Effects Of SIRT1 On Oxidative Damage During RPE Cells Stimulated By High Glucose And In Retina Of Diabetic Mice

Objective:Our aim was to investigate the role of SIRT1 in the pathogenesis of DR and whether SIRT1 plays the role by regulating PGC-1α. The goal of this study is to investigate the effect of SIRT1 on oxidative damage in vivo and in vitro, in order to provide new theoretical basis for early prevention and treatment of DR.Methods:1. effects of high glucose on SIRT1 m RNA and PGC-1αm RNA expression in RPE cells.RPE cells were randomly divided into 5 groups: The five groups were cultured by high glucose(4.5g / L) for 0, 12, 24, 48 and 72 hours respectively, and then RPE cells were harvested. The m RNA levels of SIRT1 and PGC1α were evaluated by Real-time q PCR.2.The effects of resveratrol and PGC-1α sh RNA on oxidative damage during RPE cells stimulated by high glucose.The RPE cells were randomly divided into 6 groups:normal glucose group(1g/L glucose, NG), high glucose group(4.5g/L glucose, HG),high glucose + empty group(4.5g/L glucose,HG), high glucose + resveratrol group(4.5g/L glucose + resveratrol50μmol/L, HG+R), high glucose + PGC-1α sh RNA group(4.5g/L glucose + PGC-1αsh RNA, HG+P) and high glucose + PGC-1α sh RNA + resveratrol group( 4.5g/L glucose+resveratrol 50μmol/L + PGC-1α sh RNA, HG+R+P). The six groups were cultured for 72 hours, and then RPE cells were harvested. The expression of SIRT1 were detected by immunocytochemistry. The m RNA levels of of SIRT1 and PGC1α were evaluated by Real-Time PCR. The cell ROS production were detected by DCHF-DA staining. The mitochondrial membrane potential was measured by JC-1 staining. RPE cells apoptosis was detected by AnnexinⅤ-FITC/PI staining.3. The effects of resveratrol and PGC-1α sh RNA on oxidative damage in retina of STZ-induced diabetic mice.Male SD mice were randomly divided into six groups: normal control group(NC),diabetic group(DM), diabetic+empty lentivirus group(DM+E), diabetic+resveratrol group(DM+R), diabetic+PGC-1α sh RNA group(DM+P) and diabetic+resveratrol+PGC-1α sh RNA group(DM+R+P). Diabetes were induced by intraperitoneal injection of streptozotocin(STZ, 55mg/kg). Individual animals with fasting blood glucose concentrations more than 16.7 m M for 72 hours after injection were confirmed as diabetes. After the diabetic model was affirmed to be successful, the mice of diabetic+resveratrol group and diabetic + resveratrol+PGC-1α sh RNA group were administered daily with resveratrol(30mg/kg) by gavage, diabetic +empty lentivirus group(DM+E) intravitreal injection with empty lentivirus, diabetic+PGC-1αsh RNA group(DM+P) and diabetic + resveratrol + PGC-1α sh RNA group(DM+R+P) were intravitreal injection with PGC-1α sh RNA. At 8 weeks after intervening treatment, mice from every group were respectively sacrificed. The expression of SIRT1 and PGC1αwere detected by immunocytochemistry. The m RNA levels of them were evaluated by Real-Time PCR. Apoptotic cells of retina were detected by means of TUNEL.Results:1. The effects of high glucose on SIRT1 and PGC-1α expression in RPE cells..Real-Time PCR results showed that SIRT1 and PGC-1α expressed in RPE cells.The expression of SIRT1 and PGC-1α were down-regulated with high glucose stimulated in time-dependent manner. The effects of high glucose on m RNA levels of SIRT1 were revealed at 12 h and peaked at 48 h. High glucose also reduced PGC-1αm RNA expression from 12 h and peaked at 72 h.2. The effects of resveratrol and PGC-1α sh RNA on oxidative damage during RPE cells stimulated by high glucose.(1)Immunocytochemical staining and Real-Time PCR showed that the expression of SIRT1 and PGC-1α obviously reduced in RPE cells stimulated by high glucose.HG+EV group had no obvious difference to the HG group. HG+R group showed that the expression of SIRT1 and PGC-1α were increased than HG group. The expression of SIRT1 in HG+P group showed no difference to HG group while the expression of PGC-1α had a further reduction than HG group. HG+R+P group had no difference to HG +R group in the expression of SIRT1 and had to a further reduction than HG group in the expression of PGC-1α.( 2)Result from FCM showed that, the ROS content in HG group is significantly higher than NG group, and there is no difference between HG group and HG+EV group.Compared with HG group, the ROS production was decreased significantly in HG+R group,while HG+P and HG+R+P group were incresed.( 3) The mitochondrial membrane potential was down-regulated in HG group compared with NG group, and there is no difference between HG group and HG+EV group. The mitochondrial membrane potential in HG+R group were more higher than that of HG group. Those of HG+P group were lower than those of HG group. Those of HG+R+P group were lower than HG+R group.(4)Apoptosis rate of RPE cells exposed to HG was increased gradually than that of NG group, and showed no significantly difference to HG+EV group. Apoptosis rate of RPE cell S in HG+R group were reduced than that of HG group. Those of HG+P group were more higher than those of HG group. Those of HG+R+P group were higher than HG+R group.3. The effects of resveratrol and PGC-1α sh RNA on oxidative damage in retina of STZ-induced diabetic mice.( 1) Immunocytochemical staining and Real-Time PCR results showed that the protein and m RNA of SIRT1 and PGC-1α expressed in retina of SD mice, and they are obviously reduced in DM group. DM+E group had no obviously difference to the DM group. DM+R group showed that, the protein and m RNA of SIRT1 and PGC-1α were increased than DM group. The protein and m RNA of SIRT1 in DM+P group showed no difference to DM group while the protein and m RNA of PGC-1α were obviously reduced and had a further reduction than DM group. DM+R+P group had no difference to DM+R group in protein and m RNA of SIRT1 and had no difference to DM+P group in protein and m RNA of PGC-1α.(2)Apoptotic cells by TUNEL were observed in retina of the diabetic mice. Apoptotic rate was significantly higher in DM group. Apoptosis rate of DM+R group was decreased gradually than that of DM group.Those of DM+P group were more higher than those of DM group. Those of DM+R+P group were higher than DM+R group.Conclusions:1.The expression of SIRT1 and PGC-1α in RPE cells stimulated by different time of high glucose were all reduced which shows to be time dependent.2.The mitochondrial membrane potential was down-regulated, the ROS production and apoptosis rate were increased in RPE cells stimulated by 72 h high glucose, which suggested that mitochondrial dysfunction might participate in the oxidative damage of retina in diabetes.3.Resveratrol inceased the expression of SIRT1 and PGC-1α both in RPE cells stimulated by high glucose and in retina of diabetic mice.4.Lentiviral packaging PGC-1α sh RNA have an effectively interference on the synthesis of PGC-1α both in RPE cells stimulated by high glucose and and in retina of diabetic mice.5.The activation of SIRT1 could relieve the oxidative damage and apoptosis in the RPE cells stimulated by high glucose and in retina of diabetic mice, which has been suppressed lentiviral packaging PGC-1α sh RNA. These maybe suggested that SIRT1 regulates the expression of PGC-1α in oxidative damage during RPE cells stimulated by high glucose and in retina of diabetic mice.