Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1

Wei Zhang,Yue Zhang,Yingxue Ma,Guanghui He,Jian Song,Song Chen,Jing Yang

doi:10.1186/s12886-018-0864-5

Abstract

BackgroundIn this study, we evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein.MethodsCultured human RPE cell line ARPE-19 cells were exposed to intermittent high glucose-induced oxidative stress. Reactive oxygen species (ROS) was determined by 2′, 7′-dichlorofluorescin diacetate (DCFH-DA); and malonyldialdehyde (MDA), superoxide dismutase (SOD) by commercial kits. Transmission electron microscopy was used to observe the generation of autophagosome. And MTT assay was used to examine the effect of autophagy on cell viability. For the inhibition experiments, cells were pre-incubated with lysosomal inhibitors NH4Cl or N-acetyl cysteine (NAC).Western blot was used to measure the expression patterns of autophagic markers, including LC3 and p62. The expression of HMGB1 was detected by immunohistochemistry.Cells were pre-incubated with HMGB1 inhibitor ethyl pyruvate (EP) ,then detected the expression pattern of autophagic markers and level of cellular ROS.ResultsWe found that intermittent high glucose significantly increased oxidative stress levels (as indicated by ROS, MDA, SOD), increased in the generation of autophagosome, decreased the level of p62, induced conversion of LC3 I to LC3 II. We further demonstrated that the NH4Cl/NAC inhibited intermittent high glucose-induced autophage by altered level of LC3 and p62. Intermittent high glucose-induced autophagy is independent of HMGB1 signaling, inhibition of HMGB1 release by EP decreased expression pattern of autophagic markers and level of cellular viability.ConclusionsUnder intermittent high glucose condition, autophagy may be required for preventing oxidative stress-induced injury in RPE. HMGB1 plays important roles in signaling for both autophagy and oxidative stress.

Highlights

[10] In this study, we evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein
We found that proliferative activity was decreased in both constant high glucose-treated cells (0.613 ± 0.077 OD) and intermittent high glucose-treated cells (0.527 ± 0.045 OD) compared with cells exposed to normal glucose (0.714 ± 0.089 OD) (p < 0.05) (Fig. 1a)
MDA and superoxide dismutase (SOD) assays, we found that intermittent high glucose resulted in dramatic significantly higher fluorescence of cellular reactive oxygen species (ROS) marker DCFDA (772.41 ± 20.352 OD) than constant high glucose (697.98 ± 27.798 OD) and normal glucose(300.04 ± 14.503 OD) (Fig. 1b, c)

Summary

Introduction

We evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein. Fluctuating glucose promotes a greater increase in inflammatory cytokine production from retinal endothelial cells than constantly high glucose through release of reactive oxygen species (ROS) which is another important trigger for DR pathogenesis [6]. The damaged autophagy or lysosome activity may lead to insufficiently remove the intracellular organelles or protein aggregates of oxidative damage, which leads to the accumulation of toxic substances within and outside the cells and damages the RPE function during DR

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