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OBJECTIVE: To assess hepatic transcriptional signatures in infants with gestational alloimmune liver disease (GALD) compared with other etiologies of neonatal acute liver failure (ALF) and older pediatric patients with ALF. STUDY DESIGN: Neonates with ALF (international normalized ratio ≥2 within 30 days of life) and deceased neonates without liver disease (<30 days of age) with available liver tissue between 2010 and 2021 were identified at Ann & Robert H. Lurie Children's Hospital of Chicago. Clinical information, liver histology, and data from RNA-sequencing analysis was compared between neonates with GALD, non-GALD etiologies of neonatal ALF, and nondiseased neonatal liver. RESULTS: Quantification of trichrome staining showed an increase in fibrosis in patients with GALD vs those with non-GALD neonatal ALF (P = .012); however, quantification of α-cytokeratin 19-positive ductules did not differ between groups (P = .244). Gene set enrichment analysis of RNA-sequencing data identified the pathways of complement activation, fibrosis, and organogenesis to be upregulated in patients with GALD with ALF. In contrast, patients with non-GALD causes of neonatal ALF had increased gene expression for interferon-driven immune pathways. Individual genes upregulated in GALD included matrix metallopeptidase 7, hepatocyte growth factor, and chemokine ligand 14. CONCLUSIONS: We have identified distinct pathways that are significantly upregulated in patients with GALD and potential disease-specific diagnostic biomarkers. Future studies will aim to validate these findings and help identify GALD-specific diagnostic biomarkers to improve diagnostic accuracy and reduce GALD-associated patient mortality.
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Fallo Hepático Agudo , Lactante , Recién Nacido , Humanos , Niño , Fallo Hepático Agudo/genética , Fibrosis , Biomarcadores/análisis , ChicagoRESUMEN
Many patients with indeterminate pediatric acute liver failure (PALF) have evidence of T-cell driven immune injury; however, the precise inflammatory pathways are not well defined. We have characterized the hepatic cytokine and transcriptional signatures of patients with PALF. A retrospective review was performed on 22 children presenting with indeterminate (IND-PALF; n = 17) or other known diagnoses (DX-PALF; n = 6) with available archived liver tissue. Specimens were stained for clusters of differentiation 8 (CD8) T cells and scored as dense, moderate, or minimal. Measurement of immune analytes and RNA sequencing (RNA-seq) was performed on whole-liver tissue. Immune analyte data were analyzed by principal component analysis, and RNA-seq was analyzed by unsupervised hierarchical clustering, differential gene expression, and gene-set enrichment analysis. Most patients with IND-PALF (94%) had dense/moderate CD8 staining and were characterized by Th1 immune analytes including tumor necrosis factor α, interferon γ (IFN-γ), interleukin (IL) 1ß, IL-12, C-X-C motif chemokine ligand (CXCL) 9, and CXCL12. Transcriptional analyses identified two transcriptional PALF phenotypes. Most patients in group 1 (91%) had IND-PALF and dense/moderate CD8 staining. This group was characterized by increased expression of genes and cell subset-specific signatures related to innate inflammation, T-cell activation, and antigen stimulation. Group 1 expressed significantly higher levels of gene signatures for regulatory T cells, macrophages, Th1 cells, T effector memory cells, cytotoxic T cells, and activated dendritic cells (adjusted P < 0.05). In contrast, patients in group 2 exhibited increased expression for genes involved in metabolic processes. Conclusion: Patients with IND-PALF have evidence of a Th1-mediated inflammatory response driven by IFN-γ. Transcriptional analyses suggest that a complex immune network may regulate an immune-driven PALF phenotype with less evidence of metabolic processes. These findings provide insight into mechanisms of hepatic injury in PALF, areas for future research, and potential therapeutic targets.
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Biliary atresia (BA) is a neonatal cholestatic liver disease that is the leading cause of pediatric liver transplantation, however, the mechanism of disease remains unknown. There are two major forms of BA: isolated BA (iBA) comprises the majority of cases and is thought to result from an aberrant immune response to an environmental trigger, whereas syndromic BA (BASM) has associated malformations and is thought to arise from a congenital insult. To determine whether B cells in BA biliary remnants are antigen driven, we examined the immunoglobulin (Ig) repertoire of diseased tissue from each BA group. Deep sequencing of the Ig chain DNA was performed on iBA and BASM biliary remnants and lymph nodes obtained from the Childhood Liver Disease Research Network (ChiLDReN) repository. Statistical analysis of the Ig repertoire provided measures of Ig clonality and the Ig phenotype. Our data demonstrate that B cells infiltrate diseased iBA and BASM biliary remnant tissue. The Ig repertoires of iBA and BASM disease groups were oligoclonal supporting a role for an antigen-driven immune response in both sub-types. These findings shift the current understanding of BA and suggest a role for antigen stimulation in early iBA and BASM disease pathogenesis.
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Atresia Biliar/clasificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoglobulinas/genética , Linfocitos B/inmunología , Atresia Biliar/inmunología , Preescolar , Humanos , Lactante , Recién Nacido , Análisis de Secuencia de ADN/métodosRESUMEN
OBJECTIVE: To assess the utility of whole-exome sequencing (WES) in a sibling pair with undetermined liver disease and describe the phenotype associated with mutations discovered therein. STUDY DESIGN: Next-generation WES was performed on 2 siblings (S1 and S2) who were born to nonconsanguineous parents of European extraction. Both siblings developed cirrhosis of indeterminate etiology and required liver transplantation; S1 at 7 months and S2 at 22 months. RESULTS: Sequencing of germline DNA identified compound heterozygous mutations in PPP1R15B resulting in increased levels of phosphorylated eukaryotic translation initiation factor 2α. CONCLUSIONS: The first demonstration of PPP1R15B associated with liver disease expands the phenotypic spectrum of PPP1R15B related diseases. Our findings validate the application of WES in the diagnosis of children with undetermined liver disease. Understanding the genetic basis of liver disease may allow the development of targeted therapies for treatment and adequate counseling of families.
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Trastornos del Crecimiento/genética , Cirrosis Hepática/genética , Mutación , Trastornos del Neurodesarrollo/genética , Proteína Fosfatasa 1/genética , Femenino , Humanos , Lactante , Fenotipo , Proteína Fosfatasa 1/deficiencia , Análisis de Secuencia de ADNRESUMEN
Gestational alloimmune liver disease (GALD) produces severe neonatal liver disease that is notable for paucity of hepatocytes, large numbers of parenchymal tubules, and extensive fibrosis. Liver specimens from 19 GALD cases were studied in comparison with 14 infants without liver disease (normal newborn liver; NNL) to better understand the pathophysiology that would produce this characteristic histopathology. GALD liver parenchyma contained large numbers of tubules comprising epithelium expressing KRT7/19, EPCAM, and SOX9, suggesting biliary progenitor status. Quantitative morphometry demonstrated that in GALD, the area density of KRT19+ tubules was 16.4 ± 6.2 versus 2.0 ± 2.6 area% in NNL (P < .0001). Functional hepatocyte mass was markedly reduced in GALD, 16.3 ± 6.2 versus 61.9 ± 11.0 area% of CPS1+ cells in NNL (P < .0001). A strong inverse correlation was established between CPS1+ area density and KRT19+ area density (r(2) = 0.66, P < .0001). Tubules showed active hedgehog signaling as determined by SHH and nuclear GLI2 expression and expressed the profibrogenic cytokine SPP1. SPP1 protein content and SPP1 expression were greater in GALD than NNL (15- and 13-fold respectively; P = .002). GALD liver contained large numbers of activated myofibroblasts and showed greater than 10-fold more fibrosis than NNL. The extent of fibrosis correlated with the area density of KRT19+ tubules (r(2) = 0.387, P = .001). The data support a pathogenic model in which immune injury to fetal hepatocytes provides a stimulus for expansion of parenchymal tubules, which, by way of Hh activation, produce fibrogenic signals leading to vibrant fibrosis.
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Enfermedades Genéticas Congénitas/diagnóstico , Proteínas Hedgehog/análisis , Hemocromatosis/diagnóstico , Hepatocitos/química , Cirrosis Hepática/diagnóstico , Hígado/química , Transducción de Señal , Células Madre/química , Biomarcadores/análisis , Biopsia , Estudios de Casos y Controles , Linaje de la Célula , Proliferación Celular , Preescolar , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/patología , Edad Gestacional , Hemocromatosis/metabolismo , Hemocromatosis/patología , Hepatocitos/patología , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Queratina-19/análisis , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Miofibroblastos/química , Miofibroblastos/patología , Osteopontina/análisis , Fenotipo , Índice de Severidad de la Enfermedad , Células Madre/patologíaRESUMEN
BACKGROUND & AIMS: Gestational alloimmune liver disease is the main cause of the neonatal hemochromatosis phenotype, wherein severe neonatal liver disease is associated with iron overload and extrahepatic tissue siderosis. How fetal liver disease produces extrahepatic siderosis is not known. We hypothesized that fetal liver injury causes deficient hepcidin production and poor regulation of placental iron flux. Under the resulting conditions of iron overload, the tissue pattern of extrahepatic siderosis is determined by the normal expression of proteins involved in the import of non-transferrin-bound iron and the export of cellular iron. METHODS: Liver and extrahepatic tissues from infants with gestational alloimmune liver disease were examined and compared to normal age-appropriate tissues. RESULTS: Serum iron indices indicate iron overload and excess non-transferrin bound iron in gestational alloimmune liver disease. The diseased liver showed significantly reduced hepcidin, hemojuvulin, and transferrin gene expression compared to the normal fetal and neonatal liver. Those extrahepatic tissues that are typically involved in pathological siderosis in neonatal hemochromatosis, whether from normal or diseased newborns, consistently expressed solute carrier family 39 (zinc transporter), member 14 (ZIP14) for non-transferrin-bound iron uptake and expressed little ferroportin for iron export. CONCLUSIONS: Excess non-transferrin-bound iron in gestational alloimmune liver disease may result from fetal liver injury that causes reduced synthesis of key iron regulatory and transport proteins. Whereas, the pattern of extrahepatic siderosis appears to be determined by the normal capacity of various tissues to import non-transferrin-bound iron and not export cellular iron.
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Hemocromatosis/etiología , Isoantígenos/inmunología , Hepatopatías/complicaciones , Complicaciones del Embarazo , Siderosis/etiología , Péptidos Catiónicos Antimicrobianos/genética , Femenino , Proteínas Ligadas a GPI/genética , Proteína de la Hemocromatosis , Hepcidinas , Humanos , Recién Nacido , Hierro/metabolismo , Embarazo , Tromboplastina/genéticaRESUMEN
OBJECTIVE: To determine whether alloimmune liver disease can be identified as a cause of fetal death. STUDY DESIGN: This is a retrospective examination of the autopsy tissue of 6 stillborn fetuses and 2 extreme preterm infants (gestational age, 20 to 34 weeks) drawn from families referred for suspected neonatal hemochromatosis. Thirteen appropriate nondisease controls and 8 cases of neonatal acute liver failure with known etiology were also examined. Liver sections were immunostained using anti-human C5b-9 complex. RESULTS: All of the study cases had died with no preceding evidence of fetal distress. Histopathology showed findings of acute liver injury, including global hepatocyte necrosis with minimal reticulum collapse and no fibrosis. Hepatocytes in cases stained strongly positively for C5b-9 complex, suggesting premortem lgG complement-mediated liver injury. Hepatocytes in acute liver failure case controls did not demonstrate a similar mechanism of liver injury. CONCLUSIONS: Alloimmune liver disease is sometimes associated with fetal death.
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Hemocromatosis/inmunología , Fallo Hepático Agudo/inmunología , Mortinato , Estudios de Casos y Controles , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Femenino , Hemocromatosis/congénito , Hemocromatosis/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Recién Nacido , Recien Nacido Prematuro , Hígado/patología , Fallo Hepático Agudo/congénito , Fallo Hepático Agudo/patología , Necrosis , Embarazo , Estudios RetrospectivosRESUMEN
UNLABELLED: Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc(+/-) ) (overly active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc(+/-) mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). CONCLUSION: OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH.
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Proteínas Hedgehog/fisiología , Cirrosis Hepática/etiología , Osteopontina/genética , Animales , Línea Celular , Deficiencia de Colina , Dieta , Hígado Graso/fisiopatología , Células Estrelladas Hepáticas , Humanos , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , Osteopontina/biosíntesis , Osteopontina/deficiencia , Regulación hacia Arriba , Alcaloides de Veratrum/farmacologíaRESUMEN
UNLABELLED: Evidence suggests that most neonatal hemochromatosis (NH) is the phenotypic expression of gestational alloimmune fetal liver injury. Gestational alloimmune diseases are induced by the placental passage of specific reactive immunoglobulin G and often involve the activation of fetal complement by the classical pathway leading to the formation of membrane attack complex (MAC) as the effector of cell injury. We examined liver specimens from cases of NH, from cases of non-NH liver disease, and from infants without liver disease to determine if they would provide evidence that MAC is involved in hepatocyte injury. Sections were immunostained with anti-human C5b-9 complex, the terminal complement cascade (TCC) neoantigen formed in the assembly of MAC. Fetal liver injury in NH cases is associated with a severe loss of hepatocytes. In all NH cases examined, most remaining hepatocytes showed intense staining for TCC neoantigen, whereas hepatocytes in non-NH liver disease cases showed variable light staining. The percentage of hepatocytes containing TCC neoantigen in NH was much greater than that in non-NH liver disease, and there was no overlap between the groups. Findings in both groups suggest that hepatocytes have mechanisms to protect against MAC, including a biliary pathway for its excretion. CONCLUSION: The finding that all cases of proven NH contained TCC neoantigen far in excess of cases of other neonatal liver diseases suggests that a single process, namely congenital alloimmune hepatitis, is the principal cause of NH. MAC-mediated alloimmune injury in congenital alloimmune hepatitis is a novel mechanism of liver injury that results from an interplay of maternal adaptive immunity and fetal innate immunity.
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Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Hemocromatosis/congénito , Hepatitis/congénito , Hepatocitos/metabolismo , Feto/metabolismo , Hemocromatosis/metabolismo , Hemocromatosis/patología , Hepatitis/metabolismo , Hepatitis/patología , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Hígado/metabolismo , Hígado/patologíaRESUMEN
Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.
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Regulación Enzimológica de la Expresión Génica , Hepatocitos/metabolismo , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno , Animales , Apoptosis , Supervivencia Celular , Cromonas/farmacología , Ácidos Grasos no Esterificados/metabolismo , Hígado Graso/patología , Peróxido de Hidrógeno/química , Ratones , Modelos Biológicos , Morfolinas/farmacología , Fosfohidrolasa PTEN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Feeding mice a methionine and choline-deficient (MCD) diet serves as an experimental animal model for nonalcoholic steatohepatitis (NASH). In the present study we examined the effect of exposing AML-12 hepatocytes to MCD culture medium in regard to mechanisms of steatosis and alanine amino-transferase (ALT) release. Cells exposed to MCD medium developed significant and progressive steatosis from 6 to 24 h and also had significantly increased loss of ALT into the medium at 18 and 24 hours of incubation. No increased oxidative injury or cell death was observed. Osteopontin (OPN) mRNA in cells and protein expression in medium were significantly increased during 6-24 hours of incubation. MCD medium treatment also resulted in activation of PI3-kinase by 30 minutes and its downstream target p-Akt within 1hour of incubation. Steatosis was associated with increased expression of microsomal triglyceride transfer protein (MTTP) mRNA and increased ALT release with over expression of ALT mRNA, all of which were completely prevented by inhibition of PI3-kinase (LY294002). Blocking OPN signaling by treating with anti-OPN or anti-beta3-integrin antibody prevented the increased ALT release while only partially prevented the increased ALT mRNA expression, but had no effect on either steatosis or MTTP expression. In conclusion, incubation of cultured hepatocytes with MCD medium results in cellular steatosis and OPN dependent ALT release. PI3-kinase plays a central role in signaling the MCD medium-induced steatosis and increased OPN expression, whereas OPN appears to play a role in signaling hepatocyte ALT release but not steatosis.
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Deficiencia de Colina/metabolismo , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Metionina/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Sialoglicoproteínas/metabolismo , Transaminasas/biosíntesis , Animales , Línea Celular , Deficiencia de Colina/complicaciones , Medios de Cultivo/metabolismo , Hígado Graso/etiología , Ratones , OsteopontinaAsunto(s)
Hemocromatosis/etiología , Hemocromatosis/inmunología , Hierro/metabolismo , Femenino , Hemocromatosis/embriología , Humanos , Inmunidad Materno-Adquirida , Inmunoglobulina G/fisiología , Inmunoglobulina G/uso terapéutico , Lactante , Recién Nacido , Hígado/química , Hígado/embriología , Hígado/patología , Embarazo , Complicaciones del EmbarazoRESUMEN
The acquired or perinatal form of biliary atresia is a Th1 fibro-inflammatory disease affecting both the extrahepatic and intrahepatic bile ducts. Osteopontin (OPN) is a Th1 cytokine implicated in several fibro-inflammatory and autoimmune diseases. We examined the expression of OPN in acquired biliary atresia in comparison to normal liver and several pediatric cholestatic liver diseases. We also assessed OPN expression by cultured human bile duct epithelial cells. We found that liver OPN mRNA and protein expression were significantly increased in biliary atresia versus normal and other cholestatic diseases. OPN expression in biliary atresia was localized to epithelium of proliferating biliary structures (ductules and/or ducts) and bile plugs contained therein. No portal biliary OPN expression could be demonstrated in normal liver, syndromic biliary atresia, biliary obstruction not due to biliary atresia, and idiopathic neonatal hepatitis. OPN expression by human bile duct epithelial cells in culture was responsive to IL-2 and TNF-alpha. Our results demonstrate an up-regulation of OPN expression by interlobular biliary epithelium in biliary atresia, which correlates with biliary proliferation and portal fibrosis. These findings suggest a role for OPN in the pathogenesis of biliary atresia.
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Conductos Biliares/metabolismo , Conductos Biliares/patología , Atresia Biliar/genética , Atresia Biliar/patología , Sialoglicoproteínas/genética , Secuencia de Bases , Atresia Biliar/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Colestasis/genética , Colestasis/metabolismo , Colestasis/patología , ADN Complementario/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibrosis , Expresión Génica/efectos de los fármacos , Humanos , Lactante , Recién Nacido , Interleucina-2/farmacología , Osteopontina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sialoglicoproteínas/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND/AIMS: Feeding mice a methionine choline deficient (MCD) diet serves as a nutritional model of non-alcoholic steatohepatitis (NASH). NASH and alcohol-induced steatohepatitis are histologically similar, suggesting a similar pathogenesis. Pentoxifylline (PTX) attenuates TNF-alpha production, acts as an antioxidant and decreases mortality in alcoholic steatohepatitis. The aim of our study is to determine if PTX attenuates MCD diet induced steatohepatitis and determine the mechanism of this effect. METHODS: Mice were placed on an MCD or control diet for 2 weeks and were treated with or without PTX. Serum ALT, liver histology, and inflammatory mechanisms were evaluated. RESULTS: PTX attenuates MCD diet induced steatohepatitis, decreasing both serum ALT levels and hepatic inflammation. Serum ALT levels were reduced approximately 50% in the MCD+PTX group compared to the MCD group. Hepatic glutathione levels were significantly higher in the MCD+PTX group compared to the MCD group. There was also a reduction in TNF-alpha mRNA in female mice treated with PTX. MCD+PTX mice had increased hepatic triglyceride content compared to the MCD mice, but less histologic evidence of inflammation despite the increased steatosis. Serum lipid and bile salt levels also were similar in PTX and vehicle control treated mice. CONCLUSIONS: PTX decreases serum ALT levels and hepatic inflammation in the MCD model of steatohepatitis, likely via increasing glutathione levels or reducing TNF-alpha expression.
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Deficiencia de Colina/complicaciones , Hígado Graso/patología , Hígado Graso/fisiopatología , Hepatitis/patología , Hepatitis/fisiopatología , Metionina/deficiencia , Pentoxifilina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Alanina Transaminasa/antagonistas & inhibidores , Alanina Transaminasa/sangre , Animales , Enfermedades Carenciales/complicaciones , Dieta , Progresión de la Enfermedad , Hígado Graso/etiología , Femenino , Glutatión/metabolismo , Hepatitis/etiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/antagonistas & inhibidores , Triglicéridos/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptin-deficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-alpha, TGF-beta, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH.
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Diabetes Mellitus Tipo 2/complicaciones , Modelos Animales de Enfermedad , Hígado Graso/etiología , Cirrosis Hepática/etiología , Obesidad/complicaciones , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular , Deficiencia de Colina/complicaciones , Enfermedades Carenciales/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Cirrosis Hepática/patología , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/genética , Obesidad/metabolismo , Osteopontina , Receptores de Superficie Celular/química , Receptores de Leptina , Sialoglicoproteínas/metabolismoRESUMEN
The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.