International immunopharmacology · 2026
USP8 attenuates ischemic stroke by inhibiting the microglial NF-κB/NLRP3 inflammatory axis via TRAF6 deubiquitination.
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Ma Lili, Cheng Zicheng, Shao Jiahui, Zhao Meiqi, Wang Jianing, Zhu Xinxia, Xu Tong, Han Zhao
Аннотация
BACKGROUND: Post-ischemic neuroinflammation is a key driver of brain injury following stroke. Dysregulation of ubiquitination plays a crucial role in this process, yet the specific deubiquitinating enzymes and their regulatory mechanisms remain unclear.
METHODS: In vivo studies were conducted using a mouse model of transient middle cerebral artery occlusion (tMCAO), and in vitro experiments were performed in BV2 microglial cells subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Gene manipulation techniques, including USP8 knockdown or overexpression and TRAF6 knockdown, were combined with molecular docking and biochemical assays to examine the expression and interactions of USP8 with key inflammatory pathway proteins, the ubiquitination level of TRAF6, and the release of pro-inflammatory cytokines. Cerebral infarct volume and neurological deficits were assessed using TTC staining and neurological function scoring, respectively.
RESULTS: Ischemic stress significantly downregulated the expression of USP8. Functional experiments demonstrated that USP8 knockdown specifically increased the K63-linked polyubiquitination of TRAF6, thereby promoting NF-κB p65 phosphorylation, nuclear translocation, and activation of the NLRP3 inflammasome, as evidenced by increased levels of NLRP3, cleaved caspase-1, and IL-1β. These changes ultimately led to the increased release of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). In vitro, TRAF6 knockdown reversed the excessive activation of the NF-κB/NLRP3 pathway induced by USP8 deficiency. In vivo, AAV9-mediated overexpression of USP8 selectively removed K63-linked polyubiquitin chains from TRAF6, suppressed downstream inflammatory signaling, and markedly attenuated cerebral injury, neurological deficits, and neuronal apoptosis in tMCAO mice.
CONCLUSIONS: These results confirm that USP8 suppresses the hyperactive microglial NF-κB/NLRP3 pathway by removing K63-linked polyubiquitin chains from TRAF6, thereby mitigating poststroke neuroinflammation.
METHODS: In vivo studies were conducted using a mouse model of transient middle cerebral artery occlusion (tMCAO), and in vitro experiments were performed in BV2 microglial cells subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Gene manipulation techniques, including USP8 knockdown or overexpression and TRAF6 knockdown, were combined with molecular docking and biochemical assays to examine the expression and interactions of USP8 with key inflammatory pathway proteins, the ubiquitination level of TRAF6, and the release of pro-inflammatory cytokines. Cerebral infarct volume and neurological deficits were assessed using TTC staining and neurological function scoring, respectively.
RESULTS: Ischemic stress significantly downregulated the expression of USP8. Functional experiments demonstrated that USP8 knockdown specifically increased the K63-linked polyubiquitination of TRAF6, thereby promoting NF-κB p65 phosphorylation, nuclear translocation, and activation of the NLRP3 inflammasome, as evidenced by increased levels of NLRP3, cleaved caspase-1, and IL-1β. These changes ultimately led to the increased release of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). In vitro, TRAF6 knockdown reversed the excessive activation of the NF-κB/NLRP3 pathway induced by USP8 deficiency. In vivo, AAV9-mediated overexpression of USP8 selectively removed K63-linked polyubiquitin chains from TRAF6, suppressed downstream inflammatory signaling, and markedly attenuated cerebral injury, neurological deficits, and neuronal apoptosis in tMCAO mice.
CONCLUSIONS: These results confirm that USP8 suppresses the hyperactive microglial NF-κB/NLRP3 pathway by removing K63-linked polyubiquitin chains from TRAF6, thereby mitigating poststroke neuroinflammation.
Предметные рубрики MeSH
AnimalsNLR Family, Pyrin Domain-Containing 3 ProteinMicrogliaUbiquitin ThiolesteraseTNF Receptor-Associated Factor 6MiceUbiquitinationMaleNF-kappa BEndopeptidasesInfarction, Middle Cerebral ArterySignal TransductionIschemic StrokeMice, Inbred C57BLCell LineDisease Models, AnimalHumansCytokinesEndosomal Sorting Complexes Required for Transport