OBJECTIVETo determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).

METHODSThirty-two male Sprague Dawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group), 8 (8 h group) 12 (12 h group) and 24 hours (24 h group) of treatment. Eight unmodeled rats served as controls. Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining. Ultrastructural changes in mitochondria were assessed by electron microscopy. The activity and expression of CS, CPT-1 and COX were measured.

RESULTSHepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group. Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group, with inflammatory cell invasion. Ultrastructural changes in mitochondria were present in the 4 h treatment group, and the 24 h treatment group showed mitochondria with completely destroyed outer membranes, which resulted in mitochondrial collapse. Activity and protein expression of CS, CPT-1 and COX were increased in the 4 h group (vs. controls), were at their peak in the 8 h group (CS:t =1.481, P less than 0.01; CPT-1:t =2.619, P less than 0.05; COX:t =1.014, P less than 0.01) and showed a decreasing trend in the 12 h group. In addition, the activities of CS, CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis, suggesting that these enzymes were involved in the initiation and development of ALF.

CONCLUSIONEnergy metabolism plays an important role in hepatocyte apoptosis during ALF.

Animals ; Apoptosis ; Carnitine O-Palmitoyltransferase ; metabolism ; Citrate (si)-Synthase ; metabolism ; Disease Models, Animal ; Electron Transport Complex IV ; metabolism ; Hepatocytes ; cytology ; enzymology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; Mitochondria ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.

METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.

RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.

CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism

OBJECTIVETo study the protective effect of Shaoganduogan (SGDG) on serum transaminase, liver pathology and hepatocyte mitochondria in rat with subacute liver injury induced by carbon tetrachloride.

METHODSubacute liver injury of rats were induced by carbon tetrachloride, and cured by different doses of SGDG through intragastric administration. The activity of serum ALT, AST, liver pathology and ultrastructure, activity of ATPase, SOD and content of MDA of hepatocyte mitochondria were observed.

RESULTSGDG can remarkably reduce the transaminase, alleviate the degeneration and necrosis of liver cells ,enhance activity of Na+ -K+ ATPase, Ca2+ ATPase, SOD, reduce content of MDA of mitochondria, alleviate ultrastructure change of mitochondria, reduce section area, perimeter equivalent diameter and average optical density perimeter of liver cells.

CONCLUSIONSGDG has obvious effect of liver protection, the mechanisms are related with alleviating mitochondria injury.

Animals ; Carbon Tetrachloride ; adverse effects ; Chemical and Drug Induced Liver Injury ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; drug effects ; pathology ; Male ; Malondialdehyde ; metabolism ; Mitochondria ; drug effects ; enzymology ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

Animals ; Female ; Ginsenosides ; pharmacology ; therapeutic use ; Hepatocytes ; drug effects ; metabolism ; ultrastructure ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Phytotherapy ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVETo investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSMononuclear cells were isolated from bone marrow of swine by density gradient centrifugation. MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized. Immunocytochemical analysis was performed to monitor the expression and distribution of ECM.

RESULTSThe purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function: including albumin secretion and urea synthesis (P < 0.01). The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture (P < 0.01). RNAi experiments verified that there was a correlation between ECM and hepatic functions.

CONCLUSIONECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.

Albumins ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Cell Separation ; methods ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Extracellular Matrix ; metabolism ; Female ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Swine ; Urea ; metabolism

OBJECTIVETo investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte.

METHODSThe oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by millIcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-4alpha, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit.

RESULT(1) The relative level of HNF-4alpha and albumin mRNA expression compared with pre-coculture: M-coculture: HNF-4a: 1.9+/-0.2, 10.7+/-1.2, 12.0+/-1.3; albumin: 5.7+/-1.6, 110.7+/-13.7, 173.6+/-22.3. S-coculture: 1.4+/-0.1, 3.2+/-0.6, 8.9+/-1.4 times; albumin: 2.9+/-1.4, 22.3+/-8.5, 96.3+/-16.3. The relative level of HNF-4a and albumin mRNA expression in coculture group (M- and S-coculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P less than 0.01); and a higher level of HNF-4a and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P less than 0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1+/-0.2, 0.2+/-0.0, 0.0+/-0.0; S-coculture group: AFP: 1.0+/-0.2, 0.2+/-0.1, 0.1+/-0.0; CK-19: 0.6+/-0.1, 0.1+/-0.0, 0.0+/-0.0; control group: AFP: 1.0+/-0.1, 1.0+/-0.1, 1.1+/-0.1, CK-19: 1.0+/-0.1, 1.1+/-0.1, 1.0+/-0.1. The relative level of AFP and CK-19 mRNA expression in coculture group (M- and S-coculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P less than 0.01). (2) The albumin secretion was detected in M-coculture: 14 day: (15.30+/-0.09) ng/ml, 21: (20.98+/-0.12) ng/ml; S-coculture: 14 day: (11.41+/-0.13) ng/ml, 21 day:(15.12+/-0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochondrion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared between oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups.

CONCLUSIONHSC can induce differentiation of oval cell into mature hepatocyte.

Albumins ; biosynthesis ; genetics ; Animals ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Hepatic Stellate Cells ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Liver ; cytology ; Male ; Microscopy, Confocal ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; metabolism ; ultrastructure ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo observe the in vivo colonization, migration, and differentiation of in vitro cultured human fetal hepatic stem cells (HSCs) following intrasplenic transplantation for treatment of acute liver injury in mice with severe combined immunodeficiency (SCID).

METHODSHuman fetal HSCs were isolated from the normal fetal liver (16-24 weeks) and purified, and the morphology of HSCs was observed under optical and transmission electron microscopes. The expressions of stem cell markers were examined in these HSCs by means of immunocytochemistry and flow cytometry. The passaged human fetal HSC suspension (0.2 ml) were injected into the spleen of SCID mice with acute liver injury induced by two-third partial hepatectomy, and 15, 30, 60, and 90 days after cell transplantation, immunohistochemistry was performed to examine the location and expressions of human hepatocytes, alpha1-AT and AFP antigen in the spleen and liver of the recipient SCID mice. PAS staining was used to examine the expression of glycogen and RT-PCR employed for detection of the expressions of AFP and albumin mRNA in the spleen of the mice on the scheduled time points.

RESULTSUnder optical microscope and transmission electron microscope, most of the HSCs were small, about 1/6 to 1/3 of the size of the hepatocyte, with relatively large nucleus-cytoplasm ratio and only small quantities of endocytoplasmic reticulum, chondriosome, and ribosome. Immunohistochemistry and flow cytometry identified positive expressions of AFP, Thy-1, C-kit, CD34 and CK19 in the HSCs, and after cell transplantation, positive expressions of human hepatocyte, alpha1-AT, and AFP antigen occurred in the liver and spleen of the recipient SCID mice. PAS staining confirmed the presence of glycogenosome in the spleen of the mice following cell transplantation. RT-PCR on days 30, 60, and 90 showed positive expressions of human AFP and albumin mRNA in the spleen of the mice.

CONCLUSIONHuman fetal HSCs can survive and settle in the spleen and liver, and migrate to the damaged liver of the recipient mice after intrasplenic transplantation, with the capacity of proliferation and differentiation into hepatocytes in the recipient target organs.

Animals ; Cells, Cultured ; Female ; Fetal Stem Cells ; cytology ; transplantation ; ultrastructure ; Flow Cytometry ; Hepatectomy ; methods ; Hepatocytes ; cytology ; metabolism ; transplantation ; Humans ; Immunohistochemistry ; Liver ; injuries ; surgery ; Mice ; Mice, Inbred BALB C ; Mice, SCID ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen ; metabolism ; surgery ; Stem Cell Transplantation ; methods ; Transplantation, Heterologous ; alpha-Fetoproteins ; analysis ; genetics

OBJECTIVETo characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro.

METHODSRat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology.

RESULTSTen weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation.

CONCLUSIONCombined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.

Albumins ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Epidermal Growth Factor ; pharmacology ; Glutathione Transferase ; biosynthesis ; genetics ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Keratin-18 ; metabolism ; Protein Array Analysis ; Pyruvate Kinase ; biosynthesis ; genetics ; RNA, Messenger ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Animals ; Cytoskeletal Proteins ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVESTo study the feasibility and possibility to diagnose Wilson disease with electronmicroscopical examination of liver biopsies.

METHODSClinical analysis, histological observation and ultrastructural examination were performed on 15 children with Wilson disease.

RESULTSAll 15 subjects had symptoms of hepatic disorders, such as jaundice. Morphological signs of hepatocyte injury in three phase, namely steatosis, mitochondrion changes and cholestasis in bile canaliculi of the early phase, nucleus injury, dilation of endoplasmic reticulum, increase of lysosomes and appearance of residual bodies of the second phase, and massive autophagy and cirrhosis of the late phase were shown. A few inflammatory cells in the liver specimens were observed. Accumulation of copper in lysosomes and autophagosomes was found by energy-dispersion X-ray.

CONCLUSIONThe diagnostic signs for Wilson disease are autophagosomes in hepatocytes, cirrhosis accompanied with a few of inflammatory cells. A certain diagnosis of the disease depends on the finding of copper accumulation in hepatocytes.

Adolescent ; Biopsy, Needle ; Child ; Copper ; metabolism ; Female ; Hepatocytes ; metabolism ; Hepatolenticular Degeneration ; diagnosis ; pathology ; Humans ; Liver ; pathology ; ultrastructure ; Male