• The Korean Journal of Physiology and Pharmacology
• /
• v.4 no.2
• /
• pp.121-127
• /
• 2000

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver diseases. The objective of this study was to determine the role of lipid peroxidation and glutathione on the bile acid-induced hepatic cell death mechanism in primary cultured rat hepatocytes. To induce hepatic cell death, we incubated primary cultured rat hepatocytes with glycochenodeoxycholic acid $(GCDC;\;0{\sim}400\;{\mu}M)$ for 3 hours. In electron microscopic examination and agarose gel electrophoresis, low concentration of GCDC treatment mainly induced apoptotic feature. Whereas $400\;{\mu}M$ GCDC treated cells demonstrated both apoptosis and necrosis. Lipid peroxidation was increased dose-dependently in GCDC treated hepatocyte. And this was also accompanied by decreased glutathione. Therefore, oxygen free radical damage may play a partial role in GCDC-induced hepatic cell death.

• Korean Journal of Veterinary Service
• /
• v.31 no.4
• /
• pp.457-463
• /
• 2008

The sex steroid hormone estradiol-$17{\beta}(E_2)$ mediate their biological effects on development, differentiation and maintenance of reproductive tract and other target tissue through gene regulation by nuclear steroid receptors. Although the importance of $E_2$ in many physiological process has been reported, but little is known about the effects of $E_2$ on primary cultured chicken hepatocyte. therefore, in the present study, we have examined the effect of $E_2$ on cell proliferation and it's related signal cascades. $E_2$ increase $[^3H]$-thymidine incorporation in time-(${\leq}8hr$) and dose-($10^{-10}M$)dependent manner and treatment of $E_2$ increased the phosphorylation of p44/43 MAPKs(p44/42 mitogen-activated protein kinase) and JNK(c-Jun N-terminal kinase) in a time dependent manner. In addition, PD98059(p44/42 blocker, $10^{-5}M$), SP600125(JNK blocker, $10^{-6}M$) blocked the estrogen-induced increase in $[^3H]$-thymidine incorporation. In conclusion, $E_2$ stimulates the proliferation of primary cultured chicken hepatocytes and this action is mediated by p44/42 MAPKs and JNK signal transduction pathway.

• YAKHAK HOEJI
• /
• v.50 no.6
• /
• pp.358-366
• /
• 2006

The aim of this study was to investigate the protective effects of glycynhizin, active glycosides of Glycyrrhizae Radix, and baicalin, bioactive flavonoid isolated from Scutellariae Radix, on hepatocyte injury induced by carbon tetrachloride(CCl$_4$, 10 mM), tert-butyl hydroperoxide (TBH, 0.5 mM), and D-galactosamine (GaIN, 30 mM). Primary cultures of rat hepatocyte (18 hr cultured) were treated with CCl$_4$, TBH, or GaIN and various concentrations (0.1, 1, 10, and 100 ${\mu}$M) of glycyrrhizin or baicalin. Activity was accessed by determining the release of lactate dehydrogenase (LDH) and aminotransferses. CCl$_4$ significantly increased the levels of LDH, alanine aminotransferase (ALT), and aspartate aminotransferase(AST) and these increases were prevented by baicalin concentrations of 0.1,1, and 100 ${\mu}$M. The increases in ALT and AST levels were reduced by glycyrrhizin concentration of 100 ${\mu}$M. The level of LDH was markedly increased by TBH, and this increase was reduced by both glycyrrhizin and baicalin. ALT and AST levels were increased by TBH, which were prevented by glycynhizin and bacalin, respectively: GaIN markedly increased the levels of LDH, ALT and AST These increases was significantly reduced by both glycyrrhizin and baicalin. These results suggest that glycynhizin and baicalin possess the hepatoprotective activity.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.3 no.2
• /
• pp.96-102
• /
• 1998

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within tow days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitase the development of a bioartificial liver support device.

• Toxicological Research
• /
• v.8 no.2
• /
• pp.285-302
• /
• 1992

Human and rat hepatocytes were isolated by nonperfusion method and cultured for longer than 5 days. Human liver biopsy sample and rat liver were used as hepatocyte source. Several physical and chemical factors which were influencing on hepatocyte isolation procedure were examined and a batch isolation procedure was established for small size sample of rat liver. Isolated hepatocytes showed normal morphlologica characteristics in microscopy and electron microscopical examinations and a morphologica response to phalloidin. Isolated cells were cultured as a monolayer and proven to have intact morphological characteristics for longer than 15 days. Because human liver sample is harder and tighter compared with rat liver, a standard procedure for rat hepatocytes was slightly modified to reduce mechanical damage. Similarly with rat hepatocytes, isolated human hepatocytes showed a normal morphological characteristics and could be cultured for longer than 15days. Human and rat hepatocytes were examined on their functional integrities including cytochrome-P450 related enzyme activity and it's inducibility, hormonal inducibility of AIB uptake and TAT activity, albumin synthesis, DNA synthesis, cellular protein maintenance. In all parameters used in the present study, human and rat hepatocytes showed normal functional characteristics.

• Animal cells and systems
• /
• v.15 no.1
• /
• pp.19-27
• /
• 2011

Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

• Archives of Pharmacal Research
• /
• v.21 no.6
• /
• pp.718-722
• /
• 1998

Scopoletin (7-hydroxy-6-methoxycoumarin), a coumarin, was isolated from the aerial part of Solanum lyratum Thunb. by the activity-guided fractionation employing carbon te trachloride-intoxicated primary cultured rat hepatocytes as a screening system. Its hepatoprotective activity was first evaluated by measuring the release of glutamic pyruvic transaminase and sorbitol dehydrogenase from carbon tetrachloride-intoxicated rat hepatocytes into the culture medium. Scopoletin significantly reduced the releases of glutamic pyruvic transaminase and sorbitol dehydrogenase from the carbon tetrachloride-intoxicated primary cultured rat hepatocytes by 53% and 58%, respectively, from the toxicity in a dose-dependent manner over concentration ranges of 1mcM to 50mcM. Further studies revealed that at the concentration of 10mcM, scopoletin significantly preserved glutathione content by 50% and the activity of superoxide dismutase by 36% and also inhibited the production of malondialdehyde to the degree as seen in the control.

• Microbiology and Biotechnology Letters
• /
• v.28 no.4
• /
• pp.209-213
• /
• 2000

The cultured mycelia of Ganoderma lucidum were extracted and the extract was separated into six fractions by organic solvent fractionation. The antihepatotoxic activity of all the fractions was evaluated by measuring activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT). Among the fractions tested, the high-polarity fractions such as aqueous and n-butanol fractions significantly reduced activities of GPT and GOT in CCl4- and galactosamine-intoxicated rat primary hepatocytes. When intracellular synthetic activities were measured by pulsing the rate primary cultured hepatocytes with [3H]-uridine and [3H]-leucine, activities of DNA, RNA and protein. When direct toxicities of the fractions were measured against human hepatoma(SK-Hep-1), the non-polarity fractions such as n-butanol and ethyl acetate fractions showed potent direct cytotoxicities even at the concentration of 1 $\mu\textrm{g}$/ml. These data showed that Ganoderma lucidum has hepatoprotective and hepatotoxic recovery principles in its mycelia.

• The Korean Journal of Physiology and Pharmacology
• /
• v.3 no.6
• /
• pp.565-570
• /
• 1999

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver injury. Due to the detergent-like effect of the hydrophobic bile acids, hepatocellular injury has been attributed to direct membrane damage. However histological findings of cholestatic liver diseases suggest apoptosis can be a mechanism of cell death during cholestatic liver diseases instead of necrosis. To determine the pattern of hepatocellular toxicity induced by bile acid, we incubated primary cultured rat hepatocytes with a hydrophobic bile acid, Glycochenodeoxycholate (GCDC), up to 5 hours. After 5 hours incubation with $400\;{\mu}M$ GCDC, lactate dehydrogenase released significantly. Cell viability, quantitated in propidium iodide stained cells concomitant with fluoresceindiacetate was decreased time- and dose-dependently. Most nuclei with condensed chromatin and shrunk cytoplasm were heavily labelled time- and dose-dependently by a positive TUNEL reaction. These findings suggest that both apoptosis and necrosis are involved in hepatocytes injury caused by GCDC.

• YAKHAK HOEJI
• /
• v.51 no.2
• /
• pp.115-125
• /
• 2007

The aim of this study was to investigate the protective activities of daidzin, daidzein, genistein or puerarin, active isoflavonoids of Puerariae Radix, on the hepatocyte injury induced by carbon tetrachloride (CCl$_4$, 10 mM), tert-butyl hydroperoxide (TBH, 0.5 mM) and D-galactosamine (GalN, 30 mM). Primary cultures of rat hepatocytes (18 hr cultured) were treated with CCl$_4$, TBH or GalN and various concentrations (0.1, 1, 10 and 100 ${\mu}$M) of daidzin, daidzein, genistein or puerarin. CCl$_4$ significantly increased the levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The increase in LDH level was attenuated by daidzein, genistein and puerarin. Puerarin also inhibited the increase in AST level induced by CCl$_4$. The increases in LDH and ALT levels induced by TBH were significantly attenuated by daidzin and genistein treatments. GalN markedly increased the levels of LDH, ALT and AST These increases were significantly attenuated by daidzein. Daidzin also inhibited the increases in LDH and AST levels induced by GalN. The increases in LDH and ALT levels were attenuated by genistein and puerarin, respectively. These results suggest that daidzin and daidzein possess hepatoprotective activities.