupffer cell activation in hepatic cells.

Morphological and functional cellular analysis of liver carcinogenesis — ultrastructure-histochemical enzyme activities and proliferative activities of hepatic cells during carcinogenesis in rats induced by diethylnitrosamine

In view of these histological, histochemical and electron microscopical findings, there were some similarities between NH and HC, whereas marked difference of proliferative activities was found between in the cells of NH and HC. These differences between morphological and functional findings of hepatocytes during carcinogenesis may indicate the complicated mechanism in development from normal cells to hepatocellular carcinama.

Drainage of the common bile duct in experimental injury to the liver

The increasing frequency of trauma to the liver and the ensuing high mortality forces a constant modifications of therapeutic methods. The use of external drainage of the common bile duct has greatly improved the results of treatment and has reduced the number of complications. The influence of drainage on the hepatic cell activity and on the process of healing of liver wounds has been investigated in experiments carried out on cats. The animals were divided into two groups: those with drainage of the common bile duct, and, as a control group, those without drainage. The right medial lobe of the liver was injured in an identical way in both groups. The animals were tested before the operation and two weeks afterwards. The conclusions are based on clinical observations, histopathologic and histochemical findings, and results of laboratory blood tests. In the animals with drainage the changes in serum protein fractions have deviated less from the norm than did those in the control group, although the histopathologic as well as the histochemical pictures of the site of injury did not show any basic differences. The clinical and experimental observations show that the drainage of the common biliary duct should be used in all extensive traumas to the liver.

The diurnal rhythm of mitotic activity in the regenerating liver during acute radiation sickness

In experiments on albino rats a study was made of the 24 h thythm in the mitotic activity of hepatic cells and in the percentage of binuclear cells in the regenerating liver following a single total exposure to X-irradiation in a dose of 600 r. The finding was that at the peak of acute radiation sickness the 24 h mitotic rhythm was retained. As in the case with reparative and physiological regeneration in nonirradiated animals the peak of mitotic activity occured during the early morning hours. The mean 24 h coefficient of the cell mitotic activity (2–3 days following operation and correspondingly 5–6 days following irradiation) did not diminish as compared to control. No regularities were revealed that would suggest that during the 24 h period the number of binuclear cells in irradiated and nonirradiated rats was changed.

Regenerative processes in the liver of pigs following partial hepatectomy

About 1/8 of the liver 9distal portions of the left lateral and central lobes) were removed in 6 young pigs aged 1.5 months. Regenerating liver was investigated on the 3rd, 14th and 63rd postoperative days. As demonstrated, regeneration of the liver at the early periods was accompanied by the rise of mitotic activity of hepatic cells and hypertrophy of hepatic lobules. Mitoses within the lobule were unevenly distributed—their greatest number was localized in the middle and peripheral zones of the lobule. On the 14th–63rd days of regeneration smaller hepatic lobules were revealed at the wound surface; the possibility of their formation anew was considered. Perhaps, however, the small lobules represented the retained and reconstructed areas of old lobules.

The stimulation of reparative regeneration of the liver by subcutaneous injection of glycogen

Regeneration of the liver (following resection of 2/3 of this organ) was stimulated by subcutaneous administration of glycogen at the period of marked drop of its content in the liver (during the first week after the operation). A more intensive regeneration in the rat liver following subcutaneous injection of glycogen is connected with increased cellular multiplication, as shown by the data on mitotic activity of hepatic cells. There were no significant changes in the glycogen content of the regenerating liver of rats following subcutaneous administration of glycogen. No deleterious effect of glycogen injections on the organism was detected.

Increase in Number of Nuclei and Mitotic Activity of Parenchymal Cells of the Rat Liver in Postnatal Growth

Mitotic indices of parenchymal cells of rat livers in postnatal growth were calculated under several assumptions from the age and the increase in number of hepatic nuclei estimated by biochemical methods with animals in various growth stages, and compared with those established by microscopic observations on the same materials. As to the general pattern of the change in mitotic index across various growth stages, the calculation proved to agree fairly well with the observation, but the former tends to give considerably lower values. The reason of such a discrepancy is not clear. Low mitotic activity of hepatic cells in adult rats can be regarded as an expression of slow increase in number of hepatic nuclei rather than of cellular turnover of liverr parenchyma.