Researches On Mechanisms Of Oxidative Injury In Human Retinal Pigment Epithelial Cells Induced By High Glucose

Diabetic retinopathy (DR) is one of the severe complications of diabetes. High glucose is a major etiological factor of DR and there is direct evidence that the progression of DR is related to poor glycemic control. Recent studies have shown that oxidative stress as a pathologic progress involved in several severe retinal diseases, many causative agents including high glucose can educe pathopoiesis effect through this way. Reactive oxygen species and reactive nitrogen species play an important role in oxidative stress damage. Retinal pigment epithelium plays a central role in retinal physiology by forming the outer blood-retinal barrier and supporting the function of the photoreceptors. It suggests that high glucose can influence the growth of RPE cells and the function of outer retina barrier. But the exact mechanism is still poorly understood. P38 mitogen activated protein kinase signaling pathway is a stress sensitive pathway, which can be activated by high glucose. Our study is to investigate the effect of high glucose on the growth of RPE cells and the molecular events involved in this bioprocess, focusing especially on the changes of iNOS and ROS induced by high glucose treatment and the effects of p38 MAPK on the level of iNOS expression.We cultured human RPE cells in vitro and the cells were divided into four group: control group: DMEM culture solution including 5.5mmol/L glucose; high glucose group: DMEM culture solution including 33mmol/L glucose; high glucose plus SB203580 group: DMEM culture solution including 33mmol/L glucose plus 10μmmol/L SB203580; mannitol group: DMEM culture solution including 5.5mmol/L glucose and 27.5 mmol/L mannitol. Cell morphous was investigated by phase contrast inverted microscope. Cell viability was assessed by the MTT assay. The changes of 3-NT, p-p38MAPK and iNOS expressed by RPE cells were studied with immunofluorescence staining method and western blotting analysis in a qualitative way. ROS change of RPE cells in response to high glucose was detected with scanning laser confocal microscope. The validity of SOD was detected by Xanthine Oxidase Enzymic method.Compared to the controls, the treatment of RPE cells with 33mmol/L glucose caused an obviously decrease of cellular viability. After pretreated with SB203580, cell viability was elevated compare with high glucose group. The phosphorylation level of p38MAPK, iNOS and 3-NT expression were enhanced obviously by high concentration glucose, ROS expression increased and SOD validity decreased in high glucose group. After pretreated with SB203580, the high glucose-evoked 3-NT and iNOS expression was significantly inhibited.Together with our studies, we concluded that: high glucose can damage RPE cells. The mechanisms are that high glucose can evoke ROS expression and disrupts the oxidative equilibrium in RPE cells and RPE cells cultured with high glucose can enhance expression of iNOS, at least in part, by activation of p38 MAPK pathway. It suggests that RPE cells under the effect of high glucose may participate in the pathological reaction of diabetic retinopathy.