Liu_2015_Zhonghua.Wei.Zhong.Bing.Ji.Jiu.Yi.Xue_27_811

OBJECTIVE: To 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, 100 mumol/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 10 mumol/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-alpha (TNF-alpha), interleukins (IL-1beta, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.
RESULTS: (1) The contents of TNF-alpha (ng/L: 605.09 +/- 57.13 vs. 34.07 +/- 8.62), IL-1beta (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 (all P < 0.05). These results indicated that the inflammatory model of rat alveolar macrophages was constructed successfully. (2) ACh with the final concentrations of 0.01, 0.1, and 1 mumol/L had less influence on the production of TNF-alpha, IL-1beta and IL-6 in the culture supernatants of alveolar macrophages stimulated with LPS compared with LPS group (all P > 0.05). Nevertheless, 10 mumol/L and 100 mumol/L ACh notably reduced the production of TNF-alpha (ng/L: 451.19 +/- 30.67, 332.19 +/- 32.19 vs. 604.96 +/- 22.56), IL-1beta (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 (all P < 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-alpha (ng/L: 183.17 +/- 35.44 vs. 451.19 +/- 30.67), IL-1beta (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 (all P < 0.05).
CONCLUSIONS: ACh with the final concentrations of 10 mumol/L and 100 mumol/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.