20-HETE Participates in Intracerebral Hemorrhage-Induced Acute Injury by Promoting Cell Ferroptosis.

Ranran Han,Sailu Munnuri,Raymond C Koehler,John R Falck,Honglei Ren,Xiaoning Han,Zeng-Jin Yang,Jieru Wan

doi:10.3389/fneur.2021.763419

Abstract

Intracerebral hemorrhage (ICH) is a highly fatal type of stroke that leads to various types of neuronal death. Recently, ferroptosis, a form of cell death resulting from iron-dependent lipid peroxide accumulation, was observed in a mouse ICH model. N-hydroxy-N′-(4-n-butyl-2-methylphenyl)-formamidine (HET0016), which inhibits synthesis of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE), has shown a protective effect after ICH. However, the underlying mechanisms of the neuroprotective effect need further investigation. We explored whether 20-HETE participates in ICH-induced ferroptosis ex vivo by using hemoglobin-treated organotypic hippocampal slice cultures (OHSCs) and in vivo by using a collagenase-induced ICH mouse model. Ex vivo, we found that the 20-HETE synthesis inhibitor HET0016 and antagonist 20-6,15-HEDGE reduced hemoglobin-induced cell death, iron deposition, and lipid reactive oxygen species levels in OHSCs. Furthermore, 20-HETE inhibition in OHSCs increased the expression of glutathione peroxidase (GPX) 4, an antioxidant enzyme that serves as a main regulator of ferroptosis. In contrast, exposure of OHSCs to the 20-HETE stable mimetic 20-5,14-HEDGE induced cell death that was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. In vivo, HET0016 treatment ameliorated focal deficits, reduced lesion volume, and decreased iron accumulation around the lesion at day 3 and 7 after ICH. In addition, lipid peroxidation was decreased and expression of GPX4 was increased in the HET0016-treated ICH group. The mitogen-activated protein kinase pathway also was inhibited by HET0016 in vivo. These results indicate that 20-HETE contributes to ICH-induced acute brain injury in part by activating ferroptosis pathways, thereby providing an upstream target for inhibiting ferroptosis.

Highlights

Intracerebral hemorrhage (ICH) is a devastating neurologic injury that accounts for 10–15% of all strokes [1]
Ferroptosis is a regulated form of nonapoptotic cell death that results from iron-dependent lipid peroxide accumulation, which can be reduced by glutathione peroxidase 4 (GPX4) activity; 20-hydroxyeicosatetraenoic acid (20-HETE)-Induced Ferroptosis After ICH
We found that [1] Hb-induced ferroptosis in organotypic hippocampal slice cultures (OHSCs) can be ameliorated by 20-HETE inhibitors; 20-HETE-Induced Ferroptosis After ICH

Summary

INTRODUCTION

Intracerebral hemorrhage (ICH) is a devastating neurologic injury that accounts for 10–15% of all strokes [1]. Therapy that targets the inhibition of ferroptosis may effectively prevent neuronal death, thereby reducing secondary brain injury after ICH [17]. CYP4A expression and 20-HETE were reported to increase in a mouse model of ICH, and its highly selective synthesis inhibitor N-hydroxy-N′-(4-n-butyl2-methylphenyl)-formamidine (HET0016) [27] was found to be protective after ICH, as assessed by lesion volume and by FluoroJade and TUNEL staining measurements of perilesion neuronal degeneration [28]. This protection was associated with decreased activation of microglia and astrocytes and by attenuated formation of protein carbonylation and nitration. We investigated whether inhibiting 20-HETE reduces biochemical markers of ferroptosis and whether blocking ferroptosis reduces toxic effects of a stable 20-HETE agonist

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ETHICS STATEMENT