[Effect of mild hypothermia on macrophage polarization in lipopolysaccharide-induced acute lung injury mice].

Abstract

Objective: To investigate the effect of mild hypothermia on macrophage polarization in lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice and to clarify its role in lung injury.

Methods: According to a random number table method, 18 male C57BL/6 mice were divided into sham operation group (Sham group), ALI normothermic model group (NT group) and ALI mild hypothermia treatment group (HT group), with 6 mice in each group. The ALI model in mice was established by the method of tracheal instillation of LPS, and temperature control was administered at 1 hour after surgery. The anus temperature in NT group was kept at 36-38?centigrade, while the anus temperature in HT group was kept at 32-34?centigrade. The target anus temperature in both groups were maintained for 6 hours and then slowly rewarmed to 36-38 centigrade. The Sham group was infused with an equal amount of physiological saline through the trachea without temperature control. After 24 hours of modeling, serum was collected and mice were sacrificed to obtain lung tissue. Pathological changes in lung tissue were observed under light microscopy and semi-quantitative lung injury score was performed. Enzyme linked immunosorbent assay (ELISA) was used to detect the serum levels of interleukins (IL-1β, IL-10). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to test the indicators of macrophage polarization, such as the mRNA expressions of CD86, IL-6, CD206 and arginase 1 (Arg1) in the lung tissue. The protein expression of M1 macrophage marker inducible nitric oxide synthase (iNOS) and M2 macrophage marker Arg1 were detected by Western blotting.

Results: Compared with the Sham group, the NT group appeared significant pulmonary hemorrhage and edema, thickened lung septum, inflammatory cell infiltration, and lung injury score was significantly increased; serum IL-1β level was significantly elevated; IL-10 level was increased without statistical significance; the expressions of CD86 mRNA, IL-6 mRNA and iNOS protein were significantly elevated, and CD206 mRNA was significantly decreased; the mRNA and protein expressions of Arg1 decreased, but there were no significant differences. Compared with the NT group, the pathological injury of lung tissue in HT group was significantly reduced, and the lung injury score was significantly decreased (4.78±0.96 vs. 8.56±1.98, P < 0.01); serum IL-1β level was decreased (ng/L: 13.52±1.95 vs. 27.18±3.87, P < 0.01), and IL-10 level was significantly increased (ng/L: 42.59±15.79 vs. 14.62±4.47, P < 0.01); IL-6 mRNA expression was decreased in lung tissue (2^-ΔΔCt: 3.37±0.92 vs. 10.04±0.91, P < 0.05), the expression of M1 macrophage markers CD86 mRNA and iNOS protein were significantly decreased [CD86 mRNA (2^-ΔΔCt): 0.52±0.16 vs. 1.95±0.33, iNOS protein (iNOS/β-actin): 0.57±0.19 vs. 1.11±0.27, both P < 0.05], the expression of M2 macrophage markers CD206 mRNA, Arg1 mRNA and Arg1 protein were significantly increased [CD206 mRNA (2^-ΔΔCt): 3.99±0.17 vs. 0.34±0.17, Arg1 mRNA (2^-ΔΔCt): 2.33±0.73 vs. 0.94±0.23, Arg1 protein (Arg1/β-actin): 0.96±0.09 vs. 0.31±0.11, all P < 0.05].

Conclusions: Mild hypothermia can alleviate the inflammatory response and protect lung tissue in ALI mice, which may be related to the inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization.