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1.
Cell Rep ; 30(5): 1310-1318.e5, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32023451

RESUMO

Pathological activation of TGF-ß signaling is universal in fibrosis. Aberrant TGF-ß signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-ß pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-ß1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-ß1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases.

2.
Cell Mol Gastroenterol Hepatol ; 9(4): 679-688, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31887435

RESUMO

BACKGROUND & AIMS: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study. METHODS: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1-/- (Casp1-/-) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids. RESULTS: Caspase-1, NLRP1, NLRP3 and IL-1ß were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1ß (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1-/- BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1-/- BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1-/- BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1-/- BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300µM TCA), neither IL-1ß induction nor procaspase-1 cleavage were detected. CONCLUSIONS: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.

3.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683554

RESUMO

In our previous research, ten antioxidant pentapeptides including FYKWP, FTGMD, GFEPY, YLPYA, FPPYERRQ, GFYAA, FSGLR, FPYLRH, VPDDD, and GIEWA were identified from the hydrolysate of miiuy croaker (Miichthys miiuy) swim bladder. In this work, their protective function on H2O2-induced oxidative damage to human umbilical vein endothelial cells (HUVECs) was studied. Results indicated that there was no significant difference in the HUVEC viability between the normal group and the treated groups with the 10 pentapeptides at the concentration of 100 µM for 24 h (p < 0.05). Furthermore, FPYLRH of 100 µg/mL extremely significantly (p < 0.001) increased the viability (80.58% ± 5.01%) of HUVECs with H2O2-induced oxidative damage compared with that of the model group. The protective mechanism indicated that FPYLRH could extremely significantly (p < 0.001) increase the levels of superoxide dismutase (SOD) (211.36 ± 8.29 U/mg prot) and GSH-Px (53.06 ± 2.34 U/mg prot) and decrease the contents of reactive oxygen species (ROS) (139.1 ± 11.8% of control), malondialdehyde (MDA) (13.66 ± 0.71 nM/mg), and nitric oxide (NO) (4.36 ± 0.32 µM/L) at the concentration of 100 µM in HUVECs with H2O2-induced oxidative damage compared with those of the model group. In addition, FPYLRH dose-dependently protected DNA in oxidative damage HUVECs model. These results suggested that FPYLRH could significantly attenuate the H2O2-induced stress injury in HUVECs and might be used as a potential natural antioxidant in the functional food industries.

4.
5.
Acta Pharmacol Sin ; 40(7): 895-907, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30573812

RESUMO

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.


Assuntos
Benzamidas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Simportadores/antagonistas & inibidores , Animais , Benzamidas/farmacocinética , Benzamidas/toxicidade , Linhagem Celular , Citocinas/metabolismo , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/patologia , Masculino , Mesocricetus , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidade
6.
Hepatol Commun ; 2(11): 1356-1368, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30411082

RESUMO

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re-induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situ hybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell-specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+ cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5-diethoxycarbony1-1,4-dihydrocollidine (DDC), and multidrug-resistant gene 2 knockout ( Mdr2 -/- ) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double-positive H19RNA+/sex-determining region Y (SRY)-box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple-positive H19 RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19 RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19 RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

7.
Gastroenterology ; 155(5): 1578-1592.e16, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30063921

RESUMO

BACKGROUND & AIMS: Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. METHODS: We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. RESULTS: Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. CONCLUSIONS: We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fatores de Crescimento de Fibroblastos/análise , Fatores de Crescimento de Fibroblastos/fisiologia , Humanos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição RelA/fisiologia
9.
JCI Insight ; 3(10)2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29769440

RESUMO

Excessive hepatic glucose production (HGP) contributes significantly to the hyperglycemia of type 2 diabetes; however, the molecular mechanism underlying this dysregulation remains poorly understood. Here, we show that fasting temporally increases the expression of H19 long noncoding RNA (lncRNA) in nondiabetic mouse liver, whereas its level is chronically elevated in diet-induced diabetic mice, consistent with the previously reported chronic hepatic H19 increase in diabetic patients. Importantly, liver-specific H19 overexpression promotes HGP, hyperglycemia, and insulin resistance, while H19 depletion enhances insulin-dependent suppression of HGP. Using genome-wide methylation and transcriptome analyses, we demonstrate that H19 knockdown in hepatic cells alters promoter methylation and expression of Hnf4a, a master gluconeogenic transcription factor, and that this regulation is recapitulated in vivo. Our findings offer a mechanistic explanation of lncRNA H19's role in the pathogenesis of diabetic hyperglycemia and suggest that targeting hepatic H19 may hold the potential of new treatment for this disease.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Fígado/metabolismo , RNA Longo não Codificante/genética , Animais , Western Blotting , Metilação de DNA , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Transcriptoma
10.
Cell Metab ; 27(2): 339-350.e3, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414684

RESUMO

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.


Assuntos
Digoxina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hepatopatia Gordurosa não Alcoólica/genética , Piruvato Quinase/metabolismo , Ativação Transcricional/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Modelos Animais de Doenças , Endotoxinas , Histonas/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Oxirredução , Ligação Proteica/efeitos dos fármacos , Piruvato Quinase/química , Células THP-1 , Transcrição Genética/efeitos dos fármacos
11.
Liver Int ; 38(6): 1128-1138, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29356312

RESUMO

BACKGROUND & AIMS: Cholestatic liver injury is mediated by bile acid-induced inflammatory responses. We hypothesized that superior therapeutic effects might be achieved by combining treatments that reduce the bile acid pool size with one that blocks inflammation. METHODS: Bile duct-ligated (BDL) rats and Mdr2(Abcb4)-/- mice were treated with all-trans retinoic acid (atRA), a potent inhibitor of bile acid synthesis, 5 mg/kg/d by gavage, or Cenicriviroc (CVC), a known antagonist of CCR2 and CCR5, 50 mg/kg/d alone or in combination for 14 days and 1 month respectively. RESULTS: All-trans retinoic acid alone reduced bile acid pool size and liver necrosis in BDL rats. However, the combination with CVC further reduced liver to body weight ratio, bile acid pool size, plasma liver enzyme, bilirubin, liver necrosis and fibrosis when compared to the atRA treatment. The assessment of hepatic hydroxyproline content further confirmed the reduced liver injury concurrent with reduction of pro-inflammatory cytokines emphasizing the synergistic effects of these two agents. Profiling of hepatic inflammatory cells revealed that combination therapy reduced neutrophils and T cells but not macrophages. The superior therapeutic effects of combination treatment were also confirmed in Mdr2-/- mice where a significant reduction in plasma liver enzymes, bilirubin, liver fibrosis, bile duct proliferation and hepatic infiltration of neutrophils and T cells and expression of cytokines were found. CONCLUSIONS: Multitargeted therapy is an important paradigm for treating cholestatic liver injury. The combination of CVC with atRA or other FXR activators may warrant a clinical trial in patients with cholestatic liver disease.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/tratamento farmacológico , Imidazóis/farmacologia , Hepatopatias/tratamento farmacológico , Tretinoína/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colestase/complicações , Colestase/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Ligadura , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Receptores de Citocinas/antagonistas & inibidores
12.
Artigo em Inglês | MEDLINE | ID: mdl-28804737

RESUMO

The mechanism of bile acid induced cholestatic liver injury remains controversial, thus hindering the development of new therapies for these diseases. In this research highlight, we briefly review the evolution of our understanding of the pathogenesis of bile acid induced liver injury, and summarize our recent findings on this topic. Our data suggests that under pathophysiological conditions bile acid induced liver injury is mediated by inflammatory responses that are initiated from stressed hepatocytes. We conclude by mentioning potential new therapeutic approaches for treating cholestatic liver injury based on these pathophysiologic concepts.

13.
Mol Aspects Med ; 56: 45-53, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28606651

RESUMO

Bile acids are synthesized in the liver and are the major component in bile. Impaired bile flow leads to cholestasis that is characterized by elevated levels of bile acid in the liver and serum, followed by hepatocyte and biliary injury. Although the causes of cholestasis have been extensively studied, the molecular mechanisms as to how bile acids initiate liver injury remain controversial. In this chapter, we summarize recent advances in the pathogenesis of bile acid induced liver injury. These include bile acid signaling pathways in hepatocytes as well as the response of cholangiocytes and innate immune cells in the liver in both patients with cholestasis and cholestatic animal models. We focus on how bile acids trigger the production of molecular mediators of neutrophil recruitment and the role of the inflammatory response in this pathological process. These advances point to a number of novel targets where drugs might be judged to be effective therapies for cholestatic liver injury.


Assuntos
Ácidos e Sais Biliares/toxicidade , Colestase/metabolismo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Fígado/metabolismo , Neutrófilos/imunologia , Animais , Ácidos e Sais Biliares/imunologia , Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colestase/genética , Colestase/imunologia , Colestase/patologia , Citocinas/genética , Citocinas/imunologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Imunidade Inata , Inflamação , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/lesões , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo
14.
Dig Dis ; 35(3): 232-234, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28249287

RESUMO

BACKGROUND: The mechanism by which bile acids induce liver injury in cholestasis remains controversial. Although high levels of bile acids are toxic when applied to liver cells, the level of toxic bile acids in the liver of most cholestatic animals and patients is <10 µM, indicating there must be alternative mechanisms. Recent studies suggest that the inflammatory response may play an important role in bile acid-induced liver injury, as pro-inflammatory cytokine expression is stimulated by bile acids in mouse hepatocyte cultures. To elucidate the mechanisms of bile acid-induced liver injury, we assessed signs of liver damage and gene expression in Abcb4-/- mice, a well-known model for cholestasis. Key Messages: Elevated plasma levels of bile acids were detected as early as 10 days after birth and at all later ages in Abcb4-/- mice compared to their wild-type littermate controls. Parallel increases in expression of Tnfα, Ccl2, Cxcl1, and Cxcl2 mRNA occurred at these early time points and throughout 12 weeks in Abcb4-/- livers. Marked hepatic neutrophil infiltration was first detected in 3-week mice, whereas histological evidence of liver injury was not detected until 6-weeks of age. Subsequent in vitro studies demonstrated that normal hepatocytes but not other non-parenchymal liver cells responded to bile acids with inflammatory cytokine induction. CONCLUSION: Bile acids induce the expression of pro-inflammatory cytokines in hepatocytes in Abcb4-/- mice that initiates an inflammatory response. This inflammatory response plays an important role in the development of cholestatic liver injury in this and other cholestatic conditions. Furthermore, understanding of these inflammatory mechanisms should lead to new therapeutic approaches for cholestatic liver diseases.


Assuntos
Ácidos e Sais Biliares/efeitos adversos , Inflamação/patologia , Fígado/patologia , Envelhecimento/metabolismo , Alanina Transaminase/sangue , Animais , Ácidos e Sais Biliares/sangue , Citocinas/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Neutrófilos/metabolismo
15.
JCI Insight ; 2(5): e90780, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28289714

RESUMO

Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in Ccl2-/- mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from Tlr9-/- mice, while liver injury was diminished both in conventional and hepatocyte-specific Tlr9-/- mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease.


Assuntos
Ácidos e Sais Biliares/fisiologia , Colestase/fisiopatologia , Hepatócitos/patologia , Inflamação/fisiopatologia , Hepatopatias/fisiopatologia , Animais , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Citocinas/fisiologia , Humanos , Mediadores da Inflamação/fisiologia , Masculino , Camundongos , Camundongos Knockout
16.
Am J Physiol Regul Integr Comp Physiol ; 312(4): R477-R484, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28077388

RESUMO

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.


Assuntos
Ácidos e Sais Biliares/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sódio/metabolismo , Simportadores/química , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Animais , Ácidos e Sais Biliares/química , Sítios de Ligação , Humanos , Fígado/metabolismo , Especificidade de Órgãos , Transportadores de Ânions Orgânicos Dependentes de Sódio/sangue , Ligação Proteica , Homologia de Sequência , Sódio/química , Especificidade da Espécie , Relação Estrutura-Atividade , Simportadores/sangue , Ácido Taurocólico/química , Distribuição Tecidual
17.
J Clin Gastroenterol ; 51(2): e11-e16, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27428727

RESUMO

GOALS: To perform an exploratory pilot study of all-trans retinoic acid (ATRA) combined with ursodeoxycholic acid (UDCA) in patients with primary sclerosing cholangitis (PSC). BACKGROUND: PSC is a progressive disorder for which there is no accepted therapy. Studies in human hepatocyte cultures and in animal models of cholestasis indicate that ATRA might have beneficial effects in cholestatic disorders. STUDY: ATRA (45 mg/m/d, divided and given twice daily) was combined with moderate-dose UDCA in patients with PSC who had incomplete response to UDCA monotherapy. The combination was administered for 12 weeks, followed by a 12-week washout in which patients returned to UDCA monotherapy. We measured alkaline phosphatase (ALP), alanine aminotransferase (ALT), bilirubin, cholesterol, bile acids, and the bile acid intermediate 7α-hydroxy-4-cholesten-3-one (C4) at baseline, week 12, and after washout. RESULTS: Fifteen patients completed 12 weeks of therapy. The addition of ATRA to UDCA reduced the median serum ALP levels (277±211 to 243±225 U/L, P=0.09) although this, the primary endpoint, did not reach significance. In contrast, median serum ALT (76±55 to 46±32 U/L, P=0.001) and C4 (9.8±19 to 7.9±11 ng/mL, P=0.03) levels significantly decreased. After washout, ALP and C4 levels nonsignificantly increased, whereas ALT levels significantly increased (46±32 to 74±74, P=0.0006), returning to baseline. CONCLUSIONS: In this human pilot study, the combination of ATRA and UDCA did not achieve the primary endpoint (ALP); however, it significantly reduced ALT and the bile acid intermediate C4. ATRA appears to inhibit bile acid synthesis and reduce markers of inflammation, making it a potential candidate for further study in PSC (NCT 01456468).


Assuntos
Colagogos e Coleréticos/administração & dosagem , Colangite Esclerosante/tratamento farmacológico , Tretinoína/administração & dosagem , Ácido Ursodesoxicólico/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Ácidos e Sais Biliares/biossíntese , Colangite Esclerosante/sangue , Colangite Esclerosante/fisiopatologia , Colestenonas/sangue , Quimioterapia Combinada , Feminino , Humanos , Fígado/fisiopatologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Resultado do Tratamento , Adulto Jovem
18.
J Hepatol ; 63(6): 1440-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26212029

RESUMO

BACKGROUND & AIMS: Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. METHODS: We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. RESULTS: Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. CONCLUSIONS: Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.


Assuntos
Colestase/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Adulto , Canalículos Biliares/metabolismo , Estudos de Casos e Controles , Colestase/etiologia , Colestase/patologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Feminino , Cálculos Biliares/complicações , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator Autócrino de Motilidade/antagonistas & inibidores , Receptores do Fator Autócrino de Motilidade/genética , Receptores do Fator Autócrino de Motilidade/metabolismo , Treonina/química
19.
Acta Pharmacol Sin ; 36(4): 473-82, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25832428

RESUMO

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.


Assuntos
Catequina/análogos & derivados , Colestase/complicações , Cirrose Hepática/complicações , Cirrose Hepática/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Catequina/uso terapêutico , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Smad/metabolismo
20.
J Pharmacol Exp Ther ; 349(1): 94-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24492652

RESUMO

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-ß and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.


Assuntos
1-Naftilisotiocianato/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colagogos e Coleréticos/uso terapêutico , Colestase/tratamento farmacológico , Fígado/efeitos dos fármacos , Tretinoína/uso terapêutico , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/sangue , Proliferação de Células/efeitos dos fármacos , Colagogos e Coleréticos/administração & dosagem , Colestase/induzido quimicamente , Colestase/genética , Colestase/patologia , Doença Crônica , Modelos Animais de Doenças , Quimioterapia Combinada , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Tretinoína/administração & dosagem , Ácido Ursodesoxicólico/administração & dosagem , Ácido Ursodesoxicólico/uso terapêutico
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