Expression of superoxide dismutase in whole lens preventscataract formation

[摘要] Purpose: Oxidative damage is a major factor causing cataracts, whichaccount for almost half of human blindness cases worldwide. In thisstudy, we wished to determine if overexpression of superoxide dismutase(SOD) in intact lenses could prevent cataract formation induced byoxidative stress.Methods: Fresh, intact lenses from 6-week-old male/female SpragueDawley rats were incubated with plasmid DNA encoding the human SOD1(Cu/Zn-SOD) gene at 37 °C in a CO2 cell culture chamber with95% air and 5% CO2. SOD1 expression was determined by westernblotting and SOD enzyme activity. Lenses with or without overexpressionof SOD1 were treated with H2O2 and cataract formation wasexamined. SOD1 regulation of protein kinase Cγ (PKCγ) wasdetermined by PKCγ enzyme activity assay. Intact lens gapjunctions were determined by dye transfer assay.Results: In the lens overexpression system, SOD1 cDNA was fused toEYFP to generate EYFP:SOD1 fusion proteins which allow detection fromendogenous SOD1. Incubation of intact lenses with plasmid DNA producedEYFP:SOD1 fusion proteins as determined by western blot using anti-GFPor anti-SOD1 antibodies. This caused significant increases in SOD enzymeactivity. Data indicated that SOD1 plasmid DNA can be expressed as afunctional enzyme in intact lenses in culture. Lenses overexpressingSOD1 remained clear after H2O2 treatment at 100 μM for 24h, similar to control. Overexpression of SOD1 diminished the effect ofH2O2 on PKCγ activation and subsequent inhibition ofgap junctions, indicating that overexpression of SOD1 may reducereactive oxygen species (ROS) production, and this would prevent thenormal H2O2 effect on cataract formation.Conclusions: Overexpression of SOD1 in whole lens preventsH2O2-induced oxidative damage (cataract formation) to thelens and subsequent control of gap junctions by protein kinase Cγ.

[学科分类] 生物化学/生物物理