Recently traditional Chinese medicine (TCM)-induced liver injury has been an unresolved critical issue which impacts TCM clinical safety. The premise and key step to reduce or avoid drug-induced liver injury (DILI) is to identify the drug source of liver injury in early stage. Then the timely withdrawal of drug and treatment can be done. However, the current diagnosis of DILI is primarily governed by exclusive method relying on administering history supplied by patients and experience judgment from doctors, which lacks objective and reliable diagnostic indices. It is obvious that diagnosis of TCM-induced liver injury is especially difficult due to the complicated composition of TCM medication, as well the frequent combination of Chinese and Western drugs in clinic. In this paper, we proposed construction of research pattern and method for objective identification of TCM-related DILI based on translational toxicology, which utilizes clinical specimen to find specific biomarkers and characteristic blood-entering constituents, as well the clinical biochemistry and liver biopsy. With integration of diagnosis marker database, bibliographic database, medical record database and clinical specimen database, an integrative diagnosis database for TCM-related DILI can be established, which would make a transformation of clinical identification pattern for TCM-induced liver injury from subjective and exclusive to objective and index-supporting mode. This would be helpful to improve rational uses of TCM and promote sustainable development of TCM industry.
Animals ; Biomarkers, Pharmacological ; metabolism ; Biopsy ; methods ; Chemical and Drug Induced Liver Injury ; diagnosis ; metabolism ; pathology ; Early Diagnosis ; Humans ; Liver ; drug effects ; pathology ; Medicine, Chinese Traditional ; adverse effects ; Rats

OBJECTIVETo explore the effects of toosendanin in inducing apoptosis of human hepatocarcinoma cell line SMMC-7721 and Hep3B, and its influence on the related genes, Bcl-2, Bax and Fas.

METHODSThe inhibitory rate of cell proliferation and cell growth curve were detected by MTT assay; morphological changes of cells were observed by inverted microscope; early stage apoptosis rate were detected by Annexin V-FITC/PI assay; relative activities of Caspase-3,-8 and-9 were analyzed by spectrophotometry; and the expressions of Bcl-2, Bax and Fas were detected using immunohistochemistry assay.

RESULTSToosendanin presented significant inhibitory effect on proliferation of hepatocarcinoma cells in a time- and dose-dependent manner. After toosendanin treatment, the amount of cells was significantly reduced, shrunk in size and rounded in shape, with decreased adhesion ability. The apoptosis rates of SMMC-7721 cells and Hep3B cells treated with 0.5 micromol/L toosendanin for 72 h were 21.55% and 18.35% respectively, which were reduced after z-VAD-fmk (inhibitor of Caspase) treatment. The activities of Caspase-3,-8 and -9 all markedly enhanced after treatment in SMMC-7721 cells, while in Hep3B cells, activities of Caspase-3 and -9 enhanced, but that of Caspase-8 unchanged. As compared with the control group, after toosendanin treatment, expression of Bcl-2 decreased, and that of Bax and Fas increased in SMMC-7721 cells; but in Hep3B cells the expression of Bcl-2 decreased, that of Bax increased, and expression of Fas unchanged.

CONCLUSIONSToosendanin could inhibit the proliferation and induce the apoptosis of both P53 and P53 human hepatocarcinoma cells, which involved the participation of mitochondria-dependent pathway. So it may be a kind of natural anti-cancer drug, playing its effect through P53 independent pathway.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mitochondria ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the effects of simvastatin on the proliferation, cell cycle and expression of cyclin-dependent kinase inhibitor p21 protein in human hepatocellular carcinoma (HepG2) cells in vitro.

METHODSHepG2 cells were administrated with simvastatin. Proliferation of the cells was detected by MTT assay, cell cycle was measured by flowcytometry and the cyclin-dependent kinase inhibitor p21 protein expression was detected by immunocytochemistry. The results were evaluated by factorial design and one-way analysis of variance.

RESULTSSimvastatin inhibited HepG2 cells growth in vitro (F(concentration) = 1264, P value less than 0.001; F(time) = 17.466, P value less than 0.001; F(concentration*time) = 35.053, P value less than 0.001) and could arrest HepG2 cells in G0/G1 phase of cell cycle. However, apoptosis of HepG2 cells was not obvious. Simvastatin could also increase cyclin-dependent kinase inhibitor p21 protein expression (F = 512.133, P value less than 0.001).

CONCLUSIONSimvastatin can inhibit the growth of HepG2 cells in vitro, which may be explained by its effects of enhancing cyclin-dependent kinase inhibitor p21 protein expression and arresting HepG2 cells at G0/G1 phase of cell cycle.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Simvastatin ; pharmacology

OBJECTIVETo investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.

METHODSIGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.

RESULTSIGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).

CONCLUSIONIGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Podophyllotoxin ; analogs & derivatives ; pharmacology ; Receptor, IGF Type 1 ; metabolism

OBJECTIVETo investigate the intervention effect of aqueous fractions from Boschniakia rossica (BRAF) on hepatic oxidative stress in mice with liver injury induced by carbon tetrachloride (CCl4).

METHODThe experimental mice were randomly assigned into the normal control group, the model group, the silymarin (positive control) group, as well as high and low dose BRAF groups. Mice were treated intragastrically with silymarin or BRAF once every day for 7 days. At the end of the experiment, CCl4 was injected intraperitoneally into the mice to establish the acute liver injury model. The pathological changes was detected with hematoxylin and eosin (HE) staining, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD) , catalase (CAT), glutathione peroxidase (GPx), Na+ -K+ -ATPase, Ca2+ -Mg2+ -ATPase, and the contents of reduced glutathione (GSH) and malondialdehyde (MDA) were detected by the colorimetric method.

RESULTBRAF significantly reduced ALT, AST and ALP activities in serum, alleviated hepatic injury induced by CCl4, increased SOD, CAT, GPx and GSH levels in liver, and SOD, Na + -K + -ATPase and Ca2+ -Mg2 + -ATPase activities in liver mitochondria, and decreased the MDA content in liver and liver mitochondria.

CONCLUSIONBRAF reduces hepatic oxidative stress in mice with acute liver injury induced by CCl4, thereby showing the protective effect on mice with acute liver injury induced by CCl4.

Animals ; Carbon Tetrachloride ; toxicity ; Chemical and Drug Induced Liver Injury ; enzymology ; metabolism ; pathology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Liver ; drug effects ; enzymology ; metabolism ; pathology ; Male ; Mice ; Mitochondria ; drug effects ; metabolism ; Orobanchaceae ; chemistry ; Oxidative Stress ; drug effects ; Solubility ; Water ; chemistry

OBJECTIVETo explore the changes and significance of the level of reactive oxygen species (ROS) and mitochondria membrane potential (Delta Psi) in HepG2 cells under the stress of cisplatin (CDDP).

METHODSHepG2 cells were incubated with CDDP. The changes in the level of ROS were determined by a probe (2,7-dichloro fluorescein-ciactate, DCFH-DA) and the changes of Delta Psi were reflected as changes of intensities of fluorescence seen under a laser scan microscope using a probe (rhodamine-123). All these changes in cells at 0 h, 24 h, 48 h, 72 h, 120 h, 168 h were dynamically observed.

RESULTSThe level of ROS was much higher after the CDDP treatment than the non-treated, and the increase lasted for 24 h and 48 h. Then it started to decrease at 72 h, gradually returning to normal level at 120 h. Under the selective pressure of CDDP, the fluorescence intensity of rhodamine-123 in HepG2 cells was decreasing at 24 h and 48 h, then gradually started to increase at 72 h. There were no such changes in the cells of the controls.

CONCLUSIONThe changes of ROS and Delta Psi in HepG2 cells under the pressure of CDDP suggest that the cells change themselves adapting to such pressures.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cisplatin ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria, Liver ; physiology ; Reactive Oxygen Species ; metabolism ; Tumor Cells, Cultured

OBJECTIVE: To investigate the antitumor activity of decoction and study its liver and kidney toxicity and its effect on the immune system in a tumor-bearing mouse model. METHODS: Hepatoma H22 tumor-bearing mouse models were randomized into model group, cyclophosphamide (CTX) group, and low-, moderate-, and high-dose decoction groups (JW-L, JW-M, and JW-H groups, respectively). The antitumor activity of decoction was assessed by calculating the tumor inhibition rate and pathological observation of the tumor tissues. Immunohistochemistry was used to detect the expressions of Bax, Bcl-2, Bax/Bcl-2 and caspase-3 in the tumors. The liver and kidney toxicity of decoction was analyzed by evaluating the biochemical indicators of liver and kidney functions. The immune function of the tumor-bearing mice were assessed by calculating the immune organ index, testing peripheral blood routines, and detection of serum IL-2 and TNF-α levels using enzyme-linked immunosorbent assay. RESULTS: Compared with that in the model group, the tumor mass in CTX, JW-M and JW-H groups were all significantly reduced ( < 0.05) with cell rupture and necrosis in the tumors. Immunohistochemistry revealed obviously up-regulated expressions of Bax and caspase-3 and down- regulated expression of Bcl-2 protein with an increased Bax/Bcl-2 ratio in CTX, JW-M and JW-H groups. Treatment with decoction significantly reduced Cr, BUN, AST and ALT levels, improved the immune organ index, increased peripheral blood leukocytes, erythrocytes and hemoglobin levels, and up-regulated the levels of TNF-α and IL-2 in the tumor-bearing mice. These changes were especially significant in JW-H group when compared with the parameters in the model group ( < 0.01). CONCLUSIONS decoction has a strong anti-tumor activity and can improve the liver and kidney functions of tumor-bearing mice. Its anti-tumor effect may be attributed to the up-regulation of Bax, caspase-3, TNF-α and IL-2 levels and the down-regulation of Bcl-2 expression as well as the enhancement of the non-specific immune function.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; immunology ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; drug effects ; Liver ; drug effects ; pathology ; Liver Neoplasms ; drug therapy ; immunology ; metabolism ; pathology ; Mice ; Necrosis ; Neoplasm Proteins ; metabolism ; Random Allocation ; Up-Regulation

To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.
Apoptosis/*drug effects ; Carrier Proteins/*metabolism ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Line ; Cells, Cultured ; Endotoxins/*pharmacology ; Liver/cytology ; Liver/*metabolism ; Liver/pathology ; Liver Failure/chemically induced ; Liver Failure/pathology ; Mitochondrial Proteins/*metabolism ; Rats, Wistar

OBJECTIVETo study the reverse effect of ligustrazine (TMP) on HepG2/ADM, a herd of hepatocellular carcinoma cell, multidrug resistance (MDR) and the influence of P-gp170 expression.

METHODThe reverse effect of ligustrazine on HepG2/ADM cell was observed, with the methods of cell culture, MTT's analyze, RT-PCR and Flow cytometric, etc.

RESULTLigustrazine could make MDR of cell line of HepG2/ADM reduce the expression of P-gp170, enhance the density of adriamycin in cell and increase the adriamycin's cytotoxicity. With the Flow cytometric, the results of RT-PCR showed the transcriptional activity of the MDR1 decreased.

CONCLUSIONLigustrazine can reverse MDR of HCC cell line of HepG2/ADM and has prospect in clinical use.

Calcium Channel Blockers ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; drug effects ; Doxorubicin ; metabolism ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Genes, MDR ; Glycoproteins ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Pyrazines ; pharmacology

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