MicroRNA-191-5p Ameliorates Aβ_1-40-mediated Retinal Pigment Epithelium Cell Injury By Suppressing The NLRP3 Inflammasome Pathway

Amyloid β(Aβ)is a crucial component of drusen,the hallmark of the early-stage of age-related macular degeneration(AMD),which can cause retinal pigment epithelium(RPE)cell damage through activation of inflammatory response.Aβ-mediated activation of the NLRP3 inflammasome is essential in the pathophysiology of RPE injury in AMD.However,there is a limited understanding of the mechanism involving Aβ in activation of the NLRP3 inflammasome.This study consists of three parts:(1)Aβ1-40 induces RPE damage by the activation of the NLRP3 inflammasome;(2)The mechanism of C/EBPβ transcription regulation on NLRP3;(3)The mechanism of miR-191-5p ameliorates Aβ1-40-mediated RPE injury.ObjectiveTo clarify the phenotype of Aβ1-40 activating the NLRP3 inflammasome and inducing RPE damage;to explore the transcriptional regulation mechanism of C/EBPβ on NLRP3;to explore the protective effect of miR-191-5p on Aβ1-40 induced RPE damage mechanisms.MethodsTo establish Aβ1-40-induced RPE injury models,C57/BL6 mice received an intravitreal injection(IVI)of Aβ1-40,and mouse primary RPE cells were cultured with Aβ1-40 to construct an in vitro damage model of RPE cell.Fundus photography,OCT and ERG were used to detect changes in retinal structure and function;Confocal immunofluorescence staining and H&E were used.was used to determine the damage of RPE cells.Realtime-PCR,western blotting and immunofluorescence staining were performed to determine the expression of the NLRP3 inflammasome pathway.bioinformatics method was used to predict the binding site of the NLRP3 promoter region and C/EBPβ motif.Luciferase reporter assays were applied to verify that C/EBPβ specifically binds to the NLRP3 promoter region and miR-191-5p was a repressor of C/EBPβ.miR-191-5p was overexpressed in vitro and in vivo and RT-PCR and western blotting were used to detect the expression of C/EBPβ and the NLRP3 pathway factors and ERG was performed to determine the protective effect of miR-191-5p.ResultsIn this study,we demonstrated Aβ1-40 induced RPE impairment through the activation of the NLRP3 inflammasome which is up-regulated by transcription factor C/EBPβ.The endogenous miR-191-5p decline induced by Aβ1-40 resulting in the increase of C/EBPβ.Overexpression of miR-191-5p,a repressor of C/EBPβ,alleviates RPE cell injury.ConclusionsThe current study elucidates a novel transcriptional regulatory mechanism of NLRP3 and indicate the anti-inflammatory effect of miR-191-5p in Aβ1-40-induced RPE impairment,shedding light on novel preventive or therapeutic approaches for RPE impairment associated AMD.