Inhibitory Effects Of NADPH Oxidase 4 Inhibitor On The Epithelial-mesenchymal Transformation Of Human RPE Cells Induced By Bevacizumab

Objective:To observe the effect of Bevacizumab(BEV)on the epithelial-mesenchymal transition(EMT)of human retinal pigment epithelial cells(ARPE-19).To investigate the inhibitory effect of reduced nicotinamide adenine dinucleotide(NADPH oxidase 4(NOX4))on the development of EMT in retinal pigment epithelial cells.To provide some new therapeutic ideas for alleviating the fibrosis caused by fundus neovascularization due to the clinical treatment with anti-vascular endothelial growth factor(VEGF).Methods:ARPE-19 cells were cultured in vitro and detected.According to the experimental requirements,the cells were divided into four groups according to different drug treatment methods : blank control group(conventional culture),BEV group(0.25g/L BEV was added to the culture medium),BEV+VAS2870 group(0.25g/L BEV and NOX4 inhibitor VAS2870 were added to the culture medium,3μmol/L),BEV + GKT137831group(0.25g/L BEV and NOX4 inhibitor GKT137831,20μmol/L).Under the above conditions,after the cells were cultured for 72 hours,the gene and protein expression levels of EMT markers [fibronectin(FN),Vimentin(vimentin),α-smooth muscle actin(α-SMA)and tight junction-related protein locking band protein-1(ZO-1)] were detected by RT-PCR and Western blot,and the gene and protein expression levels of NOX4 in each group were also detected.Then,the expression of EMT marker protein and NOX4 in cells was verified by cellular immunofluorescence,so as to observe the inhibition of NOX4 on BEV-induced EMT in cells.Results:After 72 hours of BEV treatment of ARPE-19 cells,compared with the blank control group,the relative m RNA expression of EMT interstitial markers FN,Vimentin andα-SMA in BEV group increased,the relative m RNA expression of epithelial marker protein ZO-1 decreased,and the m RNA expression of NOX4 increased,all with statistical significance(P < 0.05).Western blot and cellular immunofluorescence showed that the relative expression of interstitial cell marker proteins FN,Vimentin and α-SMA in BEV group increased,while the relative expression of epithelial cell marker protein ZO-1decreased and the relative expression of NOX4 increased,all with statistical significance(P < 0.05).The results indicated that BEV induced EMT in ARPE-19 cells,and the expression of NOX4 in cells increased.Compared with BEV group,the relative expression of m RNA and protein of NOX4 was significantly lower than that of BEV group after NOX4 was added to inhibit VAS2870 or GKT137831(BEV+ VAS2870 group and BEV+ GKT137831 group).(all p < 0.05).The relative m RNA expression of FN,Vimentin and α-SMA in BEV+ VAS2870 group and BEV+ GKT137831 group were lower than that in BEV group,while the relative m RNA expression of ZO-1 was higher than that in BEV group,and the differences were statistically significant(all p < 0.01).Western blot and cellular immunofluorescence staining showed that compared with BEV group,the relative expression levels of FN,Vimentin and α-SMA in BEV+VAS2870group and BEV+GKT137831 group were lower than those in BEV group,while the relative expression levels of ZO-1 were significantly higher than those in BEV group,and the differences were statistically significant(all P < 0.01).The results indicated that NOX4 inhibited VAS2870 and GKT137831 effectively inhibited EMT induced by BEV in ARPE-19 cells.Conclusion:NOX4 is involved in bevacizumab-induced EMT in RPE cells,and inhibitors of NOX4 attenuate the EMT in bevacizumab-induced human RPE cells.