Pro-EGCG Inhibits HIF-1α/VEGF/VEGFR2 Pathway And M1 Macrophages/Microglia Activation In Choroidal Neovascularization

Purpose:The main purpose of this study is to investigate the roleand potential and potential of prodrug of pigallocatechin-3-gallate(Pro-EGCG)alleviates choroidal neovascularization(CNV)by down-regulating the HIF-1α/VEGF/VEGFR2 pathway and M1 type macrophage/microglia activation.Methods:First,EGCG was used as the starting material,and treated with acetic anhydride and pyridine overnight to obtain Pro-EGCG.A mouse CNV model was induced by laser photocoagulation and randomly divided them into normal group,blank group,PBS group,Pro-EGCG group(different density).From day 0 to day 7,the latter two groups were given the same volume of PBS and Pro-EGCG drugs via gavage needle daily.Fundus fluorescein angiography(FFA)and Indocyanine green angiography(ICGA)was used to detect the effect of Pro-EGCG on CNV generation in mice.Western blot and immunohistochemistry stain assay were used to detect the blocking of HIF-1 α/VEGF/VEGFR2signaling pathway in mouse CNV model by Pro-EGCG.Choroidal flat-mounts and RT-PCR experiments were used to detect the inhibition of CNV generation by Pro-EGCG after blocking M1 type macrophage/microglia polarization.The murine brain microvascular endothelial cell line B-End3 were used to establish an in vitro model.Cell counting Kit-8(CCK-8)assay and 5-Ethynyl-2’-deoxyuridine(EdU)assay were used to detect the effect of Pro-EGCG on the proliferation of B-End3 cells,Transwell migration assay were used to detect B-End3 cells migration capacity,Tube formation assay was conducted to detect the tube formation ability of endothelial cells.Results:Western blot showed that the expression levels of HIF-1α,VEGF,and VEGFR2 proteins in the CNV region of mice reached a peak on the 7 day.F4/80 and Ibal antibody fluorescence staining results showed that M1 type macrophages and microglial cells increased expression.After After gavage of Pro-EGCG,FFA and ICGA results showed CNV leakage and area reduction;Western blot and immunohistochemistry staining showed HIF-1α/VEGF/VEGFR2 pathway was inhibited and the infiltration of M1 type macrophages and microglia cells was reduced.Pro-EGCG reduces proliferation,migration,and tube formation ability of B-End3 cells.Conclusion:Pro-EGCG slowed down the laser-induced CNV leakage area in mice by down-regulating the HIF-1α/VEGF/VEGFR2 signaling pathway and polarization of M1 macrophages and microglia.It provides a new potential therapy for age-related macular degeneration patients.