基于Th1免疫应答增强探讨穴位贴敷诱发接触性皮炎对哮喘气道炎症的反向调节机制

Point application is an effective treatment to prevent and reduce the recurrence of asthma. Most of the patients had contact dermatitis on the pasting site after point application. The occurrence of contact dermatitis had positive correlation with clinical efficacy. The mechanism has been unknown till now that contact dermatitis induced by point application can improve the airway inflammation in asthma. Moreover, how long the effect maintains is unclear after the point application. It is believed that inducing contact dermatitis can cause the skin Th1 immunoenhancement and the airway Th2 immunosupression through systemic immune response. And the immune balance of Th1/Th2 is kept which causes the improving of airway inflammation in asthma. It was found in the previous studies that point application could improve airway inflammation in asthma. And Th1 immunoenhancement was found in contact dermatitis induced by point application in asthma.On the basis of previous studies, we are going to establish mice model with chronic asthma, and to induce its contact dermatitis by point application. From the aspect that turning Th1/Th2 immune balance to Th1 immunoenhancement, we tend to study the mechanism of that contact dermatitis caused by point application could improve airway inflammation. From the aspect that the dynamic changes of Th1/Th2 ratio in the blood after point application, we tend to observe the lasting time of improving airway inflammation induced by point application. In addition, we tend to clarify the close relation between lung and skin according to immune regulation mechanism between contact dermatitis caused by point application and airway inflammation.

穴位贴敷在预防和减轻哮喘反复发作中疗效确切。多数患者在贴敷部位出现接触性皮炎改变，皮炎的发生与疗效呈正向关系。但穴位贴敷对哮喘气道炎症改善的作用机制不明，且贴敷结束后对气道炎症改善的维持时间仍不清楚。诱发接触性皮炎可使皮肤中Th1免疫应答增强，通过全身免疫应答导致气道Th2免疫应答抑制，保持Th1/Th2免疫平衡，从而减轻气道炎症。前期研究发现：穴位贴敷可改善哮喘气道炎症，其诱发的接触性皮炎存在Th1免疫应答增强现象。本研究拟在前期研究的基础上，建立慢性哮喘小鼠模型，穴位贴敷诱发其接触性皮炎。从Th1/Th2免疫平衡中Th1免疫应答增强层面，研究穴位贴敷诱发接触性皮炎对气道炎症影响的作用机制。从穴位贴敷结束后血中Th1/Th2比值动态变化层面，明确穴位贴敷改善气道炎症的维持时间。进而从穴位贴敷诱发接触性皮炎以气道炎症的免疫调节角度部分阐释“肺外合皮毛”的科学内涵。

本研究建立卵清蛋白（OVA）诱导的慢性哮喘气道炎症小鼠模型，运用咳喘停贴剂背部穴位贴敷治疗，诱发了小鼠皮肤出现接触性皮炎。从Th1/Th2免疫平衡中Th1免疫应答增强层面，研究咳喘停穴位贴敷诱发接触性皮炎对气道炎症影响的作用机制。.运用超高效液相-四级杆飞行时间质谱（UHPLC-Q-TOF-MS/MS）技术检测咳喘停药物的化学成分，并采用高效液相色谱法（HPLC）指纹图谱其进行质量控制；对治疗后哮喘小鼠抓鼻和打喷嚏的症状进行行为学评价；小鼠肺组织HE病理观察；小鼠外周血、肺组织中Th1、Th2和Th1/Th2的水平流式细胞术的检测；小鼠外周血、支气管肺泡灌洗液（BALF）中OVA特异性IgE、IL-4、IL-5、IL-13和INF-γ等细胞因子水平进行酶联免疫吸附测定（ELISA）检测；小鼠肺组织中OVA特异性IgE、IL-4、IL-5、IL-13和INF-γ mRNA水平进行聚合酶链式反应（PCR）检测。.研究发现：（1）通过咳喘停贴剂穴位贴敷药物的HPLC指纹图谱将芥子碱硫氰酸盐、大戟二烯醇、细辛脂素、延胡索乙素和6-姜辣素作为其质量控制标准；（2）咳喘停穴位贴敷能明显减轻OVA诱导的哮喘小鼠气道炎症反应，其作用与布地奈德相当；（3）咳喘停穴位贴敷可减少哮喘小鼠喷嚏和搔鼻次数，提高外周血清中INF-γ和肺组织中IFN-γmRNA的水平，降低外周血清中IL-4、OVA特异性IgE和肺组织中IL-4、IL-5、IL-13 mRNA的水平；（4）咳喘停穴位贴敷还增加了肺组织中Th1细胞的百分率，降低了Th2细胞的百分率，调节Th1/Th2的免疫失衡状态；（5）INF-γ治疗组和咳喘停穴位贴敷组具有相近的疗效，咳喘停穴位贴敷+INF-γ抗体中和治疗后，哮喘小鼠气道炎症无明显减轻，提示咳喘停穴位贴敷可能通过上调Th1的细胞因子INF-γ，增强了Th1免疫应答，从而抑制Th2免疫应答，使得Th1/Th2平衡向Th1转移，从而改善慢性气道炎症.