Abstract
Purpose:
Autophagy is an important lysosomal protein degradation pathway in cells responsible for removing and recycling damaged proteins and molecules to maintain cell integrity. Autophagy has been described to be altered in retinal cells under diabetic conditions together with endoplasmic reticulum stress. Apoptosis appears to play an important role in the pathogenesis of diabetic complications. The aim of this study was to investigate the role of autophagy on Muller cell apoptosis under diabetic conditions.
Methods:
Rat Muller cells (rMCs) were cultured in normal glucose (5.5 mM), high glucose (30 mM) and under oxidative stress conditions (H2O2; 10 µM) for 24, 48, 72h in presence of different pharmacologycal inhibitors or siRNA. The markers of autophagosome formation, Beclin1 and LC3, and autophagosome-lysosome fusion, p62, and those of ER stress, peif2α and CHOP, were measured by western blot (WB). Intracellular reactive oxygen species (ROS) were evaluated by 2'-7' dichlorofluorescein (DCFH(2)) assay and apoptosis was assessed by TUNEL and extrinsic apoptotic pathway by caspase 8 activity.
Results:
In rMCs, HG and/or H2O2 increased Beclin1, LC3 and p62 levels, p-eif2α and CHOP (p<0.05) as well as the activation of caspase 8 and rate of cellular apoptosis (p<0.05). Inhibition of autophagic flux through 3 methyladeninel and bafilomycin under HG increased further p62 and p-eif2α levels, caspase 8 activation and cellular apoptosis (p<0.05). p62 siRNA was able to prevent these changes (p<0.05). The presence of PERK Iinhibitor, which inhibits the expression of protein kinase RNA-like endoplasmic reticulum kinase, decreased the activation of caspase 8 (p<0.05). N acetylcysteine (NAC), an antioxidant compound, was able to prevent the increase of intracellular ROS production (p<0.05) and reversed the increments of Beclin1, LC3 and p62, caspase 8 activity and cellular apoptosis (p<0.05).
Conclusions:
rMCs exposed to HG displayed a reduction of autophagic flux as demonstrated by increased levels of p62 for a defective clearance of autophagosomes by lysosome system. The accumulation of p62 levels causes activation of caspase 8, via ER stress, leading to rrMCs apoptosis. The modulation of p62 levels could be a possible therapeutic target to prevent retinal Muller cell apoptosis under diabetic milieu conditions.