OTUD1 Inhibits Inflammation In Cerebral Ischemic Injury By Regulating Ubiquitination Of RIP2

Stroke,the second leading cause of death worldwide,is divided into ischemic stroke and hemorrhagic stroke.Ischemic stroke accounted for most cases of stroke,no other effective method is available for ischemic stroke except the thrombolytic therapy and mechanical thrombectomy in current.Therefore,it is pivotal to deeply explore the pathological mechanism of cerebral ischemia.Inflammation mediated by innate immune plays an important role in the pathological mechanism of ischemic brain injury.Receptor-interacting protein kinase 2(RIP2)is a key molecular in nucleotide-binding oligomerization domain-containing protein(NOD)1/2 signal transduction,and plays a key role in the regulation of inflammation.We previously reported that NOD 2 was involved in the inflammatory responses to cerebral ischaemic insult.It suggests that regulation of RIP2 activity can be a potential treatment of cerebral ischemia.Ubiquitination is an important post-translational modification of RIP2.OTUD1,a member of the OTU deubiquitinase family,is an interferon-stimulating gene widely involved in innate immunity.However,whether OTUD1 supresses RIP2 ubiquitination and modulates the immune inflammatory response after cerebral ischemia remain unclear.In this study,and middle cerebral artery occlusion(MCAO)in mice and oxygen and glucose deprivation(OGD)models in cells were used to explore the role and mechanism of OTUD1 and RIP2 in cerebral ischemia and the regulation of OTUD1 on RIP2.Firstly,we found that RIP2 protein levels were significantly increased in microglia and astrocytes after cerebral ischemia.Administration of RIP2 inhibitor significantly reduced the inflammation and improved cerebral ischemic injury.These results suggested that RIP2-mediated inflammatory response plays an important role in cerebral ischemia.Meanwhile,OTUD1 protein level was also upregulated after cerebral ischemia both in vivo and vitro.Furthermore,we proved that OTUD1 interacted with RIP2 and removed K63 ubiquitin from RIP2.We further observed OTUD1 knockout aggravated ischemic brain injury by increasing RIP2 ubiquitination level and enhancing inflammation.In conclusion,our study proved that OTUD1 represses inflammatory response and protects ischemic brain injury by removing K63 ubiquitination of RIP2.This may provide a potential drug target for the treatment of cerebral ischemia.