Reduced Bile Flow Research Articles

Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells.

Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells. Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells. Expression of hepatobiliary transporters varies during hypoxia and reoxygenation. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.

Collodion Baby And Cholestasis: A Rare Case Report

Collodion baby with cholestasis is an uncommon condition. The term "collodion baby" refers to newborns with thick skin covering their entire body. Neonatal cholestasis is a form of conjugated cholestasis hyperbilirubinemia caused by reduced bile flow. The condition causes persistent jaundice (over two weeks) and elevated conjugated bilirubin. Genetic abnormalities or gene mutations have been reported in neonatal ichthyosis syndrome with cholestasis. A case report of a collodion baby, a rare congenital condition characterized by a parchment-like membrane covering the entire body, was brought to Dr. Soetomo General Academic Hospital. Prolonged jaundice was one of the other findings. A skin biopsy found characteristics consistent with the diagnosis of collodion in a newborn. The paediatric team managed this case, which was treated in collaboration with a dermatologist. This case showed that newborn collodion syndrome required skin care, cholestasis screening, and jaundice management from birth.

Angiotensin II type 1 receptor localizes at the blood–bile barrier in humans and pigs

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin–angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood–bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability.

Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14−/− and car14+/+ mice was cannulated and hepatic HCO3− output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14−/− and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3− output were significantly reduced in car14−/− mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14−/− mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3− output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3− against luminal noxi to the cholangiocyte.

Downregulation of TLR4-NF-?B-p38 MAPK Signalling in Cholestatic Rats Treated with Cranberry Extract.

<b>Background and Objective:</b> Cholestasis is a liver disease that occurs when bile flow is restricted or blocked. Estrogen-induced cholestasis is marked by a reduction in bile flow and the accumulation of bile acids in the liver as well as liver damage. The aim was to evaluate the hepatoprotective effect on EE-induced cholestasis in rats of Cranberry Water Extract (CWE). <b>Materials and Methods:</b> Adult albino rats weighing approximately 150±10 g were divided into six groups of six animals each. As control groups, three groups (I, II and IV) and three experimental groups were used (III, V, VI). <b>Results:</b> Oral administration for 15 days of CWE (150 mg kg<sup>1</sup> b.wt.) in EE-treated rats (100 μg kg<sup>1</sup> 5 days b.wt.) improved serum cholesterol, bile acid and TBIL as well as hepatic SOD and GPx significantly. Also, CWE inhibited ALP, ALT, γ-GT activity as well as levels of TNF-α, NO, MMP-2 and MMP-9 and MDA in comparison with the EE treatment rats. On the other hand, the liver TLR4, NF-κB and p38MAPK gene expression was down regulated group of rats administrated with cranberry extract when compared with the EE-treated rats. CWE's prophylactic action II is more pronounced than prophylactic one. The hepatoprotective effects of cranberry in restoring normal liver functional ability were also supported by histopathological examination of liver tissues. <b>Conclusion:</b> The results show clearly that cranberry extract has a strong prophylactic effect in EE-induced cholestasis by normalizing the levels of TLR4, NF-κB and p38MAPK gene expression.

The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX, BCL-2, BAD, PGC-1α and TFAM Genes in the Hippocampus of Male Wistar Rats.

Cholestasis is a bile flow reduction that is induced following Bile Duct Ligation (BDL). Cholestasis impairs memory and induces apoptosis. Apoptosis consists of two pathways: intrinsic and extrinsic. The intrinsic pathway is modulated by BCL-2 (B cell lymphoma-2) family proteins. BCL-2 (a pro-survival BCL-2 protein) has anti-apoptotic effect, while BAD (BCL-2-associated death) and BAX (BCL-2-associated X), the other members of BCL-2 family have pro-apoptotic effect. Furthermore, TFAM (mitochondrial transcriptional factor A) is involved in transcription and maintenance of mitochondrial DNA and PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) is a master regulator of mitochondrial biogenesis. On the other hand, NeuroAid is a Traditional Chinese Medicine with neuroprotective and anti-apoptosis effects. In this study, we evaluated the effect of cholestasis on spatial memory and expression of BCL-2, BAD, BAX, TFAM, and PGC-1α in the hippocampus of rats. Additionally, we assessed the effect of NeuroAid on cholestasis-induced cognitive and genetic alterations. Cholestasis was induced by BDL surgery and NeuroAid was injected intraperitoneal at the dose of 0.4mg/kg. Furthermore, spatial memory was evaluated using Morris Water Maze (MWM) apparatus. The results showed cholestasis impaired spatial memory, increased the expression of BAD and BAX, decreased the expression of TFAM and PGC-1α, and did not alter the expression of BCL-2. Also, NeuroAid decreased the expression of BAD and BAX and increased the expression of TFAM, PGC-1α, and BCL-2. In conclusion, cholestasis impaired spatial memory and increased the expression of pro-apoptotic genes. Also, cholestasis decreased the expression of TFAM and PGC-1α. Interestingly, NeuroAid restored the effects of cholestasis.

The effect of Crocin on TFAM and PGC-1α expression and Catalase and Superoxide dismutase activities following cholestasis-induced neuroinflammation in the striatum of male Wistar rats

Bile secretion is a physiological function that is disrupted following Bile Duct Ligation (BDL) and induces cholestasis. Cholestasis is a bile flow reduction that induces apoptosis, oxidative stress, and inflammation, and alters the expression of genes. Evidence shows the relationship between cholestasis and neuroinflammation. Cholestasis via attenuating mitochondrial biogenesis and anti-oxidant activity can induce neuroinflammation and apoptosis. Mitochondrial transcriptional factor A (TFAM) and Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) are involved in mitochondrial biogenesis, and TFAM, PGC-1α, Catalase (CAT), and Superoxide dismutase (SOD) have a role in upregulating antioxidant pathways. On the other hand, many studies have shown the neuroprotective effects of Crocin, the water-soluble carotenoid of Saffron (Crocus sativus L.). In this study, we aimed to investigate the effect of Crocin on the level of TFAM, PGC-1α, CAT, and SOD following cholestasis-induced neuroinflammation in the rat's striatum. Cholestasis was induced by BDL surgery and administration of Crocin was intraperitoneal, at the dose of 30mg/kg every day, 24h after BDL surgery up to thirty days. The results showed that TFAM, PGC-1α, and SOD were decreased following cholestasis; while, CAT was increased. In addition, Crocin restored the effects of cholestasis on the level of TFAM, PGC-1α, and SOD. In conclusion, Crocin may have improvement effects on cholestasis-induced neuroinflammation in the rat's striatum.

The effects of ursodeoxycholic acid on sepsis-induced cholestasis management in an animal model

ObjectivesCholestasis refers to a reduction in bile flow from the liver into the biliary system. Ursodeoxycholic acid (UDCA) is commonly used for the treatment of hepatic cholestasis. This study aimed to explore the role of UDCA in the treatment and prevention of lipopolysaccharide (LPS)-induced cholestasis. MethodsSixty male albino rats were randomly classified into five groups of 12 rats each: the control group (received saline and water), UDCA group (received UDCA), LPS group (received LPS), treatment group (received LPS followed by UDCA), and prevention group (received UDCA followed by LPS). Changes in gamma-glutamyl transferase (GGT), plasma aspartate transferase (AST), plasma alkaline transferase (ALT), plasma alkaline phosphatase (ALP), total bilirubin (TBIL), hepatocyte apoptosis, immunomodulatory activity, plasma pro-inflammatory cytokines (TNF-α, IL-1α, and IL-4), and liver histology were assessed. ResultsUDCA improved serum liver chemical markers (GGT, ALP, and AST) in both the prevention and treatment groups (p < 0.05 and p < 0.05, respectively). CD3 count was higher in the UDCA treatment group compared to the LPS group (p < 0.001). UDCA caused a reduction in plasma TNF-α in the prevention group (P < 0.05); however, it had no effect on the treatment group, as compared to the LPS group. Similarly, UDCA had no effect on IL-1α or IL-4. UDCA treatment resulted in improved liver histological features and a significant reduction in liver tissue apoptosis in both the treatment and prevention groups, as compared to the LPS group (p = 0.013 and p = 0.002, respectively). ConclusionsThis study provides evidence of the effectiveness of UDCA for the treatment and prevention of sepsis-induced cholestasis.

Possible Protective Effect of Onion Supplementation on Hepatic Functional and Structural Alterations Induced by Cholestasis

Abstract Background Cholestasis is the obstruction or the reduction in bile flow that results in intrahepatic accumulation of bile constituents, which progresses to develop liver pathology. Common bile duct ligation (BDL) in rodents is an experimental model of cholestasis that has been carried out in research for many years. BDL model of cholestatic liver injury involves other mechanisms, including oxidative stress, inflammation, and fibrogenesis. Antioxidant, antiinflammatory or antiapoptotic properties gained much interest for the amelioration of liver dysfunction. Aim the aim of this study is to assess the possible protective effects of onion supplementation on hepatic structural and functional alterations induced by BDL in rats, which reflect the effects of cholestasis resulting from intrahepatic accumulation of bile. Methods Thirty adult female Wistar rats were randomly and equally allocated into three groups: (1) control group, (2) BDL group; subjected to ligation of the common bile duct and (3) Onion-supplemented BDL groups (O-BDL). Both control and BDL groups received distilled water (solvent for onion powder) daily by gavage for 4 weeks. Onion-supplemented BDL group (O-BDL); subjected to ligation of the common bile duct and then received 500 mg/kg of onion powder dissolved in distilled water, daily by gavage for 4 weeks. At the end of the experimental period, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin, total proteins, total antioxidant capacity (TAC), tumor necrosis factor-α (TNF-α), and hepatic tissue level of malondialdehyde (MDA) and transforming growth factor-β (TGF-β) were measured for all groups. In addition, histopathological examination of liver tissue samples was performed for the three groups. Results Plasma levels of ALT, AST, ALP, direct bilirubin, TNF-α and hepatic tissue levels of MDA and TGF-β were significantly increased and TAC was significantly decreased in the BDL group compared to the control group. In addition, altered architecture was detected in hepatic tissue samples of BDL group. Onion supplementation significantly decreased the plasma levels of ALT, AST, ALP, direct bilirubin, TNF-α and hepatic tissue levels of MDA and TGF-β in the O-BDL group when compared to the BDL group. Total proteins level was not significantly different among all the studied groups. In addition in O-BDL group, histopathological examination of liver revealed near normal structure of hepatic tissue. Conclusion BDL induces hepatic structural alterations and functional disturbances. Onion supplementation inhibits inflammation and oxidative insults that associate BDL, and subsequently protects against BDL-induced liver injury.

Effect of cholestasis and NeuroAid treatment on the expression of Bax, Bcl-2, Pgc-1α and Tfam genes involved in apoptosis and mitochondrial biogenesis in the striatum of male rats.

Cholestasis means impaired bile synthesis or secretion. In fact, it is a bile flow reduction following Bile Duct Ligation (BDL). Cholestasis has a main role in necrosis and apoptosis. Apoptosis is a form of programmed cell death that has intrinsic and extrinsic pathways. The intrinsic pathway is mediated by Bcl-2 (B cell lymphoma-2) proteins which integrate death and survival signals. Bcl-2 has anti-apoptotic and Bax has pro-apoptotic effects. Also, striatum is one of the brain regions that has high expressions of Bcl-2 proteins. Moreover, Tfam and Pgc-1α are involved in mitochondrial biogenesis. On the other hand, NeuroAid, is a drug that has neuroprotective and anti-apoptosis effects. In this study, using quantitative PCR, we measured the expression of all these genes in the striatum of male rats following BDL and NeuroAid administration. Results showed, BDL increased the expression of Bax and Tfam and decreased the expression of Bcl-2. NeuroAid restored the effect of BDL on the expression of Bax, while did not alter the effect of BDL on Bcl-2. In addition, it increased the expression of Tfam that was previously elevated by BDL and raised the expression of Tfam in normal rats. Both BDL and NeuroAid, had no effect on Pgc-1α. In conclusion, cholestasis increased the expression of Bax and decreased the expression of Bcl-2, and this effect may have related to enhanced susceptibility of mitochondrial pathways following oxidative stress. Tfam expression was increased following cholestasis and this effect may have related to cellular compensatory mechanisms against high accumulation of free radicals or mitochondrial biogenesis failure. Furthermore, NeuroAid may play a role against apoptosis and can be used to increase mitochondrial biogenesis.

Possible Protective Effect of Onion Supplementation on Hepatic Functional and Structural Alterations Induced by Cholestasis

AbstractBackground: Cholestasis is the obstruction or the reduction in bile flow that results in intrahepatic accumulation of bile constituents, which progresses to develop liver pathology. Common bile duct ligation (BDL) in rodents is an experimental model of cholestasis that has been carried out in research for many years. BDL model of cholestatic liver injury involves other mechanisms, including oxidative stress, inflammation, and fibrogenesis. Antioxidant, anti-inflammatory or antiapop-totic properties gained much interest for the amelioration of liver dysfunction.Aim of Study: The aim of this study is to assess the possible protective effects of onion supplementation on hepatic struc-tural and functional alterations induced by BDL in rats, which reflect the effects of cholestasis resulting from intrahepatic accumulation of bile.Material and Methods: Thirty adult female Wistar albino rats were randomly and equally allocated into three groups: (1) Control group, (2) BDL group; subjected to ligation of the common bile duct and (3) Onion-supplemented BDL groups (O-BDL). Both control and BDL groups received distilled water (solvent for onion powder) daily by gavage for 4 weeks. Onion-supplemented BDL group (O-BDL); subjected to ligation of the common bile duct and then received 500mg/kg of onion powder dissolved in distilled water, daily by gavage for 4 weeks. At the end of the experimental period, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin, total proteins, total antioxidant capacity (TAC), tumor necrosis factor-a (TNF-a), and hepatic tissue level of malondialdehyde (MDA) and transforming growth factor-b1 (TGF-b1) were measured for all groups. In addition, his-topathological examination of liver tissue samples was per-formed for the three groups.Results: Plasma levels of ALT, AST, ALP, direct bilirubin, TNF-a and hepatic tissue levels of MDA and TGF-b1 were significantly increased and TAC was significantly decreased in the BDL group compared to the control group. In addition, altered architecture was detected in hepatic tissue samples of BDL group. Onion supplementation significantly decreased the plasma levels of ALT, AST, ALP, direct bilirubin, TNF-a and hepatic tissue levels of MDA and TGF-b1 in the O-BDL group when compared to the BDL group. Total proteins level was not significantly different among all the studied groups. In addition in O-BDL group, histopathological exam-ination of liver revealed near normal structure of hepatic tissue.Conclusion: BDL induces hepatic structural alterations and functional disturbances. Onion supplementation inhibits inflammation and oxidative insults that associate BDL, and subsequently protects against BDL-induced liver injury.

Abstract 2333: Aspirin may extend biliary tract cancer survival: Results from population-based cohort

Abstract Background: Biliary tract cancers (BTCs) are rare, but lethal cancers with a median survival of &amp;lt;12 months. Because of this poor prognosis, there is a critical need for treatments that extend survival. Cyclooxygenase-2 (COX-2) is an enzyme responsible for producing prostaglandin E (PGE). PGEs increase mucin production and reduce bile flow in the gallbladder, leading to gallstones, a precursor to gallbladder cancer. Overexpression of COX-2 due to chronic inflammation from cholangiocarcinoma has been found to enhance PGE production and promote tumor growth. Previous research suggests that COX-2 inhibitors, such as aspirin, may slow cancer cell growth. We investigated aspirin use following diagnosis and overall survival in a large cohort of patients with BTCs. Methods: All patients diagnosed with incident biliary tract cancer between 1990 to 2017 were selected from the population-based United Kingdom’s Clinical Practice Research Datalink. Site-specific associations between aspirin use after BTC diagnosis and overall survival was analyzed using Cox proportional hazards models with aspirin use modeled as time-dependent, adjusting for age at diagnosis, sex, comorbidities, history of aspirin use, and use of statins, using follow-up time as the time scale. Results: We identified 3,211 biliary tract cancer cases in which 2,694 (84%) deaths occurred after an overall median follow-up time of 5 months. The table presents the number of cases by cancer subtype, deaths, and median survival time. Aspirin use following a diagnosis of BTC was associated with a reduction in mortality for each of the BTC sites: gallbladder, HR 0.41 (95% CI: 0.28, 0.61); cholangiocarcinoma, HR 0.46 (95% CI: 0.36, 0.60); ampulla of Vater, HR 0.24 (95% CI: 0.11, 0.51); and mixed, HR: 0.51 (95% CI: 0.33, 0.78). Conclusions: The use of aspirin after BTC diagnosis was associated with a lower risk of overall mortality. These results warrant additional studies to understand the mechanisms underlying improved survival with aspirin use. Table.Association between post-BTC diagnosis aspirin use versus no use and survival in the UK CPRD datasetCancer siteBTC patients N (%)Mortality N (%)Median survival time in months (interquartile range)HR (95% CI)*Gallbladder668 (23)555 (83)3.9 (2 - 11)0.41 (0.28, 0.61)Cholangiocarcinoma1,560 (53)1321 (85)4.8 (2 - 12)0.46 (0.36, 0.60)Ampulla of Vater224 (8)142 (63)9 (4 - 21)0.24 (0.11, 0.51)Mixed484 (16)401 (83)5 (2 - 13)0.51 (0.33, 0.78)*Adjusted for age at diagnosis, comorbidities, prior aspirin use, and use of statins.Abbreviations: CPRD, Clinical Practice Research Datalink; CI, confidence interval; HR, hazard ratio; N, number; and UK, United Kingdom. Citation Format: Sarah S. Jackson, Bin Zhu, Ruth Pfeiffer, Zhiwei Liu, Shahinaz Gadalla, Jill Koshiol. Aspirin may extend biliary tract cancer survival: Results from population-based cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2333.

Resveratrol modifies biliary secretion of cholephilic compounds in sham-operated and cholestatic rats.

AIMTo investigate the effect of resveratrol on biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.METHODSResveratrol (RSV) or saline were administered to rats by daily oral gavage for 28 d after sham operation or reversible bile duct obstruction (BDO). Bile was collected 24 h after the last gavage during an intravenous bolus dose of the Mdr1/Mrp2 substrate azithromycin. Bile acids, glutathione and azithromycin were measured in bile to quantify their level of biliary secretion. Liver expression of enzymes and transporters relevant for bile production and biliary secretion of major bile constituents and drugs were analyzed at the mRNA and protein levels using qRT-PCR and Western blot analysis, respectively. The TR-FRET PXR Competitive Binding Assay kit was used to determine the agonism of RSV at the pregnane X receptor.RESULTSRSV increased bile flow in sham-operated rats due to increased biliary secretion of bile acids (BA) and glutathione. This effect was accompanied by the induction of the hepatic rate-limiting transporters for bile acids and glutathione, Bsep and Mrp2, respectively. RSV also induced Cyp7a1, an enzyme that is crucial for bile acid synthesis; Mrp4, a transporter important for BA secretion from hepatocytes to blood; and Mdr1, the major apical transporter for xenobiotics. The findings were supported by increased biliary secretion of azithromycin. The TR-FRET PXR competitive binding assay confirmed RSV as a weak agonist of the human nuclear receptor PXR, which is a transcriptional regulator of Mdr1/Mrp2. RSV demonstrated significant hepatoprotective properties against BDO-induced cirrhosis. RSV also reduced bile flow in BDO rats without any corresponding change in the levels of the transporters and enzymes involved in RSV-mediated hepatoprotection.CONCLUSIONResveratrol administration for 28 d has a distinct effect on bile flow and biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.

Adenovirus-mediated human aquaporin-1 expression in hepatocytes improves lipopolysaccharide-induced cholestasis.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Genetic loss of the muscarinic M3 receptor markedly alters bile formation and cholestatic liver injury in mice.

Hepatic innervation represents a potentially underestimated regulator of liver function and regeneration. The muscarinic 3 receptor (M3 -R) is the primary cholangiocyte receptor for the afferent parasympathetic innervation of bile ducts. We aimed to determine the specific role of the M3 -R in bile formation and models for cholestatic liver disease in mice. We compared bile flow and composition in M3 -R knock-out mice (M3 -R-/- ) and wild type littermates (WT). Furthermore, we compared liver inury of M3 -R-/- and WT mice after 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding, a well-characterized preclinical model of cholestatic liver disease. To analyze the possible role of the M3 -R as a therapeutic target, we treated 4-week-old Mdr2-/- mice, a preclinical model for sclerosing cholangitis, with the M3 -R agonist bethanechol for 4 weeks. M3 -R-/- mice showed significantly reduced bile flow compared to WT mice, most likely due to decreased biliary HCO3- secretion. However, even aged M3 -R-/- mice did not spontaneously develop liver injury or cholestasis. Challenging M3 -R-/- and WT littermates with DDC feeding showed substantially aggravated liver injury in M3 -R-/- mice. After 4 weeks bethanechol treatment, Mdr2-/- mice showed less liver injury compared to controls. Our experimental findings suggest that M3 -R-signalling significantly influences bile formation. Loss of the M3 -R increases susceptibility to cholestatic injury in DDC-fed mice. Since treatment of Mdr2-/- mice with a M3 -R agonist decreases liver injury, M3-R signaling may represent a therapeutic target in specific cholangiopathies.

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis

Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-β-muricholic acid and taurocholic acid. Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Some biological indicators relevant for the management of cholestasis in children

Cholestasis is a multifactorial disorder with various biological, infectious, toxic, genetic and metabolic manifestations, its principal feature presented as reduced bile flow or abnormalities in bile formation. It has lately been accepted that some specific biological markers would shorten the period needed to establish a positive diagnosis, as currently it is necessary to navigate through a complex diagnostic protocol for this disorder. The purpose of this study was to establish some biological parameters and biomarkers useful for cholestasis management in children. Two hundred thirty-two children with cholestasis were selected, during a six-year study. The biological indicators followed were serum bilirubin, gamma-glutamyl transpeptidase, aspartate transaminase, alkaline phosphatase, lactate dehydrogenase, serum cholesterol and triglycerides. Our data showed that certain biological parameters are more often involved in the various forms of cholestasis, and the conclusions of this study could be useful in the early detection of cholestasis and appropriate disease management.

Adenoviral transfer of human aquaporin-1 gene to rat liver improves bile flow in estrogen-induced cholestasis.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per μmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

Claudin 2 Deficiency Reduces Bile Flow and Increases Susceptibility to Cholesterol Gallstone Disease in Mice

Bile formation and secretion are essential functions of the hepatobiliary system. Bile flow is generated by transepithelial transport of water and ionic/nonionic solutes via transcellular and paracellular pathways that is mainly driven by osmotic pressure. We examined the role of tight junction-based paracellular transport in bile secretion. Claudins are cell-cell adhesion molecules in tight junctions that create the paracellular barrier. The claudin family has 27 reported members, some of which have paracellular ion- and/or water-channel-like functions. Claudin 2 is a paracellular channel-forming protein that is highly expressed in hepatocytes and cholangiocytes; we examined the hepatobiliary system of claudin 2 knockout (Cldn2(-/-)) mice. We collected liver and biliary tissues from Cldn2(-/-) and Cldn2(+/+) mice and performed histologic, biochemical, and electrophysiologic analyses. We measured osmotic movement of water and/or ions in Cldn2(-/-) and Cldn2(+/+) hepatocytes and bile ducts. Mice were placed on lithogenic diets for 4 weeks and development of gallstone disease was assessed. The rate of bile flow in Cldn2(-/-) mice was half that of Cldn2(+/+) mice, resulting in significantly more concentrated bile in livers of Cldn2(-/-) mice. Consistent with these findings, osmotic gradient-driven water flow was significantly reduced in hepatocyte bile canaliculi and bile ducts isolated from Cldn2(-/-) mice, compared with Cldn2(+/+) mice. After 4 weeks on lithogenic diets, all Cldn2(-/-) mice developed macroscopically visible gallstones; the main component of the gallstones was cholesterol (>98%). In contrast, none of the Cldn2(+/+) mice placed on lithogenic diets developed gallstones. Based on studies of Cldn2(-/-) mice, claudin 2 regulates paracellular ion and water flow required for proper regulation of bile composition and flow. Dysregulation of this process increases susceptibility to cholesterol gallstone disease in mice.

Altered biliary flow rate and bile composition following consumption of ethanolic fruit extract of Dennettia in rats

Background:Dennettia tripetala is a tropical plant with some ethno-medicinal uses; it enhances uterine contraction/involution in pregnant and post-partum women, it is also a mild laxative, with anti-bacteria, antifungal properties etc.,Aims:This study sought to elucidate the impact of D. tripetala fruit extract intake on biliary flow rate and bile composition in rats.Materials and Methods:A total of 18 albino Wistar rats were randomly assigned into three groups of 6 rats each and fed on normal rat chow + drinking water and/or 85 mg/kg, 170 mg/kg body weight of D. tripetala extract for 14 days.Results:The rate of bile secretion in the control, low dose (LD) and high dose (HD) D. tripetala extract treated rats was 4.40 ± 0.24 ml/h, 3.20 ± 0.20 ml/h and 4.60 ± 0.25 ml/h respectively. Showing a significant (P < 0.05) decrease in LD and an increase in HD. Na+ concentration increased significantly (P < 0.01) in the LD extract recipients, but was reduced in the HD. LD of the extract increased K+ significantly (P < 0.001) while HD decreased it (P < 0.05). Both low and HD of the extract reduced Cl– concentration significantly (P < 0.05 and P < 0.001 respectively). HCO3– increased significantly (P < 0.05) in the HD extract recipients. However, total cholesterol, total and conjugated bilirubin concentrations were not significantly altered by D. tripetala fruit extracts.Conclusion:In conclusion, this study showed that LD of D. tripetala may reduce bile flow rate while HD may increase it without altering the saturation of cholesterol and bilirubin.