Role of X-box Binding Protein 1 in Acrolein-induced Oxidative Injury of Retinal Pigment Epithelium / 中山大学学报(医学科学版)

ABSTRACT

[Objective]The oxidative injury of retinal pigment epithelium(RPE)plays a key role in the pathogenesis of age-related macular degeneration(ARMD). This study is to investigate the effects of endoplasmic reticulum stress and the vital transcriptional factor X-box binding protein 1(XBP1)in acrolein-induced oxidative damage of RPE.[Methods]RPE cells were treated with acrolein (75μmol/L)for 2~24 h,expression of glucose regulated protein 78(GRP78)and XBP1 was determined by Western blot analysis. After being transfected with XBP1 siRNA with 24 h,the expression of XBP1 was knocked-down in RPE cells. Protein level of Nrf2 and SOD2 was then determined by Western blot analysis and intracellular Reactive Oxygen Species(ROS)generation was determined by DCF staining. Acrolein was added for 8 h after being transfected with XBP1 siRNA or control siRNA for 24 h. Apoptosis was detected by TUNEL assay before and after the treatment. Subretinal injection of Cre or GFP adenovirus was performed in XBP 1flox mice. After 1 week the mice were sacrificed. Total RNA was extracted from mice eyecups using TRIzol and real-time RT-PCR was performed to determine the two XBP1 down-stream genes,ERdj4 and p58IPK. Cryosectioning and immunofluorescent staining were performed to look at the expression of XBP1,Nrf2 and SOD2 in mice RPE.[Results]Protein level of GRP78 was significantly un-regulated after exposure to acrolein for 2 and 4 h. XBP1 was activated after acrolein treatment for 6 h. Knock-down of XBP1 by siRNA down-regu?lates anti-oxidant genes expression and increased ROS generation in RPE cells. Loss of XBP1 exacerbates acrolein-induced cell apoptosis. XBP1 was knocked-down in the RPE of XBP1flox mice after subretinal injection of Cre adenovirus. Decreased mRNA level of ERdj4 and p58IPK,and decreased Nrf2 and SOD2 expression were seen in the Cre-injected group.[Conclusions]Acrolein induces ER stress and activates XBP1 in RPE cells. Knock-down of XBP1 down-regulates anti-oxidant genes expression ,increases ROS generation,and exacerbates acrolein-induced cell apoptosis. XBP1 plays a role in the anti-oxidant defense in the RPE cells.