Abstract
BackgroundOur study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells).MethodsThe morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment.ResultsARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells.ConclusionAutophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Highlights
Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells)
High glucose treatment After the cells were exposured to high glucose for 48 h, the ARPE-19 cells treated with 50 mM (68.48 ± 4.57%) or 70 mM (36.69 ± 8.94%) d-glucose had significantly lower cell viability (p < 0.05)
Structural changes and reactive oxygen species (ROS) generation induced by high glucose The transmission electron microscopy revealed that the ARPE-19 cells incubated with normal glucose (5.5 mM; control) or high glucose (30 mM; HG) for 48 h had a significant increase in the number of double-membrane vacuoles, which were typical of autophagosomes (Fig. 2a)
Summary
Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). Diabetic retinopathy (DR), the most common visual complication of diabetes, is the leading cause of blindness among. DR is mainly caused by hyperglycemia, hypertension, dyslipidemia and defects of insulin signaling pathways [6, 7]. Autophagy is a lysosomal degradation pathway that controls cellular bioenergetics and cytoplasmic quality [8]. Autophagy is regulated by multiple forms of cellular stress, such as endoplasmic reticulum (ER) stress, nutrient or growth factor deprivation, hypoxia, cytotoxicity. Mitophagy is a specialized process of autophagy that could reduce cellular damage and eliminate the need to support ineffective organelles by removing damaged mitochondria where increased levels of reactive oxygen species (ROS) are generated [11]. Accumulating data have suggested that autophagy have important effects on the pathobiology of DR [7, 12, 13]
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