Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

AIM： To investigate the effect of six bile salts： glycocholate （GC）, glycochenodeoxycholate （GCDC）, glycodeoxycholate （GDC）, taurocholate （TC）, taurochenodeoxycholate （TCDC）, taurodeoxycholate （TDC）, and their mixture on cultured human normal esophageal rnucosal epithelial cells. METHODS： Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium. 3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry （FCI） with propidium iodide （PI） staining and annexin V-FITC conjugated with PI staining. Apoptotic DNA ladders on agarose gel electrophoresis were observed. RESULTS： Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 μmol/L and their mixture at 1 500 μmol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500 μmol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 μmol/L GCDC, TCDC, GDC, TDC, and 1 500 μmol/L mixture for 24 h, respectively, which were higher than that of the control （1.5%）. The percentage was 1.4% in cells with 500 μmol/L GC for 24 h. DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 μmol/L TC for 2 h and i 500 μmnol/L mixture for 24 h. CONCLUSION： All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.

Bile salts inhibit growth and induce apoptosis of human esophageal cancer cell line

AIM： To explore the effect of six bile salts, including glycocholate （GC）, glycochenodeoxycholate （GCEX：）, glycodeoxycholate （GDC）, taurocholate （TC）, taurochenodeoxycholate （TCDC）, taurodeoxycholate （TDC）, and two bile acids including cholic acid （CA） and deoxycholic acid （DCA） on esophageal cancer Eca109 cell line. METHODS： Eca109 cells were exposed to six bile salts, two bile adds and the mixed bile salts at different concentrations for 24-72 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay was used to detect the cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. Sub-G1 DNA fragmentations and early apoptosis cells were assayed by flow cytometry （FCM） with propidium iodide （PI） staining and annexin V-FITC conjugated with PI staining. Apoptosis DNA ladders on agarose were observed. Activation of caspase-3 was assayed by FCM with FITC-conjugated monodonal rabbit anti-active caspase- 3 antibody and expressions of Bcl-2 and Bax proteins were examined immunocytochemically in 500μmol/L-TCinduced apoptosis cells. RESULTS： Five bile salts except for GC, and two bile acids and the mixed bile salts could initiate growth inhibition of Ecal09 cells in a dose- and time-dependent manner. TUNEL, FCM, and DNA ladder assays all demonstrated apoptosis induced by bile salts and bile acids at 500 μmol/L, except for GC. Early apoptosis cell percentages in Eca109 cells treated with GCDC, GDC, TC, TCDC, TDC, CA at 500 μmol/L for 12 h, DCA at 500 μmol/L for 6 h, and mixed bile salts at 1 000 μmol/L for 12 h were 7.5%, 8.7%, 14.8%, 8.9%, 7.8%, 9.3%, 22.6% and 12.5%, respectively, all were significantly higher than that in control （1.9%）. About 22% of the cell population treated with TC at 500 μmol/L for 24 h had detectable active caspase-3, and were higher than that in the control （1%）. Immunocytochemical assay suggested that TC down-regulated Bcl-2 protein level and up-regulated Bax protein level. CONCLUSION： GCDC, GDC, TC, TCDC, TDC, CA and DCA, except for GC, can inhibit growth and induce apoptosis of esophageal cancer Eca109 cells. Activation of caspase-3, decreased Bcl-2 protein and increased Bax protein are involved in TC-induced apoptosis of Eca109 cells.

Effect of bile salts and bile acids on human gastric mucosal epithelial cells

Objective：To explore the effect of bile salt and bile acid on cultured eternalized human gastric mucosa epithelium GES-1 cells. Methods：Cultured eternalized human gastric mucosa epithelium GES-1 cells were treated with media containing 6 different kinds of bile salts and 3 different kinds of bile acids and their mixture with different concentrations： GCDC（glycochenodeoxychoμte）, GDC （glycodeoxychoμte）, GC（glycochoμte）, TCDC（taurochenodeoxychoμte）, TDC（taurodeoxychoμte）, TC （taurochoμte）, LCA （lithocholicacid）, CA（cholic acid）, DCA（deoxycholic acid）（50 μ mol/L,250 μ mol/L,500 μ mol/L,1000 μ mol/L）, DY（mixture of bile salts） and DS（mixture of bile acids）（250 μ mol/L,500 μ mol/L,1000 μ mol/L,1500 μ mol/L, 2000 μ mol/L）, in comparison with the control group（in normal media without bile salts and bile acids）. Cell proliferation was assessed by MTT（3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide） assay for 72 hours with different concentrations and the apoptotic cells were assayed by flow cytometry （FCM） with Annex V-FITC conjugated with propidium iodide（PI） staining for 24 hours with different concentrations（1500,2000 μt mol/L）. Results：There was no significant difference in morphology and cell proliferation in GC group after 24-72 h. Low concentration（50 μ mol/L） of GCDC, GDC, TCDC, TDC and TC accelerated gastric epithelial cell growth in a dosage-time dependent manner. At middle concentration （250-500 μ mol/L）, it showed positive effect after 24-48 h, while negative effect after 72 h. At high concentration（1000 μ mol/L）, it accelerated gastric epithelial cell growth after 24h and show consistent inhibition even leading to necrosis after 48-72 h. LCA and CA showed a positive effect on the concentration of 50 μ mol/L after 24-72 h, while 250-1000 μ mol/L showed a trend towards apoptosis after 24-72 h. At 50-500 μmol/L, DCA showed proliferation after 24 h and apoptosis after 48-72 h, but showed necrosis after 24-72 h at 1000 μmol/L. DY and DS could facilitate normal gastric mucosa epithelial cell growth at low concentration （250-500 μ mol/L）, however at 1000-2000 μ mol/L the trend shifted from apoptosis to necrosis. FCM with Annexin-V conjugated with PI staining revealed that GCDC, GDC, GC, TCDC, TDC, TC, LCA, CA, DCA, DY and DS induced apoptosis of human gastric mucosal epithelial cells. They were all significantly higher than that of the control（P 〈 0.05）, but there was no significant difference in GC group （P 〉 0.05）. The bile salts induced apoptosis in a time-dose-dependent manner. Conclusion：Our results suggested that bile acid and bile salt is the trigger of injury in human gastric mucosal epithelial cells.

Interaction of Soybean Phospholipids Based Lipo-Vesicle and Surfactants of Bile Salts In Vitro

Liposomes were prepared with natural soybean phospholipids by extrusion method after rotating-film evaporating technique. Transmission electron micrography was used to detect the appearances of the prepared liposomes, and the liposome diameter was also measured. The prepared liposomes were sphere in shape with the mean diameter of 217 nm and span of 0.838. The phospholipid bilayer structure, suitable for entrapping various effector molecules, could be seen clearly under transmission electron microscopy. The bile salts of sodium cholate and sodium deoxycholate were used as the surfactants to investigate their interaction with liposomes. The turbidities for the mixture of bile salts and liposomes were evaluated by the visible spectrometry method at the wavelength of 500 nm. And the diameter changes of liposomes were also tested to examine the effect of bile salts on liposomes. At the beginning, the diameters and turbidities of liposomes increased a little as the result of mixed micelles formation during the different stages for the structure changes of surfactant-liposomes micelles. The further added bile salts decreased the diameters and turbidities of liposomes. The liposome suspension underwent several rearrangements before small mixed micelles formed. And the diameter of liposomes changed regularly. The interaction of bile salts and liposomes is important for the further study of the behaviors of liposomes in vivo. The drug loaded and release properties of liposomes can also be well reflected by the interaction of liposomes and surfactants.

Selective binding of bile salts by β-cyclodextrin derivatives with appended quinolyl arms

Two β-cyclodextrin derivatives bearing appended quinolyl and isoquinolyl arms,i.e.mono-（6-quinolyl- 6-deoxy）-β-cyclodextrin（1） and mono-（6-isoquinolyl-6-deoxy）-β-cyclodextrin（2） were synthesized in satisfactory yields and fully characterized.Their original conformations and binding behaviors toward four bile salt guests,that is,sodium cholate（CA）,sodium deoxycholate（DCA）,sodium glycocholate （GCA）,and sodium taurocholate（TCA）,were investigated by means of fluorescence,circular dichroism and 2D NMR spectroscopy.The study of solution structures revealed that both quinolyl and isoquinolyl arms were located outside the cyclodextrin cavity.The results obtained from the fluorescence titrations showed that the binding abilities of hosts 1 and 2 with selected bile salts varied in an order of DCA 〉 CA 〉 GCA.The selective binding of hosts toward bile salt guests was discussed from the viewpoints of induced-fit and multiple binding.

Gastric and small intestinal lipid digestion kinetics as affected by the gradual addition of lipases and bile salts

We studied the effect of evolving from static in vitro digestion conditions towards the gradual addition of essential digestive compounds for lipid digestion.Two oil-in-water emulsions were considered:a low(5%w/w)and high(20%w/w)triolein-based emulsion with identical surfactant-to-oil ratio(0.2).Emulsions were subjected to in vitro static digestion conditions or gradual gastric lipase addition,gradual pancreatic lipase addition,and/or gradual bile salt addition.For these three latter cases,similar amounts of gastric/pancreatic lipase and/or bile salts were provided as in the static case,however divided over 4 doses added during the first 30 min of each digestive phase.For the low-lipid emulsion,gradually adding lipases and bile salts did not significantly affect lipolysis kinetics.This can be related to the sufficient amounts of digestive compounds present even in the smallest initial dose.For the high-lipid emulsion,the gradual addition of bile salts significantly reduced the lipolysis rate.Bile salts are essential to remove lipid digestion products from the interface and thus allow the continuation of the lipid digestion process at the interface.Oppositely,the lipolysis extent after 2 h of small intestinal phase was not significantly influenced by the digestion approach.This is again explained by the simple nature of the emulsions studied and the excess of lipase even in the smallest initial dose.Overall,this work showed that evolving towards more(semi-)dynamic digestion conditions can impact(lipid)digestion kinetics,even for relatively simple food compositions,and is of interest to obtain more physiological relevant digestion kinetics.

Effects of bile salts and divalent cations on the adsorption of norfloxacin by agricultural soils

The effects of bile salts （sodium cholate and sodium deoxycholate, 0-20 mmol/L）, divalent cations （Ca^2＋, Mg^2＋, Cu^2＋ and Zn^2＋, 0-20 mmol/L） or pH （3.0-10.0） on the adsorption of norfloxacin by three selected soils （Paddy_H, Paddy_G and Red_J） were systematically studied. Soil adsorption of norfloxacin follows a pseudo second-order kinetics model, and the maximum adsorption capacity has been determined from the nonlinear fit of the Langmuir isotherm model to be 88.8, 88.1 and 63.0 μmol/g for the adsorption onto Paddy_H, Paddy_G and Red_J, respectively. The results indicate that norfloxacin has a high adsorption affinity for the agricultural soils tested and that the organic content of these soils have at least a slight influence on this adsorption. The adsorption of norfloxacin to soils was strongly dependent on pH and exhibited a maximum at approximately pH 6. The presence of divalent cations prominently suppressed the adsorption of norfloxacin by paddy soils, which followed an order of Cu^2＋ 〉 Mg^2＋ 〉 Ca^2＋ 〉 Zn^2＋, and by red soil, which followed an order of Cu^2＋ 〉 Zn^2＋ 〉 Ca^2＋ 〉 Mg^2＋. The adsorption of norfloxacin （by the soils studied） sharply decreased as the amount of bile salts was increased. For uncharged norfloxacin at environmentally relevant pH values, such factors as soil type, exogenous divalent cations and macromolecules significantly altered the environmental fate and transport of norfloxacin between aquatic and soil interfaces.

Alleviative effects of Bacillus coagulans strains on irritable bowel syndrome-unraveling strain specificity through physiological and genomic analysis

The high intraspecies heterogeneity of Baciillus coagulans leads to significant phenotypic differences among different strains.Thus,6 B.coagulans strains were tested in the present study using an irritable bowel syndrome(IBS)animal model to determine whether the IBS-alleviating effects of B.coagulans strains are strain-specific.The results of this study showed that the ingestion of B.coagulans GBI-30,6086,and B.coagulans CCFM1041 significantly alleviated IBS symptoms in mice.In contrast,other B.coagulans strains showed no or limited alleviating effects on IBS symptoms.According to our experimental results,the two main common features of these strains were as follows:1)The resistance of vegetative cells to bile salts,and 2)ability to synthesize specific lipids and secondary metabolites.Screening strains based on these two indicators may greatly reduce costs and provide a basis for mining new functional B.coagulans strains.Our results also suggest that administration of B.coagulans could significantly regulate microbiota dysbiosis in animal models.Moreover,the close relationships between the gut microbiota,gut microbiota metabolites,and IBS were further confirmed in this study.

High-fat-induced intestinal permeability dysfunction associated with altered fecal bile acids

AIM： To investigate whether high-fat-feeding is associ- ated with increased intestinal permeability via altera- tions in bile acid metabolism.METHODS： Male C57BI/6J mice were fed on a high-fat （n = 26） or low-fat diet （n = 24） for 15 wk. Intestinal permeability was measured from duodenum, jejunum, ileum and colon in an Ussing chamber system using 4 kDa FITC-labeled dextran as an indicator. Fecal bile ac- ids were analyzed with gas chromatography. Segments of jejunum and colon were analyzed for the expression of farnesoid X receptor （FXR） and tumor necrosis factor

Influence of gut microbiome composition on effect variations of anti-cholesterol treatment among individual mice

This study investigated if the variation in the effect of anti-cholesterol(AC)treatment on individual mice are related to gut microbiome composition.The bile salt hydrolase(BSH)activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment.Mice were fed to different diets for 6 weeks:high-fat(60%of total calories from fat;D1),high-dietary fibre(20%cellulose;D2),and low-fat(17.2%of total calories from fat;D3)diets to change their gut microbiomes.Subsequently,faecal microbiome was transplanted(FMT)into mice treated with high cholesterol diet contained 2%cholesterol,followed by AC or non-AC(sterile tap water,STW)treatments.Control groups with normal(NC)and highcholesterol diets(PC)were prepared for both AC and STW treatment.All experimental groups were subjected to serum and liver cholesterol,cholesterol metabolism-related(CMR)gene expression,and intestinal microbiome analyses.D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment.Moreover,upregulation of the Cyp7a1 gene expression was observed in this group.Furthermore,the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium,Bifidobacterium,and Parabacteroides in the D3-FMT+AC group compare to others,potentially contributing to increased bile acid synthesis.

Bile salt derivatives with novel skeleton from sea lamprey function as putative pheromone

OBJECTIVE The invasive sea lamprey(Petromyzon marinus)has devastated the ecosystem of the Laurentian Great Lakes.Application of pheromones to manipulate adult sea lamprey behavior is among the options considered for alternative sea lamprey control techniques.The male sea lamprey sex pheromone is hypothesized to be possess multiple functions through actions of multiple components,some of which have yet to be characterized.Our objective is to isolate and characterize the bioactive components from water conditioned with sexually mature male sea lamprey.METHODS The water conditioned with sexually mature male sea lamprey was extracted by solid phase extraction and concentrated in vacuo.The compounds were isolated by liquid chromatography and elucidated by spectrometry and spectroscopy.Their biological activities were evaluated by electro-olfactogram recordings and two-choice maze behavioral assays.RESULTS Five novel bile salts,petromyzene A and B and petromyzone A-C,have been characterized.Petromyzene A and B featured either a unique,rearranged side chain or a rare cis-11,12-diol on the steroidal B-ring.Petromyzone A-C represented three novel highly oxidized sulfated bile alcohols possessing different hydroxylation,oxidation,and double bond patterns,which exemplify the chemical diversity of bile salts.These five bile salts were potent odorants that stimulated the adult sea lamprey olfactory epithelium in a concentration dependent manner and showed detection thresholds between 10–13mol·L^(-1) and 10^(–11)mol·L^(-1)(paired t-test,P<0.05).Experiments in the two-choice maze showed that all isolated compounds induced behavioral responses in ovulated females.CONCLUSION The five novel compounds are likely additional components of pheromones released by sexually mature male sea lamprey,and may provide useful behavioral manipulation tools to be implemented with the integrated management of the destructive and invasive sea lamprey in the Laurentian Great Lakes.

Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T>C polymorphism in the bile salt export pump

AIM： To study the association of three common ABCB11 and ABCC2 polymorphisms （ABCB11： 1331T〉C→V444A; ABCC2： 3563T〉A → V1188E and 4544G 〉A → C1515Y） with intrahepatic cholestasis of pregnancy （ICP） and contraceptive-induced cholestasis （CIC）. METHODS： ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele. RESULTS： The ABCB11 1331T〉C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls： C allele 76.2% （CI, 58.0-94.4） vs 51.3% （CI 35.8-66.7）, respectively （P = 0.0007）; and CC allele 57.1% （CI 36.0-78.3） vs 20% （CI 7.6-32.4）, respectively （P = 0.0065）. All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype. CONCLUSION： Our data support a role for the ABCB11 1331T〉C polymorphism as a susceptibility factor for the development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and 7-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.

Partial external biliary diversion in bile salt export pump deficiency: Association between outcome and mutation

To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency. METHODSMutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney (HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatography-tandem mass spectrometry. Wild-type and mutant BSEP transport of [3H]-labeled taurocholate (TC) and taurochenodeoxycholate (TCDC) was assessed by vesicular transport assays. RESULTSA girl (at 2 mo) presented with pruritus, jaundice and elevated serum bile salts (BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919del and the nonsense mutation p.R1235X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy (age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives (< 5%). The patients’ native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919del and p.G1032R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively. CONCLUSIONIn summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2 (PFIC-2).

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.

Cloning and Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum M1-UVS29

We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.

Pathogenic role of oxidative and nitrosative stress in primary biliary cirrhosis

Primary biliary cirrhosis is a multifactor autoimmune disease characterized by hepatic and systemic manifestations,with immune system dysregulation and abnormalities in the hepatic metabolism of bile salts,lipids,and nutrients,as well as destruction of membrane lipids and mitochondrial dysfunction.Both oxidative and nitrosative stress are associated with ongoing manifestations of the disease.In particular,abnormalities in nitric oxide metabolism and thiol oxidation already occur at early stages,thus leading to the hypothesis that these biochemical events play a pathogenic role in primary biliary cirrhosis.Moreover,the association of these metabolic abnormalities with the progression of the disease may indicate some biochemical parameters as early diagnostic markers of disease evolution,and may open up the potential for pharmacological intervention to inhibit intra-and extra-cellular stress events for resuming hepatocellular functions.The following paragraphs summarize the current knowledge by outlining molecular mechanisms of the disease related to these stress events.

Dynamic localization of hepatocellular transporters in health and disease

Vesicle-based traffi cking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal,submembrane compartment,followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane,the transporters usually undergo recycling between the plasma membrane and the endosomal compartment,which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence,it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters,which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis,and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation,with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition,the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally,several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP,tauroursodeoxycholate) are described.

Molecular overview of progressive familial intrahepatic cholestasis

Cholestasis is a clinical condition resulting from the imapairment of bile flow.This condition could be caused by defects of the hepatocytes,which are responsible for the complex process of bile formation and secretion,and/or caused by defects in the secretory machinery of cholangiocytes.Several mutations and pathways that lead to cholestasis have been described.Progressive familial intrahepatic cholestasis(PFIC)is a group of rare diseases caused by autosomal recessive mutations in the genes that encode proteins expressed mainly in the apical membrane of the hepatocytes.PFIC 1,also known as Byler’s disease,is caused by mutations of the ATP8B1 gene,which encodes the familial intrahepatic cholestasis 1 protein.PFIC 2 is characterized by the downregulation or absence of functional bile salt export pump(BSEP)expression via variations in the ABCB11 gene.Mutations of the ABCB4 gene result in lower expression of the multidrug resistance class 3 glycoprotein,leading to the third type of PFIC.Newer variations of this disease have been described.Loss of function of the tight junction protein 2 protein results in PFIC 4,while mutations of the NR1H4 gene,which encodes farnesoid X receptor,an important transcription factor for bile formation,cause PFIC 5.A recently described type of PFIC is associated with a mutation in the MYO5B gene,important for the trafficking of BSEP and hepatocyte membrane polarization.In this review,we provide a brief overview of the molecular mechanisms and clinical features associated with each type of PFIC based on peer reviewed journals published between 1993 and 2020.

The bcl-2 mRNA Expression in GCDC-induced Obstructive Jaundice in Rats and Its Implication in Hepatocellular Apoptosis

The modulatory role of bcl 2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC) induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl 2 mRNA in the hepatocytes was detected by RT PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct ligation for 14 days. 100 μmol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl 2 mRNA was not detectable in the hepatocytes of normal rats by RT PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100 μmol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl 2. During obstructive jaundice, expression of bcl 2 from the hepatocytes can inhibit the bile salt induced hepatocellular apoptosis.

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct.The absorption,distribution and elimination of drugs are impaired during this pathology.Prolonged cholestasis may alter both liver and kidney function.Lactam antibiotics,diuretics,non-steroidal anti-inflammatory drugs,several antiviral drugs as well as endogenous compounds are classified as organic anions.The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds.It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions.The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis,such as multidrug resistanceassociated protein 2,organic anion transporting polypeptide 1,organic anion transporter 3,bilitranslocase,bromosulfophthalein/bilirubin binding protein,organic anion transporter 1 and sodium dependent bile salt transporter.The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.