n-(4-(n-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide and Non-alcoholic-Fatty-Liver-Disease

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD.. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD.. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation 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, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL 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 activity.. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD.. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Topics: Acrylamides; Aged; Animals; Diet, High-Fat; Disease Models, Animal; Female; Gene Knockout Techniques; Hepatocytes; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Necroptosis; Non-alcoholic Fatty Liver Disease; Protein Kinase Inhibitors; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Sulfonamides; Treatment Outcome

Receptor-interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non-alcoholic fatty liver disease (NAFLD).. Hepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High-fat diet was fed to wild-type and MLKL-knockout (KO) mice for 12 weeks. Brown adipose fat tissue was measured by [. Hepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High-fat diet-fed MLKL-KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P < 0.001), steatosis score (3.0 vs 1.8, P < 0.001), inflammation, and ballooning degeneration compared with wild-type mice. SREBP1c, fatty acid synthase, and SCD-1 expressions decreased in MLKL-KO mice. Adipose tissue F4/80-positive crown-like structures were also reduced in MLKL-KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD-1 expressions. Stimulation of necroptosis using lipopolysaccharide + caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide + zVAD-induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment.. Mixed lineage kinase domain-like pseudokinase inhibition has protective effects in non-alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C-X-C motif) ligand expressions.

Topics: Acrylamides; Adipose Tissue, Brown; Animals; Case-Control Studies; Chemokines, CXC; Diet, High-Fat; Energy Metabolism; Gene Deletion; Hep G2 Cells; Humans; Ligands; Lipids; Liver; Mice, Inbred C57BL; Mice, Knockout; Necroptosis; Non-alcoholic Fatty Liver Disease; Protein Kinase Inhibitors; Protein Kinases; Sulfonamides; U937 Cells