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

BACKGROUND/AIMS: Infectious mononucleosis (IM) is the clinical presentation of primary infection with Epstein-Barr virus. Although the literature contains a massive amount of information on IM, most of this is related specifically to only children or adults separately. In order to distinguish any differences between preschool children and youth patients, we retrospectively analyzed their demographic and clinical features. METHODS: Records of patients hospitalized from December 2001 to September 2011 with a diagnosis of IM were retrieved from Peking University First Hospital, which is a tertiary teaching hospital in Beijing. The demographic data and clinical characteristics were collected. RESULTS: IM was diagnosed in 287 patients during this 10-year period, with incidence peaks among preschool children (< or =7 years old, 130/287, 45.3%) and youth patients (>15 and <24 years old, 101/287, 35.2%). Although the complaints at admission did not differ between these two patient groups, the incidence of clinical signs (tonsillopharyngitis, lymphadenopathy, hepatomegaly, and edema of the eyelids) was much higher in preschool children. The incidence of liver lesion and percentage of atypical lymphocytes were significantly higher in the youth group (P<0.001), and the average hospital stay was longer in this group. Pneumonia was the most common complication, and there was no case of mortality. CONCLUSIONS: The incidence of IM peaks among preschool children and youth patients in Beijing, China. The levels of liver enzymes and atypical lymphocytes increase with age.
Adolescent ; Adult ; Alanine Transaminase/blood ; Aspartate Aminotransferases/blood ; Child ; Child, Preschool ; Demography ; Fever/etiology ; Humans ; Incidence ; Infant ; Infectious Mononucleosis/*diagnosis/enzymology/epidemiology/pathology ; Liver/*enzymology ; Lymphocytes/cytology/*immunology/metabolism ; Pharyngitis/etiology ; Retrospective Studies ; Young Adult ; gamma-Glutamyltransferase/blood

The hepatoprotective activity of the ethanol extract of Astragalus kahiricus (Fabaceae) roots against ethanol-induced liver apoptosis was evaluated and it showed very promising hepatoprotective actions through different mechanisms. The extract counteracted the ethanol-induced liver enzymes leakage and glutathione depletion. In addition, it demonstrated anti-apoptotic effects against caspase-3 activation and DNA fragmentation that were confirmed by liver histopathological examination. Moreover, the phytochemical study of this extract led to the isolation of four cycloartane-type triterpenes identified as astrasieversianin II (1), astramembrannin II (2), astrasieversianin XIV (3), and cycloastragenol (4). The structures of these isolates were established by HRESI-MS and 1D and 2D NMR experiments. The antimicrobial, antimalarial, and cytotoxic activities of the isolates were further evaluated, but none of them showed any activity.
Animals ; Apoptosis ; drug effects ; Astragalus Plant ; chemistry ; Caspase 3 ; metabolism ; Chemical and Drug Induced Liver Injury ; drug therapy ; genetics ; physiopathology ; prevention & control ; DNA Fragmentation ; drug effects ; Ethanol ; toxicity ; Female ; Humans ; Liver ; cytology ; drug effects ; enzymology ; Plant Extracts ; administration & dosage ; Plant Roots ; chemistry ; Protective Agents ; administration & dosage ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).

METHODSTwo recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.

RESULTSThe recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.

CONCLUSIONSFAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.

Animals ; Blotting, Western ; Cell Adhesion ; Cell Line ; Cell Movement ; Down-Regulation ; Fibronectins ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Genetic Vectors ; Hepatic Stellate Cells ; cytology ; enzymology ; Liver Cirrhosis ; pathology ; prevention & control ; Plasmids ; genetics ; Polymerase Chain Reaction ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; Rats ; Transfection

AIMTo study the stereoselectivity of skin carboxylesterase metabolism and its molecular biological foundation for improving drug percutaneous absorption.

METHODSKetoprofen ethyl ester was used as a model drug, and skin homogenate was applied for studying the stereoselectivity of carboxylesterase metabolism. Human liver L02 cell was used as control of carboxylesterase expression, and RT-PCR was used for studying the expression of carboxylesterase.

RESULTSThe main metabolite of ketoprofen ethyl ester in human skin homogenate was R-ketoprofen. Human carboxylesterase-2 was highly expressed in skin and its cells. However, the expression of human carboxylesterase-1 was very weak or not detectable.

CONCLUSIONHuman carboxylesterase-2 is the main hydrolytic enzyme of prodrugs in percutaneous absorption, and shows metabolic stereoselectivity to prodrugs with chiral esters.

Adult ; Carboxylesterase ; genetics ; metabolism ; Cell Line ; Cells, Cultured ; Humans ; Ketoprofen ; metabolism ; Liver ; cytology ; enzymology ; Prodrugs ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Skin ; enzymology ; Stereoisomerism

AIMTo prepare TK-gene nanoparticles and investigate its expression.

METHODSBiodegradable and biocompatible polymer polylactic-co-glycolic acid (PLGA) was used to prepare recombinant plasmid pEGFP-AFP nanoparticles by double-emulsion evaporation technique. The characteristics of the nanopticicles including morphology, entrapment efficiency was investigated. The expression of TK gene was also investigated by MTT assay, which could determine the dying cells after the addition of gancyclovir (GCV). The enhanced green fluorescent protein (EGFP) expression in human hepatocarcinoma SMMC-7221 cells and human normal parenchymal Chang liver cells were assessed by flow cytometric analysis.

RESULTSThe resulting plasmid-nanoparticles had regular spherical surface and a narrow particle size with a mean diameter of (72 +/- 12) nm, The average entrapment efficiency was 91.25%, the enhanced transfection efficiency and ability protecting plasmid DNA from degraded by nuclease or sonication due to nanoparticles encapsulation.

CONCLUSIONDNA-nanoparticles need further study as gene delivery system.

Biocompatible Materials ; Carcinoma, Hepatocellular ; metabolism ; Cell Line, Tumor ; Drug Delivery Systems ; Ganciclovir ; pharmacology ; Genes, Reporter ; Green Fluorescent Proteins ; Herpesvirus 1, Human ; enzymology ; Humans ; Lactic Acid ; Liver ; cytology ; metabolism ; Liver Neoplasms ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Nanotechnology ; Particle Size ; Plasmids ; Polyglycolic Acid ; Polymers ; Recombinant Proteins ; genetics ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; alpha-Fetoproteins ; genetics ; metabolism

OBJECTIVETo investigate the inhibiting effect of salianic-acid B (SA-B) on mitogen-activated protein kinase (MAPK) Signaling in activated rat hepatic stellate cells (HSCs).

METHODSHSCs were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method. HSCs were primarily cultured on uncoated plastic for 7 days. Then cells were stimulated with 10ng/ml transforming growth factor-beta1 (TGF-beta1) after incubated with 10-6 M/L SA-B. The effects of SA-B on Extracellular-regulated kinase (ERK) expression and its phosphorylation. Transforming growth factor beta1 receptor I (TbetaR I) and transforming growth factor beta1 receptor II (TbetaR II) on HSCs, type I collagen expression in HSC Induced by TGF-beta1 were detected with western blot assay. Quantity of Type I collagen in the medium of HSCs was detected by ELISA. Matrix metalloproteinase 2, 9, 13 (MMP-2, MMP-9 and MMP-13) in the medium of HSCs was tested by Zymography.

RESULTSThe phosphorylation of ERK1/2 in HSCs with or without TGF-beta1 was inhibited by SA-B. The expression of TbetaR I and TbetaR II on HSCs can not be affected by SA-B. The synthesization of Type I collagen in HSCs was decreased by SA-B; The synthesization and secretion of type I collagen in HSCs with TGF-beta1 were reduced by SA-B too. SA-B had no effect on the activity of MMP-2 and MMP-13, but induced the activity of MMP-13.

CONCLUSIONSA-B inhibits ERK signaling induced by TGF-b1 in HSC. This inhibition has no association with the expression of TbetaR I and TbetaR II on HSCs. SA-B reduces the synthesization and secretion of Type I collagen in HSC by means of inhibiting TGF-beta1 signaling, which might be not related to the degrading activities of MMPs.

Animals ; Benzofurans ; pharmacology ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Liver ; cytology ; drug effects ; enzymology ; Male ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients.

METHODSAn online database SYFPEITHI was applied to predict HLA-A*0201 restricted epitopes which located in PDC-E2 30-50 aa and 150-190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates.

RESULTSFive potential epitopes were predicted by database. Of the 5 candidates, two peptides 159-167 aa and 165-174 aa, with highly binding activity to HLA-A*0201 molecules, could stimulate PBMCs from most HLA-A*0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity.

CONCLUSIONPeptides of KLSEGDLLA (159-167 aa) and LLAEIETDKA (165-174 aa) in the inner lipoyl domain of PDC-E2 are HLA-A*0201 restricted CD8+ CTL immunodominant epitopes in PBC.

Antibody-Producing Cells ; cytology ; Autoantigens ; immunology ; Autoimmunity ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; metabolism ; Cell Line ; Dihydrolipoyllysine-Residue Acetyltransferase ; Epitope Mapping ; Epitopes, T-Lymphocyte ; immunology ; HLA-A Antigens ; immunology ; HLA-A2 Antigen ; Humans ; Liver Cirrhosis, Biliary ; enzymology ; genetics ; immunology ; Phenotype ; Protein Binding ; Pyruvate Dehydrogenase Complex ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo study the effect of p38 MAPK activity on isolated rabbit liver during the period of cold preservation and reperfusion.

METHODSBased on the cold preservation-reperfusion model of isolated rabbit livers, according to the concentration of SB202190 in the preservation solution which was a specific p38 MAPK inhibitor, the isolated livers were divided into 4 groups, six in each A, B, C and D. Liver tissue samples and blood samples were harvested at different time points: before and end of cold preservation, reperfusion for 5, 10, 15, 30, 60, and 120 minutes. The activity levels of p38 MAPK were detected by both western blot and immunoprecipitation. The function markers of isolated livers were detected with automatic biochemistry analyzer, and the levels of total bile after reperfusion were measured.

RESULTSIn normal rabbit liver tissues, p38 MAPK had low activity. During the cold preservation period, the activity of p38 MAPK elevated slightly. But during the reperfusion period, the activity of p38 MAPK changed markedly which elevated rapidly at the early stage and reached its peak value at 10 minutes, then decreased gradually to the normal level (7.6 +/-0.9) at 120 minutes. SB202190 could inhibit the activity in a dose-dependent manner. The peak values of p38 MAPK activity in group B,C and D were 42.5 +/-2.4, 10.1+/-1.4, and 7.6 +/-0.6 respectively, while 78.6 +/-6.1 in group A. During the reperfusion period, the levels of serum ALT, AST and ALP were higher in group A than those in any other group, alike the p38 MAPK activity, especially at 15 and 30 minutes. On the other hand, the total bile secretion volume was (10.2 +/-2.9) ml/100 g, (13.9 +/-1.3) ml/100 g, (15.6 +/-1.2) ml/100 g, and (16.0 +/-1.3) ml/100 g in group A, B, C and D respectively.

CONCLUSIONSDuring the cold preservation- reperfusion period, p38 MAPK specific inhibitor SB202190 can lower the p38 MAPK activity, and ameliorate the isolated liver injury. Activation of p38 pathway is one of the important mechanisms to cause ischemia-reperfusion injury of isolated liver.

Animals ; Enzyme Inhibitors ; pharmacology ; Female ; Hepatocytes ; cytology ; physiology ; Imidazoles ; pharmacology ; In Vitro Techniques ; Liver ; blood supply ; cytology ; Male ; Protein Kinase C ; metabolism ; Pyridines ; pharmacology ; Rabbits ; Reperfusion Injury ; enzymology ; physiopathology ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.
Albumins ; secretion ; Animals ; Bioreactors ; Cell Culture Techniques ; methods ; Cell Separation ; Cytochrome P-450 CYP1A1 ; metabolism ; Hepatocytes ; cytology ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Male ; Mitochondria, Liver ; enzymology ; Mitochondrial Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors