藏药佐太中汞在肠壁跨膜转运中的价态、配位形式及分子行为机制研究

The relation between heavy metals and human health is the focus of attention in the modern society, and many people are on "mercury phobia". However, mercuric sulfide preparations in many traditional medicines still be wildly used today and appear non-toxic and effective in clinical, such as traditional Tibetan medicine Zuotai (gTso thal). So, this conflict between modern understanding of mercury and Traditional experience is a great challenge to the survival of national medicine. In fact, the toxicity of mercury is greatly relative to its chemical speciation: organic mercury is highly toxic, but the toxic of mercuric sulfide is not yet proved conclusively so far. Therefore, to explore the chemical species of mercuric sulfide in intestinal transmembrane absorption is the key that solving the toxic or non-toxic problem of Tibetan medicine Zuotai. Our previous study found that mercuric sulfide is main component in Zuotai, and confirmed it could produce mercury accumulation in rat organs. In present research, we try the interdisciplinary research of high-energy physics and traditional Tibetan medicine, using Synchrotron Radiation X-ray Absorption Fine Structure (XAFS), micro X -ray Fluorescence (μXRF) and other technologies to detect the chemical valence, ligand and the local spatial structure of mercury in intestine and blood in situ, and using isotope labeling and chemical fluorescent tracing technique to study visually the molecular behavior dynamic of mercury of Zuotai in intestinal transmembrane transport. Moreover, we try reveal the roles of hydrophobic diffusion of plasma membrane and carrier-mediated assist transit (aminoacid/peptide transporters, divalent metals transporters, organic anion transporters and multi-drug resistance-associated proteins) in the intestinal transmembrane absorption of converted species of mercuric sulfide in Zuotai using the phospholipid bilayer model, intestinal cell monolayer model and others. In conclusion, this research will provide new ideas and methods for the safety and effectiveness evaluation of heavy metals in traditional medicines.

重金属与人体健康的关系备受社会关注，人们更是"谈汞色变"，但另一方面含汞传统药物如藏药佐太等在临床上却广泛使用，并被认为"无毒且有效"，这种现代认识与传统经验的冲突对民族医药生存已形成极大挑战。研究表明，汞的毒性与其赋存形态有很大关系，有机汞极毒，而硫化汞尚未有文献确切证明其有毒。因此，准确探明佐太中硫化汞的跨膜吸收形态及转变，就成为解决其有毒无毒问题的关键。在我们前期大鼠器官分布蓄积研究证明佐太中汞可以被吸收的基础上，1）采用同步辐射X射吸收精细结构（XAFS）与微区X射线荧光(μXRF)技术，原位探测佐太中汞在肠壁和血液中价态、配基及局域空间结构；2）运用放射性同位素标记、化学荧光背底示踪技术研究汞跨肠转运分子行为动态；3）利用磷脂双层、肠细胞单层模型，探明疏水性质膜扩散和载体蛋白介导的协助转运在汞跨膜中的作用，以期为科学阐释重金属传统药物的安全性与有效性提供新思路与新方法。

重金属与人体健康的关系备受社会关注，人们更是“谈汞色变”，但另一方面含汞传统药物如藏药佐太等在临床上却广泛使用，并被认为“无毒且有效”，这种现代认识与传统经验的冲突对民族医药生存已形成极大挑战。前期研究表明，汞的毒性与其赋存形态有很大关系，有机汞极毒，而硫化汞尚未有文献确切证明其有毒。因此，准确探明佐太中硫化汞的跨膜吸收形态及转变，就成为解决其有毒无毒问题的关键。为此，本项目从3个方面研究：（1）肠壁中汞的化学种态与微区分布。利用同步辐射微区X-射线荧光谱（μ-XRF），揭示了佐太中汞在大鼠十二指肠中主要分布在肠腔和粘膜层，少量分布在粘膜下层，并发现对佐太和β-HgS的吸收远远小于氯化汞；同时，利用同步辐射X-射线吸收精细结构谱（XFAS），发现佐太中汞在肠壁组织中主要为HgCys2和β-HgS等，提示其可能是佐太中汞跨膜吸收的主要形态。（2）肠壁跨膜转运分子行为机制探索。探明佐太、β-HgS和α-HgS中汞在模拟肠液的溶出高于在纯水的溶出，以及巯基生物分子（Cys和GSH）能促进佐太中汞在模拟肠液中的溶出；发现佐太中汞在大鼠十二指肠道环境中的化学形态，主要为β-HgS、HgCys2、游离二价汞离子（Hg2+）等；探明佐太中汞转化形态HgCys2和游离Hg2+在10-1000 μg/L Hg浓度下在十二指肠中的吸收差异特征（后者高于前者），并对吸收时间具动态依赖性；弄清了在Cys浓度0-1000 μg/mL范围对佐太、β-HgS和α-HgS中汞在离体大鼠肠囊中的吸收干预特征——浓度越高对汞的跨膜转运促进越大，从侧面验证了氨基酸/多肽转运载体参与汞转运的猜想。（3）血液中汞的结合形态。揭示了藏药佐太、β-HgS和HgCl2中汞血液中的价态和配位形式；利用蛋白分离纯化与鉴定技术，发现转铁蛋白是是汞在血浆中的主要结合蛋白，并计算出Hg2+在转铁蛋白Fe3+结合位点的配位参数。本研究促进了传统药物中汞的安全性评价由原来的单纯含量测定提升到价位、形态、动态的综合评价，可为佐太进入机体后“无毒或有效”以及形态结构与功能关系研究开启一扇大门。