Remote Ischemic Preconditioning Protects the Liver from Hemorrhagic Shock/Resuscitation by Activation of Nrf2/Heme-Oxygenase 1 and Autophagy
Author(s):
Chung Ho Leung, University of Toronto; Christopher Caldarone, Hospital for Sick Children; Ori Rotstein, St. Michael's Hospital
Background: Oxidative stress resulting from hemorrhagic shock/resuscitation contributes a major role to the development of organ injury. We have previously reported that transient ischemia/reperfusion in the limb known as remote ischemic preconditioning (RIPC) conferred organ protection following a murine model of hemorrhagic shock/resuscitation. To examine the potential mechanisms, we evaluated the role of antioxidant proteins and autophagy in RIPC mediated liver protection.
Hypothesis: Activation of the transcription factor Nrf2 and its downstream antioxidant proteins, and autophagy are protective mechanisms of RIPC.
Methods: Male C57BL/6 mice were subjected to hemorrhagic shock at MAP of 30 mmHG for two hours followed by resuscitation with shed blood plus lactated Ringer’s solution. RIPC was performed prior to hemorrhage by occlusion of the left hindlimb with a tourniquet for four cycles of 5-minute ischemia and 5-minute reperfusion. Serum alanine aminotransferase (ALT), expression of Nrf2 and antioxidant proteins, and activity of autophagy were evaluated in the liver at two hours after resuscitation.
Results: Hemorrhagic shock/resuscitation resulted in liver injury with a significant rise in serum ALT (606 ± 176 U/L). By contrast, antecedent RIPC markedly attenuated this increase (220 ±44 U/L, P < 0.05). Total cell and nuclear protein expression of Nrf2 were significantly higher in mice with RIPC. The induction of Nrf2 was strongly associated with a profound increase in the mRNA and protein expression of the antioxidant protein heme oxygenase 1 with RIPC. mRNA levels of antioxidant proteins MnSOD, GPX, and NQO1 were not significantly different. The activity of autophagy was higher with RIPC as evident by an elevated conversion of LC3-I to LC3-II and increased formation of autophagosomes as observed by electron microscopy. Furthermore, the augmented antioxidant capacity and autophagic activity were associated with a significant increase in the phosphorylation of the pro-survival kinases AKT and ERK-1/2.
Conclusions: RIPC prevents organ injury by evoking a robust Nrf2 antioxidant pathway and autophagy in response to hemorrhagic shock. Endogenous induction of antioxidant capacity by remote ischemic conditioning has potential to improve outcome in trauma patients.