Molecular Mechanism Of Delphinidin Protecting Retinal Photochemical Damage By Regulating Oxidative Stress And Pyroptosis

Previous research found that the black rice anthocyanidins(BRACs)can effectively protect retina photochemical damage(RPD)through antioxidant and anti-pyroptosis pathways.Delphinidin(DP)is the active ingredient with the highest content of anthocyanins and has strong antioxidant capacity.There is oxidative damage at an early stage in the pathological process of RPD,but it is not clear whether photoreceptor cells have pyroptosis.It is unclear whether delphinidin monomers can protect RPD through antioxidant and anti-pyroptosis mechanism.This study aims to confirm the role of delphinidin in protecting RPD by regulating oxidative stress and pyroptosis,and to elucidate its molecular mechanism.ObjectiveTo explore the molecular mechanism of delphinidin's protective effect on RPD mediated by oxidative stress and pyroptosis,then provide experimental data for the prevention and treatment of RPD diseases by flavonoids.Methods(1)661W photoreceptor cells were used as the research object to establish the RPD in vitro model,and different concentrations of delphinidin(5,10,20 ?mol / L)were separately treated the cells;(2)CCK-8 method was used to detect the proliferation of 661 W photoreceptor cells;Light microscopy was used to observe cell morphology;LDH kit was used to assess cytoxicity;ELIS A was used to detect peroxidation products MDA,TBARS and biochemiacal methods were used to determine the activity of antioxidant enzymes SOD,GSH-Px,GST;CRISPR / Cas9 gene editing technology was used to knock out Caspase-1 in 661 W photoreceptor cells;661W cells and Cas1 KO cells were used to establish RPD model in vitro,then Observed and compared the cell morphology;CCK-8 method was used to detect the cell proliferation ability of each group;LDH kit was used to evaluate the cytotoxicity of each group;the mRNA and protein levels of the key factors in pyroptosis pathway(NLRP3,ASC,Caspase-1,GSDMD,IL-1?,IL-18)were measured by RT-qPCR and Western Blot;(3)661W cells(RPD in vitro model)were treated with 5 ?mol / L delphinidin;Flow cytometry was used to detect the amount of ROSproduced in each group of cells;Scanning electron microscope was used to observe the morphological changes of the cell surface in each group;Hoechst / PI double staining was used to compare the degree of cell damage in each group;RT-qPCR and Western Blot were used to detect the mRNA and protein expression levels of key factors(NLRP3,ASC,Caspase-1,GSDMD,IL-1?,IL-18)in the pyroptosis pathway of each group;ELISA was used to detect the activities of IL-1? and IL-18 secreted of cells.Results(1)Delphinidin can effectively protect the 661 W photoreceptor cell proliferation after photodamage in a dose-dependent manner;photodamaged 661 W cells fall off and lyse,surface shrinkage,and delphinidin treatment can protect the morphology of light-damaged cells;delphinidin can effectively reduce the 661 W photoreceptor cytotoxicity after light damage in a dose-dependent manner;delphinidin can effectively inhibit the contents of cell peroxidation products MDA and TBARS after light damage;delphinidin can effectively increase the activity of SOD,GSH-Px,and GST in cell antioxidant enzymes after photodamage.(2)the Cas1 KO cell line was successfully constructed by using CRISPR / Cas9 gene editing technology;the light treatment of Cas1 KO cells was intact and in good condition;Cell viability and proliferative capacity increased in the light treatment of Cas1 KO cells;LDH detection assessed the cytotoxicity of each group,and the amount of LDH released from light damage of Cas1 KO cells decreased;mRNA and protein levels of key factors(GSDMD,IL-1?,IL-18)in the pyroptosis pathway were significantly reduced;(3)delphinidin treatment significantly reduced ROS production in 661 W cells;Scanning electron microscope was used to observe the ultrastructure of 661 W cell membrane.The surface morphology of the photo-damaged cells treated with delphinidin was complete,and the number of Pyroptotic bodies was reduced;Hoechst / PI double staining was used to compare the degree of cell damage in each group.After delphinidin treatment of photo-damaged cells,PI fluorescence expression is significantly reduced,and the number of necrotic cells is reduced;delphinidin reduced the mRNA and pyroptosis level of pyroptosis related proteins(NLRP3,ASC,Caspase-1,GSDMD,IL-1?,IL-18)in 661 W cells after light exposure;delphinidin reduces the activity of IL-1? and IL-18 secreted by 661 W cells after light exposure.ConclusionDelphinidin regulates oxidative stress to anti-RPD;there is a phenomenon of cell death in the pathological mechanism of RPD,Knock out Caspase-1 in 661 W photoreceptor cells can block pyroptosis,which can effectively protect cells;delphinidin may protect retina by anti-pyroptosis.