Abstract
Purpose :
Age-related macular degeneration (AMD) is a degenerative eye disease which may lead to central vision loss of patient. Researchers have investigated the relationship between AMD and autophagy and found that patients with AMD had dysregulated autophagy on their retinal pigment epithelium layer (RPE). Recently, ROCK inhibitors have been suggested by several studies that it may enhance autophagy and survival of RPE cells by altering their biochemical properties. Therefore, the aim of this study is to investigate if ROCK inhibitor enhances autophagy at the gene level using human ARPE-19 cell line.
Methods :
The study was done by incubating ARPE-19 cell line with or without different concentrations of Y-39983, a ROCK inhibitor, for 48 hours. Cell viability and morphology were examined. Cytoskeletal change was also explored by staining F-actin on the treated cells. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed for studying the expression level of various autophagy genes. Autophagy reflux assay was performed using flow cytometry approach.
Results :
Viability test showed that all the concentrations of Y39983 (0.01 μM, 0.1 μM and 1 μM) did not affect the cell viability. ARPE-19 cells treated with 0.1 μM and 1 μM Y39983, but not 0 or 0.01 μM Y39983 displayed stellate morphology. Our results demonstrated that 1 μM of Y-39983 significantly increased mRNA expression level of multiple autophagy-related genes (p< 0.05, Student’s t-test), including ATG5, ATG7, TFEB, NRF2 and p62. Moreover, green dye signal of the autophagy flux assay increased about 20%, indicating that Y39983 induced autophagy in ARPE-19 cells.
Conclusions :
Our findings suggest that Y39983 does not affect ARPE-19 cell viability, indicating the absence of toxicity. Y39983 is found to alter ARPE-19 intracellular cell architecture. Our results also suggest that ROCK inhibitor Y-39983 enhanced autophagy of ARPE-19 cells at the gene level and increased the autophagy flux. It provides us future research direction on how ROCK inhibitor involved in the signalling pathway of autophagy in RPE and apply ROCK inhibitor as treatment to facilitate autophagy and slow down degeneration of RPE cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.