Syringin Exerts Neuroprotective Effects In A Rat Model Of Cerebral Ischemia Through The FOXO3a/NF-κB Pathway

Background:The inflammatory cascade response plays an important role in cerebral ischemia-reperfusion injury.The transcription factor NF-κ B(Nuclear factor-κB)is the hub of the inflammatory response and intracellular signal transduction pathway,which is involved in the process of cerebral ischemiareperfusion injury.NF-κB is activated in neuronal cells,cerebrovascular endothelial cells and glial cells during cerebral ischemia,and the activated NF-κB can contribute to the upregulation of cytokine expression such as IL-1,IL-6 and TNF-α,while high levels of IL-1β,IL-6 and TNF-αexacerbate the organ damage after ischemia-reperfusion.Therefore,finding a drug that inhibits NF-κ B is the key to reduce cerebral ischemiareperfusion injury.Syringin(Syringin/Eleutheroside B,SYR),also known as syringoside B,extracted from Syringin,is a lignan-like compound with strong immunomodulatory and anti-inflammatory effects of its own,and has been closely related to NF-κB in previous reports,while its counterpart can inhibit protein phosphorylation of FOXO3a to affect cell injury and death.In a model of lipopolysaccharide-induced hepatic ischemia-reperfusion injury,eugenolide could inhibit lipopolysaccharide-induced NF-κB activation and affect downstream inflammatory cytokines.However,studies on the role of Syringin in cerebral ischemia injury have not been reported.Objective:To investigate the effect of syringin on cerebral ischemia-reperfusion injury in rats,and to further clarify whether it exerts anti-inflammatory effects through the NF-κB/FOXO3a pathway.Methods:(1)The middle cerebral artery embolism(MCAO)model was established in SD rats using LONGA’s vascular wire embolization method,and the experimental animals were randomly divided into 6 groups: sham group,MCAO group,MCAO+DMSO group,5mg/kg,10mg/kg,20mg/kg SYR+MCAO group.Both the drug-treated and MCAO groups used a wire plug to insert the left middle cerebral artery for 1h,and the drug-treated group injected different concentrations of drugs into the SD rats intraperitoneally after perfusion.After 24 h,intact brain tissues were collected from each group for subsequent TTC staining experiments to screen for the best drug concentration.The subsequent experimental groups were divided into sham group,MCAO group,MCAO + DMSO group and MCAO + SYR.Neurological function scores(5-point scale)were performed on SD rats to assess neurological deficits in each group;brain water content was measured to assess the degree of brain edema in each group;HE and Nissl staining were used to assess neuronal cell damage.(2)WB experiments were used to determine the expression of inflammatory response-related proteins IL-1β,IL-6 and TNF-α in Sham group,model group,solvent control group and drug-treated group,and enzyme activities of IL-1β,IL-6 and TNF-α were determined by enzymelinked immunosorbent assay(ELISA).Myeloperoxidase(MPO)immunofluorescence staining was also used to observe and count neutrophil infiltration.(3)WB was used to determine the expression of NF-κB-related proteins in the sham group,MCAO group,MCAO+DMSO group and MCAO +SYR group.The specific proteins were as follows: NF-κ B total protein,cytoplasmic protein of NF-κB,cytosolic protein of NF-κB,FOXO3a total protein,FOXO3a phosphorylated protein,and FOXO3a cytoplasmic cytosolic protein.(4)Randomly grouped into 7 groups: sham group,MCAO group,MCAO+DMSO group,MCAO +SYR group,MCAO+control si RNA group,MCAO+FOXO3a sirna group and MCAO+FOXO3a sirna +SYR group.The FOXO3a si RNA was screened in a pre-experiment to select the fragment with the best interference efficiency,and the experimental animals were injected into the Lateral ventricle before MCAO.Protein expression of NF-κB,IL-1β,IL-6 and TNF-α was detected using Western blot in randomized7 groups.The entry and exit of NF-κB p65 into and out of the nucleus was observed using immunofluorescence staining.(5)Co-localization of NF-κ B with FOXO3a was detected using immunofluorescence co-localization.And the binding level of NF-κB to FOXO3a was further detected using immunoprecipitation(C0-IP)method in the sham group,MCAO group,MCAO+DMSO group and SYR+MCAO group.Results:(1)SYR can effectively reduce the volume of cerebral infarction.The optimal drug concentration of 10 mg/kg was selected as the dosing concentration for subsequent experiments.Meanwhile,SYR treatment could improve the neurological function of rats,reduce brain edema and decrease the cell damage by cerebral ischemia.(2)Compared with the MCAO group,the expression of inflammationrelated factors IL-1β,TNF-α and IL-6 was significantly reduced in the drugtreated group,and the infiltration of neutrophils was effectively reduced.(3)Treatment with SYR induced phosphorylation of FOXO3a,thereby preventing its translocation to the nucleus and inhibiting the expression and activity of NF-κB.(4)The fragment with the best knockdown effect was screened.Knockdown of FOXO3a reversed the inhibitory effects of SYR on NF-κB,IL-1β,IL-6 and TNF-α,and demonstrated that FOXO3a was involved in the regulation of NF-κB by SYR.(5)SYR promoted the interaction of NF-κB with FOXO3a.Conclusions:(1)SYR treatment significantly reduced rat cerebral ischemia damage and improved nerve function damage.(2)SYR attenuates cerebral ischemic injury through the FOXO3a/NF-κB axis.