基于肥大细胞TCRPs筛选中药芍药多靶点抗过敏分子机制及其物质基础研究

Traditional Chinese Medicine (TCM) has been used to treat allergic disease for long history, however is lack of research of based-on molecular technology and theory. Recently the biosensor technology for monitoring living cell biological function has many features such as large scale, real-time, free-label and multi-parameters, which has been widely used as screening of small molecular compounds, but not of the natural products. In this work, we apply a novel high-throughput cell sensor chip strategy that uses electronic impedance readouts for dynamic monitoring of dose-dependent mast cell degranulation process (TCRPs). The utility of this approach was used to screen and characterize multi-targets and active ingredients in natural anti-inflammatory Chinese herbs combined with biochemical methods and whole animal experiments.Finally it was to investigate molecular mechanism and its material basis deeply. We have screened several nature products interfering IgE-mediated mast cell degranulation in our pre-experiments based-on mast cell TCRPs. To demostrate the feasibility of novel methodology for natural products, we used a variety of well-studied molecular and cell biological tools to detecte FcεRI-mediated downstream signaling to characterize anti-allergic mechanism and identify active ingredients of the representative Chinese herb Paeonia lactiflora . In this study, we will construct anti-allergic screening assay targeted for mast cell degranulation and make clear the theory and evidence of the TCRPs assay screening for anti-allergic natural products.

中医药治疗变态反应有上千年历史，但基于现代分子技术理论与实践非常匮乏。近年来以活细胞为敏感元件的细胞传感器具有大规模、实时、无创及多参分析的特点，已广泛用于小分子化合物筛选，但在天然药物领域尚缺乏研究报道。本课题运用新型高通量活细胞传感芯片，以变态反应的效应细胞为模型，构建时间剂量依赖性图谱（TCRPs）动态监控肥大脱颗粒过程，同时结合整体动物、生化手段用于抗变态反应的多靶点中药药效分析与组分筛选，进而深入探讨其分子机制及物质基础。基于前期大量预试验数据，本课题将筛选若干以调控IgE介导脱颗粒为靶向的中草药，通过分子检测及FcεRI介导下游多信号通路进一步明确以芍药为代表的抗炎中草药的药效分子机制，同时基于组分分离探索其多靶点药效的物质基础。本研究预期成果将建立以IgE介导肥大细胞脱颗粒为靶向的抗过敏多靶点筛选模型，从而明确TCRPs技术用于抗炎中医药研究理论与实践依据。

肥大细胞是过敏性炎症的主要效应细胞，激活后脱颗粒，释放组胺、类胰蛋白酶、细胞因子、细胞毒素及蛋白酶等细胞毒活性物质，使平滑肌收缩和微血管通透性升高，并募集炎症细胞，引起组织损伤和变态反应。我们运用新型高通量活细胞传感芯片来建立时间剂量依赖性图谱（TCRPs）动态监控肥大脱颗粒过程，同时结合整体动物、生化手段用于抗变态反应的多靶点中药白芍药效分析与组分筛选，进而深入探讨其分子机制及有效物质基础。课题组建立了IgE介导的肥大细胞脱颗粒TCRP体系并用于抗过敏中药的筛选，同时构建了肥大细胞囊泡颗粒转运的腺苷TCRP、骨架屏障解除过程的佛波酯TCRP 两个不同信号通路的子模型。这监测方法可帮助我们更加客观的理解细胞反应过程，对细胞反应过程进行如此精细的监测，是目前终点实验所无法实现。我们通过测定β氨基己糖苷酶活性、炎症因子表达和整体迟发型变态反应模型观察白芍抗过敏药效，论证了基于IgE介导的TCRP作为抗过敏筛选模型的可行性。同时，我们构建了adenosine TCRP 和PMA TCRP作为快速预测白芍抗过敏作用机理筛选模型，用WB和钙流验证了白芍作用于Gab2/AKT/Erk信号通路从而影响囊泡转运和抑制钙流从而影响微丝重排和囊泡释放，从而进一步在方法上论证了TCRP技术用于传统中草药抗过敏作用筛选的可行性。也证明了中药白芍具有多靶点抗过敏作用特性：既能影响囊泡转运过程又能通过抑制钙流而影响囊泡释放；同时我们发现了白芍抗过敏的有效组分，可改善小鼠免疫性紫癜模型血小板各项指标，同时可调节T reg 细胞功能和平衡Th1/Th2功能；有效组分可改善小鼠过敏性哮喘气道高反应和肺脏炎症情况，为后期白芍有效组分进一步机制研究和产品开发提供了坚实基础。