NLRP3 Inflammasome Activation By All-Trans-Retinal Underlies The Death Of Retinal Pigment Epithelium Cells

Objective:To investigate the mechanism of atRAL-induced retinal pigment epithelium(RPE)cell death.Methods:Experimental research.Human RPE cell lines were used for this study.Cellular toxicities were assayed by MTT analysis and we selected the appropriate concentration and time to explore the mechanism.Real-time PCR was used to detect changes of inflammatory factors.Protein levels of NLRP3,ASC,Caspase-1 p20,pro-IL-1?,and pro-IL-18 were measured by Western blotting.Secreted IL-1? and IL-18 in culture supernatant were analyzed by immunoblotting and enzyme-linked immunosorbent assay(ELISA).NLRP3 expression,caspase-1 activity and lysosomal cathepsin localization were determined by immunofluorescence staining.Results:atRAL induces the death of ARPE-19 cells in a time-and concentration-dependent manner.The expression levels of NLRP3,ASC,caspase-1 p20,IL-1? and IL-18 in ARPE-19 cells were significantly increased by atRAL.atRAL induced the increase of apoptosis markers PPAR and Caspase-3 in ARPE-19 cells and the increase of pyroptosis marker cleaved GSDME,but no significant change of cleaved GSDMD protein.The combined treatment of atRAL with AC-YVAD-CMK,an inhibitor of caspase-1 activity,can significantly reduce the toxicity of atRAL to ARPE-19 cells.NLRP3siRNA and atRAL co-treatment significantly inhibited the overexpression of NLRP3 induced by atRAL,and the levels of cleaved Caspase-1 and mature IL-1?and IL-18 proteins after NLRP3siRNA and atRAL co-treatment were significantly lower than those induced by atRAL alone.The activation of NLRP3 inflammasome was significantly inhibited after Ca-074-Me and Pepstain A,the inhibitors of Cathepsins treated ARPE-19 cells working together with atRAL compared with atRAL treatment.Conclusion:atRAL induced ARPE-19 cell death by activating NLRP3 inflammasome.atRAL impaired mitochondrias and lysosomes of ARPE-19 cells and activated NLRP3 inflammasome,which released ROS,cathepsin B and D,respectively.Both apoptosis and pyroptosis were involved in the process of cell death induced by atRAL...