[α7 nicotinic acetylcholine receptor agonist attenuated the lipopolysaccharide-induced inflammatory response via inhibiting the activation of nuclear factor-ΚB] - PubMed

Objective: To investigate the effects of α7 nicotinic acetylcholine receptor (α7nAChR) on the inflammatory response induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and its molecular mechanisms.

Methods: RAW264.7 macrophages were cultured in vitro. Inflammatory cell model was constructed by LPS stimulation. Cells were challenged by LPS (1, 10, 100 and 500 μg/L) for 5 hours or 100 μg/L LPS for 0, 2, 4, 8, 12, 24, 48 and 72 hours, and the release of tumor necrosis factor-α (TNF-α) was detected by the enzyme linked immunosorbent assay (ELISA). The location of α7nAChR was examined in RAW264.7 macrophages by immunofluorescence. Then the cell proliferation and toxicity kit (CCK-8) was used to detect 1, 10, 100, 1?000 μmol/L GTS-21, a α7nAchR agonist, on the cell viability after LPS stimulation. ELISA was used to detect 1, 10, 100, 1?000 μmol/L GTS-21 on the levels of TNF-α, interleukin 1β (IL-1β) after LPS stimulation. Cells were challenged with 100 ?g/L LPS and 100 ?mol/L GTS-21, then, the level of high mobility group box 1 (HMGB1) was detected by Western Blot and the intracellular location of HMGB1 and nuclear factor-ΚB p65 (NF-ΚB p65) was tested by immunofluorescence.

Results: LPS increased the level of TNF-α to a peak at the concentration of 100 μg/L and at 24 hours after stimulation. The α7nAChR expressed on the macrophages. The cell viability was decreased in a dose-dependent manner [(96.2±1.0)%, (92.0±1.1)% vs. (86.5±2.2)%, both P < 0.05]. Compared with the control group, the levels of TNF-α and IL-1β in the supernatant of LPS group were significantly increased [TNF-α (ng/L): 453.0±60.6 vs. 100.8±3.2, IL-1β (μg/L): 8.21±0.31 vs. 0.87±0.16, both P < 0.05]. TNF-α and IL-1β were significantly decreased by 10 μmol/L and 100 μmol/L GTS-21 in a dose-dependent manner [TNF-α (ng/L): 227.5±17.5, 81.0±8.8 vs. 453.0±60.6; IL-1β (μg/L): 4.86±0.72, 2.32±0.45 vs. 8.21±0.31, all P < 0.05]. GTS-21 significantly reduced the expression of HMGB1 which was induced by LPS management (gray value: 0.788±0.130 vs. 2.061±0.330, P < 0.05) and reversed LPS-induced HMGB1 cytoplasmic transfer. GTS-21 also reversed LPS-induced nuclear translocation of NF-ΚB p65.

Conclusions: GTS-21 reduces the inflammatory response via inhibiting the activation of NF-ΚB.