An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells

摘要

DMEM/F12 nutrient mixture, a recommended media for ARPE-19 culture, contains glucose concentration of 17.5mM. But, several recent studies employed normal glucose media (5.5mM) that was shown to affect the growth and function of ARPE-19 cells. Here, we set a protocol to study the effect of hyperglycemia on intracellular oxidative stress and redox status in ARPE-19 using DMEM/F12 as control. The WST-1 assay was performed to analyze the viability of ARPE-19 upon glucose treatment. The intracellular oxidative stress was measured by a dichlorofluorescein assay. The mitochondrial membrane potential (MMP) was monitored by using a JC-10 MMP assay kit. The expression of antioxidant marker proteins was analyzed by western blotting. Exogenous addition of glucose (7.5 and 12.5mM) for 24 and 48h did not change the viability and morphology of ARPE-19 cells. Hyperglycemia increased intracellular ROS level and decreased MMP in a dose-dependent manner. High-glucose treatment for 24h down-regulated the protein expression of redox-specific transcription factors Nrf-2, XBP-1 and NF-B, and subsequently decreased the expression of HO-1, catalase, and SOD-2. This study offers baseline information for the subsequent use of DMEM/F12 nutrient mixture to study glucose-mediated changes in intracellular oxidative stress and redox status of ARPE-19 without affecting its basic functions.