亚低温对脂多糖诱导急性肺损伤小鼠巨噬细胞极化的影响

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

目的探讨亚低温对脂多糖(LPS)诱导急性肺损伤(ALI)小鼠巨噬细胞极化的影响,从而阐明其对肺损伤的作用。方法将18只雄性C57BL/6小鼠按照随机数字表法分成假手术组(Sham组)、ALI常温模型组(NT组)和ALI亚低温治疗组(HT组),每组6只。采用经气管滴注LPS的方法制备小鼠ALI模型,术后1 h给予温度控制,其中NT组将小鼠肛温控制在36~38℃,HT组将小鼠肛温控制在32~34℃,两组需要维持目标肛温6 h,控温6 h后缓慢复温到36~38℃。Sham组则经气管滴入等量生理盐水,不予温度控制。制模后24 h留取血清并处死小鼠取肺组织,光镜下观察肺组织病理学改变并进行半定量肺损伤评分;采用酶联免疫吸附试验(ELISA)检测血清中白细胞介素(IL-1β、IL-10)含量;采用实时定量聚合酶链反应(RT-qPCR)检测肺组织巨噬细胞极化相关指标CD86、IL-6、CD206和精氨酸酶1(Arg1)的mRNA表达;采用蛋白质免疫印迹试验(Western blotting)检测M1型巨噬细胞指标诱导型一氧化氮合酶(iNOS)和M2型巨噬细胞指标Arg1的蛋白表达。结果与Sham组相比,NT组肺组织充血、水肿明显,肺间隔明显增厚,炎症细胞浸润,肺损伤评分显著升高;血清IL-1β水平显著升高,IL-10水平升高但差异无统计学意义;肺组织CD86 mRNA、IL-6 mRNA和iNOS蛋白表达明显升高,CD206 mRNA表达明显降低,Arg1 mRNA和蛋白表达降低但差异无统计学意义。与NT组相比,HT组肺组织病理损伤明显减轻,肺损伤评分明显降低(分:4.78±0.96比8.56±1.98,P<0.01);血清中细胞因子IL-1β水平明显降低(ng/L:13.52±1.95比27.18±3.87,P<0.01),IL-10水平显著升高(ng/L:42.59±15.79比14.62±4.47,P<0.01);肺组织中IL-6 mRNA表达水平明显降低(2^(-ΔΔCt):3.37±0.92比10.04±0.91,P<0.05),M1型巨噬细胞指标CD86 mRNA和iNOS蛋白表达明显降低〔CD86 mRNA(2^(-ΔΔCt)):0.52±0.16比1.95±0.33,iNOS蛋白(iNOS/β-actin):0.57±0.19比1.11±0.27,均P<0.05〕,M2型巨噬细胞指标CD206 mRNA、Arg1 mRNA和Arg1蛋白表达明显升高〔CD206 mRNA(2^(-ΔΔCt)):3.99±0.17比0.34±0.17,Arg1 mRNA(2^(-ΔΔCt)):2.33±0.73比0.94±0.23,Arg1蛋白(Arg1/β-actin):0.96±0.09比0.31±0.11,均P<0.05〕。结论亚低温可减轻ALI小鼠的炎症反应,保护肺组织,这可能与其抑制M1型巨噬细胞和促进M2型巨噬细胞的极化有关。

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℃,while the anus temperature in HT group was kept at 32-34℃.The target anus temperature in both groups were maintained for 6 hours and then slowly rewarmed to 36-38℃.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-1βlevel 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-1βlevel 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].Conclusion 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.