Study On The Role Of REL Family Members In Regulating Aβ_1-40-mediated Pro-inflammatory Cytokine Secretion By Retinal Pigment Epithelial Cells

Purpose:Retinal pigment epithelium（RPE）cells is the key pathological changes of age-related macular degeneration（AMD）.Previous studies indicated that after RPE cells’degeneration,the inflammatory microenvironment caused by impaired immune regulation function of RPE cells plays a critical role in AMD progress.Recent researches found that amyloid beta（Aβ）,an important component of AMD’s hallmark“drusen”,is likely to induce inflammatory microenvironments by inducing inflammatory cytokines’secretion in RPE cells.In this study,Aβ-mediated RPE degeneration model was established to explore the possible mechanism and intervention target of the inflammatory microenvironment.Methods:Intravitreal injection of Aβ_1-40 in C57BL/6 mice was performed to establish the Aβ_1-40-mediated RPE degeneration model in vivo.Intravitreal injection of PBS was performed in control group.The function and morphological damage of RPE cells were evaluated by electrophysiological and hematoxylin-eosin（HE）staining,respectively.The mRNA level of IL-1β,IL-18,IL-12b,IL-8,IL-6 and REL family members was detected by qPCR.At the same time,Aβ_1-40-mediated RPE degeneration model in vitro was created by introducing Aβ_1-40 to ARPE-19 cells and primary mouse RPE cells respectively.Western blot was used to detect REL expression level both in the nucleus and cytoplasm.Immunofluorescence was performed to investigate REL distribution.REL family’s activation form was evaluated by using immune Co-Immunoprecipitation（Co-IP）.The mRNA levels of IL-1β,IL-18,IL-12b,IL-8,IL-6were detected after the expression of REL family was silenced respectively by siRNA.The statistical method used was Student’s t test and P<0.05 was considered statistically significant.Results:After the application of Aβ_1-40-40 intravitreal injection,we found that the amplitude of a wave and b wave in the model group was lower than that in the PBS group（P<0.05）,and the morphology of RPE cells was altered.Meanwhile,the mRNA level of IL-1β,IL-18,IL-12b,IL-8,IL-6 and REL family in RPE cells increased in model group（P<0.05）.In addition,the level of REL family expression was elevated both in RPE cell nucleus and cytoplasm after stimulation of Aβ_1-40（P<0.05）.We observed that REL mainly distributed in the nucleus of the family of proteins,indicating the activated state of REL family.Then,we also found that after the REL family was activated,they would mediate the transcription of downstream inflammatory factors in the form of dimer.When the expression of REL protein family was silenced,the level of pro-inflammatory cytokines was decreased（P<0.05）.Conclusion:The effect of Aβ_1-40 is multi-targeted.REL family members regulates Aβ_1-40-mediated RPE cell injury secretion inflammatory factor.When REL family members are activated after the stimulation of Aβ_1-40,they form dimer with each other,transfer into the nucleus,and combine with the promoter of downstream pro-inflammatory cytokines.At the same time,the regulation between REL family members leads to a positive feedback of retinal inflammation.REL family members may become new intervention targets of Aβ_1-40-mediated RPE degeneration.