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中华重症医学电子杂志

中华重症医学电子杂志 ›› 2020, Vol. 06 ›› Issue (02) : 198 -205. doi: 10.3877/cma.j.issn.2096-1537.2020.02.019

所属专题： 文献；

基础研究

miR-155-5P在脓毒症急性肺损伤时调控肺泡巨噬细胞IL-6、MIP-2的表达对中性粒细胞迁移的影响
刘凯1, 贾玲玲1, 刘倩1, 孙洁2, 赵希伟1, 张文凯2,†()   
  1. 1. 030001　太原，山西医科大学
    2. 030001　太原，山西医科大学第二医院重症医学科三组
  • 收稿日期:2020-02-17 出版日期:2020-05-28
  • 通信作者: 张文凯
  • 基金资助:
    山西省面上自然基金项目(201801D121329)

miR-155-5P regulates interleukin-6 and macrophage inflammatory protein-2 expression in alveolar macrophages to influence neutrophil migration during acute lung injury in sepsis

Kai Liu1, Lingling Jia1, Qian Liu1, Jie Sun2, Xiwei Zhao1, Wenkai Zhang2,†()   

  1. 1. Shanxi Medical University, Taiyuan 030001, China
    2. Department of ICU (Division III), Second Hospital of Shanxi Medical University, Taiyuan 030001, China
  • Received:2020-02-17 Published:2020-05-28
  • Corresponding author: Wenkai Zhang
  • About author:
    Corresponding author: Zhang Wenkai, Email:
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引用本文：

刘凯, 贾玲玲, 刘倩, 孙洁, 赵希伟, 张文凯. miR-155-5P在脓毒症急性肺损伤时调控肺泡巨噬细胞IL-6、MIP-2的表达对中性粒细胞迁移的影响[J]. 中华重症医学电子杂志, 2020, 06(02): 198-205.

Kai Liu, Lingling Jia, Qian Liu, Jie Sun, Xiwei Zhao, Wenkai Zhang. miR-155-5P regulates interleukin-6 and macrophage inflammatory protein-2 expression in alveolar macrophages to influence neutrophil migration during acute lung injury in sepsis[J]. Chinese Journal of Critical Care & Intensive Care Medicine(Electronic Edition), 2020, 06(02): 198-205.

目的

探讨脓毒症急性肺损伤（ALI）时miR-155-5P对肺泡巨噬（MH-S）细胞白介素6、巨噬细胞炎性蛋白2（MIP-2）表达的调控作用及中性粒细胞迁移的影响。

方法

培养小鼠MH-S细胞并将其分为对照组、脓毒症组和miR-155-5P抑制物组，miR-155-5P抑制物组预先通过miR-155-5P antagomir完成转染，采用脂多糖干预脓毒症组和miR-155-5P抑制物组12 h，应用RT-PCR检测3组MH-S细胞miR-155-5P、白介素6 mRNA和MIP-2 mRNA的表达，酶联免疫吸附实验检测细胞悬液中白介素6和MIP-2的浓度；再次将MH-S分为上述3组培养在transwell板的下室，CFSE荧光染料标记的中性粒细胞培养在上室，脂多糖干预后应用流式细胞术分别检测上室与下室细胞的荧光值，计算中性粒细胞的迁移率。

结果

脓毒症组MH-S细胞的miR-155-5P（8.04±0.65）、白介素6 mRNA（57.05±6.88）和MIP-2 mRNA（52.98±2.58）较对照组（1.00±0.00、1.00±0.00、1.00±0.00）表达明显上调（P<0.05）；白介素6［（50.85±0.12）pg/ml］、MIP-2［（69.96±1.40）pg/ml］的浓度和中性粒细胞迁移率（64.36%）较对照组［（38.58±0.13）pg/ml、（56.00±0.29）pg/ml、30.98%］均显著增加（P<0.05）。而miR-155-5P抑制物组细胞的miR-155-5P表达（0.19±0.35）低于对照组（P<0.05），白介素6 mRNA（39.66±3.65）、MIP-2 mRNA（31.01±2.88）的表达及白介素6［（42.45±1.08）pg/ml］、MIP-2［（59.01±1.63）pg/ml］的浓度也高于对照组（P<0.05），而低于脓毒症组（P<0.05）。

结论

脓毒症ALI时，肺泡巨噬细胞miR-155-5P基因高度表达，促炎因子白介素6 mRNA、MIP-2mRNA的表达均明显上调，其促进白介素6、MIP-2的产生，使中性粒细胞大量趋化迁移至肺内，造成肺损伤；而抑制miR-155-5P的表达，可以抑制白介素6、MIP-2的产生，从而减少中性粒细胞的趋化迁移，起到肺保护作用。

关键词: miR-155-5P, 白细胞介素6, 巨噬细胞炎性蛋白类, 巨噬细胞, 中性粒细胞, 急性肺损伤
Objective

To determine the regulatory effect of miR-155-5P on the expression of interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) in alveolar macrophages and the influence on neutrophil migration during acute lung injury (ALI) in sepsis.

Methods

Mouse alveolar macrophages (MH-S) were cultured and divided into a control group, sepsis group, and miR-155-5P inhibitor group. The miR-155-5P inhibitor group was transfected with miR-155-5P antagomir in advance. Lipopolysaccharide (LPS) was used to intervene the sepsis group and miR-155-5P inhibitor group for 12 h. RT-PCR was used to detect the expression of miR-155-5P, IL-6 mRNA, and MIP-2 mRNA in alveolar macrophages of the three groups, and ELISA was used to detect the concentrations of IL-6 and MIP-2 in cell suspension. Again, MH-S was divided into three groups and cultured in the lower compartment of transwell plate. Neutrophils labeled with CFSE were cultured in the upper compartment. After LPS intervention, fluorescence values of cells in the upper compartment and lower compartment were detected by flow cytometry, respectively, to calculate the migration rate of neutrophils.

Results

The expression of miR-155-5P (8.04±0.65), IL-6 mRNA (57.05±6.88), and MIP-2 mRNA (52.98±2.58) in MH-S cells in the sepsis group were significantly up-regulated compared with the control group (1.00±0.00, 1.00±0.00, and 1.00±0.00, respectively; P<0.05). The concentrations of IL-6 [(50.85±0.12) pg/ml] and MIP-2 [(69.96±1.40) pg/ml] and neutrophil migration rate (64.36%) were significantly higher sepsis group than in the control group [(38.58±0.13) pg/ml, (56.00±0.29) pg/ml, and 30.98%, respectively; P<0.05]. The expression of miR-155-5P in the miR-155-5P inhibitor group (0.19±0.35) was lower than that in the control group (P<0.05). The expression of IL-6 mRNA (39.66±3.65) and MIP-2 mRNA (31.01±2.88), and the concentrations of IL-6 [(42.45±1.08) pg/ml] and MIP-2 [(59.01±1.63) pg/ml] in the miR-155-5P inhibitor group were significantly higher than those of the control group (P<0.05), but lower than those of the sepsis group (P<0.05).

Conclusion

In ALI secondary to sepsis, miR-155-5P is highly expressed in alveolar macrophages, which is positively correlated with the expression of pro-inflammatory factors IL-6 and MIP-2. miR-155-5P promotes the production of IL-6 and MIP-2, and causes a large number of neutrophils to migrate into the lung, thus causing lung injury. Inhibiting the expression of miR-155-5P can inhibit the production of IL-6 and MIP-2, thus reducing the chemotactic migration of neutrophils and protecting the lung.

Key words: miR-155-5P, Interleukin-6, Macrophage inflammatory proteins, Macrophages, Neutrophils, Acute lung injury
表1 MiR-155-5P抑制物组、脓毒症组和对照组肺泡巨噬细胞miR-155-5P、白介素6 mRNA和MIP-2 mRNA表达水平的比较（±s）
组别 miR-155-5P 白介素6 mRNA MIP-2 mRNA
对照组 1.00±0.00 1.00±0.00 1.00±0.00
脓毒症组 8.04±0.65 57.05±6.88 52.98±2.58
MiR-155-5P抑制物组 0.19±0.35 39.66±3.65 31.01±2.88
F值 398.61 122.13 409.17
P值 <0.001 <0.001 <0.001
表2 MiR-155-5P抑制物组、脓毒症组和对照组肺泡巨噬细胞白介素6和MIP-2表达水平比较（pg/ml，±s）
组别 白介素6 MIP-2
对照组 38.58±0.13 56.00±0.29
脓毒症组 50.85±0.12 69.96±1.40
MiR-155-5P抑制物组 42.45±1.08 59.01±1.63
F值 295.58 103.03
P值 <0.001 <0.001
图1 显微镜下的中性粒细胞　图1a为瑞氏染色，放大100倍，图b为瑞氏染色，放大400倍，图1c为台盼蓝染色，放大100倍
图2 CFSE标记的中性粒细胞　图2a为明场，图2b为暗场
图3 对照组、脓毒症组、miR-155-5P抑制物组和transwell板上室（图3a、3c、3e）与下室（图3b、3d、3f）中性粒细胞的荧光值测定
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