| The prevalence,mortality and disability rate of ischemic stroke remain high,which has become one of the focuses of the global medical community.At present,its treatment is still vascular recanalization,and the accompanying cerebral ischemia-reperfusion injury(C I/R I)is inevitable.The pathophysiological mechanism of C I/R I is complex,involving oxidative stress,calcium ion overload,free radical damage,and toxic effects of excitatory amino acids,among which inflammatory reaction plays an important role.Inflammatory reaction is mediated by inflammatory factors,which are produced in large quantities under the activation of transcription factor NF-κB.Related research shows that the activation of NF-κB is involved in the occurrence of inflammatory reaction in C I/R I.In C I/R I rats,nuclear translocation of NF-κB occurs,which mediates the production of inflammatory factors and promotes the inflammatory cascade reaction.Dexmedetomidine,DEX)can exert neuroprotective effiect by regulating neuroinflammation.Research evidence shows that DEX inhibits the expression of inflammatory factors and protects nerves in C I/R I rats.However,the specific mechanism of DEX inhibiting inflammation in C I/R I rats is not clear.Dendritic cell-associated lectin-1(Dectin-1)activates downstream Splenic tyrosine kinase(Syk)by recognizing various damage-related molecular patterns(DAMPS)including C I/R I,thus activating transcription factor NF-κB and mediating downstream inflammatory cascade.Our previous results showed that Dectin-1/Syk/NF-κB signaling pathway was significantly up-regulated in C I/R I rats.At the same time,it is verified that Dectin-1,as an upstream regulatory protein,can regulate NF-vB through Syk.However,the specific regulatory mechanism of Dectin-1/Syk on the activation of NF-κB downstream is still unclear.Therefore,by establishing the focal ischemia-reperfusion(I/R)model of middle cerebral artery occlusion(MCAO),this study explored whether dexmedetomidine preconditioning has protective effect on cerebral ischemia-reperfusion injury,and whether it can inhibit the expression of Dectin-1/syk/NF-κB signal pathway and the nuclear translocation of NF-κB,thus alleviating the inflammatory reaction mechanism.Methods:In this study,54 healthy adult male SD rats weighing 250±15g were randomly divided into sham operation(Sham)group,ischemia reperfusion(I/R)group and dexmedetomidine preconditioning+ischemia reperfusion(DEX+I/R)group,with 18 rats in each group.I/R group underwent middle cerebral artery embolization(MCAO).DEX+I/R group was given intraperitoneal injection of low dose DEX(30 ug/kg)before MCAO operation in rats.Sham group only underwent anatomical separation without arterial embolization.The grasping power of forelimbs and the degree of nerve function injury(mNSS score and Longa score)were measured before and after operation.Samples were taken 24 hours after ischemia reperfusion,and the water content of brain tissue and the volume ratio of cerebral infarction were measured.Western blot was used to detect the protein expressions of Dectin-1,Syk and NF-κB in brain tissue at 24h,48h and 72h after ischemia-reperfusion,as well as the expressions of NF-κB p65,NF-κB p50 and their phosphorylated proteins in brain nuclei.RT-qPCR was used to detect the mRNA expressions of Dectin-1,Syk,NF-κB and TNF-α.The content of TNF-α in brain tissue was detected by Elisa.The immunofluorescence co-expression of NF-κB and microglia marker Iba-1 was observed and counted by immunofluorescence technique.Results:1.Comparison of the forelimb grip of three groups of rats Compared with Sham group,the forelimb grip of rats in I/R group and DEX+I/R group decreased significantly at 24 h,48 h and 72 h after ischemia and reperfusion(P<0.05).Compared with I/R group,DEX+I/R group significantly improved the grip of forelimbs(P<0.05),and it was time-dependent(P<0.05).2.Compared with I/R group,the neurological deficit scores of DEX+ I/R group decreased significantly at 24 h,48 h and 72 h after ischemia/reperfusion(P<0.05).Compared with 24 hours after ischemia reperfusion,the neurological dysfunction score of DEX+I/R group decreased significantly at 72 hours after ischemia reperfusion(P<0.05).3.Compared with sham group,the water content and infarct volume of brain tissue in I/R group and DEX+I/R group increased significantly(P<0.05).Compared with I/R group,the water content and infarct volume of brain tissue in DEX+I/R group decreased(P<0.05).4.Western Blot was used to compare the expression of Dectin-1/Syk/NF-κB p65 protein in rat brain.Compared with sham group,the expression levels of Dectin-1,Syk and NF-κB p65 in brain tissue of I/R group were all increased(P<0.05).Compared with I/R group,the protein expression levels of Dectin-1,Syk and NF-κB p65 in brain tissue of DEX+I/R group decreased(P<0.05).In DEX+I/R group,the protein expression levels of Dectin-1,Syk and NF-κB p65 decreased with time(P<0.05).5.Comparison of expression of NF-κB p65,NF-κB p50 and phosphorylated protein isolated from the nucleus and cytoplasm of rat brain tissue.Western Blot method was used.Compared with sham group,the expression levels of NF-κB p65,NF-κB p50 and their phosphorylated proteins in brain nuclei of I/R group and DEX+I/R group increased,while the expression levels of NF-κB p65,NF-κB p50 and their phosphorylated proteins in cytoplasm decreased.Compared with I/R group,the expression levels of nuclear NF-κB p65,NF-κB p50 and their phosphorylated proteins in DEX+I/R group decreased,while the expression levels of NF-κB p65,NF-κB p50 and their phosphorylated proteins in cytoplasm increased(P<0.05).6.Detect the expression of Dectin-1/Syk/NF-κB p65 and TNF-α mRNA in rat brain tissue by RT-qPCR.Compared with sham group,the mRNA expressions of Dectin-1,Syk,NF-κB p65 and TNF-α in brain tissue of I/R group and DEX+I/R group increased(P<0.05).Compared with I/R group,the expressions of Dectin-I,Syk,NF-κB p65 and TNF-α mRNA in DEX+I/R group decreased(P<0.05).In DEX+I/R group,the expressions of Dectin-1,Syk,NF-κB p65 and TNF-α mRNA decreased significantly with the prolongation of reperfusion time(P<0.05).7.Comparison of TNF-α and IL-6 contents in rat brain tissue.Detection by ELISA.Compared with sham group,the contents of TNF-α and IL-6 in brain tissue of I/R group and DEX+I/R group increased(P<0.05).Compared with I/R group,the contents of TNF-α and IL-6 in DEX+I/R group decreased(P<0.05).The contents of TNF-α and IL-6 in DEX+I/R group decreased significantly with the prolongation of reperfusion time(P<0.05).8.The expression of NF-κB p65 与 Iba-1 in rat brain was compared by immunofluorescence detection.Detection of the number of cells co-expressing NF-κB p65与Iba-1 in cerebral cortex can reflect the activation degree of inflammatory reaction to some extent.Compared with sham group,the number of cells co-expressing NF-κB p65 与 Iba-1 in I/R group,DEX+I/R group increased significantly(P<0.05).Compared with I/R group,the number of cells co-expressing NF-κB p65 与 Iba-1 in DEX+I/R group decreased significantly(P<0.05).Conclusions:1.Dexmedetomidine intraperitoneal injection pretreatment can reduce the volume of cerebral infarction and the degree of cerebral edema in rats,reduce the release of inflammatory factors,improve the neurological deficit score and forelimb grip,and have a protective effect on cerebral ischemia-reperfusion injury.2.The mechanism of dexmedetomidine in reducing inflammatoiy response may be related to the inhibition of Dectin-1/syk/NF-κB signaling pathway expression and NF-κB nuclear translocation. |
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