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1.
World J Gastroenterol ; 25(32): 4580-4597, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31528088

RESUMO

Chronic delta hepatitis is the most severe form of viral hepatitis affecting nearly 65 million people worldwide. Individuals with this devastating illness are at higher risk for developing cirrhosis and hepatocellular carcinoma. Delta virus is a defective RNA virus that requires hepatitis B surface antigen for propagation in humans. Infection can occur in the form of a co-infection with hepatitis B, which can be self-limiting, vs superinfection in a patient with established hepatitis B infection, which often leads to chronicity in majority of cases. Current noninvasive tools to assess for advanced liver disease have limited utility in delta hepatitis. Guidelines recommend treatment with pegylated interferon, but this is limited to patients with compensated disease and is efficacious in about 30% of those treated. Due to limited treatment options, novel agents are being investigated and include entry, assembly and export inhibitors of viral particles in addition to stimulators of the host immune response. Future clinical trials should take into consideration the interaction of hepatitis B and hepatitis D as suppression of one virus can lead to the activation of the other. Also, surrogate markers of treatment efficacy have been proposed.


Assuntos
Antivirais/uso terapêutico , Coinfecção/tratamento farmacológico , Hepatite B Crônica/tratamento farmacológico , Hepatite D Crônica/tratamento farmacológico , Superinfecção/tratamento farmacológico , Terapias em Estudo/métodos , Antivirais/farmacologia , Coinfecção/epidemiologia , Coinfecção/virologia , Quimioterapia Combinada/métodos , Carga Global da Doença , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Hepatite D Crônica/epidemiologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Literatura de Revisão como Assunto , Superinfecção/epidemiologia , Superinfecção/virologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Resultado do Tratamento , Montagem de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
2.
PLoS Genet ; 15(8): e1008073, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31465442

RESUMO

The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions.


Assuntos
Ácidos e Sais Biliares/metabolismo , Variação Biológica da População/genética , Microbioma Gastrointestinal/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Locos de Características Quantitativas/genética , Simportadores/genética , Animais , Ácidos e Sais Biliares/sangue , Camundongos de Cruzamento Colaborativo , Feminino , Firmicutes/crescimento & desenvolvimento , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Modelos Animais , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Verrucomicrobia/crescimento & desenvolvimento
3.
Life Sci ; 232: 116643, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31299237

RESUMO

AIMS: Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. MAIN METHODS: Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. KEY FINDINGS: sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. SIGNIFICANCE: For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Endoglina/sangue , Endoglina/fisiologia , Homeostase , Fígado/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Colesterol/sangue , Fezes , Inflamação/sangue , Masculino , Camundongos , Camundongos Transgênicos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Estresse Oxidativo , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Simportadores/metabolismo , Regulação para Cima
4.
Curr Drug Metab ; 20(5): 377-389, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31258056

RESUMO

BACKGROUND: Sodium Taurocholate Co-transporting Polypeptide (NTCP) and Bile Salt Export Pump (BSEP) play significant roles as membrane transporters because of their presence in the enterohepatic circulation of bile salts. They have emerged as promising drug targets in related liver disease. METHODS: We reviewed the literature published over the last 20 years with a focus on NTCP and BSEP. RESULTS: This review summarizes the current perception about structure, function, genetic variation, and regulation of NTCP and BSEP, highlights the effects of their defects in some hepatic disorders, and discusses the application prospect of new transcriptional activators in liver diseases. CONCLUSION: NTCP and BSEP are important proteins for transportation and homeostasis maintenance of bile acids. Further research is needed to develop new models for determining the structure-function relationship of bile acid transporters and screening for substrates and inhibitors, as well as to gain more information about the regulatory genetic mechanisms involved in the processes of liver injury.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Hepatopatias/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Humanos
5.
Biol Chem ; 400(10): 1371-1384, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31256060

RESUMO

The solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriers in vivo needs further investigation.


Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Animais , Transporte Biológico , Dimerização , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sódio/metabolismo
6.
Emerg Microbes Infect ; 8(1): 879-894, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179847

RESUMO

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Hepatócitos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Animais , Membrana Celular/genética , Proliferação de Células , Feminino , Células Hep G2 , Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Simportadores/metabolismo
7.
Food Funct ; 10(7): 3839-3850, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31210195

RESUMO

Cholestatic liver injury induced by estrogen is a common clinical syndrome in women undergoing oral administration of contraceptives, pregnancy or hormone replacement therapy. Estrogen-induced cholestasis is associated with the accumulation of endogenous bile acids, which play critical roles in the disease progression and symptoms. In the present study, we described the protective effect of auraptene, a simple coumarin present in the peels of citrus fruits, such as grapefruit, against 17α-ethinylestradiol (EE)-induced cholestasis, and further elucidated the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect. Auraptene treatment alleviated EE-induced cholestasis through increasing the bile flow and biliary bile acid output. The mechanism underlying the alleviated cholestasis by auraptene was associated with the increased efflux and inhibited hepatic uptake of bile acids via an induction of efflux transporters (Bsep and Mrp2) and downregulation of Ntcp. Furthermore, auraptene reduced the bile acid synthesis through repressing Cyp7a1 and Cyp8b1, and increased the bile acid metabolism through an induction in the gene expression of Sult2a1. The mentioned genes involved in the bile acid homeostasis were modulated by FXR. We further demonstrated that the changes in transporters and enzymes, as well as ameliorated liver histology by auraptene, were abrogated by the FXR antagonist guggulsterone. In conclusion, auraptene alleviated EE-induced cholestasis due to FXR-mediated gene regulation.


Assuntos
Colestase/tratamento farmacológico , Colestase/prevenção & controle , Citrus/química , Cumarínicos/farmacologia , Extratos Vegetais/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas , Colestase/induzido quimicamente , Colesterol 7-alfa-Hidroxilase , Fígado/lesões , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Esteroide 12-alfa-Hidroxilase/metabolismo , Sulfotransferases/metabolismo , Simportadores/metabolismo
8.
Tohoku J Exp Med ; 248(1): 57-61, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31142693

RESUMO

Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-related liver disorder. Although the etiology of ICP is not fully understood thus far, some genetic factors might contribute to the development of this condition. Sodium-taurocholate cotransporting polypeptide (NTCP), the protein encoded by the gene Solute Carrier Family 10, Member 1 (SLC10A1), is the primary transporter expressed in the basolateral membrane of the hepatocyte to uptake conjugated bile salts from the plasma. NTCP deficiency arises from biallelic SLC10A1 mutations which impair the NTCP function and cause intractably elevated levels of total bile acids (TBA) in the plasma (hypercholanemia). In this study, all the SLC10A1 exons and their flanking sequences were analyzed by Sanger sequencing to investigate the etiology for hypercholanemia in two male infants aged 2 and 20 months, respectively, from two unrelated families. As a result, both patients are homozygous for the reported pathogenic variant c.800C>T (p.Ser267Phe) that could impair the NTCP function to uptake bile acids, and the diagnosis of NTCP deficiency was thus made. Their mothers are also homozygotes of the same variant and both had been diagnosed to have ICP in the third trimester, with one of them undergoing cesarean section. The father of the first patient in this paper has the same SLC10A1 genotype c.800C>T/c.800C>T, also exhibiting slight hypercholanemia with a plasma TBA level of 21.5 µmol/L. In conclusion, we suggest that with hypercholanemia being a common laboratory change, NTCP deficiency may be a genetic factor leading to ICP and even cesarean section in clinical practice.


Assuntos
Colestase Intra-Hepática/patologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/deficiência , Complicações na Gravidez/patologia , Simportadores/deficiência , Sequência de Bases , Feminino , Humanos , Lactente , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Gravidez , Simportadores/genética , Simportadores/metabolismo
9.
Cell Physiol Biochem ; 52(6): 1427-1445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31088037

RESUMO

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.


Assuntos
Membrana Celular/metabolismo , Ácido Glicoquenodesoxicólico , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Ácido Taurocólico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glicoquenodesoxicólico/metabolismo , Ácido Glicoquenodesoxicólico/farmacologia , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacologia
10.
Nat Commun ; 10(1): 2032, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048734

RESUMO

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.


Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Transportadores de Sulfato/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Deinococcus , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SLC4A/química , Proteínas SLC4A/metabolismo , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/isolamento & purificação
11.
Phytomedicine ; 59: 152916, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978651

RESUMO

BACKGROUND: Shengmai Formula (SMF) is widely used to treat cardiovascular disease such as chronic heart disease, coronary atherosclerotic heart disease, viral myocarditis, and others. Our previous studies have shown that OATP1B1/1B3 mediates the interactions between ophiopogon D and ginsenoside Rb1/Rd, which are the major active components in SMF. The herb-drug interactions that involve sodium taurocholate co-transporting polypeptide (NTCP) have been drawing increasing amounts of attention. PURPOSE: The aim of the present study was to investigate the interactions of the major effective components in SMF mediated by NTCP. METHODS: By using NTCP-overexpressing HEK293T cells and liquid chromatograph-mass spectrometer (LC-MS) analytical methods, we investigated the impact of the four main effective fractions and the 12 main effective components in SMF on NTCP-mediated sodium taurocholate (TCNa) uptake. The interactions of these effective components in SMF mediated by NTCP were further studied. RESULTS: The main effective fractions, ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), all exhibited a certain inhibitory effect on the uptake of TCNa. Among the 12 main effective components, only ginsenoside Rg1, ophiopogon D', and schizandrin A showed inhibition of TCNa uptake, with IC50 values of 50.49 ± 4.24 µM, 6.71 ± 0.70 µM, and 45.80 ± 3.10 µM, respectively. Additionally, we found that ginsenoside Re and schizandrin B could be transported by NTCP-overexpressing HEK293T cells, and that the uptake of ginsenoside Re was significantly inhibited by OTS, OTF, STL, ginsenoside Rg1, ophiopogon D', and schizandrin A. The uptake of schizandrin B was significantly inhibited by GTS, OTS, OTF, and ophiopogon D'. CONCLUSION: Ginsenoside Rg1, ophiopogon D', and schizandrin A are potential inhibitors of NTCP and may interact with clinical drugs mediated by NTCP. Ginsenoside Re and schizandrin B are also potential substrates of NTCP, and their uptake mediated by NTCP was inhibited by the other components in SMF. The interaction of complex components based on NTCP may be one of the important compatibility mechanisms in SMF.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cromatografia Líquida , Células HEK293 , Humanos , Espectrometria de Massas
12.
Gut Liver ; 13(5): 569-575, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30919600

RESUMO

Background/Aims: Cholangiocytes are capable of reabsorbing bile salts from bile, but the pathophysiological significance of this process is unclear. To this end, we detected the expression and distribution of bile acid transport proteins in cholangiocytes from normal rat liver and analyzed the possible pathophysiological significance. Methods: Bile duct tissues of Sprague-Dawley rats were isolated by enzymatic digestion and mechanical isolation, and then divided into large and small bile duct tissues. Immunohistochemistry, real-time polymerase chain reaction and Western blotting were used to determine the expression of the apical sodium-dependent bile acid transporter (ASBT), ileal bile acid binding protein (IBABP), and basolateral organic solute transporter α (Ostα) in the biliary tract system of rats. Differences in the expression and distribution of these proteins were analyzed. Results: In cholangiocytes, ASBT and IBABP were mainly expressed in cholangiocytes of the large bile ducts, in which the expression of both was significantly higher than that in the small ducts (p<0.05). Ostα was simultaneously expressed in cholangiocytes of both the large and small bile ducts, showing no significant difference in expression between the two groups of bile ducts (p>0.05). Conclusions: Bile acid transporters are expressed and heterogeneously distributed in rat bile ducts, indicating that bile acid reabsorption by cholangiocytes might mainly occur in the large bile ducts. These findings may help explore the physiology of bile ducts and the pathogenesis of various cholangiopathies.


Assuntos
Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Células Epiteliais/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Ratos Sprague-Dawley
13.
Xenobiotica ; 49(7): 852-862, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30132394

RESUMO

A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17ß-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Hepatócitos/metabolismo , Modelos Biológicos , Fator 1 de Transcrição de Octâmero/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Adulto , Animais , Atorvastatina/farmacocinética , Atorvastatina/farmacologia , Transporte Biológico Ativo , Estradiol/análogos & derivados , Estradiol/farmacocinética , Estradiol/farmacologia , Feminino , Hepatócitos/citologia , Humanos , Masculino , Metformina/farmacocinética , Metformina/farmacologia , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley
14.
MAGMA ; 32(1): 163-171, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30387017

RESUMO

In up to 50% of people diagnosed with a common ailment, diarrhea-predominant irritable bowel syndrome, diarrhea results from excess spillage of bile acids into the colon-data emerging over the past decade identified deficient release of a gut hormone, fibroblast growth factor 19 (FGF19), and a consequent lack of feedback suppression of bile acid synthesis as the most common cause. 75Selenium homotaurocholic acid (SeHCAT) testing, considered the most sensitive and specific means of identifying individuals with bile acid diarrhea, is unavailable in many countries, including the United States. Other than SeHCAT, tests to diagnose bile acid diarrhea are cumbersome, non-specific, or insufficiently validated; clinicians commonly rely on a therapeutic trial of bile acid binders. Here, we review bile acid synthesis and transport, the pathogenesis of bile acid diarrhea, the reasons clinicians frequently overlook this disorder, including the limitations of currently available tests, and our efforts to develop a novel 19F magnetic resonance imaging (MRI)-based diagnostic approach. We created 19F-labeled bile acid analogues whose in vitro and in vivo transport mimics that of naturally occurring bile acids. Using dual 1H/19F MRI of the gallbladders of live mice fed 19F-labeled bile acid analogues, we were able to differentiate wild-type mice from strains deficient in intestinal expression of a key bile acid transporter, the apical sodium-dependent bile acid transporter (ASBT), or FGF15, the mouse homologue of FGF19. In addition to reviewing our development of 19F-labeled bile acid analogue-MRI to diagnose bile acid diarrhea, we discuss challenges to its clinical implementation. A major limitation is the paucity of clinical MRI facilities equipped with the appropriate coil and software needed to detect 19F signals.


Assuntos
Ácidos e Sais Biliares/química , Diarreia/diagnóstico por imagem , Imagem por Ressonância Magnética de Flúor-19 , Animais , Transporte Biológico , Testes Diagnósticos de Rotina , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Vesícula Biliar/efeitos dos fármacos , Humanos , Intestinos , Masculino , Teste de Materiais , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Radioisótopos de Selênio/química , Simportadores/metabolismo , Ácido Taurocólico/química
15.
Biomater Sci ; 7(1): 322-335, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30474653

RESUMO

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Heparina/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Lipossomos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Antibióticos Antineoplásicos/farmacologia , Sítios de Ligação , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/química , Hepatócitos/efeitos dos fármacos , Humanos , Lipossomos/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Peptídeos/química , Simportadores/metabolismo
16.
Can J Physiol Pharmacol ; 96(12): 1328-1336, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30383974

RESUMO

The enterohepatic circulation of bile acids (BAs) critically depends on BA transporters and enzymes, which can be affected by inflammatory bowel disease. Diarrhea in colitis is believed to result in part from BA malabsorption. The work aimed to investigate whether diarrhea in colitis was associated with the expression of BA transporters, enzymes, and nuclear receptors. RT-qPCR and Western blot techniques were used to evaluate the gene and protein levels of Cyp7a1, Asbt, SHP, FXR, Ostß in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of colitis. The total BAs (TBAs) levels were assayed using ELISA kits, and the individual BAs were measured by LC-MS/MS. Results showed that the fecal excretions of TBAs were significantly increased by 1.6-fold in acute stage of colitis. In ileum, Asbt was significantly decreased; however, there was a compensatory increase in Cyp7a1 level in liver. Moreover, FXR has a decreased tendency and the downstream target gene SHP was downregulated. Contrary to acute stage, molecular changes were completely reversible during the remission phase. Our results indicated that the expression of Asbt and Cyp7a1 were altered in acute colitis, which performed vital roles in maintaining BA homeostasis. Early medical manipulation of BA transporters and enzymes may help prevent diarrhea.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colite/metabolismo , Diarreia/metabolismo , Doença Aguda , Animais , Colesterol 7-alfa-Hidroxilase/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Homeostase/fisiologia , Íleo/metabolismo , Fígado/metabolismo , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Ratos , Ratos Sprague-Dawley , Simportadores/metabolismo
17.
J Vet Intern Med ; 32(6): 1918-1926, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30315593

RESUMO

BACKGROUND: Intestinal absorption of bile acids is mediated by the apical sodium-dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). OBJECTIVE: Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. ANIMALS: Twenty-four dogs with CIE and 11 control dogs. METHODS: The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography-mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. RESULTS: In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = -0.40; Pcorr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.


Assuntos
Doenças do Cão/metabolismo , Doenças Inflamatórias Intestinais/veterinária , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Ácidos e Sais Biliares/análise , Estudos de Casos e Controles , Doença Crônica , Colo/metabolismo , Colo/patologia , Doenças do Cão/patologia , Cães , Fezes/química , Feminino , Íleo/metabolismo , Íleo/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Masculino
18.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30232184

RESUMO

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocyte cell lines appeared suboptimal. In the present study, we constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1α and HNF4α, which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B virus infection.IMPORTANCE HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we constructed NTCP-expressing HepG2 and AML12 cell lines that are highly permissive to HBV infection. More significantly, we found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to the cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards the ultimate elimination of chronic hepatitis B virus infection.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Hepatócitos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Técnicas de Cultura de Células , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Hidrocortisona/farmacologia , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética
19.
Pflugers Arch ; 470(12): 1777-1786, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30140954

RESUMO

Obstructive nephropathy is characterized by alterations in renal function that depends on the degree and type of obstruction. To increase the knowledge about the physiopathological mechanisms involved in the renal damage associated with bilateral ureteral obstruction (BUO), we studied the renal expression and function (as urinary citrate excretion) of sodium-dependent dicarboxylate cotransporter (NaDC1) in rats. In addition, we evaluated the urinary excretion of NaDC1 as a candidate biomarker for this pathology. Male Wistar rats underwent bilateral ureteral obstruction for 1 (BUO1), 2 (BUO2), 5 (BUO5), and 24 (BUO24) h or sham operation. After 24 h of ureteral releasing, traditional parameters of renal function and citrate levels were determined, and NaDC1 levels were evaluated in total renal homogenates, apical plasma membranes, and urine by electrophoresis and Western blotting. Traditional parameters of renal function were only modified in BUO5 and BUO24. The renal expression of NaDC1 was decreased in BUO5 and BUO24, with a concomitant increase in urinary excretion of citrate. Moreover, the urinary excretion of NaDC1 increased after short times of ureteral obstruction (BUO1 and BUO2) and was positively correlated with the time elapsed after obstruction. The results obtained from the renal expression of NaDC1 could explain an adaptive mechanism to prevent the formation of kidney stones by increasing the levels of citrate, a calcium chelator. The urinary excretion of NaDC1 could be postulated as an early biomarker of obstructive nephropathy that also gives information about the duration of the obstruction.


Assuntos
Transportadores de Ácidos Dicarboxílicos/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Obstrução Uretral/metabolismo , Animais , Biomarcadores/urina , Citratos/urina , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/urina , Nefropatias/etiologia , Nefropatias/urina , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/urina , Ratos , Ratos Wistar , Simportadores/genética , Simportadores/urina , Obstrução Uretral/complicações , Obstrução Uretral/urina
20.
J Hepatol ; 69(6): 1231-1241, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30142426

RESUMO

BACKGROUND & AIMS: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. METHODS: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. RESULTS: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5-12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. CONCLUSIONS: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. LAY SUMMARY: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.


Assuntos
Coinfecção/virologia , DNA Circular/metabolismo , DNA Viral/metabolismo , Genoma Viral/fisiologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Dimetil Sulfóxido/metabolismo , Meia-Vida , Células Hep G2 , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Polietilenoglicóis/metabolismo , RNA Viral/metabolismo , Simportadores/metabolismo , Replicação Viral
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