烟碱型乙酰胆碱受体α7基因对非酒精性脂肪性肝炎小鼠肝脏炎症反应的作用及其机制  

Effect of nicotinic acetylcholine receptor α7 subunit gene on liver inflammatory reaction in mice with nonalcoholic steatohepatitis and related mechanisms

作　　者：李富强 吴小翠[2] 徐丽娜[2] 陈小梅[3] 陆思[2] 唐翠兰[3] 

机构地区：[1]绍兴市中心医院感染科,312030 [2]浙江中医药大学第二临床医学院,杭州310053 [3]浙江中医药大学附属第二医院感染科,杭州310005

出　　处：《中华肝脏病杂志》2016年第10期767-771,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金资（81100279）;浙江省新苗人才计划项目（2015R410013）

摘　　要：目的探讨烟碱型乙酰胆碱受体α7（α7nAChR）基因对非酒精性脂肪性肝炎（NASH）小鼠肝脏炎症的作用及其机制。方法C57BL／6J小鼠和α7nAChR基因敲除小鼠饲养24周建立NASH模型，然后处死小鼠、分离培养原代肝脏巨噬细胞；给予烟碱以及内毒素处理后，酶联免疫吸附法（ELISA）检测细胞上清液中炎症因子白细胞介素（IL）-6、肿瘤坏死因子（TNF）α的水平；细胞间接免疫荧光和Westemblot观察其对信号通路核因子（NF）-κB的影响；定量PCR检测巨噬细胞中Toll样受体（TLR）-4mRNA表达水平。多组间均数比较采用方差分析。结果ELISA结果表明细胞培养上清液中IL-6、TNFα浓度在基因敲除NASH小鼠内毒素＋烟碱组分别为（1599±65）pg／ml、（1567±66）pg／ml，较C57NASH小鼠内毒素＋烟碱组[（1465±45）pg／ml、（1433±50）pg／ml]明显升高（P值均〈0．05）。Wesetem blot结果表明磷酸化NF-κB、TLR-4的蛋白相对表达水平在基因敲除NASH小鼠内毒素＋烟碱组分别为5825±60、6387±68，较C57NASH小鼠内毒素＋烟碱组的5863±20、6198±63明显升高（P值均〈0．05）；细胞间接免疫荧光结果也显示，基因敲除NASH小鼠内毒素＋烟碱组的NF-κB荧光强度明显高于C57NASH小鼠内毒素＋烟碱组。PCR结果表明TLR-4mRNA相对表达水平在基因敲除NASH小鼠内毒素＋烟碱组为4．13±0．13，较C57NASH小鼠内毒素＋烟碱组的2．93±0．14明显升高（P〈0．05）。结论α7nAChR基因敲除使NASH炎症反应程度加重，其作用机制可能与NF-κB信号通路不能被抑制从而加重炎症反应有关。Objective To investigate the effect of nicotinic acetylcholine receptor α7 （α7nAChR） subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis （NASH） and related mechanisms.Methods C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model, and the mice were sacrificed to isolate and culture the primary liver macrophages. After the treatment with nicotine and endotoxin, ELISA was used to measure the levels of the inflammatory factors interleukin-6 （IL-6） and tumor necrosis factor-it （TNF-α） in supematant; indirect immunnfluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway, and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 （TLR-4） in macrophages. An analysis of variance was used for comparison of means between multiple groups. Results The results of ELISA showed that compared with the endotoxin＋nicotine group of C57 NASH mice, the endotoxin＋nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-αin supernatant （IL-6：1 599±65 pg/ml vs 1 465±45 pg/ml, P 〈 0.05; TNF-α： 1 567±66 pg/ml vs 1 433±50 pg/ml, P 〈 0.05）. The results of Western blot showed that compared with the endotoxin＋nicotine group of C57 NASH mice, the endotoxin＋nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR- 4 （NF-κB： 69 425±600 vs 51 133±200, P 〈 0.05; TLR-4：93 387±684 vs 64 198±630, P 〈 0.05）. The results of indirect immunofluorescence assay showed that the endotoxin＋nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin＋nicotine group of C57 NASH mice. The results of PCR showed that the endotoxin＋nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin＋nicotine group of C57 NASH mice （4.13±0.13 vs

关 键 词：脂肪肝 α7-AChR基因敲除 枯否细胞 

分 类 号：R575.1[医药卫生—消化系统]