Calcium ; metabolism ; Hepatocytes ; metabolism ; Homeostasis ; Humans

Animals ; Hepatocytes ; metabolism ; Humans ; Lipoproteins ; metabolism ; Protein Binding ; Proteins ; metabolism

Hepatocytes ; metabolism ; pathology ; Liver Diseases ; genetics ; metabolism ; pathology

Aquaporin-9 (AQP9) is a membrane-span transport protein expressed in the liver. It is located in the cytoplasm membrane of hepatic cells. In addition to water, it is also permeable to glycerol, urea, and other small solutes. Several evidences have revealed that AQP9 is involved in multiple physiological and pathological process of the liver. This paper summarized the expression of AQP9 in the liver and the effect on the physiological and pathological processes of the liver. AQP9 may be defined as a novel target for diagnosis and treatment of hepatic diseases.
Aquaporins ; metabolism ; Cell Membrane ; metabolism ; Glycerol ; Hepatocytes ; metabolism ; Humans ; Liver ; metabolism ; Urea

Carcinoma, Hepatocellular ; metabolism ; pathology ; Hepatocytes ; metabolism ; pathology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; NF-kappa B ; metabolism

Primary human hepatocytes (PHH) are the gold standard of in vitro human liver model for drug screening. However, a problem of culturing PHH in vitro is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) "sandwich" co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×10⁶ /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D in vitro liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.
Albumins/metabolism* ; Animals ; Cytochrome P-450 Enzyme System/metabolism* ; Hepatocytes/metabolism* ; Humans ; Liver ; Mice ; Urea/metabolism*

OBJECTIVETo investigate the effect of N,N-dimethylformamide (DMF) on calcium homeostasis and calpain I and II gene expression in human hepatocytes (HL-7702).

METHODSHL-7702 cells were exposed to different concentrations of DMF (10, 25, 50, 100, or 200 mmol/L); other HL-7702 cells, which were used as a control group, were exposed to the equal volume of DMEM; the intracellular Ca(2+) concentration was monitored using the calcium fluorescent probe (fluo-3/AM). After 24-h exposure to DMF (10, 25, 50, 100, 150, or 200 mmol/L), the morphology of hepatocytes was observed under an inverted phase contrast microscope, and the cell viability was measured by MTT assay. After 24-h exposure to DMF (10, 25, 50, 100, or 150 mmol/L), the mRNA expression levels of calpain I and II in hepatocytes were measured by real-time quantitative PCR.

RESULTSThere were significant differences in cell viability among different exposure groups (P < 0.01); the 50, 100, 150, and 200 mmol/L DMF exposure groups had a significantly lower cell viability than the control group (P < 0.05). Under the inverted phase contrast microscope, HL-7702 cells gradually lost the original shape, with swelling and shrinking, as the dose of DMF increased, and those treated with 150 mmol/L DMF even became round and floated. The fluorescence density of fluo-3 in hepatocytes increased as the dose of DMF rose, demonstrating a dose-response relationship, and there were significant differences among these exposure groups (P < 0.05). There were significant differences in mRNA expression levels of calpain I and II among these exposure groups (P < 0.01), and the expression increased as the dose of DMF rose; but DMF did not promote the mRNA expression of calpain I at a concentration of 150 mmol/L.

CONCLUSIONDMF can cause damage to hepatocytes, which is related to intracellular calcium increase and calpain mRNA increase.

Calcium ; metabolism ; Calpain ; metabolism ; Cell Line ; Dimethylformamide ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Humans

OBJECTIVETo investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).

METHODSHepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting.

RESULTSThe ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment.

CONCLUSIONEthanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Calcium ; metabolism ; Calcium Channels ; metabolism ; Ethanol ; adverse effects ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans

OBJECTIVETo investigate the effects of farnesoid X receptor (FXR) on lipid metabolism in human hepatic L02 cells.

METHODSA steatosis model and an intervention model were established by treating human hepatocyte line L02 cells with sodium oleate or sodium oleate and sodium chenodeoxycholate (a natural agonist of FXR) respectively. Non-treated L02 cells served as controls. At three time points of 24, 48 and 72 hours, the accumulation of lipid droplets in the hepatocytes was observed by optical microscopy after oil red O staining, and the the expression of FXR and SREBP-1c receptors was detected by RT-PCR and Western blot.

RESULTSCompared with the controls, expressions of FXR mRNA and protein were down-regulated gradually in the steatosis model at 24, 48 and 72 hours, FXR mRNA/beta-actin mRNA was 0.186+/-0.02, 0.182+/-0.028 and 0.181+/-0.022, FXR protein/beta-tubulin protein was 0.105+/-0.016, 0.103+/-0.012 and 0.103+/-0.018, F from 0.01 to 0.14; 24 h vs 48 h, 48 vs72 h: P more than 0.05. The expressions of SREBP-1c mRNA and protein were increased gradually. At 24, 48 and 72 hours, SREBP-1c mRNA/beta-actin mRNA was 0.495+/-0.062, 0.579+/-0.064 and 0.612+/-0.067, SREBP-1c protein/beta-tubulin protein was 0.394+/-0.044, 0.488+/-0.066 and 0.543+/-0.064, F from 0.80 to 4.66, 24 h vs 48 h, 48 vs 72 h: P less than 0.05. In the intervention model, expressions of FXR mRNA and protein were increased markedly compared with the steatosis model. At 24, 48 and 72 hours, FXR mRNA/beta-actin mRNA was 0.253+/-0.041, 0.298+/-0.042 and 0.334+/-0.051, and FXR protein/beta-tubulin protein was 0.221+/-0.022, 0.313+/-0.041 and 0.341+/-0.046, F from 6.41 to 50.93, intervention models vs steatosis models at the same time points: P less than 0.05-0.01. Expressions of SREBP-1 c mRNA and protein were significantly reduced. At 24, 48 and 72 hours, SREBP-1c mRNA/beta-actin mRNA was 0.296+/-0.038, 0.328+/-0.037 and 0.341+/-0.055, and FXR protein /beta-tubulin protein was 0.295+/-0.038, 0.334+/-0.047 and 0.355+/-0.054, F from 8.84 to 48.46; intervention models vs steatosis models at the same time point: P less than 0.01. Both in the steatosis model and the intervention model, content of TG and lipids accumulations were much more than those in the controls. Compared with the intervention model, levels of TG and lipids accumulation were markedly increased in the steatosis model at 24, 48, 72 hours. At 24, 48 and 72 hours, TG/cellular total protein in microg/mg was 173.0+/-20.5, 253.4+/-36.1 and 361.2+/-50.7 in the steatosis model, while in the intervention model the data was 84.1+/-17.2, 113.0+/-14.5 and 127.2+/-20.1, F from 38.70 to 268.13, intervention models vs steatosis models at the same time point: P less than 0.01.

CONCLUSIONExpression of FXR is closely associated with lipid homeostasis in hepatocytes. Up-regulation of the expression of FXR may improve lipidosis in L02 cells. Its possible mechanism involves reduction of SREBP-1c expression and lipogenesis in hepatocytes.

Cell Line ; Fatty Liver ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Lipid Metabolism ; Receptors, Cytoplasmic and Nuclear ; Up-Regulation

OBJECTIVESTo study the dynamic change of hepatic oval cells (HOC) in the process of rat liver cirrhosis formation induced by dimethylnitrosamine (DMN), and to explore its pathophysiology significance.

METHODSA rat cirrhosis model was established by using DMN. Microscopical and electronmicroscopical changes of HOC were examined. Thy1.1 was detected by immunohistochemical method at different times. The ratio of HOC was checked using image pattern analysis and Western blot. The number of HOC was counted microscopically.

RESULTSAt the 4th week after DMN administration, the liver fibrosis was at its peak, with false lobules formation combined with large areas of hemorrhage and necrosis. The fibrosis started to minimize at the 6th week, and also the inflammatory changes at the 8th week. Thy1.1 positive stained cells dispersed at the 2nd week; increased at the 4th week around fiber septa; reached its peak at the 6th week, then decreased at the 8th week. The results of image pattern analysis, cell counting under light microscope and Western blot were constant, with the highest cell numbers at the 6th week, and dropped at the 8th week. The ultrastructure of HOC was characterized by their small sized, oval nuclei, and higher nucleus/plasma ratio.

CONCLUSIONDuring the formation and reduction of rat cirrhosis caused by DMN, Thy1.1 stained HOC showed notable dynamic change, which may play an important role in the cirrhotic process.

Animals ; Dimethylnitrosamine ; Hepatocytes ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Male ; Rats ; Rats, Wistar ; Thy-1 Antigens ; metabolism