Effects Of Bevacizumab On Apoptosis Of Human Retinal Pigment Epithelial Cells

Objective:To observe the effect of anti-neovascular endothelial growth factor(VEGF)monoclonal antibody on the apoptosis of human retinal pigment epithelium(RPE)cells in order to explore the possible mechanism of atrophic formation of macular after the long-term anti-VEGF in age-related macular degeneration.Methods:Human retinal pigment epithelial cells(ARPE-19)were cultured in DMEM/F12 medium containing 0.25g.L~-11 bevacizumab,according to the different incubation time divided into 1 week dosing group,2 weeks dosing group,respectively,set up control group divided into 1 week control group,2 weeks control group.At different time points after bevacizumab treatment,the morphology of ARPE-19 cells in each experimental group was observed under an optical microscope.Flow cytometry was used to detect the apoptosis rate of ARPE-19 cells treated with bevacizumab for different time.Reverse transcriptase polymerase chain reaction(RT-PCR)was used to detect the mRNA expression level of P53,TP53INP1,Bax and apoptosis suppressor gene Bcl-2 in ARPE-19 cells treated with bevacizumab at different time points.The protein expression levels of above genes were detected by Western blot.The difference of expression was analyzed by one-way ANOVA and LSD-t test.Results:The light microscope showed that the ARPE-19 cells in the 1 week control group and the 2 weeks control group showed typical epithelial cell morphology with paved stone-like morphology and monolayer adherent growth.Compared with the control group,part of the cell morphology after bevacizumab treatment changed slightly,the cells became rounded,the cell body was elongated and the boundary was poor.Flow cytometry showed that bevacizumab could induce time-dependent apoptosis of ARPE-19 cells,the apoptotic rates of the 1 week control group,1 week dosing group,2 weeks control group and 2 weeks dosing group were(5.57±1.46)%,(6.39±1.25)%,(6.88±1.10)%and(13.34±1.94)%,there was no significant difference in the apoptotic rate between the 1 week control group and the 2weeks control group(P>0.05),the apoptotic rate of the 2 weeks dosing group was significantly higher than that of the 2 weeks control group and the 1 week dosing group,the differences were statistically significant(P<0.01).RT-PCR and Western blot results showed that the expression of P53,TP53INP1 and Bax mRNA and protein in the two dosing groups were higher than those in the control group,the expression of Bcl-2 mRNA and protein decreased,the differences were statistically significant(P<0.05).In the dosing group,the expression of apoptotic-promoting factors mRNA and protein in the group at 2 weeks were higher than that of the group at 1 week,while the mRNA and protein of Bcl-2 decreased,the difference was statistically significant(P<0.05).In the two control groups,there was no significant difference in the expression of the above factors(P>0.05).Conclusion:Bevacizumab can upregulate ARPE-19 cell apoptosis,expression of apoptosis-promoting factors,and down-regulate the expression of apoptosis inhibitory factor,which induces apoptosis of ARPE-19 cells in a time-dependent manner.RPE apoptosis may be one of the causes of macular atrophy after long-term anti-VEGF therapy.