Protective Effect Of Edaravone Against Glutamate-induced Neurotoxicity In PC12 Cells

Objective: Edaravone is an oxygen free radical scavenger that inhibits lipid peroxidation and inhibits oxidative stress damage in vascular endothelial cells and nerve cells.Glutamate(Glu)is the main excitatory amino acid in brain tissue and can produce toxic effects on neurons under pathological conditions.PC12 cells are a cloned cell strain of rat adrenal pheochromocytoma and are widely used in the experimental study of physiological functions of nerve cells.Therefore,the effects of edaravone on the damage of PC12 cells induced by Glu were explored.Methods: The PC12 cells cultured in vitro in logarithmic growth phase were divided into 6 groups.Only DMEM complete medium was added to the control group.The final concentrations of Glu in the experimental group were 2.5,5,10,20,40 mmol/L,37°C,and 5% CO2 conditions,respectively.After 24 hours of culture,cell viability was measured by MTT assay.The selection of cell survival rate was reduced to 50% to 60% of Glu concentration in the control group as follow-up modeling conditions.24 hours after the cells were inoculated,edaravone was added at concentrations of 20,40,60,80,100,and 120 ?mmol/L,respectively.At the same time,the control group was added with DMEM complete medium,and a total of 7 concentration groups were selected.40 ?mol/L was selected.80?mol/L(peak effect concentration)was used as a follow-up model for the low and high concentration groups.Logarithmic phase cells were divided into 4 groups,Glu group cells were added 10mmol/LGlu,edaravone concentration group cells were added 10mmol/LGlu and then were added 40?mmol/L,80?mmol/L edaravone,the control group cells were added Equal amounts of DMEM were added to each group of cells and incubated for 24 hours.The viability of the cells was measured by MTT assay.Apoptosis was detected by flow cytometry.The mitochondrial membrane potential,reactive oxygen species(ROS),and microplate reader were measured by confocal microscopy.The color method was used to detect superoxide dismutase(SOD)and malondialdehyde(MDA)Results:1.MTT experimental results After Glu treatment for 24 h,the cells in the control group grew well.Compared with the control group,the survival rate of PC12 cells decreased in turn as the Glu concentration increased.At 10 mmol/L Glu,the cell viability was 52.40±4.67% in the control group.Addition of edaravone increased the viability of PC12 cells,but it was still lower than that of the control group.When 80 ?mol/L edaravone was used,the effect peaked at 66.73±4.29%.Continued increase of edaravone concentration resulted in a relative decrease in cell viability.2.Changes in apoptosis After Glu treatment for 24 h,compared with Control,the apoptosis rate of other three groups of PC12 cells was significantly higher,and the apoptosis rate of Glu+EDA1 group and Glu+EDA2 group was significantly lower than that of Glu group,and the Glu+EDA2 group was more significantly decreased.The difference was statistically significant(all P<0.05).3.Effect of edaravone on reactive oxygen species ROS in PC12 cells Compared with the control group,Glu could significantly increase the ROS level in PC12 cells.The fluorescence intensity was the highest and the number of cells was the highest under the confocal microscope.The ROS levels in the Glu+EDA1 group and the Glu+EDA2 group were lower than those in the Glu group.Compared with Glu+EDA1 group,the Glu+EDA2 group decreased more significantly,and the difference was statistically significant(all P<0.05).4.Effect of edaravone on mitochondrial membrane potential in PC12 cells Compared with the control group,the mitochondrial membrane potential of PC12 cells in each drug group was significantly decreased.The mitochondrial membrane potential of Glu+EDA1 group and Glu+EDA2 group was significantly higher than that of Glu group,which were 0.73±0.06,0.93±0.07,and Glu+EDA2,respectively.The increase was more significant in the Glu+EDA1 group than in the Glu+EDA1 group(all P<0.05).5.Effect of Edaravone on SOD Enzyme Activity and MDA Content in PC12 Cells Compared with the Control group,the activity of SOD enzyme in the cells of each drug group was significantly reduced,and there was no significant difference between the Glu+EDA1 group and the Glu group,and there was no significant difference.The Glu+EDA2 group was significantly lower than the Glu.Compared with the control group,the MDA content in each group was significantly increased,and the Glu+EDA2 group was significantly lower than the Glu+EDA1 group.The difference was statistically significant.Conclusion:1.Edaravone has protective effects on Glu-induced injury in PC12 cells,and its mechanism may be related to the antagonism of edaravone against Glu-induced oxidative stress.2.ROS-mediated reduction of mitochondrial membrane potential participates in Glu-induced neuronal apoptosis.