Induction of Heme Oxygenase-1 in Human Hepatocytes to Protect Them From Ethanol-induced Cytotoxicity

Background/Aim We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.

Effects of plasma from patients with acute on chronic liver failure on function of cytochrome P450 in immortalized human hepatocytes

BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability. METHODS: Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay. RESULTS: After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable. CONCLUSIONS: Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver. (Hepatobiliary Pancreat Dis Int 2010; 9:611-614)

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Learning and memory changes in rats following exogenous human hepatocyte growth factor gene injection into cerebral ischemic penumbra

Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.

EFFECTS OF SALVIA MILTIORRHIZA BUNGE（SMB）ON LIPID PEROXIDATION AND PLASMA MEMBRANE FLUIDITY OF CULTU

In order to investigate the effect of salvia miltiorrhiza hunge(SMB)on the plasma membrane fluidity and the relationship between the lipid peroxidation and the Plasma membrane fluidityin cultured human fetdal hepatocytes,the plasma membrane fluidity,using 1,6-dipheny-1,3,5-hexatriene(DPH)as a fluorescence probe, malondialdehyde(MDA)production as well as alanine aminotransferase(ALT)release of human fetal hepatocytes cultured in Presence of carbon tetrachloride(CCl4)or SMB puls CCl4 were estimated. In the cultured hepatocytes injured by CCl4,significant increments of the MDA production and the ALT release,and significant decrease in the plasma membrane fluidity were observed.when the culture medium was supplied with SMB prior to the additionof CCl4,the CCl4 induced increments in MDA production and ALT release was suppressed signifi cantly and a concomitant raise of plasma membrane fluidity towards normal occurred.The resultssuggested that SMB could suppress the lipid peroxidation in bepatocytes,thereby normal membranefluidity might be retained.