Effects Of Oxidative Stress Induced By Intermittent High Glucose On Autophagy In ARPE-19 Cells And Its Underlying Mechanisms

Objective:To observatethe level of intracellular oxidative stress and autophagyinhuman retinal pigment epithelium(ARPE-19) cell under the condition ofintermittent high glucose.The relationship between oxidative stress and autophagy was studied,and the specific pathway was elucidatedto provide theoretical basis for the treatment of diabetic retinopathy(DR) by regulating autophagy.Methods:1.According to glucose concentration, the ARPE-19 cells were cultured in normal ?group, high glucose(HG) group, intermittent high glucose(IHG) group, and hyperosmotic(H) group for 48 hours. Cell viabilitywas measured by MTT under different glucose concentrations. The levels of oxidative stress aboved groups were detected by different oxidative stress kits.2.The formation of autophagosome was observed by transmission electron microscope( TEM) under the condition of IHG.Western blot(WB) was used to detecte the level of intracellular autophagy. Autophagy flux was also detected by WB after pretreatment with ammonium chloride(NH4Cl) which was one of autophagy blocker.The level of intracellular autophagy was again detected by WB after pretreatment with N-acetyl cysteine(NAC) which was one of antioxidant for further to study the relationship between oxidative stress and autophagy.3.The distribution of high mobility group protein 1(HMGB1) in the cells was observed under the condition of IHG.WB was used to detect the level of intracellular autophagy after pretreatment with ethyl pyruvate(EP) which was one of HMGB1 blocker.ROS kit was used to detect the level of intracellular ROS.Results:1. Cell morphology: Cells which were in order showed long spindle shape or triangle or a small amount of irregular shape in C group.A few black particles were seen in cytoplasm. In H group, there was no significant difference compared with the C group. However, the shape of cells was multiplicity, irregularity and irregular cells increased significantly in HGor IHG group. The changes of cells showed more obviously in IHG group. 2.Cell viability: There was a decline of cell viability under abnormal high blood glucose. The decline was the most obvious in IHG group. The differences among groups were statistical significance(p<0.05). There was no significant difference in H group compared with C group(p>0.05).3.Oxidative stress:Compared with C group, the levels of ROS and MDA were significantly increased in HG and IHG groups, while the levels of SOD were obviously decreased. The difference was statistically significant(p<0.05). However,it had no significant difference in H group compared with C group(p<0.05). Compared with HG group, the levels of ROS and MDA were significantly higher in IHG group, and the difference was statistically significant(p<0.05), but the levels of SOD was not statistically significant(p>0.05). 4.Autophagy: Autophagosomesin IHG group were significantly more than that in C group observed by TEM. FOR protein expression: Compared with C group, the expression of LC3 was significantly increased in IHG group, while p62 was obviously decreased, and the difference was statistically significant(p<0.05). After pretreatment with NH4 Cl, the expression of LC3 was decreased, while p62 was increased, and the difference was statistically significant(p<0.05). After pretreatment with NAC, the expression of LC3 wasalso decreased, and p62 was increased, and the difference was statistically significant(p<0.05). 5.Immunofluorescence:Compared with C group, HMGB1 released from the nucleus to the cytoplasm, which was significantly morein IHG group.Punctiform strong green fluorescence was seen in cytoplasm under fluorescence microscope. After pretreatment with EP,the expression of LC3 was significantly decreased, while p62 was increased,and the difference was statistically significant(p<0.05).At the same time, the level of ROS in the cells was increased after pretreatment with EP, and the difference was also statistically significant(p<0.05).Conclusions:1.There can be a certain degree of damage for ARPE-19 cell under abnormal high blood glucose environment, especially under the condition of IHG. 2.The levels ofoxidative damage for ARPE-19 cellwere obviously increased under environment of IHG, then, the oxidative damage was able to induce autophagy. 3.ARPE-19 cell through the HMGB1 to start autophagy under the condition of IHG. The autophagy which played a protective role can reduce the level of intracellular oxidative damage.