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Differential Expression and Distinct Roles of Proteinase-Activated Receptor 2 in Microglia and Neurons in Neonatal Mouse Brain After Hypoxia-Ischemic Injury

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Abstract

Regulation of microglial activation and neuroinflammation are critical factors in the pathogenesis of ischemic brain injury. Interest in protease-activated receptor 2 (PAR2) as a pharmaceutical target for various diseases is creasing. However, it is unclear the expression and functions of PAR2 in hypoxia-ischemic (HI) brain injury. Mice with HI and cells with oxygen–glucose deprivation and reoxygenation (OGD/R) were studied. Immunoblot and qRT-PCR were used to study the differential gene expression in cultured microglia and neurons. Immunofluorescent staining was used to study the expression pattern of PAR2 in the HI brain and phagocytotic activity of microglia after OGD/R. In neonatal mice brain after HI, we found PAR2 expression was abundant in neurons, but barely in microglia from the contralateral side of cortex and hippocampus. Conversely, PAR2 expression was barely in neurons while significantly increased in activated microglia from the ipsilateral side of cortex and hippocampus. The activations of PAR2 were increased in both microglia and neuron in a cell model of OGD/R. PAR2 activation mediated the cross-talk between microglia and neurons including the following: microglial PAR2 mediated inflammatory responses that induced neuronal damage; neuronal PAR2 regulated chemokines that recruited activated microglia to damage area; microglia PAR2 controlled the phagocytosis of degenerating neurons. These data suggested differential expression and distinct roles of PAR2 in microglia and neurons after HI injury; thereby, interventions targeting PAR2 may provide insights into the inflammatory-related diseases.

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Data Availability

The data used in this study are available from the corresponding authors upon reasonable request.

Abbreviations

CatB:

Cathepsin B

CCK-8:

Cell-Counting Kit

CCL2:

C–C motif chemokine ligand 2

CLSM:

Confocal laser scanning microscope

CNS:

Central nervous system

CXCL1:

C-X-C motif chemokine ligand 1

DDW:

Double-distilled water

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

FSR:

FSLLRY-NH2

HAND:

HIV-associated neurocognitive disorders

HE:

Hematoxylin–eosin

HI:

Hypoxia–ischemia

HRP:

Horseradish peroxidase

IL-1β:

Interleukin-1β

MCM:

Microglia-conditioned medium

NF-κB:

Nuclear factor kappa B

OGD/R:

Oxygen–glucose deprivation and reoxygenation

PAR2:

Protease-activated receptor 2

PBS:

Phosphate buffer saline

PEI:

Polyethyleneimine

PFA:

Paraformaldehyde

SD:

Standard deviation

SDS:

Sodium dodecyl sulfate

TNF-α:

Tumor necrosis factor-α

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Funding

This work was supported by the National Natural Science Foundation of China (32070954 and 82001167); the Beijing Natural Science Foundation (7212066); the Beijing Institute of Technology Research Fund Program (2020CX04166); and the Zhejiang Provincial Natural Science Foundation (Q20H140040).

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Authors and Affiliations

  1. Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing, 100081, China

    Yicong Liu, Hui Li, Hong Qing & Junjun Ni

  2. Stomatology Hospital, School of Stomatology, Clinical Research Center for Oral Diseases of Zhejiang Province, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Zhejiang University School of Medicine, Hangzhou, 310006, China

    Yicong Liu

  3. Department of Prosthodontics, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, 100081, China

    Jiangqi Hu

  4. Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, 812-8582, Japan

    Zhou Wu

  5. Department of Neurology, West China Hospital, Sichuan University, Chengdu, 610041, China

    Jie Meng

  6. Department of Physiology, Nihon University School of Dentistry, Tokyo, 101-8310, Japan

    Yoshinori Hayashi

  7. Department of Pharmacology, Faculty of Pharmacy, Yasuda Women’s University, Hiroshima, 731-0153, Japan

    Hiroshi Nakanishi

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Contributions

Y.L. conducted most of the experiments, analyzed the data, and wrote the manuscript. H.L. and J.H. conducted most of the experiments and analyzed the data. Z.W., J.M., Y.H., and H.N. provided valuable advice and unpublished reagents/analytic tools. H.Q. designed part of the study. J.N. designed the whole study and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Hong Qing or Junjun Ni.

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Yicong Liu, Hui Li, and Jiangqi Hu contributed equally to this work.

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Liu, Y., Li, H., Hu, J. et al. Differential Expression and Distinct Roles of Proteinase-Activated Receptor 2 in Microglia and Neurons in Neonatal Mouse Brain After Hypoxia-Ischemic Injury. Mol Neurobiol 59, 717–730 (2022). https://doi.org/10.1007/s12035-021-02594-5

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