title: Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.
abstract: Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: Marta B
authors.initials: MB
authors.lastname: Afonso
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Rodrigues
authors.affiliation: Histology and Comparative Pathology Laboratory, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, Edificio Egas Moniz, 1649-028 Lisbon, Portugal.
authors.forename: Tânia
authors.initials: T
authors.lastname: Carvalho
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: Marta
authors.initials: M
authors.lastname: Caridade
authors.affiliation: ESTEsL, Av. D. João II, 1990-096 Lisbon, Portugal Instituto de Anatomia Patológica, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-035 Lisbon, Portugal Hospital Cuf Descobertas, R. Mário Botas, Parque das Nações, 1998-018 Lisbon, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Borralho
authors.affiliation: Department of Gastroenterology, Hospital Santa Maria, Faculty of Medicine, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-035 Lisbon, Portugal.
authors.forename: Helena
authors.initials: H
authors.lastname: Cortez-Pinto
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: Rui E
authors.initials: RE
authors.lastname: Castro
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal cmprodrigues@ff.ulisboa.pt.
authors.forename: Cecília M P
authors.initials: CM
authors.lastname: Rodrigues
mesh_terms: D000818:Animals; D016022:Case-Control Studies; D016923:Cell Death; D002796:Choline Deficiency; D059305:Diet, High-Fat; D004195:Disease Models, Animal; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008297:Male; D008715:Methionine / Q000172:deficiency; D051379:Mice; D008810:Mice, Inbred C57BL; D065626:Non-alcoholic Fatty Liver Disease / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress; D019308:Palmitic Acid; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019308:Palmitic Acid; D008715:Methionine; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases
keywords: chronic liver diseases; mixed lineage kinase domain-like (MLKL); necroptosis; non-alcoholic steatohepatitis (NASH); oxidative stress; receptor-interacting protein 3 (RIP3)