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P2X7 Participates in Intracerebral Hemorrhage-Induced Secondary Brain Injury in Rats via MAPKs Signaling Pathways

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Abstract

This study aimed to study the role of P2X7 in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) and the underlying mechanisms. An autologous blood injection was used to induce ICH model in Sprague–Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin to mimic ICH in vitro. siRNA interference and over-expression of P2X7, agonists and antagonists of P2X7, p38 MAPK and ERK were exploited. The protein levels were assessed using Western blotting and immunofluorescence staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and Fluoro-Jade B were conducted to detect apoptotic and degenerating neurons. The protein levels of P2X7, phosphorylated p38, ERK, active caspase-3 and NF-κB were significantly increased by ICH, which could be further increased by BzATP (P2X7 agonist) and reduced by BBG (P2X7 antagonist). And BzATP demonstrated a significant increase in cell death ratio and brain water content, while BBG led to a reverse results. In addition, Over- P2X7 increased the levels of P2X7, phosphorylated p38, ERK, active caspase-3 and NF-κB, and aggravated cell apoptosis, while si P2X7 resulted in opposite effects. Finally, the protein levels of phosphorylated P38 and active caspase 3 were decreased by BzATP plus Hydrochloride (p38 MAPK antagonist) and increased vy BBG plus Asiatic acid (p38 MAPK agonist), while the protein levels of phosphorylated ERK and NF-κB were decreased with BzATP plus Nimbolide (ERK antagonist) and increased with BBG plus Saikosaponin C (ERK agonist). This study demonstrates that inhibition of P2X7 could prevent ICH-induced SBI via MAPKs signaling pathway.

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Abbreviations

ICH:

Intracerebral hemorrhage

SBI:

Secondary brain injury

CNS:

Central nervous system

ATP:

Adenosine triphosphate

OxyHb:

Oxyhemoglobin

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick end labeling staining

FJB:

Fluoro-Jade B

MAPKs:

Mitogen-activated protein kinases

ERK:

Extracellular signal-regulated kinases

JNK:

Jun amino-terminal kinases

SD:

Sprague–Dawley

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Funding

This work was supported by Jiangsu Provincial Key Medical Center, Medical Innovation Team, Medical Talent, and Medical Youth Talent, Suzhou Key Medical Center (Szzx201501), Grants from the National Natural Science Foundation of China (No. 81571121), Scientific Department of Jiangsu Province (No. BL2014045), Suzhou Government (No. SZS201413, SYS201608, and LCZX201601), Jiangsu Province (Nos. BRA2016529 and 16KJB320008).

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    Authors and Affiliations

    1. Department of Neurosurgery and Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China

      Zunjia Wen, Binbin Mei, Haiying Li, Yang Dou, Xiaodi Tian, Meifen Shen & Gang Chen

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    1. Zunjia Wen
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    2. Binbin Mei
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    Correspondence to Meifen Shen or Gang Chen.

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    The authors declare that they have no conflict of interest.

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    All animal experiments are strictly in accordance with the guideline of Soochow University institutional Animal Care and Use Committee.

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    Zunjia Wen and Binbin Mei have contributed equally to this work.

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    Wen, Z., Mei, B., Li, H. et al. P2X7 Participates in Intracerebral Hemorrhage-Induced Secondary Brain Injury in Rats via MAPKs Signaling Pathways. Neurochem Res 42, 2372–2383 (2017). https://doi.org/10.1007/s11064-017-2257-1

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