Adult ; Arsenic Poisoning ; pathology ; Chronic Disease ; Hemosiderin ; metabolism ; Hemosiderosis ; metabolism ; pathology ; Humans ; Hypertension, Portal ; chemically induced ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; Male ; Pancytopenia ; chemically induced ; metabolism ; pathology ; Splenomegaly ; chemically induced ; metabolism ; pathology

OBJECTIVETo study the mechanism of liver fibrogenesis and to find new non-invasive biomarkers.

METHODIn this study, we used subcellular proteomic technology to study the plasma membrane proteins related to immune or alcohol induced liver fibrosis. Rat liver fibrosis models were induced by pig serum or alcohol injection. The liver fibrogenesis were detected by James's staining in the rat models after 2, 4, 6 and 8 weeks of treatment. The liver plasma membrane (PM) of the 2- and 8-week treatment model rats were enriched by two-step sucrose density gradient centrifugation. The purity of PM was verified by western blotting, and the plasma membrane proteins were extracted and analyzed by 2 DE. The differentially expressed proteins were identified by LC-MS/MS. Cellular location and function of these identified differential protein were classified.

RESULTSImmune or alcohol induced liver fibrosis rat models were successfully established. Liver plasma membrane was significantly enriched after sucrose density ultracentrifugation treatment. 87 differential protein spots were find out by 2DE combined with LC-MS/MS from the liver plasma membrane proteins of the 2- and 8-week treatment rat models, which corresponded to 30 non-redundant proteins including annexin A2, keratin 8 and keratin 18.

CONCLUSIONSA list of differentially expressed proteins relate to liver fibrosis were successfully identified. Differential proteins such as annexin A2, keratin 8 and keratin 18 could be new biomarkers for liver fibrosis diagnosis.

Alcohols ; adverse effects ; Animals ; Female ; Keratin-18 ; metabolism ; Keratin-8 ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; immunology ; metabolism ; pathology ; Male ; Proteome ; metabolism ; Rats ; Rats, Sprague-Dawley

Animals ; Carbon Tetrachloride ; H(+)-K(+)-Exchanging ATPase ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the expression of integrin alpha 6 in hepatic sinusoidal capillaration.

METHODSThe rat hepatic fibrosis model was established by injection of carbon tetrachloride subcutaneously. Then the expression of laminin and integrin alpha 6 subunit was observed by immunohistochemistry and dot immuno-blotting.

RESULTSWe observed sinusoidal capillaration formed by deposition of laminin along sinusoids in Disse interspace by immunohistochemistry staining. In normal rat the expression of integrin alpha 6 was restricted to portal vascular endothelial cells and bile duct epithelial cell membranes. No expression was observed in sinusoidal endothelial cell membranes. When capillaration integrin alpha 6 was detected in a continuous pattern along the sinusoids, the content of integrin alpha 6 was significantly higher in fibrotic liver tissues than in normal liver tissues as measured by dot immuno-blotting (P<0.05).

CONCLUSIONSDuring fibrogenesis, laminin continuously accumulate in liver tissues and form basement membrane resulting in sinusoidal capillaration, and then induce the expression of integrin alpha 6 on SEC membranes.

Animals ; Antigens, CD ; biosynthesis ; Carbon Tetrachloride ; Immunoblotting ; Immunohistochemistry ; Integrin alpha6 ; Laminin ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar ; Time Factors

BACKGROUND/AIMS: Oxidative stress is one of the important underlying mechanisms of hepatic fibrosis. DA-9601, the ethanol extracts of Artemisia asiatica, has been reported to possess strong antioxidative and cytoprotective actions. We tried to evaluate whether antioxidant can ameliorate dimethylnitrosamine (DMN)-induced hepatic fibrosis. METHODS: Rat hepatic fibrosis was induced by intraperitoneal administrations of 10 mg DMN six times. Additionally, rats of one group were started daily with DA-9601 30 mg/kg containing diets and another group was fed a pellet diet containing DA-9601 100 mg/kg. The immunohistochemical studies for collagen, alpha-smooth muscle actin (alpha-SMA), and fibronectin, the measurements of hepatic malondialdehyde (MDA) and collagens, and the changes of liver function profiles were performed. Hepatic stellate cells (HSC) were isolated and in vitro effects of DA-9601 on HSC activations were measured. RESULTS: DA-9601 significantly attenuated the loss of body weights (p<0.05), the reduction of liver wet weights (p<0.05), and the elevation of liver enzymes provoked by DMN administrations. DMN injections caused the severe fibrosis of portal tract, hepatic inflammation, and significant oxidative damages, but DA-9601 treatment significantly reduced the mean scores of hepatic fibrosis, the amounts of hepatic collagens, and hepatic MDA levels. The prominent decreases in the expressions of collagens type I and III, alpha-SMA, and fibronectin or hepatic inflammations were observed in DA-9601-treated groups dose-dependently and similar efficacy was also proven in in vitro HSC experiment. CONCLUSIONS: DA-9601 effectively protected rat liver tissues against the DMN-induced hepatic fibrosis. Antioxidant could be considered as a supplementary therapeutic for alleviating the hepatic fibrosis.
Animals ; Antioxidants/*pharmacology ; Artemisia ; Dimethylnitrosamine ; English Abstract ; Immunohistochemistry ; Liver/drug effects/metabolism/pathology ; Liver Cirrhosis, Experimental/chemically induced/metabolism/*pathology ; Plant Extracts/*pharmacology ; Rats ; Rats, Sprague-Dawley

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.
Actins ; metabolism ; Alanine Transaminase ; blood ; Animals ; Antineoplastic Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Bile ; chemistry ; Bilirubin ; blood ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; blood ; chemically induced ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Liver Neoplasms, Experimental ; blood ; chemically induced ; pathology ; Male ; Powders ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ursidae

OBJECTIVETo investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM).

METHODSThirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay.

RESULTSSix weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis.

CONCLUSIONIntraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.

Animals ; Carbon Tetrachloride ; Connective Tissue Growth Factor ; metabolism ; Extracellular Matrix ; metabolism ; Gene Silencing ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Male ; RNA, Small Interfering ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the relationship between mast cell and hepatic fibrosis by histopathological method and semi-quantitative measurement.

METHODSSeventy-two Wistary male rats, the control group and the normal group of each only 16, experimental group of 40 rat liver fibrosis was induced by injection of DMN and was sampled at eight different time points. HE, histochemistry, immunohistochemistry (ABC method) and immunofluorescence were performed. The size of fibrosis and the number of mast cells were counted. The expression of MMP-2 and TIMP-2 was documented and electron microscopic examination was performed.

RESULTSAfter injection of DMN, the fibrosis was the most severe in the 2 week (3.72%) and the first month (3.73%, P = 0.2626), and then gradually diminished, although residual fibrosis was still present at 12 months (1.42%, P = 0.0003). The appearance of mast cells began at 2 weeks (1.73 per 200 power field in average by light microscope) after the injection and reached the peak at 4 months (3.06, P = 0.008). Residual amount of mast cells were present at 12 months (1.04, P = 0.045). However, the degree of fibrosis was not proportional or overlapping with the number of mast cells in this experiment model. Mast cells expressed MMP-2 but not TIMP-2.

CONCLUSIONSIn the DMN-induced rat liver fibrosis model, mast cell may be an integral player in the pathogenesis of liver fibrosis and may contribute to the degradation of fibrosis by synthesizing and secreting MMP-2.

Actins ; metabolism ; Animals ; Cell Count ; Dimethylnitrosamine ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; Male ; Mast Cells ; metabolism ; pathology ; ultrastructure ; Matrix Metalloproteinase 2 ; metabolism ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Tryptases ; metabolism

Cell cycle regulating proteins are known to have close relation with the proliferation of the mammalian cells. In injured liver, the number of HSCs is increased from proliferation. However, the expression of cell cycle proteins of HSCs during proliferation remains unevaluated. Therefore, cell cycle protein profiles of HSCs were studied in dimethyl-nitrosamine (DMN)-induced rat liver fibrosis model. Sprague-Dawley rats were intraperitoneally injected of DMN and the animals were sacrificed every week up to 4 weeks. HSCs were separated and the number of the cells in S phase was counted to evaluate the cell proliferation by flow cytometry. The expression of cyclin A, cyclin B, cyclin D1, cdk2, cdk4, cdc2, proliferating cell nuclear antigen (PCNA), p21Cip/WAF1, and p27 was examined with immunoblotting analysis. Portion of S-phase cells peaked 7days after DMN injection. At that time, cyclin A, and PCNA showed significant increase in HSCs compared to untreated HSCs (114% and 116%, respectively, P<0.001). p21Cip/WAF1 was decreased significantly in DMN-treated HSCs compared to control cells (88%, P<0.001). The increase of cyclin A, and PCNA and the decrease of p21Cip/WAF1 seem to play important roles in the proliferation of HSCs during the early period of DMN treatment.
Animals ; Cell Cycle Proteins/*metabolism ; Cell Proliferation ; Dimethylnitrosamine ; Liver/*cytology/metabolism/pathology ; Liver Cirrhosis/chemically induced/*metabolism/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; S Phase

OBJECTIVETo investigate the effects of genistein, a tyrosine protein kinase inhibitor, on the fenestrae, proliferation and nitric oxide (NO) synthesis of the liver sinusoidal endothelial cells from CCl(4)-induced hepatic fibrosis rats in vitro.

METHODSBy in situ collagenase perfusion and two-step percoll gradient centrifugation, SECs were isolated and cultured from normal and CCl(4)-treated Wistar rats. The fenestrae of SECs were observed by the scanning electron microscopy, and the SECs cell proliferation was determined by the MTT assay. The concentrations of NO in the cultured medium of SECs were detected indirectly by measurement of nitrates and nitrites (the stable products of NO) using the nitrate reduction method.

RESULTSScanning electron microscopic studies revealed that the number of fenestrae in SECs from all stage of hepatic fibrotic rats was decreased markedly as compared with the SECs from normal controls; however, no obvious changes in the fenestrae of SECs were observed after treated with genistein (100 mumol/L) for 24 hours. After treated with 100 mumol/L genistein for 24 hours, the cell proliferation rates of SECs from all stages of hepatic fibrosis were decreased significantly was compared with the control group (P<0.05). The synthesis of NO by SECs from all stages of hepatic fibrosis was markedly lower than those of normal controls. Treatment with 100 mumol/L genistein for 24 hours could increase the synthesis of NO by SECs from the early stage (stage I) of fibrosis; however, this effect of genistein was not observed in SECs from stage II or III of fibrosis at this concentration.

CONCLUSIONSThe results suggest that genistein may play an important role in regulating the function of SECs.

Animals ; Carbon Tetrachloride ; Cell Division ; drug effects ; Endothelium ; drug effects ; metabolism ; Genistein ; pharmacology ; Growth Inhibitors ; pharmacology ; Liver ; pathology ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; Male ; Nitric Oxide ; metabolism ; Rats ; Rats, Wistar