摘要
目的 研究α7烟碱型乙酰胆碱受体（α7nAchR）激动剂对脂多糖（LPS）诱导巨噬细胞炎症反应的调控作用及其分子机制.方法 体外培养RAW264.7巨噬细胞,用LPS诱导炎性细胞模型进行实验.用1、10、100和500μg/L LPS刺激细胞5 h或用100μg/L LPS刺激细胞0、2、4、8、12、24、48、72 h;酶联免疫吸附试验（ELISA）检测细胞上清液中肿瘤坏死因子-α（TNF-α）表达,免疫荧光法检测α7nAchR在巨噬细胞的定位,细胞增殖与毒性试剂盒（CCK8）检测1、10、100、1000μmol/Lα7nAchR激动剂（GTS-21）对LPS刺激后细胞活性的影响;ELISA检测不同浓度GTS-21对LPS刺激后促炎因子TNF-α、白细胞介素-1β（IL-1β）的表达.用100μg/L LPS和100μmol/L GTS-21刺激巨噬细胞,蛋白质免疫印迹试验（Western Blot）检测高迁移率族蛋白B1（HMGB1）表达,免疫荧光法检测HMGB1、NF-κB p65在细胞内的位置变化.结果 100μg/L LPS刺激巨噬细胞24 h时TNF-α表达达高峰.免疫荧光法检测显示,巨噬细胞膜上有α7nAchR存在.α7nAchR激动剂GTS-21无细胞毒性作用,且可呈剂量依赖性地反转LPS诱导细胞活性下降〔（96.2±1.0）%、（92.0±1.1）%比（86.5±2.2）%,均P〈0.05〕.与对照组比较,LPS组细胞上清液中TNF-α（ng/L：453.0±60.6比100.8±3.2）和IL-1β（μg/L：8.21±0.31比0.87±0.16）水平均明显升高（均P〈0.05）;用10μmol/L和100μmol/L GTS-21干预后可剂量依赖性地减少上清液中TNF-α（ng/L：227.5±17.5、81.0±8.8比453.0±60.6）和IL-1β（μg/L：4.86±0.72、2.32±0.45比8.21±0.31）表达水平（均P〈0.05）.GTS-21可明显降低LPS诱导的HMGB1表达水平（灰度值：0.788±0.130比2.061±0.330,P〈0.05）,并反转了LPS诱导的HMGB1胞质转移;同时GTS-21还可逆转LPS诱导的NF-κB p65核转位.结论 α7nAchR激动剂GTS-21通过抑制NF-κB活化途径而减轻LPS诱导的巨噬细胞炎症反应.
Objective To investigate the effects of α7 nicotinic acetylcholine receptor （α7nAChR） on the inflammatory response induced by lipopolysaccharide （LPS） in RAW264.7 macrophages and its molecular mechanisms. Methods RAW264.7 macrophages were culturedin vitro. Inflammatory cell model was constructed by LPS stimulation. Cells were challenged by LPS （1, 10, 100 and 500μg/L） for 5 hours or 100μg/L LPS for 0, 2, 4, 8, 12, 24, 48 and 72 hours, and the release of tumor necrosis factor-α （TNF-α） was detected by the enzyme linked immunosorbent assay （ELISA）. The location of α7nAChR was examined in RAW264.7 macrophages by immunofluorescence. Then the cell proliferation and toxicity kit （CCK-8） was used to detect 1, 10, 100, 1000μmol/L GTS-21, a α7nAchR agonist, on the cell viability after LPS stimulation. ELISA was used to detect 1, 10, 100, 1000μmol/L GTS-21 on the levels of TNF-α, interleukin 1β （IL-1β） after LPS stimulation. Cells were challenged with 100μg/L LPS and 100μmol/L GTS-21, then, the level of high mobility group box 1 （HMGB1） was detected by Western Blot and the intracellular location of HMGB1 and nuclear factor-κB p65 （NF-κB p65） was tested by immunofluorescence.Results LPS increased the level of TNF-α to a peak at the concentration of 100μg/L and at 24 hours after stimulation. Theα7nAChR expressed on the macrophages. The cell viability was decreased in a dose-dependent manner [（96.2±1.0）%, （92.0±1.1）% vs. （86.5±2.2）%, bothP 〈 0.05]. Compared with the control group, the levels of TNF-α and IL-1βin the supernatant of LPS group were significantly increased [TNF-α （ng/L）： 453.0±60.6 vs. 100.8±3.2, IL-1β（μg/L）： 8.21±0.31 vs. 0.87±0.16, bothP 〈 0.05]. TNF-α and IL-1β were significantly decreased by 10μmol/L and 100μmol/L GTS-21 in a dose-dependent manner [TNF-α （ng/L）： 227.5±17.5, 81.0±8.8 vs. 453.0±60.6;IL-1β （μg/L）： 4.86±0.72, 2.32±0.45 vs. 8.21±0.31, allP 〈 0.05]. GTS-21 significantly reduced the expression of HMGB1 which was induced by LPS management （gray value： 0.788±0.130 vs. 2.061±0.330,P 〈 0.05） and reversed LPS-induced HMGB1 cytoplasmic transfer. GTS-21 also reversed LPS-induced nuclear translocation of NF-κB p65. Conclusion GTS-21 reduces the inflammatory response via inhibiting the activation of NF-κB.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2017年第4期300-305,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金（81272135）
