α7 nicotinic acetylcholine receptor agonist attenuated the lipopolysaccharide-induced inflammatory response via inhibiting the activation of nuclear factor-κB
10.3760/cma.j.issn.2095-4352.2017.04.003
- VernacularTitle:α7烟碱型乙酰胆碱受体激动剂通过抑制核转录因子-κB活化可减轻脂多糖诱导的巨噬细胞炎症反应
- Author:
Xingxing SHI
;
Jianhua YAO
;
Cheng WANG
;
Xijing ZHANG
- Keywords:
α7 nicotinic acetylcholine receptor;
Cholinergic anti-inflammatory pathway;
Inflammatory factor;
Nuclear factor-κB;
Macrophages
- From:
Chinese Critical Care Medicine
2017;29(4):300-305
- CountryChina
- Language:Chinese
-
Abstract:
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 culturedin vitro. Inflammatory cell model was constructed by LPS stimulation. Cells were challenged by LPS (1, 10, 100 and 500μg/L) for 5 hours or 100μg/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, 1000μmol/L GTS-21, a α7nAchR agonist, on the cell viability after LPS stimulation. ELISA was used to detect 1, 10, 100, 1000μmol/L GTS-21 on the levels of TNF-α, interleukin 1β (IL-1β) after LPS stimulation. Cells were challenged with 100μg/L LPS and 100μmol/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 100μg/L and at 24 hours after stimulation. Theα7nAChR 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)%, bothP < 0.05]. Compared with the control group, the levels of TNF-α and IL-1βin 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, bothP < 0.05]. TNF-α and IL-1β were significantly decreased by 10μmol/L and 100μmol/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, allP < 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. Conclusion GTS-21 reduces the inflammatory response via inhibiting the activation of NF-κB.
