款冬酮对过敏性鼻炎模型小鼠肥大细胞活性的抑制作用  

Inhibitory effect of tussilagone on mast cell activity in mice with allergic rhinitis

作　　者：刘水霞 陈菁华 朱旭利 王丹 高旭栋 赵玉祥[2] 李安 LIU Shuixia;CHEN Jinghua;ZHU Xuli;WANG Dan;GAO Xudong;ZHAO Yuxiang;LI An(Department of Otolaryngology,North-West Women and Children’s Hospital,Xi’an 710061,China;Department of Otorhinolaryngology-Head and Neck Surgery,Shaanxi Provincial People’s Hospital)

机构地区：[1]西北妇女儿童医院耳鼻咽喉科,西安710061 [2]陕西省人民医院耳鼻咽喉头颈外科

出　　处：《山西医科大学学报》2023年第11期1488-1495,共8页Journal of Shanxi Medical University

基　　金：陕西省重点研发计划项目(2020SF-198)。

摘　　要：目的探究款冬酮(tussilagone,TUS)对过敏性鼻炎模型(allergic rhinitis,AR)小鼠肥大细胞的抑制作用及其分子机制。方法将BALB/c小鼠随机分为对照组、模型组、TUS组和Ly294002组,每组10只。AR小鼠模型采用腹腔注射卵清蛋白(OVA)联合鼻腔滴注OVA方法构建。造模完成后第1~7天,TUS组小鼠按照5 ml/kg的剂量每天腹腔注射8×10-2 mol/L款冬酮药剂;Ly294002组小鼠每天腹腔注射6.0 mg/kg的PI3K/AKT信号通路抑制剂Ly294002溶液。采用HE染色检测小鼠鼻黏膜组织病理情况,Giemsa染色检测鼻黏膜组织嗜酸性粒细胞和肥大细胞浸润;采用ELISA法检测致敏因子IgE、组胺、白三烯、IL-4、IL-5和IFN-γ水平;流式细胞术检测小鼠脾脏组织CD4^(+)CD25^(+)Foxp3^(+)Treg/CD4^(+)T细胞水平。Western blot分析PI3K、p-PI3K、PKB和p-PKB蛋白表达水平。将RBL-2H3细胞分为对照组、模型组、款冬酮组(C-TUS组)和Ly294002组(C-Ly294002组)。采用100 ng/ml的DNP-IgE处理12 h致敏细胞后,C-TUS组细胞加入1μl浓度为8×10^(-2) mol/L的款冬酮处理2 h,C-Ly294002组细胞加入10μl浓度为25 mol/L的Ly294002溶液处理2 h。采用ELISA法检测β-己糖氨酸酶和组胺水平,采用Fura-2/AM钙离子荧光探针检测细胞内钙离子浓度,Western blot分析PI3K、p-PI3K、PKB和p-PKB蛋白表达水平。结果与对照组比较,模型组小鼠鼻黏膜组织出现了大量炎性细胞浸润,肥大细胞和嗜酸性粒细胞数量均增加;TUS组和Ly294002组小鼠鼻黏膜组织炎性浸润较模型组显著改善,肥大细胞和嗜酸性粒细胞数量均减少。与对照组比较,模型组小鼠血清中致敏因子IgE、组胺、白三烯、IL-4和IL-5的水平均升高(P<0.05),IFN-γ水平降低(P<0.05),脾脏组织中CD4^(+)CD25^(+)Foxp3^(+)Treg/CD4^(+)T细胞比例降低(P<0.01),鼻黏膜组织中PI3K和PKB蛋白磷酸化水平升高(P<0.05);与模型组比较,TUS组和Ly294002组小鼠血清中致敏因子IgE、组胺、白三烯、IL-4和IL-5的水平均降低Objective To explore the inhibitory effect of tussilagone(TUS)on mast cells of allergic rhinitis(AR)model mice and its molecular mechanism.Methods BALB/c mice were randomly divided into control group,model group,TUS group and Ly294002 group,with 10 mice in each group.AR model mice were constructed by intraperitoneal injection of ovalbumin(OVA)combined with nasal infusion of OVA.At day 1-7 after the modeling was completed,the mice in TUS group were intraperitoneally injected with 8×10-2 mol/L tussilagone at a dose of 5 ml/kg every day,while the mice in Ly294002 group were intraperitoneally injected with PI3K/AKT signaling pathway inhibitor Ly294002 solution at a dose of 6.0 mg/kg every day.The pathological degree of nasal mucosa in mice was detected by HE staining,and the infiltration of eosinophils and mast cells was observed by Giemsa staining.The levels of IgE,histamine,leukotriene,IL-4,IL-5 and IFN-γwere detected by ELISA.The ratio of CD4^(+)CD25^(+)Foxp3^(+)Treg cells to CD4^(+)T cells in spleen of mice were determined by flow cytometry.The protein expression levels of PI3K,p-PI3K,PKB and p-PKB were analyzed by Western blot.RBL-2H3 cells were divided into control group,model group,tussilagone group(C-TUS group)and Ly294002 group(C-Ly294002 group).After RBL-2H3 cells were treated with 100 ng/ml DNP-IgE for 12 h to induce the cell hypersensitivity,the RBL-2H3 cells were treated with 1μl of 8×10^(-2 )mol/L tussilagone for 2 h in C-TUS group,and 10μl of 25 mol/L Ly294002 for 2 h in C-Ly294002 group.The levels ofβ-hexosaminidase and histamine were detected by ELISA.The intracellular Ca 2^(+)concentration was detected by Fura-2/AM calcium ion fluorescence probe.The protein expression levels of PI3K,p-PI3K,PKB and p-PKB were analyzed by Western blot.Results Compared with control group,a large number of inflammatory cells infiltrated the nasal mucosa of mice in model group,and the numbers of mast cells and eosinophils were increased.Compared with model group,the inflammatory infiltration of nasal mucosa in TUS

关 键 词：款冬酮 过敏性鼻炎 肥大细胞 免疫 PI3K/PKB信号通路 

分 类 号：R765.21[医药卫生—耳鼻咽喉科]