Metabolic profiling of endogenous bile acids: a novel method to assess hepatoprotective effect of Tanreqing capsule on carbon-tetrachloride-induced liver injury in rats / 中国天然药物

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.

Metabolic profiling of endogenous bile acids: a novel method to assess hepatoprotective effect of Tanreqing capsule on carbon-tetrachloride-induced liver injury in rats / 中国天然药物

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.

Efecto sobre la viabilidad celular de una nueva serie de espirosteroides sintéticos en células PC12 / Effect of a new series of synthetic spiroteroids on the PC12 cell line viability

Introducción: la diosgenina y sus derivados se han descrito como potentes inhibidores de la proliferación en varias líneas tumorales. Sin embargo otras moléculas relacionadas estructuralmente con dichos derivados, se han reportado como candidatos terapéuticos y otras de ellas se incluyen en alimentos de consumo humano. Objetivo: el presente trabajo evalúa el efecto sobre la viabilidad celular de una nueva serie de espiroesteroides sintéticos derivados de la diosgenina, en células tipo neurales PC12. Métodos: la viabilidad de los cultivos de PC12 se determinó mediante el ensayo de MTT y se calcularon descriptores moleculares teóricos como la lipofilicidad (logP virtual) y la superficie de área polar (SAP), con el objetivo de establecer relaciones estructura-actividad. Resultados: nuestros resultados demuestran que solo el acido taurodesoxicólico disminuye de manera significativa la viabilidad celular. Más aun, dicha molécula presenta valores menores y mayores de logP virtual y SAP, respectivamente, respecto al resto de los esteroides de la serie. Conclusiones: los resultados anteriores avalan el estudio del acido taurodesoxicólico como potencial inhibidor de la proliferación celular y al resto de las moléculas de la serie como candidatos neuroprotectores a evaluar en esta misma línea celular y dosis de tratamiento

Introduction: diosgenin and its derivatives have been described as potent anti-proliferative compounds in several tumor cell lines. However, other structurally-related compounds are reported to exert neuroprotective activity and are also included in food for human consumption. Objective: to evaluate the effect of a novel series of diogesin-derived synthetic spirosteroids on cellular viability of neuron-like PC12 cell line. Methods: cellular viability was determined by the MTT assay along with some theorical molecular descriptors, such as lipophilicity and polar surface area, in order to establish the structure-activity relationships. Results: the results demonstrated that only taurodeoxycholic acid significantly decrease PC12 cell culture viability. Moreover, this molecule presents lower virtual logP values and higher polar surface area values than the rest of spirosteroid series. Conclusions: those results endorse future studies of taurodeoxycholic acid as a potential anti-tumor candidate and of the rest of the molecules in this series as potential neuroprotective agents to be evaluated in this PC12 cell line and similar therapeutic dose

Role of endoplasmic reticular stress in aortic endothelial apoptosis induced by intermittent/persistent hypoxia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Accumulated 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.</p><p><b>METHODS</b>Twenty-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.</p><p><b>CONCLUSION</b>Hypoxia, especially intermittent, can induce endothelial cell apoptosis possibly through endoplasmic reticulum stress pathway, which can be attenuated by taurodeoxycholic acid.</p>

Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids / 药学学报

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.

Epicatechin abolished TDCA-induced apoptosis in Huh7 cell by inhibiting Bax, p38 MAPK and ROS production / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms involved in anti-apoptotic effects of epicathechin in liver cells.</p><p><b>METHOD</b>Human 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.</p><p><b>RESULT</b>TDCA-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.</p><p><b>CONCLUSION</b>Epicatechin 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.</p>

Isolation of cholesterol-lowering lactic acid bacteria from human intestine for probiotic use

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.

Effect of Tauroursodeoxycholic acid on cytochrome C-mediated apoptosis in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Morphologic 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.</p><p><b>RESULTS</b>Incubating 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).</p><p><b>CONCLUSIONS</b>TUDCA 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.</p>

Alteration of Phospholipase D Activity in the Rat Tissues by Irradiation / 대한치료방사선과학회지

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.