Lentivirus-mediated overexpression of OTULIN ameliorates microglia activation and neuroinflammation by depressing the activation of the NF-κB signaling pathway in cerebral ischemia/reperfusion rats

Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-κB) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-κΒ signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models.

OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.

Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting.

In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-α, IL-1β, and IL-6 by suppressing the NF-κB pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-α, IL-1β, and IL-6 via depressing the NF-κB pathway in both PM and N9 cells. Conclusions: OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.