PURPOSE: In prior study, we synthesized 99mTc-galactosylated chitosan (GC) and performed in vivo biodistribution study, showed specific targeting to hepatocyte. The aim of this study is to evaluate the labeling efficiency and cytotoxicity of modified galactosylated chitosan compounds, galactosyl methylated chitosan (GMC) and HYNIC-galactosylated chitosan (GCH). MATERIALS AND METHODS: GC, GMC and GCH were synthesized and radiolabeled with 99mTc. Then, they were incubated for 6 hours at room temperature and human serum at 37 degrees C. Labeling efficiencies were determined at 15, 30 m, 1, 2, 3 and 6 h after radiolabeling. To evaluate cytotoxicity, MTT assay was performed in HeLa and HepG2 cells. RESULTS: In comparison with them of 99mTc-GC, labeling efficiencies of 99mTc-GMC were significantly improved (100, 97 and 89% in acetone and 96.3, 95.8 and 75.6% in saline at 15 m, 1 and 6 h, respectively). Moreover, 99mTc-GCH showed more improved labeling efficiencies (> 95% in acetone and human serum and > 90% in saline at 6 h). In MTT assay, cytotoxicity was very low and not different from that of controls. CONCLUSION: These results represent that these compounds are radiochemically compatible radiopharmaceuticals, can be used in hepatocyte specific imaging study and in vivo gene or drug delivery monitoring.
Acetone ; Chitosan* ; Hep G2 Cells ; Hepatocytes ; Humans ; Radiopharmaceuticals

No abstract available.
Hepatocytes*

OBJECTIVETo determine the effect of intermittent hypoxia on lipid metabolism in liver cells and to explore the possible molecular pathways involved in this process.

METHODSAn intermittent hypoxia cell model system was established by incubating the human hepatic cell lines L02 and HepG2 in an atmosphere of 2% O₂, 5% CO₂ and 93% N₂ for 8 hours per day over a period of 1, 2, 3, 4 or 5 days. Cells cultured in normoxia conditions (21% O₂) served as controls. Changes in intracellular lipid droplets and triglyceride (TG) levels were assessed by biochemical assays and oil red staining. Changes in intracellular reactive oxygen species (ROS) were assessed by inverted fluorescence microscopy and flow cytometry. Changes in expression of hypoxia-inducible factor (HIF)-1a and HIF-2a proteins, and the downstream ADFP, SREBP-1c and FAS proteins, were detected by western blotting.

RESULTSFor both L02 and HepG2 cell lines, the cells grown under hypoxic conditions showed significantly higher lipid droplet accumulation and TG content than the cells grown under normoxic conditions (F(L02) =61.83, FHepG2 =104.19, P less than 0.01). Both oxygen concentration and time appeared to be correlated with these lipid-related changes (F(L02) =39.60, FHepG2 =76.39, P less than 0.01). The ROS fluorescence index was significantly increased after 2 days of intermittent hypoxia L02: 0.703 ± 0.129 vs. 3.310 ± 0.198, t =22.0637 and HepG2:0.617 ± 0.156 vs. 2.33 ± 0.42, t =7.2003, P < 0.05); in addition, increasing trends were observed in the ROS content and intensity of green fluorescence in conjunction with increased time of exposure to intermittent hypoxia (F(L02) =1021.84, FHepG2 =49.89, P less than 0.01). Compared with their respective control groups, the L02 and HepG2 cells both showed significantly upregulated expression of HIF-1a ADFP, SREBP-1c and FAS (L02:FHIF-1a =371.19, FsREBP-1c =204.49, FFAS =38.20, FADFP =154.31, P less than 0.05 and HepG2:FHF-1a =150.84, FSRERBP-1c =107.35, FFAs =279.71, FADFP =352.06, P less than 0.01). In contrast, the HIF-2a level was markedly decreased in the cells after 4 and 5 days of exposure to intermittent hypoxia (F(L02) =125.29, FHcpG2 =10.68, P less than 0.05).

CONCLUSIONUnder intermittent hypoxic conditions, ROS may regulate the expression of hypoxia-inducible factors and the adipose differentiation-related protein,as well as influence fatty acid metabolism via a HIF-1 a-SREBP-1 c-FAS signal and upregulation of the ADFP protein, in liver cells.

Cell Hypoxia ; Cell Line ; Hep G2 Cells ; Hepatocytes ; metabolism ; Humans ; Lipid Metabolism ; Reactive Oxygen Species ; metabolism

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

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.
Animals ; Calcium ; Cytosol ; Fluorescence ; Hep G2 Cells ; Hepatocytes ; Humans ; In Vitro Techniques ; Lipid Peroxidation ; Liver ; Membrane Potential, Mitochondrial

OBJECTIVETo study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines.

METHODSGenomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing.

RESULTSIt was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05).

CONCLUSIONPEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.

Carcinoma, Hepatocellular ; genetics ; Gene Expression ; Genomic Imprinting ; Hep G2 Cells ; Hepatocytes ; metabolism ; Humans ; Liver Neoplasms ; genetics ; Proteins ; genetics

OBJECTIVE: To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937. METHODS: We cultured the liver cell lines HepG2 and immune cell lines U937, and stimulated them with HMGB1 (400 ng/mL) for 20 h. Then the supernatant was collected. Ultrafiltration centrifugation, CM-Sepharose cation, DEAE-Sepharose anion exchange chromatography, Sephadex G75-gel filtration chromatography, and immunoprecipitation were used for purification. The molecular weight and identity of HMGB1 was confirmed by SDS-PAGE and Western blot. RESULTS: A sharp stained protein band with a molecular weight of about 26 kD was obtained by SDS-PAGE analysis and shown to be HMGB1 confirmed by Western blot. CONCLUSION High purified HMGB1 can be separated from these two cell lines.
Cell Culture Techniques ; Electrophoresis, Polyacrylamide Gel ; methods ; HMGB1 Protein ; isolation & purification ; metabolism ; Hep G2 Cells ; Hepatocytes ; metabolism ; Humans ; Monocytes ; metabolism ; U937 Cells

Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH.
AMP-Activated Protein Kinases ; Animals ; Cytokines ; Fatty Liver* ; Gene Expression ; Glucose ; Hep G2 Cells ; Hepatocytes ; In Vitro Techniques ; Inflammation* ; Lipid Metabolism ; Lipogenesis* ; Lipoproteins ; Liver ; Macrophages ; Metabolism ; Mice ; Palmitic Acid

This study was aimed to investigate the effect of rhIL-6 and rhEPO on hepcidin mRNA expression in HepG2 cells and human primary hepatocytes, and mechanism of rhEPO in treatment of anemia of chronic disease (ACD). The HepG2 cells and human primary hepatocytes were cultured with medium containing different concentrations of rhIL-6 and rhEPO for a certain time, then mRNA was isolated and its RT-PCR was performed, the bands were photographed and analyzed by UVI band, the hepcidin and G3PDH mRNA ratio were semi-quantitatively analyzed. The expression levels of hepcidin in GepG2 cells and human primary hepatocytes at different conditions were compared. The results showed that the hepcidin mRNA expression in HepG2 cells and human primary hepatocytes could be enhanced by rhIL-6, the rhEPO could inhibit rhIL6-induced hepcidin mRAN expression. The rhEPO alone basically did not influence hepcidin mRNA expression in HepG2 cells. It is concluded that Hepcidin mRNA expression in HepG2 cells and human primary hepatocytes can be elevated by rhIL-6 with concentration- and time-dependent manner in certain range. rhEPO can inhibit this effect of rhIL-6.
Antimicrobial Cationic Peptides ; genetics ; metabolism ; Erythropoietin ; pharmacology ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Hepcidins ; Humans ; Interleukin-6 ; pharmacology ; RNA, Messenger ; genetics ; Recombinant Proteins ; pharmacology

OBJECTIVETo study the influence of hepatitis B virus (HBV) replication and expressions of different viral genes on CDC37 level in hepatocytes.

METHODSWe amplified and cloned 6 HBV genes (P, preS1, preS2, S, C and X) into pCMV expression vectors, which were transfected in Huh7 and HepG2 hepatoma cell lines, and CDC37 expression level in the cells was detected using Western blotting. Wealso cloned the promoter sequence of CDC37 into pGL3 vector, and co-transfected pGL3 with pCMV recombinant plasmids into Huh7 and HepG2 cells and the fluorescent signals were detected. To study the influence of HBV replication on CDC37 expression, we constructed 1.28-copy overlength genomes of HBV genotypes B, C, D and CD recombinant. The overlength HBV genomes were transformed into Adeasier-1 cells for recombination and into 293 cells for packaging. Huh7 and HepG2 cell lines infected with the packaged HBV recombinant adenoviruses were examined for CDC37 expression with Western blotting.

RESULTSWestern blotting showed that the expression of different HBV genes did not obviously affect the protein level of CDC37 in the hepatocytes. The protein expression of HBV genes had no effect on the activity of CDC37 promoter. Huh7 and HepG2 cells infected with 1.28-copy HBV replicon showed no significant changes in the expression level of CDC37.

CONCLUSIONHBV replication and its gene expression have no effect on the level of CDC37 in hepatocytes in vitro.

Adenoviridae ; Cell Cycle Proteins ; metabolism ; Chaperonins ; metabolism ; Gene Expression Regulation, Viral ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; virology ; Humans ; Transfection ; Virus Replication