右美托咪定抑制炎症反应中巨噬细胞迁移

Inhibitory effect of dexmedetomidine on migration function of macrophage during the inflammatory reaction

目的观察右美托咪定(dexmedetomidine,DEX)对炎症反应中巨噬细胞迁移功能的影响,并初步探讨其可能的作用途径。方法利用Transwell方法观察细胞迁移功能,使用10μg/ml脂多糖(LPS)作用于RAW264.7巨噬细胞建立体外炎症反应模型,在此基础上观察不同浓度(0.01,0.1,1μmol/L)DEX对细胞迁移功能的影响。在0.1μmol/L DEX作用的基础上使用肾上腺素受体(α_2-AR)特异性阻断剂(育亨宾)进一步判断DEX影响细胞迁移能力的作用是否通过激动α_2-AR发挥作用。利用免疫细胞荧光法检测巨噬细胞是否有α_2-AR的表达;使用Western blot法检测巨噬细胞迁移功能抑制后下游的蛋白激酶Cζ(PKCζ)的表达情况。结果 10μg/ml LPS处理后可明显增加RAW264.7巨噬细胞迁移数量,较未使用LPS处理组增加(14.24±0.85)倍(P<0.01);在LPS作用的基础上,0.01,0.1,1μmol/L的DEX可以浓度依赖性地减少巨噬细胞迁移数量,分别为未使用LPS处理组的(11.19±1.33)倍,(4.34±0.68)倍和(2.56±0.69)倍(与LPS组相比,P<0.05)。0.1μmol/L DEX抑制RAW264.7巨噬细胞迁移的作用可以被α_2-AR阻断剂育亨宾逆转(P<0.05)。DEX能够下调巨噬细胞中PKCζ的表达,并且能够被α_2-AR阻断剂育亨宾抑制。结论右美托咪定通过激活α_2-AR抑制巨噬细胞的迁移功能,该作用可能是右美托咪定发挥抗炎作用的机制之一。

Objective To investigate the effect of dexmedetomidine（ DEX） on migration function of macrophage during the inflammation,and further explore the possible mechanism. Methods The inflammatory reaction model in vitro was established by adding 10μg/ml LPS to the cultured RAW264. 7 macrophage cells,and the cells were treated with 0. 01,0. 1,1 μmol/L DEX,respectively.The effect of different concentrations of DEX on the migration function of macrophage was observed by Transwell assay. Furthermore,the selective α2-AR antagonist yohimbine was applied in the 0. 1 μmol/L DEX-treated cells to further identify whether α2-AR activation was involved in the inhibitory effect of migration function of macrophage. The immunocytochemistry was used for identifying whether α2-AR expressed in the macrophage cells,and Western blotting was applied to detect the PKCζ expression. Results The 10 μg/ml LPS significantly increased the RAW264. 7 macrophage migration（ fold change： 14. 24 ± 0. 85 vs 1. 00 ± 0. 02,P〈0. 01）. DEX（ 0. 01,0. 1,1 μmol/L） dose-dependently decreased the number of migration cell after LPS stimulation（ fold change： 11. 19 ± 1. 33,4. 34 ±0. 68,2. 56 ± 0. 69,P〈0. 05）. The result of immunocytochemistry showed that α2-AR was expressed both in the membrane and cytoplasm of macrophage. The inhibitory effect of DEX on the migration function of macrophage were reversed by α2-AR antagonist yohimbine（ P〈0. 05）. Western blot data demonstrated that DEX downregulated the PKCζ expression after LPS stimulation,which also was reversed by 10 μmol/L yohimbine. Conclusion DEX could inhibit the migration function of macrophage via α2-AR activation,which is one of the mechanisms underlying the anti-inflammatory role of DEX.