黄花蒿花粉破坏鼻黏膜上皮细胞紧密连接的实验研究  

Study on the damage of the tight junctions of nasal mucosal epithelial cells by artemisia annua pollen

作　　者：雒红霞 孟亚萍 王浩江[2] 韩海阳 乔瑞红[1] 张小宁 冯彦[1] 王彤[3] Luo Hongxia;Meng Yaping;Wang Haojiang;Han Haiyang;Qiao Ruihong;Zhang Xiaoning;Feng Yan;Wang Tong(Department of Otorhinolaryngology Head and Neck Surgery,the First Hospital of Shanxi Medical University,Taiyuan 030001,China;Basic Medical College,Shanxi Medical University,Taiyuan 030001,China;School of Public Health,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院耳鼻咽喉头颈外科,太原030001 [2]山西医科大学基础医学院,太原030001 [3]山西医科大学公共卫生学院,太原030001

出　　处：《中华耳鼻咽喉头颈外科杂志》2020年第5期465-471,共7页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：山西省软科学研究一般项目计划(2018041032-5);山西省归国留学人员科研基金(晋财社[2019]91号)。

摘　　要：目的探讨黄花蒿花粉对鼻黏膜上皮细胞(HNEpC)紧密连接的损伤作用及机制。方法体外培养HNEpC,采用不同质量浓度黄花蒿花粉(0、20、40、80、100、160、200μg/ml)分别干预细胞24 h,CCK-8法检测细胞增殖活性;p38丝裂原素活化蛋白激酶(p38MAPK)抑制剂SB203580干预HNEpC前后,Western Blot法检测p38MAPK信号通路表达及其磷酸化水平;免疫荧光化学染色法、Western Blot法和实时荧光定量聚合酶链反应(qPCR)观察细胞紧密连接Occludin、Claudin-1的表达和分布情况。采用SPSS 21.0软件分析数据。结果CCK-8结果显示,与对照组相比,不同质量浓度黄花蒿花粉干预6 h后,HNEpC增殖活性均增高(P值均<0.05);干预12 h后,20、40、80、100、160μg/ml组的HNEpC增殖活性未见明显改变(P值均>0.05),200μg/ml组降低(P<0.05);干预24 h后,20、40μg/ml组的细胞增殖活性未见明显改变(P值均>0.05),80、100、160、200μg/ml组降低(P值均<0.05)。免疫荧光染色可见正常对照组Occludin和Claudin-1蛋白定位于细胞膜上,表达多,围绕细胞膜形成环状结构;而在高浓度黄花蒿花粉干预下,其表达水平下降,出现断裂、模糊,分布不均匀。Western Blot和qPCR实验结果表明,干预24 h后,黄花蒿花粉(40、80、100、160、200μg/ml)干预组HNEpC Claudin-1蛋白及其mRNA表达水平较对照组降低(mRNA表达量分别为0.567±0.214、0.443±0.109、0.462±0.160、0.497±0.134、0.388±0.076比1.001±0.067,P值均<0.05),而Occludin蛋白及其mRNA仅在200μg/ml黄花蒿花粉处理组降低(mRNA表达量为0.631±0.109比1.016±0.026,P<0.05),其他处理组均增高(mRNA表达量分别为1.258±0.134、1.827±0.1034、2.429±0.077、1.707±0.085、1.477±0.066比1.016±0.026,P值均<0.05)。Western Blot结果表明,100、160、200μg/ml黄花蒿花粉干预24 h后,磷酸化p38MAPK(p-p38MAPK)表达增加。SB203580能够抑制黄花蒿花粉引起的紧密连接蛋白Occludin表达下降(mRNA表达量为1.255±0.179比0.631±0.109,P<0.05Objective To investigate the damage and mechanism of artemisia annua pollen on tight junction of human nasal mucosa epithelial cells(HNEpC).Methods HNEpC were cultured in vitro.Different concentrations of artemisia annua pollen(0,20,40,80,100,160,200μg/ml)were used to intervene the cells for 24 h,and the cell proliferation activity was detected by the CCK-8 method.The expression and phosphorylation of p38MAPK signaling pathway were detected by Western Blot before and after the intervention of SB203580,a p38MAPK inhibitor in HNEpC.Immunofluorescence chemical staining,Western Blot and quantitative real-time PCR(qPCR)were used to observe the expression and distribution of tight junctions Occludin and Claudin-1.SPSS 21.1 software was used for statistical analysis.Results CCK-8 results showed that,compared with the control group,the proliferation activity of HNEpC increased after 6 h intervention with different concentrations of artemisia annua pollen(all P<0.05).After 12 h of intervention,the proliferation activity of HNEpC in the 20,40,80,100 and 160μg/ml groups was not significantly changed(all P>0.05),while that in the 200μg/ml group was decreased(P<0.05).After the intervention for 24 h,the proliferation activity of cells in the 20 and 40μg/ml groups was not significantly changed(all P>0.05),while that in the 80,100,160 and 200μg/ml groups was decreased(all P<0.05).Immunofluorescence staining showed that the Occludin and Claudin-1 proteins in the normal control group were localized on the cell membrane and expressed more and formed a ring structure around the cell membrane.However,under the intervention of high concentration artemisia annua pollen,its expression level decreased,appeared broken,fuzzy,and nonuniform distribution.Western Blot and qPCR results showed that after 24 h of intervention,the expression levels of HNEpC Claudin-1 protein and its mRNA in the pollen groups(40,80,100,160,200μg/ml)of artemisia annua decreased compared with those of those of the control group(mRNA expression levels were 0.56

关 键 词：鼻炎 变应性 连接蛋白类 闭锁蛋白 P38丝裂原活化蛋白激酶类 

分 类 号：R28[医药卫生—中药学]