Role of HIF-1α in hydrogen-induced inhibition of lipopolysaccharide-induced inflammatory responses in mouse macrophages / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the role of hypoxia-inducible factor-1α (HIF-1α) in hydrogen-induced inhibition of lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages.Methods:The mouse RAW264.7 macrophages cultured in vitro were divided into 4 groups ( n=24 each) according to the random number table method: control group (C group), LPS group (L group), hydrogen-rich solution plus LPS group (H+ L group), and hydrogen-rich solution plus LPS plus HIF-1α inhibitor 2-methoxyestradiol (2ME2) group (H+ L+ M group). LPS 1 μg/ml was added, and the cells were incubated for 6 h in group L. In group L+ H, LPS was added first, the medium was changed to 0.6 mmol/L hydrogen-rich solution, and cells were incubated for 6 h. In group H+ L+ M, 2ME2 10 μmol/L was given first, cells were then incubated for 30 min, LPS and hydrogen-rich solution were added, and cells were incubated for 6 h. Western blot was used to determine the expression of HIF-1α, Beclin-1, Bcl-2/E1B-19 kDa interacting protein 3 (BNIP3) and LC3.Enzyme-linked immunosorbent assay was used to detect the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the supernatant.The number of autophagosomes was observed using a transmission electron microscope. Results:Compared with group C, the concentrations of TNF-α, IL-6 and IL-1β in the supernatant were significantly increased, the expression of HIF-1α, Beclinl and BNIP3 in macrophages was up-regulated, the ratio of LC3Ⅱ/LC3Ⅰ was increased, and the number of autophagosomes was increased in group L ( P<0.05). Compared with group L, the concentrations of TNF-α, IL-6 and IL-1β were significantly decreased, the expression of HIF-1α, Beclin-1 and BNIP3 in macrophages was up-regulated, LC3Ⅱ/LC3Ⅰ ratio was increased, and the number of autophagosomes was increased in group H+ L ( P<0.05). Compared with group H+ L, the concentrations of TNF-α, IL-6 and IL-1β in the supernatant were significantly decreased, the expression of HIF-1α, Beclin-1, and BNIP3 in macrophages was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased, and the number of autophagosomes was decreased in group H+ L+ M ( P<0.05). Conclusion:HIF-1α-mediated activation of autophagy is involved in the process of hydrogen-induced inhibition of LPS-induced inflammatory responses in mouse macrophages.