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Zoe D. Fonseca-Kelly, Reas K. Sulaimankutty, Pegah Safabakhsh, Kenneth S. Shindler; SIRT1 Activators Reduce Reactive Oxygen Species Produced During Oxidative Stress In RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5909.
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© ARVO (1962-2015); The Authors (2016-present)
Optic neuritis (ON) is an inflammatory demyelinating disease of the optic nerve. We identified several SIRT1 activators that prevent retinal ganglion cell (RGC) loss in mice with ON. SIRT1 is a member of a conserved gene family (sirtuins) encoding NAD+-dependent deacetylases. Sirtuins deacetylate many protein targets, and it has been shown in other systems that activation increases mitochondrial function and reduces oxidative stress. We hypothesize that treatment with SIRT1 activators during ON increases SIRT1 activity in RGCs, thereby increasing mitochondrial function and reducing accumulation of reactive oxygen species (ROS) that can lead to cell death. Here, we examine whether SIRT1 activators can reduce ROS and promote mitochondrial function in retinal cells in vitro.
Cultured RGC-5 cells were stressed by removal of serum or addition of doxorubicin (50µM) for 24 hrs and subsequently treated with vehicle or increasing concentrations of SIRT1 activators (0 - 100µM) for 24 hrs. Cells were stained with MitoSOXTM Red (Invitrogen) to detect the ROS superoxide, and imaged by fluorescent microscopy. Cells were also harvested for protein extraction or RNA isolation and cDNA synthesis. Expression levels of SIRT1 and PGC-1α, a co-enzyme involved in mitochondrial function, were measured by Real Time PCR and Western blotting.
Removal of serum from the cell culture medium resulted in oxidative stress, as detected by accumulation of MitoSOX Red positive staining ROS in RGC-5 cells. There was a significant reduction in the levels of MitoSOX Red positive ROS in RGC-5 cells that were treated with SIRT1 activators for 24 hrs compared to untreated cells that were stressed by removal of serum. Similar oxidative stress is induced with doxorubicin administration. Treatment of RGC-5 cells with SIRT1 activators for 24 hours after doxorubicin exposure led to an increase in both SIRT1 and PGC-1α expression compared to untreated cells.
Results show that SIRT1 activators reduce ROS levels in a retinal cell line following induction of oxidative stress. Treatment with SIRT1 activators under oxidative stress conditions resulted in an increase in the level of SIRT1 gene expression, as expected, and also induced increased PGC-1α expression under the same conditions. These results support the hypothesis that SIRT1 activators reduce ROS accumulation in RGCs by promoting increased mitochondrial function.
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