Effects Of Ozone On Oxidative Stress And Mitochondrial Apoptosis In SH-SY5Y Cells During Ischemia-reperfusion Injury

Objective:In this study,human neuroblastoma cells（SH-SY5Y）were treated with oxygen and glucose deprivation to establish an ischemia-reperfusion injury model.Through ozone treatment at different concentrations,the possible mechanism of ozone treatment on neuronal antioxidant protection and inhibition of mitochondrial apoptosis was explored.Methods:SH-SY5Y cells were selected as the research model,and high glucose containing 10%fetal bovine serum（FBS）,various amino acids and glucose（DMEM）medium were used to culture these cells for 24hours in an box at 37℃,5%CO₂ saturated humidity and aseptic conditions.The culture medium was changed every 48 hours,and the cells were passaged after 80%cell fusion.The cells were passaged after 80%confluence.Tetrazolium salt（MTT）method was used to detect the viability of SH-SY5Y cells treated with ozone at different concentrations（20、40、80μg/mL）and at different times（2、6、12、24hours）,and enzyme-linked immunosorbent assay Assay（ELISA）method was used to detect the lactate dehydrogenase（LDH）level of SH-SY5Y cells after treatment with different ozone concentrations（0、20、40、80μg/mL）for 2 hours,A suitable ozone treatment concentration and time were obtained（20μg/mL,2h）.The cultured SH-SY5Y cells were then divided into four groups:control group（Control）,ozone group（Ozone）,ischemia-reperfusion（I/R）group,I/R+ozone treatment group:Control group:SH-SY5Y cells without any treatment;Ozone group:SH-SY5Y nerve cells were treated with Ozone concentration of 20ug/mL for 2 hours;I/R group:SH-SY5Y nerve cells were treated by oxygen-free deprivation of glucose and hypoxia to establish an ischemic model,and then normal cell culture was restored to become a model of reperfusion injury;the I/R+ozone treatment group:SH-SY5Y nerve cells were treated by oxygen-free deprivation of glucose and hypoxia to establish an ischemic model,and then normal cell culture was restored to become a model of reperfusion injury,treated the reperfusion injury model with ozone concentration（20μg/mL）for 2 hours at last.Antioxidant enzyme-related indicators,apoptosis rate,cell mitochondrial membrane potential level and mitochondrial activity indicators were detected by using antioxidant enzyme kit,flow cytometry technology,mitochondrial membrane potential detection kit jc-1 and other detection technologies,to bserve effects of ozone on oxidative stress and mitochondrial apoptosis of SH-SY5Y cells with ischemia-reperfusion injury.Results:1.Effects of ozone on the viability and injury of SH-SY5Y cells:The low,medium and high concentration ozone groups（20、40、80ug/ml）all showed a dose and time-dependent decrease（P<0.05）,but the cell activity in the low concentration ozone group（20ug/ml）was significantly higher than that in the medium and high concentration ozone groups（P<0.05）.The lactate dehydrogenase（LDH）leakage rate increased with the increase of ozone concentration.The LDH leakage rate in the medium and high concentration ozone groups were higher than that in the control group（P<0.05）,indicating the degree of cell damage is high.There was no significant difference between the low ozone concentration group and the control group in the LDH leakage rate（P>0.05）.2.Effects of ozone on the antioxidant enzyme activities of MDA,glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and catalase（CAT）of SH-SY5Y cells:with I/R treatment significantly decreased SOD and CAT activities and increased MDA and GSH-PX activities.Ozone reduced the effect of I/R on MDA and SOD,and increased the activity of CAT and GSH-PX.There was a significant difference between theI/R group and the I/R+ozone group（P<0.01）.The activity of GSH-Px,SOD and CAT in I/R+ozone group was significantly higher than that in I/R group（P<0.01）.The MDA level in the I/R+ozone group was significantly lower than that in the I/R group（P<0.01）.In addition,ozone treatment only increased CAT and SOD activities（P<0.01）,indicating statistical significance;but it has no statistical significance in MDA and GSH-Px（P>0.05）.3.Analysis of apoptosis rate detected by flow cytometry:Compared with the control group,apoptotic cells in the I/R group increased by about37.1%;the apoptosis rate decreased to 28.4%after ozone treatment,suggesting that ozone might reduce I/R injury by inhibiting SH-SY5Y cells apoptosis to reduce I/R injury（P<0.05）.At the same time,compared with the control group,there was no significant difference after ozone treatment（P>0.05）,and ozone had no significant effect on normal SH-SY5Y function.4.Effects of ozone on mitochondrial function of SH-SY5Y cells:the control group showed obvious red fluorescence and slight green fluorescence,while I/R treatment significantly reduced red fluorescence and increased green fluorescence.QuantitativeΔΨm analysis of red-green fluorescence ratio showed that the mitochondrial membrane potential（ΔΨm）of SH-SY5Y cells in I/R group decreased,and I/R+ozone could partially reverse the decrease inΔΨm（P<0.05）,which was statistically significant.Compared with the control group,the cytoplasmic and mitochondrial expression of cytochrome C（Cyt-c）cells in the I/R group respectively increased and decreased significantly（P<0.05）,which was statistically significant,but had no significant effect on normal nerve cells.Conclusion:1.Low concentration ozone has little effect on SH-SY5Y cell activity and LDH leakage rate.2.Low concentration ozone treatment can increase the activity of antioxidant enzymes in SH-SY5Y cells,thereby alleviating oxidative damage.3.Low concentration ozone treatment can reduce mitochondrial damage and inhibit I/R-induced apoptosis of nerve cells.