Role of autophagy in dexmedetomidine-induced reduction of lipopolysaccharide-caused inflammatory responses in macrophages of mice

Abstract

Objective To evaluate the role of autophagy in dexmedetomidine-induced reduction of lipopolysaccharide(LPS)-caused inflammatory responses in macrophages of mice. Methods Mouse macrophage cell line RAW264.7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups (n=20 each) when cell confluence reached 60% using a random number table method: control group (group Con), LPS group, LPS plus dexmedetomidine group (group LPS+ DEX), and LPS plus dexmedetomidine plus autophagy inhibitor 3-MA group (group LPS+ DEX+ 3-MA). PBS was added and cells were cultured for 12 h in group Con.LPS at the final concentration of 1 000 ng/ml was added and cells were incubated for 12 h in group LPS.LPS at the final concentration of 1 000 ng/ml was added, and then dexmedetomidine at the final concentration of 1 μmol/L was immediately added, and cells were incubated for 12 h in group LPS+ Dex.In group LPS+ Dex+ 3-MA, 3-MA at the final concentration of 2 mmol/L was added and cells were incubated for 1 h, LPS at the final concentration of 1 000 ng/ml was added, and then dexmedetomidine at the final concentration of 1 μmol/L was immediately added, and cells were incubated for 12 h. Cell viability was detected by CCK-8 assay, and the concentrations of nitrous oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in the supernatant were determined by enzyme-linked immunosorbent assay, and the expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ), LC3Ⅱ, P62 and Bcelin-1 was detected by Western blot.LC3Ⅱ/LC3Ⅰ ratio was calculated. Results Compared with group Con, the cell viability was significantly decreased, the concentrations of TNF-α, IL-1β and NO and LC3Ⅱ/LC3Ⅰ ratio were increased, and the expression of P62 and Beclin1 was up-regulated in group LPS (P<0.05). Compared with group LPS, the cell viability was significantly increased, the concentrations of TNF-α, IL-1β and NO were decreased, LC3Ⅱ/LC3Ⅰ ratio was increased, the expression of P62 was down-regulated, and the expression of Beclin1 was up-regulated in group LPS+ DEX (P<0.05). Compared with group LPS+ Dex, the cell viability was significantly decreased, the concentrations of TNF-α, IL-1β and NO were increased, LC3Ⅱ/LC3Ⅰ ratio was decreased, the expression of P62 was up-regulated, and the expression of Beclin1 was down-regulated in group LPS+ Dex+ 3-MA(P<0.05). Conclusion Enhanced autophagy is involved in dexmedetomidine-induced reduction of LPS-caused inflammatory responses in macrophages of mice. Key words: Dexmedetomidine; Autophagy; Endotoxins; Macrophages; Inflammation