Figure 2

IL-17A further activates the inflammatory signaling pathway Act1/TRAF6/IKK/NF-κB in both HG-treated retinal Müller cells and Akita mouse retinal tissue. (a) Expression of IL-17A-related signaling molecules in primarily cultured retinal Müller cells. Retinal Müller cells were exposed to HG (25 mM) for 24 h and subsequently treated with IL-17A (25 ng ml⁻¹) and/or the IKK inhibitor Wedel (10 μM) for 24 h. For silencing the Act1 gene, Müller cells were infected with Ad-Act1-shRNA (or Ad-GFP as a control) for 24 h in the HG condition; the adenoviruses were subsequently removed, and the cells were cultured for an additional 24 h. Following the treatments, the proteins of the cells were extracted for western blot analysis. (b) Expression of IL-17RA downstream signaling molecules in the retinas. At 3 months after the onset of diabetes, Akita mice were subjected to intravitreal injection of IL-17A (8 or 40 ng per 2 μl in PBS per eye) or Ad-Act1-shRNA (or Ad-GFP as a control). At 2 days after the IL-17A intravitreal injection or 2 weeks after the Ad-Act1-shRNA intravitreal injection, the proteins of the retinas were extracted for western blot analysis. **P<0.01 vs control or wild type (WT); ⁺P<0.05, ⁺⁺P<0.01 vs HG or Akita; ^&&P<0.01 vs HG+IL-17A+Ad-GFP (or HG+IL-17A) or Akita+Ad-GFP; NS (no significance) vs HG+IL-17A or Akita. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; HG, high glucose; IL, interleukin; NF-κB nuclear factor-κB; PBS, phosphate-buffered saline; TRAF6, tumor necrosis factor receptor-associated factor-6.