Zhang Bixia, Jiang Liangyan, Huang Lichuang, Hu Juntao, Tang Zhanhong
Department of Critical Care Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China. Corresponding author: Tang Zhanhong, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2024 May;36(5):514-519. doi: 10.3760/cma.j.cn121430-20231129-01017.
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.
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.
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: 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): 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): 3.99±0.17 vs. 0.34±0.17, Arg1 mRNA (2): 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].
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.
探讨轻度低温对脂多糖（LPS）诱导的急性肺损伤（ALI）小鼠巨噬细胞极化的影响，并阐明其在肺损伤中的作用。
采用随机数字表法，将18只雄性C57BL/6小鼠分为假手术组（Sham组）、ALI常温模型组（NT组）和ALI轻度低温治疗组（HT组），每组6只。采用气管内滴注LPS的方法建立小鼠ALI模型，并于术后1小时进行体温控制。NT组肛门温度维持在36-38℃，HT组肛门温度维持在32-34℃。两组目标肛门温度均维持6小时，然后缓慢复温至36-38℃。Sham组经气管注入等量生理盐水，不进行温度控制。造模24小时后，采集血清并处死小鼠获取肺组织。在光学显微镜下观察肺组织病理变化并进行半定量肺损伤评分。采用酶联免疫吸附测定（ELISA）检测血清白细胞介素（IL-1β、IL-10）水平。采用实时定量聚合酶链反应（RT-qPCR）检测肺组织中巨噬细胞极化指标，如CD86、IL-6、CD206和精氨酸酶1（Arg1）的mRNA表达。采用蛋白质印迹法检测M1巨噬细胞标志物诱导型一氧化氮合酶（iNOS）和M2巨噬细胞标志物Arg1的蛋白表达。
与Sham组相比，NT组出现明显的肺出血、水肿，肺间隔增厚，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水平升高但无统计学意义；CD86 mRNA、IL-6 mRNA和iNOS蛋白表达显著升高，CD206 mRNA显著降低；Arg1的mRNA和蛋白表达降低，但无显著差异。与NT组相比，HT组肺组织病理损伤明显减轻，肺损伤评分显著降低（4.78±0.96 vs. 8.56±1.98，P<0.01）；血清IL-1β水平降低（ng/L：13.52±1.95 vs. 27.18±3.87，P<0.01），IL-10水平显著升高（ng/L：42.59±15.79 vs. 14.62±4.47，P<0.01）；肺组织中IL-6 mRNA表达降低（2-ΔΔCt：3.37±0.92 vs. 10.04±0.91，P<0.05），M1巨噬细胞标志物CD86 mRNA和iNOS蛋白表达显著降低[CD86 mRNA（2-ΔΔCt）：0.52±0.16 vs. 1.95±0.33，iNOS蛋白（iNOS/β-actin）：0.57±0.19 vs. 1.11±0.27，均P<0.05]，M2巨噬细胞标志物CD206 mRNA、Arg1 mRNA和Arg1蛋白表达显著升高[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蛋白（Arg1/β-actin）：0.96±0.09 vs. 0.31±0.11，均P<0.05]。
轻度低温可减轻ALI小鼠的炎症反应并保护肺组织，这可能与抑制M1巨噬细胞极化和促进M2巨噬细胞极化有关。
