基于ABCB1甲基化的肾阳虚与肝肾阴虚绝经后骨质疏松症患者骨碎补总黄酮代谢差异机制的研究

The significant difference of blood concentration of effective components of Total Flavone of Rhizoma Drynariae (TFRD) between kidney Yang deficiency syndrome patients (treated with correspondence of prescription and syndrome) and patients with other syndromes was discovered by a finished study, but the mechanism is not clear yet. The flavones in TFRD, such as Naringi and Hesperidin, are all ABCB1 substrates, and their pharmacokinetics is regulated by ABCB1. According to the literature analysis, epigenetic factors of ABCB1 may be the key metabolic differences of TFRD between the two syndromes. Thus, we propose a hypothesis that the difference of methylation on promoter region results the metabolic differences of TFRD between liver and kidney Yin deficiency syndrome and kidney Yang deficiency syndrome in menopausal osteoporosis. In this study, we plan to compare the difference of blood steady state trough concentration of Naringenin, which is the common metabolite of Flavonoid glycosides, between the two syndromes patients after taking TFRD. The combined bisulfite restriction analysis (COBRA) is applied to analyze the methylation degree of promoter region of ABCB1, and support vector machines and GLM model are applied to fit the complex relationship between ABCB1 methylation, syndromes and plasma concentration. Through this project, we may discover the mechanism of metabolic differences of TFRD between liver and kidney Yin deficiency syndrome and kidney Yang deficiency syndrome in menopausal osteoporosis, which can provide a strong scientific basis for the theory of treatment based on syndrome differentiation.

本课题组前期进行不同证候绝经后骨质疏松症患者骨碎补总黄酮群体药代动力学研究，发现肾阳虚（方证相应）患者的骨碎补总黄酮有效成分血药浓度显著低于非肾阳虚（方证不相应）患者，但其机制尚不明确。骨碎补总黄酮中柚皮苷等黄酮类物质均是ABCB1的底物，药动学过程受ABCB1的调节。根据文献分析，ABCB1的表观遗传因素可能是两种证候患者骨碎补总黄酮代谢差异的机制。因此，本研究拟围绕“肾阳虚与肝肾阴虚绝经后骨质疏松症患者骨碎补总黄酮代谢差异的机制是两种证候患者的ABCB1启动子区域甲基化程度不同”的假说，以黄酮苷类共同代谢产物柚皮素为指标，研究两种证候患者服药后柚皮素稳态谷浓度的差异；采用COBRA法分析ABCB1启动子区域甲基化程度；用SVM结合GLM模型建立ABCB1甲基化、证候及血药浓度的复杂关系，从而揭示肾阳虚与肝肾阴虚绝经后骨质疏松症患者骨碎补总黄酮代谢差异机制，为辨证论治提供有力的科学依据。

背景：课题组前期开展了不同证候绝经后骨质疏松症患者骨碎补总黄酮群体药代动力学研究，发现肾阳虚证（方证相应）患者的骨碎补总黄酮有效成分血药浓度与非肾阳虚证（方证不相应）患者相比有显著性差异，但其机制尚不明确。骨碎补总黄酮中的柚皮苷、橙皮苷等黄酮类物质均是ABCB1的底物，药动学过程受ABCB1的调节。根据文献分析，ABCB1的表观遗传因素可能是两种证候患者骨碎补总黄酮代谢差异的机制。. 方法：本研究拟围绕“肾阳虚证与肝肾阴虚证绝经后骨质疏松症患者骨碎补总黄酮代谢差异的机制是两种证候患者的ABCB1启动子区域甲基化程度不同”的假说，以黄酮苷类共同代谢产物苷元（柚皮素）为指标，研究两种证候患者服药后柚皮素血药浓度的差异；采用Massarray方法分析ABCB1启动子区域甲基化程度；运用NONMEM模型，分析证候、ABCB1甲基化程度对柚皮素血药浓度的影响，并采用T检验的统计方法，观察证候、ABCB1甲基化之间的关系，进而，分析肾阳虚证与肝肾阴虚证患者骨碎补总黄酮代谢差异的机制。. 结果：收集了PMOP患者103名，其中，肝肾阴虚证53名，肾阳虚证50名。统计学检验的结果显示，ABCB1基因启动子不同位点的甲基化程度与辨证分型（肝肾阴虚或肾阳虚）之间无显著相关性；辩证分型对柚皮素的药物动力学特征无显著影响；ABCB1启动子13、14位点的甲基化水平与柚皮素的吸收速率常数呈正相关，其甲基化程度较高时，柚皮素的吸收速率常数更大。即ABCB1甲基化程度越高，ABCB1的表达越低，导致柚皮素的吸收增多，血药浓度增高。. 结论：肝肾阴虚证与肾阳虚证PMOP患者骨碎补总黄酮血药浓度无显著性差异；PMOP患者骨碎补总黄酮血药浓度差异机制是ABCB1甲基化程度，主要的影响位点是CpG岛的13、14位点，这一结论可为临床个体化用药提供理论依据。