OBJECTIVETo study the effects of hypoxia and hyperoxia on the expression and activity regulation of matrix metalloproteinase-2 (MMP-2) of the hepatic stellate cell (HSC).

METHODSThe expression of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC under hypoxic or hyperoxic conditions were detected with immunocytochemistry (LSAB method), the contents of MMP-2, TIMP-2 in culture supernatant with ELISA and the activity of MMP-2 in supernatant with zymography.

RESULTS(1) In the situation of hypoxia for 12 h, the expression of MMP-2 increased (hypoxia group positive indexes: 5.7 +/- 2.0; control: 3.2 +/- 1.0; P < 0.01), while TIMP-2 decreased (hypoxia group positive indexes: 2.5 +/- 0.7; control: 3.6 +/- 1.0; P < 0.05) in HSC, and the activity of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922; control: 17.277 +/- 7.424; P < 0.01). At the different time courses of hypoxia, the change of expression and activity of MMP-2 was most notable at 6 h. (2) In the situation of hyperoxia for 12 h, the protein contents of MMP-2, TIMP-2 in supernatant were both higher than those of the control, especially the TIMP-2 (hyperoxia group A(450): 0.050 +/- 0.014; control: 0.022 +/- 0.010; P < 0.01), and so was the activity of MMP-2 (hyperoxia group total A: 5.252 +/- 0.771; control: 4.304 +/- 1.083; P < 0.05). The expression of MT1-MMP was also increased.

CONCLUSIONSThe HSC is sensitive to the oxygen. Hypoxia accelerates the expression of MMP-2 and the effect is more marked at the early stage. Hyperoxia increases the activity of MMP-2.

Animals ; Cell Hypoxia ; physiology ; Hyperoxia ; enzymology ; Liver ; cytology ; enzymology ; Male ; Matrix Metalloproteinase 2 ; analysis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-2 ; analysis

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

To evaluate the induction of preneoplastic hepatic foci in relation to natural killer cell (NK) activity, we sequentially analyzed glutathione S-transferase placental form positive (GST-P+) hepatocytes and NK activity during diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis in Sprague-Dawley rats. Previous studies have shown that NK activity can modulate the carcinogenic process induced by chemical carcinogens. Newborn females were initially given a single intraperitoneal injection of 15 mg DEN/kg and three weeks later, they were treated with 500 ppm phenobarbital (PB). From week 3, PB was administered in drinking water for 9 weeks. Interim and terminal sacrifices were performed at weeks 12, 15 and 30. GST-P+ hepatocytes increased with age in DEN-treated rats, especially in the population of more than two GST-P+ hepatocytes. The NK activity of DEN-treated rats did not significantly differ from that of control rats until week 12, but it progressively decreased from week 15 to 30. These results indicate that changes of NK activity inversely correlated with the induction of preneoplastic hepatic foci. This strong correlation of decreased NK activity with enhanced induction of GST-P+ foci suggests that NK activity is important in the early progression of hepatocarcinogenesis in rats.
Animal ; Body Weight ; Carcinogens/pharmacology* ; Diethylnitrosamine/pharmacology* ; Female ; Glutathione Transferase/metabolism* ; Killer Cells, Natural/immunology* ; Liver/enzymology* ; Liver/cytology ; Liver Neoplasms/physiopathology* ; Organ Weight ; Placenta ; 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

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

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.
ATP Citrate (pro-S)-Lyase/*genetics ; Animal ; Base Sequence ; CHO Cells ; Cells, Cultured ; Female ; *Gene Expression Regulation, Enzymologic ; Glucose/pharmacology ; Hamsters ; Liver/cytology/enzymology ; Molecular Sequence Data ; Promoter Regions (Genetics) ; Rats ; Support, Non-U.S. Gov't ; Transcription, Genetic

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

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

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

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 ; Bioreactors ; Cell Culture Techniques/*methods ; Cell Separation ; Cytochrome P-450 CYP1A1/metabolism ; Hepatocytes/*cytology ; Hepatocytes/metabolism ; L-Lactate Dehydrogenase/metabolism ; Mitochondria, Liver/enzymology ; Mitochondrial Proteins/metabolism ; Rats, Sprague-Dawley ; Time Factors