Accumulation Of Hydrophobic Bile Acids Research Articles

Bear bile powder attenuates senecionine-induced hepatic sinusoidal obstruction syndrome in mice.

Hepatic sinusoidal obstruction syndrome (HSOS) via exposure to pyrrolizidine alkaloids (PAs) is with high mortality and there is no effective treatment in clinics. Bear bile powder (BBP) is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis, inflammation, and fibrosis. Here, we aim to evaluate the protective effect of BBP against HSOS induced by senecionine, a highly hepatotoxic PA compound. Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently, which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells, alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells, as well as decreased conventional serum liver function indicators. In addition, BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts, two well-known markers positively associated with the severity of PA-induced HSOS. Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules. BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines, in which tauroursodeoxycholic acid played an important role. What's more, BBP treatment also decreased the accumulation of hydrophobic bile acids, such as cholic acid, taurocholic acid, glycocholic acid, as well. We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis, preventing liver fibrosis, and alleviating liver inflammation. Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.

Farnesoid X Receptor as Target for Therapies to Treat Cholestasis-Induced Liver Injury.

Recent studies on liver disease burden worldwide estimated that cirrhosis is the 11th most common cause of death globally, and there is a great need for new therapies to limit the progression of liver injuries in the early stages. Cholestasis is caused by accumulation of hydrophobic bile acids (BA) in the liver due to dysfunctional BA efflux or bile flow into the gall bladder. Therefore, strategies to increase detoxification of hydrophobic BA and downregulate genes involved in BA production are largely investigated. Farnesoid X receptor (FXR) has a central role in BA homeostasis and recent publications revealed that changes in autophagy due to BA-induced reactive oxygen species and increased anti-oxidant response via nuclear factor E2-related factor 2 (NRF2), result in dysregulation of FXR signaling. Several mechanistic studies have identified new dysfunctions of the cholestatic liver at cellular and molecular level, opening new venues for developing more performant therapies.

Bile Acid Toxicity and Protein Kinases.

If the bile acids reach to pathological concentrations due to cholestasis, accumulation of hydrophobic bile acids within the hepatocyte may result in cell death. Thus, hydrophobic bile acids induce apoptosis in hepatocytes, while hydrophilic bile acids increase intracellular adenosine 3',5'-monophosphate (cAMP) levels and activate mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways to protect hepatocytes from apoptosis.Two apoptotic pathways have been described in bile acids-induced death. Both are controlled by multiple protein kinase signaling pathways. In mitochondria-controlled pathway, caspase-8 is activated with death domain-independent manner, whereas, Fas-dependent classical pathway involves ligand-independent oligomerization of Fas.Hydrophobic bile acids dose-dependently upregulate the inflammatory response by further stimulating production of inflammatory cytokines. Death receptor-mediated apoptosis is regulated at the cell surface by the receptor expression, at the death-inducing signaling complex (DISC) by expression of procaspase-8, the death receptors Fas-associated death domain (FADD), and cellular FADD-like interleukin 1-beta (IL-1β)-converting enzyme (FLICE) inhibitory protein (cFLIP). Bile acids prevent cFLIP recruitment to the DISC and thereby enhance initiator caspase activation and lead to cholestatic apoptosis. At mitochondria, the expression of B-cell leukemia/lymphoma-2 (Bcl-2) family proteins contribute to apoptosis by regulating mitochondrial cytochrome c release via Bcl-2, Bcl-2 homology 3 (BH3) interacting domain death agonist (Bid), or Bcl-2 associated protein x (Bax). Fas receptor CD95 activation by hydrophobic bile acids is initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) signaling. However, activation of necroptosis by ligands of death receptors requires the kinase activity of receptor interacting protein1 (RIP1), which mediates the activation of RIP3 and mixed lineage kinase domain-like protein (MLKL). In this chapter, mainly the effect of protein kinases signal transduction on the mechanisms of hydrophobic bile acids-induced inflammation, apoptosis, necroptosis and necrosis are discussed.

Predictive Value of Cellular Accumulation of Hydrophobic Bile Acids As a Marker of Cholestatic Drug Potential.

Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals.

Progressive and Preferential Cellular Accumulation of Hydrophobic Bile Acids Induced by Cholestatic Drugs Is Associated with Inhibition of Their Amidation and Sulfation.

Drug-induced intrahepatic cholestasis is characterized by cellular accumulation of bile acids (BAs), whose mechanisms remain poorly understood. The present study aimed to analyze early and progressive alterations of BA profiles induced by cyclosporine A, chlorpromazine, troglitazone, tolcapone, trovafloxacin, and tacrolimus after 4-hour, 24-hour, and 6-day treatments of differentiated HepaRG cells. In BA-free medium, the potent cholestatic drugs cyclosporine A, chlorpromazine, and troglitazone reduced endogenous BA synthesis after 24 hours, whereas the rarely cholestatic drugs tolcapone, trovafloxacin, and tacrolimus reduced BA synthesis only after 6 days. In the presence of physiologic serum BA concentrations, cyclosporine A, chlorpromazine, and troglitazone induced early and preferential cellular accumulation of unconjugated lithocholic, deoxycholic, and chenodeoxycholic acids that increased 8- to 12-fold and 47- to 50-fold after 24 hours and 6 days, respectively. Accumulation of these hydrophobic BAs resulted from strong inhibition of amidation, and in addition, for lithocholic acid reduction of its sulfoconjugation, and was associated with variable alterations of uptake and efflux transporters. Trovafloxacin also caused BA accumulation, especially after 6 days, whereas tolcapone and tacrolimus were still without effect. However, when exogenous BAs were added to the medium at cholestatic serum concentrations, a 6-day treatment with all drugs resulted in cellular BA accumulation with higher folds of chenodeoxycholic and lithocholic acids. At the tested concentration, tolcapone had the lowest effect. These results bring the first demonstration that major cholestatic drugs can cause preferential and progressive in vitro cellular accumulation of unconjugated toxic hydrophobic BAs and bring new insights into mechanisms involved in drug-induced cellular accumulation of toxic BAs.

Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis.

Accumulation of hydrophobic bile acids in the liver contributes to cholestatic liver injury. Inflammation induced by excessive bile acids is believed to play a crucial role, however, the mechanisms of bile acids triggered inflammatory response remain unclear. Recent studies have highlighted the effect of NLRP3 inflammasome in mediating liver inflammation and fibrosis. In this study, we for the first time showed that chenodeoxycholic acid (CDCA), the major hydrophobic primary bile acid involved in cholestatic liver injury, could dose-dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1β in macrophages by promoting ROS production and K+ efflux. Mechanistically, CDCA triggered ROS formation in part through TGR5/EGFR downstream signaling, including protein kinase B, extracellular regulated protein kinases and c-Jun N-terminal kinase pathways. Meanwhile, CDCA also induced ATP release from macrophages which subsequently causes K+ efflux via P2X7 receptor. Furthermore, in vivo inhibition of NLRP3 inflammasome with caspase-1 inhibitor dramatically decreased mature IL-1β level of liver tissue and ameliorated liver fibrosis in bile duct ligation (BDL) mouse model. In conclusion, excessive CDCA may represent an endogenous danger signal to activate NLRP3 inflammasome and initiate liver inflammation during cholestasis. Our finding offers a mechanistic basis to ameliorate cholestatic liver fibrosis by targeting inflammasome activation.

Lithocholic acid feeding results in direct hepato-toxicity independent of neutrophil function in mice

Lithocholic acid (LCA) supplementation in the diet results in intrahepatic cholestasis and bile infarcts. Previously we showed that an innate immune response is critical for cholestatic liver injury in the bile duct ligated mice. Thus, the purpose of this study was to investigate the role of neutrophils in the mechanism of liver injury caused by feeding mice a diet containing LCA. C57BL/6 mice were given control or 1% LCA containing diet for 24–96h and then examined for parameters of hepatotoxicity. Plasma ALT levels were significantly increased by 48h after LCA feeding, which correlated with both neutrophil recruitment to the liver and upregulation of numerous pro-inflammatory genes. The injury was confirmed by histology. Deficiency in intercellular adhesion molecule-1 (ICAM-1) expression or inhibition of neutrophil function failed to protect against the injury. Bile acid levels were quantified in plasma and bile of LCA-fed mice after 48 and 96h. Only the observed biliary levels of taurochenodeoxycholic acid and potentially tauro-LCA caused direct cytotoxicity in mouse hepatocytes. These data support the conclusion that neutrophil recruitment occurs after the onset of bile acid-induced necrosis in LCA-fed animals, and is not a primary mechanism of cell death when cholestasis occurs through accumulation of hydrophobic bile acids.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis

Intrahepatic cholestasis of pregnancy is a high-risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis. CoQ, vitamin E and malondialdehyde were measured in plasma and/or tissues by HPLC-UV method whereas serum bile acids by capillary electrophoresis in rats with ethinyl estradiol-induced cholestasis and women with pregnancy cholestasis. CoQ and vitamin E plasma levels were diminished in both rats and women with intrahepatic cholestasis. Furthermore, reduced CoQ was also found in muscle and brain of cholestatic rats but no changes were observed in heart or liver. In addition, a positive correlation between CoQ and ursodeoxycholic/lithocholic acid ratio was found in intrahepatic cholestasis suggesting that increased plasma lithocholic acid may be intimately related to CoQ depletion in blood and tissues. Significant CoQ and vitamin E depletion occur in both animals and humans with intrahepatic cholestasis likely as the result of increased hydrophobic bile acids known to produce significant oxidative stress. Present findings further suggest that antioxidant supplementation complementary to traditional treatment may improve cholestasis outcome.

692 NOS-3 REGULATION BY OXIDATIVE STRESS IN A CELLULAR MODEL OF CHOLESTATIC DAMAGE

Background: During cholestatic liver disease, excessive accumulation of hydrophobic bile acids exerts a cytotoxic effect leading to cell death and tissue damage. Oxidative stress plays a key role in this process by promoting the development of fibrosis, cirrhosis, portal hypertension and chronic liver failure. Furthermore, in cellular models of cholestatic damage it has been established a cytoprotective role for nitric oxide (NO). The aim of the study was to evaluate the regulation of endothelial nitric oxide synthase (NOS-3) in a cellular model of cytotoxicity by glycochenodeoxycholic acid (GCDCA) and its relationship with the oxidative stress and cell death. Materials and Methods: A kinetic study was performed (0–24 hours) for induction of cell death by GCDCA (0.5 mM) in the human hepatocarcinoma cell line HepG2. The compound Mn (III) tetrakis (4-benzoic acid) porphyrinchloride (MnTBAP, 1mg/mL) was tested as an antioxidant molecule. The detection of reactive oxygen species and assessment of cell death was performed spectrophotometrically by using the probes 2,7-dichlorofluorescein diacetate and dihydroetidium, and by measuring caspase-3 activation and lactate dehydrogenase cellular release, respectively. NOS activity was determined by analyzing nitrite and nitrate accumulation in the extracellular medium. NOS-3 expression was measured by RT-qPCR and western-blot. The promoter activity of Nos-3 gene (1601 bp) was assessed using the luciferase activity assay. The identification of transcription factors (TFs) that could be involved in the NOS-3 regulation was performed using prediction programs. Chromatin immunoprecipitation assay and western-blot were used for further analysis and for the identification of the TFs binding sites in the Nos-3 promoter. Results: GCDCA administration was associated to oxidative stress increase and Nos-3 promoter activity decrease, with a reduction in NOS-3 expression and cellular NO production. The expression and the binding of TFs cJun, cFos and SP1 to the Nos-3 promoter (identified positions), as well as the phosphorylation of protein kinases JNK and ERK1/2, were related to GCDCA-induced hepatocellular damage. MnTBAP treatment prevented the cellular effects of GCDCA. Conclusions: GCDCA-induced cell death was associated to NOS-3 expression/activity decrease by oxidative-stress. This fact was related to JNK and ERK1/2 phosphorilation, and Nos-3 promoter binding increase of TFs cJun, cFos and SP1.

Conjugated Hyperbilirubinemia in Children

A variety of anatomic, infectious, autoimmune, and metabolic diseases can lead to conjugated hyperbilirubinemia, both in the newborn period and later in childhood. The pediatric practitioner is most likely to encounter conjugated hyperbilirubinemia in the neonatal period.It is crucial to maintain a high degree of suspicion for cholestasis in the persistently jaundiced newborn. The goal is recognition of conjugated hyperbilirubinemia between 2 and 4 weeks after birth, allowing for the prompt identification and management of infants who have biliary atresia, which remains the most common cause of neonatal cholestasis.

The effect of ursodeoxycholic acid on oxidative stress level and DNase activity in rat liver after bile duct ligation

Accumulation of hydrophobic bile acids (BAs) during cholestasis plays an important role in apoptosis initiation as well as oxidative stress increase in liver cells. Ursodeoxycholic acid (UDCA) acts as a protector in BA-induced cell injury.The aim of the study was to evaluate the effect of UDCA on oxidative stress level and DNase I and II activity caused by liver injury in bile duct ligation (BDL) rats.Wistar rats were divided in four groups: group 1, control (sham-operated); group 2, sham-operated and injected with UDCA (30 mg/kg); group 3,animals with BDL; and group 4,UDCA-treatedcholestatic rats. Animals were sacrificed after 9 days. Malondialdehyde (MDA; lipid peroxidation end-product) level and protein-molecule oxidative modification (carbonyl group content) significantly increased in BDL rat liver. Catalase (CAT) activity in liver tissue was found to be decreased in BDL rats. In addition, xanthine oxidase (XO) activity, which is thought to be one of the key enzymes producing reactive oxygen species, was found to be increased in the cholestatic group. The apoptotic effect in cholestasis was probably triggered by the increased activation of DNase I and II. The protective effect of UDCA on liver tissue damage in BDL rats, in comparison to cholestatic liver, were 1) decrease of MDA levels, 2) increased CAT activity, 3) reduced XO activity, and 4) effect on terminal apoptotic reaction, shown as a decrease in DNase I and II activity.Therefore, UDCA may be useful in the preservation of liver function in cholestasis treatment.

Mitochondrial dysfunction in cholestatic liver diseases

Cholestatic liver diseases are characterized by blockade of bile flow from the liver to the intestine, and accumulation of hydrophobic bile acids in the liver and plasma. As a consequence an inflammatory response evolves associated with increased apoptosis, oxidative stress, and eventually fibrosis. Cholestasis is associated with profound metabolic changes, alterations in the mitochondrial function, decreased fatty acid oxidation, and increased glycolisis. Mitochondria play a central role in the development of this liver disease because they mediate death receptor signaling - triggered by inflammatory cytokines or bile acids - and contribute to oxidative damage, metabolic disorder, and onset of fibrosis. During the pathogenesis of biliary cirrhosis mitochondria's need for renewal is hampered by a blunted mitochondrial biogenesis. Lack of stimulation of mitochondrial renewal helps to explain mitochondrial impairment in long-term cholestasis. The marked depletion of mitochondrial DNA and occurrence of mitochondrial DNA deletions are probably relevant contributors to the progression of this severe disease. All these findings certainly support the consideration of long-term cholestasis as a secondary mitochondrial hepatopathy.

Mitochondrial dysfunction in cholestatic liver diseases

Cholestatic liver diseases are characterized by blockade of bile flow from the liver to the intestine, and accumulation of hydrophobic bile acids in the liver and plasma. As a consequence an inflammatory response evolves associated with increased apoptosis, oxidative stress, and eventually fibrosis. Cholestasis is associated with profound metabolic changes, alterations in the mitochondrial function, decreased fatty acid oxidation, and increased glycolisis. Mitochondria play a central role in the development of this liver disease because they mediate death receptor signaling - triggered by inflammatory cytokines or bile acids - and contribute to oxidative damage, metabolic disorder, and onset of fibrosis. During the pathogenesis of biliary cirrhosis mitochondria’s need for renewal is hampered by a blunted mitochondrial biogenesis. Lack of stimulation of mitochondrial renewal helps to explain mitochondrial impairment in long-term cholestasis. The marked depletion of mitochondrial DNA and occurrence of mitochondrial DNA deletions are probably relevant contributors to the progression of this severe disease. All these findings certainly support the consideration of long-term cholestasis as a secondary mitochondrial hepatopathy.

Yin-Chen-Hao-Tang ameliorates obstruction-induced hepatic apoptosis in rats

The accumulation of hydrophobic bile acids in the liver is considered to play a pivotal role in the induction of apoptosis of hepatocytes during cholestasis. Thus, factors that affect apoptosis may be used to modulate liver fibrosis. Yin-Chen-Hao-Tang (YCHT) decoctions have been recognised as a hepatoprotective agent for jaundice and various types of liver diseases. We used an experimental rat model of bile-duct ligation (BDL) to test whether YCHT plays a regulatory role in the pathogenesis of hepatic apoptosis. BDL-plus-YCHT groups received 250 or 500 mg kg (-1) YCHT by gavage once daily for 27 days. YCHT significantly ameliorated the portal hypertensive state and serum TNF-alpha compared with the vehicle-treated control group. In BDL-plus-YCHT-treated rats, hepatic glutathione contents were significantly higher than than in BDL-only rats. BDL caused a prominent liver apoptosis that was supported by an increase in Bax and cytochrome c protein and increased expression of Bax and Bcl-2 messenger RNA. The normalising effect of YCHT on expression of Bax and Bcl-2 mRNA was dependent on the dose of YCHT, 500 mg kg (-1) having the greater effect on both Bax and Bcl-2 of mRNA levels. Additionally, YCHT treatment down-regulated both hepatic caspase-3 and -8 activities of BDL rats. This study demonstrates the anti-apoptotic properties of YCHT and suggests a potential application of YCHT in the clinical management of hepatic disease resulting from biliary obstruction.

The effect of capillarisin on glycochenodeoxycholic acid-induced apoptosis and heme oxygenase-1 in rat primary hepatocytes

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. Glycochenodeoxycholate acid (GCDC) triggers a rapid oxidative stress response as an event of glutathione (GSH) depletion and nuclear factor kappa B (NF-kappaB) activation. We therefore investigated whether the bioactivity of the antioxidant capillarisin (Cap) prevents GCDC-induced hepatocyte damage. Isolated rat hepatocytes were co-incubated with 100 muM GCDC and 0.5 mg/ml Cap for 4 h. GSH depletion and thiobarbituric acid-reactive substances (TBARS, measure of lipid peroxidation) increased after GCDC exposure, but were markedly suppressed by Cap treatment. Cap protected hepatocytes from a GCDC-induced increase in reactive oxygen species (ROS) generation and mitochondrial membrane potential induction, as measured by flow cytometry analysis. In addition, Cap was shown to inhibit GCDC-mediated NF-kappaB activation by using electrophoretic mobility shift assays (EMSA). In contrast to GCDC, Cap not only significantly decreased cytochrome c release and caspase-3 enzyme activity, but also suppressed heme oxygenase-1 protein and mRNA expression in hepatocytes. These results demonstrate that Cap function as an antioxidant reduced hepatocyte injury caused by hydrophobic bile acids, perhaps by preventing generation of ROS and release of cytochrome c, thereby minimizing hepatocytes apoptosis.

Role of Caspases-9 and -4 in Glycochenodeoxycholic Acid-induced Apoptosis in HepG2 Cells

It is believed that hepatocellular damage in human chronic cholestatic liver diseases is caused by the accumulation of hydrophobic bile acids, such as glycochenodeoxycholic acid(GCDCA). Previous reports have shown that GCDCA-induced apoptosis is promoted by both mitochondria-mediated and endoplasmic reticulum(ER)stress-associated pathways in HepG2 cells and that these two pathways are linked by the action of caspase-8 on BAP31. However, the interdependence of multiple pathways remains poorly understood and the role of caspases is unclear. Thus, in the present study, we investigated the interactions among the executioner caspases in GCDCA-induced apoptotic HepG2 cells. Cells were treated for 1-24 h with GCDCA(300μM), in the presence or absence of inhibitors of caspase-9, -4 or -3(each at 30μM). Pretreatment of cells with the caspase-9 inhibitor significantly suppressed GCDCA-induced apoptosis ; however, pretreatment of cells with the caspase-4 inhibitor had no effect. Furthermore, pretreatment of cells with the caspase-9 inhibitor significantly reduced GCDCA-induced increases in caspase-3 and -4 activity, as well as the mRNA expression of BiP, a molecular chaperone located in the ER. In contrast, pretreatment of cells with the caspase-4 inhibitor had no effect. These results suggest that, in GCDCA-treated HepG2 cells, caspase-4 acts downstream of both the proapoptotic Bcl-2 protein Bax and caspase-9. Because the major mechanism underlying GCDCA-induced apoptosis involves a mitochondria-mediated pathway, it is unlikely that caspase-4 has a major role in the initiation and promotion of GCDCA-induced apoptosis.

Involvement of endoplasmic reticulum in glycochenodeoxycholic acid-induced apoptosis in rat hepatocytes

In chronic cholestatic liver diseases, accumulation of hydrophobic bile acids is thought to damage hepatocytes. The mechanism of how cells die has been an open debate, but apoptotic pathways are known to involve activation of death receptors and mitochondrial dysfunction. Recently apoptosis via an endoplasmic reticulum (ER) stress-mediated pathway was also found. In this study, we examined whether ER stress is induced in rat hepatocytes by treatment with glycochenodeoxycholic acid (GCDCA, 50–300 μM for 1–24 h), and if so, whether ER stress-mediated apoptosis occurs in this system. We determined mobility of intracellular calcium ion, activities of calpain and caspase-12, specific to ER stress-mediated apoptosis, and Bip and Chop mRNA expressions, biomarkers of ER stress. We found that GCDCA induces ER-related calcium release within about ten seconds. Significant increases in activities of calpain and caspase-12 were observed after 15 h of GCDCA treatment. Bip and Chop mRNA expressions were increased with the treated GCDCA dose and incubation time. Cytochrome c release from mitochondria peaked in about 2 h of incubation. These results suggest that ER stress is actually induced by GCDCA, though its role in hepatocellular apoptosis may be smaller than mitochondria-mediated pathway. The presence of ER stress might be important in pathogenesis of cholestatic liver diseases.

Licorice Compounds Glycyrrhizin and 18β-Glycyrrhetinic Acid Are Potent Modulators of Bile Acid-induced Cytotoxicity in Rat Hepatocytes

The accumulation of hydrophobic bile acids results in cholestatic liver injury by increasing oxidative stress, mitochondrial dysfunction, and activation of cell signaling pathways. Licorice root and its constituents have been utilized as antihepatotoxic agents. The purpose of this study was to evaluate the potential modulation by a primary component of licorice root, glycyrrhizin (GL), and its metabolite, 18beta-glycyrrhetinic acid (GA), in a hepatocyte model of cholestatic liver injury. Preincubation of fresh rat hepatocyte suspensions with GL or GA reduced glycochenodeoxycholic acid (GCDC)-dependent reactive oxygen species generation, with GA more potent than GL. Interestingly, GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3, and PARP; all were inhibited by GA but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA but not GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the mitochondrial permeability transition, reactive oxygen species generation, and cytochrome c release at submicromolar concentrations. Results from this study suggest that GL exhibits pro-apoptotic properties, whereas GA is a potent inhibitor of bile acid-induced apoptosis and necrosis in a manner consistent with its antioxidative effect.

Interactions between Hepatic Mrp4 and Sult2a as Revealed by the Constitutive Androstane Receptor and Mrp4 Knockout Mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats.

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-alpha1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of alpha-smooth muscle actin and transforming growth factor-beta and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.