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

Rui Zeng,Zhaoxing Ou,Yuqing Lan,Jiahao Luo,Liqun Yang,Si Zhang,Jinmiao Li,Zijing Li,Haijun Gong,Rachida Bouhenni

doi:10.1155/2020/8206849

Rui Zeng, Zhaoxing Ou + Show 8 more

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https://doi.org/10.1155/2020/8206849

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Abstract

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.

Highlights

Glaucoma is the second leading cause of blindness in the world according to the World Health Organization [1]
A large amount of the DMAPA-Glyp/Cy3-Small interfering RNA (siRNA) complexes were found in the ciliary muscle after intracameral injection which was localized by α-smooth muscle actin (α-SMA)-positive immunofluorescence staining
The results showed that matrix metalloproteinases (MMPs)-2 activity changed insignificantly in the three IκBα-siRNA-transfected groups only at 24 h after transfection compared with the three control groups (F = 1:691, df = 23, P > 0:05)

Summary

Introduction

Glaucoma is the second leading cause of blindness in the world according to the World Health Organization [1]. Pathological intraocular hypertension is the main risk factor leading to optic nerve damage in glaucoma. Lowering intraocular pressure (IOP) is currently the only method that has been strictly proven to be an effective approach to glaucoma treatment [2]. The IOP-lowering eye drops currently in clinical use must be administered at least once per day and require long-term use, which may damage the ocular surface and cause various ocular symptoms. The compliance of patients often declines, leading to irreversible impairment of visual function. It is essential to find a means of lowering IOP that offers a better pressurelowering and more long-lasting effect with fewer side effects

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