Gene Silencing of NALP3 Protects Against Liver Ischemia–Reperfusion Injury in Mice

Abstract

Liver ischemia-reperfusion (I/R) injury is a multifactorial process that affects graft function after liver transplantation. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18, have been shown to play key roles in the pathophysiology of liver I/R injury. Studies have indicated that NALP3 (NACHT domain, leucine-rich repeat [LRR] domain, and pyrin domain [PYD]-containing protein-3) inflammasome is pivotal in the processing and releasing of IL-1β and IL-18. The aim of this study was to test whether NALP3 silencing has a protective effect in murine liver I/R injury. Using a partial lobar liver warm ischemia model, mice were hydrodynamically injected with pNALP3shRNA, pshRNANC, or saline 48 hr before ischemia. Those mice pretreated with pNALP3shRNA showed decreased serum alanine aminotransferase levels; inhibited production of proinflammatory cytokines such as IL-1β, IL-18, TNF-α, and IL-6 by downregulation of caspase-1 activation and NF-κB activity; as well as decreased release of HMGB1 (high-mobility group box-1) and inflammatory cell infiltration, leading to the prevention of liver I/R injury, when compared with controls. Histology revealed that pretreatment with pNALP3shRNA significantly ameliorated hepatocellular damage after I/R. Thus, by using a small hairpin RNA approach, our study confirms that NALP3 signaling is involved in liver I/R and that silencing of NALP3 can protect the liver from I/R injury by reducing IL-1β, IL-18, TNF-α, IL-6, and HMGB1 release through downregulation of caspase-1 activation and NF-κB activity.

Inflammasome activation and function in liver disease

“…136,137 In studies using mouse models of ischaemiareperfusio n injury, Nlrp3 deficiency prevented inflammasome activation, release of Il-1β, Il-18 and other inflammatory cytokines, and reduced the extent of ischaemia-reperfusion injury. 138,139 A similar level of protection was observed in another study using mice genetically deficient in the inflammasome components Asc or Casp-1, 139,140 or treated with YVAD, a specific Casp-1 inhibitor. 141 These data were further supported by studies in animals in which pre-treatment with Il-1ra, delivery of Il-1ra cDNA or administration of Il-18-neutralizing antibodies in the liver substantially reduced ischaemia-reperfusion-related liver damage, i nflammation and mortality.…”

HMGB1 in health and disease

“…Inflammasomes have recently been shown to play an important role in mediating HMGB1 release from activated immune cells (Barlan et al, 2011; Craven et al, 2009; Lamkanfi et al, 2010; Lippai et al, 2013; Willingham et al, 2009; Willingham et al, 2007) or cancer cells (Miller et al, 2014). Thus, inflammasome-deficient mice or inflammasome inhibitor can inhibit HMGB1 release and protects mice against sepsis or I/R injury (Kamo et al, 2013; Xiang et al, 2011; Zhu et al, 2011b). LPS can induce ATP release and P2Y2 receptor upregulation, which in turn triggers the activation of inflammasome and enhanced HMGB1 release (Eun et al, 2014).…”

Inflammasomes in liver diseases

“…The characteristics of I/R are hepatocyte death, release of DAMPs, inflammatory cell infiltration, Kupffer cell activation, ROS production, and disruption of liver sinusoidal endothelial cells (LSEC) that can all lead to inflammasome activation [88]. Silencing NLRP3 ameliorated I/R-induced hepatocellular injury and reduced IL-1b, IL-18, HMGB1, IL-6, and TNFa release via inhibition of caspase-1 and NFjB activity [89]. Consistent with this, decreased caspase-1 activation was found in the presence of the antioxidant N-acetylcysteine, in I/R injury [75].…”