Role Of NF-κB-mediated Pyroptosis In Neonatal Sevoflurane-induced Neuroinflammation And Neurocognitive Dysfunction

Background: Sevoflurane is the most common used inhalational general anesthetics（Gas）for pediatric patients.Increasing evidence has demonstrated that sevoflurane causes neuroinflammation and neuronal death in the developing brain,leading to neurodevelopmental cognitive deficits and psychiatric disorders later in life.Pyroptosis is a novel inflammatory form of programmed cell death,triggered through the canonical caspase-1 pathway and non-canonical caspase-4/5/11 pathway.Both of the canonical and non-canonical pathways can be activated by the NF-κB signaling.Pyroptosis has been implicated in the occurrence and development of many neurological diseases,including neurodegenerative diseases,epilepsy,stroke and traumatic intracerebral hemorrhage.However,its role in sevoflurane-induced neuroinflammation and cognitive deficits is unknown.This study established the sevoflurane GA model both in vivo and in vitro and aimed to investigate the effects of NF-κB inhibitor BAY 11-7082 on NF-κB pathway,neuroinflammation,pyroptosis,synaptic formation and cognitive function in neonatal rats under sevoflurane anesthesia.Methods: Primary hippocampal neuronal cultures on days in vitro 9 and Sprague-Dawley rat pups at postnatal day 6 were randomly assigned to one of following four treatment protocols: control + vehicle（Con group）,control + BAY 11-7082（Con+BAY group）,sevoflurane + vehicle（Sev group）,and sevoflurane + BAY11-7082（Sev+BAY group）.According to grouping,the neurons or rat pups received3% sevoflurane or control gas exposure for 2?h daily for three consecutive days at37?°C.BAY 11-7082 or equal volume of vehicle was given 30?min before GA exposure according to group assignment.The protein levels of NF-κB-p65,IκBα,NLRP3,caspase-1,caspase-11,GSDMD,IL-1β,IL-18,Synapsin-1 and PSD-9 were determined by Western Blotting and ELISA.m RNA levels of NLRP3,caspase-1 and caspase-11 was determined by RT-PCR.The intensity of GSDMDimmunofluorescence and the neuronal morphology and network were determined by immunocytochemistry（ICC）staining.t Te neuronal viability was determined by cell viability assay.The number of GSDMD-possitive cells in CA1 and DG regions was calculated by immunohistochemical（IHC）staining.Cognitive function was evaluated by open field test,Morris water maze test and fear conditioning test at PND 40,50 and 60,repectively.Results: When compared the Sev+BAY group with the Sev group,the levels of Pro-GSDMD,GSDMD-N,IL-1β and IL-18 and the intensity of GSDMD-immunofluorescence were significantly reduced while the neuronal viability was increased in vitro study.Moreover,the neuronal morphology and network were largely improved.In addition to the in vitro study,the in vivo study showed that the freezing time of context test was significantly increased,the escape latency in training test was shorterned and the target quadrant time and crossing platform times were increased.Moreover,the protein levels of cytoplasm NF-κB-p65,IκBα,Synapsin-1and PSD-95 were significantly increased while the protein levels of nuclear NF-κBp65,p-IκBα,NLRP3,Pro-caspase-1,Cleaved caspase-1,Pro-caspase-11,Cleaved caspase-11,Pro-GSDMD,GSDMD-N,IL-1β and IL-18 and m RNA levels of NLRP3,caspase-1 an caspase-11 m RNA were significantly reduced.Conclusion: NF-κBmediated pyroptosis may be involved in neuroinflammation,cell death and cognitive impairment after repeated neonatal sevoflurane.When inhibiting the activation of NF-κB by Bay 11-7082,the pyroptosis mediated by canonical and non-canonical inflammatory caspases was significantly alleviated.Our study provides a promising strategy for the treatment of cognitive deficits in the developing brain involving pyroptosis.