Authors: Jiang Wenjing, Zhang Lina, Yu Xiao, Niu Yingjun
The light damage model of retinal pigment epithelium (RPE) cells is a research direction of retinal degeneration diseases, and RPE cell apoptosis induced by light damage and inflammation is an important pathologic basis of light-induced RPE cell damage.However, whether endoplasmic reticulum stress (ERS) paticipates in light-induced RPE cell damage is rarely reported.
This study was to explore the effects of ERS on light-induced RPE cell damage.
Human RPE cell line (ARPE-19) was cultured, and light damage models were created by irradiating the cells for 3-, 6-, 12- and 24-hours with white fluorescent lamp with the intensity of (2 000±500)lx for the selection of optimal irradiating time, and the cells in the normal control group were cultured in the dark environment.The cells were divided into normal control group, light exposure group and 4-phenylbutyric acid (4-PBA) pretreated+ light exposure group.The cells from 4-PBA pretreated+ light exposure group were cultued firstly with 4-PBA for 30 minutes and followed by light exposure for 12 hours.The apoptisis rate of the cells and intracellular reactive oxygen species (ROS) content were detected by flow cytometry; the concentrations of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were assyed by ELISA.The relative expressing levels of activating transcription factor 6 (ATF-6), C/enhancer binding protein homologous protein (CHOP) and caspase-12 mRNA and protein in the cells were detected by real-time quantitative PCR and Western blot, respectively.
The cultured cells showed a long spindle shape, the border was not clear, the cytoplasm was degranulation, and the cell fragments increased.Flow cytometry showed that compared with the normal control group, the ROS content in the cells and the apoptosis rate were evidently increased with the lapse of light exposure time (F=763.00, 119.30, both at P<0.01). ELISA results showed that the concentrations of IL-1β and TNF-α in the cell supernatant were significantly higher in the light exposure 6-hour group than those in the normal control group with the peak value in the light exposure 12-hour group.Compared with the normal control group, the relative expression levels of ATF-6, CHOP and caspase-12 mRNA and protein in the cells were elevated in the light exposure group and peaked in the light exposure 12-hour group.In addition, the relative expression levels of ATF-6 mRNA, CHOP mRNA and caspase-12 mRNA in the cells were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=281.69, 473.88, 308.45, all at P<0.01), and their proteins were also significantly reduced (F=47.86, 57.93, 106.59, all at P<0.01). The apoptosis rate, concentrations of IL-1β and TNF-α in the cell supernatant were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=88.64, 245.47, 101.01, all at P<0.01).
The light exposure at (2 000±500)lx induces intracellular ROS accumulation and activates the ERS response, which results in apoptosis and inflammatory process of human RPE cells.4-PBA, a inhibitor of ERS, can suppress light-induced ERS response and therefore reduces the apoptosis rate and inhibits inflammatory process.