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Targeting iNOS Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage via Promoting Ferroptosis of M1 Microglia and Reducing Neuroinflammation

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Abstract

Numerous studies have demonstrated the role of neuroinflammation in mediating acute pathophysiological events of early brain injury after subarachnoid hemorrhage (SAH). However, it is not clear how to target this inflammatory cascade after SAH. M1 activation of microglia is an important pathological mechanism driving neuroinflammation in SAH, which is considered aggressive, leading to cytotoxicity and robust inflammation related to the release of proinflammatory cytokines and chemokines after SAH. Thus, reducing the number of M1 microglia represents a potential target for therapies to improve outcomes after SAH. Previous studies have found that inducible nitric oxide synthase (iNOS/NO•) plays an essential role in promoting the survival of M1 microglia by blocking ferroptosis. Ferroptosis is a new type of iron-dependent cellular procedural death associated with pathological cell death related to mammalian degenerative diseases, cerebral hemorrhage, and traumatic brain injury. Here, we investigated the effect of L-NIL, an inhibitor of iNOS, on M1 microglia, neuroinflammation, neuronal cell death, brain edema, and neurological function in an experimental SAH model in vivo and in vitro. We found that L-NIL reduced the number of M1 microglia and alleviated neuroinflammation following SAH. Notably, treatment with L-NIL relieves brain edema and neuronal injury and improves outcomes of neurological function after SAH in rats. Mechanistically, we found that L-NIL inhibited the expression of iNOS and promoted ferroptosis of M1 microglia by increasing the expression of ferroptosis-related proteins and lipid peroxidation in an in vitro model of SAH, which was reversed by a ferroptosis inhibitor, liproxstatin-1. In addition, inhibiting iNOS had no significant effect on ferroptosis of neurons after oxyhemoglobin stimulation in vitro. Thus, our research demonstrated that inhibition of iNOS might represent a potential therapeutic strategy to improve outcomes after SAH by promoting ferroptosis of M1 microglia and reducing neuroinflammation.

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Data Availability

All data generated or analyzed during this study are included in this article and its supplementary information files.

Abbreviations

SAH:

Subarachnoid hemorrhage

EBI:

Early brain injury

iNOS:

Inducible nitric oxide synthase

Lip-1:

Liproxstatin-1

qRT–PCR:

Quantificational real-time polymerase chain reaction

FJB:

Fluoro-Jade B

BBB:

Blood–brain barrier

CNS:

Central nervous system

ROS:

Reactive oxygen species

L-NIL:

L-N (6)-iminoethyl-lysine

RNS:

Reactive nitrogen species

GPX4:

Glutathione peroxidase 4

OxyHb:

Oxyhemoglobin

CSF:

Cerebrospinal fluid

Lipid-ROS:

Lipid peroxidation-reactive oxygen species

DAPI:

4’, 6-Diamidino-2-phenylindol

H2O2 :

Hydrogen peroxide

DAB:

3,3-Diaminobenzidine

CCK8:

Cell Counting Kit-8

TMB:

3, 3, 5,5-Tetramethylbenzidine

MWM:

Morris Water Maze

STEAP3:

Six-transmembrane epithelial antigen of the prostate 3

DMT1:

Divalent metal transporter 1

Fth:

Ferritin heavy chain

Ftl:

Ferritin light chain

GSH:

Glutathione

Cys:

Cysteine

Cox2:

Cyclooxygenase 2

TfR1:

Transferrin receptor 1

Tf:

Transferrin

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Funding

This study was supported by the National Nature Science Foundation of China (Nos. 81571121 and 81971163).

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Author notes

  1. Wenhao Qu and Ying Cheng contributed equally.

Authors and Affiliations

  1. Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, 215031, Jiangsu Province, China

    Wenhao Qu, Wei Peng, Yan Wu & Jian Zhang

  2. Department of Forensic Medicine, Medical College of Soochow University, Suzhou, 215123, Jiangsu Province, China

    Ying Cheng, Tongyu Rui & Chengliang Luo

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  1. Wenhao Qu
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Contributions

W.Q. and Y.C. contributed equally to this paper. J.Z. and C.L. comprehended the study, provided critical suggestions, contributed to manuscript preparation, oversaw the research program, and wrote the main manuscript.

W.Q., Y.C., and T.R. performed the Western blot, qRT–PCR, and immunostaining experiments and analyzed the data.

W.Q., Y.C. and Y.W. performed the lipid peroxidation (lipid ROS) assay and malondialdehyde (MDA) content measurement and analyzed the data.

W.Q., Y.C., T.R., and W.P. performed the Nissl staining, FJB staining, the behavioral experiment, and the brain edema test and analyzed the data.

All the authors listed in the manuscript have agreed upon and reviewed the manuscript and provided feedback.

Corresponding authors

Correspondence to Chengliang Luo or Jian Zhang.

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Qu, W., Cheng, Y., Peng, W. et al. Targeting iNOS Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage via Promoting Ferroptosis of M1 Microglia and Reducing Neuroinflammation. Mol Neurobiol 59, 3124–3139 (2022). https://doi.org/10.1007/s12035-022-02788-5

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