RESUMEN
The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.
Asunto(s)
Estradiol , Hepatocitos , NADPH Oxidasas , Especies Reactivas de Oxígeno , Animales , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratas , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Estradiol/farmacología , Estradiol/metabolismo , Estradiol/análogos & derivados , Femenino , Colestasis/inducido químicamente , Colestasis/metabolismo , Colestasis/patología , Ratas Wistar , Acetofenonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Cultivadas , Antioxidantes/farmacología , Antioxidantes/metabolismo , Colestasis Intrahepática , Complicaciones del Embarazo , Transportadoras de Casetes de Unión a ATPRESUMEN
BACKGROUND: Skeletal muscle is sensitive to bile acids (BA) because it expresses the TGR5 receptor for BA. Cholic (CA) and deoxycholic (DCA) acids induce a sarcopenia-like phenotype through TGR5-dependent mechanisms. Besides, a mouse model of cholestasis-induced sarcopenia was characterised by increased levels of serum BA and muscle weakness, alterations that are dependent on TGR5 expression. Mitochondrial alterations, such as decreased mitochondrial potential and oxygen consumption rate (OCR), increased mitochondrial reactive oxygen species (mtROS) and unbalanced biogenesis and mitophagy, have not been studied in BA-induced sarcopenia. METHODS: We evaluated the effects of DCA and CA on mitochondrial alterations in C2C12 myotubes and a mouse model of cholestasis-induced sarcopenia. We measured mitochondrial mass by TOM20 levels and mitochondrial DNA; ultrastructural alterations by transmission electronic microscopy; mitochondrial biogenesis by PGC-1α plasmid reporter activity and protein levels by western blot analysis; mitophagy by the co-localisation of the MitoTracker and LysoTracker fluorescent probes; mitochondrial potential by detecting the TMRE probe signal; protein levels of OXPHOS complexes and LC3B by western blot analysis; OCR by Seahorse measures; and mtROS by MitoSOX probe signals. RESULTS: DCA and CA caused a reduction in mitochondrial mass and decreased mitochondrial biogenesis. Interestingly, DCA and CA increased LC3II/LC3I ratio and decreased autophagic flux concordant with raised mitophagosome-like structures. In addition, DCA and CA decreased mitochondrial potential and reduced protein levels in OXPHOS complexes I and II. The results also demonstrated that DCA and CA decreased basal, ATP-linked, FCCP-induced maximal respiration and spare OCR. DCA and CA also reduced the number of cristae. In addition, DCA and CA increased the mtROS. In mice with cholestasis-induced sarcopenia, TOM20, OXPHOS complexes I, II and III, and OCR were diminished. Interestingly, the OCR and OXPHOS complexes were correlated with muscle strength and bile acid levels. CONCLUSION: Our results showed that DCA and CA decreased mitochondrial mass, possibly by reducing mitochondrial biogenesis, which affects mitochondrial function, thereby altering potential OCR and mtROS generation. Some mitochondrial alterations were also observed in a mouse model of cholestasis-induced sarcopenia characterised by increased levels of BA, such as DCA and CA.
Asunto(s)
Colestasis , Sarcopenia , Animales , Ratones , Sarcopenia/metabolismo , Sarcopenia/patología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mitocondrias , Modelos Animales de Enfermedad , Colestasis/metabolismo , Colestasis/patologíaRESUMEN
Chronic liver diseases are a group of pathologies affecting the liver with high prevalence worldwide. Among them, cholestatic chronic liver diseases (CCLD) are characterized by alterations in liver function and increased plasma bile acids. Secondary to liver disease, under cholestasis, is developed sarcopenia, a skeletal muscle dysfunction with decreased muscle mass, strength, and physical function. CCL5/RANTES is a chemokine involved in the immune and inflammatory response. Indeed, CCL5 is a myokine because it is produced by skeletal muscle. Several studies show that bile acids induce CCL5/RANTES expression in liver cells. However, it is unknown if the expression of CCL5/RANTES is changed in the skeletal muscle of mice with cholestatic liver disease. We used a murine model of cholestasis-induced sarcopenia by intake of hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC diet), in which we detected the mRNA levels for ccl5. We determined that mice fed the DDC diet presented high levels of serum bile acids and developed typical features of sarcopenia. Under these conditions, we detected the ccl5 gene expression in diaphragm muscle showing elevated mRNA levels compared to mice fed with a standard diet (chow diet). Our results collectively suggest an increased ccl5 gene expression in the diaphragm muscle concomitantly with elevated serum bile acids and the development of sarcopenia.
Asunto(s)
Colestasis , Hepatopatías , Sarcopenia , Ratones , Animales , Sarcopenia/patología , Diafragma/metabolismo , Diafragma/patología , Regulación hacia Arriba , Quimiocina CCL5/metabolismo , Colestasis/complicaciones , Colestasis/metabolismo , Colestasis/patología , Hígado/metabolismo , Ácidos y Sales Biliares , Hepatopatías/metabolismo , Expresión Génica , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Skeletal muscle is sensitive to bile acids (BA) because it expresses the TGR5 receptor for BA. Cholic (CA) and deoxycholic (DCA) acids induce a sarcopenia-like phenotype through TGR5-dependent mechanisms. Besides, a mouse model of cholestasis-induced sarcopenia was characterised by increased levels of serum BA and muscle weakness, alterations that are dependent on TGR5 expression. Mitochondrial alterations, such as decreased mitochondrial potential and oxygen consumption rate (OCR), increased mitochondrial reactive oxygen species (mtROS) and unbalanced biogenesis and mitophagy, have not been studied in BA-induced sarcopenia.METHODS: We evaluated the effects of DCA and CA on mitochondrial alterations in C2C12 myotubes and a mouse model of cholestasis-induced sarcopenia. We measured mitochondrial mass by TOM20 levels and mitochondrial DNA; ultrastructural alterations by transmission electronic microscopy; mitochondrial biogenesis by PGC-1α plasmid reporter activity and protein levels by western blot analysis; mitophagy by the co-localisation of the MitoTracker and LysoTracker fluorescent probes; mitochondrial potential by detecting the TMRE probe signal; protein levels of OXPHOS complexes and LC3B by western blot analysis; OCR by Seahorse measures; and mtROS by MitoSOX probe signals. RESULTS: DCA and CA caused a reduction in mitochondrial mass and decreased mitochondrial biogenesis. Interestingly, DCA and CA increased LC3II/LC3I ratio and decreased autophagic flux concordant with raised mitophagosome-like structures. In addition, DCA and CA decreased mitochondrial potential and reduced protein levels in OXPHOS complexes I and II. The results also demonstrated that DCA and CA decreased basal, ATP-linked, FCCP-induced maximal respiration and spare OCR. DCA and CA also reduced the number of cristae. In addition, DCA and CA increased the mtROS. In mice with cholestasis-induced sarcopenia, TOM20, OXPHOS complexes I, II and III, and OCR were diminished. Interestingly, the OCR and OXPHOS complexes were correlated with muscle strength and bile acid levels. CONCLUSION: Our results showed that DCA and CA decreased mitochondrial mass, possibly by reducing mitochondrial biogenesis, which affects mitochondrial function, thereby altering potential OCR and mtROS generation. Some mitochondrial alterations were also observed in a mouse model of cholestasis-induced sarcopenia characterised by increased levels of BA, such as DCA and CA.
Asunto(s)
Animales , Ratones , Colestasis/metabolismo , Colestasis/patología , Sarcopenia/metabolismo , Sarcopenia/patología , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Modelos Animales de Enfermedad , MitocondriasRESUMEN
Bile acids (BA) are recognized by their role in nutrient absorption. However, there is growing evidence that BA also have endocrine and metabolic functions. Besides, the steroidal-derived structure gives BA a toxic potential over the biological membrane. Thus, cholestatic disorders, characterized by elevated BA on the liver and serum, are a significant cause of liver transplant and extrahepatic complications, such as skeletal muscle, central nervous system (CNS), heart, and placenta. Further, the BA have an essential role in cellular damage, mediating processes such as membrane disruption, mitochondrial dysfunction, and the generation of reactive oxygen species (ROS) and oxidative stress. The purpose of this review is to describe the BA and their role on hepatic and extrahepatic complications in cholestatic diseases, focusing on the association between BA and the generation of oxidative stress that mediates tissue damage.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colestasis/patología , Animales , Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/farmacología , Colestasis/metabolismo , Hígado/metabolismo , Músculo Esquelético/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The mechanism of damage of the biliary epithelium remains partially unexplored. However, recently many works have offered new evidence regarding the cholangiocytes' damage process, which is the main target in a broad spectrum of pathologies ranging from acute cholestasis, cholangiopathies to cholangiocarcinoma. This is encouraging since some works addressed this epithelium's relevance in health and disease until a few years ago. The biliary tree in the liver, comprised of cholangiocytes, is a pipeline for bile flow and regulates key hepatic processes such as proliferation, regeneration, immune response, and signaling. This review aimed to compile the most recent advances on the mechanisms of cholangiocellular damage during cholestasis, which, although it is present in many cholangiopathies, is not necessarily a common or conserved process in all of them, having a relevant role cAMP and PKA during obstructive cholestasis, as well as Ca2+-dependent PKC in functional cholestasis. Cholangiocellular damage could vary according to the type of cholestasis, the aggressor, or the bile ducts' location where it develops and what kind of damage can favor cholangiocellular carcinoma development.
Asunto(s)
Procedimientos Quirúrgicos del Sistema Biliar/efectos adversos , Sistema Biliar/patología , Colestasis/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Sistema Biliar/metabolismo , Proliferación Celular , Colestasis/metabolismo , Colestasis/cirugía , Humanos , Ligadura , Transducción de SeñalRESUMEN
INTRODUCTION AND OBJECTIVES: Free and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters' increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters' mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. METHODS: The expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. RESULTS: The results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. CONCLUSION: Primary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Colestasis/genética , Colestasis/patología , Fármacos Gastrointestinales/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Técnicas de Cultivo de Célula , Colestasis/metabolismo , Células Hep G2 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Mensajero/metabolismoRESUMEN
A busca pelo corpo perfeito pode gerar graves consequências para a população que faz uso indiscriminado de substâncias visando a resultados rápidos. O caso relatado se refere a um pa- ciente de 21 anos, do sexo masculino, na cidade de São Paulo (SP), que apresentou quadro de síndrome colestática 15 dias após uso do anabolizante estanazolol para fins estéticos na ativi- dade física, evoluindo com hepatite medicamentosa grave, com aumento de transaminases, hiperrubilinemia às custas de bilirrubina direta e fatores de coagulação, sem resposta satis- fatória ao tratamento de suporte convencional, com melhora significativa após introdução de corticoterapia.
Searching for the perfect body image can cause severe conse- quences to the population using substances indiscriminately to reach results fast. The case reported refers to a male patient, 21 years old, from the city of São Paulo (SP), who developed choles- tatic syndrome 15 days after the use of the steroid Stanazol for aesthetic purposes during physical activity, progressing with se- vere drug-induced hepatitis, transaminases, bilirubin, and coagu- lation factors increase with no satisfactory response to the con- ventional support treatment, and significant improvement after the introduction of corticotherapy.
Asunto(s)
Humanos , Masculino , Adulto , Adulto Joven , Estanozolol/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Anabolizantes/toxicidad , Ácido Ursodesoxicólico/administración & dosificación , Bilirrubina/sangre , Biopsia , Colagogos y Coleréticos/uso terapéutico , Prednisona/administración & dosificación , Colestasis/diagnóstico , Colestasis/patología , Colesterol/sangre , Resina de Colestiramina/administración & dosificación , Enfermedad Catastrófica , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Transaminasas/sangre , Hidroxizina/administración & dosificación , Hígado/patología , Anticolesterolemiantes/uso terapéutico , Antipruriginosos/uso terapéuticoRESUMEN
BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.
Asunto(s)
Anemia de Células Falciformes/complicaciones , Bilis/metabolismo , Colestasis/etiología , Insuficiencia Hepática/etiología , Hígado/patología , Adolescente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Conductos Biliares Intrahepáticos/diagnóstico por imagen , Conductos Biliares Intrahepáticos/patología , Colestasis/patología , Colestasis/prevención & control , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Hemoglobina Falciforme/genética , Insuficiencia Hepática/patología , Insuficiencia Hepática/prevención & control , Humanos , Microscopía Intravital , Hígado/diagnóstico por imagen , Masculino , Ratones , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
OBJECTIVE: To prospectively assess the diagnostic performance of ultrasound shear wave elastography (SWE) and hepatobiliary laboratory biomarkers for discriminating biliary atresia from other causes of neonatal cholestasis. STUDY DESIGN: Forty-one patients <3 months of age with neonatal cholestasis (direct bilirubin >2 mg/dL) and possible biliary atresia were prospectively enrolled. Both 2-dimensional (2D) and point ultrasound SWE were performed prior to knowing the final diagnosis. Median 2D (8) and point (10) shear wave speed measurements were calculated for each subject and used for analyses. The Mann-Whitney U test was used to compare shear wave speed and laboratory measurements between patients with and without biliary atresia. Receiver operating characteristic curve analyses and multivariable logistic regression were used to evaluate diagnostic performance. RESULTS: Thirteen subjects (31.7%) were diagnosed with biliary atresia, and 28 subjects (68.3%) were diagnosed with other causes of neonatal cholestasis. Median age at the time of ultrasound SWE was 37 days. Median 2D (2.08 vs 1.49 m/s, P = .0001) and point (1.95 vs 1.21 m/s, P = .0014) ultrasound SWE measurements were significantly different between subjects with and without biliary atresia. Using a cut-off value of >1.84 m/s, 2D ultrasound SWE had a sensitivity = 92.3%, specificity = 78.6%, and area under the receiver operating characteristic curve (AuROC) of 0.89 (P < .0001). Using a cut-off value of >320 (U/L), gamma-glutamyl transferase (GGT) had a sensitivity = 100.0%, specificity = 77.8%, and AuROC of 0.85 (P < .0001). Multivariable logistic regression demonstrated an AuROC of 0.93 (P < .0001), with 2 significant covariates (2D ultrasound SWE [OR = 23.06, P = .01]; GGT [OR = 1.003, P = .036]). CONCLUSIONS: Ultrasound SWE and GGT can help discriminate biliary atresia from other causes of neonatal cholestasis.
Asunto(s)
Atresia Biliar/diagnóstico por imagen , Colestasis/diagnóstico por imagen , Alanina Transaminasa/sangre , Atresia Biliar/patología , Biomarcadores/sangre , Colestasis/etiología , Colestasis/patología , Diagnóstico por Imagen de Elasticidad , Femenino , Humanos , Lactante , Recién Nacido , Hígado/diagnóstico por imagen , Hígado/patología , Masculino , Valor Predictivo de las Pruebas , Estudios Prospectivos , Ultrasonografía , gamma-Glutamiltransferasa/sangreRESUMEN
In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (JOH-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 ± 0.3 vs. 3.1 ± 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 ± 0.3 vs. 3.5 ± 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.
Asunto(s)
Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Cloruros/metabolismo , Colestasis/metabolismo , Regulación hacia Abajo , Hepatocitos/metabolismo , Animales , Colestasis/patología , Modelos Animales de Enfermedad , Hepatocitos/patología , Transporte Iónico , Masculino , Ratas , Ratas WistarRESUMEN
We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert-butylhydroperoxide (tBuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by tBuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background.
Asunto(s)
Antioxidantes/farmacología , Colestasis/prevención & control , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemina/farmacología , Hígado/efectos de los fármacos , Estrés Oxidativo , Animales , Bilis/metabolismo , Bilirrubina/metabolismo , Catalasa/metabolismo , Colestasis/inducido químicamente , Colestasis/enzimología , Colestasis/patología , Modelos Animales de Enfermedad , Inducción Enzimática , Glutatión/metabolismo , Hígado/enzimología , Hígado/patología , Masculino , Ratas Wistar , Superóxido Dismutasa/metabolismo , terc-ButilhidroperóxidoRESUMEN
INTRODUCTION AND AIM: Neonatal cholestasis constitutes for 19 to 33% of all chronic liver disease in India. Cholestasis leads to fibrosis of liver and ultimately cirrhosis. There are various methods of diagnosis of fibrosis of liver like fibroscan, APRI index, FIB-4, fibro index, forns index, heap score, magnetic elastography. Here we are comparing APRI index with METAVIR index in patients with neonatal cholestasis without biliary atresia and determining whether APRI index can be used as a tool to determine fibrosis in these patients. MATERIAL AND METHODS: Patients with neonatal cholestasis without biliary atresia were included in the study. This retrospective analysis was done between 2009 and 2015. All patients underwent a liver biopsy and METAVIR index was calculated. APRI at the time of liver biopsy was determined. RESULTS: Forty-eight patients were included in this study with mean age of 3.5 ± 2.8 months with a male: female ratio of 35:13. Metavir Index F0 was seen in was 32 (66.67%) patients, F1 in 6(12.5%), F2 in 4(8.33%), F3 in 0 and F4 in 6(12.5%) patients respectively. Mean APRI for F0-F3 was 1.38 and for F4 was 3.74 respectively. With an APRI of 1.38, the sensitivity and specificity to detect fibrosis/cirrhosis was 100% and 21.43% respectively. CONCLUSION: APRI is not an effective tool to measure fibrosis or cirrhosis in patients with non-BA neonatal cholestasis in Indian children.
Asunto(s)
Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Plaquetas , Colestasis/diagnóstico , Técnicas de Apoyo para la Decisión , Enfermedades del Recién Nacido/diagnóstico , Cirrosis Hepática/diagnóstico , Bilirrubina/sangre , Biomarcadores/sangre , Biopsia , Colestasis/sangre , Colestasis/diagnóstico por imagen , Colestasis/patología , Pruebas Enzimáticas Clínicas , Diagnóstico por Imagen de Elasticidad/métodos , Femenino , Humanos , India , Lactante , Recién Nacido , Enfermedades del Recién Nacido/sangre , Enfermedades del Recién Nacido/diagnóstico por imagen , Enfermedades del Recién Nacido/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/patología , Imagen por Resonancia Magnética , Masculino , Recuento de Plaquetas , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios RetrospectivosRESUMEN
Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS: Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.
Asunto(s)
Traslocación Bacteriana , Colestasis/metabolismo , Colestasis/microbiología , Microbioma Gastrointestinal , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Intestinos/microbiología , Mucina 2/metabolismo , Animales , Colestasis/patología , Modelos Animales de Enfermedad , Hepatitis/metabolismo , Hepatitis/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Intestinos/patología , Hígado/metabolismo , Hígado/microbiología , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/microbiología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Masculino , Permeabilidad , Ratas Wistar , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 ß-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.
Asunto(s)
Colestasis/metabolismo , Endocitosis , Hepatocitos/metabolismo , Hígado/metabolismo , Microdominios de Membrana/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Colestasis/inducido químicamente , Colestasis/patología , Modelos Animales de Enfermedad , Femenino , Hepatocitos/patología , Hígado/patología , Microdominios de Membrana/patología , Ratas , Ratas WistarRESUMEN
Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Comunicación Celular , Colestasis/terapia , Citocinas/metabolismo , Hepatocitos/metabolismo , Cirrosis Hepática/terapia , Animales , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/metabolismo , Colestasis/complicaciones , Colestasis/genética , Colestasis/patología , Colágeno/metabolismo , Citocinas/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Adenoviridae/genética , Acuaporina 1/genética , Ácidos y Sales Biliares/metabolismo , Colestasis/terapia , Hepatocitos/metabolismo , Sepsis/terapia , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Adenoviridae/metabolismo , Animales , Acuaporina 1/metabolismo , Transporte Biológico , Colestasis/inducido químicamente , Colestasis/genética , Colestasis/patología , Colesterol/metabolismo , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hepatocitos/patología , Humanos , Lipopolisacáridos , Hígado/metabolismo , Hígado/patología , Masculino , Ratas , Ratas Wistar , Reología , Sepsis/inducido químicamente , Sepsis/genética , Sepsis/patología , TransgenesRESUMEN
Bile acids (BA) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades there have been great advances in the understanding of the role of a number of specific transport proteins present at the sinusoidal and canalicular membrane domains of hepatocytes and cholangiocytes in generating and maintaining bile flow. Also, a clearer understanding on how BA regulate their own synthesis and the expression and/or function of transporters has been reached. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches including new hydrophilic BA such as the conjugation-resistant nor- ursodeoxycholic acid, nuclear receptors (FXR, PPAR-alpha) agonists, FGF19 analogues, inhibitors of the apical sodium-dependent bile acid transporter [ASBT] and modulators of the inflammatory cascade triggered by BAs are being studied as novel treatments of cholestasis. In the present review we summarize recent experimental and clinical data on the role of BAs in cholestasis and its treatment.
Asunto(s)
Ácidos y Sales Biliares/uso terapéutico , Conductos Biliares/efectos de los fármacos , Colestasis/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , Hígado/efectos de los fármacos , Animales , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/inmunología , Conductos Biliares/metabolismo , Conductos Biliares/patología , Colestasis/inmunología , Colestasis/metabolismo , Colestasis/patología , Humanos , Inmunidad Innata/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Percutaneous drainage of the bile ducts is an established procedure for malignant obstructions, in which a histological diagnosis is often not obtained. We describe the biopsy technique of obstructive lesions through biliary drainage access, using a 7F endoscopic biopsy forceps, widely available; some are even reusable. This technique applies to lesions of the hepatic ducts, of the common hepatic duct and of all extension of the common bile duct. RESUMO A drenagem percutânea das vias biliares é um procedimento estabelecido para obstruções malignas, nos quais, muitas vezes, não se consegue um diagnóstico histológico. Descrevemos a técnica de biópsia da lesão obstrutiva através do acesso de drenagem biliar, utilizando um fórcipe de biópsia endoscópica 7F, amplamente disponível e alguns reutilizáveis. Esta técnica aplica-se a lesões dos ductos hepáticos, do hepático comum e de toda extensão do colédoco.
Asunto(s)
Neoplasias de los Conductos Biliares/patología , Neoplasias de los Conductos Biliares/complicaciones , Biopsia/métodos , Colestasis/etiología , Colestasis/patología , HumanosRESUMEN
ABSTRACT Percutaneous drainage of the bile ducts is an established procedure for malignant obstructions, in which a histological diagnosis is often not obtained. We describe the biopsy technique of obstructive lesions through biliary drainage access, using a 7F endoscopic biopsy forceps, widely available; some are even reusable. This technique applies to lesions of the hepatic ducts, of the common hepatic duct and of all extension of the common bile duct.
RESUMO A drenagem percutânea das vias biliares é um procedimento estabelecido para obstruções malignas, nos quais, muitas vezes, não se consegue um diagnóstico histológico. Descrevemos a técnica de biópsia da lesão obstrutiva através do acesso de drenagem biliar, utilizando um fórcipe de biópsia endoscópica 7F, amplamente disponível e alguns reutilizáveis. Esta técnica aplica-se a lesões dos ductos hepáticos, do hepático comum e de toda extensão do colédoco.