Mechanism Study Of Edaravone Regulating Cells Autophagy After Cerebral Ischemia-Reperfusion

ObjectiveTo investigate the effect of Edaravone on cerebral autophagy in rats with Cerebral ischemia reperfusion injury（CIRI）through the PINK1/Parkin1 pathway,and further explore the mechanism of its function.MethodsSD rats were randomly divided into 4 groups:Sham gruop,MCAO group,Edaravone group,and 3-methyladenine group（3-MA group）.Sham group,model group and Edaravone group were injected intraperitoneally one week in advance,and 3-m A group was injected into lateral ventricle.Except for the sham group,the middle cerebral artery occlusion method was used to establish a focal ischemia-reperfusion model in rats.The edaravone group and the 3-MA group were given edaravone 3ml·kg^-1,3-MA 75μg,and the other two groups were given the same amount of normal saline.After the operation,the model and efficacy were verified by neurological deficit score,triphenyltetrazolium chloride（TTC）staining and brain edema detection.The mitochondrial autophagy of the brain tissue was observed using fluoroscopic electron microscopy.The JC-1 method was used.Observe the changes in cell membrane potentials;PINK1/Parkin1 pathway related factors were detected by immunoblotting in each group.Results（1）Model identification and drug efficacy test:Sham group rats showed no neurological deficits,cerebral infarction and cerebral edema.Model group,3-MA group and Edaravone group had different degrees of neurological deficits,cerebral infarction and cerebral edema.Compared with the Model group,the above indicators were significantly reduced in the Edaravone group,while the 3-MA group was higher than the Model group,but there was no statistical difference.The results suggest that the symptoms of neurological deficits are not relieved after3-MA inhibition of autophagy,but the symptoms are significantly reduced after treatment with Edaravone.（2）Electron microscope observation showed that normal mitochondria and mitochondrial crest structure were intact in the Sham operation group.In the Model group and Edaravone group,different degrees of autophagosomes were seen.The statistical results showed that compared with the Sham group,the number of autophagosomes in the Model group,Edaravone group,and 3-MA group increased significantly.Compared with the Model group,the number of Edaravone groups increased significantly,but the number of autophagosomes in the 3-MA group decreased significantly.It is suggested that Edaravone induces autophagy response to ischemia-reperfusion,while 3-MA inhibits cell autophagy.（3）Analysis of JC-1 detection results using a fluorescence microplate reader showed that compared with the Sham group,the JC-1 polymer to monomer ratio in the Model group,3-MA group,and Edaravone group was significantly reduced;compared with the Model group,the Edaravone group Significantly increased,but there was no statistical difference between the 3-MA group and the model group.This result indicates that the mitochondrial membrane potential does not improve significantly after 3-MA inhibits autophagy,but the mitochondrial membrane potential is significantly improved after Edaravone treatment.（4）Western Blot test results showed that PINK1,Parkin1,and LC3B expression bands were obvious in each group.Compared with the Sham group,the levels of PINK1,Parkin1,and LC3B in the Model and Edaravone groups were significantly increased;compared with the Model group,the levels of PINK1,Parkin1,and LC3B in the Edaravone group were significantly increased,but the levels of the 3-MA group were significantly reduced;suggesting that Edaravone induced ischemia Reperfusion of PINK1,Parkin1,and LC3B expression levels induces mitochondrial autophagy,while 3-MA inhibits the expression of PINK1,Parkin1 and LC3B to inhibit mitochondrial autophagy.Conclusions1.Edaravone can induce mitochondrial autophagy after cerebral ischemia-reperfusion,improve mitochondrial membrane potential,reduce neurological deficit score,cerebral infarction area and brain edema content,and play a protective role after cerebral ischemia-reperfusion injury.2.Edaravone may induce mitochondrial autophagy by up regulating the expression of PINK1/Parkin1 pathway and LC3B,a marker of autophagy,after cerebral ischemia-reperfusion.