The Effect Of Gigantol On The Interaction Between The Target Proteins AR And INOS Related To Diabetic Cataract

ObjectiveAldose reductase (AR) and nitric oxide synthase (iNOS) are the target proteins of gigantol against diabetic catarat. the aim of this study is to investigate the interaction between AR and iNOS as well as the effect of gigantol on their interaction, so as to further clarify the functional mechanism of gigantol against DC and lay a foundation for its development and utilization as molecular targeted agent.Methods1. Co-immunoprecipitation (Co-IP) was performed to confirm the interaction between AR and iNOS, in which iNOS antibody was applied for immune coprecipitation reactions and then western blot was used to detect the presence of AR both with and without the presence of gigantol.2. Using EGFP labelled AR (EGFP-AR) as donor and Cy3 labelled iNOS (Cy3-iNOS) as the receptor, receptor bleaching technology of fluorescence resonance energy transfer (FRET) was used to detect their interaction to obtain the FRET efficiency and the distance with and without the presence of gigantol.Result1. After immunoprecipitation with iNOS antibody, AR protein was detected with AR antibody by western blot. Co-IP result demonstrated AR and iNOS interact with each other. The intensity of AR in blot increased after the addition of gigantol, implying the latter enforces the interaction.2. AR and iNOS coexpressed and colocalized in all the HLEC cells. FRET phenomenon was observed in the colocalization area. When the fluorescence of the receptor Cy3-iNOS was bleached, the fluorescence of the donor EGFP-AR was enhanced obviously. Rate of energy transfer between AR and iNOS was calculated as 38.37±4.76%(n=10) and increased to 43.12±2.69%(n=10) after the stimulation of gigantol. The values are significantly different from those of the negative control (4.23±1.04%(n=4)) with p value less than 0.01. The distance between AR and iNOS was calculated as 6.50±0.22 nm and reduced to 6.28±0.12nm after the stimulation of gigantol, which are also significantly different from the negative control (10.14±0.44nm) with p value less than 0.01.ConclusionOur study demonstrated that AR and iNOS are colocalized in whole HLEC cells. A direct interaction was observed between AR and iNOS with the distance less than 10nm. Their interaction could be intensified with gigantol.