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. 2021 Nov 12:12:763419.
doi: 10.3389/fneur.2021.763419. eCollection 2021.

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

Ranran Han  1 , Jieru Wan  1 , Xiaoning Han  1 , Honglei Ren  1 , John R Falck  2 , Sailu Munnuri  2 , Zeng-Jin Yang  1 , Raymond C Koehler  1
Affiliations

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

Ranran Han et al. Front Neurol. .

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.

Keywords: 20-hydroxyeicosatetraenoic acid; ferroptosis; glutathione peroxidase; intracerebral hemorrhage; lipid peroxide.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
20-HETE participates in hemoglobin (Hb)-induced cell death in organotypic hippocampal slice cultures (OHSCs). (A) OHSCs were exposed to 10 μM Hb. HET0016 (10 μM), a 20-HETE synthesis inhibitor, or 20-6,15-HEDGE (10 μM), a 20-HETE antagonist, was added along with Hb for 18 h. OHSCs treated with HET0016 or 20-6,15-HEDGE alone and a vehicle-treated group served as controls. Propidium iodide (PI) staining was performed before (P0h) and 18 h after (P18h) reagent treatment. Scale bar is 500 μm. (B) Quantification of (A). Cell death was evaluated as PI uptake percentage. Hb-induced cell death was significantly lowered by both HET0016 and 20-6,15-HEDGE. n = 8–14 slices/group. (C) Hb significantly increased 20-HETE content in OHSC lysates at 18 h, but HET0016 prevented this increase. n = 5–6 from more than 25 slices per group. (D) OHSCs were exposed to Hb with or without HET0016 or 20-6,15-HEDGE for 18 h, after which iron deposition was assessed. Representative images are shown along with examples of iron-positive cells at high magnification (insets). Arrows indicate iron-positive cells. Scale bar is 50 μm. Both HET0016 and 20-6,15-HEDGE significantly reduced Hb-induced iron accumulation. n = 5 slices/group. #P < 0.05, ##P < 0.01 vs. Control group; *P < 0.05, **P < 0.01 vs. Hb group. One-way ANOVA followed by appropriate post hoc test was applied. Data are expressed as mean ± SD.
Figure 2
Figure 2
20-HETE promotes ferroptosis in ex vivo ICH model. Organotypic hippocampal slice cultures (OHSCs) were exposed to 10 μM hemoglobin (Hb) with or without 10 μM HET0016 or 10 μM 20-6,15-HEDGE. (A) Lipid peroxidation level was detected with BODIPY 581/591 C11 reagent 6 h after incubation with different reagents. Representative fluorescent images and fluorescent intensity quantification relative to control slices showed an increased lipid peroxidation state with Hb exposure that was attenuated by HET0016 or 20-6,15-HEDGE co-treatment. Scale bar is 200 μm. n = 5–8 slices/group. (B,C) Western blotting of OHSC total protein extract was carried out to assess the level of 4-hydroxy-2-nonenal (4-HNE) adducted protein (B) and glutathione peroxidase (GPX) 4 (C) 18 h after incubation with different reagents. The expression was normalized relative to the control group. 20-HETE inhibition decreased 4-HNE adducted protein level and increased GPX4 expression. n = 4–5 from at least 20 slices/group. (D) Immunofluorescence result of GPX4 expression at 18 h after exposure to different reagents in fixed slices. Bar is 30 μm. n = 5 slices/group. #P < 0.05, ##P < 0.01 vs. Control group; *P < 0.05, **P < 0.01 vs. Hb group. One-way ANOVA followed by appropriate post hoc test was applied. Data are expressed as mean ± SD.
Figure 3
Figure 3
20-HETE–induced cell death is mitigated by inhibiting ferroptosis. (A) The stable 20-HETE mimetic 20-5,14-HEDGE at indicated concentrations or vehicle was added to organotypic hippocampal slice cultures (OHSCs), and cell death was assessed by PI staining 18 h later. All three doses of 20-5,14-HEDGE induced significant cell death. The dose of 10 μM was chosen for subsequent experiments because it produced cell death similar to that produced by Hb. n = 5–9 slices/group. (B) Fixed slices were immunostained with NeuN, Iba1, and GFAP (green) to assess the distribution of neurons, microglia, and astrocytes among PI-labeled dead cells. The percentage of PI+ neurons (PI+NeuN+) was more than PI+ microglia (PI+Iba1+) and PI+ astrocytes (PI+GFAP+). Bar is 50 μm. n = 6 slices/group. (C) The effect of ferrostatin-1 (Fer-1; 10 μM) on 20-5,14-HEDGE-induced cell death was measured by PI staining. Fer-1 significantly alleviated 20-HETE mimetic-induced cell death. Bar is 500 μm. n = 9–12 slices/group. (D) Effect of cell death inhibitors caspase 3 inhibitor VII (apoptosis inhibitor, 25 μM), necrostatin-1 (Nec-1, necroptosis inhibitor, 10 μM), and a combination of Fer-1, caspase 3 inhibitor, and Nec-1 on 20-5,14-HEDGE–induced cell death was assessed by PI staining. n = 5–6 slices/group. *P < 0.05, **P < 0.01 vs. 20-5,14-HEDGE Vehicle group; ##P < 0.01 vs. Control group. One-way ANOVA followed by appropriate post-hoc test was applied. Data are expressed as mean ± SD.
Figure 4
Figure 4
HET0016 ameliorates ICH injury severity in mice. (A) HET0016 treatment improved neurological deficit scores of mice at 1, 3, and 7 days after ICH. n = 8–10/group. (B) HET0016-treated ICH mice exhibited longer latency to fall in the wire-hanging test than did vehicle-treated mice at 1, 3, and 7 days after ICH. n = 8–10/group. (C) Examples of cresyl violet (CV)/luxol fast blue–stained serial brain sections from vehicle- and HET0016-treated mice 3 days after ICH. Lesion volume was similar on day 1 but significantly less on days 3 and 7 in the HET0016-treated group. n = 8–10/group. (D) Representative images of 3,3′-diaminobenzidine (DAB)-enhanced Perls' staining shows iron deposition at 3 and 7 days after ICH. Scale bar is 200 μm. The number of iron-positive cells was lower in the HET0016-treated group than in the vehicle-treated group. n = 6–9/group. *P < 0.05, **P < 0.01. Mann–Whitney U-test (A,B) or unpaired t-test (C,D) was applied between vehicle and HET0016 groups at each time point. Data are expressed as median with confidence interval (A,B) or mean ± SD (C,D).
Figure 5
Figure 5
HET0016 decreases lipid peroxidation level after ICH mice. Glutathione (GSH) (A), malondialdehyde (MDA) (B), and 4-hydroxy-2-nonenal (4-HNE) (C) content in brain 3 days after ICH. HET0016 reversed the ICH-induced reduction in GSH and increases in MDA and 4-HNE. n = 9 (A,C) or 5 (B) per group. (D) Brain tissue surrounding the hematoma was harvested 3 days after ICH, and total protein was extracted for western blotting. After ICH, cyclooxygenase-2 (COX-2) levels increased to a similar extent in the vehicle and HET0016 groups relative to that in the Sham group. n = 7/group. *P < 0.05, **P < 0.01 vs. Vehicle group. Mann–Whitney U-test (B) or unpaired t-test (A,C,D) was applied. Data are expressed as median with confidence interval (B) or mean ± SD (A,C,D).
Figure 6
Figure 6
HET0016 affects ferroptosis pathway after ICH in mice. (A) Brain tissue surrounding the hematoma was harvested 1 and 3 days after ICH, and total protein was extracted for western blotting. Glutathione peroxidase (GPX) 4 expression was significantly higher at day 1 post-ICH than it was in the sham group and then declined to baseline at day 3 post-ICH. n = 4 in sham group, n = 6 in ICH groups. (B) Western blot analysis shows GPX4 expression in brain tissues from different groups at day 3 after ICH. GPX4 level was significantly higher in the HET0016-treated group than in the vehicle-treated group. n = 12/group. (C) Immunofluorescence staining was carried out 3 days after ICH for GPX4 (red) and nuclei (blue). Arrows indicate the GPX4–positive cells. Scale bar is 30 μm. Quantification of GPX4 positive cell density is shown. (D) Total and phospho-P38/ERK1/2/JNK expression 3 days after ICH. Values were calculated as ratios of phospho- to total-p38/ERK1/2/JNK and further normalized to vinculin or β-actin loading controls. The ratios of p-p38/p38, p-ERK1/2/ERK1/2, and p-JNK/JNK were significantly lower in the HET0016-treated group than in the vehicle-treated group. n = 7/group. #P < 0.05, ##P < 0.01 vs. Sham group; *P < 0.05, **P < 0.01 vs. Vehicle group. One-way ANOVA followed by appropriate post-hoc test was applied for multiple comparison and t-test for two group comparison. Data are expressed as mean ± SD.
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