白芍调控P-gp介导生物碱类成分血脑屏障转运机制研究

For those toxicity drugs acting on the nervous system can cause toxic on central nervous system, the literature studies have shown that Radix Paeoniae Alba often combined with medicine containing alkaloid, which have central nervous system toxicity, such as Radix Aconite, Radix Aconiti Lateralis and Strychni semen for restricting toxicity. The preliminary experimental results indicated that Radix Paeoniae Alba could reduce central nervous system drugs strychnine concentration in the brain. Putting this type of central nervous system toxicity herbs alkaloids-containing as model drug, we will simulate and predict the regulation of the active components on action of blood-brain barrier in alkaloids affected by P-gp in Radix Paeoniae Alba at the molecular level. Using MDCK-MDR1cell model, with the technology of FQ-RT-PCR and protein electrophoresis, we could not only study the effect of active substance of Radix Paeoniae Alba on the express of P-gp, P-gp drug binding site, the ATPase activity, the cell membrane fluidity, but also illustrate the material base, the mechanism and structure relevance of Radix Paeoniae Alba on regulating drugs transport that affected by P-gp. The research will provide new ideas to solve the central nervous system toxicity alkaloids of traditional Chinese medicine, and lay a scientific foundation for development on parties in toxic medicine, as well as clinical drug safety.

针对作用于神经系统的有毒中药易引起的中枢神经毒性，文献研究表明白芍常与中枢神经毒性川乌、附子、马钱子等含生物碱类中药配伍，以制约毒性；前期实验表明白芍与马钱子配伍能够降低中枢神经毒性中药马钱子中马钱子碱在脑中浓度。以此类中枢神经毒性中药生物碱类成分为模型药，对白芍活性成分调控此类生物碱类成分透血脑屏障(BBB)作用进行分子对接技术模拟预测；运用MDCK-MDR1细胞模型，对调控透BBB性能进行研究；采用荧光定量PCR、蛋白电泳等技术，研究白芍活性成分与生物碱类配伍对P-gp表达、与药物结合位点的关系、对ATP酶活性及细胞膜流动性的影响；并对白芍调控BBB作用与生物碱类成分结构类型进行关联，阐明白芍调控P-gp介导有毒中药生物碱类成分透BBB的物质基础、作用机制和结构选择关联性。从而为解决中药生物碱类成分在临床上中枢神经毒性提供新思路，为含有毒中药类方研究开发及临床用药安全性提供科学依据。

含生物碱类中药是临床常用的中药。关于中药引起中枢神经系统毒性临床报道较多的是马钱子、乌头类中药等，毒性以生物碱类成分为主。白芍为一种临床常用中药，临床上常将白芍与此类含生物碱类中药配伍使用，以制约其毒性。项目考察白芍对生物碱类成分引起大鼠神经毒性的抑制作用；通过网络药理学和分子对接技术探讨白芍和生物碱类成分作用神经毒性靶点的相互作用；采用分子生物学技术，运用MDCK-MDR1细胞模型研究白芍活性成分（芍药苷、白芍药苷）对生物碱类成分（士的宁、马钱子碱、乌头碱、次乌碱和培胺）跨膜转运的影响，以及两者配伍对P-gp蛋白表达、ATP酶活性、P-gp功能活性和MDR1 mRNA表达水平的影响。整体动物研究表明，芍药苷明显降低了生物碱类成分对中枢神经系统的抑制作用，且大脑皮层神经元损伤减轻，前额叶皮质区、海马、纹状体、小脑、下丘脑及血清TPH、TH、BDNF、NGF表达显著增加，显著改善生物碱类成分引起的大鼠中枢神经毒性损伤；分子对接研究表明，生物碱类成分均能通过碱基苯环、N原子与P-gp、MRP1、Claudin、ZO-1等蛋白结合位点的氨基酸残基作用产生氢键、范德华力、阳离子-π等作用力。并因与蛋白结合，从而外排到细胞外；细胞实验表明白芍活性成分可通过上调P-gp表达水平、诱导P-gp ATP酶活性、增强P-gp外排功能和提高MDR1 mRNA表达来促进生物碱类成分外排，从而减少药物浓度，降低其毒性。项目从分子-细胞-动物水平，阐释白芍调控P-gp介导有毒中药生物碱类成分透血脑屏障的转运机制与结构关联，为含有毒中药类药方研究开发及临床用药安全性提供新策略，对丰富配伍给药内涵具有积极意义。