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Updated: Jul 29, 2025
Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction
Published on: May 7, 2015
DDX3X Deficiency Attenuates Pyroptosis Induced by Oxygen-glucose Deprivation/Reoxygenation in N2a Cells
Yong Liu1,2, Yanlin Gui3, Hao Tang4
1Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China.
Background:
NOD like 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/R induced NLRP3 inflammasome activation and pyroptosis, which implies that DDX3X may become a potential therapeutic target for cerebral I/R injury.
Insights
DDX3X deficiency reduces cell death and pyroptosis in brain injury models. This finding suggests DDX3X as a potential therapeutic target for cerebral ischemia/reperfusion injury.
Area of Science:
- Neuroscience
- Cellular Biology
- Biochemistry
Background:
- NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis is linked to cerebral ischemia/reperfusion (I/R) injury.
- DDX3X, an ATPase/RNA helicase, is known to promote NLRP3 inflammasome activation.
Purpose of the Study:
- To investigate if DDX3X deficiency can mitigate NLRP3 inflammasome-mediated pyroptosis in an in vitro model of cerebral I/R injury.
Main Methods:
- Mouse neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) with DDX3X knockdown.
- Assays included CCK-8, LDH cytotoxicity, double immunofluorescence, transmission electron microscopy (TEM), and Western blotting to assess cell viability, pyroptosis, and protein expression.
Main Results:
- OGD/R treatment increased cell death, pyroptosis, and expression of pyroptosis-related proteins (NLRP3, cleaved-Caspase1, GSDMD-N).
- DDX3X knockdown significantly improved cell viability, reduced pyroptosis markers, and inhibited pyroptosis-associated membrane pore formation and GSDMD translocation.
Conclusions:
- DDX3X knockdown effectively attenuates OGD/R-induced NLRP3 inflammasome activation and pyroptosis in N2a cells.
- DDX3X emerges as a potential therapeutic target for mitigating cerebral I/R injury.
