The Regulation And Mechanism Of Allograft Inflammatory Factor-1 On The Biological Function In Choroid-retinal Endothelial Cells

Objective:This study aims to discuss the role of Allograft inflammatory factor-1（AIF-1）and related signaling pathway in rhesus choroid-retinal endothelial cell line（RF/6A）chemical hypoxia model.The interaction between AIF-1 and vascular endothelial growth factor（VEGF）in RF/6A under hypoxic condition was investigated as well,which provided a new theoretical and experimental basis for the study of the pathogenesis of MNV and the exploration of anti-neovasculogenic therapy.Methods:1.The chemical anoxic agent cobalt chloride（Co Cl₂）was added into the complete medium to simulate the stress injury of hypoxia induced endothelial cells（ECs）.In vitro chemical hypoxia model of RF/6A was established.2.Western blot was used to detect the expression trend of AIF-1 and VEGF protein in RF/6A cell samples at 0,1h,3h,6h,12h and 24h under hypoxia condition,and compared with the control group.3.After 6h of hypoxia,cells were divided into four groups according to the experimental design:（1）normal control group;（2）hypoxia group;（3）Hypoxia+scramble si RNA group:RF/6A transfected scramble si RNA before hypoxia stimulation;（4）Hypoxia+AIF-1 si RNA group:AIF-1 si RNA was transfected with RF/6A before hypoxia stimulation.AIF-1si RNA was used to silence the AIF-1 expression,and then the AIF-1 and VEGF protein expression levels were detected by Western blot.3.In the co-culture system of ARPE-19（Human Retinal Epithelial Cells）and RF/6A cells,Transwell migration experiment was used to explore the migration ability of RF/6A cells transfected with AIF-1 si RNA under hypoxia conditions,and compared with the hypoxia group.Cell counting kit（CCK-8）cell proliferation experiment and tube formation experiment were used to explore the influences of different treatments on proliferation and tube formation ability of RF/6A under hypoxia conditions.4.Cell viability（live and dead cell staining,Calcein AM,PI method）assay was used to verify the influence of AIF-1 si RNA transfection on the apoptotic ability of ECs.5.After AIF-1 si RNA inhibited the expression of AIF-1,the expression levels of p-ERK1/2 and ERK1/2 proteins were detected by Western blot.6.The ERK1/2 specific pathway inhibitor（SCH772984）blocked the ERK1/2 signaling pathway,and the expression levels of AIF-1,VEGF,p-ERK1/2 and total ERK1/2 protein were detected by Western blot.Results:1.In the cytochemical hypoxia model in vitro,Western Blot results showed that compared with the normal control group,the protein expression levels of AIF-1 and VEGF were up-regulated in parallel,gradually increased with the extension of hypoxia time（P<0.05）,peak at 6 h,and then decreased.2.RF/6A was transfected with AIF-1si RNA for hypoxia induction,and cell samples were collected for Western Blot analysis.The results showed that,compared with the hypoxia group,AIF-1 si RNA can silence AIF-1 expression and down-regulate VEGF protein expression at the same time（P<0.05）.3.In the ARPE-19 and RF/6A cell co-culture system,the number of migrating cells was significantly decreased after AIF-1 si RNA transfection,which inhibited the migration ability of RF/6A under hypoxia condition（P<0.05）.The results of cell proliferation experiment showed that the number of cells decreased with the silenced expression of AIF-1 compared with the normal group（P<0.05）,indicating that AIF-1 si RNA can effectively reduce the proliferation ability of ECs under hypoxia conditions.Similarly,the link point between ECs was also significantly decreased in the tube formation experiment（P<0.05）,and the tube forming ability of ECs under hypoxia condition could also be inhibited by AIF-1 si RNA4,The cell survival state displayed under different wave lengths was observed under fluorescence microscope,and it was found that the proportion of dead cells increased after transfection of AIF-1si RNA into RF/6A,indicating that AIF-1si RNA promoted the apoptosis of RF/6A in the condition of hypoxia.5.After transfection of AIF-1 si RNA into RF/6A under hypoxia condition,the expression level of p-ERK1/2 protein decreased compared with that in the hypoxia group.5.The ERK1/2specific pathway inhibitor（SCH772984）significantly decreased the protein expression levels of VEGF and P-ERK1/2（P<0.05）under hypoxia,but the expressions of AIF-1and total ERK1/2 were not affected（P>0.05）.Conclusions:AIF-1 and VEGF were expressed in RF/6A cell hypoxia model.Inhibition of AIF-1 expression under hypoxia can significantly reduce the proliferation,migration and tube formation ability of RF/6A,and enhance the apoptosis of RF/6A.AIF-1regulates ECs biological activity and VEGF expression through ERK1/2 signaling pathway.