Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.057
Filtrar
1.
Gut ; 69(1): 133-145, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31409605

RESUMO

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.


Assuntos
Proteína do X Frágil de Retardo Mental/fisiologia , Hepatite Viral Animal/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/patologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Morte Celular/fisiologia , Células Cultivadas , Colestase/imunologia , Colestase/metabolismo , Colestase/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Hepatite Viral Animal/patologia , Hepatite Viral Animal/prevenção & controle , Hepatócitos/patologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Vírus da Coriomeningite Linfocítica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia
2.
Nat Med ; 25(12): 1885-1893, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792455

RESUMO

Early disease diagnosis is key to the effective treatment of diseases. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D (three-dimensional) structural changes resulting from functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of morphometric cellular and tissue parameters correlated with disease progression, and discover profound topological defects in the 3D bile canalicular (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and micro-cholestasis, consistent with elevated cholestatic biomarkers in patients' sera. Our spatially resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multiparametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.


Assuntos
Colestase/patologia , Imagem Tridimensional , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Canalículos Biliares/patologia , Sistema Biliar/patologia , Colestase/diagnóstico , Simulação por Computador , Progressão da Doença , Diagnóstico Precoce , Humanos , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/diagnóstico
3.
Yonsei Med J ; 60(11): 1045-1053, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637886

RESUMO

PURPOSE: To explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis. MATERIALS AND METHODS: The mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice. RESULTS: The mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects. CONCLUSION: Our findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.


Assuntos
Colestase/metabolismo , Resistência a Múltiplos Medicamentos , Fator de Transcrição E2F1/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Animais , Colestase/sangue , Colestase/genética , Colestase/patologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/sangue
4.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31455001

RESUMO

The purpose of this study was to investigate whether Ger-Gen-Chyn-Lian-Tang (GGCLT) suppresses oxidative stress, inflammation, and angiogenesis during experimental liver fibrosis through the hypoxia-inducible factor-1α (HIF-1α)-mediated pathway. Male C57BL/6 mice were randomly assigned to a sham-control or bile duct ligation (BDL) group with or without treatment with GGCLT at 30, 100, and 300 mg/kg. Plasma alanine aminotransferase (ALT) levels were analyzed using a diagnostic kit. Liver histopathology and hepatic status parameters were measured. Compared to control mice, the BDL mice exhibited an enlargement in liver HIF-1α levels, which was suppressed by 100 and 300 mg/kg GGCLT treatments (control: BDL: BDL + GGCLT-100: BDL + GGCLT-300 = 0.95 ± 0.07: 1.95 ± 0.12: 1.43 ± 0.05: 1.12 ± 0.10 fold; p < 0.05). GGCLT restrained the induction of hepatic hydroxyproline and malondialdehyde levels in the mice challenged with BDL, further increasing the hepatic glutathione levels. Furthermore, in response to increased hepatic inflammation and fibrogenesis, significant levels of ALT, nuclear factor kappa B, transforming growth factor-ß, α-smooth muscle actin, matrix metalloproteinase-2 (MMP-2), MMP-9, and procollagen-III were found in BDL mice, which were attenuated with GGCLT. In addition, GGCLT reduced the induction of angiogenesis in the liver after BDL by inhibiting vascular endothelial growth factor (VEGF) and VEGF receptors 1 and 2. In conclusion, the anti-liver fibrosis effect of GGCLT, which suppresses hepatic oxidative stress and angiogenesis, may be dependent on an HIF-1α-mediated pathway.


Assuntos
Colestase/complicações , Medicamentos de Ervas Chinesas/farmacologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Animais , Biomarcadores , Biópsia , Colestase/patologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacocinética , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Resultado do Tratamento
5.
Adv Pharmacol ; 85: 1-30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31307583

RESUMO

Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/patologia , Animais , Desdiferenciação Celular , Células Cultivadas , Colestase/patologia , Humanos , Fígado/patologia
6.
J Ethnopharmacol ; 245: 112103, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31336134

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis is a clinical syndrome caused by toxic bile acid retention that will lead to serious liver diseases. Ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) are the only two FDA-approved drugs for its treatment. Thus, there is a clear need to develop new therapeutic approaches for cholestasis. Here, anti-cholestasis effects of the lignans from a traditional Chinese herbal medicine, Schisandra sphenanthera, were investigated as well as the involved mechanisms. MATERIALS AND METHODS: Adult male C57BL/6J mice were randomly divided into 9 groups including the control group, LCA group, LCA with specific lignan treatment of Schisandrin A (SinA), Schisandrin B (SinB), Schisandrin C (SinC), Schisandrol A (SolA), Schisandrol B (SolB), Schisantherin A (StnA) and Schisantherin B (StnB), respectively. Mice were treated with each drug (qd) for 7 days, while the administration of lithocholic acid (LCA) (bid) was launched from the 4th day. Twelve hours after the last LCA injection, mice were sacrificed and samples were collected. Serum biochemical measurement and histological analysis were conducted. Metabolomics analysis of serum, liver, intestine and feces were performed to study the metabolic profile of bile acids. RT-qPCR and Western blot analysis were conducted to determine the hepatic expression of genes and proteins related to bile acid homeostasis. Dual-luciferase reporter gene assay was performed to investigate the transactivation effect of lignans on human pregnane X receptor (hPXR). RT-qPCR analysis was used to detect induction effects of lignans on hPXR-targeted genes in HepG2 cells. RESULTS: Lignans including SinA, SinB, SinC, SolA, SolB, StnA, StnB were found to significantly protect against LCA-induced intrahepatic cholestasis, as evidenced by significant decrease in liver necrosis, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activity. More importantly, serum total bile acids (TBA) and total bilirubin (Tbili) were also significantly reduced. Metabolomics analysis revealed these lignans accelerated the metabolism of bile acids and increased the bile acid efflux from liver into the intestine or feces. Gene analysis revealed these lignans induced the hepatic expressions of PXR-target genes such as Cyp3a11 and Ugt1a1. Luciferase reporter gene assays illustrated that these bioactive lignans can activate hPXR. Additionally, they can all upregulate hPXR-regulate genes such as CYP3A4, UGT1A1 and OATP2. CONCLUSION: These results clearly demonstrated the lignans from Schisandra sphenanthera exert hepatoprotective effects against LCA-induced cholestasis by activation of PXR. These lignans may provide an effective approach for the prevention and treatment of cholestatic liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colestase/tratamento farmacológico , Lignanas/uso terapêutico , Receptor de Pregnano X/genética , Substâncias Protetoras/uso terapêutico , Schisandra , Animais , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/induzido quimicamente , Colestase/metabolismo , Colestase/patologia , Fezes/química , Células HEK293 , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Lignanas/farmacologia , Ácido Litocólico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Substâncias Protetoras/farmacologia
7.
Eur J Pharmacol ; 857: 172461, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31220436

RESUMO

Estrogen-induced cholestasis is a common etiology of hepatic diseases in women with contraceptives administration, pregnancy or hormone replacement therapy. Farnesoid X receptor (FXR) is a member of nuclear receptor super family of ligand-activated transcription factors that is highly expressed in liver. FXR is acknowledged to contribute to the bile acid homeostasis, as well as the pathogenesis and progression of cholestasis. Specific targeting of FXR is an innovative approach for the treatment of cholestasis. The current study aimed to verify the anti-cholestasis effect of yangonin that is a natural product isolated from Kava via FXR signaling pathway in vivo and in vitro. The analyses of FXR gain- or loss-of-function were performed. Yangonin treatment ameliorates estrogen-induced cholestasis through increasing bile flow and biliary bile acid output. The mechanisms were an induction in the hepatic efflux transporters (Bsep and Mrp2) and an inhibition in hepatic uptake transporter (Ntcp) by yangonin. Likewise, yangonin through repressing Cyp7a1, Cyp8b1 and inducing Sult2a1 expression suppressed bile acid synthesis and promoted bile acid metabolism. Furthermore, yangonin improved estrogen-induced inflammatory cell infiltration and the inflammation gene expression. In vitro experiments further consolidated that yangonin alleviated estrogen-caused cholestasis via FXR activation. Noteworthily, the effects of yangonin were enhanced by FXR expression plasmids but abrogated by FXR siRNA. In conclusion, yangonin alleviates estrogen-induced cholestasis, due to FXR-mediated gene regulation.


Assuntos
Colestase/metabolismo , Colestase/prevenção & controle , Estrogênios/efeitos adversos , Pironas/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Sistema Biliar/efeitos dos fármacos , Sistema Biliar/metabolismo , Colestase/induzido quimicamente , Colestase/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
Zhonghua Gan Zang Bing Za Zhi ; 27(5): 321-324, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31177654

RESUMO

Intrahepatic cholestasis is a pathological condition in which the synthesis, secretion, and excretion of bile are blocked, and thus the bile does not flow normally into the duodenum and bloodstream. According to cytological damage site, it can be divided into hepatocellular cholestasis, biliary duct cell cholestasis and mixed cell cholestasis. The two kinds of pathophysiological models [ascending or upstream (damage begins with cholangiocytes and then extends to the hepatocytes) and descending or downstream (the damage starts from the liver cells and then extends to the bile duct cells)] has distinct features in the process of disease occurrence and development. This article mainly elaborates the "descending" pathophysiological model of cholestatic liver disease (hepatocytic damage progresses to biliary duct cell), and further explores its etiology, pathogenesis and treatment methods.


Assuntos
Colestase Extra-Hepática/fisiopatologia , Colestase Intra-Hepática/fisiopatologia , Colestase/etiologia , Colestase/patologia , Bile , Ácidos e Sais Biliares , Ductos Biliares/patologia , Humanos
9.
Int J Hematol ; 110(3): 381-384, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31093933

RESUMO

Obstructive jaundice is an initial symptom in 1-2% of diffuse large B cell lymphoma (DLBCL) cases. The major cause of bile duct obstruction in patients with DLBCL is extrinsic compression by enlarged lymph nodes. In such cases, the existence of bile duct invasion of lymphoma is rarely mentioned or observed pathologically, so the ratio of bile duct invasion to the total cases of obstructive jaundice, and its significance remains unknown. We report two cases of DLBCL presenting as an obstructive jaundice, in which we demonstrated bile duct invasion pathologically by biopsy from the wall of common bile duct with endoscopic retrograde cholangiopancreatography (ERCP). Endoscopic stent placement is a minimally invasive procedure to relieve cholestasis and is effective for diagnosing bile duct invasion. This procedure should thus be performed in all cases of obstructive jaundice caused by lymphoma to evaluate for bile duct invasion. Our cases suggest that ERCP may be useful as a diagnostic procedure for bile duct invasion.


Assuntos
Colangiopancreatografia Retrógrada Endoscópica , Colestase , Icterícia Obstrutiva , Linfoma Difuso de Grandes Células B , Idoso , Colestase/diagnóstico por imagem , Colestase/patologia , Colestase/fisiopatologia , Humanos , Icterícia Obstrutiva/diagnóstico por imagem , Icterícia Obstrutiva/patologia , Icterícia Obstrutiva/fisiopatologia , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Linfonodos/fisiopatologia , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/fisiopatologia , Masculino , Invasividade Neoplásica
10.
Medicine (Baltimore) ; 98(22): e15708, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31145285

RESUMO

RATIONALE: Cholestasis in pediatric patients has diverse etiologies and can be broadly classified as intrahepatic or extrahepatic. The common causes of extrahepatic cholestasis are bile duct calculus, inflammation, or pancreatitis. Malignant tumor is a rare cause of bile ducts obstruction in adolescent. Here we report a 14-year-old male patient with cholestasis due to poorly differentiated adenocarcinoma. PATIENT CONCERNS: A 14-year-old male patient with cholestasis was admitted because of jaundice, weakness, weight loss, and stomach pain for 2 months. The patient had been diagnosed with epilepsy 4 years previously and was being treated with sodium valproate and oxcarbazepine. On admission, laboratory studies showed elevated levels of aspartate aminotransferase (271 IU/L), alanine aminotransferase (224 IU/l), γ-glutamyltransferase (1668.9 IU/L), total bilirubin (66.4 µmol/L), and direct bilirubin (52.6 µmol/L). Additional laboratory tests eliminated common causes of cholestasis such as bacterial/viral infection, autoimmune liver disease, Wilson disease, Alagille syndrome, or progressive familial intrahepatic cholestasis type 3. The results of laboratory investigations showed no improvement after 10 days of treatment with ursodeoxycholic acid and vitamins A, D, and K1. Enhanced magnetic resonance imaging demonstrated a tumor of 22 mm diameter in the duodenal lumen and dilatation of the common bile duct. Endoscopic retrograde cholangiopancreatography detected a tumor in the duodenal lumen. DIAGNOSIS: Considering the clinical features, imaging manifestation, endoscopic findings, and pathologic characteristic, the patient was diagnosed with poorly differentiated adenocarcinoma. INTERVENTIONS: The patient underwent pancreaticoduodenectomy and chemotherapy. OUTCOME: The patient recovered well. Elevated levels of tumor biomarkers or abnormal liver function tests have not occurred during the 2-year follow-up. CONCLUSION: Cholestasis resulting from primary duodenal papillary carcinoma is rare in pediatric patients but should be considered in the differential diagnosis.


Assuntos
Ampola Hepatopancreática/patologia , Carcinoma Papilar/complicações , Colestase/etiologia , Neoplasias Duodenais/complicações , Adolescente , Carcinoma Papilar/patologia , Colestase/patologia , Neoplasias Duodenais/patologia , Humanos , Masculino
11.
J Ethnopharmacol ; 238: 111860, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-30965080

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis, caused by hepatic accumulation of bile acids, is a serious manifestation of liver diseases resulting in liver injury, fibrosis, and liver failure with limited therapies. Da-Huang-Xiao-Shi decoction (DHXSD) is a representative formula for treating jaundice and displays bright prospects in liver protective effect. AIM OF THE STUDY: This study was designed to assess the effects and possible mechanisms of DHXSD against alpha-naphthylisothiocyanate-induced liver injury based on ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) metabonomic approach. MATERIALS AND METHODS: The effects of DHXSD on serum indices (TBIL, DBIL, AST, ALT, ALP, TBA, and γ-GT) and the histopathology of the liver were analyzed. Moreover, UHPLC-Q-Orbitrap MS was performed to identify the possible effect of DHXSD on metabolites. The pathway analysis was conducted to illustrate the pathways and network by which DHXSD treats cholestasis. RESULTS: The results demonstrated that DHXSD could significantly regulate serum biochemical indices and alleviate histological damage to the liver. Twelve endogenous components, such as glycocholic acid, taurocholic acid and indoleacetaldehyde, were identified as potential biomarkers of the therapeutic effect of DHXSD. A systematic network analysis of their corresponding pathways indicates that the anti-cholestatic effect of DHXSD on alpha-naphthylisothiocyanate-induced cholestasis rats occurs mainly through regulating primary bile acid biosynthesis, arginine and proline metabolism, and arachidonic acid metabolism. CONCLUSIONS: DHXSD has exhibited favorable pharmacological effect on serum biochemical indices and pathological observation on cholestatic model by partially regulating the perturbed pathways. Moreover, these findings may help better understand the mechanisms of disease and provide a potential therapy for cholestasis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestase/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , 1-Naftilisotiocianato , Animais , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/induzido quimicamente , Colestase/patologia , Cromatografia Líquida de Alta Pressão , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Espectrometria de Massas , Ratos Sprague-Dawley
12.
J Surg Res ; 241: 254-263, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31035140

RESUMO

BACKGROUND: Clinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet. MATERIALS AND METHODS: Mouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence. RESULTS: Although cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice. CONCLUSIONS: Unlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy.


Assuntos
Autofagia , Colestase/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cirrose Hepática Experimental/imunologia , Fígado/patologia , Animais , Ductos Biliares/cirurgia , Colestase/etiologia , Colestase/patologia , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Ligadura , Fígado/citologia , Fígado/imunologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , Necrose/imunologia , Necrose/patologia , Fosforilação/imunologia , Tioacetamida/toxicidade
13.
Methods Mol Biol ; 1981: 117-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016651

RESUMO

The blockage of bile flow, cholestasis, could lead to serious clinical outcomes, including severe liver injury. Accumulation of the cytotoxic molecules, such as bile acids, during cholestasis, not only impairs liver function, but also affects other organs, including the kidneys. Although the precise mechanisms of cytotoxicity and organ injury in cholestasis are far from clear, oxidative stress and its subsequent events seem to play a central role in this complication. Oxidative stress acts as a signaling path which could finally lead to cell death and organ injury. At the cellular level, mitochondria are major targets affected by cytotoxic molecules. Mitochondrial impairment could lead to severe outcomes, including cellular energy crisis and release of cell death mediators from this organelle. Therefore, targeting oxidative stress and mitochondrial dysfunction might serve as a therapeutic point of intervention against cholestasis-associated organ injury. In this protocol, an animal model of cholestasis is described, and the techniques for liver mitochondria isolation, evaluating mitochondrial indices of functionality, and assessing biomarkers of oxidative stress in the liver tissue are outlined.


Assuntos
Colestase/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose , Ácidos e Sais Biliares/metabolismo , Colestase/patologia , Humanos , Mitocôndrias/patologia , Estresse Oxidativo/fisiologia
14.
Methods Mol Biol ; 1981: 149-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016653

RESUMO

Necroptosis is emerging as a critical pathogenic mechanism in several liver diseases, including cholestatic disorders. Necroptosis was recently described as a novel cell death subroutine, activated downstream of death receptor stimulation and dependent on receptor-interacting serine/threonine-protein kinase 3 activity and mixed lineage kinase domain-like oligomerization and translocation to cell membrane. Here, we describe a combination of methods to evaluate necroptosis triggering in in vitro and in vivo models of cholestasis. Particularly, we detail alternative protocols to isolate total and soluble/insoluble protein extracts from tissues and cell cultures, as well as in vitro receptor-interacting serine/threonine-protein kinase 3 kinase activity assays, and subsequent Western blot analysis.


Assuntos
Colestase/metabolismo , Colestase/patologia , Animais , Apoptose/fisiologia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Necrose/metabolismo , Necrose/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/fisiologia
15.
Methods Mol Biol ; 1981: 237-247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016658

RESUMO

Cholestatic liver disease covers a range of biliary disorders marked by an impaired bile duct flow. Various conditions can result in bile obstruction including choledocholithiasis, surgical trauma, and autoimmune disorders. Cholestatic liver disease can be mild but generally progresses to more severe conditions with increased hepatobiliary injury, cholangitis, and ultimately liver fibrosis and cirrhosis. An extensively used experimental model to investigate the pathophysiology of biliary cirrhosis and potential novel therapies is the common bile duct ligation in mice and rats. Common bile duct ligation induces the different stages of cholestatic-induced liver disease being cholestasis, subsequently accompanied by inflammation and finally liver fibrosis and cirrhosis. In this protocol, an outline of the surgical procedures to conduct common bile duct ligation in mice is provided. The major steps include the isolation of the common bile duct, followed by ligation and dissection.


Assuntos
Cirrose Hepática Biliar/metabolismo , Animais , Colestase/metabolismo , Colestase/patologia , Ducto Colédoco/metabolismo , Ducto Colédoco/patologia , Modelos Animais de Doenças , Hipertensão Portal/metabolismo , Hipertensão Portal/patologia , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/patologia , Camundongos , Ratos
16.
Methods Mol Biol ; 1981: 273-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016661

RESUMO

Cholestasis is a condition that impairs bile flow, resulting in retention of bile fluid in the liver. It may cause significant morbidity and mortality due to pruritus, malnutrition, and complications from portal hypertension secondary to biliary cirrhosis. The zebrafish (Danio rerio) has emerged as a valuable model organism for studying cholestasis that complements with the in vitro systems and rodent models. Its main advantages include conserved mechanisms of liver development and bile formation, rapid external development, ease of monitoring hepatobiliary morphology and function in live larvae, and accessibility to genetic and chemical manipulations. In this chapter, we provide an overview of the existing zebrafish models of cholestatic liver diseases. We discuss the strengths and limitations of using zebrafish to study cholestasis. We also provide step-by-step descriptions of the methodologies for analyzing cholestatic phenotypes in zebrafish.


Assuntos
Colestase/patologia , Modelos Animais de Doenças , Hepatopatias/patologia , Peixe-Zebra , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colestase/metabolismo , Fígado/metabolismo , Fígado/patologia , Hepatopatias/metabolismo
17.
Methods Mol Biol ; 1981: 291-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016662

RESUMO

Since HepaRG cells can differentiate into well-polarized mature hepatocyte-like cells that synthesize, conjugate, and secrete bile acids, they represent an appropriate surrogate to primary human hepatocytes for investigations on drug-induced cholestasis mechanisms. In this chapter, culture conditions for obtaining HepaRG hepatocytes and the main methods used to detect cholestatic potential of drugs are described. Assays for evaluation of bile canaliculi dynamics and morphology are mainly based on time-lapse and phase-contrast microscopy analysis. Bile acid uptake, trafficking, and efflux are investigated using fluorescent probes. Individual bile acids are quantified in both culture media and cell layers by high-pressure liquid chromatography/tandem mass spectrometry. Preferential cellular accumulation of toxic hydrophobic bile acids is easily evidenced when exogenous primary and secondary bile acids are added to the culture medium.


Assuntos
Colestase/metabolismo , Colestase/patologia , Hepatócitos/citologia , Linhagem Celular , Células Cultivadas , Humanos , Microscopia de Contraste de Fase
18.
Methods Mol Biol ; 1981: 313-323, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016663

RESUMO

Cholestasis remains a major challenge in drug-induced liver injury, and therefore warrants identification of chemical entities that may lead to cholestasis. Recent advances in cell culture methods enable 3D spheroid models to remain viable for much longer periods of time than conventional sandwich cultures of primary human hepatocytes while maintaining native tissue-like functionality, such as drug metabolism activity, receptor signaling functionality, and physiological relevance. These spheroid models enable us to study repeated exposure effects associated with chemicals and their metabolites that may ultimately progress to cholestasis and liver injury. HepaRG cells cultured as spheroids are viable for more than 4 weeks with cytochrome P450 enzymatic activities comparable to ranges observed in freshly isolated/cryopreserved suspensions of primary human hepatocytes. HepaRG spheroids form bile canalicular structures with potential application as a model to study biliary excretion processes and intrahepatic obstruction of bile flow, leading to hepatocellular damage and death. In this chapter, we describe methods to culture 3D spheroids of HepaRG cells with extensive bile canalicular structures/networks, image transport of bile acid (cholyl-lysyl-fluorescein) to the bile canaliculi, and measure cholestatic drug-induced cytotoxicity.


Assuntos
Colestase/metabolismo , Colestase/patologia , Hepatócitos/citologia , Fígado/citologia , Canalículos Biliares/metabolismo , Canalículos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
19.
Methods Mol Biol ; 1981: 335-350, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016665

RESUMO

Drug-induced cholestasis poses a major hurdle for the pharmaceutical industry as it is one the primary mechanisms of drug-induced liver injury. Hence, detection of drug-induced cholestasis during the early stages of drug development is of utmost importance. The most commonly used in vitro models rely on the extent of inhibition of bile salt export pump-mediated taurocholic acid transport, thereby assuming that drug-induced cholestasis mechanisms are merely restricted to the interaction with this sole hepatic transporter. Sandwich-cultured human hepatocytes represent a more holistic in vitro tool to investigate drug-induced cholestasis as they preserve all relevant disposition pathways and cellular functions involved in bile acid homeostasis. We developed and validated a sandwich-cultured human hepatocytes-based in vitro assay which is able to identify compounds causing cholestasis by altering bile acid disposition. The in vitro cholestatic potential is expressed by calculating a drug-induced cholestasis index value, which reflects the relative residual urea formation of sandwich-cultured human hepatocytes co-incubated with bile acids and test compound as compared to sandwich-cultured human hepatocytes treated with test compound alone. In addition, a safety margin can be calculated to determine the in vivo risk for cholestasis based on the determination of the drug-induced cholestasis index at various concentrations and the peak plasma concentration of the drug candidate. This chapter outlines the various steps involved in performing our sandwich-cultured human hepatocytes-based in vitro assay.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/metabolismo , Colestase/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Células Cultivadas , Humanos
20.
Methods Mol Biol ; 1981: 351-362, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016666

RESUMO

To mimic (human) cholestasis in vitro requires multiple triggers to establish a diseased phenotype. However, this is currently not simulated by existing in vitro models. Therefore, there is a high need for multicellular systems similar to the human physiology. In such an in vitro model, cell-cell interactions and intact bile canaliculi with functional bile flow should be present and preserved during long-term culture. Precision-cut liver slices represent an ex vivo tissue culture technique that replicates most of the multicellular characteristics of a whole liver in vivo. This chapter describes the preparation and culturing of (human) precision-cut liver slices. Furthermore, a protocol to use the precision-cut liver slices technique to predict drug-induced cholestatic liver injury is described.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/metabolismo , Colestase/patologia , Canalículos Biliares/metabolismo , Canalículos Biliares/patologia , Humanos , Fígado/metabolismo , Fígado/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA