Effect of hypoxia on autophagy in R28 cells under low and high glucose conditions

Abstract

Purpose:Retinal ischemia/reperfusion (I/R) injury occurs in various ocular diseases, such as diabetic retinopathy, and is a common cause of visual impairment and blindness. There has been increasing interest in autophagy as a potential therapeutic target for ocular diseases but the role of autophagy in retinal I/R injury remains controversial. Our previous in vivo research showed that retinal I/R injury (middle cerebral artery occlusion stroke model) resulted in autophagy upregulation in the inner retinae of Akita (type I diabetic) and wild-type mouse. In this study, we used an in vitro model to further investigate the role of autophagy in retinal cells exposed to hypoxic conditions under different glucose levels. Methods:The R28 retinal precursor cell line (R28) was chosen for its rat retinal origin and heterogenous nature, which reflects the diversity of cell types found in the retina. R28 cells were cultured in low glucose DMEM medium with 10% fetal bovine serum and pCPT-cyclic AMP to induce cell differentiation into a more neuronal-like phenotype. To mimic a hyperglycemic state, cells were incubated with additional glucose. Hypoxia was chemically induced in both low and high glucose-treated cells using cobalt (II) chloride (CoCl2) for 24 hours. Cell viability was determined using the MTS assay. Autophagy was assessed through the detection of microtubule-associated light chain protein LC3 (an autophagosome marker) by Western blot. Results:Cells subjected to CoCl2-induced hypoxia had lower cell viability as compared with untreated cells (control group). Hypoxia resulted in significant autophagic upregulation in both low glucose and high glucose-treated cells. LC3 expression was higher in high glucose-treated cells as compared with low glucose-treated cells after hypoxia induction. Conclusions:CoCl2-induced hypoxia induces autophagy activation in retinal cells. Both our in vivo and in vitro results suggest that elevated glucose levels may further exacerbate retina I/R injury-induced autophagy. Autophagy modulation may represent a potential therapeutic strategy for retinal I/R injury related diseases.