RIP1-Dependent Programmed Necrosis is Negatively Regulated by Caspases During Hepatic Ischemia-Reperfusion

Abstract

Programmed necrosis (necroptosis), a newly discovered form of cell death, is mediated by receptor-interacting protein 1 (RIP1) and plays a pivotal role after myocardial, renal, and cerebral ischemia-reperfusion (I/R). The relevance of necroptosis in the postischemic liver remains, however, unclear. The aim of this study was to analyze the role of programed necrosis during hepatic I/R. C57BL6 mice were subjected to warm hepatic I/R (90 min/240 min). The animals were pretreated with either the RIP1 inhibitor necrostatin-1 (Nec-1, 3.5 μg kg) or vehicle (Nec-1inactive, 3.5 μg kg) administered systemically before ischemia. Sham-operated animals served as controls (n = 6 each group). The inflammatory response was evaluated by intravital microscopy. The hepatic transaminases alanine aminotransferase/aspartate aminotransferase in plasma as well as the activity of caspase-3 in tissue were determined as markers of hepatocellular injury. Leukocyte recruitment to the liver, sinusoidal perfusion failure, as well as the transaminase activities were strongly increased on I/R as compared with the sham-operated mice. Inhibition of the RIP1-dependent pathway with Nec-1, however, did not attenuate I/R-induced leukocyte migration, perfusion failure, and hepatocellular injury. Western blot analysis showed a baseline RIP1 expression in livers from sham-operated mice, whereas RIP1 expression was not detectable in both Nec-1-treated and vehicle-treated I/R group. Caspase-3 activity was significantly elevated after I/R in both postischemic groups. Our in vivo data show that RIP1-mediated necroptosis is not present in the postischemic liver and that I/R-induced caspase activation is associated with loss of RIP1 expression. Because caspases are able to cleave RIP1, we hypothesize that I/R-triggered caspase activation negatively regulates necroptosis and, thereby, determines apoptosis as a preferred route of cell death after hepatic I/R.

Galangin Alleviates Liver Ischemia-Reperfusion Injury in a Rat Model by Mediating the PI3K/AKT Pathway

“…I/R injury is caused by a complex series of events including cell death resulting from oxygen deprivation and adenosine triphosphate depletion during ischemia and subsequent activation of cells of the innate immune system by inflammatory responses during reperfusion [26]. In the process of I/R injury, redox balance is dysregulated and the normal functions and integrity of tissues are damaged, resulting in the accumulation of reactive oxygen species (ROS).…”

The in vivo evidence for regulated necrosis

“…A causative role for the TNFα/TNFR1 axis was established, as TNFR1-KO but not Fas-KO animals showed protection. 106 While 3.5 mg/kg Nec-1 before 90 minutes ischemia/240 minutes reperfusion was not protective, 107 1.65 mg/kg before 60/240 minutes I/R reduced necrosome formation, TNFα, IL-6, and ALAT levels, respectively. 108 Therefore, and because we lack solid genetic evidence, this IRI model is difficult to evaluate.…”

“…106 While 3.5 mg/kg Nec-1 before 90 minutes ischemia/240 minutes reperfusion was not protective,107 1.65 mg/kg before 60/240 minutes I/R reduced necrosome formation, TNFα, IL-6, and ALAT levels, respectively. A causative role for the TNFα/TNFR1 axis was established, as TNFR1-KO but not Fas-KO animals showed protection.…”

Necroptosis: Mechanisms and Relevance to Disease

“…Several lines of evidence suggest that necroptosis is intimately involved in the etiology of various hepatic conditions with a prominent inflammatory component but may not contribute to hepatic I/R (169). Thus, the Ripk3 −/− genotype, the intraperitoneal delivery of Ripk3 -targeting antisense oligonucleotides, or the administration of dabrafenib (an FDA-approved inhibitor of oncogenic BRAF that also suppresses RIPK3 kinase activity) provides considerable hepatoprotection in mouse models of acetaminophen toxicity (134, 170, 171), concanavalin A–driven hepatitis (134), ethanol and dietary intoxication (172, 173), and nonalcoholic steatohepatitis (NASH) (174, 175).…”