基于动态生物网络体系的马钱子致神经毒性“物质基础-毒性效应-损伤机理”和白芍干预作用的研究

Our study is conducted under the guidance of Chinese medical theory, modern analytical science and technology, focusing on the core issues of the neurotoxicity induced by Semen strychni and the protective effects of Radix Paeoniae Alba. The toxic (effect) components and mechanism are to be investigated integratively and innovatively based on an idea of dynamic bio-network system in the perspective of the interaction of herb and body..With the direction of Cell-based toxic screening and pharmacological models in vivo, the toxic part (components) of Semen strychni extract is going to be screened out. Then the quantification of dynamic profiles of the extract of Semen strychni and Radix Paeoniae Alba in vitro and in vivo (blood and different brain regions) is to be carried out to clarify the potential toxic (effect) substances. Furthermore, a set of biomarkers which are related to neurotoxicity and used for early diagnosis and clinical prevention, will be obtained from the quantification of sensitive endogenous substances in the blood and brain. These results will be helpful to identify the metabolic pathways and target regions in the brain involved in neurotoxicity. .According to the research on the cellular and molecular mechanisms mediated by Semen strychni (Radix Paeoniae Alba), the changes of the expression levels of specific proteins and cytokines in PC12 cells are traced at the molecular level in order to elucidate metabolic pathways and investigate the neurotoxicity(intervention) mechanism induced by Semen strychni (Radix Paeoniae Alba). Ultimately, the goals will be obtained to recognize its toxic (effect) components, identify the brain target regions and demonstrate its toxic (effect) mechanism from an integral view of material basis, toxic effects and damage mechanism. Our findings are promising to provide beneficial information for further understanding and better using of Semen strychni and might be helpful to develop new antidotal herb in clinical.

以中医药理论为指导，现代科学技术为支撑，针对马钱子致神经毒性作用和白芍配伍减毒效应核心问题，从药物对机体影响和机体对药物处置两方面，以整体动物-靶器官-细胞关联角度，集成创新性地提出了动态生物网络体系研究思路。.首先，以细胞毒理效应和体内模型为指导，筛选毒性部位（成分）；其次，对致神经毒性作用马钱子和回调药白芍的化学成分及血-各脑区移行成分进行动态、定量指纹图谱分析，阐明毒性（药效）物质基础；进而，对中毒大鼠血、脑内源性物质变化追踪，确认敏感、特异性生物标记物，推断代谢通路和脑部靶向性；进一步，在细胞和分子水平上对马钱子（白芍）调控细胞网络分子机理研究，追踪细胞内特定蛋白、细胞因子表达水平变化，揭示马钱子（白芍）神经毒性（干预作用）相关通路，探讨神经损伤可能机制。最终，从“物质基础-毒性效应-损伤机理角度，达到物质组分清晰、作用靶点明确、机制清楚目标，为安全用药和解毒药资源开发提供科学依据

本项目以马钱子为研究对象，建立体内、外神经毒性评价模型，综合应用血清药理学、分子生物学、生物统计学等多学科理论与分析技术，对马钱子总生物碱的神经毒性和白芍总苷的干预作用进行了研究，本研究项目的所有研究内容为马钱子的临床合理、安全用药提供了依据，并为神经保护药物的开发提供了有价值的参考。. 根据体内、 外药理模型，确定了马钱子致神经毒性作用的毒性部位为马钱子总生物碱，白芍的神经保护有效部位为白芍总苷。对毒性化学部位进行提取分离，获得了马钱子总生物碱提取物中的5个单体化合物。采用Q Exactive Focus UPLC-MS方法，准确鉴定了含马钱子总生物碱血清中的7种原型成分和21种代谢产物成分，以及给药后脑组织中的3种原型成分；对白芍总皂苷干预作用的入血成分进行分析，确定了血清中的13个化学成分。采用HPLC-MS/MS 方法测定大鼠血浆和脑中毒性成分士的宁和马钱子碱的浓度，分析了对白芍总苷马钱子总生物碱主要毒性成分入血、入脑吸收率和代谢规律的影响。结果表明，给予白芍总苷后，士的宁的脑血分配系数减小，透过血脑屏障的能力降低。. 建立了LC-MS/MS、ELISA等方法，对马钱子中毒后大鼠血清和脑中包括神经递质、神经营养因子、神经内分泌激素、雌性激素、能量代谢产物和磷脂与鞘脂代谢物在内的30余种内源性物质的变化进行了测定，并结合Lasso-Logistic回归模型筛选出了13种代表性的內源性生物标志物。将PC12细胞在含有马钱子总生物碱的培养基中孵育24 h，以细胞形态、细胞存活率、细胞生化指标、细胞凋亡率为毒效指标，建立评价马钱子总生物碱致神经毒性和白芍总苷干预作用的细胞模型。结果表明白芍总苷能够减轻马钱子总生物碱引起的氧化损伤，提高细胞存活率，对马钱子总生物碱毒性具有干预作用。采用Western bloting分析技术测定了细胞内Nrf2和HO-1蛋白表达量，结果表明白芍总苷可激活Nrf2/ARE信号通路发挥其干预马钱子总生物碱引起的神经毒性。