Effects Of Sevoflurane Post-conditioning In Cerebral Alchemist-repercussion Injury Via TLR4/NF-κB Pathway In Rats

BackgroundIschemia-reperfusion injury is one of the main causes of brain damage and severe long-term disability.Ischemia-reperfusion stroke impairs neuronal function and leads to cell death,especially in hippocampal pyramidal neurons.It occurs when a major cerebral artery is blocked,and middle cerebral artery occlusion(MCAO)is a widely accepted animal model used to study ischemia mechanisms.Although ischemia-reperfusion stroke causes high mortality and morbidity in clinical,there is still no effective treatment for lessening the resulting neurological damage.Toll-like receptor-4(TLR4)is the first line of defense in the brain,which is mainly located on glial cells,including microglia,astrocytes and oli-godendrocytes.Toll-like receptors(TLRs)are starting to be known as the primary non antigen-specific innate defense immune mechanism.They represent a family of receptors that,upon binding of their ligands,service to recognize molecular patterns associated with pathogens.They also induce activation of several kinases and nuclear factor-kappa B(NF-κB)..NF-κB,as a central regulator of inflammatory response,also affected the signal pathway TLR4 and induced many pro-inflammatory mediators involved in innate immunity.In previous study,TLR4/NF-κB pathway was closely related to the myocardial ischemia reperfusion injury by releasing of inflammatory cytokines.In recent years,the role of sevoflurane in ischemia-reperfusion injury attracts more and more people’s attention.Studies have shown that sevoflurane post-conditioning may enhance immunity by decreasing serum tumor necrosis factor-alpha(TNF-a),interleukin-1 beta(IL-1β),nitric oxide(NO),nitric oxide synthase(NOS).This reduced TLR2 and TLR4 expression as well as inflammatory markers in human endothelial cells.Autophagy and apoptosis following different types of brain injury.Autophagy is a self-destructive process and is implicated in the pathology of many neurodegenerative diseases.Mounting evidence supports that autophagy damaged intracellular proteins and organelle,and autophagy always activating apoptosis to promote cell death.Caspase-3 and B cell lymphoma 2(Bcl-2)are important apoptotic regulators,which can determine the fate of cells.Furthermore,1 light chain 3(LC3)and Beclin-1 are participate in the regulation of neuronal autophagy.Because disturbances in inflammatory or autophagic pathways or both are observed in many serious human diseases,information on the interaction between these pathways may be important for elucidating not only the molecular basis of human disorders,but also general cellular signaling pathways.Previous studies have shown that apoptosis of brain cells can be improved by inhibiting TLR4/NF-kB signaling pathway.However,it is not yet clear whether TLR4/NF-κB signaling pathway is involved in MCAO-induced I/R brain injury.Sevoflurane is a volatile anesthetic,which is a viable anesthetic for neurosurgery.Recently,a number of studies have demonstrated that sevoflurane post-conditioning exhibits neuroprotective effects.Previous studies also have revealed that sevoflurane post-conditioning displayed anti-information effects via the TLR4-NF-κB signaling pathway.However,the mechanisms underlying the protective effects of sevoflurane are not fully understood.Therefore,in this study,we hypothesize that sevoflurane exerts neuroprotective effects by inhibiting autophagy and apoptosis during cerebral ischemia.We also sought to verify whether sevoflurane improves the brain damage of MCAO rats through TLR4-NF-κB pathway.OBJECTIVE:1.The aim of the research was to investigate the anti-inflammatory effect ofSeverance post-conditioning on cerebral alchemist-repercussion injury in rats.2.Our study aimed to investigate the effects of sevoflurane post-conditioning in a rat brain cerebral ischemia-reperfusion model and examine its possible mechanism.MethodsPart 1Thirty Sprang Daley(SD)rats were randomly divided into 3 groups:sham operation group(Sham),Alchemist/repercussion injury(I/R)group and Severance post-conditioning group(Se).Hematologist(HE)staining was used to observe the inflammatory response in the brain tissue.The levels of INF-a,IL-1β,IL-6 in serum were measured by ELISA.The mRNA and protein expression of TLR4 and NF-κ B p65 were detected by CRT and Western blot in the brain tissue.Part 2Forty rats were randomly divided into four groups:sham control group(Sham),ischemia-reperfusion group(I/R),sevoflurane group(Se),and QNZ(EVP4593)inhibitor group(QNZ).The rat brain cerebral ischemia-reperfusion model induced by middle cerebral artery.occlusion(MCAO)and the rats in different groups were given different treatments.Morris water maze test and TTC staining were used to investigate the ischemia/reperfusion injury.The nerve cell apoptosis and autophagy in the hippocampus were detected by TUNEL and transmission electron microscopy,respectively.The expression of TLR4 and NF-κB proteins were observed by immunohistochemical.In addition,the expression of autophagy and apoptotic associated proteins were assayed by western blot.ResultsPart 1The post-conditioning of Severance decreased the level of inflammatory reaction in chemise-repercussion rat cerebral infarction area and reduced the content of pro-inflammatory cytokines such as INF-a,IL-1β,IL-6 in rats with alchemist-repercussion injury.In addition,after treatment with Severance,the mRNA and protein expression of TLR4 and NF-κBp65 in TLR4/NF-κ B pathway was inhibited.Part 2Sevoflurane post-conditioning improved the learning and memory dysfunction caused by cerebral ischemia-reperfusion injury.The cerebral infarction area,nerve cell apoptosis and formation of autophagic vacuoles were reduced after sevoflurane administration.Sevoflurane post-conditioning also inhibited the expression of TLR4 and NF-κB proteins in hippocampus.Moreover,the expression of LC3Ⅰ,LC3Ⅱ,beclin-1,Bad and Caspase-3 proteins were inhibited and the expression of Bcl-2 protein was up-regulated after sevoflurane administration.Conclusion1.Severance post-conditioning can decrease the inflammatory reaction incerebral infarct area induced by cerebral alchemist-repercussion injury in rats.The overprotective effect mechanism of Severance may be related to TLR4/NF-κ B signaling pathway.2.Our study demonstrated that sevoflurane post-conditioning could protect MCAO-induced brain injury rats by inhibiting autophagy and apoptosis,and its mechanism is related to TLR4-NF-κB signaling pathway.