Study On Role Of Mitochondrial Reactive Oxygen Species In Diabetic Retinopathy And Possible Mechanism

Diabetic retinopathy is one of the main causes of blindness in developed country. As for its molecular mechanism, there are four hypotheses: glucose-induced activation of protein kinase C isoform; increased formation of glucose-derived advanced glycation end-products; increased glucose flux through the polyol pathway; and increased flux through hexosamine pathway. Though each of these pathways is supported by animal studies, only some pathogenesis processes can be explained by these four hypotheses. A new hypothesis about pathogenesis of diabetic retinopathy--mitochondrial superoxide overproduction is placed at the heart of pathogenesis of diabetic microvascular complications. Mitochondria are the key site of reactive oxygen species production. Mitochondrial reactive oxygen species overproduction is induced by hyperglycemia. Then the other processes are switched on. Normalizing mitochondrial superoxide production can block other pathways of hyperglycemic damage. How could mitochondrial superoxide increase in hyperglycemia? Is the increasing related to the weakened mitochondrial antioxidation mechanism? Manganese superoxide dismutase is an important antioxidation enzyme in mitochondria. Is its expression inhibited in hyperglycemia? Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier gene family and, as suggested by their name, can uncouple ATP production from mitochondrial respiration by causing proton leak. As an elevated proton electrochemical potential gradient favors superoxide production, limiting the magnitude of this gradient should decrease superoxide production. Five different UCPs have already been identified-UCP1-5. These proteins are expressed in different tissue. Retinal capillary cells including endothelial cells and pericytes are main targets of hyperglycemia damage. Which UCPs isomers are expressed on them? Will their expression be modified in hyperglycemia?So in this study, bovine retinal endothelial cells and pericytes were cultured in different concentration glucose. Mitochondrial ROS quantification, its effects and possible causes were studied. The expression of UCPs and MnSOD in the retinal capillary cells and their modification in high glucose concentration were also studied. The role of ROS and UCPs on the pathogenesis of diabetic retinopathy wasinvestigated.Section 1 Reactive oxygen species changes in mitochondria of retinal endothelial cells and pericytes induced by high glucose and pathogenesis Objective To study reactive oxygen species (ROS) changes in mitochondria of bovine retinal endothelial cells and pericytes cultured in high-glucose. To study the causes and effects of these changes, and to further explore their pathogenesis. Methods ①Bovine retinal endothelial cells and pericytes were cultured with selective culture media. ROS changes in mitochondria of retinal endothelial cells and pericytes in different glucose concentration were detected with scanning laser confocal microscope. ②The mitochondria membrane potential (MMP) and cell death rate (CDR) were measured with flowing cytometer. ③Uncoupling protein (UCP)1-5 isomers expression in capillary cells were detected by immunocytochemical and immunofluorescence methods. Expression of MnSOD and UCP1-3 and changes in different glucose were detected with reverse-transcriptase PCR (RT-PCR). Results ①in high-glucose, ROS in mitochondria of retinal endothelial cells and pericytes increased. ②MMP and CDR of endothelial cells increased in high-glucose, too. No changing tendency was found in pericytes.③ UCP1,2 were detected positively by both immunocytochemical and immunofluorescence methods. UCP1, 2 mRNA was expressed in cultured cells whereas UCP3 was not. The expression of UCP1, 2, MnSOD mRNA changed with the changing of glucose concentration. Conclusions Increasing of ROS production in mitochondria of retinal endothelial cells and pericytes can be induced by high glucose. There is a feedback accommodation mechanism between ROS increasing and MMP elevation in endothelial cells. In high-glucose, expression of UCP1,2,MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared as glucose concentration was too high. The different features of endothelial cells and pericytes in high glucose demonstrate their effects in pathogenesis of diabetic retinopathy are different.Section 2 Expression changes of UCPs,MnSOD in retina of diabetic rats induced by streptozotocinObjective To study ocular characteristics of diabetic rats induced by STZ including the occurrence of diabetic cataract and retinopathy. To explore a three-dimensional examination method for retinal vessels. To study expression of uncoupling proteins homologues in retina of rats. To observe changes of mRNA expression of UCPs and MnSOD in different courses of diabetes. The molecular mechanism of diabetic retinopathy was discussed.Methods ①Diabetic Sprague-Dawley rats were induced by STZ. Ocular features were studied by OCT and FFA.②The rats were sacrificed and perfused till eyeballs pale. Then 4% paraformaldehyde was perfused for vascular internal fixation. Eyeballs were fixed in 4% paraformaldehyde for 24hr, and the retinal vessels were isolated by collagenase digest technique. After immunofluorescence staining, the retinal vessels were observed with laser scanning confocal microscope.③ To detect UCPs homologues 1～5 expression in normal retinal capillary net of SD rats by immunofluorescence. To evaluate UCP1-5 mRNA content in normal SD retina with cerebral cortex, skeleton muscle and lens acted as positive control. Diabetic rats induced by STZ were humanely sacrificed 2,4,6,8 and 12 weeks after the onset of diabetes. The mRNA expression of UCPs and MnSOD in retina was detected with RT-PCR.Results ①10/19 rats (52.6%) exhibited cataract 12 weeks after the onset of diabetes. At 16 weeks after the onset of diabetes, 9 rats among 13 rats (69.2%) exhibited cataract. Fundus exam, OCT and FFA found no prominent diabetic retinopathy. ② Ideal complete retinal vascular digest preparations were obtained after collagenase digestion. Three-dimensional characteristics of retinal vessels can be observed by laser scanning confocal microscope. ③By immunofluorescence tests, UCP1 and UCP2 were positive in retinal capillary net whereas UCP3, UCP4 and UCP5 were negative. UCP2,4 and 5 mRNA can be detected in retina of normal rats. Its content is lower than that of cerebral cortex. UCPs,MnSOD mRNA expression changed with the courses of disease. The expression increased at first and then decreased gradually. Conclusions ①Diabetic retinopathy remained at background stage 16 weeks after the onset of diabetes. It was suitable for early pathology study of diabetic retinopathy. Occurrence rate of cataract is high in diabetes model induced by STZ. It can be used in the study of diabetic cataract. ②Modified retinal vascular digest preparations combined with laser scanning confocal microscope provided us a method for retinal vascular three-dimensional structure observation. ③UCP1 and UCP2 were expressedin normal SD retinal capillary net. UCPs isomers expressed in normal retinal were similar to those of cortex. UCP4 and 5 are expressed specifically in neuro-tissure of retina. The mRNA expression of UCPs and MnSOD changed with the courses of diabetes and which is related to the occurrence of diabetic retinopathy. It is supposed that the mitochondrial ROS overproduction is the key pathway.