Nicotinamide Inhibits Epithelial-mesenchymal Transition Of ARPE-19 Cells By Attenuating Oxidative Stress

Purpose:To investigate the effects of nicotinamide?NAM?on bevacizumab?BEV?-induced epithelial-mesenchymal transition?EMT?of a human retinal pigment epithelial cell line?ARPE-19?and to explore the potential mechanisms.Furthermore,this study may also provide novel solutions for alleviating neovascular fibrosis of ocular fundus after anti-vascular endothelial growth factor?VEGF?therapy.Methods:ARPE-19 cells were cultured in medium and treated with different agents in vitro to conduct relevant detections.First,ARPE-19 cells were treated with BEV for 24,48,and 72h and EMT-related markers?three mesenchymal markers:fibronectin,?-SMA and vimentin;an epithelial marker:ZO-1?were then assessed by Western Blot to determine the optimal treatment time for subsequent experiments.Second,NAM was added to the medium,with or without BEV,during the optimal treatment time,and the mRNA and protein levels of the aforementioned EMT-related markers were then measured by RT-qPCR,Western Blot and Immunofluorescent Staining.The effects of NAM on BEV-induced EMT could be observed in the above analyses.The accumulation of reactive oxygen species?ROS?and H₂O₂ and the total antioxidant capacity of the cells were also measured to evaluate the level of oxidative stress under the above treatment conditions.The impacts of NAM on oxidative stress could be estimated in these detections.The quantitative data were compared with single factor analysis of variance.A P-value<0.05 was considered statistically significant.Results:Firstly,the EMT model was successfully established when ARPE-19 cells were treated with BEV.Although the protein expressions of fibronectin and vimentin were slightly elevated,there were no significant differences?P=0.61 and P=0.58?when cells were treated with BEV for 24h.Meanwhile,the protein expressions of?-SMA and ZO-1 were increased and reduced respectively,the differences were statistically significant?P=0.006 and P=0.001?.When cells were treated with BEV for 48h,the protein expressions of fibronectin,?-SMA and vimentin were increased significantly,and the expression level of ZO-1 was decreased distinctly,the differences were statistically significant?P<0.001?.When the BEV-treated time reached 72h,the changes of aforementioned EMT-related markers became more obviously,and the differences were statistically significant?P<0.001?.Therefore,72h as an optimal treatment time was used to investigate the functions of NAM in the following experiments.Secondly,the RT-qPCR and Western Blot results indicated that NAM markedly inhibited BEV-induced EMT in ARPE-19 cells by downregulating fibronectin,?-SMA and vimentin,the differences were statistically significant?P<0.001?,and upregulating ZO-1,the differences were statistically significant?P<0.001 and P=0.004?.Meanwhile,Immunofluorescent Staining indicated that BEV-induced higher levels of?-SMA was attenuated by NAM.The above results indicated that NAM effectively inhibited BEV-induced EMT of ARPE-19 cells.Besides,BEV promoted oxidative stress by increasing the levels of ROS and H₂O₂,and reducing the total antioxidant capacity.However,NAM reversed above phenomena in ARPE-19 cells,which exhibited the low levels of ROS and H₂O₂,and the high level of cellular total antioxidant capacity,the difference was statistically significant?P<0.001?.Conclusions:This study demonstrates that NAM inhibit BEV-induced EMT in ARPE-19 cells by attenuating oxidative stress.Hence,NAM may be a novel therapeutic agent for alleviating neovascular fibrosis of the ocular fundus after anti-VEGF therapy.