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

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

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

To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.
Animals ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Down-Regulation ; Extracellular Matrix ; metabolism ; Gene Expression Regulation ; Gene Silencing ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hyaluronic Acid ; metabolism ; Laminin ; metabolism ; Liver Cirrhosis ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta ; metabolism

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

Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA + MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA + MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA + MLB group as compared to TAA only group. Hepatic mRNA expression of alpha-smooth muscle actin (alpha-SMA), TGF-beta1, and collagen alpha1(I) was significantly decreased in TAA + MLB group as compared to TAA only group. Incubation with HSCs and MLB (> or =100 microM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-kappaB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H2O2-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.
Actins/genetics/metabolism ; Animals ; Antioxidants/*administration & dosage ; Cell Proliferation/drug effects ; Collagen Type I/genetics/metabolism ; Drugs, Chinese Herbal/*administration & dosage ; Fibrosis/prevention & control ; Hepatic Stellate Cells/*drug effects/metabolism/pathology ; Liver/*drug effects/metabolism/pathology ; Liver Cirrhosis, Experimental/chemically induced/*drug therapy/physiopathology ; Male ; NF-kappa B/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Salvia miltiorrhiza/immunology ; Thioacetamide/administration & dosage ; Transcriptional Activation/drug effects

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

This study is to investigate the therapeutic effect and mechanism of indole-3-carbinol (I3C) on pig serum-induced liver fibrosis of rats. The liver fibrotic model of rats was induced by pig serum. After models were successfully established, rats in the treatment groups were administered with I3C through intraperitoneal injection or curcumin by intragastric administration, daily for 17 days. Hepatic hydroxyproline (Hyp) content was measured. The liver histology and immunohistochemistry with a-smooth muscle actin (alpha-SMA) were assayed. Hepatic stellate cells line, HSC-T6 was incubated with different concentrations of I3C (25, 50, and 100 micromol x L(-1)) for 24 h. The effect of I3C on cell apoptosis was identified by FITC-Annexin V/PI double labeled assay. And the mRNA expressions of Bax and Bcl-2 were measured by real time RT-PCR. The results showed that hepatic content of Hyp decreased by I3C treatment, as compared with the fibrotic model control. Histopathological changes, such as steatosis, necrosis, deposition of collagenous fiber reduced remarkably and the expression of alpha-SMA was significantly down-regulated in the I3C-treated groups (P < 0.01). Apoptosis analysis showed that I3C significantly increased HSC-T6 apoptosis rate and the expressional ratio of Bax to Bcl-2. The results indicated that I3C could effectively cure pig serum-induced liver fibrosis in vivo by inducing HSC apoptosis and promoting ECM degradation.
Actins ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Line ; Collagen ; metabolism ; Curcumin ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Hepatic Stellate Cells ; cytology ; Hydroxyproline ; metabolism ; Indoles ; pharmacology ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Male ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Serum ; Swine ; blood ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo explore the mechanism of Danggui Buxue Decoction (, DBD) on the liver fifibrosis related to hepatic lipid peroxidation and matrix metalloproteinases (MMP) -2/9 activities.

METHODSThe liver fifibrosis in 28 rats was induced by an injection of carbon tetrachloride (CCl(4)) and fed with high lipid and low protein diet for 6 weeks, the model rats were randomly divided into the model group and DBD treated group, 14 in each group, and another 10 rats as the normal group were observed as well. Rats in the DBD group were administered with DBD at the dose of 6 g/kg body weight for 6 weeks since CCl(4) intoxication. The hepatic inflammation and fibrosis were examined with HE and Sirius red stain. The liver function including serum alanine aminotransamine (ALT), aspartate transamine (AST), albumin (Alb) and total bilirubin (TBIL), liver triglyceride (TG) and malondialdehyde (MDA) contents, superoxide dismutase (SOD) activity were assayed. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The alpha-SMA expression was analyzed by immunohistochemistry and the Western blot. Liver MMP-2 mRNA was analyzed with Real-time PCR, and MMP-2/9 activities were measured with gelatin zymography and in situ zymography.

RESULTSCompared with the normal group, the levels of ALT, AST and TBIL, the content of Hyp, TG and MDA were remarkably increased, the Alb content and SOD activity were signifificantly decreased in the model group (P<0.05), and higher levels of MMP-2 mRNA and MMP-2/9 activities (P<0.01), the hepatic fatty degeneration and collagen accumulation and fifibrosis at liver were observed. Compared with the model control, DBD group showed slighter hepatic fatty degeneration and collagen deposition, and had lower levels of ALT, AST and TBIL activities, lower contents of MDA, TG and Hyp, but higher SOD level and Alb content (P<0.05), and DBD also down-regulated MMP-2 mRNA expression and decreased MMP-2/9 activities in the fifibrotic livers (P<0.01).

CONCLUSIONThe action of DBD against liver fibrosis is related to prevent lipid peroxidation and inhibit MMP-2/9 activities in the fibrotic livers.

Animals ; Carbon Tetrachloride ; toxicity ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hepatic Stellate Cells ; drug effects ; Lipid Peroxidation ; drug effects ; Liver ; drug effects ; pathology ; physiopathology ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Matrix Metalloproteinase Inhibitors ; Rats ; Rats, Wistar

Acetaldehyde ; pharmacology ; Animals ; Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Flow Cytometry ; Hepatic Stellate Cells ; drug effects ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Liver Cirrhosis, Alcoholic ; pathology ; prevention & control ; Rats ; Signal Transduction ; drug effects