Role of Nrf2 in the Protective Effect of Remote Ischemic Conditioning in Hemorrhagic Shock/Resuscitation

Author(s):
Chung Ho Leung; Rui Guan; Xiao-Yan Wen; Christopher A. Caldarone; Ori Rotstein

Background:

Oxidative stress and immune cell activation remain the major contributors to the development of secondary complications in resuscitated trauma patients. Anti-oxidant interventions targeting these inflammatory events have potential to improve outcomes. We have previously demonstrated that remote ischemic conditioning (RIC) exerts hepatoprotection in a murine model of hemorrhagic shock/resuscitation (HS/R) that is mediated by antioxidant response and elaboration of humoral factors. In the current study, we examined the mechanisms of RIC using murine and zebrafish models of injury.

Hypothesis:

Humoral factors released by RIC exerts remote organ protection that is mediated by the anti-oxidant transcription factor Nrf2.

Methods:

Wildtype (WT) C57/BL6 or Nrf2 KO mice were subjected to hemorrhagic shock for 2h at 30mmHG MAP and resuscitated with blood taken from donor WT or Nrf2 KO mice that had undergone RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) and two volumes of Ringer’s Lactate. Mice were sacrificed at 2h post resuscitation for evaluation of liver injury by serum ALT. In zebrafish studies, zebrafish were subjected to morpholino knockdown of Nrf2a and microinjected with plasma (5%) taken from WT or Nrf2 KO mice after RIC. Neutrophil migration was assessed after tailfin transection and survival studies were conducted under LPS (100ug/ml) or H₂O₂ (2mM) treatment. Zebrafish Oxidative Stress RT² Profiler PCR Array was used to investigate the effect of RIC plasma on oxidative gene expression.

Results:

WT mice resuscitated with WT RIC blood significantly reduced liver injury. However, the protective effect of WT RIC blood was abolished when resuscitated in Nrf2 KO mice. Furthermore, WT mice resuscitated with Nrf2 KO RIC blood failed to exert hepatoprotection. In parallel, WT zebrafish microinjected with WT RIC plasma had significantly lower neutrophil migration towards the site of injury – an effect that was abrogated in zebrafish with Nrf2a knockdown or when WT zebrafish were microinjected with Nrf2 KO RIC plasma. In addition, zebrafish microinjected with WT RIC plasma significantly improved survival following incubation with LPS or H₂O₂. The mRNA levels of anti-oxidant genes (myoglobin, hmox1) were up regulated and pro-oxidant gene (duox) was down regulated in zebrafish microinjected with WT RIC plasma.

Conclusions:

Nrf2 is a central mediator in the elaboration of humoral factors and organ protection of RIC against HS/R. Up regulation of cytoprotective antioxidant genes and down regulation of oxidative genes may contribute to the downstream mechanisms of RIC. RIC represents a potential therapeutic for trauma patients.