探讨益气温阳方抑制变应性鼻炎小鼠鼻黏膜细胞自噬及保护ZO-1及occludin的作用  

Yiqi Wenyang Prescription inhibits autophagy and protects ZO⁃1 and occludin in nasal mucosal cells of an allergic rhinitis murine model

作　　者：顾文哲 史军[3] 严道南[3] 沈政洁 GU Wenzhe;SHI Jun;YAN Daonan;SHEN Zhengjie(Zhangjiagang Hospital of Traditional Chinese Medicine,Zhangjiagang 215600,China;Zhangjiagang Affiliated Hospital of Nanjing University of Chinese Medicine,Zhangjiagang 215600,China;Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]张家港市中医医院,张家港215600 [2]南京中医药大学张家港附属医院 [3]南京中医药大学附属医院

出　　处：《北京中医药大学学报》2024年第3期364-373,共10页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.82104939);苏州市卫生青年骨干人才“全国导师制”培训项目(No.Qngg2022035)。

摘　　要：目的 观察益气温阳方(黄芪、党参、干姜、桂枝、麻黄、辛夷、五味子、地龙、甘草)对变应性鼻炎小鼠模型的治疗效果和对鼻黏膜细胞抑制自噬、保护紧密连接关键蛋白——闭锁连接蛋白1(ZO-1)及闭合蛋白(occludin)的影响。方法 将30只C57BL/6小鼠按照随机数字表法分为6组:空白组,模型组,益气温阳方低、中、高剂量组(7、14、28 g/kg)及西替利嗪组(1.667 mg/kg),每组5只。卵清蛋白腹腔注射加鼻部激发致敏造模,鼻部激发致敏前1 h灌胃给药,共7 d。对小鼠行为进行评分。采集小鼠鼻黏膜组织,HE染色观察鼻黏膜炎症反应,过碘酸希夫染色观察鼻黏膜杯状细胞增生情况,酶联免疫吸附测定法检测鼻腔灌洗液IgE含量,免疫组化法检测空白组、模型组、益气温阳方高剂量组鼻黏膜中ZO-1及occludin蛋白表达,蛋白质印迹法检测鼻黏膜中自噬微管相关蛋白轻链β3(LC3B)、P62及Beclin1蛋白表达。结果 与空白组比较,模型组小鼠行为学总分升高(P<0.01),挠鼻、喷嚏、流涕各项评分增加。鼻黏膜上皮中断、被破坏,大量炎性细胞浸润,杯状细胞增生,黏液分泌增多,黏膜肿胀明显,鼻腔灌洗液中IgE含量增加(P<0.01)。鼻黏膜中ZO-1、occludin蛋白表达减少(P<0.05)。LC3BⅡ/Ⅰ、Beclin1蛋白表达增加(P<0.05),P62蛋白表达减少(P<0.05)。给药干预后,与模型组比较,益气温阳方高剂量组和西替利嗪组小鼠行为学总分降低(P<0.01),鼻黏膜肿胀减轻,炎性细胞浸润和杯状细胞数量减少,鼻腔灌洗液中IgE含量减少(P<0.01),鼻黏膜中ZO-1、occludin蛋白表达增加(P<0.05)。益气温阳方高剂量组小鼠LC3BⅡ/Ⅰ、Beclin1蛋白表达减少(P<0.05),P62蛋白表达增加(P<0.05)。结论 益气温阳方对变应性鼻炎小鼠具有良好的治疗效果,该作用机制可能与其抑制鼻黏膜细胞自噬从而促进鼻黏膜修复相关。Objective We aimed to(i)observe the therapeutic effect of Yiqi Wenyang Prescription(YQWYP,which consists of milkvetch root,tangshen,dried ginger,cassig twig,ephedra,biond magnolia flower⁃bud,Chinese magnoliavine fruit,earthworm,and liquorice root)on an allergic rhinitis(AR)murine model and the protective effect on the tight junction(TJ)key proteins zonula occludens⁃1(ZO⁃1)and occludin and(ii)elucidate whether YQWYP exerts these effects by inhibiting autophagy in nasal mucosal cells.Methods Thirty C57BL/6 mice were randomly divided into six groups with 5 mice per group:(i)the blank group,(ii)the model group,(iii⁃v)the YQWYP low⁃dose,mid⁃dose,and high⁃dose groups(7,14,28 g/kg),and(vi)the cetirizine group(1.667 mg/kg).The model was established by intraperitoneal injection and nasal stimulation of ovalbumin.1 hour before the nasal stimulation,mice in the blank group or in the model group were given normal saline by gavage,mice in the YQWYP groups and those in the cetirizine group were given the corresponding medicines solution by gavage.After 7 days,mice behavior was scored.After that,the mice were sacrificed and the nasal mucosa tissues were harvested.The inflammatory reaction of the nasal mucosa was observed by HE staining,the proliferation of goblet cells in the nasal mucosa was observed by PAS staining,the IgE content in nasal lavage fluid was determined by ELISA,the expression levels of ZO⁃1 and occludin were analyzed by immunohistochemistry,and the expression levels of LC3B,P62,and Beclin1 in the nasal mucosa of the blank group,model group,and YQWYP high⁃dose group were analyzed by Western blotting.Results After the successful establishment of the AR model,compared with the blank group,the symptom scores of nose scratching,sneezing,and runny nose in the model group were higher,while the total scores was significantly higher(P<0.01).In the model group,the nasal mucosal epithelium was disrupted and destroyed,a large number of inflammatory cells were infiltrated,goblet cells showed hyperplasia,mu

关 键 词：益气温阳方 变应性鼻炎 自噬 紧密连接 闭锁连接蛋白1 闭合蛋白 小鼠 

分 类 号：R285.5[医药卫生—中药学]