Effect Of Bevacizumab On The Function Of Anti-oxidative Stress In Human Retinal Pigment Epithelium

Purpose:To investigate the effect of bevacizumab on the antioxidative function of human retinal pigment epithelium(RPE),in order to explore the possible mechanism of macular atrophy induced by the application of anti-vascular epithelial growth factor(VEGF)agents in age-related macular degeneration.Methods:Human retinal pigment epithelial cells were incubated in DMEM/F12 medium containing 0.25 g·L-1 bevacizumab and divided into 5 groups according to incubation period: 0 hour(control),12 hours,24 hours,48 hours and 72 hours,and then the oxidative stress was induced by adding H2O2.Cell viability was measured by the CCK8 assay.MitoSox Red was used to determine mitochondrial reactive oxygen species(mtROS)production.Mitochondrial membrane potential was measured using the JC-1 assay.The expression levels of NOX4,HO-1,NQO-1 and SOD2 were detected by RT-PCR and Western blot,respectively.Results:CCK8 assay determination showed that the above treatment had no significant effect on cell viability,the cell viability of 0 hours,12 hours,24 hours,48 hours and 72 hours were(100.2±3.3)%?(99.2±2.7)%,(102.5±6.4)%?(103.9±3.7)%?(103.6±3.3)%,the difference was not statistically significant(all P>0.05).Compared with the control group,the levels of mtROS increased at 12 hours,24 hours,48 hours and 72 hours,the difference was statistically significant(all P<0.05).Mitochondrial membrane potential at 12 hours,24 hours,48 hours,72 hours were lower than the control group,the difference was significant,48 h reached the lowest,72 hours significantly increased,but still lower than the control group.RT-PCR and western blot results demonstrated that the expression of NOX4 mRNA and protein increased at 12 hours,24 hours,48 hours and 72 hours,and reached the highest at 24 hours,then decreased significantly,but still higher than the control group,the difference was statistically significant(all P<0.01).Compared with the control group,the expression of HO-1 mRNA decreased at 24 hours,48 hours and 72 hours,while the expression of HO-1 protein decreased at 48 hours and 72 hours,the difference was statistically significant(all P<0.05).Compared with the control group,the expression of NQO-1 mRNA and protein decreased at 24 hours,48 hours and 72 hours,the difference was statistically significant(all P<0.05).Compared with the control group,the expression of SOD2 mRNA and protein did not change significantly at 12 hours,24 hours,48 hours and 72 hours,the difference was not statistically significant(all P>0.05)Conclusions:The clinical concentration of bevacizumab can reduce the anti-oxidative function of RPE cells,which may be one of the causes of progressive atrophy of macula after long-term anti-VEGF therapy.