The objective of this study was to analyse the response of hepatocytes on various concentrations of 17ß-oestradiol (17ß-E) under iron-induced oxidative stress in vitro. Isolated by in situ collagenase perfusion hepatocytes were cultured in DMEM/HAMS-12 (v/v) medium without any additional agents (control), with Fe³⁺ alone, and with Fe³⁺ aild 0.2%, 0.02%, and 0.002% solution of 17ß-E (17ß-EI, 17ß-EII, and 17ß-EIII, respectively). After 24, 48, and 72 h, medium malonylodialdehyde (MDA), haptoglobin (Hpt) concentration and proliferative activity were determined. In comparison to control samples, and samples collected at 24 and 72 h, hepatocytes exposition to Fe³⁺, caused a significant increase in MDA (0.056 ±0.011 nM/mL) only after 48 h of incubation. Each of 17ß-E concentrations resulted in a decrease in MDA in samples obtained after 24 and 48 h. In comparison to the first 24 h, Fe³⁺ alone and together with 17ß-EI, 17ß-EII, and 17ß-EIII caused a significant augmentation of Hpt level in 48 h and 72 h of the experiment. Each of the 17ß-E concentrations added to the culture medium resulted in inhibition of hepatic proliferative activity, especially in the 72 h of cell culture.
The apoptotical effect of nitric oxide on effector apoptotical caspase 3 in rats' hepatocytes was examined. The experiment was performed on 16 white Wistar female rats divided into two equal groups. The rats from the experimental group received orally L-arginine in a dose of 40 mg/kg b.w. every other day for 2 weeks. The rats from the control group received orally 2 ml of distilled water in the same manner as the experimental group. All the rats were decapitated after 3 weeks of the experiment. After decapitation, specimens from the liver were collected, fixed in 10% formalin, and then embedded in paraffin blocks. Protein caspase 3 on slides was detected using the standard three-step immunohistochemical method. The quantitative evaluation of caspase 3 expression showed that the area occupied by positive caspase 3 reaction in the liver of the experimental group (128.11 µm²±96.54) was comparable to that in the control group (212.18 µm² ±1 16.59) (P=0.25). The dose of L-arginine used was similar to that applied in pregnant women treated for gestosis. The study shows that L-arginine as a donor of exogenous nitric oxide has no an apoptotic effect on rats' hepatocytes.
Under normal physiological conditions, the majority of hepatocytes are in the functional state (G0 phase). After injury or liver partial hepatectomy (PH), hepatocytes are rapidly activated to divide. To understand the mechanism underlying hepatocyte G0/G1 transition during rat liver regeneration, we used the Rat Genome 230 2.0 Array to determine the expression changes of genes, then searched the GO and NCBI databases for genes associated with the G0/G1 transition, and QIAGEN and KEGG databases for the G0/G1 transition signaling pathways. We used expression profile function (E t ) to calculate the activity level of the known G0/G1 transition signal pathways, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the interactions among these signaling pathways. The results of our study show that the activity of the signaling pathways of HGF, IL-10 mediated by p38MAPK, IL-6 mediated by STAT3, and JAK/STAT mediated by Ras/ERK and STAT3 are significantly increased during the priming phase (2–6 h after PH) of rat liver regeneration. This leads us to conclude that during rat liver regeneration, the HGF, IL-10, IL-6 and JAK/STAT signaling pathways play a major role in promoting hepatocyte G0/G1 transition in the regenerating liver.
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The development of the nase liver was examined under light and electron microscopes from the moment of hatching until the juvenile stage. Three phases of hepatocyte differentiation were observed during the organogenesis of nase livers. In the first phase, from hatching until day 4, the hepatoblasts of the primordial liver are morphologically undifferentiated and divided by sinus vessels. They also store glycogen. In the second phase, from the moment when the mouth cavity becomes passable until the resorption of the yolk sac, organelles typical of the structure of hepatocytes appear and begin to function. At the end of this phase signs of bile lipid synthesis and secretion become visible. The third phase is when exogenous nutrition begins and is characterized by the increased activity of the significant organelles engaged in protein synthesis and secretion, such as the endoplasmic reticulum and the Golgi apparatus.
The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4°C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 ± 6.5% and 34.8 ± 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reduction rate (6.4 ± 3.9% and 25.1 ± 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 106 cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4°C in UW at a density of 12.5 x 106 cells/ml for at least 24 h without significant decrease in functional integrity.
ABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.
The aim of this study was to establish the optimal conditions for the culture of equine hepatocytes in a monolayer configuration. The obtained results show that the rate of MTT metabolism correlated with the number of cultured cells and a linear increase of MTT reduction rate was observed in cases when the cell density varied between 1.25 × 10⁴ to 5 × 10⁴ viable cell/well of 96-well plate. Hepatocytes reached the optimal cell attachment rate and MTT reduction at a cell density of 5 × 10⁴ cells/well. The number of attached cells to a plastic culture dish was also related to incubation time. The greatest ability of hepatocytes to attach to the culture dish was observed after 10 h of incubation and it was found to be 84.1 ± 2.5% of seeded hepatocytes. It was also found that fetal bovine serum was more efficient than horse serum for the attachment of equine isolated hepatocytes in a monolayer culture. The highest rate of cell attachment (assessed microscopically and with MTT reduction test) was observed when cells were plated with the culture medium supplemented with FBS or HS at a concentration of 5%. However, medium supplementation with higher than 5% serum concentration (10% of FBS or HS) significantly decreased MTT reduction rate. The rate of MTT metabolism and cell attachment in hepatocytes cultured in WE supplemented with FBS or HS was also dependent on the plating time and were the highest after 10 h of seeding.
The examinations were conducted on hepatocvtes isolated by means of enzymatic method from the liver of three-month-old Wistar rats. The cells were incubated in medium with addition of theophylline and/or N-acetylcysteine. Significant changes in the activity of SOD, GPx, and GR in hepatocvtes incubated in the presence of the compounds in comparison with control cells demonstrated that theophylline and/or N-acetylcysteine disturb oxidative-reductive homeostasis of the cells. Changes in concentrations of ketone bodies, resulting in disturbances of acetoacetate to ß-hydroxybutyrate molar ratio, point to an unfavourable interference of theophylline into ketogenesis, which is equivalent with the disturbance of the balance between NAD⁺ and NADH+H⁺ in hepatocvtes. N-acetylcysteine simultaneously present with theophylline in incubation medium exerted a protective action on ketogenesis.