Non-alcoholic fatty liver disease (NAFLD) is a significant public health concern, owing to its high prevalence, progressive nature and lack of effective medical therapies. NAFLD is a complex and multifactorial disease involving the progressive and concerted action of factors that contribute to the development of liver inflammation and eventually fibrosis. Here, we summarize fundamental molecular mechanisms underlying the pathogenesis of non-alcoholic steatohepatitis (NASH), how they are interrelated and possible translation to clinical applications. We focus on processes triggering and exacerbating apoptotic signalling in the liver of NAFLD patients and their metabolic and pathological implications. Indeed, liver injury and inflammation are cardinal histopathological features of NASH, a duo in which derailment of apoptosis is of paramount importance. In turn, the liver houses a very high number of mitochondria, crucial metabolic unifiers of both extrinsic and intrinsic signals that converge in apoptosis activation. The role of lifestyle options is also dissected, highlighting the management of modifiable risk factors, such as obesity and harmful alcohol consumption, influencing apoptosis signalling in the liver and ultimately NAFLD progression. Integrating NAFLD-associated pathologic mechanisms in the cell death context could provide clues for a more profound understating of the disease and pave the way for novel rational therapies.

Acute kidney injury (AKI) is a destructive clinical condition induced by multiple insults including ischemic reperfusion, nephrotoxic drugs and sepsis. It is characterized by a sudden decline in renal function, in addition to excessive inflammation, oxidative stress and programmed cell death of renal tubular epithelial cells. RIPK1-mediated necroptosis plays an important role in AKI. In the present study, we evaluated the treatment effects of Compound-71 (Cpd-71), a novel RIPK1 inhibitor, by comparing with Necrostatin-1 (Nec-1), a classic RIPK1 inhibitor, which has several drawbacks like the narrow structure–activity relationship (SAR) profile, moderate potency and non-ideal pharmacokinetic properties, in vivo and in vitro . Our results showed that pretreatment of Cpd-71 attenuated cisplatin-induced renal injury, restored renal function and suppressed renal inflammation, oxidative stress and cell necroptosis. In addition, Cpd-71 inhibited renal damage while reducing the up-regulated serum creatinine (Cr) and blood urea nitrogen (BUN) levels in established AKI mice model. Consistently, we confirmed that Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1, by physically binding to the allosteric type III ligand binding site of RIPK1, thereby reduced RIPK1 kinase activity, RIPK1/RIPK3 complex formation and phosphor-MLKL membrane translocation by molecular docking, Western blot, co-immunoprecipitation and cellular thermal shift assay (CETSA). Taken together, we currently showed that targeting RIPK1 with Cpd-71 may serve as a promising clinical candidate for AKI treatment.

A dramatic increase in the incidence of inflammatory bowel disease (IBD) has been observed in the past two decades, mainly in developed countries and also in developing regions. Necroptosis has been found to play an important role in the pathogenesis of IBD, suggesting its inhibitors are promising in clinic. However, clinical drugs targeting necroptosis are seriously lacking. Through screening a clinical compound library that contains 611 inhibitors, a pan-RAF inhibitor LY3009120 was found to be promising as a necroptosis inhibitor. LY3009120 inhibited necroptosis in vitro , and its inhibition against necroptosis was independent of its well-known activity to inhibit RAF. Surprisingly, LY3009120 prevented phosphorylation of receptor interacting serine/threonine kinase 1 (RIPK1) and subsequently phosphorylation of receptor interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) which happened during necroptosis. In vivo , LY3009120 significantly alleviated dextran sulfate sodium (DSS)-induced colitis as indicated by prevention of body weight loss, colon shortening, and decreased mortality. Furthermore, LY3009120 inhibited necroptosis of intestinal epithelial cells (IECs) and prevented intestinal barrier function loss. Consistently, LY3009120 decreased DSS-induced colonic inflammation, as indicated by decreased infiltration of macrophages and neutrophils, and decreased colonic TNF-α, IL-6, and IL-1β level in DSS treated mice. These results indicate that an anti-cancer pan-RAF inhibitor LY3009120 is a necroptosis inhibitor and may serve as a potential therapeutic drug for colitis.

Background and aims : TLR9 deletion protects against steatohepatitis due to choline–amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods : We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type ( Wt ), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9 −/− , and Tlr9 −/− . foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results : Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9 −/− mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9 −/− . foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9 +/+ .foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9 −/− mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt . Livers from Ath-fed Tlr9 −/− mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt , and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology. Conclusion: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis.

The liver is constantly exposed to a host of injurious stimuli. This results in hepatocellular death mainly by apoptosis and necrosis, but also due to autophagy, necroptosis, pyroptosis and in some cases by an intricately balanced combination thereof. Overwhelming and continuous cell death in the liver leads to inflammation, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Although data from various disease models may suggest a specific (predominant) cell death mode for different aetiologies, the clinical reality is not as clear cut. Reliable and non-invasive cell death markers are not available in general practice and assessment of cell death mode to absolute certainty from liver biopsies does not seem feasible, yet. Various aetiologies probably induce different predominant cell death modes within the liver, although the death modes involved may change during disease progression. Moreover, current methods applicable in patients are limited to surrogate markers for apoptosis (M30), and possibly for pyroptosis (IL-1 family) and necro(pto)sis (HMGB1). Although markers for some death modes are not available at all (autophagy), others may not be specific for a cell death mode or might not always definitely indicate dying cells. Physicians need to take care in asserting the presence of cell death. Still the serum-derived markers are valuable tools to assess severity of chronic liver diseases. This review gives a short overview of known hepatocellular cell death modes in various aetiologies of chronic liver disease. Also the limitations of current knowledge in human settings and utilization of surrogate markers for disease assessment are summarized.

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.