朱砂、氯化汞和甲基汞的肾汞蓄积差异及其肾转运体机制

Cinnabar (HgS) is frequently included in traditional medicines, either as the presumed main active ingredient or as an auxiliary agent to assist the efficacy of the given therapy. However, mercury (Hg) is a highly toxic substance and the allowable Hg contents in cinnabar-containing Chinese medicines are over the safety limit and are forbidden in Western world. It should be noted that only cinnabar is used in traditional medicines, but it is frequently mistaken with mercury chloride (HgCl2) or methylmercury (MeHg) for risk assessment. Kidney is a major target of Hg toxicity, and our prior work indicate that marked differences in renal toxicity and Hg accumulation are found between cinnabar and HgCl2 or MeHg. Some studies recent show that the renal transporters including Oat1 and Oat3 play an important role in renal accumulation.The objective of this application is to determine the transport mechanisms of cinnabar, HgS, HgCl2 and MeHg accumulation in kidney. We hypothesize that cinnabar affects renal transporters differentially than HgCl2 and MeHg. Renal transporters can be grouped as basolateral uptake from the blood, efflux into the urine, reabsorption from the tubular lumen, and transport back into the blood. We will use mice and HEK293 cells to determine the effects of cinnabar, HgCl2 and MeHg on renal transport expression and to explore the relationships of cinnbar, HgCl2 and MeHg with renal mercury accumulation and renal toxicity after its exposure. Our proposed study is novel, in that it will for the first time elucidate the renal transport mechanisms for differential nephrotoxicity of cinnabar, HgCl2 and MeHg and the reveal potential Hg-drug interactions on renal excretion. Given the appealing nature of cinnabar in traditional medicines, our proposed study is significant in providing scientific basis for safety evaluation of cinnabar, and could be a paradigm shift in the way the toxic potentials of cinnabar, HgS, HgCl2 and MeHg will be viewed.

朱砂在中国传统医药中已有上千年药用史，涉及数百种中成药，但因其总汞含量超标，欧美国家对含朱砂中成药禁止销售。而申请者研究表明：朱砂和含朱砂中药的肾毒性远低于氯化汞和甲基汞，其肾汞蓄积差异达上百倍。因此提出仅用总汞来评价含朱砂中成药的安全性不尽完善。最新研究提示：肾汞蓄积与汞的肾毒性以及肾Oat1和Oat3转运体密切相关。为了进一步探索朱砂与常见汞化合物肾汞蓄积巨大差异的分子生物学基础，项目拟采用小鼠探索朱砂与氯化汞和甲基汞暴露后，观察肾汞蓄积和尿汞排泄差异与肾脏转运体的关系；采用RNA沉默法干扰HEK293细胞膜Oat1和Oat3转运体后朱砂与氯化汞和甲基汞引起的细胞毒性和细胞内汞蓄积的变化。阐明朱砂与常见汞化合物氯化汞和甲基汞的肾汞蓄积差异的肾转运体机制，为完善朱砂和含朱砂中成药安全性评价打下基础，对含朱砂中成药的合理应用和走向世界具有重要意义。

朱砂在中国传统医药中已有上千年药用史，涉及数百种中成药，但因其总汞含量超标，欧美国家禁止销售含朱砂中成药。而申请者研究表明：朱砂和含朱砂中药的肾毒性远低于氯化汞和甲基汞，其肾汞蓄积差异达上百倍。因此提出仅用总汞来评价含朱砂中成药的安全性不尽完善。最新研究提示：肾汞蓄积与汞的肾毒性以及肾Oat1和Oat3转运体密切相关。为了进一步探索朱砂与常见汞化合物肾汞蓄积巨大差异的分子生物学基础，项目采用小鼠探索朱砂与氯化汞和甲基汞暴露后，观察肾汞蓄积与肾脏转运体的关系，发现氯化汞、甲基汞和朱砂肾汞蓄积程度不同；本项目还采用RNA沉默法分别干扰293T细胞、Caco-2细胞的Oat1、Oat3和PEPT1转运体，观察朱砂与氯化汞和甲基汞引起的细胞毒性和细胞内汞蓄积的变化以及朱砂、硫化汞和氯化汞在Caco-2细胞模型中的转运水平，发现朱砂与氯化汞、甲基汞和硫化汞的肾汞蓄积不同即朱砂的转运与转运体OAT1和OAT3没有直接关系；朱砂、硫化汞的肠道转运水平低于氯化汞；发现PEPT1 介导了朱砂在 Caco-2 细胞模型中的转运。为了进一步研究朱砂和其他含汞化合物肾脏的摄入和外排差异，我们采用UPLC-ICP/MS检测293T细胞内外无机汞和甲基汞含量的变化，发现朱砂入胞速度远低于HgCl2和MeHg，并发现HgS和朱砂的外排可能与肾脏转运体Mdr1、Mrp2有关；同时我们还研究不同生长发育大鼠的转运体与年龄之间的关系，发现肾脏转运体的表达受个体发育和衰老的影响，这一研究揭示儿童和老人对药物和毒物的代谢与成年人不同，同时也为儿童和老人用药提供科学依据。本项目阐明了朱砂与常见汞化合物氯化汞和甲基汞的肾汞蓄积差异的肾转运体机制，为完善朱砂和含朱砂中成药安全性评价打下基础，对含朱砂中成药的合理应用具有重要意义。