RESUMO
The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.
Assuntos
Células Estreladas do Fígado/metabolismo , Inflamassomos/metabolismo , Cirrose Hepática/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Biomarcadores/metabolismo , Feminino , Lipopolissacarídeos , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/metabolismoRESUMO
The NLRP3 inflammasome, a caspase-1 activation platform, plays a key role in the modulation of liver inflammation and fibrosis. Here, we tested the hypothesis that interleukin 17 (IL-17) and tumor necrosis factor (TNF) are key cytokines involved in amplifying and perpetuating the liver damage and fibrosis resulting from NLRP3 activation. To address this hypothesis, gain-of-function Nlrp3A350V knock-in mice were bred onto il17a and Tnf knockout backgrounds allowing for constitutive Nlrp3 activation in myeloid derived cells in mice deficient in IL-17 or TNF. Livers of Nlrp3A350V knock-in mice exhibited severe liver inflammatory changes characterized by infiltration with neutrophils, increased expression of chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL2 chemokines, activated inflammatory macrophages, and elevated levels of IL-17 and TNF. Mutants with ablation of il17a signal showed fewer neutrophils when compared to intact Nlrp3A350V mutants, but still significant inflammatory changes when compared to the nonmutant il17a knockout littermates. The severe inflammatory changes associated with mutant Nlrp3 were almost completely rescued by Tnf knockout in association with a marked decrease in circulating IL-1ß levels. Intact Nlrp3A350V mutants showed changes in liver fibrosis, as evidenced by morphometric quantitation of Sirius Red staining and increased mRNA levels of profibrotic genes, including connective tissue growth factor and tissue inhibitor of matrix metalloproteinase 1. Il17a lacking mutants exhibited amelioration of the aforementioned fibrosis, whereas Tnf-deficient mutants showed no signs of fibrosis when compared to littermate controls. Conclusion: Our study uncovers key roles for TNF and, to a lesser extent, IL-17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid-derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2018;67:736-749).
Assuntos
Hepatite/etiologia , Interleucina-17/fisiologia , Cirrose Hepática Experimental/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células Estreladas do Fígado/fisiologia , Macrófagos/fisiologia , Camundongos , Infiltração de Neutrófilos , Transdução de SinaisRESUMO
Circulating oxidized linoleic acid (LA) metabolites (OXLAMs) are increased in patients with nonalcoholic steatohepatitis (NASH) and their levels correlate with disease severity. However, the mechanisms by which OXLAMs contribute to NASH development are incompletely understood. We tested the hypothesis that LA or OXLAMs provided directly through the diet are involved in the development of hepatic injury. C57BL/6 mice were fed an isocaloric high-fat diet containing low LA, high LA, or OXLAMs for 8 weeks. The livers of OXLAM-fed mice showed lower triglyceride concentrations, but higher FA oxidation and lipid peroxidation in association with increased oxidative stress. OXLAM-induced mitochondrial dysfunction was associated with reduced Complex I protein and hepatic ATP levels, as well as increased mitochondrial biogenesis and cytoplasmic mitochondrial DNA. Oxidative stress increased thioredoxin-interacting protein (TXNIP) in the liver and stimulated the activation of mitochondrial apoptosis signal-regulating kinase 1 (ASK1) leading to apoptosis. We also found increased levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and Caspase-1 activation in the livers of OXLAM-fed mice. In vitro, OXLAMs induced hepatocyte cell death, which was partly dependent on Caspase-1 activation. This study identified key mechanisms by which dietary OXLAMs contribute to NASH development, including mitochondrial dysfunction, hepatocyte cell death, and NLRP3 inflammasome activation.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamassomos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/metabolismoRESUMO
BACKGROUND & AIMS: Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. METHODS: Arginase 2-knockout (Arg2(-/-)) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2(-/-) mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS: Unexpectedly, Arg2(-/-) mice showed profound changes in their livers at baseline, characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked mRNA level increases of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2(-/-) mice. CONCLUSIONS: This study identifies arginase 2 as a novel link between innate immune responses, hepatic lipid deposition, and liver injury.
Assuntos
Arginase/metabolismo , Fígado Gorduroso/imunologia , Hiperargininemia/complicações , Imunidade Inata , Células de Kupffer/imunologia , Metabolismo dos Lipídeos , Lipogênese/imunologia , Animais , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hiperargininemia/imunologia , Hiperargininemia/metabolismo , Immunoblotting , Células de Kupffer/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND/AIMS: Hepatocyte cell death is a key feature of nonalcoholic steatohepatitis (NASH). As the contribution of specific caspases remains unclear, our aim was to ascertain the effect of caspase 3 suppression on liver injury and fibrogenesis. METHODS: C57BL/6 wild-type (WT) and caspase 3 knock out (Casp3 (-/-)) mice were placed on a methionine- and choline-deficient (MCD) diet for 6 weeks to induce steatohepatitis and liver fibrosis. Thereafter, liver injury, liver fibrosis and hepatocellular apoptosis were quantified in liver sections. Additionally, expression of proteins associated with liver inflammation and fibrogenesis was analyzed. RESULTS: WT mice fed MCD diet showed marked activation of caspase 3 in hepatocytes, in conjunction with steatohepatitis and increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3 (-/-) mice fed the MCD diet showed similar serum aminotransferase levels and NAFLD activity scores (NAS) compared with WT MCD-fed mice. However, Casp3 (-/-) mice on the MCD diet showed a marked reduction in expression of transcripts for profibrogenic genes, which translated into reduced hepatic collagen deposition. These changes were associated with decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling. Casp3 (-/-) mice on the MCD showed a reduction in expression of chemokine receptor 2 (CCR2) leading to ameliorated infiltration of inflammatory lymphocyte antigen 6 complex, locus C1 (Ly6c) positive monocytes. CONCLUSION: These findings support a prominent role for hepatocyte caspase 3 activation in NASH-related apoptosis, fibrogenesis and fibrosis which in part is mediated via CCR2-dependent infiltration of Ly6c positive monocytes.
Assuntos
Caspase 3/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Apoptose , Caspase 3/genética , Colina/administração & dosagem , Colina/farmacologia , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Células Estreladas do Fígado , Cirrose Hepática/enzimologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Metionina/administração & dosagem , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Receptores CCR2/genética , Receptores CCR2/metabolismoRESUMO
Non-alcoholic steatohepatitis (NASH) is typically associated with pro-apoptotic caspase activation. A potential role for pro-inflammatory caspases remains incompletely understood. Our aims were to examine a potential role of caspase-1 in the development of liver damage and fibrosis in NASH. C57BL/6 wild type (WT) developed marked steatohepatitis, activation, fibrosis and increased hepatic caspase-1 and interleukin-1ß expression when placed on the methionine- and choline-deficient (MCD) diet. Marked caspase-1 activation was detected in the liver of MCD-fed mice. Hepatocyte and non-parenchymal fractionation of the livers further demonstrated that caspase-1 activation after MCD feeding was mainly localized to non-parenchymal cells. Caspase-1-knockout (Casp1(-/-)) mice on the MCD diet showed marked reduction in mRNA expression of genes involved in inflammation and fibrogenesis (tumor necrosis factor-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; F4/80 was 1.5-fold greater in WT vs Casp1(-/-) MCD-fed mice; α-smooth muscle actin was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice; collagen 1-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; transforming growth factor-ß was 2.4-fold greater in WT vs Casp1(-/-) MCD-fed mice; cysteine- and glycine-rich protein 2 was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp1(-/-) MCD-fed mice compared with WT MCD-fed animals. However, serum alanine aminotransferase levels, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells were similar in Casp1(-/-) and WT mice on the MCD diet. Selective Kupffer cell depletion by clodronate injection markedly suppressed MCD-induced caspase-1 activation and protected mice from fibrogenesis and fibrosis associated with this diet. The conclusion of this study is that it uncovers a novel role for caspase-1 in inflammation and fibrosis during NASH development.
Assuntos
Caspase 1/metabolismo , Colágeno Tipo I/metabolismo , Fígado Gorduroso/metabolismo , Células Estreladas do Fígado/metabolismo , Inflamação/metabolismo , Células de Kupffer/metabolismo , Cirrose Hepática/metabolismo , Actinas/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Caspase 1/deficiência , Caspase 1/genética , Caspase 3/sangue , Caspase 3/metabolismo , Deficiência de Colina/complicações , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Ácido Clodrônico/farmacologia , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Interleucina-1beta/metabolismo , Células de Kupffer/efeitos dos fármacos , Proteínas com Domínio LIM/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Ethanol metabolism by liver generates short lived reactive oxygen species that damage liver but also affects distal organs through unknown mechanisms. We hypothesized that dissemination of liver oxidative stress proceeds through release of biologically active oxidized lipids to the circulation. We searched for these by tandem mass spectrometry in plasma of rats fed a Lieber-DeCarli ethanol diet or in patients with established alcoholic liver inflammation, steatohepatitis. We found a severalfold increase in plasma peroxidized phosphatidylcholines, inflammatory and pro-apoptotic oxidatively truncated phospholipids, and platelet-activating factor, a remarkably potent and pleiotropic inflammatory mediator, in rats chronically ingesting ethanol. Circulating peroxidized phospholipids also increased in humans with established steatohepatitis. However, reactive oxygen species generated by liver ethanol catabolism were not directly responsible for circulating oxidized phospholipids because the delayed appearance of these lipids did not correlate with ethanol exposure, hepatic oxidative insult, nor plasma alanine transaminase marking hepatocyte damage. Rather, circulating oxidized lipids correlated with steatohepatitis and tumor necrosis factor-alpha deposition in liver. The organic osmolyte 2-aminoethylsulfonic acid (taurine), which reduces liver endoplasmic reticulum stress and inflammation, even though it is not an antioxidant, abolished liver damage and the increase in circulating oxidized phospholipids. Thus, circulating oxidized phospholipids are markers of developing steatohepatitis temporally distinct from oxidant stress associated with hepatic ethanol catabolism. Previously, circulating markers of the critical transition to pathologic steatohepatitis were unknown. Circulating oxidatively truncated phospholipids are pro-inflammatory and pro-apoptotic mediators with the potential to systemically distribute the effect of chronic ethanol exposure. Suppressing hepatic inflammation, not ethanol catabolism, reduces circulating inflammatory and apoptotic agonists.
Assuntos
Alcoolismo/sangue , Etanol/administração & dosagem , Fosfolipídeos/sangue , Administração Oral , Alcoolismo/complicações , Alcoolismo/patologia , Animais , Dieta , Etanol/metabolismo , Fígado Gorduroso/sangue , Fígado Gorduroso/complicações , Fígado Gorduroso/patologia , Comportamento Alimentar/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Ratos , Ratos Wistar , Taurina/administração & dosagem , Taurina/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal-regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.
Assuntos
Morte Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cirrose Hepática/metabolismo , Fígado/lesões , Fígado/metabolismo , MAP Quinase Quinase Quinase 5/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Inibidores Enzimáticos/administração & dosagem , Feminino , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cirrose Hepática/patologia , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosforilação , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Autoimmune pancreatitis is highly responsive to steroid therapy, but because it mimics pancreatic cancer, it often precipitates unnecessary surgery. Adequate diagnostic tests are needed to permit appropriate medical therapy. Lymphocytic and obliterative phlebitis are reported in the majority of cases, as are elevated IgG4-positive plasma cells, indicating their high sensitivity. Their specificities, especially when used in conjunction, however, remain largely unknown. Movat pentachrome vascular and IgG4 immunohistochemical stains were performed on a total of 15 autoimmune pancreatitis cases (11 pancreatic resections and 4 biopsies), 39 usual-type alcoholic or idiopathic chronic pancreatitis cases, 35 pancreatic ductal adenocarcinoma-associated chronic pancreatitis cases, and 29 normal pancreata. Marked and diffuse lymphocytic and obliterative venulitis were detected in all 15 cases of autoimmune pancreatitis on Movat staining (100% sensitivity). Only a single carcinoma-associated chronic pancreatitis case among all of the controls showed diffuse benign venulitis that was nonobliterative (99% specificity). Nine of 13, 9 autoimmune pancreatitis cases showed marked IgG4 immunopositivity at >or=10 positive plasma cells per x 400 field (69% sensitivity). No increased IgG4 plasma cells were found in any of 103 controls (100% specificity). In combination, all of the autoimmune pancreatitis cases had at least one (13/13) and most had both markers (9/13), whereas none of the controls had both markers. Overall, these combined stains show very promising diagnostic utility and should be considered in combination with clinical and serologic analyses in the evaluation of chronic pancreatitis suspicious for malignancy. Future validating studies on preoperative biopsies with outcome data following steroid therapy will be essential.
Assuntos
Doenças Autoimunes/diagnóstico , Imunoglobulina G , Pancreatite/diagnóstico , Coloração e Rotulagem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are growing public health problems, and are strongly associated. The link between the two conditions remains poorly understood. Hepatic interleukin-6 (IL-6), a major proinflammatory cytokine, expression is increased in animal models of NAFLD, while in mice, selective sustained upregulation of IL-6 in the liver results in systemic insulin resistance. The extent and clinical significance of hepatic IL-6 expression in human NAFLD, as well as potential mechanisms by which steatosis may increase IL-6 production in the liver, have not been examined. AIMS: To ascertain the occurrence and significance of IL-6 expression in the liver in human NAFLD. PATIENTS AND METHODS: Plasma was obtained at time of liver biopsy from 50 consecutive patients with suspected NAFLD. Histology was assessed blindly. Hepatic IL-6 expression was assessed by immunohistochemistry, while plasma IL-6 levels were determined by an enzyme-linked immunosorbent assay. RESULTS: IL-6 expression was markedly increased in the livers of patients with nonalcoholic steatohepatitis (NASH) as compared to patients with simple steatosis (P < 0.005) or normal biopsies (P < 0.010), confirming the presence of hepatic IL-6 expression in human NASH. A positive correlation was observed between hepatocyte IL-6 expression and degree of inflammation and stage of fibrosis. Furthermore, liver IL-6 expression positively correlated with plasma IL-6 levels and degree of systemic insulin resistance. Culture of liver cells with saturated, but not mono- or polyunsaturated, FFA resulted in a significant increase in IL-6 messenger RNA (mRNA) and protein expression. CONCLUSION: Collectively, these data suggest that increased hepatic IL-6 production may play an important role in NASH development, as well as in systemic insulin resistance and diabetes.
Assuntos
Fígado Gorduroso/metabolismo , Hepatite/metabolismo , Interleucina-6/metabolismo , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Ácidos Graxos não Esterificados/fisiologia , Fígado Gorduroso/patologia , Feminino , Hepatite/patologia , Humanos , Resistência à Insulina/fisiologia , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
AIM: Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. MATERIALS & METHODS: RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. RESULTS: The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. CONCLUSION: These findings uncover RNP as a novel potential NASH therapy.
Assuntos
Inflamação/tratamento farmacológico , Nanopartículas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Oxirredução , Animais , Antioxidantes/química , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Perfilação da Expressão Gênica , Células Estreladas do Fígado/citologia , Hepatócitos/citologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Nanomedicina , Nanopartículas/química , Estresse Oxidativo , Polímeros/química , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/químicaRESUMO
BACKGROUND&AIMS: Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The triggers for HSCs activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype. METHODS: EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSCs activation, migration and proliferation were assessed. Loss- and gain-of-functions studies were performed to explore the potential role of PPAR-γ-targeting miRNAs carried by EVs into HSC. RESULTS: Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of pro-fibrogenic genes (Collagen-I, α-SMA and TIMP-2), proliferation, chemotaxis and wound healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSC. Hepatocyte-derived EVs miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression with miR-128-3p being the most effectively transferred. Furthermore loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation. CONCLUSION: Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel anti-fibrotic targets for NAFLD and other fibrotic diseases.
RESUMO
BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. DESIGN: Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. RESULTS: We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2â=â0.64, p<0.05), fibrosis (r2â=â0.66, p<0.05) and pathological angiogenesis (r2â=â0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. CONCLUSIONS: These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Fígado/metabolismo , MicroRNAs/sangue , Hepatopatia Gordurosa não Alcoólica/patologia , Proteoma/metabolismo , Animais , Biomarcadores/sangue , Micropartículas Derivadas de Células/genética , Deficiência de Colina/complicações , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Exossomos/genética , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Nonalcoholic steatohepatitis (NASH) is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1(-/-)) mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1ß. Both wild-type and Casp1(-/-) mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1(-/-) mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1(-/-) mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1(-/-) mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1(-/-) mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH.
Assuntos
Caspase 1/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/enzimologia , Fígado Gorduroso/etiologia , Adiposidade , Animais , Biomarcadores/metabolismo , Caspase 1/deficiência , Caspase 1/genética , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fibrose , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Interleucina-1beta/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/enzimologia , Obesidade/etiologia , Obesidade/genética , Obesidade/patologiaRESUMO
Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic steatohepatitis (NASH). However, the relevant pathways that contribute to oxidative damage in vivo remain poorly understood. Here we explore the gene-expression patterns related to oxidative stress, antioxidant defense, and reactive oxygen metabolism in an established dietary murine model of NASH. C57BL/6 mice were placed on either a methionine- and choline-deficient (MCD) or a control (CTL) diet for 6 weeks. Hepatic oxidative damage and the development of NASH were monitored by biochemical and histologic indices. Analysis of 84 oxidative stress-related genes was performed by real-time reverse transcription polymerase chain reaction (PCR) in the livers of the two groups of mice. Mice on the MCD diet showed increased ALT, histologic features of NASH, and oxidative liver damage with increases in 4-hydroxynonenal and 3-nitrotyrosine. Of the genes analyzed, the GPx family were most significantly upregulated, whereas SCD1 was most significantly downregulated. Other genes that were significantly upregulated included Fmo2 and peroxiredoxins, whereas genes downregulated included Catalase and Serpinb1b. Our data demonstrate that oxidative stress-related genes are differentially expressed in the livers of mice with diet-induced NASH. These findings have important implications for NASH pathogenesis and the development of novel therapeutic strategies for patients with this condition.
Assuntos
Antioxidantes/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Perfilação da Expressão Gênica , Estresse Oxidativo , Transcrição Gênica , Animais , Sequência de Bases , Primers do DNA , Camundongos , Reação em Cadeia da PolimeraseRESUMO
The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.
Assuntos
Neoplasias da Mama/genética , Genes erbB-2 , Hibridização In Situ/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Compostos de Prata , Feminino , Humanos , Hibridização in Situ Fluorescente , Variações Dependentes do Observador , Reprodutibilidade dos TestesRESUMO
BACKGROUND & AIMS: Many patients with nonalcoholic fatty liver disease (NAFLD) have a benign clinical course, but a subgroup of patients progress to advanced fibrosis and cirrhosis. However, there are no available clinical tools to predict fibrosis progression in this population. Activated hepatic stellate cells (HSCs) are the source of collagen deposition in the liver. We aimed at determining whether an HSC activation score predicts fibrosis progression in NAFLD patients. METHODS: The cohort consisted of 39 untreated patients with NAFLD with paired liver biopsies performed 5-59 months apart (mean, 22 months). Patients were divided into 2 groups on the basis of whether fibrosis progression was noted on their second liver biopsy. Liver tissue was immunostained for alpha-smooth muscle actin, and the HSC score was determined independently by 2 pathologists in the NAFLD population and in control subjects without liver disease. RESULTS: The HSC activation score was significantly increased in patients with fibrosis progression versus patients in whom no fibrosis progression was observed (4.8 +/- 0.5 vs 1.8 +/- 0.6, respectively; P < .001). The HSC score was accurate in predicting fibrosis progression, with a positive predictive value of 90%, specificity of 94%, and an area under the receiver operating characteristic curve of 0.82. However, the negative predictive value and sensitivity were 56% and 41%, respectively. The inter-pathologist agreement for the HSC score was excellent (kappa coefficient, 0.95). CONCLUSIONS: These findings suggest that the HSC activation score is a suitable clinical tool to determine the risk of fibrosis progression in patients with NAFLD.
Assuntos
Fígado Gorduroso/patologia , Hepatócitos/patologia , Cirrose Hepática/patologia , Adulto , Idoso , Biópsia por Agulha , Estudos de Coortes , Progressão da Doença , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Probabilidade , Medição de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
Only a subset of hepatitis C virus (HCV)-infected patients develop progressive hepatic fibrosis after liver transplantation (LT). Hepatic stellate cell (HSC) activation is a pivotal step in hepatic fibrosis and precedes clinically apparent fibrosis. We determined whether early HSC activation, measured in 4-month protocol post-LT biopsies, is predictive of subsequent development of more histologically severe recurrence of HCV. Early (4 month) post-LT HSC activation, as measured by alpha-smooth muscle actin (alpha-SMA) staining, was determined in liver biopsies from recipients with severe (fibrosis score > or = 2, n = 13) and with mild (fibrosis score of 0, n = 13) recurrence of HCV at one-year post-LT. Immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) was used to generate HSC activation scores (regional and total). Total HSC activation scores at 4 months were similar in patients with severe and mild HCV recurrence (3.9 +/- 2.0 vs. 2.7 +/- 2.2, P = 0.2). Regional HSC activation, assessed as parenchymal (zones 1, 2, and 3) or mesenchymal (portal tracts and fibrous septa), was different between the study groups, with higher mesenchymal scores predictive of progression. No patients in the mild recurrence group had detectable mesenchymal alpha-SMA staining vs. 46% (6/13) of patients with severe recurrence (P < 0.01). Mesenchymal activation of HSC had a specificity and positive predictive value of 100% for development of progressive fibrosis in liver allografts of patients with hepatitis C. In conclusion, early activation of mesenchymal HSCs is a marker for progressive fibrosis in patients with hepatitis C post-LT and may help select patients who would benefit from HCV or HSC-targeted therapy.