The Protective Mechanism And Optimal Dosage Of Picrosides On SH-SY5Y Cell Models With Oxygen-glucosedeprivation/reoxygenation Injury

Objective:To explore the protective mechanism and optimal dosage of picrosides on human neuroblastoma SH-SY5Y cell models with oxygen-glucose deprivation/reoxygenation（OGD/R）injury.Methods:OGD/R models of SH-SY5Y cells were established by inducing hypoxia and hypoglycemia for 4 h and reoxygenation for 24 h in vitro.The cells damaged by OGD/R were divided into different concentration groups of the combination of picrosideⅠ,picrosideⅡ,picrosideⅢand model groups,adding the drug according to the set dose,5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L,during reoxygenation.The optimal doses of picrosideⅠ,picrosideⅡ,picrosideⅢwere selected for the factorial design study of 2×2×2 factorial design study.Observe the morphological changes of cells with the light microscope.Detect cell viability by CCK-8 assay（CCK-8）.The release of lactate dehydrogenase（LDH）was detected by the lactate dehydrogenase release method.Intracellular reactive oxygen species（ROS）was determined by fluorescence probe DCFH-DA.Comprehensively evaluate its protective effect according to the dosage and various results.After determining the optimal concentration of picrosides,SH-SY5Y cells were randomly divided into 5 groups:normal control group（conventional culture,no OGD treatment）,ODG/R group（sugar-free medium,1%O₂）incubating the cells in the box for 4 hours in reoxygenated and regenerated sugar for 24 hours),the positive control group（Ligustrazine）,the LR290042 group,and the picroside group.Observe the morphological changes of cells with a light microscope.The CCK-8 assay was used to detect cell viability.The lactate dehydrogenase release method was used to detect the amount of LDH.The level of ROS in the cells was measured using a fluorescent probe DCFH-DA.The Annexin V-FITC/PI double staining method was used to detect the apoptosis rate.Results:Compared with the control group,the morphological structure of SH-SY5Y cells changed after OGD/R,the number of apoptotic cells increased,the model group cells became round or swollen,the protrusions retracted or disappeared,the cell membrane was wrinkled or damaged,and the number of cells was significantly reduced.Floating cells increased and cell viability decreased;compared with the model group,the cell damage of the picroside group was improved to varying degrees,and cell viability increased.Among them,the three drugs of picrosideⅠ,picrosideⅡand picrosideⅢwere the best,and the effect was best at 20μmol/L each.The improvement is most obvious.Compared with the normal control group,the cell survival rate of the model group decreased,while the content of LDH release in the supernatant increased（P<0.05）.Compared with other groups,the combined application group of picrosides increased significantly cell survival rate and improved cell damage,decreased LDH content and ROS level,and reduced the apoptosis rate（P<0.05）.Conclurions:Picroside could play neuroprotective effects on OGD/R injury of SH-SY5Y cells with the best doses of picrosideⅠ,ⅡandⅢat 20μmol/L.The may play a neuroprotection and anti-apoptosis through anti-oxidative stress.