Protective Effect Of Ozone Oxidation Preconditioning On Hypoxia/Reoxygenation Injury Of HL-7702 Hepatocytes

Objective:To investigate the role of ozone preconditioning in protecting HL-7702 hepatocytes against hypoxia/reoxygenation injury.Methods:1.HL-7702 cell hypoxia/reoxygenation(H/R)model was established by hypoxia workstation to simulate the ischemia-reperfusion injury process of human liver.The HL-7702 cells were divided into 7 groups according to the different density of ozone,which consisted Normal Control(Control)group,H/R group,H/R+AIR group,H/R+20μg/m L ozone preconditioning(H/R+OP20)group,H/R+50 μg/m L ozone preconditioning(H/R+OP50)group,H/R+80 μg/m L ozone preconditioning(H/R+OP80)group and H/R+50 μg/m L ozone preconditioning+HSP70 inhibitor(H/R+OP50+Q25)group.The cell viability was assayed by cell counting kit-8(CCK-8)and the cell apoptotic rate was detected by flow cytometry.The relative rate of cell viability which decreased by 40% was applied to access the establishment of hypoxia/reoxygenation model.2.SOD activity,MDA content and LDH activity were used to evaluate the oxidative stress of HL-7702 cell after hypoxia and reoxygenation in Control group,H/R group,H/R+OP50 group and H/R+OP50+Q25 group by colorimetric method.3.The expression of HSP70 protein and HSP70 mRNA of Control group,H/R group,H/R+OP50 group and H/R+OP50+Q25 group were detected by Western Blot and RT-q PCR.Results:1.The effect of ozone preconditioning on cell viabilityCompared with the Control group,the cell viability of HL-7702 cells in the H/R group was decreased by 40%.Compared with the H/R group,the cell viability of20,50,80 μg/m L ozone preconditioning groups were increased(P<0.05).Interestingly,the cell viability of the H/R+OP50 group was higher than other ozone preconditioning groups(P>0.05).Compared with H/R+OP50 group,the cell viability of the H/R+OP50+Q25 group was decreased(P<0.05).2.The effect of ozone preconditioning on the cells apoptotic rateCompared with the H/R group,the cell apoptotic rate of 20、50、80 μg/m L ozone preconditioning groups were decreased(P<0.05),the cell apoptotic rate of the H/R+OP50 group was lower than other ozone preconditioning groups(P<0.05).Compared with the H/R+OP50 group,the cell apoptotic rate of the H/R+OP50+Q25group was decreased(P<0.05).3.The effect of ozone preconditioning on cellular oxidative stressCompared with the H/R group,the H/R+OP50 group reduced the content of MDA,strengthen the activity of SOD and decrease the LDH leakage rate of HL-7702 cells after hypoxia and reoxygenation(P<0.05).4.The effect of ozone preconditioning on HSP70 protein expressionCompared with the H/R group,the H/R+OP50 group can increase the expression of HSP70 protein(P<0.05),quercetin can inhibit the expression of HSP70(P<0.05).5.The effect of ozone preconditioning on HSP70 mRNA TranscriptionCompared with H/R group,the H/R+OP50 group can increase the transcription of HSP70 mRNA in HL-7702 cells(P<0.05).Conclusion:Ozone preconditioning can attenuate hypoxia-reoxygenation injury of HL-7702 cells,and the protective mechanism may be related to the up-regulation of HSP70 protein transcription and expression,inhibition of apoptosis and reduction of oxidative stress.