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Current Neurovascular Research

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ISSN (Print): 1567-2026
ISSN (Online): 1875-5739

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Research Article

DDX3X Deficiency Attenuates Pyroptosis Induced by Oxygen-glucose Deprivation/Reoxygenation in N2a Cells

Author(s): Yong Liu, Yanlin Gui, Hao Tang, Jianping Yu, Zhengzhou Yuan, Lei Liu, Xuntai Ma and Changqing Li*

Volume 20, Issue 2, 2023

Published on: 14 June, 2023

Page: [197 - 206] Pages: 10

DOI: 10.2174/1567202620666230522155944

Price: $65

Abstract

Background: NODlike receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis is strongly related to cerebral ischemia/reperfusion (I/R) injury. DDX3X, the DEAD-box family's ATPase/RNA helicase, promotes NLRP3 inflammasome activation. However, whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis induced by cerebral I/R injury.

Objectives: This study investigated whether DDX3X deficiency attenuates NLRP3 inflammasomemediated pyroptosis in N2a cells after oxygen-glucose deprivation/ reoxygenation (OGD/R) treatment.

Methods: In vitro model of cerebral I/R injury, mouse neuro2a (N2a) cells subjected to OGD/R were treated with the knockdown of DDX3X. Cell counting kit-8 (CCK-8) assay and Lactate dehydrogenase (LDH) cytotoxicity assay were conducted to measure cell viability and membrane permeability. Double immunofluorescence was performed to determine the pyroptotic cells. Transmission electron microscopy (TEM) was used to observe morphological changes of pyroptosis. Pyroptosis-associated proteins were analyzed by Western blotting.

Results: The OGD/R treatment reduced cell viability, increased pyroptotic cells and released LDH compared to the control group. TEM showed membrane pore formation of pyroptosis. Immunofluorescence showed that GSDMD was translocated from the cytoplasm to the membrane after OGD/R treatment. Western blotting showed that the expression of DDX3X, and pyroptosis-related proteins (NLRP3, cleaved-Caspase1, and GSDMD-N) were increased after OGD/R treatment. Nevertheless, DDX3X knockdown markedly improved cell viability and reduced LDH release, expression of pyroptosis-related proteins, and N2a cells pyroptosis. DDX3X knockdown significantly inhibited membrane pore formation and GSDMD translocation from cytoplasm to membrane.

Conclusion: This research demonstrates for the first time that DDX3X knockdown attenuates OGD/Rinduced NLRP3 inflammasome activation and pyroptosis, which implies that DDX3X may become a potential therapeutic target for cerebral I/R injury.

Keywords: DDX3X, NLRP3, OGD/R, pyroptosis, cell viability, immunofluorescence.

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