Chemical structure of astragalin (A) and viability of BEAS-2B cells treated with 1–20 μM astragalin for 24 h (B). Cell viability was measured by MTT assay and viability data are mean ± SEM (n = 5, cell viability of untreated controls = 100%). Inhibitory effects of astragalin on TLR4 expression in LPS-exposed BEAS-2B cells (C and D). BEAS-2B cells were cultured with 2 μg/ml LPS in the absence and presence of 1–20 μM astragalin for 8 h. Cell lysates were prepared for Western blotting with a primary antibody against TLR4 (C). β-Actin protein was used as an internal control. The bar graphs (means ± SEM, n = 3) represent quantitative results of the upper bands obtained from a densitometer. Fluorescent images for the TLR4 induction were obtained by binding with a Cy3-conjugated IgG and by counterstaining with DAPI (D). For the measurement of ROS formation, cells were incubated with 10 μM DCF-DA. At the end of DCF-DA incubation, cells were lysed with NaOH and fluorescence was measured (E). Fluorescent images (F) were obtained with a fluorescence microscopy. Image magnification: x200. Means in bar graphs (mean ± SEM, n = 3) not sharing a common superscript refer to significant different at P < 0.05.