RESUMO
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.
Assuntos
Anemia Falciforme/complicações , Bile/metabolismo , Colestase/etiologia , Insuficiência Hepática/etiologia , Fígado/patologia , Adolescente , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Animais , Ductos Biliares Intra-Hepáticos/diagnóstico por imagem , Ductos Biliares Intra-Hepáticos/patologia , Colestase/patologia , Colestase/prevenção & controle , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Hemoglobina Falciforme/genética , Insuficiência Hepática/patologia , Insuficiência Hepática/prevenção & controle , Humanos , Microscopia Intravital , Fígado/diagnóstico por imagem , Masculino , Camundongos , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Hemorrhagic shock (HS) is a life-threatening condition associated with tissue hypoperfusion and often leads to injury of multiple organs including the liver. Pregnane X receptor (PXR) is a species-specific xenobiotic receptor that regulates the expression of drug-metabolizing enzymes (DMEs) such as the cytochrome P450 (CYP) 3A. Many clinical drugs, including those often prescribed to trauma patients, are known to activate PXR and induce CYP3A. The goal of this study is to determine whether PXR plays a role in the regulation of DMEs in the setting of HS and whether activation of PXR is beneficial or detrimental to HS-induced hepatic injury. PXR transgenic, knockout, and humanized mice were subject to HS, and the liver injury was assessed histologically and biochemically. The expression and/or activity of PXR and CYP3A were manipulated genetically or pharmacologically in order to determine their effects on HS-induced liver injury. Our results showed that genetic or pharmacological activation of PXR sensitized wild-type and hPXR/CYP3A4 humanized mice to HS-induced hepatic injury, whereas knockout of PXR protected mice from HS-induced liver injury. Mechanistically, the sensitizing effect of PXR activation was accounted for by PXR-responsive induction of CYP3A and increased oxidative stress in the liver. The sensitizing effect of PXR was attenuated by ablation or pharmacological inhibition of CYP3A, treatment with the antioxidant N-acetylcysteine amide, or treatment with a PXR antagonist. Conclusion: We have uncovered a function of PXR in HS-induced hepatic injury. Our results suggest that the unavoidable use of PXR-activating drugs in trauma patients has the potential to exacerbate HS-induced hepatic injury, which can be mitigated by the coadministration of antioxidative agents, CYP3A inhibitors, or PXR antagonists.
Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Insuficiência Hepática/patologia , Receptor de Pregnano X/genética , Choque Hemorrágico/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Insuficiência Hepática/etiologia , Insuficiência Hepática/genética , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Prognóstico , Distribuição Aleatória , Medição de Risco , Choque Hemorrágico/complicações , Choque Hemorrágico/tratamento farmacológico , Taxa de Sobrevida , Resultado do Tratamento , Regulação para CimaRESUMO
The induction of cytochrome P450 (P450) enzymes in response to drug treatment is a significant contributing factor to drug-drug interactions, which may reduce therapeutic efficacy and/or cause toxicity. Since most studies on P450 induction are performed in adults, enzyme induction at neonatal, infant, and adolescent ages is not well understood. Previous work defined the postnatal ontogeny of drug-metabolizing P450s in human and mouse livers; however, there are limited data on the ontogeny of the induction potential of each enzyme in response to drug treatment. Induction of P450s at the neonatal age may also cause permanent alterations in P450 expression in adults. The goal of this study was to investigate the short- and long-term effects of phenytoin treatment on mRNA and protein expressions and enzyme activities of CYP2B10, 2C29, 3A11, and 3A16 at different ages during postnatal liver maturation in mice. Induction of mRNA immediately following phenytoin treatment appeared to depend on basal expression of the enzyme at a specific age. While neonatal mice showed the greatest fold changes in CYP2B10, 2C29, and 3A11 mRNA expression following treatment, the levels of induced protein expression and enzymatic activity were much lower than that of induced levels in adults. The expression of fetal CYP3A16 was repressed by phenytoin treatment. Neonatal treatment with phenytoin did not permanently induce enzyme expression in adulthood. Taken together, our data suggest that inducibility of drug-metabolizing P450s is much lower in neonatal mice than it is in adults and neonatal induction by phenytoin is not permanent.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fenitoína/farmacologia , Animais , Indução Enzimática/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: To employ metabolomics-based pathway and network analyses to evaluate the cerebrospinal fluid metabolome after severe traumatic brain injury in children and the capacity of combination therapy with probenecid and N-acetylcysteine to impact glutathione-related and other pathways and networks, relative to placebo treatment. DESIGN: Analysis of cerebrospinal fluid obtained from children enrolled in an Institutional Review Board-approved, randomized, placebo-controlled trial of a combination of probenecid and N-acetylcysteine after severe traumatic brain injury (Trial Registration NCT01322009). SETTING: Thirty-six-bed PICU in a university-affiliated children's hospital. PATIENTS AND SUBJECTS: Twelve children 2-18 years old after severe traumatic brain injury and five age-matched control subjects. INTERVENTION: Probenecid (25 mg/kg) and N-acetylcysteine (140 mg/kg) or placebo administered via naso/orogastric tube. MEASUREMENTS AND MAIN RESULTS: The cerebrospinal fluid metabolome was analyzed in samples from traumatic brain injury patients 24 hours after the first dose of drugs or placebo and control subjects. Feature detection, retention time, alignment, annotation, and principal component analysis and statistical analysis were conducted using XCMS-online. The software "mummichog" was used for pathway and network analyses. A two-component principal component analysis revealed clustering of each of the groups, with distinct metabolomics signatures. Several novel pathways with plausible mechanistic involvement in traumatic brain injury were identified. A combination of metabolomics and pathway/network analyses showed that seven glutathione-centered pathways and two networks were enriched in the cerebrospinal fluid of traumatic brain injury patients treated with probenecid and N-acetylcysteine versus placebo-treated patients. Several additional pathways/networks consisting of components that are known substrates of probenecid-inhibitable transporters were also identified, providing additional mechanistic validation. CONCLUSIONS: This proof-of-concept neuropharmacometabolomics assessment reveals alterations in known and previously unidentified metabolic pathways and supports therapeutic target engagement of the combination of probenecid and N-acetylcysteine treatment after severe traumatic brain injury in children.
Assuntos
Acetilcisteína/uso terapêutico , Lesões Encefálicas Traumáticas/líquido cefalorraquidiano , Lesões Encefálicas Traumáticas/tratamento farmacológico , Probenecid/uso terapêutico , Adjuvantes Farmacêuticos , Adolescente , Lesões Encefálicas Traumáticas/metabolismo , Criança , Pré-Escolar , Método Duplo-Cego , Quimioterapia Combinada , Humanos , Escala de Gravidade do Ferimento , MetabolômicaRESUMO
UNLABELLED: The aryl hydrocarbon receptor (AHR), also known as the dioxin receptor, was originally characterized as a xenobiotic receptor that senses xenotoxicants. We investigated the endobiotic and hepatic role of AHR in fatty liver and energy metabolism and identified the endocrine factor that mediates the metabolic function of AHR. Wild-type and liver-specific constitutively activated human AHR transgenic mice were used to investigate the role of AHR in fatty liver and energy homeostasis. Adenovirus expressing short hairpin RNA targeting fibroblast growth factor 21 (FGF21) were used to determine the involvement of FGF21 in the metabolic effect of AHR. We showed that, despite their severe fatty liver, the transgenic mice were protected from diet-induced obesity and type 2 diabetes. We identified the endocrine hormone FGF21 as a mediator for the metabolic benefit of AHR and established FGF21 as a direct transcriptional target of AHR. Interestingly, the transactivation of FGF21 by AHR contributed to both hepatic steatosis and systemic insulin hypersensitivity, both of which were largely abolished upon FGF21 knockdown. CONCLUSIONS: The AHR-FGF21 endocrine signaling pathway establishes AHR as a pivotal environmental modifier that integrates signals from chemical exposure in the regulation of lipid and energy metabolism.