The effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell

Abstract

Objective To observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3). Methods Cultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β , IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Results Blue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like doubleconcave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04), IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P<0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P<0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P<0.001). Conclusion The expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells. Key words: Retinal pigment epithelium/physiopathology; Exosomes; Inflammasomes; Photic stimulation/adverse effects