烟碱激活α7烟碱型乙酰胆碱受体调控DBA/1小鼠巨噬细胞迁移的作用研究

Nicotine regulates macrophage migration through activating alpha 7 nicotinic acetylcholine receptors in DBA/1 mice

目的 通过观察α7烟碱型乙酰胆碱受体（α7nAChR）激动剂烟碱及阻断剂甲基牛扁亭碱（MLA）对巨噬细胞迁移的影响,初步探讨胆碱能抗炎通路抑制DBA/1小鼠滑膜巨噬细胞浸润的机制.方法 分离DBA/1小鼠原代脾脏巨噬细胞,将其分为对照组、白细胞介素（IL）-6组、烟碱预处理组（Nic组）和MLA组.采用Transwell小室法检测巨噬细胞的迁移能力,免疫荧光法检测巨噬细胞纤维状肌动蛋白（F-actin）的形成,Western blot法检测趋化因子受体2（CCR2）的表达量.结果 与对照组比较,IL-6组、Nic组及MLA组从Transwell小室上层迁移到背膜的细胞数明显增多,而Nic组迁移至背膜的细胞数明显低于IL-6组及MLA组（P＜0.05）.与对照组比较,IL-6组、Nic组及MLA组F-actin表达量增加（P＜0.05）,且IL-6组及MLA组边缘出现片状及丝状伪足;Nic组F-actin的表达量明显低于IL-6组及MLA组（P＜0.05）,且片状及丝状伪足减少.IL-6组、Nic组及MLA组CCR2的表达量明显高于对照组（P＜0.05）,IL-6组及MLA组CCR2的表达量高于Nic组（P＜0.05）.结论 烟碱通过α7nAChR抑制巨噬细胞的迁移,这可能与其抑制F-actin的表达和分布所致的伪足形成以及CCR2的表达有关.

Objective To observe effect of alpha 7 nicotinic acetylcholine receptor （a7nAChR） agonists nicotine and antagonists methyllycaconitine （MLA） on macrophage migration and explore the mechanism of cholinergic anti-inflammtory pathway （CAP） on macrophage infiltration in synovialis of DBA/1 mice.Methods DBA/1 murine spleen macrophages were isolated and cultured.These macrophages were divided into control group,interleukin（IL）-6 group,nicotine pretreated group（Nic group） and MLA group.Transwell assays were used to examine ability of macrophage migration.The alteration of F-actin was detected by indirect immunofluorescence staining.Western blotting was used to detect the expression of C-C motif chemokine receptor 2 （CCR2）.Results Compared with the control group,the number of cells transmitting from the upper layer to the back layer of the Transwell chamber significantly increased in IL-6 group,Nic group and MLA group,while the number of them was significantly lower in Nic group than that in IL-6 group and MLA group （P 〈 0.05）.Compared with the control group,the expression of F-actin increased in IL-6 group,Nic group and MLA group （P 〈 0.05）,and lamellipodia and filopodia appeared in IL-6 group and MLA group.While the expression of F-actin was lower in nicotine pretreatment group than that in IL-6 group and MLA group （P 〈 0.05）,and lamellipodia and filopodia diminished in Nic group.The expressions of CCR2 in IL-6 group,Nic group and MLA group were significantly higher than that in control group（P 〈 0.05）,and the expression of CCR2 in IL-6 group and MLA group was higher than that in Nic group （P 〈 0.05）.Conclusion Nicotine inhibits the migration of macrophages which is related to pseudopodium formation caused by inhibition of F-actin distribution and expression and CCR2 expression though activatingα7nAChR.