基于“代谢吡咯-DNA加合物”致毒机制的电化学发光传感器的千里光安全性评价研究

Traditional Chinese medicines (TCMs)-based products are becoming more and more popular over the world. Consequently, safety evaluation of Chinese medicinal materials has been an important issue, especially for the development of analytical system based on their in vivo toxic mechanism. Taking Senecionis Scandentis Hebra as an example, it has been demonstrated that pyrrolizidine alkaloids (PAs) exert carcinogenicity via a set of pyrrole-DNA adducts. The levels of pyrrole-DNA adducts correlate well with the severity of liver damage. Therefore, the pyrrole-DNA adducts could be considered as in vivo toxic mechanism-based biomarker for assessing the PAs-induced carcinogenicity. However, due to the lack of practical analytical methods for quantifying the pyrrole-DNA adducts in vivo, the safety evaluation of PAs-containing herbs was severely limited. Our previously developed electrochemiluminescence (ECL) sensor could potentially quantify pyrrole−DNA adducts. Thus, we will conduct the following studies: (i) improve the fabrication of ECL sensor with high-sensitivity, visualization and portability; (ii) validate the correlation between the levels of pyrrole-DNA adducts formed in vivo with the toxic potency of PAs treated animals in a dose-responsive manner; (iii) validate the practicality and reliability of mechanism-based ECL sensor in assessing the toxic risk posed by Senecionis Scandentis Hebra. This project aims at providing a novel thought and method for safety evaluation of TCMs.

中药安全性问题是制约中医药现代化和国际化的瓶颈，建立基于致毒机制的安全性评价方法是解决问题的关键。千里光因含有肝毒性成分吡咯里西啶类生物碱（PAs）而备受关注。代谢吡咯-DNA加合物是PAs在体内的致毒形式，但目前尚缺乏体内代谢吡咯-DNA加合物的有效测定方法。申请者前期初步构建了基于PAs与CYP450酶代谢反应的电化学发光传感器，发现其具有测定代谢吡咯-DNA加合物的潜力。在此基础上，本项目拟1）基于“代谢吡咯-DNA加合物”致毒机制，构建高灵敏、可视化、便携式的电化学发光传感器，并进行系统的性能优化、机理探讨及方法学考察；2）采用传感器检测不同剂量下动物样本中代谢吡咯-DNA加合物的量，通过多元统计分析，构建加合物的量与毒理指标的相关性；3）基于致毒机制的电化学发光传感器用于千里光安全性评价，阐明其致毒物质基础。本项目旨在为解决中药安全评价问题提供新方法和新思路。

中药安全性问题是制约中医药现代化和国际化的瓶颈，建立基于致毒机制的安全性评价方法是解决问题的关键。本项目从千里光为研究对象，针对体内代谢吡咯-DNA加合物的有效测定方法的缺乏，将电化学发光和微流控液滴引入安全性评价研究，解决了以下关键科学问题1）基于“代谢吡咯-DNA加合物”致毒机制，进行PAs处理，若PAs与DNA形成了加合物，则会破坏G-四链体的结构，导致其过氧化物酶活性降低，以此实现DNA加合物损伤的检测，构建了高灵敏、可视化、便携式的传感器，解决代谢吡咯-DNA加合物的有效测定方法缺乏的难题；2）构建基于微流控液滴技术的药物安全性评价平台；合成3D胶原液滴，包埋肝细胞，构建细胞液滴库；构建PAs液滴库；将药物液滴和细胞液滴融合，基于之前建立的DNA加合物损伤测定方法，研究PAs对细胞的肝毒性；采用搭建的荧光分选系统，分选出有肝毒性的细胞液滴做进一步分析；3）构建基于微流控液滴技术的药物安全性评价平台用于千里光安全性评价研究，实现阐明其致毒物质基础。本项目为解决中药安全评价问题提供新方法和新思路。