OTULIN is a new target of EA treatment in the alleviation of brain injury and glial cell activation via suppression of the NF-\u03baB signalling pathway in acute ischaemic stroke rats

Hongbei Xu,You Wang,Yong Luo

doi:10.1186/s10020-021-00297-0

Abstract

ObjectiveOvarian tumour domain deubiquitinase with linear linkage specificity (OTULIN) is a potent negative regulator of the nuclear factor-κB (NF-κB) signalling pathway, and it plays a strong neuroprotective role following acute ischemic stroke. Electroacupuncture (EA) is an effective adjuvant treatment for reducing brain injury and neuroinflammation via the inhibition of NF-κB p65 nuclear translocation, but the underlying mechanism is not clear. The present study investigated whether OTULIN was necessary for EA to mitigate brain injury and glial cell activation in a transient middle cerebral artery occlusion (tMCAO) model in rats.MethodsAn acute ischaemic stroke model was established via tMCAO surgery in Sprague–Dawley (SD) rats. EA was performed once daily at “Baihui (GV 20)”, “Hegu (LI 4)”, and “Taichong (LR 3)” acupoints. The effect of EA on the spatiotemporal expression of OTULIN in the ischaemic penumbra of the cerebral cortex was detected within 7 days after reperfusion. The effects of OTULIN gene silencing on EA neurological deficits, cerebral infarct volume, neuronal damage, the activation of microglia and astrocytes, the contents of tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), and the expression of p-IκBa, IκBa and nucleus/cytoplasm NF-κB p65 protein were assessed.ResultsEA treatment increased endogenous OTULIN expression, which peaked at 48 h. Enhanced OTULIN was primarily located in neurons, but a small amount of OTULIN was detected in microglia. OTULIN silencing obviously reversed EA neuroprotection, which was demonstrated by worsened neurobehavioural performance, cerebral infarct volume and neuronal injury. The inhibitory effect of EA on the NF-κB pathway was also attenuated by enhanced IκBα phosphorylation and NF-κB p65 nuclear translocation. EA partially inhibited the transformation of microglia and astrocytes from resting states to activated states and reduced the secretion of TNF-α, IL-1β and IL-6. However, these preventive effects were reversed after the silencing of OTULIN expression.ConclusionsOTULIN provides a new potential therapeutic target for EA to alleviate acute ischaemic stroke-induced brain injury and the activation of glial cells, which are related to suppression of the NF-κB signalling pathway.

Highlights

Ischaemic stroke-induced cell injury initiates the release of danger signals that activate the immune system in the central nervous system (CNS) (Corps et al 2015)
OTULIN provides a new potential therapeutic target for EA to alleviate acute ischaemic stroke-induced brain injury and the activation of glial cells, which are related to suppression of the nuclear factor-κB (NF-κB) signalling pathway
EA enhanced OTULIN expression following focal cerebral ischaemia/reperfusion injury To detect the effects of EA on the time course of OTULIN expression, OTULIN mRNA and protein in the ischaemic penumbra of the cerebral cortex (Fig. 2a) were measured from 2 h to 7 days after reperfusion using Real-time quantitative polymerase chain reaction (RTqPCR) and Western blotting, respectively

Summary

Introduction

Ischaemic stroke-induced cell injury initiates the release of danger signals that activate the immune system in the central nervous system (CNS) (Corps et al 2015). The subsequent sterile inflammatory response primarily involves the innate immune system, with the activation of resident immune cells of the central nervous system (CNS) and a rapid infiltration of peripheral immune cells (Li et al 2020; Lin et al 2020; Shan et al 2020; Wang et al 2020). Following activation by upstream kinases after ischaemia onset, IκBα is degraded and phosphorylated, which leads to the nuclear translocation of the p65 and binding to NF-κB-responsive genes (Mattson and Camandola 2001). Inhibition of NF-κB activation is an effective strategy for reducing acute ischaemic stroke-induced brain injury and inflammation (Li et al 2019; Liang et al 2019; Tan et al 2019)

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Results

Conclusion