Hyperbranched Cationic Glycogen Derivative-Mediated IκBα Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

Abstract EN

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative.

The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group).

The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls.

The mRNA and protein expression of IκBα, NF-κBp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography.

Nuclear translocation of NF-κBp65 was analyzed by immunofluorescence.

Rat intraocular pressure (IOP) was monitored pre- and postinjection.

Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated.

After RNA interference (RNAi), IκBα mRNA and protein expression were significantly inhibited.

NF-κBp65 mRNA and protein expression showed no significant differences.

Nevertheless, nuclear translocation of NF-κBp65 occurred in the DMAPA-Glyp+siRNA group.

Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group.

IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi.

The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time.

At 7 and 14 d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex.

Taken together, our results suggest that downregulation of IκBα expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats.

IκBα may become a new molecular target for lowering IOP in glaucoma.

The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.