Inhibition of neuronal ferroptosis protects hemorrhagic brain
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
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Research Article Neuroscience

Inhibition of neuronal ferroptosis protects hemorrhagic brain

Abstract

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell–derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

Authors

Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang

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Figure 3

Fer-1 reduces Hb-induced lipid ROS and ameliorates glutathione peroxidase (GPx) activity reduction in OHSCs.

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Fer-1 reduces Hb-induced lipid ROS and ameliorates glutathione peroxidas...
Organotypic hippocampal slice cultures (OHSCs) were treated with 10 μM ferrostatin-1 (Fer-1) and 20 μM hemoglobin (Hb) alone or in combination for 16 hours. (A) ROS were measured by quantifying fluorescence intensity after incubation with hydroethidine (HEt). Representative images and quantification are shown. **P < 0.01 versus control; ###P < 0.001 versus Hb. (B) Lipid ROS were measured with BODIPY 581/591 C11 reagent, and images were taken under a fluorescence microscope. Slices were then lysed and fluorescence intensity measured on a microplate reader. Cumene hydroperoxide (CHP) treatment was used as a positive control. Representative images and quantification are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; #P < 0.05 versus Hb. (C) Lipid ROS were measured with a malondialdehyde (MDA) assay. *P < 0.05 versus control; #P < 0.05 versus Hb. (D) GPx activity was measured with a GPx assay kit. **P < 0.01, ***P < 0.001 versus control; #P < 0.05 versus Hb. (E) mRNA was extracted from the OHSCs, and reverse transcriptase real-time PCR was carried out with different primers. GAPDH was used as an internal control, and results are shown as fold change of control. *P < 0.05 versus control; #P < 0.05 versus Hb. Results are represented as box-and-whisker plots (A–C; the middle horizontal line within the box represents the median, boxes extend from the 25th to the 75th percentile, and the whiskers represent 95% confidence intervals) or mean ± SD (D and E). Statistical tests used were 1-way ANOVA followed by Dunn’s multiple comparison post test (A–C, and E) and repeated measurement followed by Tukey’s multiple comparison (D). Results are from at least 3 independent experiments. Scale bars: 1 mm (A), 100 μm (B).