Picroside Ⅱ Exerts Neuroprotective Effect Against Cerebral Ischemia-Reperfusion Injury By Regulating AMPK-mTOR-ULK1 Autophagy Signaling Pathway

Objective: To investigate whether picroside Ⅱ can protect neurons from cerebral ischemia-reperfusion injury by regulating ROS-mediated AMPK-mTOR-ULK 1autophagy signaling pathway.Methods: The middle cerebral artery occlusion/reperfusion(MCAO/R)model was established in healthy adult SD rats(body mass 230±20 g)by inserting a monofilament thread,and the rats were randomly divided into sham operation group,model group,and picroside Ⅱ group.The modified neurological deficit score(mNSS)was used to evaluate the neurobehavioral function of rats;the volume of cerebral infarction was detected by triphenyltetrazolium chloride(TTC)staining.The morphological changes and apoptosis of nerve cells in brain tissue were detected by HE staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL).The expression of autophagy-related proteins LC 3,Beclin 1 and p62 were detected by Western blot.The oxygen-glucose deprivation/ reoxygenation(OGD/R)model was established by SH-SY5 Y cells,and the cells were randomly divided into control group,model group,picroside Ⅱ group and Compound C group.Compound C was used as an inhibitor of AMPK.The morphology of the cells was observed by light microscopy.The activity of the cells was detected by CCK-8 kit.The apoptosis was detected by flow cytometry.The level of reactive oxygen species(ROS)in the cells was detected by reactive oxygen species kit.The expression of LC3,Beclin 1,p62,phospho-AMPK,phospho-mTOR,phospho-UL1 were detected by Western Blot.The expression of LC3 B,phospho-AMPK,phospho-mTOR were detected by immuno-fluorescence.The dynamic process of autophagy(autophagy flux)was monitored by GFP-mRFP-LC3 autophagy adenovirus.Results: After MCAO/R in rats,compared with sham operation,the mNSS score increased significantly,the volume of cerebral infarction increased,the number of deformed cells and apoptotic cells in cerebral cortex increased,the expression of LC 3and Beclin 1 increased,and the expression of p62 decreased(P<0.05).After treatment with picroside Ⅱ,the expression of autophagy-related proteins LC 3 and Beclin 1decreased,the expression of p62 increased,the mNSS score decreased,the infarct volume decreased,and the number of denatured cells and apoptotic cells decreased compared with the model group(P<0.05).In the OGD/R model: compared with the control group,abnormal morphological cells and apoptotic cells increased,cell viability decreased,and expression of LC 3,Beclin 1,and phospho-AMPK increased,p62,phospho-mTOR,and phospho-ULK 1.The autophagy flux and the level of ROS in the cells increased(P<0.05).After intervention with picroside Ⅱ,the expression of LC 3 and Beclin 1 decreased,and the expression of p62 increased.The number of abnormal cells,the level of ROS and and autophagy flux decreased,the number of apoptotic cells and cell viability increased,the expression of phospho-AMPK decreased,and the expression of phospho-mTOR and phospho-ULK 1 increased(P<0.05).Conclusion: Picroside Ⅱ exerts neuroprotective effects in cerebral ischemia-reperfusion injury by inhibiting ROS-mediated activation of the AMPK-mTOR-ULK1 autophagy signaling pathway.