Drug Screening And Mechanism Of Inhibiting Ferroptosis In Retinal Pigment Epithelial

Objective:1.Explore the effect and mechanism of ferroptosis in sodium iodate induced retinal pigment epithelial cell injury model.2.Preliminary screening out potential small molecule compounds against retinal pigment epithelial cell injury.Methods:Firstly,the injury model of ARPE-19 cells induced by NaIO₃was established.ARPE-19cells were cultured in vitro with different concentrations of NaIO₃.The proliferation activity was detected by CCK-8.Cell morphology was observed with inverted phase contrast microscope in 5m M NaIO₃over time.Eighteen ferroptosis inhibitors were selected from previously published research,CCK-8 was used to detect the cell proliferation activity for preliminary drug screening.The concentration of NaIO₃was gradually increased to screen out effective drugs to protect RPE cells.Finally,the expression of ferroptosis related proteins in the drug-treated and NaIO₃-treated groups were detected with Western blot.Results:1.The effect on the proliferation activity in ARPE-19 cells by NaIO₃:The damage induced by NaIO₃is concentration-dependent.Cell damage is obvious in the concentration of NaIO₃is 5m M.The IC50 is 5.25 m M.With the increase of NaIO₃concentration,cell proliferation activity significantly decreased,and the difference was statistically significant（P<0.01）.2.The effect on the morphology of ARPE-19 cells by NaIO₃:After NaIO₃5 m M injury for24 h,cell morphology was gradually elongated and irregular,cell growth was slow,and cell fragments was observed in the medium with the phase contrast microscope.With the time is longer,the intercellular adhesion was lost,cell volume was reduced,and abnormal morphological cells gradually increased.Moreover,when the concentration of NaIO₃was increased to 10 m M,cell damage was significant after 24 hours,the intracellular melanin granules disappeared,and abnormal morphological cells were significantly increased,cell mitochondria was shrink,mitochondrial structural integrity disappeared under transmission electron microscope.3.Screening small molecule compounds that inhibit cell damage:five drugs with better protection（proliferation activity≥80%）in the 5 m M NaIO3 cell damage model after 24 hours of preliminary screening by CCK-8 are:Baicalein,Genistein,Ferrostatin-1,Quercetin and SRS16-86.Then,the concentration of NaIO₃was gradually increased to 20 m M,the drugs with cell proliferation activity still above 50%were screened out:Ferrostatin-1、Genistein and SRS16-86,and cell proliferation activity of the drug-treated groups were significantly higher than that of the NaIO3-treated group（P<0.01）.4.Effects of NaIO3-treated group and drug-treated group on the expression of ferroptosis related proteins:The level of HO-1 in ARPE-19 cells was increased and SLC7A11、GPX4 were decreased in NaIO₃-treated group.The drug-treated groups or the normal control group were opposite.In addition,the level of HO-1 and SLC7A11 in normal control group significantly decreased.Conclusion:1.The cell model,cell morphology and mitochondrial morphology induced by NaIO₃are similar to ferroptosis characteristics and expressed ferroptosis marker proteins,suggesting that this model is closely indeed related to ferroptosis.2.Discover 3 drugs that may inhibit cell damage from ferroptosis inhibitors which still have cell protection effects at high concentrations.