OTULIN is a New Target for EA Treatment in Alleviating Brain Injury and The Activation of Glial Cells By Depressing NF-κB Signalling Pathway in Acute Ischaemic Stroke Rats
Abstract Objective: Ovarian tumour domain deubiquitinase with linear linkage specificity (OTULIN), a potent negative regulator of nuclear factor-κB (NF-κB) signalling pathway, exerted strong neuroprotective role following acute ischemic stroke. Electroacupuncture (EA) is an effective adjuvant treatment for reducing brain injury and neuroinflammtion through inhibiting the nuclear translocation of NF-κB p65, while the underlying mechanism remains unclear. This study investigated whether OTULIN was necessary for EA to mitigate brain injury and the activation of glial cells, following by a transient middle cerebral artery occlusion (tMCAO) model in rats.Methods: Acute ischaemic stroke model was established by performing tMCAO surgery in Sprague-Dawley (SD) rats. EA was performed once daily at “Baihui (GV 20)”, “Hegu (LI 4)”, and “Taichong (LR 3)” acupoints. EA’s effect on the spatiotemporal expression of OTULIN in the ischaemic penumbra of cerebral cortex was detected within 7 d after reperfusion. Following gene interference to silence OTULIN expression, the effects of OTULIN on EA neurological deficits, cerebral infarct volume, neuronal damage, the activation of microglia and astrocytes, the contents of tumor 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. Results: EA treatment increased endogenous OTULIN expression, with a peak at 48 h. Enhanced OTULIN was mainly located in neurons, and little OTULIN was detected in microglia as well. OTULIN silencing obviously reversed EA neuroprotection as demonstrated by worsened neurobehavioural performance, cerebral infarct volume and neuronal injury. Besides, the inhibitory effect of EA on NF-κB pathway was also attenuated by enhanced IκBα phosphorylation and NF-κB p65 nuclear translocation. Furthermore, EA partly inhibited the transformation of microglia and astrocytes to from resting states to activated states, and reduced the secretion of TNF-α, IL-1β and IL-6, but this preventive effects were reversed after silencing OTULIN expression. Conclusions: OTULIN provides a new potential therapeutic target for EA to alleviate acute ischaemic stroke induced brain injury and the activation of glial cells, which is related to depression of NF-κB signalling pathway.