中药细辛治疗难治性慢性咳嗽的药效物质与多靶点作用机制研究

The pathogenesis of cough hypersensitivity syndrome (CHS) includes cough receptor activation, neurogenic airway inflammation, abnormal immunoregulation and oxidative stress injury. This complicated mechanism makes CHS a challenge for refractory chronic cough treatment. The cough receptor and neuropeptide antagonists has been developed in western countries, unfortunately, the property of these drugs, such as single-target and unstable structure, limit their clinical use. Therefore, there is still lack of effective drugs for CHS treatment currently. In our previous study, we observed that the polysaccharide and lignan extracts from Asarum Heterotropoides (Xi Xin), showed antitussive, anti-inflammatory and anti-oxidative stress effects in animal model. This finding indicates that A. Heterotropoides might exert its effect through multiple therapeutic targets. But the effective compounds of A. Heterotropoides for multi-target and the underlying mechanism still remained unknown. Thus, based on the former finding, we will further isolate the components and compounds of A. Heterotropoides and then screen effective ingredients in several in-vitro models, such as cigarette smoking induced A549 and SH-SY5Y cell, TRPV1/TRPA1 stable transcription HEK293 cell, OVA induced HMC cell, LPS induced THP-1 cell. Subsequently, we will evaluate the efficacy of multi-target complex in CHS guinea pig model. This project aims to illuminate the effective components of A. Heterotropoides for CHS treatment and explore the underlying mechanism, and encourage the new drug research and development.

咳嗽高敏综合征（CHS）发病机制复杂，涉及气道咳嗽受体活化、神经源性炎症、免疫调节异常及氧化应激损伤等多个靶点，是难治性慢性咳嗽治疗的难点。国外针对咳嗽受体及神经肽受体等靶点已开发相应拮抗剂，但由于靶点单一或结构不稳定等，目前尚无有效治疗药物。我们前期研究发现细辛总多糖、总木脂素等具有良好镇咳、抗炎及抗氧化活性，揭示细辛可能具有多靶点作用，但针对每个靶点的活性成分及机制仍不明确。因此，本项目在前期基础上首先从细辛中系统分离相关组分及成分，其次通过已构建的多个靶点细胞模型（香烟烟雾诱导的A549与SH-SY5Y细胞模型、TRPV1/TRPA1稳转的HEK 293细胞模型、OVA诱导的HMC细胞模型、LPS诱导的THP-1细胞模型等）高效筛选相应的活性成分，继而再以CHS豚鼠模型综合评价多个靶点成分组合物的药效，阐明细辛治疗CHS的药效物质与多靶点作用机制。本项目将为细辛新药开发研究提供依据。

1 细辛各总提物的慢性镇咳活性及安全性初步评价.研究结果表明：非挥发性部位细辛水提物及细辛乙醇提取物均可以明显降低咳嗽高敏感性（CHS）豚鼠模型的咳嗽次数，延长咳嗽潜伏期。此外细辛水提物在最大给药浓度10 g/kg剂量下仍没有表现任何毒性，细辛乙醇提取物毒性亦较低。.2 细辛水提物中细辛总多糖（PSA）的制备及慢性镇咳活性研究.我们通过水提醇沉、乙醇洗涤、减压干燥等制备工艺得到PSA部位，通过硫酸苯酚法测定多糖含量；通过凝胶色谱法测定PSA样品分子量分布。多糖水解衍生化后GC-MS分析其主要单糖组成；药效结果表明PSA可以显著抑制豚鼠的咳嗽次数并延长其咳嗽潜伏期，具有较好的镇咳活性；PSA还可以降低肺组织中炎症介质及神经肽含量，具有降低气道炎症的作用，迄今尚未见有关细辛总多糖止咳活性的报道。.3 细辛醇提物中总木脂素（TLF）的制备及慢性镇咳活性研究.首次通过大孔树脂纯化工艺从中分离得到了TLF部位。药效结果表明细辛TLF可以明显抑制CHS豚鼠的咳嗽次数，具有较好的镇咳活性；TLF还具有明显的抗氧化及抵抗气道炎症的作用。.4 细辛挥发油制备新工艺及止咳药效物质.通过优化超临界CO2（SFE-CO2）萃取条件所制得的挥发油，GC-MS比较分析鉴定了17个可能的急性镇咳活性单体化合物；研究发现细辛脂素、芝麻素等非挥发性成分应是细辛发挥慢性镇咳药效的活性成分之一。.5 细辛脂素、芝麻素慢性镇咳活性及作用机制研究.从细辛SFE-CO2挥发油中得到3.8 g无色针状结晶，经1H-NMR、13C-NMR、HR-MS色谱测定为细辛脂素；新制备工艺大大简化了实验步骤，可以为后续活性及机制研究提供大量的受试前体药物；CHS豚鼠镇咳药效结果表明其具有一定的止咳活性；体外实验表明其可以显著抑制香烟提取物诱导的A549细胞模型TRPV1、TRPA1 mRNA的过表达，说明细辛可能是通过抑制咳嗽受体的表达发挥止咳作用。目前未见有采用制备细辛挥发油的方法制备得到细辛非挥发性成分细辛脂素的报道，亦未见有关细辛脂素止咳活性的报道。