Plasma obtained following murine hindlimb ischemic conditioning protects against oxidative stress in zebrafish models through activation of nrf2a and downregulation of duox.

Eric A Shelden,Youdong Wang,Caterina Di Ciano-Oliveira,Farhad Karbassi,Si Yuan Dai,Rui Guan,Xiao-Yan Wen,Chung Ho Leung,Stephen Lam,Ori Rotstein,Antonio Mauro

doi:10.1371/journal.pone.0260442

Abstract

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.

Highlights

Cellular injury resulting from sequential ischemia/reperfusion (I/R) of tissues is a pathophysiological process which contributes to morbidity and mortality in a wide range of medical settings, including ischemic stroke, myocardial infarction, organ transplantation and traumainduced hemorrhagic shock [1]
In order to exclude the possibility that the decrease of neutrophils in the tail wound by remote ischemic preconditioning (RIC) was due to the decrease of neutrophils in the whole fish body, we counted total body neutrophils and found that there was no significant difference in neutrophil counts between saline (237 ± 29, n = 12), Ctrl (229 ± 12, n = 12) and RIC plasma injected fish larvae
Work by Niethammer and colleagues [20] demonstrated that the directional migration and tissue infiltration of neutrophils are mediated by H2O2 gradient, we further investigated whether RIC might exert an effect on the level of reactive oxygen species (ROS) generation at the tail transection region

Summary

Introduction

Cellular injury resulting from sequential ischemia/reperfusion (I/R) of tissues is a pathophysiological process which contributes to morbidity and mortality in a wide range of medical settings, including ischemic stroke, myocardial infarction, organ transplantation and traumainduced hemorrhagic shock [1]. The balance between metabolic supply and demand is altered due to hypoxia within the ischemic organ and subsequent reperfusion further exacerbates the metabolic imbalance by enhancing the activation of immune responses and a cascade of inflammatory processes [2, 3]. Neutrophils in the circulation perform an essential host-defense function by directly migrating to the site of injury for tissue repair, but excess neutrophils may cause tissue damage, in part through the generation of oxidative stress [4]. ROS are able to directly injure cells, but may promote pro-inflammatory signaling cascades which exacerbate injury [2, 5, 6]. As a part of the host response, oxidative stress is known to induce a broad antioxidant response through the generation of neutralizing antioxidant proteins. The transcription factor nuclear factor erythroid 2-related factor (nrf2) is a known regulator of the antioxidant response, by virtue of its ability to induce antioxidant response element-dependent genes

Methods

Results

Discussion

Conclusion