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Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition,and explore the protection role of the 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) on Müller cells.Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly,including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B).High glucose group with 45,60,70 μmol/L CPDT and cultured them 72 hour was set as group C,D and E.Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability.Flow cytometry was used to measure the active oxygen and apoptosis index.The expression of nuclear factor erythroid 2-related factor 2 (Nrf2),hemeoxygenase-1 (HO-1),Bcl-2 and Bax protein were measured by Western blot.Results Compared with group A,the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05).Compared with the group B,the viability of Müller cells had changes in group C (t=0.97,P＞0.05),but recovered in group D and E (t=-4.17,-7.52;P＜0.05).Compared with group A,the FCM showed that the mitochondrial ROS levels was higher in group B (t=-30.99,P＜0.05).Compared with group B,the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05).Compared with group A,Bax,Nrf2 and HO-1 increased (t=-11.03,-63.17,-11.44;P＜0.05),while the bcl-2 decreased in group B (t=7.861,P＜0.05).Compared with the group B,Nrf2,HO-1 and Bax decreased (t=15.11,26.59,6.27;P＜0.05),while the bcl-2 increased in group D (t=-6.53,P＜0.05).Conclusions Under the high glucose,CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2,HO-1 and Bax protein of Müller cells.It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
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The pathogenesis of diabetic retinopathy is complicated.The vast network of multiple factors including unifying mechanism,inflammatory reaction,neuron degeneration and metabolic memory of glucose,and the four established pathogenic molecular pathways are hotspots of mechanism research for diabetic retinopathy.Nevertheless,these researches may be only one corner of the iceberg of DR mechanism,and we still face enormous challenges in DR mechanism research.Collaboration with multiple disciplines to study the relationship between DR and diabetes and other systemic diseases,search novel therapy targets may increase the result in an unexpected windfall for DR basic research.
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ObjectiveTo investigate Kir4.1 expressions in Müller cells under high glucose conditions and treatment of pigment epithelium derived factor (PEDF).Methods Cultured rat Müller cells were divided into control group (5 mmol/L glucose),high glucose group (25 mmol/L glucose),PEDF treatment group (25 mmol/L glucose+ 100 ng/ml PEDF) and intervention control group(25 mmol/L glucose +phosphate buffer solution). Kir4.1 expressions were measured by Western blot and real-time reverse transcription polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) productions were measured using 2′7′-dichlorofluorescin diacetate and glutathione peroxidase (GPx)expressions were studied by real-time RT-PCR.ResultsBy Western blot and real-time RT-PCR,it was found the expressions of Kir4.1 decreased obviously under high glucose conditions (real-time RT-PCR:t=4.12,P＜0.05; Western blot:t=3.53,P＜0.05) ; simultaneously,ROS generation was increased (t=3.76,P＜0.05) and GPx level was decreased (t=3.18,P＜0.05).PEDF treatment inhibited the high glucose-induced Kir4.1 down regulation (real-time RT-PCR:t =3.66,P＜0.05 ; Western blot:t =6.43,P＜0,01 ) and decreased ROS generations (t=4.11,P＜0.05) and increased GPx levels (t=5.12,P＜0.01).ConclusionsThe high glucose can supress Kir4.1 expressions in Müller cells by oxidative stress,and PEDF can ameliorate these effects.
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Objective To observe the effect of diabetic retinopathy on endothelial progenitor cells (EPCs)from peripheral blood.Methods Sixty male Wistar rats were divided into control group and diabetes group.The rats in diabetes group were induced with streptozotocin(STZ)injection for diabetic retinopathy model.Flow cytometry was used to identify and count the number of EPCs from peripheral blood at 1 week.1,3 and 6 months after injection.All eyeballs were examined by hematoxylin and eosin (HE)staining,periodic acid-Schiffs(PAS)staining of trypsin-digested retinal vessels flat preparation and transmission electron microscope.EPCs count,and the relationship between DR morphological changes and EPCs count were compared and analyzed.Results The quantity of EPCs from peripheral blood at 1 week,1,3 and 6 months after STZ injection were 25±7,28±8,39±7,43±7 cells per 200 000 monocytes respectively,which decreased compared with the control group 45±4 cells per 200 000 monocytes(F=8.933,P＜0.0 1).The quantity of EPCs was gradually increased at 1 week,1,3 and 6 months after STZ injection,accompanied with responsive pathological changes of retinal structure and vessels.The thickness of retina at 1 week and 1 month after injection were reduced slightly.The number of retinal ganglion cells reduced,with the time passing by.Endothelial cells were edema,mitochondrial was swollen,capillary basement membrane was thicken,lumen was significant stenosis,lumen occlusion and retinal artery aneurysm were observed at 6 months after STZ injection.Conclusion The number of EPCs increases gradually throughout the development of DR.
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Objective To investigate the amounts of endothelial progenitor cells(EPCs)in peripheral blood of patients with proliferative diabetic retinopathy(PDR).Methods Forty patients with PDR(PDR group),thirty patmnts with type 2 diabetes mellitus(DM)without DR(DM group),and twenty agematched normal subjects(control group)were enrolled in this study.Blood samples were treated bv repeated centrifugation and stained with monoclonal antibodies.At least 2 × 105 cells were analyzed bv flow cytometry.EPCs were identified by CD34 and CD133 antibody.The correlation between EPCs numbers and DR duration,glycosylated hemoglobin,serum lipids was analyzed.Results The number of EPCs in PDR,DM and control group were(49±12)、(35±11)、(90±25)cells/ml respectively,the difference was statistically significant(F=56.260,P=0.000).There was a positive correlation between EPCs numbers and DR duration(r=0.564,P＜0.05).However there was no correlation between EPCs numbers and glycosylated hemoglobin(r=-0.170,P>0.05)or triglyceride levels(r=0.261,P>0.05).Conclusions The number of EPCs in peripheral blood of PDR patients was decreased. EPCs might play an important role in the pathogenesis of PDR.