Study Of The Interaction Mode Of Dendrobium-extacted Sysingic Acid And Gigantol With AR Gene And INOS Gene To Exert Its Anti-cataract Activity

ObjectiveTo characterize the interaction mechanism between gigantol/Sysingic acid and the AR gene/iNOS gene. It is provided a more comprehensive theoretical basis for the use of Dendrobium for the treatment of diabetic cataract from the molecular leve and a great effect on the design of more effective new drugs.Methods1. Using the wavelength of UV-visible absorption spectroscopy to scan and observe the change in the maximum absorption peak position and intensity of the AR gene/iNOS gene before and after Gigantol/Sysingic acid acted on, and their binding constants were measured.2. Using the atomic force microscope (AFM) to direct the observation of the changes of surface morphology、structure and height of AR gene/iNOS gene before and after adding Gigantol/Sysingic acid at the single molecule.3. Surface-enhanced Raman spectroscopy (SERS) was tested the spectral peak under a 632.8 nm excitation at the wavelength of 40cm-1-4000cm-1 about the interaction of Gigantol/Sysingic acid with AR gene/iNOS gene and the interaction sites were determined.4. Under the condition of living cells, fluorescence resonance energy transfer (FRET) was performed to real-time detect the distance and the energy transfer rate when gigantol/sysingic acid combined with AR gene/iNOS gene.Results1. The interaction studies of Gigantol with AR gene:UV-visible absorption spectra can initially determine the manner of gigantol interaction with AR gene may be combined with a groove. According to the absorbance value at the maximum absorption of the AR gene as gigantol ratio in the concentration of the concentration of the AR gene, the action between gigantol and AR gene have many binding sites. And the binding constant was 1.85 X 103L/mol. Atomic force microscopy further study the interaction showed that gigantol can cause AR gene molecular aggregation wound, the average height increasing 0.3-0.6nm, and further confirms its interaction mode.2. Gigantol and iNOS gene interactions study:UV spectroscopy results showed gigantol primarily by binding to the major groove or minor groove role in iNOS gene. A linear relationship between absorbance and concentration gigantol and iNOS gene at a ratio of maximum absorption gene indicates the presence of multiple binding between gigantol and iNOS gene. The binding constant was 7.2×102L/mol. The change of surface appearance figure of AFM showed gigantol could make iNOS gene cause chain wound contraction, aggregation, the number of nodes and the average height increasing.3. The interaction research of Syringic acid with AR gene:From UV-visible absorption spectrum, it could be seen that the maximum absorption wavelength of the AR gene with increasing concentrations of Syringic acid exhibits grace red shift phenomenon, which inferred that the binds way was groove. There also were more binding sites, and the binding constant was 2.85 X 103L/mol. Atomic force microscopy showed that Syringic acid and AR gene cause genetic molecule binding and wrapping aggregation, average height increasing. The results of surface enhanced raman spectroscopy further confirmed the interaction. With the increase of the Syringic acid concentration, the raman spectral of AR gene lined in 1493.24cm-1 moved to the high wavenumber (1495.51cm-1).ConclusionGigantol/Syringic acid could bind the target gene (AR gene/iNOS gene). Through the groove function and gene molecules inhibit its expression, which may lead to a corresponding amount of protein expression.