基于毒代动力学改变的朱砂-马钱子配伍降低神经毒性的物质基础和机理研究

Shang-Ke-Jie-Gu Tablet (SKJGT) is a kind of Chinese patent medicine, which has been extremely widely used in the current fracture treatment. In our previous study, the combination of cinnabar and Semen Strychini in SKJGT was first found to be of great significance, that cinnabar markedly attenuated the convulsions induced by Semen Strychni. With the support of National Natural Science Foundation of China, a series of experiments were conducted, leading to the preliminary conclusion that the cinnabar-induced alternation in toxicokinetics of Semen Strychini components may account for the toxicity-attenuation effect. Previous investigations suggested that the toxicokinetic alternation of Semen Strychini components could be caused by changes in their absorption, enterohepatic metabolism, and transportation. Cinnabar-Semen Strychini combination is usually used in bone fracture presciption, which might be a new pair in detoxication. Thus, the material basis along with the compatibility of the formula remained need to be clarified furtherly. Besides, the soluble mercury was found to be the active constituent for sendation in serum according to our previous study and reports, whether it is the active constituent for detoxication needs to be studied more. Therefore, sustainable funding is now eagerly applied to state the above issues. With the support, experiments will be performed to illustrate the material basis and impact of cinnabar on the absorption, transportation and metabolism of the toxic alkaloid in Semen Strychni. Then, the mechanism of toxicity-attenuation effect of cinnabar-Semen Strychni combination will be defined, reclaiming the vital role played by cinnabar in SKJGT, thereby enriching the prescription compatibility theory as well as the application theory of mineral medicine, providing the important proof for the application of cinnabar in chinese herbal compound prescriptions.

伤科接骨片是目前骨伤科销售量最大的中成药，前期研究发现，方中朱砂可极大地降低马钱子所引起的神经毒性，是未见报道的配伍减毒药物组合。在国家自然基金主任基金的资助下，我们发现朱砂减马钱子毒的主要原因是改变了其毒性生物碱的毒代动力学性质，这可能与朱砂影响了马钱子生物碱的吸收、转运以及肝肠代谢有关。且朱砂-马钱子配伍组合经常出现于骨伤中药方剂中，可能是新的配伍减毒药对，它们的作用物质基础和配伍规律应该阐明。据文献及前期研究发现，可溶性汞为朱砂吸收入血、起镇静作用的主要成分，它是否为降低马钱子毒性的有效成分也有待于进一步研究确认。因此，我们拟申请继续资助，深入阐释朱砂减马钱子毒的物质基础及朱砂-马钱子配伍减毒的规律，并基于朱砂影响马钱子毒性生物碱的吸收、转运和代谢等方面研究它们配伍减毒的机理，进而丰富中药配伍减毒药对理论及矿物药应用研究结果，重新评价朱砂在中成药制剂中不可或缺的作用。

本项目对伤科接骨片中朱砂减马钱子神经毒性的物质基础和机理进行了研究，取得的主要成果如下：.1. 发现朱砂中可溶性汞是降低马钱子神经毒性的关键物质。与单独给予马钱子（SS）相比，给予朱砂-马钱子（CSS）、朱砂提取物-马钱子（CESS）和HgCl2-马钱子（HSS）均可使大鼠血浆中士的宁和马钱子碱的最大吸收浓度（Cmax）与药时曲线下面积（AUC0-∞）显著降低，其中士的宁的Cmax 分别降低 1.71、1.90和 2.52倍。.2. 发现不同给药剂量与周期下，朱砂均可以促进马钱子中有毒生物碱的代谢。通过对比发现，连续15天给予马钱子与单次给予马钱子相比，大鼠血浆中士的宁与马钱子碱显著升高，提示马钱子连续服用可能会产生蓄积毒性。通过更改给药剂量（1倍，19倍）和周期（1天，15天），我们发现与单独给药马钱子相比，朱砂-马钱子配伍后均能够显著降低大鼠血浆中士的宁和马钱子碱的含量，进而降低马钱子的毒性，说明朱砂配伍减毒的必要性。.3. 以Caco-2细胞为模型，未发现朱砂影响马钱子的吸收，提示朱砂降低马钱子毒性可能与影响其肠道吸收无关。.4. 利用SD大鼠肝微粒体、分子探针以及western blot等方法研究了朱砂对肝代谢关键酶的分子调节作用，发现朱砂可以上调CYP3A2的表达进而提高马钱子中毒性生物碱的代谢达到减毒的作用，从肝代谢的角度解析了朱砂降低马钱子生物利用度的机理，明确了朱砂减马钱子毒是基于CYP450 酶减毒的机理。.5. 采用16S rRNA宏基因测序技术，对朱砂-马钱子配伍前后的肠道菌群进行分析，发现大鼠肠道中的螺杆菌科 NK4A136 菌、乳酸杆菌和普雷沃菌发生显著性变化，可能参与了朱砂对马钱子的减毒作用，包括影响神经递质释放与马钱子的代谢。.本项目阐明了朱砂降低马钱子神经毒性的物质基础，配伍规律与机制，重新评价朱砂在中成药制剂中不可或缺的作用，丰富了中药配伍减毒理论，为矿物药应用提供了理论基础。