The Function And Mechanism Of PIDD In Regulating Irradiated BV-2Cells’ Inflammatory Response

【Background and purpose】: PIDD(P53-induced protein with a death domain),as one of the target genes of tumor supressor P53, plays a critical role in theregulating of cell cycle as well as apoptosis. However, the research of the expressionand function of PIDD in the irradiated microglia’s inflammatory response haven’tbeen reported yet. We conducted this study to investigate the expression and functionof PIDD in the irradiated microglia’s inflammatory response.【Methods】:1, We designed three distinct anti-PIDD-si-RNA(antisenseoligonucleotides targeting murine PIDD mRNA).2,GFP-labled blank plasmids andsiRNA were co-transfected into BV-2cells, and the transfection efficiency wasassessed by observing the green fluorescece using a fluorescence microscopy;Real-Time PCR was used to choose the anti-PIDD-si-RNA with the mostdownregulating effects to carry out the following expriments.3,BV-2cells weretransfected with obtained anti-PIDD-si-RNA and its negative control.12hours aftertransfection, BV-2cells were stimulated by radiation(0,32Gy).4, The PIDD, IL-1β,TNF-α mRNA expression level of different time(0h,3h,6h,12h,24h) after irradiationwas determined by Real-Time PCR.5, Western blotting was used to detect the PIDD and NF-κB protein.6, Confocal microscopy was applied to detect the expression ofIba-1, CD68(the expression of these two protein represents the activation ofmicroglia).7, The ability of migration of BV-2was analysed by transwell assay.8,Electron microscope was used to assess the changes of cellular organelles.【Results】:1, the transfection efficiency of siRNAwas approximately50%-60%,and the anti-PIDD-si-RNA2possesses the most downregulating effects.2, irradiationwas able to elevate the mRNA expression of PIDD and the inflammatory cytokinessuch as IL-1β, TNF-α while the anti-PIDD-si-RNA2can significantly downregulatethe mRNA expression of PIDD, IL-1β and TNF-α after irradiation.3,the expression ofPIDD protein and the NF-κB protein of irradiated BV-2cells was elevated within24hours while the anti-PIDD-si-RNA2can significantly downregulate them.4,irradiation was able to activate microglia cell with expression of Iba-1, and CD68,However, anti-PIDD-si-RNA2was significantly inhibit the expression of of Iba-1,and CD68.5,the ability of migration of activated microglial was raised by irradiationwhile the anti-PIDD-si-RNA2can decrease it.6, irradiated BV-2cells present moresecondary lysosomes and severe degeneration of endoplasmic reticulums andmitochondrions while the BV-2cells transfected with anti-PIDD-si-RNA2present lesssecondary lysosomes and mild degeneration of endoplasmic reticulums andmitochondrions.【Conclusions】: Downregulation of PIDD can significantlyinhibit the activationof radiated microglia cells and the expression of inflammatory cytokines.PIDD/NF-κB signal pathway may be involved.