基于汞元素金属组学的朱砂中枢神经毒性机制研究

Cinnabar (HgS) is a traditional Chinese material medica with wide applications. Since the mercury compounds are extremely toxic, and the “settling fright, tranquilizing the mind” efficacies of cinnabar are highly similar to the central nervous system side effects of mercury poisoning, the safety of cinnabar arose great concerns recently. In vitro studies have demonstrated that cinnabar was much less toxic than the other mercuric species, such as mercury chloride, methylmercury, etc. But cinnabar can be converted into the highly toxic methylmercury due to in vivo metabolism, posing a significant risk to the central nervous system. However, the mechanisms of cinnabar-induced central neurotoxicity remain elusive. Cinnabar is still formulated in almost 4.5 percent of Traditional Chinese Medicines included in Chinese Pharmacopoeia to “settle fright, tranquilize the mind”, even in some of the pediatric medicines..Therefore, this project will study on the mechanisms of the cinnabar-induced central neurotoxicity through the Metallomics of mercury in rat’s brain. The key laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) in situ imaging method will be established for the distribution analysis of mercury in rat’s brain after administration of cinnabar. HPLC-Cold Atomic Vapor-Atomic Fluorescence Spectroscopy method will be established for the mercuric species analysis of each brain region. Combined with the toxicity evaluations, the material basis of cinnabar-induced central neurotoxicity will be revealed. Gel Electrophoresis/LA-ICP-MS techniques will be used to screen and separate the mercury-binding proteins for the protein identification using mass spectrometry, illustrating the molecular targets in the brain. And finally, the mechanisms of cinnabar-induced central neurotoxicity will be clarified, which can provide scientific references for the safety evaluation and rational use of cinnabar.

朱砂(HgS)是传统中药材，应用广泛。但由于朱砂含有大量的重金属汞，其“镇惊安神”的功效与汞对神经系统的毒副作用高度相似，不良反应又时有发生，其安全性备受质疑。研究表明，朱砂体外毒性远小于氯化汞、甲基汞等汞形态，但经体内代谢可能产生剧毒甲基汞，存在显著的中枢毒性风险。然而，朱砂神经毒性机制尚不明确，在中国药典中却仍约4.5%的成方制剂中配伍使用，甚至包括数十种儿童专用制剂。因此，本项目拟针对朱砂中枢神经毒性，采用金属组学研究策略，以激光剥蚀-电感耦合等离子体质谱(LA-ICP-MS)原位成像关键技术，精准测定摄入朱砂后汞元素在大鼠脑内的分布；利用色谱-光谱联用技术分析各个脑区的汞形态，结合毒性观测，揭示朱砂神经毒性的物质基础；通过凝胶电泳/LA-ICP-MS技术筛选汞结合蛋白进行质谱分析，明确朱砂的脑内分子靶点；从而阐明朱砂中枢神经系统毒性机制，为朱砂的安全性评价和合理使用提供科学依据。

朱砂(HgS)是传统中药材，应用广泛。但由于朱砂含有大量的重金属汞，其“镇惊安神”的功效与汞对神经系统的毒副作用高度相似，安全性备受质疑。然而朱砂神经毒性机制尚不明确。本研究切合临床应用，根据实际用药方案，设置了合理的朱砂染毒方案，利用神经行为学和组织形态学方法综合评价朱砂神经毒性，通过比较不同染毒剂量与染毒周期对神经毒性的影响，明确了朱砂的“量-时-毒”关系：服用时间越长，剂量越高，神经毒性越强，长期过量服用朱砂会导致大鼠学习记忆能力显著下降，提示临床使用时要避免长期、过量服用朱砂，以免引起神经中毒反应。采用金属组学技术分析了大鼠摄入朱砂后体内的汞形态变化，结合毒性观测，通过多种数据模型分析明确了HgII与MeHg是朱砂导致中枢神经毒性的物质基础，并且二者的内暴露量安全限值分别为27.15 ng/mL、20.91 ng/mL，可用于临床监测朱砂潜在毒性。利用脂质组学研究策略，分析了脑组织正常生理功能所依赖的关键脂质分子变化，大鼠口服朱砂后会打破脂质稳态，MeHg可引起TAG类脂质表达上调，HgⅡ可引起PC、SM类脂质上调，并经过多种数据模型筛选得到TAG54:2和TAG58:7是朱砂神经毒性的关键效应分子，可作为朱砂神经毒性的监测指标之一。利用LA-ICP-MS对脑组织中汞分布进行原位成像分析，大鼠口服朱砂后汞能够穿透血脑屏障分布至脑组织中，且易富集于血供丰富的皮质与海马CA1区，导致神经毒性；并对脑组织中关键蛋白进行分析，明确了大脑皮质及海马突触后膜损伤是导致朱砂神经毒性的关键原因。通过上述研究可为朱砂的安全性评价和合理使用提供科学依据。