Hypoxia-inducible factor-1 alpha is involved in RIP-induced necroptosis caused by in vitro and in vivo ischemic brain injury

Xiao-Sa Yang,Cheng Yang,Sai Zhang,Ze-Qi Yu,Tai-Long Yi,Hong-Tao Sun,Shi-Xiang Cheng,Yue Tu,Zhong-Wei Xu

doi:10.1038/s41598-017-06088-0

Abstract

Necroptosis, a novel type of programmed cell death, is involved in stroke-induced ischemic brain injury. Although studies have sought to explore the mechanisms of necroptosis, its signaling pathway has not yet to be completely elucidated. Thus, we used oxygen-glucose deprivation (OGD) and middle cerebral artery occlusion (MCAO) models mimicking ischemic stroke (IS) conditions to investigate mechanisms of necroptosis. We found that OGD and MCAO induced cell death, local brain ischemia and neurological deficit, while zVAD-fmk (zVAD, an apoptotic inhibitor), GSK’872 (a receptor interacting protein kinase-3 (RIP3) inhibitor), and combined treatment alleviated cell death and ischemic brain injury. Moreover, OGD and MCAO upregulated protein expression of the triggers of necroptosis: receptor interacting protein kinase-1 (RIP1), RIP3 and mixed lineage kinase domain-like protein (MLKL). The upregulation of these proteins was inhibited by GSK’872, combination treatments and RIP3 siRNA but not zVAD treatment. Intriguingly, hypoxia-inducible factor-1 alpha (HIF-1α), an important transcriptional factor under hypoxic conditions, was upregulated by OGD and MCAO. Similar to their inhibitory effects on aforementioned proteins upregulation, GSK’872, combination treatments and RIP3 siRNA decreased HIF-1α protein level. These findings indicate that necroptosis contributes to ischemic brain injury induced by OGD and MCAO and implicate HIF-1α, RIP1, RIP3, and MLKL in necroptosis.

Highlights

Ischemic stroke (IS) is the second leading cause of death worldwide, with an annually increasing incidence rate, and it poses an enormous threat to public health[1]
A growing body of literature suggests that oxygen and glucose deprivation within normal cells is a useful model of ischemia and reperfusion injury in vitro[16]
It revealed that the viability of cells exposed to 4, 8 and 12 h of Oxygen-glucose deprivation (OGD) decreased to 94.36 ± 1.94% (p < 0.05), 81.11 ± 3.52% and 56.40 ± 1.13%, respectively, whereas cells exposed to 14 and 16 h of OGD suffered dramatically with cell viabilities of 39.57 ± 0.68% and 17.86 ± 0.73%, respectively; 16 h of OGD resulted in excessive cell death (Fig. 1b), which was in agreement with morphologic alterations

Summary

Introduction

Ischemic stroke (IS) is the second leading cause of death worldwide, with an annually increasing incidence rate, and it poses an enormous threat to public health[1]. Recent evidence indicates that necroptosis, a novel form of programmed cell death, plays an important role in the brain, contributing to the pathological processes of cerebral ischemia/reperfusion injury and traumatic brain injury. It was previously shown that necroptosis is mediated by the assembly of RIP1 and RIP3, forming the necrosome complex[6], which recruits several molecules to execute necroptosis Of these recruited molecules, mixed lineage kinase domain-like protein (MLKL) is considered to be an essential downstream protein, and its interaction with necrosome leads to necroptosis activation in IS7. MCAO model is performed by occluding the unilateral cerebral middle artery of animals with a suture to induce local ischemic brain injury, removing the suture to execute reperfusion[11]. Numerous studies have validated that OGD and MCAO models are feasible to mimic the pathogenesis of IS and provide the experimental basis for the mechanism study of IS12, 13

Methods

Results

Conclusion