No abstract

Background & Aims: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis, a regulated form of necrotic cell death mediated by the receptorinteracting protein kinase (RIPK) 1. We herein assessed the potential of RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL), as therapeutic targets and markers of activity in NAFLD. Methods: C57/BL6J-mice were fed a normal chow diet (NCD) or high fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in either a prophylactic or a curative treatment of HFD-fed mice, and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. Results: Both prophylactic and curative treatments of HFD-fed mice with RIPA-56, caused a down-regulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide (NSA), a specific inhibitor of human MLKL, and by knocking out (KO) MLKL in fat-loaded AML-12 mouse hepatocytes. MLKL KO in steatotic hepatocytes, caused an activation of the mitochondrial respiration, and an increase in b-oxidation. Along with MLKL decreased activation, RIPK3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with the activity. Conclusion: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis by an MLKL-dependent mechanism that involves at least partly an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD.