Anti-inflammatory effect of acetylcholine on lipopolysaccharide induced inflammatory response of alveolar macrophages / 中华危重病急救医学

ABSTRACT

ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.