基于PK-PD相关性及其ADME-Act分析研究甘草协同祖师麻治疗类风湿性关节炎的配伍机制

glycyrrhiza was extensively applied to tranditional chinese medicine prescription, but its mechanism of synergistic interaction was unknown. In the previous study, the effect of zushima on rheumatoid arthritis was enhanced by compatibility of glycyrrhiza, and the bioavailability of daphnetin, 7-OH daphnetin, the major active ingredients was improved obvioulsy. Therefore, the hypothesis about whether the related targets of rheumatoid arthritis was effected by active ingredients in glycyrrhiza and zushima simuteneaously, whether distribution was effected to result in the pharmacokenitics of active ingredients by transporters and enzymes changed based on absorption and metabolism, and whether synergistic effect resulted from active components groups effect on the mecular targets of network effect, need all to be investigated. The key tegrets of rheumatoid arthritis were selected to study the expression level of key inflammatory factors such as MIF、TNF、IL-1, ect pre-and post compability. It was also studied the difference of the activity of metabolic enzyme and transporters and mRNA expression level and protein expression pre-and post compability based on transporters and metabolic enzymes, and established the correlation of PK-PD based on active ingredients groups of ADME to elucidate the compatibility mechanism of rheumatoid arthritis of synergistic effect of glycyrrhiza combined with zushima.

中医方剂广泛配伍应用甘草，其协同增效机制尚不明了。本项目基于前期研究发现，治疗类风湿性关节炎的中药祖师麻因配伍甘草而活性增强、主要活性成分瑞香素、7-羟基香豆素生物利用度显著提高，提出甘草配伍祖师麻协同增效机制在于祖师麻与甘草主要活性成分作用于类风湿性关节炎相关作用靶点，介导体内吸收与代谢相关的转运蛋白和代谢酶，影响药物分布，导致主要活性成分的药代动力学发生变化，综合表现为活性成分群以适宜血药浓度作用于网络效应分子靶点产生协同增效作用的科学假说。选择类风湿性关节炎关键靶点，考察配伍前后MIF、TNF、IL-1等关键炎症因子表达水平的差异；以转运蛋白和代谢酶为切入点，考察配伍前后代谢酶和转运蛋白活性、mRNA水平的表达及蛋白表达量的差异，建立基于主要活性成分群ADME相互影响的PK-PD相关性，阐明甘草配伍祖师麻治疗类风湿性关节炎协同增效机制。

本课题采用整体动物模型阐明甘草协同祖师麻治疗RA的配伍机制，并筛选最佳配伍比例；当祖师麻与甘草配伍比为3:2时，对抑制大鼠体重减轻，足肿胀和AI的增加，血清中TNF-α、IL-1β水平异常升高以及降低滑膜组织中VEGF、MIF的表达有较好的效果。推测甘草配伍祖师麻治疗RA可能是通过调节TNF-α、IL-1β、VEGF和MIF等细胞因子而发挥疗效。体内药代动力学模型考察了甘草提取物对祖师麻中瑞香素药代动力学参数的影响以及甘草中主要活性成分对瑞香素药代动力学参数的影响。甘草提取物能够显著增加祖师麻中瑞香素的AUC、Tmax，降低CL；甘草酸配伍瑞香素后，对瑞香素药动学参数无明显影响；甘草苷配伍瑞香素，能够增大瑞香素的AUC、Cmax、Tmax，降低瑞香素CL与MRT。由此可见，甘草提取物增加瑞香素生物利用度的原因可能与甘草苷有关。Caco-2细胞模型研究了甘草提取物对祖师麻中瑞香素吸收的影响，甘草提取物对瑞香素在Caco-2单层细胞模型的转运没有显著影响，提示甘草提取物对祖师麻中瑞香素在小肠上皮细胞吸收可能影响不显著。体外肝微粒体模型研究了甘草及其主要活性成分对祖师麻中瑞香素代谢的影响。甘草能明显抑制祖师麻中瑞香素的代谢。甘草提取物中主要成分对瑞香素在肝微粒体Ⅰ相代谢中没有显著影响；甘草酸、甘草苷及甘草素对瑞香素在肝微粒体Ⅱ相代谢中有抑制作用，甘草次酸对瑞香素在肝微粒体Ⅱ相代谢中有促进作用。建立了同时测定祖师麻—甘草中9种活性成分（瑞香素、西瑞香素、7-羟基香豆素、甘草苷、甘草素、异甘草苷、异甘草素、甘草酸和甘草次酸）的LC-MS/MS方法，并用于比较其在健康和疾病大鼠体内的药代动力学差异。与正常组相比，AA大鼠血浆中瑞香素、甘草苷、异甘草苷和甘草酸的生物利用度降低，甘草酸的生物利用度增加；异甘草苷、异甘草素和甘草酸Tmax显著增加，延缓了其体内吸收。采用血浆代谢组学的手段分析了祖师麻—甘草治疗RA的作用机制，从生理指标和内源性变化的角度描述AA大鼠的疾病过程。鉴定了30个与疾病相关的代谢物，RA能引起体内包括氨基酸、脂类在内的多种代谢通路紊乱，祖师麻—甘草主要调控了其中12个异常代谢物，对佐剂关节炎干预效果良好。