Figure 5.

Cholinergic stimulation attenuates LPS-induced IL-8 response in SW480 cells via a7nAChR. Expression of ACHRs in SW480 cells (A). SW480 cells (5 x 10⁴/200µl) were stimulated with LPS (100ng/ml) in the presence of different agonist and antagonists. IL-8 was measured in cell-free supernatants 20h after LPS stimulation. Cells were stimulated with LPS 20min prior to acetylcholine (ACh, 10µM), nicotine (10µM), and muscarine (10µM) stimulation (B). Cells were pretreated (20min) with the non-selective nAChR antagonist Mecamylamine (Mec, 100µM) followed by treatment with ACh (10µM), 20min prior to LPS stimulation (C). Cells were pretreated (20min) with the selective a7nAChR antagonist alpha-Bungarotoxin (a-BTX, 100µM) followed by treatment with GTS-21 (100µM), 20min prior to LPS stimulation (D). Cells were pretreated (20min) with GTS-21 (100µM) followed by stimulation with LPS. Phospho (p)-NFĸB-p65 was measured 4h after LPS stimulation (E). Cells were pretreated (20min) with DMSO (0.01%, control) or tyrphostin AG490 (1µM) or wortmannin (1µM) followed by stimulation with LPS (100ng/ml), 20min prior to treatment with a7nAChR agonist GTS-21 (20min) (F). Cell viability was assessed by colorimetric MTS assay (G). Each point in the scatter plots with bar represent triplicates or quadruplicates ± SEM. Data are representative of three independent experiments. Significance was determined using one-way ANOVA multiple comparison analysis (A–D) and paired t test (E, F) with *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005. ns, not significant. n.d., not detectable.