BRCC3-mediated Ubiquitination Of NLRP6 Affects Its Inflammasome Activation And Regulates Inflammatory Response In Cerebral Ischemia/Reperfusion Injury

Objective: Ischemic stroke is associated with high disability rate and fatality rate,which seriously endangers human life and health.Cerebral ischemia/reperfusion injury involves complex pathophysiological mechanisms in which inflammatory cascade plays a key role.As part of innate immunity,NLR(Nod-like receptor)family is involved in the formation of inflammasome.As a new member of NLR family,NLRP6(Nod-like receptor protein Six)has been confirmed to be involved in the formation of inflammasome in our previous studies.Therefore,it is particularly important to explore the mechanism of regulating the activation of NLRP6 inflammasome.As a deubiquitination enzyme,BRCC3(human homologous BRCC36)can specifically recognize and hydrolyse the polyubiquitin chain formed by the K63 site connection between ubiquitin,which plays an important role in angiogenesis,tumorigenesis,inflammation and other processes.NLRP6 inflammasome has been shown to influence its activation through ubiquitination modifications in intestinal inflammation.Therefore,this study focused on the mechanism by which BRCC3 regulates NLRP6 inflammasome activation,which may be a potential therapeutic target for cerebral ischemia/reperfusion injury.Methods:(1)To determine the effects of BRCC3 on NLRP6 inflammosome activation,downstream inflammatory factors and pyroptosisafter cerebral ischemia/reperfusion injury,The in vivo model of cerebral ischemia/reperfusion injury(C57BL/6J mouse middle cerebral artery embolization for 1h/ reperfusion for 24h)and in vitro model of cerebral ischemia/reperfusion injury(HT22 cells oxygen-glucose deprivation for 4h/ reoxygenation for 24h)were prepared by using healthy male C57BL/6J mice and HT22 cells,respectively.On this basis,small interfering RNA(si RNA)was used to knock down BRCC3 protein expression in the cerebral cortex and HT22 cells by lateral ventricular injection and transfection,respectively.(2)To explore the relationship between BRCC3 and NLRP6,neurological deficits,NLRP6 inflammosome and downstream inflammatory factors,and pyroptosis indicators were detected after BRCC3 intervention.(3)In order to explore the effect of BRCC3 on inflammosome activation and pyroptosis through NLRP6,we used NLRP6 si RNA for the recovery experiment based on the overexpression of BRCC3.(4)To explore the mechanism by which BRCC3 regulates NLRP6,we constructed BRCC3,NLRP6,Ub and ASC plasmids as well as BRCC3-N and BRCC3-C truncates.Results:(1)Knockdown of BRCC3 not only significantly reduced the volume of cerebellar infarction and neurological deficit score,but also improved the HE and Nissl staining results of cerebral cortex.It also significantly inhibited activation of NLRP6 inflammasome and protein expression of cleaved-caspase-1 and cleaved-IL-1β,as well as pyroptosismarker GSDMD-N.At the same time,in neurons with a major distribution of BRCC3,activation of NLRP6 inflammosome and expression of downstream inflammatory cytokines(cleaved-caspase-1 and cleaved-IL-1β)and pyroptosis indicator GSDMD-N were reduced after transfection of BRCC3 si RNA.(2)On the basis of overexpression of BRCC3,NLRP6 was knocked down,and the downstream inflammatory factors and pyroptosis index GSDMD-N recovered.(3)In neuron cell lines,BRCC3 and NLRP6 showed co-localization.(4)Exogenous BRCC3 and NLRP6 interacted,and both the br CC3-N and BRCC3-C ends of truncated body interacted with NLRP6.(5)BRCC3 can significantly reduce the ubiquitination level of NLRP6.(6)BRCC3 can promote the combination of NLRP6 and ASC.Conclusion: BRCC3 can promote the activation of NLRP6 inflammosome and induce inflammatory response and pyroptosis in vitro and in vivo.BRCC3 may mediate activation of NLRP6 inflammasome through ubiquitination.