“分子-细胞-动物”三维活性评价方法结合药物代谢组学技术研究车前子治疗痛风的药效物质基础及作用机制

Scientifically explain the pharmacodynamic material basis and function mechanism of traditional Chinese medicine is the bottleneck in the modernization of traditional Chinese Medicine. As a common medicine for gout treatment, the pharmacodynamic material basis and function mechanism of Plantaginis Semen are still not very clear. In order to solve the existing problems, we propose to study on the pharmacodynamic material basis and function mechanism of Plantaginis Semen for treating gout based on three-dimensional activity evaluation method of “molecular-cell-animal” coupled with pharmaco-metabonomics technology. Firstly, the different fractions of Plantaginis Semen are prepared by column chromatography and the effective fractions are screened by three-dimensional activity evaluation method. Secondly, the in vivo chemical substances and the effects of active components on the main pathways of disease and endogenous biomarkers were studied by pharmaco-metabonomics technology. Finally, the “drug-biomarker-target-disease” interactions network was established based on the in vivo chemical substances and endogenous biomarkers. And the key targets in network are verified by western-blot and RT-PCR methods. The in vivo pharmacodynamic material basis is verified by ultrafiltration mass spectrometry method. According to the above-mentioned results, the pharmacodynamic material basis and multi-component, multi-target function mechanism of Plantaginis Semen for treating gout will be clarified. This project will provide the methodological reference and technical support for the study of pharmacodynamic material basis and multi-component, multi-target function mechanism of traditional Chinese medicine.

科学阐释中药药效物质基础和作用机制是中药现代化亟待突破的瓶颈问题。中药车前子作为治疗痛风的常用药，其药效物质基础和作用机制尚不清楚。针对该问题，本项目将基于中医药理论的整体观，采用“分子-细胞-动物”三维活性评价方法结合药物代谢组学技术，研究车前子治疗痛风的药效物质基础及作用机制。首先，通过柱色谱制备结合三维活性筛选，得到车前子活性组分。其次，利用药物代谢组学技术，阐明活性组分的体内化学物质组及其对疾病主要通路及内源性生物标志物的影响。在此基础上，辅以网络药理学方法，构建“药效物质—生物标志物—靶点—疾病”作用网络；采用Western-blot和RT-PCR技术对网络中的关键靶点进行确认和验证；针对关键靶点，利用超滤质谱方法对体内药效物质进行确认和验证；最终阐明车前子治疗痛风的药效物质基础和整体作用机制。本项目将为痛风治疗中药的药效物质基础及整体作用机制研究提供一种新的研究方法。

针对车子前治疗痛风的药效物质不清，作用机制不明的关键科学问题，本项目利用“分子-细胞-动物”三维活性评价方法与药物代谢组学研究方法相结合的组合技术，辅以网络药理学研究方法，对车前子治疗痛风的药效物质基础及作用机制进行了深入研究。首先利用大孔树脂柱分离并制备了车前子的不同极性部位，并利用液质联用和离子淌度质谱组合技术，从不同部位中分析并鉴定了86种化学成分，其中包括离子淌度结合理论计算鉴定出的一组同分异构体；然后从痛风的核心病理环节出发，从抑制尿酸产生、抑制尿酸重吸收和抑制炎症三个方面设计实验，构建了“分子-细胞-动物”三维活性评价方法，综合评价车前子总提取物及其不同部位对痛风的治疗效果，最后筛选出车前子50%洗脱部位（P2）是综合活性最佳的部位；接下来，利用细胞膜色谱和超滤质谱方法，对P2部位中主要的活性成分进行了筛选及鉴定，并利用黄嘌呤氧化酶抑制实验和尿酸重吸收实验对活性成分进行了确证，明确了P2部位的主要活性成分为毛蕊花糖苷、车前草苷等7种成分；利用“体外-体内”逐层递进的方法对车前子P2部位的主要入血成分进行研究，共鉴定出7种原型成分和27种代谢产物，并明确了主要原型成分的作用靶点。利用尿液代谢组学方法，对车前子P2部位可以调控的主要生物标志物及其靶点网络进行了研究。最终，将入血成分与尿液代谢组学进行关联分析，构建了“药效物质—生物标志物—靶点—疾病”整体作用网络，基于该网络，初步确定体内真正发挥疗效的物质及关键作用靶点。并通过Western Blot从蛋白质水平对关键靶点进行验证。综上，从中药多成分、多靶点协同作用出发，阐明了车前子治疗痛风的药效物质基础和整体作用机制。