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H. Nambu, E. Kubo, T. Miyazawa, Y. Takamura, S. Tsuzuki, Y. Akagi; Abrasion of Aldose Reductase Gene Suppresses High Glucose Induced–Apoptosis and Oxidative Stress in Rat Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2555.
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© ARVO (1962-2015); The Authors (2016-present)
One of major contributory factors of diabetic cataract is increased aldose reductase (AR) activity in polyol pathway. Activation of AR generates both osmotic and oxidative stress, which have been implicated in the pathogenesis of diabetic complications. We studied the effect of RNA interference of AR gene on the high glucose–induced cell death, oxidative stress and the activation of NF–ΚB in rat lens epithelial cells (RLECs) and explored the association between polyol pathway and high glucose mediated cell death and activation of NF–ΚB.
Small interference RNAs (siRNAs) were designed to target the coding sequence of rat AR–siRNAs were chemically synthesized, purified, and annealed. RLECs were cultured in either 5 (control) or 50mM (high) D–glucose. AR– and control– siRNAs were transfected using TransIT®–KO transfection reagent (Mirus) into RLECs. Western analysis was performed to monitor AR expressions. MTS and TUNEL assays were used to detect apoptotic cell death. Intra cellular reactive oxygen species (ROS) were assessed using H2DCFDA probe. Activation of NF–ΚB was measured using TransAMTM NF–ΚB p65 Chemi Kit (Active Motif).
Cell survival gradually decreased in RLECs from 72 to 120 hrs incubated with 50 mM glucose and number of TUNEL positive cells increased significantly. Activation of NF–ΚB was increased in cells under high glucose culture.ROS level was increased significantly under high glucose culture. Activation of NF–ΚB was increased in cells under high glucose culture. Inhibition of AR expression by siRNA decreased high glucose–induced apoptosis, ROS and activation of NF–ΚB.
Present findings reveal that the abrasion of AR suppresses high glucose–induced apoptosis in RLECs. Hence, the inhibition of AR expression by siRNA appears to be effective in diminishing high glucose–induced apoptosis in RLEC.
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