从调控上皮细胞易感性探讨玉屏风散益气固表防治过敏性疾病的机制

Current therapy for allergic diseases only manage symptoms and do not alter the natural course of disease. Yu-ping-feng-san(YPFS), a typical formulae of Yi-qi-gu-biao method in Chinese medicine, can alter the natural course of disease and prevent the recurrence of the allergic diseases in clinic. Improved understanding of the epithelium's susceptibility to environmental insults in allergic diseases has provided important mechanistic insight into how allergy is initiated and perpetuated. Our previous study showed that Th2 mediated allergic contact dermatitis(ACD)was inhibited and epithelial cell(EC) derived TSLP was significantly reduced by YPFS.Moreover,epithelial cell extraction combined with HPLC-MS was used to screen potential chemicals which can bind to ECs and five chemicals were found out. Herein, the hypothesis is proposed that the Yi-qi-gu-biao mechanism of YPFS is closely related to regulation of EC's susceptibility with ameliorating PRRs activation and epithelial barriar defect by active components in YPFS. To verify this, the following research would be conducted. Firstly, the effect of YPFS on susceptibility of ECs(PRRs related pathway and epithelial barrier protein) in mice OVA induced asthma and FITC induced ACD would be explored in vivo. Then we will study the effect of YPFS and its ingredients on PRRs activation,integrity of ECs and related signaling pathway in vitro. With some key factors being illustrated, the inhibitor, antibody or RNAi would be employed to demonstrate the role of these molecules in the effect of YPFS. Finally,the network of multi-component and multi-targets effect of YPFS will be analysed by software cytoscape. Through this study, it is expected that the regulatory mechanism of YPFS on susceptibility of ECs could be revealed and this would explain how the Yi-qi-gu-biao method works. In addition, potential new targets might be raised by this research.

益气固表方玉屏风散广泛用于过敏性疾病，结合过敏性疾病的现代认识及前期工作基础，提出假说：玉屏风散通过调控上皮细胞易感性，抑制促过敏微环境的形成，从而改善过敏体质、控制疾病进程并减少复发；该效应是由作用于上皮细胞的成分群通过影响模式识别受体PRRs相关通路及细胞间连接的结构蛋白而实现的。研究1）玉屏风散对整体过敏性疾病模型中上皮细胞易感性的影响；2）玉屏风散及单体成分对离体上皮细胞PRRs通路和细胞连接蛋白的影响，提示其可能的机制；3）针对上述发现的玉屏风散成分群产生影响的关键分子群，采用特异性抑制剂或基因沉默等干预手段，在离体上皮细胞及整体模型上，考察分子在YPFS效应机制中的作用，验证成分群作用的主要通路和靶分子。4）分析玉屏风散多成分多靶点调控上皮细胞易感性的作用机制。通过以上研究试图阐明玉屏风散多成分多靶点防治过敏性疾病的机制与调控上皮细胞易感性的相关性，探索益气固表治病求本的内涵。

益气固表代表方玉屏风散广泛用于过敏性疾病，具有治病求本、抗复发的临床优势，在前期工作的基础上，提出玉屏风散通过“益气固表”减少过敏性疾病复发的机制与调控上皮细胞易感性密切相关。主要研究发现包括，1）建立多种小鼠过敏性疾病模型及特征指标分析：FITC、MC903诱导的小鼠ACD模型及复发模型不同时期特征基因芯片分析、HDM诱导的小鼠哮喘及复发模型；2）基于网络药理学预测YPFS抗过敏作用靶点， YPFS的预测靶标主要与PPRs、T细胞和B细胞受体通路，趋化因子通路以及自然杀伤细胞通路有关；3）YPFS有明显的缓解ACD的作用，且显著缓解复发，其中的效应成分AS-IV、Calycosin、Cimifugin和Formononetin分别通过增加连接蛋白CLDND1、CLDN-1、Occludin的表达，修复上皮细胞间E-Cadherin表达及分布，进而影响TSLP/IL-33的分泌，调控DCs和ILC2s的百分含量，从而对ACD模型有显著的抗过敏炎症效应及发挥缓解ACD复发的作用；4） YPFS可明显的缓解HDM诱导的哮喘，且显著缓解复发，其效应成分或可通过激动GPER受体，或分别影响EGFR、A20、呼吸道菌群、NK细胞等的活性，改善连接蛋白Occludin、CLDN1和E-Cadherin的表达，抑制TSLP/IL-33的分泌，进而控制小鼠过敏性哮喘炎症，缓解哮喘复发；5）YPFS全方可有效抑制由刺激剂诱导的离体EC表达TSLP和IL-33，Calycosin通过调控TJs和HIF-1α的表达干预EC分泌TSLP/IL-33，Cimifugin通过调控TJs和miR-155-5p的表达干预EC分泌TSLP/IL-33，Formononetin通过调控TLR3和TLR4信号通路和E-cadherin的表达干预EC分泌TSLP/IL-33。.以上研究阐明YPFS抗过敏性疾病及复发机制与其参与调控上皮细胞易感性密切相关，并由其效应成分分别作用于PRRs相关分子信号通路活化或结构连接完整性的相关靶点而完成。本研究揭示了YPFS益气固表法治病求本的本质可能与对调控过敏性疾病上皮细胞易感性有关，同时为上皮细胞PRRs相关分子信号通路和结构连接完整性成为药物干预过敏性疾病的靶点提供证据。