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*

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

BACKGROUND/OBJECTIVE: Although Angelica keiskei (AK) has widely been utilized for the purpose of general health improvement among Asian, its functionality and mechanism of action. The aim of this study was to determine the protective effect of ethanol extract of AK (AK-Ex) on acute hepatotoxicity induced by acetaminophen (AAP) in HepG2 human hepatocellular liver carcinoma cells and HepaRG human hepatic progenitor cells. MATERIALS/METHODS: AK-Ex was prepared HepG2 and HepaRG cells were cultured with various concentrations and 30 mM AAP. The protective effects of AK-Ex against AAP-induced hepatotoxicity in HepG2 and HepaRG cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, lactate dehydrogenase (LDH) assay, flow cytometry, and Western blotting. RESULTS: AK-Ex, when administered prior to AAP, increased cell growth and decreased leakage of LDH in a dose-dependent manner in HepG2 and HepaRG cells against AAP-induced hepatotoxicity. AK-Ex increased the level of Bcl-2 and decreased the levels of Bax, Bok and Bik decreased the permeability of the mitochondrial membrane in HepG2 cells intoxicated with AAP. AK-Ex decreased the cleavage of poly (ADP-ribose) polymerase (PARP) and the activation of caspase-9, -7, and -3. CONCLUSIONS: These results demonstrate that AK-Ex downregulates apoptosis via intrinsic and extrinsic pathways against AAP-induced hepatotoxicity. We suggest that AK could be a useful preventive agent against AAP-induced apoptosis in hepatocytes.
Acetaminophen ; Angelica* ; Apoptosis ; Asian Continental Ancestry Group ; Blotting, Western ; Caspase 9 ; Ethanol* ; Flow Cytometry ; Functional Food ; Hep G2 Cells ; Hepatocytes ; Humans ; L-Lactate Dehydrogenase ; Liver ; Mitochondrial Membranes ; Permeability ; Stem Cells

BACKGROUND/OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease and is closely associated with metabolic syndrome. In the present study, we observed the effect of ethanol extract of Allium fistulosum (EAF) on NAFLD and have suggested the possibility of using EAF as a natural product for application in the development of a treatment for NAFLD. MATERIALS/METHODS: The preventive effect on hepatic lipid accumulation was estimated by using an oleic acid (OA)-induced NAFLD model in vitro and a Western diet (high-fat high-sucrose; WD)-induced obese mouse model. Animals were divided into three groups (n = 7): normal diet group (ND), WD group, and WD plus 1% EAF group. RESULTS: EAF reduced OA-stimulated lipid accumulation in HepG2 cells in the absence of cellular cytotoxicity and significantly blocked transcriptional activation of sterol regulatory element-binding protein 1 and fatty acid synthase genes. Subsequently, we investigated these effects in vivo in mice fed either ND or WD in the presence or absence of EAF supplementation. In comparison to the ND controls, the WD-fed mice exhibited increases in body weight, liver weight, epididymal fat weight, and accumulation of fat in hepatocytes, and these effects were significantly attenuated by EAF supplementation. CONCLUSIONS: Allium fistulosum attenuates the development of NAFLD, and EAF elicits anti-lipogenic activity in liver. Therefore, EAF represents a promising candidate for use in the development of novel therapeutic drugs or drug combinations for the prevention and treatment of NAFLD.
Allium* ; Animals ; Body Weight ; Diet ; Diet, Western ; Drug Combinations ; Ethanol* ; Hep G2 Cells ; Hepatocytes ; In Vitro Techniques ; Lipogenesis ; Liver ; Liver Diseases ; Mice ; Mice, Obese ; Non-alcoholic Fatty Liver Disease* ; Oleic Acid ; Sterol Regulatory Element Binding Protein 1 ; Transcriptional Activation

We investigated whether the combination of phytochemicals and acetic acid in the form of fruit vinegar provides an additive effect on changes of mRNA levels related to fatty acid oxidation in human hepatocyte (HepG2). Among the seven fruit vinegars (Rubuscoreanus, Opuntia, blueberry, cherry, red ginseng, mulberry, and pomegranate) studied, treatment of HepG2 with pomegranate vinegar (PV) at concentrations containing 1 mM acetic acid showed the highest in vitro potentiating effect on the mRNA expression levels of peroxisome proliferator-activated receptor alpha, carnitinepalmitoyl transferase-1, and acyl-CoA oxidase compared to the control group (P < 0.05). Reversed-phase liquid chromatography in combination with quadrupole time-of-flight mass spectrometry analysis revealed four potential compounds (punicalagin B, ellagic acid, and two unidentified compounds) responsible for altered gene expression in HepG2 cells treated with PV as compared with the others. Further investigations are warranted to determine if drinking PV beverages may help to maintain a healthy body weight in overweight subjects.
Acetic Acid ; Acyl-CoA Oxidase ; Beverages ; Blueberry Plant ; Body Weight ; Chromatography, Reverse-Phase ; Drinking ; Ellagic Acid ; Fruit ; Gene Expression ; Hep G2 Cells ; Hepatocytes ; Humans ; Mass Spectrometry ; Morus ; Opuntia ; Overweight ; Panax ; PPAR alpha ; Prunus ; Punicaceae ; RNA, Messenger

Background/Airns: Plasma membrane fatty acid binding protein (FABPpm), a long chain fatty acid (FFA) transporter, is reported to be identical with mitochondrial (m) AST. Alcoholic liver disease (ALD) is characterized by increased plasma level of AST>ALT. In ALD, the elevated AST consists mainly of m-AST which are originated from the leakage of the morphologically abnormal mitochondria. However, increase in plasma AST is often found without mitochondrial injury. Moreover, since m-AST leaking from mitochondria first enters the cytoplasm, it is unclear why m-AST should preferentially escape from the cytoplasm into the circulation. METHODS: We established four long term HepG2 cell cultures in 0, 20, 40 or 80 mM ethanol. In these cultures, plasma membrane expression of FABPpm was assessed by immunofluorescence, Western blotting of plasma membrane extracts: m-AST mRNA by slot blotting/scanning densitornetry; AST leakage into the medium enzymatically; and mitochondrial morphology by EM. The FFA uptake kinetics were quantified with [3H]-oleate. RESULTS: HepG2 cells at all ethanol levels displayed FABPpm on the plasma membrane, with a progressive, ethanol related increase. This paralleled an increase in m-AST mRNA. Leakage of total AST into the culture medium per 24 hours was similar in cells cultured in 0 and 20 mM ethanol but the amount of leakage was increased 6 to 14 times in cells cultured at 40 and 80 mM ethanol. No gross alteration was studied. The Vmax for oleate uptake was closely correlated with m-AST mRNA level (p<0.01). CONCLUSIONS: Ethanol upregulated with m-AST synthesis and the m-AST sorting to the plasma membrane. Increased plasma m-AST in ALD may not be derived from mitochondria, but from the plasma membrane FABPpm. Increased plasma membrane FABPpm mediates increased FFA uptake in hepatocyte, which may be a contributing factor to alcoholic fatty liver.
Blotting, Western ; Cell Membrane ; Cytoplasm ; Ethanol* ; Fatty Acid-Binding Proteins ; Fatty Liver, Alcoholic ; Fluorescent Antibody Technique ; Hep G2 Cells* ; Hepatocytes ; Kinetics ; Liver Diseases, Alcoholic ; Mitochondria ; Oleic Acid ; Plasma ; RNA, Messenger ; United Nations

BACKGROUND AND OBJECTIVES: Excretion of bile acid and free cholesterol of bile was important to maintain cholesterol homeostasis. ATP-binding cassette transporter G5 (ABCG5) and G8 (ABCG8) promoted biliary cholesterol excretion. In previous study, hepatic secretion of cholesterol and ABCG5/G8 expression are strongly stimulated in hypophysectomized rats during treatment with thyroid hormone. In this study, we aimed to evaluate the effect of thyroid hormone to expression of ABCG5 and G8 in mouse liver. MATERIALS AND METHODS: We administered thyroid hormone (T3) to C57BL/6 mice and then RNA and protein was isolated from liver. We isolated primary hepatocyte and administered T3 to evaluate in vitro effect. HepG2 cells were cotransfected with either a control plasmid or expression plasmids for human thyroid hormone receptor (hTR)beta/human retinoid X receptor (hRXR)alpha or human liver X receptor (hLXR)alpha in combination with reporter plasmids TK-LXRE3-LUC with or without T3. RESULTS: Serum total cholesterol was decreased after 5 days of T3 treatment. Expression of ABCG5/8 mRNA and ABCG5 protein was increased after T3 treatment. In primary hepatocytes, T3 also increased ABCG5/8 mRNA expression. LXRalpha mRNA was not increased by T3. However, when we cotransfected liver X receptor response element (LXRE) construct and TRbeta/RXRalpha with T3, the activity of LXRE containing construct was markedly increased. CONCLUSION: We confirmed that thyroid hormone increased expression of ABCG5/8. This result suggested that thyroid hormone played an important role in decreasing serum cholesterol through bile excretion.
Animals ; Bile ; Cholesterol ; Hep G2 Cells ; Hepatocytes ; Homeostasis ; Humans ; Liver ; Mice ; Orphan Nuclear Receptors ; Plasmids ; Rats ; Receptors, Thyroid Hormone ; Response Elements ; Retinoid X Receptors ; RNA ; RNA, Messenger ; Thyroid Gland ; Thyroid Hormones

No abstract available.
Animals ; Hepatocytes* ; Organelles* ; Rats*

No abstract available.
Hemodynamics* ; Hepatocytes* ; Perfusion*