基于SFKs-HEMT生物芯片的过敏康方治疗过敏性鼻炎的药效物质基础及作用机制研究

Allergic rhinitis (AR) is a common disease seriously affecting human health. Previous studies have indicated that the key pathogenesis of AR is mast cell degranulation mediated by IgE, of which the initial stage is regulated by Src family kinases (SFKs). As a famous formula of traditional Chinese medicine (TCM), Guominkang shows a significant effect on the treatment of AR, but its action mechanism is still unclear. Through the previous investigations, it is found that some inhibitors of SFKs own anti-allergic activity. Thus, it could be inferred that the possible mechanism of Guominkang formula treating AR is that mast cell degranulation is regulated by pharmacodynamic components in the formula via acting on SFKs. Moreover, HEMT biochip is especially suitable for the screening of trace amount of pharmaceutical substances owing to its high sensitivity and specificity, as well as the ability for direct detection of active ingredients. In view of this, this project would screen the affinity substances of SFKs in serum after administration by constructing SFKs-functionalized HEMT biochips, and determine the potential pharmacodynamic components through combining other methods including virtual screening and UPLC-MS identification. After that, pure products of pharmacodynamic components would be prepared by application of a variety of techniques, followed by activity evaluation and mechanism mining. Finally, the pharmacodynamic material basis and action mechanism of Guominkang formula treating AR would be clarified. The implementation of this project would establish a systematic research strategy as “component screening - pure compounds preparation - activity evaluation - mechanism mining”, which would provide new idea for explaining the scientific connotation of TCM formula, and also give methodological guidance for discovering pharmacodynamic substances and developing new drugs.

过敏性鼻炎（AR）是严重影响人类的疾病，已有研究表明其关键发病机理是IgE介导肥大细胞脱颗粒，而该过程的起始受Src家族激酶（SFKs）调控。中药名方过敏康对AR的治疗效果显著，但作用机制不明。前期研究发现，部分SFKs抑制剂显示抗过敏活性，由此推断，过敏康方治疗AR的可能机制是其药效成分作用于SFKs调控肥大细胞脱颗粒。另外，HEMT生物芯片因其灵敏、特异及能直接检测活性成分等优势，特别适用于微量药物的筛选。鉴于此，本项目拟构建SFKs功能化的HEMT生物芯片筛选给药后体内SFKs亲和物质，并联合虚拟筛选及液质联用鉴定等确定潜在药效成分；之后，运用多种技术制备纯品以进行活性评价和机制挖掘，最终阐明过敏康方治疗AR的药效物质基础和作用机制。本项目的实施将建立“成分筛选-纯品制备-活性评价-机制挖掘”的系统研究策略，为阐释中药复方的科学内涵提供新思路，也为发现药效物质和研发新药提供方法指导。

过敏性鼻炎（AR）是严重影响人类健康的常见疾病之一，也是目前临床上亟待解决的难题。过敏康是一种中药经验复方，在临床上对AR显示良好的疗效，但其药效物质基础和作用机制至今不明。本课题首先采用UHPLC-MS对过敏康粗提物的化学组成及入血成分进行表征，从粗提物中共鉴定出94种化合物，发现其中15种为入血成分。其次，借助网络药理学，对过敏康的作用靶点及其主要信号通路进行预测，共得到35个作用靶点以及88个生物通路。接着，对过敏康的体内、外抗过敏活性和作用机制进行研究。通过PCA实验，发现过敏康可剂量依赖性地降低小鼠耳廓血管的渗透性，有效减轻耳廓水肿并减少肥大细胞的数量，表明其可有效抑制I型过敏反应；构建AR模型评价疗效，发现过敏康能显著改善小鼠搔鼻和打喷嚏症状，降低血浆中OVA-sIgE、IL-4和TNF-α含量，并减轻小鼠鼻粘膜形态改变和炎症。以RBL-2H3为炎症细胞模型，发现过敏康可显著抑制IgE/Ag诱导的肥大细胞脱颗粒，并降低组胺、IL-4和TNF-α的含量，有效抑制细胞钙内流，并能抑制PLCγ1、Lyn、Syk、IκBα和NF-κBp65的磷酸化，其机制可能是抑制FcεRI和NF-κB 相关蛋白的激活。最后，构建高灵敏度、高选择性的Syk/Lyn/Fyn-HEMT生物芯片，对AR相关的靶蛋白与过敏康中关键入血成分进行亲和检测和筛选，发现亥茅酚可选择性地作用于Syk和Lyn蛋白，大黄素可选择性作用于Lyn蛋白，表明这两个活性成分均可通过直接与这些靶点发生亲和相互作用来抑制过敏反应；经体内、外实验验证及分子对接模拟，首次发现亥茅酚可有效抑制肥大细胞脱颗粒，减少组胺和炎症因子的释放，抑制钙内流，并显著抑制PLCγ1、Lyn、Syk、Fgr、IκBα和PKCδ/θ的磷酸化。总之，本研究表明过敏康在体内和体外均可显著减轻AR病症，而亥茅酚和大黄素则可能是其主要入血活性成分。其中，亥茅酚的抗过敏作用可能与抑制FcεRI、MAPK及NF-κB通路磷酸化的激活有关。本研究可为过敏康方的临床应用提供理论依据，可为其它中药复方的研究在技术或方法上提供借鉴。