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S. T. Lee, N. R. Prabhu, N. Kumar, S. B. H. Bach, C. Mullens, R. D. Glickman; Phytochemicals Reduce NF-B Phosphorylation at 24 Hr But Not at 1 Hr Following Light Exposure in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4390.
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Certain phytochemicals, such as the stilbene, resveratrol (Rsvt, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in various berries and medicinal plants), have antioxidant, anti-inflammatory, and anti-tumor effects. Here we report the effects of these two compounds on pro-inflammatory responses in retinal pigment epithelial (RPE) cells, following light exposure that has previously been demonstrated to cause NF-ΚB nuclear translocation in these cells (Glickman et al., Proc. SPIE 5695:260, 2005).
Experiments were conducted with the hTERT-RPE cell line. In one series of experiments, cells were loaded with 10 µM dichlorodihydrofluorescein (DCFH) to detect presence of reactive oxygen species (ROS). In the second series, cells were incubated for one hour prior to light exposure in phenol-free medium alone, or in medium supplemented with 2 µM Rsvt (MP Bio) or UA (Sigma). The cells were then exposed for 7.5 min to the broadband output of a 150 W Hg vapor arc lamp, at an irradiance of 18 mW/cm2. Following post-exposure incubation of 1 hr or 24 hr, cells were harvested, and the presence of phosphorylated NF-ΚB (p65 component) was assayed by ELISA (Cell Signaling Tech.) according to the manufacturer’s protocol.
Similar to our previous findings, in untreated cells, light exposure produced a biphasic increase in NF-ΚB activation, i.e. at 1 hr and 24 hr. In cells treated with Rsvt or UA, the phospho-NF-ΚB levels were comparable to untreated cells at 1 hr post-exposure; however, by 24 hr post-exposure, NF-ΚB levels were lower than in the untreated cells. The DCFH assay indicated elevated levels of ROS in Rsvt-treated cells at 1 hr post-exposure.
The early increase in ROS in the presence of Rsvt during light exposure indicates a possible photochemical interaction, possibly due to the known photoisomerization of cis to trans-Rsvt. In contrast, by 24 hr post -exposure, Rsvt or UA treatment appears to exert a possible anti-inflammatory effect by lowering NF-kB activation.
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