Maintenance of mixed-function oxidase and conjugation enzyme activities in hepatocyte cultures prepared from normal and diseased human liver

Isolated hepatocytes were prepared from normal and diseased human livers and maintained in primary monolayer culture for up to 96 h. The viability and yields of cell preparations obtained from diseased livers did not differ significantly from those obtained from normal livers. During the culture period a significant increase in cell protein/DNA ratio was observed in both normal and diseased hepatocytes. The maintenance of a number of drug metabolising enzyme activities was determined in these hepatocytes during 96 h of culture. In normal hepatocytes the maintenance pattern of mixed-function oxidase activities (ethoxycoumarin-O-deethylase and ethoxyresorufin-O-deethylase) was clearly different from that of the conjugating enzymes (sulfotransferase and glutathione transferase). Whereas ethoxycoumarin-O-deethylase and ethoxyresorufin-O-deethylase activities declined sharply over the first 24 h in culture and then either totally or partially recovered, sulfotransferase and glutathione transferase activities were found to be relatively more stable initially but thereafter decline progressively. In diseased hepatocytes mixed-function oxidase activities were maintained less well than the corresponding activities in normal hepatocytes whereas conjugation enzyme activities were maintained to a similar extent.     

Asialoglycoprotein receptor in human isolated hepatocytes from normal liver and its apparent increase in liver with histological alterations

We have characterized a binding site for galactosyl terminal glycoproteins in hepatocytes isolated from human biopsies. The binding of asialoorosomucoid on hepatocytes previously treated by Triton X-100 was saturable, calcium-dependent and highly affine (Ka = 1.11 +/- 0.87.10(9) M-1) thus corresponding to a ligand-receptor binding. The total number of receptors in the normal human liver was 140,000 +/- 65,000 sites per cell. This corresponded to the value obtained in the human hepatoma cell line HepG2, but was significantly lower than for isolated rat hepatocytes. Furthermore, in hepatocytes isolated from livers with histological features of either fibrosis, cirrhosis, hepatocarcinoma with cirrhosis or nodular regenerative hyperplasia, the number of asialoglycoprotein receptors per cell was increased, while the binding affinity was unchanged.     

Functional properties of isolated hepatocytes from ethanol-treated rat liver

Gluconeogenesis and palmitate incorporation into triacylglycerols and phosphatidylcholine were measured in isolated hepatocytes from control and ethanol-treated rats. Basal gluconeogenesis and its hormonal response decreased in hepatocytes from ethanol-treated animals; palmitate incorporation into triacylglycerols increased. In ethanol-treated rat liver, 45Ca2+ uptake and methylating capacity were reduced, and the hormonal response exhibited differences in binding parameters of insulin.     

Biochemical studies of isolated rat hepatocytes from normal and phenobarbital-treated liver as obtained by rate zonal centrifugation.

Cell suspensions from normal and phenobarbital treated livers were analysed by rate zonal centrifugation in ficoll gradients. No difference in the microsomal cytochrome P-450 and NADPH-cytochrome C reductase content and in the incorporation rate of radioactive leucine was noted between light and heavy hepatocytes. After centrifugation of a mixture of radioactive phenobarbital-treated cells and normal liver cells, the specific radioactivity was constant, indicating that the hepatocytes from drug-treated liver had the same density as the normal cells.     

COVID-19 in normal,diseased and transplanted liver

Starting from December 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extended in the entire world giving origin to a pandemic. Although the respiratory system is the main apparatus involved by the infection, several other organs may suffer coronavirus disease 2019 (COVID-19)-related injuries. The human tissues expressing angiotensin-converting enzyme 2 (ACE2) are all possible targets of viral damage. In fact myocarditis, meningo-encephalitis, acute kidney injury and other complications have been described with regard to SARS-CoV-2 infection. The liver has a central role in the body homeostasis contributing to detoxification, catabolism and also synthesis of important factor such as plasma proteins. ACE2 is significantly expressed just by cholangiocytes within the liver, however transaminases are increased in more than one third of COVID-19 patients, at hospital admission. The reasons for liver impairment in the course of this infection are not completely clear at present and multiple factors such as: Direct viral effect, release of cytokines, ischemic damage, use of hepatotoxic drugs, sepsis, and others, may contribute to damage. While COVID-19 seems to elicit just a transient alteration of liver function tests in subjects with normal hepatic function, of concern, more severe sequelae are frequently observed in patients with a reduced hepatic reserve. In this review we report data regarding SARS-CoV-2 infection in subjects with normal or diseased liver. In addition the risks of COVID-19 in immunosuppressed patients (either transplanted or suffering for autoimmune liver diseases) are also described.     

Binding of plasminogen to hepatocytes isolated from injured mice liver and nonparenchymal cell-dependent proliferation of hepatocytes

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.     

阿魏酸钠对分离过程中肝细胞的活性、功能影响及机制探讨

目的观察阿魏酸钠（SF）对分离过程中肝细胞活性、功能的影响,并探讨其机制。方法12只SD大鼠随机分为A、B组,均采用改良的四步胶原酶分离技术分离肝细胞,而B组在第一步灌注液中加入SF。台盼蓝检测分离肝细胞收获量及成活率,PAS染色检测其纯度,并收集不同时期的培养上清,MTT法观察肝细胞增殖活性,全自动生化分析仪检测白蛋白（ALB）、乳酸脱氢酶（LDH）水平,ELLSA法检测丙二醛（MDA）及超氧化物歧化酶（SOD）水平。结果与A组比较,B组成活率高,6h、1d ALB合成增加,LDH释放减少,MDA水平显著下降,SOD水平显著上升,P均〈0．05。结论SF减轻分离过程导致的肝细胞活性和功能损伤;其机制与SF诱导肝细胞自身的抗氧化及清除氧自由基能力有关。     

Hepatic production of insulin-like growth factors in normal and diseased liver

BACKGROUND/AIMS: IGF-I levels are reduced in cirrhotic patients. However, it is not known whether this decreased level is the result of reduced hepatic production or modified bioavailability secondary to decreased binding proteins. We determined the hepatic production of IGF-I and IGF-II and their receptors in normal and diseased liver. METHODOLOGY: Twenty-five patients included, 11 controls with normal liver and 14 with either chronic hepatitis or cirrhosis. mRNA for IGF-1, IGF-II and their receptors were measured. Immunohistochemical staining was performed to localize the IGF-producing cells. RESULTS: In 11 normal livers, the IGF-I mRNA levels were 4.95 +/- 1.8; in the 14 diseased livers, the levels were 1.22 +/- 0.69 (p < 0.001). IGF-II mRNA levels were 3.78 +/- 1.45 for the control and 5.11 +/- 2.15 in the diseased livers (NS). IGF-I receptor levels were 1.15 +/- 0.83 in the normal and 0.31 +/- 0.22 in the liver disease group (p < 0.05). There was no statistical difference between the two groups for IGF-II receptor. CONCLUSIONS: Patients with chronic liver disease have a significant reduction in their hepatic production of IGF-I, whereas IGF-II tends to be elevated. Treatment with recombinant IGF-I in patients with metabolic or endocrine complications of cirrhosis might prove useful.     

Decreased uptake of taurocholate and ouabain by hepatocytes isolated from cirrhotic rat liver

To differentiate between the "intact" and "sick" cell hypothesis explaining decreased clearance of endo- and xenobiotics, we measured uptake of taurocholate and ouabain in hepatocytes isolated from cirrhotic rat liver. Cirrhosis was induced by chronic exposure of male Sprague-Dawley rats to phenobarbital and carbon tetrachloride. Uptake of [14C]taurocholate and [3H]ouabain was measured by a rapid filtration technique. Hepatocytes from cirrhotic liver were as viable as control hepatocytes--as judged by trypan blue exclusion and lactate dehydrogenase release--but consumed 28% less oxygen. Vmax of both taurocholate (3.16 +/- 0.95 vs. 0.40 +/- 0.35 nmoles X min-1 X 10(6) cells-1; p less than 0.001) and ouabain (2.16 +/- 0.78 vs. 0.83 +/- 0.26 nmoles X min-1 X 10(6) cells-1; p less than 0.005) was significantly reduced. These results are compatible with the "sick" cell hypothesis.     

Hepatitis C virus receptor expression in normal and diseased liver tissue

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. CONCLUSION: HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.     

Analysis of backscattered ultrasound from normal and diseased arterial wall

Intra-arterial ultrasonic imaging has several features which affect the feasibility of clinical tissue characterization when compared with trans-thoracic ultrasound. The short distance from transducer to tissue, fluid path, high frequencies, and special characteristics of the tissues of interest all contribute to making practical tissue characterization by measurement of the backscattered signal more probable in intra-arterial imaging. The properties of backscattered ultrasound, and methods of characterizing such signals, are discussed with special reference to intra-arterial applications.     

Glutamine transport in isolated human hepatocytes and transformed liver cells

The transport of l-glutamine was examined in isolated adult and fetal human hepatocytes as well as in the human hepatoma cell lines HepG2 and SK-Hep. In all cells studied, glutamine uptake was at least 85% Na⁺-dependent. Kinetic analysis of the Na⁺-dependent component indicated mediation by a single transporter in three human hepatocyte preparations and in SK-Hep cells, whereas two transporters appeared to be responsible for glutamine uptake in HepG2 cells and in hepatocytes from the liver of one male patient. Amino acid inhibition analysis showed primary mediation by System N in fetal and adult hepatocytes, whereas System ASC was principally responsible for glutamine uptake in transformed cells. Similar to the rat transporter, human System N was pH-sensitive, stereospecific, and responsive to treatment with steroid hormones. Although the human carrier was less tolerant of Li⁺- for Na⁺ substitution, glutamine transport in primary human hepatocytes was stimulated by treatment with hypotonic buffer (cell swelling), as reported in rat parenchymal cells. In contrast, glutamine transport in hepatoma cells was relatively insensitive to changes in extracellular pH and failed to show enhanced activity in response to hypoosmotic challenge. Collectively, the data suggest that markedly distinct plasma membrane transporters mediate the concentrative uptake of glutamine in normal and transformed human hepatocytes, and that the salient properties of System N have been largely conserved from rat to man.     

Endothelin-induced vasoconstriction in isolated perfused liver preparations from normal and cirrhotic rats

Isolated, perfused rat liver preparations (IPRL), obtained from rats with carbon tetrachlorideinduced cirrhosis and normal controls, were used to investigate responses to the vasoactive peptide endothelin-1 (ET-1). The mean perfusion resistance (R) of cirrhotic IPRL was significantly greater than that of controls (2.63 ± 0.24 vs 1.54 ± 0.14 mmHg/mL per min per g; P < 0.01). Both control and cirrhotic IPRL demonstrated a concentration-related increase in resistance (ΔR) in response to ET-1, with a minimum effective concentration of approximately 3 × 10⁻¹¹ mol/L. The EC₅₀ (-log of the 50% effective concentration) was not significantly different between cirrhotic and control IPRL (8.48 ± 0.19 and 8.79 ± 0.11, respectively); however, the maximum response to ET-1 was significantly greater in cirrhotic preparations (R: 10.4 ± 2.2 vs 4.4 ± 0.5 mmHg/mL per min per g, P < 0.01; DR, 7.8 ± 2.1 vs 2.8 ± 0.4 mmHg/mL per min per g, P < 0.01). Following maximal stimulation by ET-1, the mean portal-hepatic venous pressure gradient at a physiological flow rate of 1 mL/min per g was approximately 90% greater across cirrhotic IPRL than that across normal IPRL (11.2 ± 2.0 vs 5.9 ± 0.9 mmHg, respectively; P < 0.05). These results support the hypothesis that endogenously released ET-1 has a significant influence on the portal vascular resistance of cirrhotic liver in vivo and has an important role in the pathogenesis of portal hypertension.     

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Conclusion: Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications.     

Sinusoidal endothelial cells from normal guinea pig liver: isolation, culture and characterization

Guinea pig nonparenchymal hepatic cells were isolated by enzymatic digestion and subsequent separation on a 17.5% metrizamide gradient. Endothelial cell and Kupffer cell-enriched fractions were separated by centrifugal elutriation. Viability of both cell fractions was approximately 80%. Endothelial cells were cultured on a substratum of guinea pig liver collagen and 1% gelatin (1:1). Freshly isolated and cultured sinusoidal endothelial cells contained Factor VIII R:antigen, angiotensin I converting enzyme activity, and they synthesized prostaglandins characteristic of other endothelial cells. Sieve plates were identified in both freshly isolated and cultured cells. Fresh endothelial cells and Kupffer cells formed Fc receptor-mediated rosettes with IgG-opsonized sheep red blood cells, but cultured endothelial cells did not. Only Kupffer cells demonstrated Fc and C3 receptor-mediated phagocytosis. These methods for isolating and culturing sinusoidal endothelial cells should permit further functional assessment of endothelial cells and their interrelationship with other sinusoidal lining cells.     

N-乙酰半胱氨酸对C3A永生化肝细胞增殖和生物转化功能的影响

目的研究N-乙酰半胱氨酸（NAc）对慢性重型肝炎肝衰竭时肝细胞增殖和生物转化功能的影响,并比较NAC和还原型谷胱甘肽（GSH）的疗效。方法体外慢性重型肝炎患者血浆培养C3A细胞,同时分别加入大、中、小三种剂量NAC及对应剂量的GSH,以正常血浆及培养基（10％FBS-MEM）为对照,分别于24h、48h、72h三个时问点检测细胞安定代谢量的变化,并用MTT法测定C3A细胞活力。结果慢性重型肝炎血浆使细胞的安定代谢量降低,细胞增殖活力提高。NAC和GSH均可提高慢性重型肝炎血浆培养C3A细胞的安定代谢量,呈一定剂量依赖性,并可使细胞增殖活力进一步提高。结论NAC可以显著改善慢性重型肝炎肝衰竭时肝细胞增殖活力和生物转化功能,GSH也有相似功效,相同剂量的NAC优于GSH。