Influence of COS on the activation of NF-κB in retina and RGC-5 cells.

<p>(A)Immunohistochemical study of the expression of the NF-κB p65 subunit in retinas. For quantitative immunohistochemistry for NF-κB p65, we first determined the p65 index, which could be measured and calculated from the following formula: Σ [(p65 staining density-threshold) × area (pixels)] / total cell number. The relative density of p65 was defined as p65 index of PQ only, COS only, PBS-treated, low-dose or high-dose COS treated groups / p65 index of normal group. Data were expressed as mean ± SD. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA with post hoc Bonferroni test; N=3 in each group). (B) COS decreased the amount of activated NF-κB translocated into nucleus in PQ-stimulated RGC-5 in a time-dependent and (C) dose-dependent manner. RGC-5 cell were stimulated with 0.5 mM PQ and treated with 50 or 100 μg/ml COS or PBS for indicated time. The amount of activated NF-κB translocated into nucleus in PQ-stimulated RGC-5 was measured by using EMSA. (D) Evaluation of the sequential change of IκB with Western blot analysis. The Y scale represents the ratio of IκB blot density to the β-actin blot density. (E) Evaluation of the sequential change of NF-κB p65 with Western blot analysis. The level of NF-κB p65 significantly reduced in RGC-5 cells after 3 hours of PQ stimulation. The Y scale represents the ratio of NF-κB p65 blot density to the β-actin blot density. Data were expressed as mean ± SD. (†<i>P</i> < 0.05 compared with PQ; ‡<i>P</i> < 0.05 compared with normal control by Student’s t-test; N=3 in each group).