基于多位点微透析和血关节囊屏障开放研究三妙丸中牛膝"引药下行"的物质基础和作用机理

To interpret "Meridian Guide" theory using modern pharmaceutical research techniques is one of the key scientific issues in the translational medicine of Chinese materia medica, which can provide some valuable scientific data for clinical application. The classical prescription of San Miao Wan is composed of Rhizoma Atractylodis, Cortex Phellodendri Chinensis, and Radix Achyranthis Bidentatae and used for eliminating heat and excreting dampness, in which the "Meridian Guide" mechanism of Radix Achyranthis Bidentatae is not clear. .In this study, microdialysis is used for synchronously sampling from blood, liver and knee joint of rat models of rheumatoid arthritis, and the alkaloids in Cortex Phellodendri Chinensis and volatile oil of Rhizoma Atractylodis in the microdialysates will be determined by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry method, respectively. Knee joint targeted distribution will be evaluated by relative targeting efficiency. Based on the dose-effect relationship, the dose distributions of multiple ingredients in Cortex Phellodendri Chinensis and Rhizoma Atractylodis in knee joint are chosen to be responsible for the meridian guide effect of Radix Achyranthis Bidentatae,and the substance basis of joint targeted drug delivery will be tracked using spectrum-effect relationship analysis. The underlying mechanism of Chinese materia medica was considered to be the interaction of two complicated biosystems, that is, the complex material system composed of active drug ingredients and the complex biological system composed of drug targets under pathological conditions, which function interactively in disease treatment..Based on the "blood-storing and tendons-governing function of the live" and the theory of "treatment of arthralgia-syndrome from liver", together with the fact that Radix Achyranthis Bidentatae mainly returns liver meridian, the substance basis of meridian guide effect on the hemorheology index and its relationship with the blood invigorating effect of Radix Achyranthis Bidentatae will be evaluated.The dual effect of substance basis of meridian guide effect on the synovial cells (increasement of drug distribution in articular cavity via F-actin, and suppression of drug efflux from articular cavity via P-glycoprotein) and its relationship with the promotion of drug-meridian-transport will also be investigated.It is the first time that the key role of "blood-joint barrier" opening in the "Meridian Guide" effect of Radix Achyranthis Bidentatae will be explored at the molecular level. The results of this project can offer a reference for the systematic study of other Meridian Guide drugs.

用现代药学研究技术阐释"引经"理论，可为引经药的临床应用提供科学依据，是转化医学中的中药关键科学问题之一。经方三妙丸由苍术、黄柏和牛膝组成，主治湿热下注之痹症，其中牛膝"引药下行"机制尚不明确。本项目以关节炎模型大鼠为对象，采用血、肝、膝关节微透析采样，以相对靶向率评价关节靶向分布；基于靶关节分布量效关系，确定苍术+黄柏多指标成分关节分布量为"牛膝引经指标"，谱效关系追踪牛膝引经物质基础，主张中药作用机制是"有效成分组成的复杂物质体系"和"病理条件下作用靶点组成的复杂生物体系"相互作用的结果。牛膝主归肝经，基于"肝藏血，主筋"和"痹证从肝论治"，研究引经成分对血液流变性指标影响与牛膝活血相关，对滑膜细胞双重作用（F-肌动蛋白增加药物关节腔分布，P-糖蛋白抑制关节腔药物外排）与牛膝促进药物经络转运相关，首次从分子水平探讨"血关节囊屏障"开放对牛膝引药下行的关键作用。为其他引经药研究提供借鉴。

“引经”理论是中药药性理论的重要组成部分，引经药可引导其他药物选择性地分布于该药物归经部位中的某脏腑组织。引经作用与协同药效的区别，关键在于引经药通过促进其他药物在靶部位分布，从而提高靶组织的药效。三妙丸源于二妙丸（治疗痛风基础方），首载于《医学正传》，由黄柏、苍术和牛膝（2：3：1）组成，黄柏为君药，牛膝“引药下行”为佐使药，可促进有效成分向下肢分布而提高疗效。.本研究梳理了中药引经理论的历史沿革，综述了各种引经药对其他药物体内过程和药效的影响，并对三妙丸方诸药进行考证。采用急性痛风关节炎模型大鼠，多次给药三妙丸（去牛膝组和含低、中、高牛膝组）后，UPLC-MS/ MS法检测大鼠血浆和组织中小檗碱和药根碱浓度，与去牛膝组比较，牛膝组可增加生物碱在下肢（膝关节、踝关节）以及肝、肾等下焦分布，减少其在心、肺等上焦分布。低、中、高剂量牛膝组的膝关节小檗碱浓度分别增加了6.53%，44.31%和212.96%，同时关节肿胀度减小，关节滑膜组织增生、炎细胞浸润等减轻。我们采用伊文思蓝法，发现牛膝可增加正常家兔血－关节囊屏障通透性，降低炎症家兔屏障通透性并修复受损屏障，即牛膝对关节囊屏障通透性具有双向调节作用。采用血、肝、膝关节多位点微透析采样技术，发现牛膝能增加游离小檗碱在正常家兔血、肝和关节液中的分布；在关节炎症状态下，关节液中游离小檗碱的分布量增加，血和肝中游离小檗碱的分布量减少。小檗碱是P-糖蛋白的底物，抑制细胞表面P-糖蛋白表达或功能可增加小檗碱的细胞内药物浓度。我们通过细胞摄取和P-糖蛋白表达和功能实验研究牛膝“引药下行”作用机制，发现牛膝水提物可显著增加人肝细胞（L02）与大鼠成纤维滑膜细胞（FLS）小檗碱摄取量，显著降低大鼠肺泡上皮Ⅱ型细胞（RLE-6TN）小檗碱摄取量。在24 h内，与单给小檗碱组比较，牛膝水提物（2, 20, 200 μg/ml）分别使FLS小檗碱摄取量增加了15.2%、48.5%、57.6%。牛膝可显著增加L02与FLS的罗丹明-123转运量，而减少RLE-6TN的罗丹明-123转运量，均呈剂量相关性，而对三种细胞的P-糖蛋白表达量没有影响。.本研究采用现代药学研究技术阐释“引经”理论，可为引经药牛膝的临床应用提供科学依据，为其他引经药研究提供借鉴。引经成分的研究利于寻找天然的靶向给药体系。