莪术活血化瘀功效与子代神经发育毒性选择性表达的分子机制研究: 基于“有故无殒，亦无殒”理论

Results obtained with normal animal could not completely demonstrate embryo-fetal developmental toxicity of Chinese medicine with drastic effect, which have restricted rational clinical applicability of it. The theory of “YOU-GU-WU-YUN” is criterion about safety of Chinese medicine during pregnancy. Therefore, we propose the working hypothesis that cold coagulation and blood stasis syndrome is determining factor for selective expression of developmental neurotoxicity of Rhizoma Curcumae or its therapentic effect of removing blood stasis and resolving static blood. To test this hypothesis, this project take brain tissue of offspring from oxidative stress injury and apoptosis induced by Rhizoma Curcumae, which is forbidden to take for pregnant women, as an entry point to evaluate the differences of developmental neurotoxicity, oxidative stress and apoptosis in offspring between normal and cold coagulation and blood stasis syndrome pregnant mice. Then, we investigate the effect of cold stress on the Nrf2-mediated anti-oxidative stress signaling pathway, and the important role played by Nrf2 in selective expression of developmental neurotoxicity of Rhizoma Curcumae was verified using Nrf2-deficient mice and tert-butylhydroquinone, Nrf2 pharmacological activators. This project will evidence the theory of toxicity-attenuating based on syndrome differentiation of Chinese medicine, and has the important theory and the practical significance for establishing research system of developmental neurotoxicity of traditional Chinese medicine.

以正常动物为对象的毒性研究结果不能全面反映峻烈中药的发育毒性，限制了其临床应用，“有故无殒，亦无殒”是中医对妊娠期用药安全的权衡准则。本项目围绕“寒凝血瘀证是莪术神经发育毒性与活血化瘀功效选择性表达的决定性因素” 的科学假说，以妊娠禁忌中药莪术的子代脑组织氧化应激损伤为切入点，比较正常状态和寒凝血瘀证模型小鼠母体妊娠期莪术暴露对子代神经发育、脑组织氧化应激和凋亡影响的差异。观察寒邪（冷应激）对核因子E2相关因子2（Nrf2）介导的抗氧化应激通路信号分子的影响，并利用Nrf2基因敲除小鼠和Nrf2激动剂叔丁基对苯二酚（tBHQ）验证Nrf2在神经发育毒性选择性表达中的关键作用。丰富“有故无殒，亦无殒”的中药辨证减毒理论的科学内涵，对构建符合中医理论和临床实际的中药神经发育毒性研究体系具有重要的理论和现实意义。

活/破血化瘀法在治疗妇科疾病方面表现出独特优势，破血化瘀中药的妊娠毒性与其应用脱离中医药理论指导有关。“有故无殒，亦无殒”是中医对妊娠期用药安全的权衡准则。本资助项目按计划比较正常状态和寒凝血瘀证模型小鼠母体妊娠期莪术暴露对子代早期神经行为发育、脑组织氧化应激和细胞凋亡影响的差异，探讨神经发育毒性差异的抗氧化机制，明确Nrf2活化在莪术神经发育毒性“有故无殒”现象中的关键作用。本资助项目研究发现：中药莪术显著降低血瘀证孕大鼠的血液黏度、血小板聚集率、血浆血栓素B2含量，升高血浆6-酮-前列腺素F1α含量。孕期莪术暴露致子代早期神经行为发育障碍在血瘀证模型小鼠和正常小鼠中选择性表达，在正常小鼠中表现出毒性效应。孕期莪术暴露致正常小鼠子代脑组织氧化应激和细胞凋亡，对血瘀证小鼠子代无显著影响。孕期莪术暴露导致正常小鼠子代脑组织NADPH氧化酶4和Bax表达上调、caspase-9和caspase-3活性升高、JNK磷酸化水平增加。血瘀证小鼠子代脑组织核因子E2相关因子2、血红素加氧酶1、谷氨酰半胱氨酸合成酶催化亚基和超氧化物歧化酶1等抗氧化酶表达较正常小鼠子代显著上调。核因子E2相关因子2基因敲除显著提高血瘀证小鼠的莪术子代神经发育毒性、认知损伤、脑组织氧化应激、细胞凋亡。2,5-二叔丁基氢醌预处理显著减弱莪术子代神经发育毒性、认知损伤、脑组织氧化应激、细胞凋亡。本资助项目研究结果部分阐明莪术神经发育毒性“有故无殒”现象的科学内涵，发现核因子E2相关因子2活化是莪术活血化瘀功效和神经发育毒性选择性表达的关键机制。本资助项目研究为传统活血化瘀中药妊娠毒性评价提供示范性研究，丰富中医辨证用药减毒理论。本资助项目研究结果发表论文4篇。