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

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

Liu Y ，Gui Y ，Tang H ，Yu J ，Yuan Z ，Liu L ，Ma X ，Li C ... -  《-》

MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation.

Cerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. An in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1β, IL-18 and LDH. MiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1β, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. Upregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.

Wang QS ，Luo XY ，Fu H ，Luo Q ，Wang MQ ，Zou DY ... -  《-》

Astragaloside IV Alleviates Cerebral Ischemia-Reperfusion Injury through NLRP3 Inflammasome-Mediated Pyroptosis Inhibition via Activating Nrf2.

As one of the fundamental components of Astragalus membranaceus, astragaloside IV (AST IV) exerts protective effects against cerebral ischemia-reperfusion injury (CIRI). Nevertheless, the underlying mechanisms have not yet been conclusively elucidated. To do so, here, we report on the regulatory effects of Nrf2 on NLRP3 inflammasome-mediated pyroptosis. CIRI was induced by middle cerebral artery occlusion-reperfusion (MCAO/R) in Sprague Dawley rats and modeled by oxygen and glucose deprivation/reoxygenation (OGD/R) in SH-SY5Y cells. Cerebral infarct volume and neurological deficit score served as indices to evaluate MCAO/R injury. In addition, the CCK-8 assay was used to assess cell viability, the LDH leakage rate was used as a quantitative index, and propidium iodide (PI) staining was used to visualize cells after OGD/R injury. The NLRP3/Caspase-1/GSDMD pathway, which produces the pores in the cell membrane that are central to the pyroptosis process, was assessed to investigate pyroptosis. Nrf2 activation was assessed by detecting Nrf2 protein levels and immunofluorescence analysis. We show that after MCAO/R of rats, the infarct volume and neurological deficit score of rats were strongly increased, and after OGD/R of cell cultures, cell viability was strongly decreased, and the LDH leakage rate and the proportion of PI-positive cells were strongly increased. In turn, MCAO/R and OGD/R enhanced the protein levels of NLRP3, Caspase-1, IL-1β, GSDMD, and GSDMD-N. Moreover, Nrf2 protein levels increased, and Nrf2 translocation was promoted after CIRI. Interestingly, AST IV (i) reduced the cerebral infarct volume and the neurological deficit score in vivo and (ii) increased the cell viability and reduced the LDH leakage rate and the proportion of PI-positive cells in vitro. AST IV reduced the protein levels of NLRP3, Caspase-1, IL-1β, GSDMD, and GSDMD-N, inhibiting NLRP3 inflammasome-mediated pyroptosis. AST IV also increased the protein levels of Nrf2 and promoted the transfer of Nrf2 to the nucleus, accelerating Nrf2 activation. Particularly revealing was that the Nrf2 inhibitor ML385 partly blocked the above effects of AST IV. Taken together, these results demonstrate that AST IV alleviates CIRI through inhibiting NLRP3 inflammasome-mediated pyroptosis via activating Nrf2.

Xiao L ，Dai Z ，Tang W ，Liu C ，Tang B ... -  《-》

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes.

Jiang T ，Li Y 《-》

Exosomes derived from hypoxic bone marrow mesenchymal stem cells rescue OGD-induced injury in neural cells by suppressing NLRP3 inflammasome-mediated pyroptosis.

Exosomes have been shown to have therapeutic potential for cerebral ischemic diseases. In this study, we investigated the neuroprotective effects of normoxic and hypoxic bone marrow mesenchymal stromal cells-derived exosomes (N-BM-MSCs-Exo and H-BM-MSCs-Exo, respectively) on oxygen-glucose deprivation (OGD) injury in mouse neuroblastoma N2a cells and rat primary cortical neurons. The proportions of dead cells in N2a and primary cortical neurons after OGD injury were significantly increased, and N-BM-MSCs-Exo (40 μg/ml) could reduce the ratios, noteworthily, the protective effects of H-BM-MSCs-Exo (40 μg/ml) were more potent. Western blotting analysis indicated that N-BM-MSCs-Exo decreased the expression of NLRP3, ASC, Caspase-1, GSDMD-N, cleaved IL-1β and IL-18 in N2a cells. However, H-BM-MSCs-Exo (40 μg/ml) was more powerful in inhibiting the expression of these proteins in comparison with N-BM-MSCs-Exo. Similar results were obtained in primary cortical neurons. Immunofluorescence assays showed that after N-BM-MSCs-Exo and H-BM-MSCs-Exo treatment, the co-localization of NLRP3, ASC, Caspase-1 and the GSDMD translocation from the nucleus to the cytoplasm and membrane after OGD injury were reduced in N2a cells and primary cortical neurons, and H-BM-MSCs-Exo had a more obvious effect. In addition, N-BM-MSCs-Exo and H-BM-MSCs-Exo significantly reduced lactate dehydrogenase (LDH) release and the IL-18 levels in cell culture medium in N2a cells and primary cortical neurons. Once again H-BM-MSCs-Exo induced these effects more potently than N-BM-MSCs-Exo. All of these results demonstrated that N-BM-MSCs-Exo and H-BM-MSCs-Exo have significant neuroprotective effects against NLRP3 inflammasome-mediated pyroptosis. H-BM-MSCs-Exo has a more pronounced protective effect than N-BM-MSCs-Exo and may be used to ameliorate the progression of cerebral ischemia and hypoxia injury in patients.

Kang X ，Jiang L ，Chen X ，Wang X ，Gu S ，Wang J ，Zhu Y ，Xie X ，Xiao H ，Zhang J ... -  《-》