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Robert J. Walker, Nancy M. Anderson, Suleiman Bahouth, Jena J. Steinle; Knockdown of TNF-α Decreases Cell Death in Retinal Müller Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5424.
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To determine the role of TNF-α in regulation of cell death in the insulin-signaling pathway in retinal Müller cells.
Retinal Müller cells (rMC-1) were cultured in DMEM medium with normal (5mM) or high glucose (25mM) conditions. Medium was supplemented with 10% FBS and antibiotics. Cells were allowed to grow until reaching 80-90% confluency. 10ug of TNF-α shRNA plasmids were transiently transfected into cells using Lipofectamine 2000. Twenty-four to forty-eight hours following transfection, cells were lysed and harvested for protein analysis using Western blotting. For salmeterol treated experiments, cells were treated with 10uM salmeterol 24 hours following TNFα shRNA transfection.
We found that knockdown of TNF-α with shRNA significantly reduced the phosphorylation of IRS-1Ser307, which was further decreased following salmeterol+TNF-α shRNA. Knockdown of TNF-α increased Akt activity, which correlated with the decrease in cell death of retinal Müller cells. Further studies found that stimulation of the β2-adrenergic receptor with a selective β2-adrenergic receptor agonist, salmeterol, restored phosphorylation of insulin receptor and Akt, as well as decreased cell death in a high glucose environment.
Decreasing the levels of TNF-α suggests a possible mechanism by which restoration of β-adrenergic receptor signaling may protect the retina against diabetes-induced damage. These studies will help to further characterize the function of β2-adrenergic receptors in the retina in diabetic retinopathy.
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