基于NF-κB信号通路对肺脾气虚变应性鼻炎大鼠鼻黏膜水通道蛋白5表达的影响探讨“培土生金”法干预机制

Excessive gland secretion is common symptom of allergic rhinitis, and its pathogenesis may be related to the NF-κB pathway, which can down-regulate the expression of Aquaporin 5.There is no effective treatment so far. Although the exact mechanism remaining unclear，the method of “Enhancing earth to supplement metal” (aslo called “Enhancing Spleen to supplement Lung”) which is the most common treatment with better efficacy，especially on clearing the nasal discharge and opening the nasal orifice.The 112 SD rats will be randomly divided into 7 groups in this research, the rat model of AR will be established by the basic ovalbumin-sensitization combined with nasal provocation, meanwhile，to establish the model of lung and spleen qi deficiency of rats with AR use fumigation combined with papain aerosol inhalation and senna leaf laxation method. After the models established successfully, Bi Min decoction will use at high dose and low dose for 2 weeks and 4 weeks respectively. The score of behavior of rats will be recorded during the treatment period. These rats will be killed after intervention and their nasal mucosa will been taken.Then immunohistochemistry and Western blot will be performed to investigate the expression levels of NF-κB and aquaporin 5 while RT-PCR will be performed to investigate the expression levels of IL-lβ,TNF-α, IL-6 and in aquaporin 5 rat nasal mucosa. Aiming to provide a scientific basis for the promotion of “Enhancing earth to supplement metal”method of Chinese medicine for prevention and treatment of AR, to explore mechanism of Bi Min decoction base on the influence of NF-κB signaling pathway to the expression of aquaporin-5 in the nasal mucosa of AR rats.

腺体过度分泌是变应性鼻炎（Allergic Rhinitis，AR）常见病征，其发病机制可能与NF-κB信号通路下调水通道蛋白5有关；尚未有较好的治疗方法。“培土生金”（健脾益肺）法治疗AR是临床最常用且疗效较好的方法，尤其在止涕通窍方面，但机制不明。本研究拟将112只SD大鼠随机分7组，采用基础致敏和鼻腔激发联合方法建立AR大鼠模型，同时烟熏法复合木瓜蛋白酶雾化吸入法和番泻叶泻下法建立肺脾气虚AR大鼠模型，造模成功后用鼻敏方高剂量、低剂量分别干预2周、4周，干预期间记录大鼠行为评分，干预后处死大鼠取鼻黏膜，HE染色涂片观察鼻黏膜病理，免疫组化和western-blot法检测NF-κB和水通道蛋白5，RT-PCR法检测IL-1β、TNF-α、IL-6和水通道蛋白5，从NF-κB信号通路对AR大鼠鼻黏膜水通道蛋白5表达的影响探讨鼻敏方的干预机制，为推广“培土生金”法中药防治AR提供科学依据。

目的：从NF-κB信号通路对肺脾气虚变应性鼻炎(allergic rhinitis，AR）大鼠鼻黏膜水通道蛋白5( Aquaporin 5,AQP5)表达的影响探讨“培土生金”法防治AR的作用机制。方法：将56只SD大鼠随机分7组:正常组、AR组、肺脾气虚AR组、鼻敏方低剂量2周组和4周组、鼻敏方高剂量2周组和4周组。AR组以卵清蛋白致敏方法建立AR模型；其余5组大鼠以烟熏加番泻叶灌胃法建立肺脾气虚证候模型，同期与AR组建立AR模型。造模过程观察大鼠的一般情况、AR行为学积分、体质量、摄食量、负重游泳时间、HE染色法观察鼻黏膜病理、ELISA检测血清IgE。造模成功后鼻敏方组分别予鼻敏方低、高剂量干预2周和4周。干预后观察大鼠的一般情况和鼻黏膜病理，免疫组化法和WB法检测NF-κB、AQP5蛋白的表达，RT-PCR法检测IL-1β、TNF-α、IL-6、AQP5 mRNA表达。结果：与正常组比较，AR组大鼠出现喷嚏、挠鼻频繁、流涕等症状，肺脾气虚AR组大鼠尚伴有气急、活动减少、毛发蓬乱发黄无泽、易掉落、精神萎靡倦怠、喜蜷缩聚堆和便溏等症状，两组AR行为学评分较高（P<0.05），肺脾气虚AR组体质量增长较慢（P<0.05）、摄食量减少（P<0.05）、负重游泳时间短（P<0.05）、血清IgE水平高（P<0.05）。与正常组比较，AR组及肺脾气虚AR组的鼻黏膜纤毛脱落，排列紊乱，腺体增多，局部炎性细胞浸润；AR组及肺脾气虚AR组NF-κB和AQP5蛋白表达上升（P<0.05），IL-1β、TNF-α、IL-6和AQP5 mRNA表达上升（P<0.05）。与AR组比较，肺脾气虚AR组的病理改变更严重，胞核NF-κB蛋白表达上升（P<0.05），TNF-α、AQP5 mRNA表达上升（P<0.05）。鼻敏方干预后AR症状及肺脾气虚症状表现明显改善；与肺脾气虚AR组比较，鼻敏方干预组鼻黏膜病理损伤改善，NF-κB和AQP5蛋白表达下降（P<0.05），IL-1β、TNF-α、IL-6和AQP5 mRNA表达下降（P<0.05）；与鼻敏方低剂量2周组比较，鼻敏方高剂量4周组胞核NF-κB蛋白表达下降（P<0.05）。结论：烟熏加番泻叶灌胃联合卵清蛋白致敏可建立肺脾气虚AR大鼠模型。鼻敏方可治疗AR并能减轻腺体过度分泌，可能与其抑制NF-κB信号通路、降低AQP5表达有关。