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Peroxynitrite-Triggered Carbon Monoxide Donor Improves Ischemic Stroke Outcome by Inhibiting Neuronal Apoptosis and Ferroptosis

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Abstract

Cerebral ischemia-reperfusion injury produces excessive reactive oxygen and nitrogen species, including superoxide, nitric oxide, and peroxynitrite (ONOO-). We recently developed a new ONOO--triggered metal-free carbon monoxide donor (PCOD585), exhibiting a notable neuroprotective outcome on the rat middle cerebral artery occlusion model and rendering an exciting intervention opportunity toward ischemia-induced brain injuries. However, its therapeutic mechanism still needs to be addressed. In the pharmacological study, we found PCOD585 inhibited neuronal Bcl2/Bax/caspase-3 apoptosis pathway in the peri-infarcted area of stroke by scavenging ONOO-. ONOO- scavenging further led to decreased Acyl-CoA synthetase long-chain family member 4 and increased glutathione peroxidase 4, to minimize lipoperoxidation. Additionally, the carbon monoxide release upon the ONOO- reaction with PCOD585 further inhibited the neuronal Iron-dependent ferroptosis associated with ischemia-reperfusion. Such a synergistic neuroprotective mechanism of PCOD585 yields as potent a neuroprotective effect as Edaravone. Additionally, PCOD585 penetrates the blood-brain barrier and reduces the degradation of zonula occludens-1 by inhibiting matrix metalloproteinase-9, thereby protecting the integrity of the blood-brain barrier. Our study provides a new perspective for develo** multi-functional compounds to treat ischemic stroke.

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Data Availability

The datasets generated and analyzed during this study are available upon reasonable request from the corresponding author.

Abbreviations

ACSL4:

Acyl-CoA synthetase long-chain family member 4

BBB:

Blood-brain barrier

CO:

Carbon monoxide

CORM-3:

Carbon monoxide releasing molecular-3

DMT1:

Divalent metal transporter 1

EDA:

Edaravone

Fer-1:

Ferrostatin-1

Fpn-1:

Ferroportin-1

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GPX4:

Glutathione peroxidase 4

I/R:

Ischemia/reperfusion

MCAO:

Middle Cerebral Artery Occlusion

MMP:

Matrix metalloproteinase

mNSS:

Modified Neurological Severity Scores

OGD/R:

Oxygen and Glucose Deprivation/Reperfusion

PDC:

Peroxynitrite decomposition catalyst

ROS:

Reactive oxygen species

TFR1:

Transferrin receptor 1

TUNEL:

TdT-mediated dUTP Nick-End Labeling

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Acknowledgements

We sincerely thank Dr. Fuxing Dong from the Public Experimental Research Center for his enthusiastic help in the experiment of laser scanning confocal microscopy.

Funding

This work was supported by Grants of Natural Science Foundation of Jiangsu Province, China (No. BK20211348), Xuzhou Basic Research Program (KC21030), Leadership program through open competition in Xuzhou Medical University (JBGS202203), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX21_2722).

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Author notes

  1. **n-Jian Guo, Lin-Yan Huang, and Shi-Tong Gong contributed equally to this work.

Authors and Affiliations

  1. School of Medical Technology, Xuzhou Key Laboratory of Laboratory Diagnostics, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    **n-Jian Guo, Lin-Yan Huang, Ming Li, Wan Wang, Jie Chen, Yi-De Zhang, Lan Luo & Su-Hua Qi

  2. Xuzhou Central Hospital, Affiliated Xuzhou Clinical College of Xuzhou Medical University, Xuzhou, Jiangsu, 221004, China

    Shi-Tong Gong

  3. State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Meilong Road 130, Shanghai, 200237, China

    **cun Lu, **aohua Chen, Youjun Yang & **ao Luo

  4. Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai, 200241, China

    **ao Luo

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Contributions

XJG, LYH and SHQ. conceived and designed the research. XJG, LYH and STG contributed equally to this work with the help of ML, WW, JC and YDZ, XG and XL performed immunofluorescence experiments. XC, LL, and YY provided technical assistance with the biological and chemical knowledge. LYH and XJG wrote the manuscript with help from XL All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to **ao Luo or Su-Hua Qi.

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The animal experiment was conducted by the national and institutional guidelines on ethics and biosafety, and the protocol was approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (License ID: 201907W079).

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ESM 1

Figure S1. Representative images of Rhodamine B and CO production in different organs from the MCAO rats treated with PCOD585. The cryosections were prepared from the rats subjected to 2 h ischemia and followed with 6 mg/kg PCOD585 intravenous transfusion. The fluorescent images were captured by invert fluorescent microscopy. Rhodamine B (red), CO (Cop-1 probe, green), DAPI (blue). Scale bar: 200 μm. Figure S2. The expression and analysis of GPX4 (green) in caudate putamen from the rats with MCAO, PDC and PCOD585. PDC: MCAO+PDC, PCOD585: MCAO+PCOD585. The data were obtained from three individual experiments. For each experiment, five fluorescent fields were selected. **P < 0. 01, ***P < 0.001. Scale bar: 200 μm. Figure S3. The effect of different reoxygenation times on the expression of GPX4 in OGD-treated HT22 cells. The data were obtained from three individual experiments. *P < 0. 05, **P < 0.01. Figure S4. The effect of PCOD585 with a different concentration on the expression of GPX4 in OGD/R-treated HT22 cells. PCOD585 were added along with reoxygenation. The data were obtained from three individual experiments. *P < 0. 05 (DOCX 1615 kb)

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Guo, XJ., Huang, LY., Gong, ST. et al. Peroxynitrite-Triggered Carbon Monoxide Donor Improves Ischemic Stroke Outcome by Inhibiting Neuronal Apoptosis and Ferroptosis. Mol Neurobiol (2024). https://doi.org/10.1007/s12035-024-04238-w

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