A Preliminary Study Of The Expression Of SIRT1 And Its Mechanism In Diabetic Retinopathy Patients Fibrovascular Membrane

Purpose:Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. The class Ⅲ Histone deacetylases SIRT1plays crucial roles in the development of DR. We aimed to investigate the role of SIRT1in the pathogenesis of neovascularization in DR, and the protective effects of resveratrol, a SIRT1activator, on RPE and the underlying mechanisms by using an in vitro model of hyperglycemia. We also investigate whether2,3,4’,5-Tetrahydroxystilbene-2-O-p-D-glucoside (TSG), a resveratrol analog, has the same effect.Method:The expression of SIRT1was assessed via immunohistochemistry in excised neovascular membranes from DR patients and epiretinal membrane from non-diabetic patients. Retinal (ARPE-19) cells were incubated with5.5mmol/L glucose,5.5mmol/L glucose and mannitol,25mmol/L glucose,25mmol/L glucose and different concentrations of resveratrol or25mmol/L glucose and different concentrations of TSG. Cell viability was determined by the CCK-8assay. The mRNA expression of SIRTl and PGC-lα was done by real-time PCR. The protein expression of acetylated NF-κB p65and AMPK activity was done by Western blot. The level of vascular endothelial growth factor (VEGF) was determined by the enzyme-linked immunosorbent assay (ELISA).Results:Formalin-fixed paraffin-embedded sections of neovascular membranes were excised from12patients with DR and epiretinal membranes from7non-diabetic patients. SIRT1was more frequently expressed in neovascular membranes than epiretinal membranes (100%vs28.6%). This difference was statistically significant after adjustments for age (p=0.021).25mmol/L glucose significantly induced the accumulation of VEGF, activation of NF-κB, reduction of SIRT1and PGC-la expression and AMPK activity. Incubation of ARPE-19cells with25mmol/L glucose in the presence of resveratrol or TSG reduced the inhibition effect of hyperglycemia on cell viability, SIRT1and PGC-1α expression, and AMPK activity. They also reduced the activation effect of hyperglycemia on NF-κB and VEGF. Moreover, the change of VEGF was consistent with that of SIRT1and NF-κB. The change of PGC-la was consistent with that of AMPK activity. Discussion:SIRT1levels appear elevated in human neovascular membranes in DR eyes compared with control eyes. These data support a potential role for SIRT1in the pathogenesis of neovasculation in DR. On the other hand,hyperglycemia inhibited the expression of SIRT1in ARPE-19. These data indicate that in vitro model may be different from the in vivo physiological conditions. It is also possible that SIRT1levels fluctuate during the course of disease. Our results also suggest that TSG can function as a SIRT1activator. Resveratrol and TSG can protect the retinal pigment epithelial cells against hyperglycemia induced damage. They efficiently reduce VEGF secretion through SIRT1—NF-κB pathway. Furthermore, the level of PGC-1α may be associated with AMPK activity.