OBJECTIVETo investigate the effect of Tauroursodeoxycholic acid (TUDCA) on Taurodeoxycholic acid (TDCA)-induced HepG2 cell apoptosis and to clarify the molecular mechanism of its anti-apoptosis effect of TUDCA.

METHODSMorphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and electron microscope. DNA fragment was detected by electrophoresis on 1.5% agarose gels. Apoptosis rate was measured by flow cytometry using PI dye. Following incubation of HepG2 cells either with TDCA alone, or coincubation with TUDCA and TDCA, the releasing level of cytochrome c from mitochondria into cytosol was determined by western blot, also the activity of caspase-3, 8, 9.

RESULTSIncubating the cells with 400 micromol/L TDCA for 12 h induced the cells apoptosis significantly. The apoptotic rate decreased from 50.35% +/- 2.20% to 13.78% +/- 0.84% after coincubation with TUDCA, and this anti-apoptotic effect of TUDCA was confirmed by morphological and DNA ladder detection. TUDCA significantly inhibited the release of cytochrome C from mitochondria into cytosol, and the activity of caspase-9, 3 (t > or = 13.00, P < 0.01), especially at 12 h, caspase-3 activity decreased by 54.9% (t = 16.88, P < 0.01) and 52.5%, however it had no obvious effect on the activity of caspase-8 (t = 1.94, P > 0.05).

CONCLUSIONSTUDCA prevents HepG2 cells apoptosis induced by TDCA through modulating mitochondrial membrane stability, inhibiting the release of cytochrome c and the activation of procaspase-9 and 3. Anti-apoptotic mechanism of TUDCA may be considered to be one of the most important reasons that TUDCA exerts significant efficacy in the treatment of cholestatic liver diseases.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; Caspase 9 ; Caspases ; metabolism ; Cytochromes c ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Taurochenodeoxycholic Acid ; pharmacology ; Taurodeoxycholic Acid ; analogs & derivatives ; pharmacology ; Tumor Cells, Cultured

Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L.
Animals ; Bile Acids and Salts ; metabolism ; Chemical and Drug Induced Liver Injury ; metabolism ; Cholic Acid ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Dioscorea ; toxicity ; Drugs, Chinese Herbal ; isolation & purification ; toxicity ; Heterocyclic Compounds, 4 or More Rings ; isolation & purification ; toxicity ; Least-Squares Analysis ; Male ; Mice ; Mice, Inbred ICR ; Plants, Medicinal ; toxicity ; Principal Component Analysis ; Rhizome ; toxicity ; Tandem Mass Spectrometry ; methods ; Taurochenodeoxycholic Acid ; metabolism ; Taurocholic Acid ; metabolism ; Taurodeoxycholic Acid ; metabolism

Cholesterol-lowering effect of lactic acid bacteria (LAB: Streptococcus, Lactobacillus and Bifidobacterium) is well-known. Thus, we investigated LAB isolated from human intestine on the cholesterol-lowering effect in vitro. Seven Streptococcus (61.1%), 11 Lactobacillus (71.8%) and 7 Bifidobacterium (27.9%) were isolated as acid (pH 2.5 and 3.0) and bile (0.3% oxgall) tolerant strains. Streptococcus HJS-1, Lactobacillus HJL-37 and Bifidobacterium HJB-4 were finally selected as probiotic strains to use through the bile salt hydrolase (BSH) activity assay by using MRS media added taurodeoxycholic acid (TDCA) and the cholesterol-lowering test by using soluble cholesterol containing MRS broth. These studies suggested that the isolated LAB had an excellent hypocholesterolemic effect.
Amidohydrolases/metabolism ; Bifidobacterium/*isolation&purification/physiology ; Cholesterol/*metabolism ; Feces/microbiology ; Female ; Humans ; Intestines/*microbiology ; Lactobacillus/*isolation&purification/physiology ; Male ; Probiotics/*therapeutic use ; Streptococcus/*isolation&purification/physiology ; Taurodeoxycholic Acid

OBJECTIVE: To compare the therapeutic effect of different animal bile powders on lipid metabolism disorders induced by high-fat diet in rats, and analyze the bioactive components of each animal bile powder. METHODS: Sixty Sprague-Dawley rats were randomly divided into 6 groups (n=10): normal diet control group, high-fat diet model group, high-fat diet groups orally treated with bear, pig, cow and chicken bile powders, respectively. Serum biochemical markers from the abdominal aorta in each group were analyzed. Changes in the body weight and liver weight were recorded. Pathohistological changes in the livers were examined. High performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to determine the composition of bioactive components in each animal bile powder. RESULTS: Treatment with different types of animal bile powders had different inhibitory effects on high-fat diet-induced increase of body weight and/or liver weight in rats, most notably in bear and pig bile powders (P<0.05). High-fat diet induced lipid metabolism disorder in rats, which could be reversed by treatment with all kinds of bile powders. Bear bile and chicken bile showed the most potent therapeutic effect against lipid metabolism disorder. Cow and bear bile effectively alleviated high-fat diet induced liver enlargement and discoloration, hepatocyte swelling, infiltration of inflammatory cells and formation of lipid vacuoles. Bioactive component analysis revealed that there were significant differences in the relative content of taurocholic acid, taurodeoxycholic acid and ursodeoxycholic acid among different types of animal bile. Interestingly, a unique component with molecular weight of 496.2738 Da, whose function has not yet been reported, was identified only in bear bile powder. CONCLUSIONS Different animal bile powders had varying therapeutic effect against lipid metabolism disorders induced by high-fat diet, and bear bile powder demonstrated the most effective benefits. Bioactive compositions were different in different types of animal bile with a novel compound identified only in bear bile powder.
Animals ; Bile/metabolism* ; Biomarkers/metabolism* ; Body Weight ; Cattle ; Diet, High-Fat ; Female ; Lipid Metabolism ; Lipid Metabolism Disorders/metabolism* ; Lipids/analysis* ; Liver/metabolism* ; Powders ; Rats ; Rats, Sprague-Dawley ; Swine ; Taurodeoxycholic Acid/metabolism* ; Ursidae/metabolism* ; Ursodeoxycholic Acid/metabolism*

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidyl choline to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. MATERIAL AND METHODS: The reaction mixture for the PLD assay contained 0.1microCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer (pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. RESULTS: Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward gamma-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. CONCLUSION: The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs.
Animals ; Attention ; Bone Marrow ; Brain ; Cell Death ; Cell Proliferation ; Choline ; Cobalt ; Female ; Hematopoiesis ; HEPES ; Humans ; Hydrolysis ; Kidney ; Liver ; Lung ; Muscle, Skeletal ; Oleic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D* ; Phospholipases* ; Radiation Effects ; Radioactivity ; Rats* ; Rats, Wistar ; Scintillation Counting ; Signal Transduction ; Sodium ; Spleen ; Taurodeoxycholic Acid ; Thymus Gland ; Whole-Body Irradiation

Tanreqing (TRQ), a traditional Chinese medicine (TCM) formula, can alleviate liver injury and improve liver function. Its pharmacological mechanisms of actions are still unclear due to its complex components and multi-target natures. Metabolomic study is an effective approach to investigating drug pharmacological actions, new diagnostic markers, and potential mechanisms of actions. In the present study, a new strategy was used to evaluate the protective effect of TRQ capsule against carbon tetrachloride (CCl)-induced hepatotoxicity in rats, by analyzing metabolic profiling of endogenous bile acids (BAs) along with biochemical and histological analyses. BAs concentrations were determined by ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS). Principal component analysis and partial least squares discriminant analysis were then employed to analyze the UPLC-MS results and compare the hepatoprotective effect of TRQ capsule in different groups at the doses of 0.36, 1.44, and 2.88 g·kg body weight, respectively. Moreover, our results suggested that taurocholic acid (TCA) and taurohyodesoxycholic acid (THDCA) were the most important biochemical markers, which were indicative of CCl-induced acute hepatic damage and hepatoprotective effect of TRQ capsule. Therefore, this new strategy would be an excellent alternative method for evaluating hepatoprotective effect and proposing potential mechanisms of action for other drugs as well.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Bile Acids and Salts ; blood ; metabolism ; Biomarkers ; blood ; Carbon Tetrachloride ; pharmacology ; Chemical and Drug Induced Liver Injury ; drug therapy ; metabolism ; pathology ; Chromatography, Liquid ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Female ; Liver ; drug effects ; pathology ; Male ; Mass Spectrometry ; Metabolome ; drug effects ; Metabolomics ; Rats ; Rats, Wistar ; Taurocholic Acid ; blood ; Taurodeoxycholic Acid ; analogs & derivatives ; blood

BACKGROUNDAccumulated evidence shows that hypoxia can induce endothelial apoptosis, however the mechanism is still unknown. We hypothesized whether intermittent or persistent hypoxia could induce endoplasmic reticular stress, leading to endothelial apoptosis.

METHODSTwenty-four 8-week male Sprague Dawley (SD) rats were divided into three groups: normoxia (NC) group, intermittent hypoxia (IH) group and persistent hypoxia (PH) group. TUNEL staining was performed to detect aortic arch endotheliar apoptosis, and immunohistochemistry for BIP, CHOP and caspase12 to test protein expression; human umbilical vein endothelial cells (HUVECs) of the line ECV304 were cultured (with or without taurodeoxycholic acid (TUDCA) 10 mmol/L, 100 mmol/L) and divided into four groups: NC group (20.8% O2 for 4 hours), PH1 group (5% O2 for 4 hours), PH2 group (5% O2 for 12 hours) and IH group (20.8% O2 and 5% O2 alternatively for 8 hours). Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in each group. The expressions of GRP78, CHOP and caspase12 were detected by real-time quantitative reverse-transcription PCR. Result Intermittent and persistent hypoxia could increase the rate of endothelium apoptosis and the expressions of GRP78, CHOP and caspase12 compared with the control, induction by intermittent hypoxia was slightly higher than persistent hypoxia. In the HUVEC experiment, TUDCA significantly reduced apoptosis and the expressions of GRP78, CHOP and caspase12.

CONCLUSIONHypoxia, especially intermittent, can induce endothelial cell apoptosis possibly through endoplasmic reticulum stress pathway, which can be attenuated by taurodeoxycholic acid.

Animals ; Apoptosis ; drug effects ; genetics ; physiology ; Caspase 12 ; genetics ; Endoplasmic Reticulum Stress ; drug effects ; genetics ; physiology ; Heat-Shock Proteins ; genetics ; Human Umbilical Vein Endothelial Cells ; Humans ; Hypoxia ; genetics ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Taurodeoxycholic Acid ; pharmacology ; Transcription Factor CHOP ; genetics

OBJECTIVETo investigate the molecular mechanisms involved in anti-apoptotic effects of epicathechin in liver cells.

METHODHuman hepatoma cell line (Huh7) was treated with 400 miromol x L(-1) taurodeoxycholic acid (TDCA) for 48 hours to induce apoptosis. Intracellular generation of reactive oxygen species (ROS) was detected with DCFH-DA assay. Caspase-3/7 activity was analyzed with EnzoLyte Homogeneous AMC kit. Cell proliferation was measured by MTT assay. The expression of Bax, Phospho-p38 MAPK and the levels of cytochrome C were assessed by Western-blot analysis.

RESULTTDCA-dependent intracellular ROS production was 8-fold higher as compared to untreated cells, consequently resulting in 45% reduction of cell viability. Interestingly, pretreatment of cells with epicatechin resulted in a dose-dependent inhibition of TDCA-induced ROS generation and reduced cell apoptosis by threefold as compared to TDCA treatment alone. In addition epicatechin reduced Bax expression with consequential inhibition of cytochrome C release from mitochondria, inhibition of caspase 3/7 activation and p38 MAPK phosphorylation.

CONCLUSIONEpicatechin protects Huh7 cells from oxidative stress and mitochondria-induced apoptosis. The molecular mechanisms of anti-apoptotic effects of epicatechin were associated with inhibition of p38 MAPK phosphorylation and Bax expression, and reduction of ROS production. These findings implicate epicathechin might have potential as protective agent against a variety of oxidative stress-mediated liver conditions.

Apoptosis ; drug effects ; physiology ; Carcinoma, Hepatocellular ; pathology ; Catechin ; pharmacology ; Cell Proliferation ; drug effects ; Cytochromes c ; antagonists & inhibitors ; Drug Interactions ; Humans ; MAP Kinase Kinase Kinases ; antagonists & inhibitors ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins ; antagonists & inhibitors ; Reactive Oxygen Species ; metabolism ; Taurodeoxycholic Acid ; pharmacology ; Tumor Cells, Cultured ; bcl-2-Associated X Protein ; antagonists & inhibitors ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo discriminate and determine of the artificial bear bile of the compound bile capsule.

METHODTaking the pharmacopoeia as reference, the artificial bear bile was discriminated and determined by HPLC.

RESULTThe compound bile capsule and the control sample had chromatographic peak at the same time from HPLC. The content of the artificial bear bile was above 10 mg per tablets.

CONCLUSIONThe artificial bear bile of compound bile capsules can be discriminated effectively and determined accurately by HPLC method.

Animals ; Bile ; chemistry ; Capsules ; analysis ; Chromatography, High Pressure Liquid ; methods ; Discriminant Analysis ; Medicine, Chinese Traditional ; Taurochenodeoxycholic Acid ; analysis ; Ursidae

No direct comparison of tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) has yet been carried out in the treatment of liver cirrhosis in China. We designed a double-blind randomized trial to evaluate the potential therapeutic efficacy of TUDCA in liver cirrhosis, using UDCA as parallel control. The enrolled 23 patients with liver cirrhosis were randomly divided into TUDCA group (n=12) and UDCA group (n=11), and given TUDCA and UDCA respectively at the daily dose of 750 mg, in a randomly assigned sequence for a 6-month period. Clinical, biochemical and histological features, and liver ultrasonographic findings were evaluated before and after the study. According to the inclusion criteria, 18 patients were included in the final analysis, including 9 cases in both two groups. Serum ALT, AST and ALP levels in TUDCA group and AST levels in UDCA group were significantly reduced as compared with baseline (P<0.05). Serum albumin levels were significantly increased in both TUDCA and UDCA groups (P<0.05). Serum markers for liver fibrosis were slightly decreased with the difference being not significant in either group. Only one patient in TUDCA group had significantly histological relief. Both treatments were well tolerated and no patient complained of side effects. It is suggested that TUDCA therapy is safe and appears to be more effective than UDCA in the treatment of liver cirrhosis, particularly in the improvement of the biochemical expression. However, both drugs exert no effect on the serum markers for liver fibrosis during 6-month treatment.
Adult ; Cholagogues and Choleretics ; therapeutic use ; Double-Blind Method ; Female ; Humans ; Liver Cirrhosis ; diagnosis ; drug therapy ; Male ; Middle Aged ; Taurochenodeoxycholic Acid ; therapeutic use ; Treatment Outcome ; Ursodeoxycholic Acid ; therapeutic use