大鼠巨噬细胞α7-乙酰胆碱受体介导的抗炎通路对内毒素所致炎症反应的作用及机制

Effect and mechanism of rat macrophage α7-acetylcholine receptor mediated anti-inflammatory ;pathway on endotoxin-induced inflammation reaction

目的：探讨大鼠巨噬细胞α7-乙酰胆碱受体（α7-AChR ）介导的胆碱能抗炎通路在内毒素所致炎症反应中的作用及机制。方法采用密度梯度离心的方法原代分离培养大鼠巨噬细胞，经流式细胞仪对细胞纯度进行鉴定。采用荧光共聚焦显微镜和蛋白质免疫印迹试验（ Western blot ）鉴定大鼠巨噬细胞α7-AChR的表达特征。烟碱预处理原代分离培养的大鼠巨噬细胞后给予内毒素脂多糖（LPS）刺激，采用酶联免疫吸附试验（ELISA）测定上清液中肿瘤坏死因子（TNF）-α的浓度变化。 Western blot检测原代培养巨噬细胞中p-NF-κB蛋白表达。采用方差分析比较不同浓度烟碱下TNF-α的水平差异。结果荧光共聚焦显微镜下观察到原代分离培养大鼠巨噬细胞膜上存在α7-AChR。给予烟碱预处理能显著降低LPS刺激后巨噬细胞培养上清中TNF-α浓度，在烟碱浓度为0、1、10、100μmol／L 时， TNF-α的浓度分别为（2123±86）、（1486±80）、（1316±83）和（1090±77） pg／mL，随着烟碱浓度的增加，TNF-α释放量减少（t＝16．33、20．18和26．83，P＜0．05）。同时，不同浓度的烟碱预处理后，巨噬细胞内的p-NF-κB水平明显下降。结论激活大鼠巨噬细胞α7-AChR可以抑制内毒素诱导的炎症因子TNF-α的释放，其作用机制为通过NF-κB信号通路抑制炎症反应。

Objective To investigate the effect of rat macrophage α7-acetylcholine receptor （α7-AChR ）-mediated cholinergic anti-inflammatory pathway on endotoxin-induced inflammation reaction . Methods Density gradient centrifugation method was used to isolate rat primary macrophages and flow cytometry was used to identify the cell purity .α7-AChR in macrophages was detected by fluorescence confocal microscopy and Western blot .After 1ipopolysaccharides （ LPS） was added to the culture media of primary culture of macrophages , the concentration of tumor necrosis factor （ TNF）-αin the supernatant was determined by enzyme linked immunosorbent assay （ELISA）.The expression of p-NF-κB protein in primary cultured macrophages was detected by Western blot .ANOVA was used to analyze TNF-αlevels after adding different concentrations of nicotine .Results α7-AChR was observed by fluorescence confocal microscope in primary macrophages .Nicotine could significantly reduce the concentration of TNF-αin culture supernatants＆amp;nbsp;of macrophages after LPS stimulation .When the concentrations of nicotine were 0, 1, 10, 100μmol/L, the concentrations of TNF-αwere （2 123 ±86）, （1486 ±80）, （1316 ±83） and （1 090 ±77）pg/mL, respectively （t=16.33, 20.18 and 26.83, P〈0.05）.The level of p-NF-κB in macrophages was also reduced when nicotine added .Conclusion Activation of macrophage α7-AChR can inhibit the endotoxin-induced release of inflammatory factor TNF-α, which may be through NF-κB signal pathway .