OBJECTIVETo optimize a compound prescription for treatment of liver fibrosis with an improved therapeutic effect and low toxicity.

METHODSIn rat models of liver fibrosis induced by thioacetamide (TAA), the optimized prescription was screened based on a uniform design with 2-factor 5-level table using Uniform Design 3.0 software and tested using liver content of Hyp as the screening index. To verify the efficacy of the optimized prescription, the rat models of liver fibrosis were randomized into normal control group, model group, colchicine group and optimized prescription group, and the changes of hepatic Hyp content, serum HA, ALT, AST, and ALB levels, and the pathology liver fibrosis were observed after corresponding treatments.

RESULTSThe optimized prescription, which contained 70 mg/kg glycyrrhizin and 70 mg/kg matrine, showed a significant therapeutic effect against liver fibrosis in rats (Plt;0.05), and the effect was equivalent to that of colchicine (P>0.05).

CONCLUSIONUniform design is a valuable method in prescription optimization. The optimized compound prescription of matrine and glycyrrhizin has a significant effect in inhibiting liver fibrosis.

Alkaloids ; administration & dosage ; Animals ; Drug Therapy, Combination ; Female ; Glycyrrhizic Acid ; administration & dosage ; Liver Cirrhosis ; chemically induced ; drug therapy ; Male ; Phytotherapy ; Quinolizines ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Thioacetamide

INTRODUCTION: Applying clinical conditions to on experimental animals forto verifverifyingy the mechanism of disease and drug effects is crucial. Cirrhotic livers induced by Hepatitis B virus are frequent, and eEspecially in Korea where a great deal of more liver-related diseases occurs, cirrhotic livers induced by Hepatitis B virus are frequent, and, such viral-induced cirrhosis, and this often impedes other medical treatments. Therefore, creating a proper elucidating properly deriveding cirrhosis method in animal model to simulate the actual pathophysiology of cirrhosis can benefit future researches. AIMS: We wanted toTe testing various hypotheticalsized methods of inducing cirrhosis in animal models, and we wanted the model to have a with higher rate of reproducibility. METHOD: To induce cirrhotic liver, thioacetamide (Sigma, St. Louis, USA) wasis given either freely via oral intaken or it wasand injected into the peritoneal space ofn Sprague-Dawley(SD) rats. The SD rats wereare divided into four groups: the oOral intake gGroup 1 ((N=10, 0.03%, 13 weeks), the oOral intake gGroup 2 (N=20, 0.04%, 30 weeks), the iIntraperitoneal Injected gGroup 1 (N=10, 300mg/kg, 12 weeks (3 times per week for first 2 weeks, 2 times per week for next 10 weeks) and the iIntraperitoneal Injected gGroup 2 (N=20, 300mg/kg, 2 times per week for 16 weeks). The mMortality rate of the tested subjects is recorded, and a visual test of the livers is performed at the end of the experiment, a visual test of the livers is performed. Also, the extracted liver cells that were dyed with Trichrome are compared to evaluate the extent of the liver cirrhosis. RESULT: For theIn oral intake group 1, no loss of occurred until wWeek 13, and 5 of the SD rats (50%) showed signs of liver cirrhosis by the Trichrome dye test. However, the extent of cirrhosis greatly differed betweenfrom each of the subjects. ForIn the oral intakae group 2, no loss occurred until wWeek 30. 20 of the SD rat (100%) in this group possessed a cirrhotic liver. However, the weight of the cirrhoscirrhotic liversis differed from a minimum of 231g to a maximum of 770g. For theIn Injected Group 1, 4 tested subjects (40%) died between wWeeks 3 and 4; however, the rest of them survived and they all revealed a signs of cirrhosis. ForIn the iInjected Group 2, only 3 tested subjects (15%) died, and after wWeek 16, 17 survivors (100%) showed a signs of cirrhosis. CONCLUSION: The short-term oral administration of thioacetamide only induced a minimal amount of cirrhosis;, thus, a longer period of consumption is suggested. Injection of thioacetamide into the peritoneum resulted in higher death rate when thoacetamide wasis injected frequently. Therefore, selecting a proper method to create a cirrhotic liver, with considering the reproducibility, on cirrhotic liver, the survival rate of the experimental animals, and the length of the experiment, isare strongly suggested for creating an animal model of cirrhotic liverfor further experiments.
Administration, Oral ; Animals ; Fibrosis ; Hepatitis B virus ; Humans ; Korea ; Liver ; Liver Cirrhosis* ; Models, Animal ; Mortality ; Peritoneum ; Rats* ; Survival Rate ; Survivors ; Thioacetamide

Background: Thioacetamide is a classic hepatotoxic reagent which leads to the reproducible hepatic fibrosis in rats. Thioacetamide-induced fibrosis is an appropriate model for cirrhosis in humans due to the long duration of course and similiar histology. Thioacetamide produces hepatotoxicity through lipid peroxidation but it is unclear whether lipid peroxidation directly correlated with hepatic fibrosis. Pentoxifylline, a derivative of the methylxanthine, showed an antifibrogenic effect in cell cultures of human fibroblasts and some animal models. But this antifibrogenic effect is controversial. Pentoxifylline revealed a hepatoprotective effect in some toxic hepatitis. This hepatoprotective effect seems to influence cell cycle regulatory protein during regeneration. This study aimed to evaluate an effect of pentoxifylline on fibrosis and cell cycle regulatory protein during liver regeneration in thioacetamide-induced rat cirrhosis. Lipid peroxidation assay was compared with collagen content so as to evaluate the correlation with fibrosis. METHOD: Liver cirrhosis was induced by 0.03% oral administration of thioacetamide. Pentoxifylline was administered simultaneously with thioacetamide. The semiquantitative fibrosis index was measured based on histologic finding. Collagen content was estimated by spectrophotometric assay. Activated hepatic stellate cells were counted using alpha-SMA immunohistochemistry. Malondialdehyde, lipid peroxidation metabolite, was estimated by thiobarbituric acid reactive substance assay. Cell cycle regulatory protein was evaluated by western blot. RESULTS: There was no difference in semiquantitative fibrosis index, collagen content and hepatic stellate cell count between thioacetamide treated rats and simultaneous pentoxifylline treated rats. Lipid peroxidation product was not correlated with collagen content. Western blot showed no difference in cell cycle regulatory protein. CONCLUSION: Pentoxifylline does not show an antifibrogenic effect in thioacetamide-induced rat cirrhosis, in which thioacetamide induced hepatocellular damage and fibrosis. Lipid peroxidation may be a secondary effect rather than primary mediating mechanism in hepatic fibrosis.
Administration, Oral ; Animals ; Blotting, Western ; Cell Culture Techniques ; Cell Cycle* ; Collagen ; Drug-Induced Liver Injury ; Fibroblasts ; Fibrosis* ; Hepatic Stellate Cells ; Humans ; Immunohistochemistry ; Lipid Peroxidation ; Liver Cirrhosis* ; Liver Regeneration ; Liver* ; Malondialdehyde ; Models, Animal ; Negotiating ; Pentoxifylline* ; Rats* ; Regeneration ; Thioacetamide

Alanine Transaminase ; blood ; Animals ; Bilirubin ; blood ; Disease Models, Animal ; Endotoxemia ; etiology ; Endotoxins ; blood ; Gastrointestinal Hormones ; blood ; Gastrointestinal Motility ; physiology ; Intestine, Small ; physiopathology ; Liver ; pathology ; Liver Failure, Acute ; blood ; etiology ; physiopathology ; Liver Function Tests ; Rats ; Thioacetamide ; administration & dosage

AIM: Silybin (SB), a major constituent of the milk thistle, has been used to treat several liver disorders. However, liver diseases were always accompanied by CYP450 dysfunction. This study was designed to explore the relationship between the hepatoprotective effect and CYP3A regulation of SB during thioacetamide (TAA)-induced rat liver injury. METHODS: Serum biochemical analysis and histopathological study were taken to evaluate the hepatoprotectinve effect of SB. α-SMA were detected by immunohistochemical analysis and cytokine release in rat liver was determined by ELISA assay. CYP3A and PXR expression were determined by RT-PCR and Western blot analysis, and CYP3A activity was based on the midazolam 4-hydroxylation reaction. Also, siRNA transfection was induced in HepG2 cells to evaluate the effect of PXR on cytotoxicity and CYP3A4 dysregulation caused by TAA. RESULTS: SB showed powerful hepatoprotective effects, and anti-inflammatory and anti-fibrosis effects, and reversed the loss of CYP3A and PXR in TAA-injured rat liver, and decreased PXR translocation into the cell nucleus. PXR silencing weakened the effect of SB on cytoprotection and CYP3A regulation. CONCLUSIONS PXR was a very important factor of CYP3A regulation and might be the target of SB in TAA-induced liver disease. Also, because of the potential interactions of SB and co-administered medicines, it might be necessary to adjust the dosage in the clinical medication of liver disease.
Animals ; Chemical and Drug Induced Liver Injury ; drug therapy ; enzymology ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Liver ; drug effects ; enzymology ; metabolism ; Male ; Milk Thistle ; chemistry ; Pregnane X Receptor ; Rats ; Rats, Sprague-Dawley ; Receptors, Steroid ; genetics ; metabolism ; Signal Transduction ; drug effects ; Silybin ; Silymarin ; administration & dosage ; Thioacetamide ; adverse effects

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

Parthenolide (PT), a sesquiterpene lactone derived from the plant feverfew, has pro-apoptotic activity in a number of cancer cell types. We assessed whether PT induces the apoptosis of hepatic stellate cells (HCSs) and examined its effects on hepatic fibrosis in an in vivo model. The effects of PT on rat HSCs were investigated in relation to cell growth inhibition, apoptosis, NF-kappaB binding activity, intracellular reactive oxygen species (ROS) generation, and glutathione (GSH) levels. In addition, the anti-fibrotic effects of PT were investigated in a thioacetamide-treated rat model. PT induced growth inhibition and apoptosis in HSCs, as evidenced by cell growth inhibition and apoptosis assays. PT increased the expression of Bax proteins during apoptosis, but decreased the expression of Bcl-2 and Bcl-XL proteins. PT also induced a reduction in mitochondrial membrane potential, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. PT inhibited TNF-alpha-stimulated NF-kappaB binding activity in HSCs. The pro-apoptotic activity of PT in HSCs was associated with increased intracellular oxidative stress as evidenced by increased intracellular ROS levels and depleted intracellular GSH levels. Furthermore, PT ameliorated hepatic fibrosis significantly in a thioacetamide-treated rat model. In conclusion, PT exhibited pro-apoptotic effects in rat HSCs and ameliorated hepatic fibrosis in a thioacetamide-induced rat model.
Animals ; Apoptosis/*drug effects ; Gene Expression/drug effects ; Hepatic Stellate Cells/*drug effects ; Humans ; Liver Cirrhosis/chemically induced/*drug therapy ; Membrane Potential, Mitochondrial/drug effects ; NF-kappa B/metabolism ; Oxidative Stress/drug effects ; Rats ; Reactive Oxygen Species/metabolism ; Sesquiterpenes/*administration & dosage ; Thioacetamide/toxicity ; Tumor Necrosis Factor-alpha/metabolism ; bcl-2-Associated X Protein/metabolism ; bcl-X Protein/metabolism

OBJECTIVETo evalueate hepatoprotective effects Feronia elephantum (F. elephantum) correa against thioacetamide (TA) induced liver necrosis in diabetic rats.

METHODSMale wistar rats were made diabetic with alloxan (160 mg/kg) on day 0 of the study. They were intoxicated with hepatotoxicant (thioacetamide, 300 mg/kg, ip) on day 9 of study to produce liver necrosis. Effects of 7 day daily once administration (day 2 to day 9) of EF (400 and 800 mg/kg, po) were evaluated on necorosis of liver in terms of mortality, liver volume, liver weight, serum aspartate aminotransferase (AST) and serum alanine transaminase (ALT), and histopathology of liver sections (for signs of necorosis and inflammation) on day-9 of the study. Separate groups of rats with treated only with alloxan (DA control), thioacetamide (TA control) and both (TA+DA control) were maintained.

RESULTSFE significantly lowered the mortality rate and showed improvement in liver function parameters in TA-induced diabetic rats without change in liver weight, volume and serum glucose levels.

CONCLUSIONSFE showed promising activity against TA-induced liver necorsis in diabetic rats and so might be useful for prevention of liver complications in DM.

Animals ; Blood Glucose ; drug effects ; Chemical and Drug Induced Liver Injury ; drug therapy ; mortality ; pathology ; prevention & control ; Diabetes Mellitus, Experimental ; Disease Models, Animal ; Liver Function Tests ; Male ; Necrosis ; Plant Extracts ; administration & dosage ; chemistry ; pharmacology ; Protective Agents ; Rats ; Rutaceae ; chemistry ; Thioacetamide ; adverse effects

Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).
Animals ; Bromodeoxyuridine/metabolism ; CDC2 Protein Kinase/drug effects/metabolism ; *CDC2-CDC28 Kinases ; Cell Cycle/drug effects/*physiology ; Cell Cycle Proteins/drug effects/metabolism ; Cyclin-Dependent Kinases/antagonists & inhibitors/drug effects/metabolism ; Cyclins/drug effects/metabolism ; Dose-Response Relationship, Drug ; G1 Phase/drug effects/*physiology ; Liver/*drug effects/pathology ; Male ; Nuclear Proteins/drug effects/metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; Protein p53/metabolism ; Protein-Serine-Threonine Kinases/antagonists & inhibitors/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Thioacetamide/administration & dosage/*pharmacology ; Tumor Suppressor Proteins/drug effects/metabolism