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Here, we report a novel truncating mutation in the ubiquitin-specific peptidase gene ( USP53 ) causing low-γ-GT (GGT) cholestasis. Genetic testing was carried out, including clinical exome sequencing for the proband and Sanger sequencing for the proband and his parents. The proband harbored a novel c.1017_1057del (p.(Cys339TrpfsTer7)) mutation in the ubiquitin carboxyl-terminal hydrolase (UCH) domain of USP53 ; we describe the clinical and laboratory features of the patient with a rare type of low-GGT cholestasis caused by this variant. The clinical presentation was found to be similar to that of phenotypes described in previous studies. However, there was an unusual presence of liver hemangiomas observed in our patient. Thus, our report reinforces the link between USP53 mutations and cholestasis. With this report, we confirm USP53 as the gene for low-GGT cholestasis and describe liver hemangiomas as a possible additional symptom of the phenotype spectrum. The inclusion of USP53 in the OMIM database and liver gene panels can further increase the effectiveness of molecular genetic studies.

Cholestasis is a pathological condition involving blockage of bile flow that results in hepatotoxicity, inflammation, and fibrosis. Although recent studies have shown that histone deacetylases (HDACs) are involved in the progression of fibrosis in various organs, the role of HDAC8 on liver fibrosis has until now remained unexplored. This study presents a newly-synthesized, selective HDAC8 inhibitor SPA3014 composed of a vinyl disulfide-sulfoxide core, and evaluates its therapeutic efficacy against cholestatic liver injury and fibrosis in bile duct-ligated (BDL) mice. We first observed the increase in HDAC8 protein levels in mice with BDL and patients with cholestatic liver disease. Mice with BDL that were pretreated with SPA3014 had lower liver damage and fibrosis, based on gross examination, histopathologic findings, and biochemical analyses, than did vehicle-treated mice. Studies with LX-2 human hepatic stellate cells showed that SPA3014 exerted protective effects by inhibiting TGF-β-mediated activation of MAPK-Smad2/3 and JAK2-STAT3 pathways and by upregulating PPARγ expression. Overall, these results strongly suggest that HDAC8 inhibition constitutes a new therapeutic strategy for treatment of cholestatic liver injury., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To assess the diagnostic and prognostic usefulness of the serum matrix metallopeptidase-7 (MMP-7) level for biliary atresia in infants with cholestasis after hepatoportoenterostomy., Study Design: We enrolled 100 infants with cholestasis (age, 43.56 ± 1.97 days; 62 males) with a direct bilirubin level of >1 mg/dL, of whom 36 (36%) were diagnosed with biliary atresisa. The MMP-7 levels in serum samples collected during the cholestasis workup and 6 months after hepatoportoenterostomy were assessed by enzyme-linked immunosorbent assay. We quantified liver fibrosis by Picro Sirius red staining of collagen in specimens from the 81 infants with cholestasis., Results: Infants with biliary atresisa had a significantly higher serum MMP-7 level than that of non-biliary atresisa infants with cholestasis of equivalent age (P < .0001). Receiver operating characteristic analysis showed that a serum MMP-7 level of >1.43 ng/mL was predictive of biliary atresisa in infants with cholestasis (diagnostic accuracy, 88%). There was a positive correlation between the serum MMP-7 level and the severity of liver fibrosis (P = .0002). Survival analysis showed that the frequency of liver transplantation was significantly higher in infants with biliary atresisa with a serum MMP-7 level of >10.30 ng/mL compared with a serum MMP-7 level of ≤10.30 ng/mL after hepatoportoenterostomy (hazard ratio, 4.22; P = .02)., Conclusions: The serum MMP-7 level, which reflects the severity of liver fibrosis and can be determined noninvasively, may facilitate the diagnosis of biliary atresisa among infants with cholestasis. Moreover, the serum MMP-7 level after hepatoportoenterostomy is associated with a need for liver transplantation in infants with biliary atresisa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert -butylhydroperoxide ( t BuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by t BuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The present study was designed to investigate the potential protective effects of diosmin (DS) and/or sildenafil against bile duct ligation (BDL). In order to achieve this goal, BDL was performed to induce liver cirrhosis, DS (100 mg/kg/day, p.o.) and sildenafil (10 mg/kg, twice daily, p.o.) were administrated alone or in combination 24 h after the surgical operation and lasted for 4 weeks. Liver function biomarkers, fibrotic markers, oxidative stress markers, mRNA expression of NF-κB-p65, P 38 -MAPK, Nrf-2, and Keap-1, as well as protein expression of cytoglobin, NF-κB-p65, Nrf-2, iNOS and eNOS were investigated concomitantly with histopathological study. The results revealed that, 4 weeks of BDL induced a significant alteration in liver functions, fibrotic and oxidative stress markers. Furthermore, up-regulation of NF-κB-p65, P 38 -MAPK, Keap-1 and iNOS concomitantly with down-regulation of Nrf-2, cytoglobin and eNOS expressions were observed after BDL. DS and/or sildenafil treatment significantly alleviated the disturbance induced by BDL. These findings were further supported by the improvement in histopathological features. Additionally, co-administration of DS and sildenafil were found to significantly improved liver defects due to BDL as compared to the individual drugs. It can be concluded that, DS and sildenafil exhibit hepatoprotective effects through modulation of Keap-1/Nrf-2 and P 38 -MAPK/NF-κB/iNOS pathway., (Copyright © 2018. Published by Elsevier Ltd.)

Hepatic fibrosis is a potential health problem that may end with life-threatening cirrhosis and primary liver cancer. Recent studies point out to the protective effects of silent information regulator1 (SIRT1), against different models of organs fibrosis. This work aimed to investigate the possible protective effect of sildenafil (SIRT1 activator) against hepatic fibrosis induced by bile duct ligation (BDL). Firstly, three different doses of sildenafil (5, 10, 20mg/kg/day) were investigated; to detect the most protective one against BDL induced liver dysfunction and hepatic fibrosis. The most protective dose is then used; to study its effect on BDL induced SIRT1 downregulation, imbalance of oxidant/antioxidant status, increased inflammatory cytokines and fibrosis. Sildenafil (20mg/kg/day) was the most protective one, it caused upregulation of SIRT1, reduction of hepatic malondialdehyde (MDA) content, increase in expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenease (HO)-1, reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hepatic content of tumor necrosis factor-α (TNF-α) and nuclear factor κB (NFκB) expression & content displayed significant reductions with sildenafil treatment, Furthermore, sildenafil caused marked reductions of transforming growth factor (TGF)-β content, expression of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), α-smooth muscle actin (α-SMA), fibronectin, collagen I (α1) and hydroxyproline content. However, sildenafil protective effects were significantly reduced by co-administration of EX527 (SIRT1 inhibitor). Our work showed, for the first time that, sildenafil has promising protective effects against BDL induced liver dysfunction and hepatic fibrosis. These effects may be, in part, mediated by up regulation of SIRT1., (Copyright © 2017. Published by Elsevier Inc.)

Aim: To ascertain whether cholestasis affects the expression of two CYP3A isoforms (CYP3A1 and CYP3A2) and of pregnane X receptor (PXR) and constitutive androstane receptor (CAR)., Methods: Cholestasis was induced by bile duct ligation in 16 male Wistar rats; whereas 8 sham-operated rats were used as controls. Severity of cholestasis was assessed on histological examination of liver sections, and serum concentrations of albumin, AST, ALT, GGT, ALPK and bilirubin. Gene and protein expressions of PXR, CAR, CYP3A1 and CYP3A2 were assessed by means of qRT-PCR and Western blot, respectively. Alterations in CYP3A activity were measured by calculating the kinetic parameters of 4-OH and 1'-OH-midazolam hydroxylation, marker reactions for CYP3A enzymes., Results: The mRNA and protein expression of CYP3A1 increased significantly in mild cholestasis ( P < 0.01). At variance, mRNA and protein expression of CYP3A2 didn't change in mild cholestasis, whereas the expression and activity of both CYP3A1 and CYP3A2 decreased dramatically when cholestasis became severe. Consistently with these observations, the nuclear expression of both PXR and CAR, which was measured because they both translocate into the cell nucleus after their activation, virtually disappeared in the late stage of cholestatic injury, after an initial increase. These results indicate that early- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently, probably as consequence of the different activation of PXR and CAR., Conclusion: Early- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently. PXR and CAR might be targeted therapeutically to promote CYP3A-mediated liver detoxification., Competing Interests: Conflict-of-interest statement: The authors have no conflict of interest to report.

Several hepatobiliary disorders have been reported in ulcerative colitis (UC) patients with primary sclerosing cholangitis (PSC) being the most specific. Primary biliary cholangitis (PBC), previously known as primary biliary cirrhosis, rarely occurs in UC. We present two PBC cases of 67 and 71 years who suffered from long-standing UC. Both patients were asymptomatic but they had increased cholestatic enzymes and high titres of antimitochondrial antibodies (AMA)-the laboratory hallmark of PBC. After careful exclusion of other causes of cholestasis by MRI/magnetic resonance cholangiopancreatography (MRCP), virological and microbiological investigations, a diagnosis of PBC associated with UC was established. The patients started ursodeoxycholic acid (13 mg/kg/day) with complete response. During follow-up, both patients remained asymptomatic with normal blood biochemistry. Although PSC is the most common hepatobiliary manifestation among patients with UC, physicians must keep also PBC in mind in those with unexplained cholestasis and repeatedly normal MRCP. In these cases, a reliable AMA testing can help for an accurate diagnosis., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Bile acids facilitate the absorption of lipids in the gut, but are also needed to maintain cholesterol homeostasis, induce bile flow, excrete toxic substances and regulate energy metabolism by acting as signaling molecules. Bile acid biosynthesis is a complex process distributed across many cellular organelles and requires at least 17 enzymes in addition to different metabolite transport proteins to synthesize the two primary bile acids, cholic acid and chenodeoxycholic acid. Disorders of bile acid synthesis can present from the neonatal period to adulthood and have very diverse clinical symptoms ranging from cholestatic liver disease to neuropsychiatric symptoms and spastic paraplegias. This review describes the different bile acid synthesis pathways followed by a summary of the current knowledge on hereditary disorders of human bile acid biosynthesis with a special focus on diagnostic bile acid profiling using mass spectrometry., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Advanced cholestatic liver disease is a leading referral to pediatric liver transplant centers. Recent advances in the genetic classification of this group of disorders promise a highly personalized management although the genetic heterogeneity also poses a diagnostic challenge. Using a next-generation sequencing-based multi-gene panel, we performed retrospective analysis of 98 pediatric patients who presented with advanced cholestatic liver disease. A likely causal mutation was identified in the majority (61%), spanning many genes including ones that have only rarely been reported to cause cholestatic liver disease, e.g. TJP2 and VIPAS39. We find no evidence to support mono-allelic phenotypic expression in the carrier parents despite the severe nature of the respective mutations, and no evidence of oligogenicity. The high-carrier frequency of the founder mutations identified in our cohort (1 in 87) suggests a minimum incidence of 1:7246, an alarmingly high disease burden that calls for the primary prevention through carrier screening., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nimesulide (NIM) is a classic nonsteroidal anti-inflammatory drug. However, some patients treated with NIM experienced cholestatic liver injury. For this reason, we investigated the potential mechanism underlying NIM-induced cholestasis by using in vivo and in vitro models. Oral administration of 100 mg/kg/day NIM to Wistar rats for 5 days increased the levels of plasma total bile acids, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase by 1.49-, 1.31-, 1.60-, and 1.29-fold, respectively. In sandwich-cultured rat hepatocytes, NIM and 4'-hydroxynimesulide (M1) reduced the biliary excretion index of d 8 -taurocholic acid (d 8 -TCA) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein in a concentration-dependent manner, indicating the inhibition of the efflux transporters bile salt export pump and multidrug resistance-associated protein 2, respectively. In suspended rat hepatocytes, NIM and M1 inhibited the uptake transporters of d 8 -TCA for Na + -taurocholate cotransporting polypeptide at IC 50 values of 21.3 and 25.0 μ M, respectively, and for organic anion-transporting proteins at IC 50 values of 45.6 and 39.4 μ M, respectively. By contrast, nitro-reduced NIM and the further acetylated metabolite did not inhibit or only marginally inhibited these transporters at the maximum soluble concentrations. Inhibitory effects of NIM and M1 on human bile acid transporters were also confirmed using sandwich-cultured human hepatocytes. These data suggest that the inhibition of bile acid transporters by NIM and M1 is one of the biologic mechanisms of NIM-induced cholestasis., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Apoptosis plays pivotal in vivo roles in not only vital processes, such as cell turnover and embryonic development, but also various inflammatory disorders. However, the role of apoptosis by vascular and hepatic cells in the respective progression of atherosclerosis and liver injury remains controversial. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that is activated through distinct mechanisms in response to various cytotoxic stressors. ASK1, ubiquitously expressed, is situated in an important upstream position for many signal transduction pathways, which subsequently induce inflammation and/or apoptosis. Our serial in vivo studies have uniquely reported that the expression of phosphorylated ASK1 is variably seen in atherosclerotic lesions or bile-duct-ligation (BDL)-induced injury livers. In mice genetically deficient of ASK1 (ASK1⁻/⁻), activated ASK1 signaling accelerates high-cholesterol-diet-induced necrotic lipid core formation by inducing macrophage apoptosis and enhances ligation injury-induced vascular remodeling via pro-inflammatory reactions and by stimulating apoptosis of smooth muscle cells. In contrast, in models of BDL-induced cholestatic liver injury, the pathogenic roles of ASK1-mediated early necro-inflammation, but not apoptosis, and the proliferation of hepatocytes and cholangiocytes are crucial in subsequent peribiliary fibrosis/fibrogenesis. These animal models of acute to chronic inflammatory diseases show that stimulated ASK1 signaling critically and diversely regulates not only hypercholesterolemia-induced atherosclerosis and injury-induced arteriosclerosis, but also the acute and subacute-to-chronic phase of BDL-induced cholestasis. We herein review the diverse, key in vivo roles of ASK1 signaling in the pathogenesis of inflammatory disorders closely related to metabolic syndrome.

Background: Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress, hepatocellular injury and fibrosis. Quercetin is a flavonoid with antifibrotic, and hepatoprotective properties. However, the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood. The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model., Methods: We divided male Wistar rats into 4 groups (n=8 for each): sham, sham+quercetin (30 mg/kg per day), BDL, and BDL+quercetin (30 mg/kg per day). Four weeks later, the rats were sacrificed, the blood was collected for liver enzyme measurements and liver for the measurement of Rac1, Rac1-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting, respectively., Results: Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes. Furthermore, the mRNA and protein expression of Rac1, Rac1-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (P<0.05); quercetin treatment reversed these variables back toward normal (P<0.05). Another interesting finding was that the antioxidant markers e.g. superoxide dismutase and catalase were elevated in quercetin-treated BDL rats compared to BDL rats (P<0.05)., Conclusion: Quercetin demonstrated hepatoprotective activity against BDL-induced liver injury through increasing antioxidant capacity of the liver tissue, while preventing the production of Rac1, Rac1-GTP and NOX1 proteins.

Estrogen-induced cholestasis occurs in many women who are susceptible due to pregnancy or hormone replacement therapy for postmenopausal syndrome. 17α-Ethinylestradiol (EE), as a synthetic estrogen, has been widely used to study the underlying mechanisms of estrogen-induced cholestasis. Recent studies have also reported that liver kinase B1 (LKB1)-mediated activation of AMP-activated protein kinase (AMPK) plays a critical role in the regulation of canalicular network formation. However, the role of AMPK in EE-induced cholestasis remains to be determined. In this study, the effects of EE (1-100 µM) on AMPK activation and the expression of farnesoid X receptor (FXR) and hepatic bile acid transporters were examined in in vitro using 3D-cultured rat primary hepatocytes and in in vivo using rat cholestasis models. We also used specific chemical agonist and antagonist of AMPK, AMPK subunit-specific antibodies and lentiviral shRNAs for AMPKα1 and AMPKα2 to delineate the role of AMPK in EE-induced cholestasis and potential cellular mechanisms. We found that EE-induced phosphorylation of AMPKα1 via extracellular signal-regulated kinases-LKB1-mediated signaling pathways and subsequent nuclear translocation accounted for the down-regulation of FXR and bile acid transporters and disruption of bile acid homeostasis. Inhibition of AMPK activation using an AMPK antagonist Compound C (2 µM) or down-regulation of AMPKα1 using gene-specific shRNA attenuated EE-induced cholestasis both in in vitro and in in vivo. In conclusion, these results revealed that activation of cAMP-ERK-LKB1-AMPKα1 signaling pathway plays a critical role in EE-mediated dysregulation of the expression of FXR and bile acid transporters. AMPKα1 may represent an important therapeutic target for estrogen-induced cholestasis., Competing Interests: The authors declare no competing financial interest.

Ethnopharmacology Relevance: Calculus Bovis, also known as Niuhuang, is a rare traditional Chinese medicine that has been widely used in China for 2000 years in pharmacology for sedation, anti-spasm, relieving fever, diminishing inflammation and recovering gallbladder functions., Aim of the Study: This study aimed to investigate the choleretic potential and molecular responses in rats to Calculus Bovis (CB) administration after 17α-ethynylestradiol (EE)-induced cholestasis., Material and Methods: CB (50 and 100mg/kg per day) was intragastrically (i. g.) given to experimental rats for five consecutive days in coadministration with EE (5mg/kg daily for five days, s.c.). The levels of serum biomarkers were determined biochemically. The histopathology of the liver tissue was evaluated. Expression of bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were studied by western blot and immunohistochemical assay. The expression of Akt and phospho-Akt (pAkt) were also measured by western blot., Results: In response to EE, CB treatment significantly prevented an increase in serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT) and total bilirubin (TBIL). CB treatment also repaired tissue lesions caused by EE. Western blots showed that EE significantly decreased the protein expression of BSEP and MRP2. EE also dramatically increased levels of pAkt and decreased levels of Akt. Compared to the EE group, CB treatment increased levels of hepatic BSEP and MRP2 while pAkt levels decreased and Akt levels increased. Immunohistochemistry also indicated that EE decreased the expression of BSEP and MRP2. LY294002 is a selective PI3K inhibitor and showed similar beneficial effects as CB. Decreased expression of BSEP and MRP2 caused by EE were also prevented by LY294002 treatment., Conclusion: Calculus Bovis administration can alleviate liver injury and up-regulate the expression of BSEP and MRP2 in 17α-ethynylestradiol-induced cholestasis by a mechanism that may involve inhibiting the activated PI3K/Akt signaling pathway., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

An LC-MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were 385.1≥159.1 and 376.4≥266.3, respectively. The linear response range of 7α-OH cholesterol was covered from 1.563 to 100.0ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Serum autotaxin (ATX) activity is significantly increased in cholestatic patients. Our study aimed to unravel the source(s) of ATX in cholestasis., Materials and Methods: ATX activity and protein were measured in sera of healthy (n=33) and cholestatic patients (n=152), including women with intrahepatic cholestasis of pregnancy. ATX mRNA and protein expression were analyzed in various tissues from mice and men. Induction of ATX activity was assessed in mouse models of extrahepatic (bile duct ligation) and intrahepatic cholestasis (Atp8b1(G308V/G308V), 0.1% cholate-supplemented diet). ATX clearance in cholestatic and control mice was assessed after intravenous injection of recombinant ATX. Human hepatic clearance was estimated by comparing ATX activity in portal and hepatic vein serum., Results: Serum ATX activity and ATX protein concentration tightly correlated under all conditions in patients and controls (p<0.0001). In humans Atx mRNA was highly expressed in small intestine, whereas in mice Atx was expressed mainly in brain and placenta but not in small intestine. Extensive ATX protein expression was identified in human, but not murine intestinal enteroendocrine cells. In murine models of cholestasis and cholestatic pregnancy plasma ATX activity was only mildly elevated (up to 2.1-fold). Atx tissue expression and rATX clearance after parenteral administration did not differ between cholestatic and control mice., Conclusion: Serum ATX activity during cholestasis and itch is enhanced by increased protein concentration rather than enzymatic induction. In mice, clearance of ATX is not affected by cholestasis. Small intestinal ATX expression by enteroendocrine cells might represent an important source of cholestasis-induced serum ATX activity in men., (Copyright © 2016. Published by Elsevier B.V.)

Bile acids have been implicated in cholestatic liver damage, primarily due to their detergent effect on membranes and induction of oxidative stress. Gangliosides can counteract these harmful effects by increasing the rigidity of the cytoplasmic membrane. Induction of haem oxygenase (HMOX) has been shown to protect the liver from increased oxidative stress. The aim of this study was to determine the changes in the synthesis and distribution of liver gangliosides following bile duct ligation (BDL), and to assess the effects of HMOX both on cholestatic liver injury and ganglioside metabolism. Compared to controls, BDL resulted in a significant increase in total as well as complex gangliosides and mRNA expression of corresponding glycosyltransferases ST3GalV, ST8SiaI and B3GalTIV. A marked shift of GM1 ganglioside from the intracellular compartment to the cytoplasmic membrane was observed following BDL. Induction of oxidative stress by HMOX inhibition resulted in a further increase of these changes, while HMOX induction prevented this effect. Compared to BDL alone, HMOX inhibition in combination with BDL significantly increased the amount of bile infarcts, while HMOX activation decreased ductular proliferation. We have demonstrated that cholestasis is accompanied by significant changes in the distribution and synthesis of liver gangliosides. HMOX induction results in attenuation of the cholestatic pattern of liver gangliosides, while HMOX inhibition leads to the opposite effect.

Matrix metalloproteinases (MMPs) are a family of proteases using zinc-dependent catalysis to break down extracellular matrix (ECM) components, allowing cell movement and tissue reorganization. Like many other proteases, MMPs are produced as zymogens, an inactive form, which are activated after their release from cells. Hepatic ischemia/reperfusion (I/R) is associated with MMP activation and release, with profound effects on tissue integrity: their inappropriate, prolonged or excessive expression has harmful consequences for the liver. Kupffer cells and hepatic stellate cells can secrete MMPs though sinusoidal endothelial cells are a further source of MMPs. After liver transplantation, biliary complications are mainly attributable to cholangiocytes, which, compared with hepatocytes, are particularly susceptible to injury and ultimately a major cause of increased graft dysfunction and patient morbidity. This paper focuses on liver I/R injury and cholestasis and reviews factors and mechanisms involved in MMP activation together with synthetic compounds used in their regulation. In this respect, recent data have demonstrated that the role of MMPs during I/R may go beyond the mere destruction of the ECM and may be much more complex than previously thought. We thus discuss the role of MMPs as an important factor in cholestasis associated with I/R injury.

Background: Paeonia lactiflora Pall. (PLP), a traditional Chinese herbal medicine, has been used for hepatic disease treatment over thousands of years. In our previous study, PLP was shown to demonstrate therapeutic effect on hepatitis with severe cholestasis. The aim of this study was to evaluate the antioxidative effect of PLP on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis by activating NF-E2-related factor 2 (Nrf2) via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway., Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the main compounds present in PLP. The mechanism of action of PLP and its therapeutic effect on cholestasis, induced by ANIT, were further investigated. Serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) were measured, and histopathology of liver was also performed to determine the efficacy of treatment with PLP. Moreover, in order to illustrate the underlying signaling pathway, liver glutathione (GSH) content and mRNA or protein levels of glutamate-cysteine ligase catalytic subunit (GCLc), glutamate-cysteine ligase modulatory subunit (GCLm), Akt, heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase 1 (Nqo1), and Nrf2 were further analyzed. In addition, validation of PLP putative target network was also performed in silico., Results: Four major compounds including paeoniflorin, albiflorin, oxypaeoniflorin, and benzoylpaeoniflorin were identified by LC-MS analysis in water extract of PLP. Moreover, PLP could remarkably downregulate serum levels of TBIL, DBIL, AST, ALT, ALP, γ-GT, and TBA, and alleviate the histological damage of liver tissue caused by ANIT. It enhanced antioxidative system by activating PI3K/Akt/Nrf2 pathway through increasing Akt, Nrf2, HO-1, Nqo1, GCLc, and GCLm expression. The putative targets network validation also confirmed the important role of PLP in activating Akt expression., Conclusion: The potential mechanism of PLP in alleviating ANIT-induced cholestasis could to be related to the induction of GSH synthesis by activating Nrf2 through PI3K/Akt-dependent pathway. This indicates that PLP might be a potential therapeutic agent for cholestasis.

Background: Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis., Objectives: In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs., Methods: We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays., Results: After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation., Conclusions: Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background & Aims: Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin-linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell (SEC) eNOS regulation and function., Methods: Primary liver SECs were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK., Results: Integrin-linked kinase expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhance eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression and ultimately NO production. In contrast, ILK overexpression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serine-threonine kinase, Akt and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation., Conclusions: Our data suggest a potential role for ILK, the cytoskeleton and ILK signalling partners including Rho in regulating intrahepatic SEC eNOS expression and function., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Levels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear., Methods: We collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured., Results: The levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p<0.05). Conversely, the expression levels of liver detoxification enzymes, UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4, were reduced by approximately 50% (p<0.05) in human obstructive cholestasis. The levels of membrane transporters, OSTβ and OCT1, were increased 10.4-fold and 1.8-fold, respectively, (p<0.05), whereas those of OSTα, ABCG2 and ABCG8 were all decreased by approximately 40%, (p<0.05) in human cholestatic livers. Hepatic nuclear receptors, VDR, HNF4α, RXRα and RARα, were induced (approximately 2.0-fold, (p<0.05) whereas FXR levels were markedly reduced to 44% of control, (p<0.05) in human obstructive cholestasis. There was a significantly positive correlation between the reduction in FXR mRNA and UGT2B4/7, SULT2A1, GSTA1, ABCG2/8 mRNA levels in livers of obstructive cholestatic patients (p<0.05)., Conclusions: The levels of hepatic detoxification enzymes were significantly decreased in human obstructive cholestasis, and these decreases were positively associated with a marked reduction of FXR levels. These findings are consistent with impaired detoxification ability in human obstructive cholestasis.

Importance: Acitretin is a systemic retinoid that is used in dermatology for a variety of conditions. A well-recognized potential adverse event from acitretin is elevated transaminases, indicating acute hepatocyte damage. Less well known is the possible cholestatic injury that can occur, signaled by elevated γ-glutamyltransferase (GGT) and alkaline phosphatase (ALP)., Observations: Our patient presented with an acute acitretin-induced hepatitis with a mixed pattern of elevated transaminases as well as GGT and ALP. A literature review demonstrated that most cases of acitretin-induced hepatitis, outside clinical trials, describe patients with a similar mixed hepatitis pattern., Conclusions: This is the first literature review on acitretin-induced hepatitis. Although acitretin-induced hepatoxicity is rare, the seemingly unusual presentation of a mixed pattern of hepatocyte injury and cholestasis may be more common than previously thought. The findings should encourage clinicians not only to monitor transaminases but also to consider measuring cholestatic enzymes (ALP/GGT) in patients with transaminase abnormalities., (© 2014 Canadian Dermatology Association.)

Cholestasis, which is a component of many liver diseases, is often associated with symptoms that resemble clinical adrenal insufficiency. This work aimed to study the histomorphometrical and electron microscopic structure of adrenocorticocytes after surgical induction of bile duct resection (BDR) in adult female albino rats. Sixty rats were randomly divided into control, BDR and sham-operated groups. Six weeks after surgery, the blood serum of the rats was examined biochemically, and the suprarenal cortexes were prepared for histological, morphometrical and statistical studies. The BDR group showed a highly significant increase in bilirubin and serum alkaline phosphatase levels, whereas aldosterone and cortisol levels were highly significantly decreased. The area percentages of positive immunoreactions for P53, cyclooxygenase II (COX-II) and inducible nitric oxide synthase (INOS) revealed highly significant increases in the BDR group. Electron microscopic examination of the BDR group showed marked cytoplasmic vacuolations, large lipid droplets, swollen mitochondria and many small dark nuclei in the adrenocorticocytes. The zona fasciculata had heterogeneously electron-dense mitochondria and dilated smooth endoplasmic reticulum. Some of the zona reticularis cells contained lipofuscin pigments. The surgical induction of BDR produced deleterious effects on the structure and function of the adrenocorticocytes. A long-term study using different animal species is recommended for further examination.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Flavin-containing monooxygenase-3 (FMO3) catalyzes metabolic reactions similar to cytochrome P450 monooxygenase, however, most metabolites of FMO3 are considered non-toxic. Recent findings in our laboratory demonstrated Fmo3 gene induction following toxic acetaminophen (APAP) treatment in mice. The goal of this study was to evaluate Fmo3 gene expression in other diverse mouse models of hepatic oxidative stress and injury. Fmo3 gene regulation by Nrf2 was also investigated using Nrf2 knockout (Nrf2 KO) mice. In our studies, male C57BL/6J mice were treated with toxic doses of hepatotoxicants or underwent bile duct ligation (BDL, 10 days). Hepatotoxicants included APAP (400 mg/kg, 24-72 h), alpha-naphthyl isothiocyanate (ANIT; 50 mg/kg, 2-48 h), carbon tetrachloride (CCl4; 10 or 30 μL/kg, 24 and 48 h) and allyl alcohol (AlOH; 30 or 60 mg/kg, 6 and 24 h). Because oxidative stress activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), additional studies investigated Fmo3 gene regulation by Nrf2 using Nrf2 knockout (Nrf2 KO) mice. At appropriate time-points, blood and liver samples were collected for assessment of plasma alanine aminotransferase (ALT) activity, plasma and hepatic bile acid levels, as well as liver Fmo3 mRNA and protein expression. Fmo3 mRNA expression increased significantly by 43-fold at 12 h after ANIT treatment, and this increase translates to a 4-fold change in protein levels. BDL also increased Fmo3 mRNA expression by 1899-fold, but with no change in protein levels. Treatment of mice with CCl4 decreased liver Fmo3 gene expression, while no change in expression was detected with AlOH treatment. Nrf2 KO mice are more susceptible to APAP (400mg/kg, 72 h) treatment compared to their wild-type (WT) counterparts, which is evidenced by greater plasma ALT activity. The Fmo3 mRNA and protein expression increased in Nrf2 KO mice after APAP treatment. Collectively, not all hepatotoxicants that produce oxidative stress alter Fmo3 gene expression. Along with APAP, toxic ANIT treatment in mice markedly increased Fmo3 gene expression. While BDL increased the Fmo3 mRNA expression, the protein level did not change. The discrepancy with Fmo3 induction in cholestatic models, ANIT and BDL, is not entirely clear. Results from Nrf2 KO mice with APAP suggest that the transcriptional regulation of Fmo3 during liver injury may not involve Nrf2., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Background & Aims: Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals., Methods: We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers., Results: Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions., Conclusions: In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short-term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC-mediated regulation of plasma membrane localization of Na+-taurocholate cotransporting polypeptide (NTCP) and multidrug resistance-associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine-rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections., (© 2014 by the American Association for the Study of Liver Diseases.)

Background: The Danning tablets (DNts) is commonly prescribed in China as a cholagogic formula. Our previous studies showed that DNts exerted the protective effect on α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis in a dose-dependent mannar. However, the detailed molecular mechanisms of DNts against ANIT-induced cholestasis are still not fully explored., Methods: Danning tablet (3 g/kg body weight/day) was administered orally to experimental rats for seven days before they were treated with ANIT (60 mg/kg daily via gastrogavage) which caused cholestasis. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and total bile acid (TBA) were measured to evaluate the protective effect of Danning tablet at 12, 24 and 48h after ANIT treatment. Meanwhile, total bilirubin or total bile acid in the bile, urine and liver were also measured at 48h after ANIT treatment. Furthermore, the hepatic or renal mRNA and protein levels of metabolic enzymes and transports were investigated to elucidate the protective mechanisms of Danning tablet against ANIT-induced cholestasis., Results: In this study, we found that DNts significantly attenuated translocation of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane into an intracellular and up-regulated the hepatic mRNA and protein expressions of metabolic enzymes including cytochrome P450 2b1(Cyp2b1) and uridine diphosphate-5¢- glucuronosyltransferase (Ugt1a1)) and transporters including bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2)) as well as renal organic solute transporter beta (Ostβ), accompanied by further increase in urinary and biliary excretion of bile acid and bilirubin., Conclusions: DNts might promote bile acid and bilirubin elimination by regulating the expressions of hepatic and renal transporters as well as hepatic metabolic enzymes.

Background and Study Aims: Measuring serum superoxide dismutase (SOD) levels in infants and children having acute or chronic liver disease of different aetiologies, and correlating these levels with disease aetiology in an attempt to clarify the role of SOD as an antioxidant in these diseases., Patients and Methods: We prospectively enrolled 58 infants and children and divided them into four groups: Group I, 24 patients with surgical cholestasis; group II, 11 patients with medical cholestasis; group III, nine patients with autoimmune chronic hepatitis; and group IV, 14 patients with viral hepatitis. Forty healthy age- and sex-matched children served as controls. Serum SOD activity was measured in all patients and controls using spectrophotometry., Results: The level of SOD showed a statistically significant increase in patients with medical cholestasis compared to healthy controls (p<0.0001). SOD activity of other groups showed no significant difference compared to controls., Conclusions: Significantly increased serum SOD in infants and children with medical cholestasis is probably consequent to its increase in liver tissue in response to the liberation of reactive oxygen species. This suggests that products of free radical reactions might be involved in the pathogenesis and/or progression of medical cholestasis, and that SOD might attempt to minimise the liver injury., (Copyright © 2014 Arab Journal of Gastroenterology. Published by Elsevier Ltd. All rights reserved.)

Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome (OMIM 208085) is an autosomal recessive disorder that is caused by mutations in 2 interacting genes VPS33B and VIPAS39. Mutations in VPS33B gene account for most cases of ARC. As low or normal gamma-glutamyl transpeptidase (GGT) activity has been described in all patients with ARC syndrome identified so far, ARC syndrome is a possible diagnosis for low GGT cholestasis. Here we describe a Chinese patient with neonatal cholestasis and a high GGT level in three consecutive tests. She had other typical manifestations of ARC syndrome, including arthrogryposis multiplex congenita, renal involvement and ichthyosis. Genetic study of the VPS33B gene further confirmed the diagnosis by identification of compound heterozygosity of two known disease-causing mutations, c.403+2T > A and c.1509-1510insG. The mechanism of high GGT in this patient is unclear. Nevertheless, this case indicates that ARC syndrome cannot be excluded from the differential diagnosis of neonatal cholestasis even if high GGT activity is found.

Cholestasis is characterized by the elevation of serum total bile acids (TBA), which leads to the production of both free radicals and oxidative stress. Although they do not share the same mechanisms, membrane glycosphingolipids (GSL) and the antioxidant enzyme heme oxygenase-1 (HMOX1) both act against the pro-oxidative effect of TBA. The aim of the study was to assess the role of HMOX on GSL redistribution and composition within hepatocytes in the rat model of estrogen-induced cholestasis. Compared to the controls, an increase of total gangliosides in the liver homogenates of the cholestatic group (P=0.001) was detected; further, it paralleled along with the activation of their biosynthetic b-branch pathway (P<0.01). These effects were partially prevented by HMOX activation. Cholestasis was accompanied by a redistribution of GM1 ganglioside from the cytoplasm to the sinusoids; while HMOX activation led to the retention of GM1 in the cytoplasm (P=0.014). Our study shows that estrogen-induced cholestasis is followed by changes in the synthesis and/or distribution of GSL. These changes are not only triggered by the detergent power of accumulated TBA, but also by their pro-oxidant action. Increases in the antioxidant defenses might represent an important supportive therapeutic measure for patients with cholestatic liver disease.

Glucuronidation, catalyzed by uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic bile acids (BAs). We aimed to (i) characterize the circulating BA-glucuronide (BA-G) pool composition in humans, (ii) determine how sex and UGT polymorphisms influence this composition, and (iii) analyze the effects of the lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and postfenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G (CDCA-3G) concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of five BA-G species, including CDCA-3G, and upregulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrated that fenofibrate stimulates BA glucuronidation in humans and thus reduces BA toxicity in the liver.

A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin-proline was used as an ECL coreagent to enhance Ru(bpy)3 (2+) ECL signals efficiently. The detection limit for proline was 2.88 × 10(-6) m (signal-to-noise ratio 3), which was equal to 9.60 × 10(-8) units of LAP being used to catalyze leucin-proline for 1 min. The Michaelis constant Km (2.07 × 10(-2) mol/L) and the maximum reaction velocity Vmax (1.06 × 10(-5) mol/L/min) of LAP for leucin-proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: The aim of this study was to evaluate the preventive and therapeutic potential of hyperbaric oxygen therapy (HBO) on the liver tissue against bile duct ligation (BDL)-induced oxidative damage and fibrosis in rats., Materials and Methods: We divided 32 adult male Sprague Dawley rats into four groups: sham, sham plus HBO, BDL, and BDL plus HBO; each group contained eight animals. We placed the sham plus HBO and BDL plus HBO groups in an experimental hyperbaric chamber in which we administered pure oxygen at 2.5 atmospheres absolute 100% oxygen for 90 min on 14 consecutive days., Results: The application of BDL clearly increased the tissue malondialdehyde level, myeloperoxidase activity, and hydroxyproline content and decreased the antioxidant enzymes (superoxide dismutase and catalase activities) and glutathione level. Hyperbaric oxygen therapy treatment significantly decreased the elevated tissue malondialdehyde level, myeloperoxidase activity, and hydroxyproline content and increased the reduced superoxide dismutase and catalase activities and glutathione level in the tissues. The changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with HBO attenuated alterations in liver histology. Alpha smooth muscle actin, cytokeratin-positive ductular proliferation, and the activity of terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling in the BDL decreased with HBO treatment., Conclusions: The data indicate that HBO attenuates BDL-induced oxidative injury, hepatocytes damage, bile duct proliferation, and fibrosis. The hepatoprotective effect of HBO is associated with antioxidative potential., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: The aim of this study was to assess the prevalence of abnormal liver function tests (LFTs) and the associated clinical profile and outcome(s) in acute decompensated heart failure (ADHF) patients. Alteration in LFTs is a recognized feature of ADHF, but prevalence and outcomes data from a broad contemporary cohort of ADHF are scarce and the mechanism(s) of ADHF-induced cholestasis is unknown., Methods and Results: We conducted a post hoc analysis of SURVIVE, a large clinical trial including ADHF patients treated with levosimendan or dobutamine. All LFTs were available in 1134 patients at baseline. Abnormal LFTs were seen in 46% of ADHF patients: isolated abnormal alkaline phosphatase (AP) was noted in 11%, isolated abnormal transaminases in 26%, and a combination of abnormal AP and transaminases in 9%. Abnormal AP was associated with marked signs of systemic congestion and elevated right-sided filling pressure. Abnormal AP had no relationship with 31-day mortality but was associated with worse 180-day mortality (23.5 vs. 34.9%, P = 0.001 vs. patients with normal AP). Abnormal transaminases were associated with clinical signs of hypoperfusion and with greater 31-day and 180-day mortality compared with normal transaminase profiles (17.6 vs. 8.4% and 31.6 vs. 22.4%, respectively; both P < 0.001). There was no additive value of abnormal AP plus abnormal transaminase on a long-term outcome., Conclusion: Abnormal LFTs were present in about a half of patients presenting with ADHF treated with inotropes. Abnormal AP and abnormal transaminases were associated with specific clinical, biological, and prognostic features, including a short-term overmortality with increased transaminases but not with biological signs of cholestasis, in ADHF patients.

Liver fibrosis results from chronic injury followed by activation of macrophages and fibrogenic cells like myofibroblasts and activated hepatic stellate cells. These fibrogenic cells express α-smooth muscle actin (α-SMA) and produce excessive extracellular matrix (ECM), with disorganization and loss of function of hepatic parenchyma. It is known that increased levels of metalloproteinases (MMPs) in liver fibrosis are associated with reduction of the pathologic ECM and fibrosis resolution. Recently, it has been shown that bone marrow mononuclear cells (BMMNCs) may reduce collagen and α-SMA expression, and ameliorate liver function in cholestatic rats. Therefore, this study aimed to analyze MMP-2, MMP-9 and MMP-13, and tissue inhibitors of MMPs (TIMPs)-1 and TIMP-2 in the liver of cholestatic rats transplanted with BMMNC. Animals were divided into normal rats, cholestatic rats obtained after 14 and 21 days of bile duct ligation (BDL), and rats obtained after 14 days of BDL that received BMMNCs and were killed after 7 days. MMP and TIMP expression was assessed by Western blotting, along with α-SMA, CD68 and CD11b expression by confocal microscopy. Western blotting analysis showed that 14-day BDL animals had significantly reduced amounts of MMP-2 and MMP-13, but increased amounts of MMP-9 compared to normal rats. After 21 days of BDL, overall MMP amounts were decreased and TIMPs were increased. BMMNC transplantation significantly increased MMP-9 and MMP-13, and decreased TIMP expression. Increased MMP activity was confirmed by zymography. MMP-9 and MMP-13 were expressed by macrophages near fibrotic septa, suggesting BMMNC may stimulate MMP production in fibrotic livers, contributing to ECM degradation and hepatic regeneration., (© 2013 S. Karger AG, Basel.)

Cholestasis develops either from a defect in bile synthesis, impairment in bile secretion, or obstruction to bile flow, and is characterized by an elevated serum alkaline phosphatase and gamma-glutamyltransferase disproportionate to elevation of aminotransferase enzymes. Key elements to the diagnostic workup include visualization of the biliary tree by cholangiography and evaluation of liver histology. The hope is that recent advances in understanding the genetic factors and immune mechanisms involved in the pathogenesis of cholestasis will lead to newer therapeutic interventions in the treatment of these diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Obstetric cholestasis (OC) is a cholestatic disorder with a prominent genetic background including variation in diverse hepatobiliary lipid transporters, such as ABCB4 (phospholipids) and ABCB11 (bile salts). Given a marked hepatocellular dysfunction in an OC patient indicated by > 40-fold rise in alanine aminotransferase activity and minor gamma-glutamyl transpeptidase increases, we performed genotyping of candidate gene variants associated with adult cholestatic phenotypes. Genetic analysis revealed the heterozygous ABCB4 mutation p.R590Q, the ABCB11 variant p.V444A and the lithogenic ABCG8 variant p.D19H. Aggregation of multiple hepatobiliary transporter variants is rare in OC, and may cooperate to negatively modulate hepatobiliary transport capacities.

Background: Nonanastomotic biliary strictures (NAS) are a serious complication after orthotopic liver transplantation (OLT). Matrix metalloproteinases (MMPs) are involved in connective tissue remodelling in chronic liver disease and complications after OLT., Aim: To evaluate the relationship between MMP-2 and MMP-9 gene polymorphisms and NAS., Methods: MMP-2 (-1306 C/T) and MMP-9 (-1562 C/T) gene promoter polymorphisms were analysed in 314 recipient-donor combinations. Serum levels of these MMPs were determined in subgroups of patients as well. NAS were identified with various radiological imaging studies performed within 4 years after OLT and defined as any stricture, dilation or irregularity of the intra- or extrahepatic bile ducts of the liver graft followed by an intervention, after exclusion of hepatic artery thrombosis and anastomotic strictures., Results: The average incidence of NAS was 15%. The major clinical risk factor for the development of NAS was PSC in the recipient. The presence of the MMP-2 CT genotype in donor and/or recipient was associated with a significantly higher incidence of NAS, up to 29% when both donor and recipient had the MMP-2 CT genotype (P=0.003). In the multivariate analyses, pre-OLT PSC (hazard ratio 2.1, P=0.02) and MMP-2 CT genotype (hazard ratio 3.5, P=0.003) were found to be independent risk factors for the development of NAS after OLT. No obvious association was found between NAS and the MMP-9 genotype and serum levels of the MMPs., Conclusion: MMP-2 CT genotype of donor and recipient is an independent risk factor, in addition to PSC, for the development of NAS after OLT., (© 2011 John Wiley & Sons A/S.)

A 75 year old woman presented with long-lasting pruritus and elevation of serum levels of alkaline phosphatase and gamma-glutamyl transpeptidase (GGT). Potentially hepatotoxic drugs were stopped; an abdominal ultra-sonography was normal. Antimitochondrial antibodies were elevated and are the serologic hallmark of primary biliary cirrhosis. The disease can progress from an asymptomatic inflammation to a cirrhosis. The disease-modifying treatment consists in ursodeoxycholic acid 13-15 mg/kg per day. Concomitant to the primary biliary cirrhosis hypothyroidism, osteoporosis, malabsorption and dyslipidemia can occur.