OBJECTIVETo evaluate the influence of Fuzhenghuayu decoction on fibrotic liver tissue and activated hepatic stellate cells (HSCs) using a carbon tetrachloride (CCl4)-induced liver cirrhosis rat model system.

METHODSSixty-four Sprague-Dawley rats were randomly divided into the following groups: normal (non-model, non-drug intervention), CCl4 liver fibrosis model, and CCl4 liver fibrosis model Fuzhenghuayu drug intervention at low dose (0.75 g/kg/d) and high dose (1.5 g/kg/d). The drug intervention was administered via oral-gastric irrigation once daily for 6 times per week over a 6-week period. Four rats from each group were sacrificed at the end of week 2, 4, and 6 for serum and liver tissue collection. Liver fibrosis was evaluated by histology, and expression of a-smooth muscle actin (a-SMA) was determined by immunohistochemistry. Liver function was assessed by measuring levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBil). Between-group comparisons were made by completely random design and ANOVA with Bonferroni correction.

RESULTSAt the end of weeks 2, 4 and 6, all four groups showed significantly different levels of ALT, AST, and TBil; in addition, the model group and drug intervention groups had significantly higher levels of ALT, AST, and TBil than the control group, the drug intervention groups showed significantly lower levels of ALT, AST, and TBil than the model group (P less than 0.01 or less than 0.05), and the differences between the low dose and high dose groups reached statistical significance (P less than 0.01 or less than 0.05). At the end of weeks 2, 4 and 6, the model group and drug intervention groups had significantly higher area ratio of liver fibrosis than the normal group (F = model: 18.68, low dose: 49.95, high dose: 82.44, P less than 0.01), but the two drug intervention groups had significantly less area ratio of liver fibrosis than the model group (P less than 0.05) and the high dose group showed the most robust decrease. In addition, the model group and drug intervention groups showed higher expression of a-SMA than the normal group (F = model: 18.68, low dose: 49.95, high dose: 82.44, P less than 0.01), but two drug intervention groups had significantly less a-SMA than the model group (F = model: 46.32, low dose: 40.30, high dose: 58.42, P less than 0.05) and the high dose group showed the most robust decrease.

CONCLUSIONThe Fuzhenghuayu decoction reduces the numbers of activated HSCs, thereby leading to down-regulated a-SMA expression and reduced degree of liver fibrosis; these effects may represent the mechanism by which this drug suppresses hepatic fibrosis.

Actins ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; Liver ; drug effects ; pathology ; Liver Cirrhosis, Experimental ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

Animals ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; Curcuma ; Extracellular Matrix ; metabolism ; Hepatic Stellate Cells ; drug effects ; pathology ; Plant Extracts ; pharmacology ; Plant Oils ; pharmacology ; Rats

Animals ; Cell Line ; Hepatic Stellate Cells ; drug effects ; metabolism ; pathology ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; Rats ; Transforming Growth Factor beta1 ; pharmacology

AIMTo investigate the inhibiting role of Salvia miltiorrhiza monomer IH764-3 in proliferation and collagen synthesis and the effect on the expression of focal adhesion kinase in hepatic stellate cells (HSCs) stimulated by H2O2 in vitro, so as to provide evidences for preventing hepatic fibrosis.

METHODSThe effects of IH764-3 on the proliferation and collagen synthesis of HSCs were examined by 3H-TdR and 3H-proline incorporation assay, respectively. The expression of FAK mRNA was examined by RT-PCR in lysates of cultured HSCs.

RESULTSThe proliferation and collagen synthesis were inhibited in dose-dependent and time-dependent manners. The expressions of FAK mRNA in the IH764-3 (10 microg/ml, 20 microg/ml, 30 microg/ml, 40 microg/ml) groups were significantly lower than that in the control group.

CONCLUSIONThe present results demonstrate that IH764-3 can inhibit HSC proliferation and collagen synthesis. Meanwhile the mRNA transcription of FAK is reduced significantly, FAK as a signal molecule, can be involved in the effect of IH764-3. This may be one of the anti-fibrotic mechanisms of Salvia Miltiorrhiza.

Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Focal Adhesion Kinase 1 ; metabolism ; Hepatic Stellate Cells ; cytology ; drug effects ; Humans ; Hydrogen Peroxide ; pharmacology ; Liver Cirrhosis ; metabolism ; pathology ; Salvia miltiorrhiza ; Signal Transduction

OBJECTIVETo investigate the underlying molecular mechanism of the cholesterol-blocking drug, simvastatin, in treating nonalcoholic fatty liver fibrosis.

METHODA rat model of nonalcoholic fatty liver fibrosis was established by feeding Wistar rats a fat-rich diet. After treatment with simvastatin (4 mg/kg/day), liver histological specimens were stained with hematoxylin-eosin and Masson's trichrome for microscopic analysis. Expression of adenosine monophosphate-activated protein kinase-alpha (AMPKa) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR; for mRNA) and Western blotting (protein). The levels of serum total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor-alpha (TNFa) were measured by standard biochemical assays. The human hepatic stellate cell line, LX-2 (quiescent or activated), was treated with transforming growth factor-beta 1 (TGF-b1) alone, simvastatin alone, or TGF-b1 + simvastatin. RT-PCR and Western blotting were used to determine changes in AMPKa mRNA and protein expression, respectively.

RESULTSIn the rat model of nonalcoholic fatty liver fibrosis, the extent of pathological changes in hepatic tissues correlated with severity of disease progression. The levels of serum TC, TG, ALT, AST and TNFa were increased significantly in model rats (vs. healthy controls; all, P less than 0.01). AMPKa mRNA expression and activity was significantly decreased in model rats (vs. healthy controls; P less than 0.01 and P less than 0.05, respectively). Simvastatin, treatment significantly improved all of these parameters in model rats (vs. untreated model rats; all, P less than 0.05). In vitro simvastatin treatment of human HSCs significantly increased AMPKa activity (quiescent LX-2: 0.93+/-0.10 vs. 0.72+/-0.09, activated LX-2: 0.72+/-0.10 vs. 0.54+/-0.10, q=7.00, 6.00; all, P less than 0.01), decreased a-smooth muscle actin expression (mRNA: 0.30+/-0.02 vs. 0.36+/-0.02, protein: 0.30+/-0.03 vs. 0.38+/-0.02, q=11.245, 11.216; all, P less than 0.01), and decreased collagen I expression (mRNA: 0.30+/-0.03 vs. 0.37+/-0.03, protein: 0.25+/-0.03 vs. 0.33+/-0.03, q=8.791, 11.163; all, P less than 0.01).

CONCLUSIONSimvastatin may improve nonalcoholic fatty liver fibrosis by inducing AMPK phosphorylation.

Adenylate Kinase ; metabolism ; Animals ; Cell Line ; Fatty Liver ; drug therapy ; metabolism ; pathology ; Hepatic Stellate Cells ; drug effects ; enzymology ; Humans ; Liver Cirrhosis ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar ; Simvastatin ; pharmacology ; therapeutic use

OBJECTIVETo observe the apoptosis of hepatic stellate cell (HSC) and hepatocyte and the effect of Qinggan Huoxue Recipe (QHR) on it in alcoholic liver fibrosis (ALF) rats.

METHODSRats were divided into six groups: the normal group, the model group, the QHR low dose (4.75 g/(kg x d)], medium dose (14.25 g/(kg x d)] and high dose (28.5 g/(kg x d)) groups, and the Essentiale (66.5 mg/(kg x d)] group. They were treated with respective drugs through gavage for 2 weeks, while the normal and model groups were given normal saline instead. The serum levels of ALT, AST and gamma-GT were measured by chromatometry, the degree of inflammation and fibrosis was observed by HE staining and Masson staining, and the apoptosis of hepatocyte and HSC were detected by TUNEL and TUNEL-alpha-SMA double immunolabeling respectively.

RESULTSCompared with those before treatment, the serum levels of ALT, AST and gamma-GT obviously decreased (P < 0.05), inflammation and fibrosis relieved (P < 0.05), and the apoptosis of hepatocyte reduced (P < 0.05), while that of activated HSC increased in the QHR groups after treatment (P <0.05).

CONCLUSIONQHR can effectively relieve hepatic fibrosis, decrease hepatocyte apoptosis caused by alcohol and induce activated HSC apoptosis in ALF rats.

Actins ; analysis ; Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hepatic Stellate Cells ; drug effects ; metabolism ; pathology ; Hepatocytes ; drug effects ; metabolism ; pathology ; Immunohistochemistry ; In Situ Nick-End Labeling ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Alcoholic ; drug therapy ; metabolism ; pathology ; Male ; Muscle, Smooth ; chemistry ; Phytotherapy ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice.

METHODSHigh performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively.

RESULTSAmygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 μg/mL, 50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05).

CONCLUSIONSXYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.

Actins ; metabolism ; Animals ; Carbon Tetrachloride Poisoning ; drug therapy ; Collagen Type I ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Endothelium ; drug effects ; pathology ; Hepatic Stellate Cells ; drug effects ; pathology ; ultrastructure ; Liver Cirrhosis ; chemically induced ; drug therapy ; pathology ; Male ; Mice, Inbred C57BL ; Microscopy, Electron, Scanning ; Myofibroblasts ; drug effects ; pathology ; ultrastructure ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Primary Cell Culture ; Rats, Sprague-Dawley

The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.
Animals ; Cell Line ; Globins ; genetics ; pharmacology ; Hepatic Stellate Cells ; cytology ; pathology ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; RNA, Small Interfering ; genetics ; Rats ; Reactive Oxygen Species ; metabolism

Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.
Alkaloids ; pharmacology ; Animals ; Cell Line ; Cyclin D1 ; metabolism ; Dimethylnitrosamine ; toxicity ; Enzyme Activation ; drug effects ; ErbB Receptors ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Hepatic Stellate Cells ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; prevention & control ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinolizines ; pharmacology ; Rats

OBJECTIVESTo observe the effects of NS-398 on proliferation of hepatic stellate cells (HSCs) in vitro, and to investigate the possible molecule mechanism.

METHODSHSCs were incubated with different concentrations of NS-398. The effects of NS-398 on cell proliferation was detected by MTT colormetric assay. The cell cycle of HSCs was analyzed by Flow Cytometry (FCM), cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) proteins in HSCs were detected by immunocytochemistry.

RESULTSAdministration of 20-160 micromol/L NS-398 significantly inhibited HSCs proliferation in dose-dependent manner compared with the control group (P less than 0.01). After treated with NS-398 at concentrations of 90, 120, and 150 micromol/L for 48 h, the number of HSCs in G(2)/M phase increased and the number of HSCs in G(0)/G(1) phase decreased (P less than 0.05); Incubated with 120 micromol/L NS-398 for 48 h, percentage of masculine cell of PCNA was 28.91%+/-0.11%, which was significantly lower than that of the control group (85.99%+/-0.13%) (P less than 0.01). Percentage of masculine cell of COX-2 was 13.80%+/-0.43%, which was not significantly different from that of the control group (14.07%+/-0.59%) (P more than 0.05).

CONCLUSIONSNS-398 could inhibit the proliferation of HSC-T6 and arrest HSCs in G2/M phase. Down-regulation of PCNA protein may partially accounted for the proliferation inhibition effect on HSCs induced by NS-398.

Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase Inhibitors ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Flow Cytometry ; Hepatic Stellate Cells ; drug effects ; metabolism ; Liver Cirrhosis ; pathology ; prevention & control ; Nitrobenzenes ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Sulfonamides ; pharmacology