Papel de x-binding protein-1 (xbp 1) en hepatocitos en un modelo preclínico de toxicidad por fármacos

• Autores: Hui Ye
• Directores de la Tesis: Francisco Javier Cubero Palero (dir. tes.)
• Lectura: En la Universidad Complutense de Madrid ( España ) en 2021
• Idioma: español
• Número de páginas: 144
• Títulos paralelos:
  • Role of hepatocytic xbp1 in a preclinical model of toxic liver injury
• Tribunal Calificador de la Tesis: Manuel Gómez del Moral (presid.), Eduardo Martínez Naves (secret.), Yulia Nevzorova (secret.), Leonard J. Nelson (voc.), Oleg Lunov (voc.), Kateryna Levada (voc.)
• Programa de doctorado: Programa de Doctorado en Investigación Biomédica por la Universidad Complutense de Madrid
• Materias:
  • Ciencias médicas
    • Medicina interna
      • Gastroenterología
    • Patología
      • Patología experimental
• Enlaces
  • Tesis en acceso abierto en: Docta Complutense
• Resumen
  • español

    Acetaminophen (APAP) hepatotoxicity has already become a public health concern in the world, accounting for 57% of cases of acute liver failure in the UK and 39% in the United States. Once the hepatotoxicity caused by APAP was recognized, its underlying mechanisms have been extensively investigated, especially in the mouse model, which recapitulates key mechanistic aspects of liver injury in humans, including mitochondrial dysfunction and inflammation. APAP-mediated hepatotoxicity also increases endoplasmic reticulum (ER) stress as a response to the accumulation of unfolded or misfolded proteins in the ER lumen, which then induces the activation of the unfolded protein response (UPR). There¿re three main signaling pathways of UPR, and IRE1¿-XBP1 arm is the most conserved one among them, which is worthy of our more attention. However, the mechanisms underlying ER stress and APAP toxicity remains poorly understood, thus dampening the chances of pharmacologic therapy for DILI patients. We want to correlate APAP hepatotoxicity with activation of UPR both in vivo and in vitro, and understand the role of hepatocytic Xbp1 in APAP-mediated hepatotoxicity through our work. HepaRG cells and WT mice were challenged with different concentrations of APAP respectively (HepaRG cells: 0-20 mM, WT mice: 0-300 mg/kg). Mice with specific deletion of Xbp1 in hepatocytes (Xbp1¿hepa) and floxed (Xbp1f/f) control mice got an intraperitoneal injection of a sublethal dose of APAP (500 mg/kg). The molecule inhibitor of Xbp1 splicing, STF-083010, was also applied in WT mice with 500 mg/kg APAP challenge. 24 h later, HepaRG cells were collected and mice were sacrificed. All the livers and serums were taken. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Our results strongly suggest that a sublethal dose of APAP both in vitro and in vivo elicited liver parenchymal cells (LPC) damage and loss of hepatocyte polarity, and impaired lipid metabolism. In addition, activation of IRE1¿-XBP1 arm and JNK1/2 was characteristic of both human and murine APAP-mediated hepatotoxicity. The increase of reactive oxygen species (ROS) and the infiltration of inflammatory cytokines were also observed in APAP-induced liver injury. Ablation of Xbp1 in hepatocytes reduced APAP-induced hepatic damage, maintained the tight junctions (TJs) and ameliorated the activation of UPR and JNK1/2. In addition, ablation of Xbp1 also reduced oxidative stress, lipid accumulation, immune cells infiltration and inflammasome activation. As a feedback of Xbp1 deficiency, hyperactivation of IRE1¿ in hepatocytes resulted in activation of regulated IRE1-dependent decay (RIDD) and suppression of cytochrome P450 activity, which would contribute to the protective effects against APAP toxicity. Moreover, we also comfirmed that this protective effect in Xbp1¿hepa mice was likely due to the activation of autophagy via AMPK activation and AKT inhibition. The degradation of P62 and the conversion of light chain 3 (LC3)-I to LC3-II were promoted in Xbp1¿hepa mice. In summary, our results show the hepatocytic Xbp1 plays a critical role in the pathogenesis of DILI and provide the basis for therapies targeting the restoration of LPC function after acute liver injury.

  • English

    La lesión hepática inducida por fármacos (DILI) es una de las principales causas de lesión hepática aguda, incluida la sobredosis de acetaminofén (APAP). El estrés del retículo endoplásmico (ER) es un mecanismo sin explorar, pero muy probablemente, desencadenante de la lesión hepática inducida por APAP. En condiciones de estrés, ER puede iniciar la respuesta de proteína desplegada. Sin embargo, los mecanismos del estrés ER y UPR siguen siendo poco conocidos. En este estudio, nuestro objetivo general fue develar la relevancia del brazo IRE1α-XBP1, el brazo más conservado de UPR, durante la insuficiencia hepática aguda inducida por APAP y proporcionar la base para la intervención terapéutica...