基于雌激素网络动态调控探讨青盐方“补肾益精”配伍规律及机制研究

Scientific interpretation of Bushen Yijing of classic prescription is one of the breakthrough to explain the traditional Chinese medicine theory of Bushen and the treatment of related disease . Qingyan formula, recorded in medical book of sheng ji zong lu, is a classic recipe of Bushen Yijing and has a composition of halitum, zanthoxyli pericarpium, cistanches herba, achyranthis bidentatae radix and crotonis semen pulveratum. Efficacy of five drugs are all played in kidney meridian. Previous studies have found that "kidney and estrogen regulation has a correlation", accordingly, we put forward the hypothesis that "Qingyan formula play the role of Bushen Yijing through the dynamic regulation of estrogen network." In this study, "kidney main reproductive and bone" as the breakthrough point, estrogen deficiency animals is studied to discuss the efficacy, status and phase interaction of　"Reproductive - Reproductive Endocrine - Bone System" by using decomposed recipes. The research is systematically studied around the estrogen dynamic regulation network from three aspects, the first is the regulation of estrogen precursors, estrogen and neurotransmitter synthesis and secretion by upstream hypothalamus - pituitary - adrenal axis, second is the downstream of estrogen-related endogenous metabolites and metabolic pathways. The last is dynamic regulation of key targets estrogen receptor signal transduction pathways ."Blood active ingredient - kidney function - estrogen network dynamic regulation association" of each drug group were implemented to explain the compatibility rules, pharmacological material basis, mechanism, scientific connotation of "Bushen Yijing" of Qingyan formula, guide the clinical medication better and also provide experimental basis for the scientific connotation of traditional Chinese medicine theory based on Bushen．

科学诠释补肾益精经典方剂是阐释中医补肾理论及相关疾病临床遣方用药科学内涵的突破口之一。青盐方源于《圣济总录》，由肉苁蓉、巴戟天、牛膝、蜀椒、青盐组成是补肾益精的经典方剂。前期研究发现“补肾与雌激素调控”具有相关性，我们据此提出研究假说：“青盐方通过对雌激素网络动态调控发挥补肾益精功效”。本研究以“肾主生殖和骨”为切入点，拆方研究对雌激素低下动物的生殖-生殖内分泌-骨系统的作用、地位及相互作用关系；围绕雌激素动态调控网络的“上游下丘脑-垂体-肾上腺轴调控性激素、雌激素前体及神经递质的合成分泌、下游雌激素介导的内源性代谢物和代谢通路的动态调节、关键靶点雌激素受体信号转导通路”进行系统研究。分析各拆方组的“入血活性成分-药效-雌激素网络动态调控关联”解析配伍规律、药效物质基础及作用机制。以期诠释青盐方“补肾益精”的科学内涵，更好地指导临床遣方用药, 亦为中医补肾经典理论的科学内涵提供实验依据。

前期证实补肾名方青盐方(QYF)的雌激素受体激动剂样作用是其归肾经药效表征之一，靶器官上的雌激素受体（ER）是归肾经的生物学靶点之一。但其补肾益精的配伍规律尚不清楚。为此按照君、臣、佐、使配伍规律进行拆方研究，采用网络药理学技术分析其治疗围绝经期综合征的配伍规律，采用低雌激素围绝经期实验动物进行生殖-内分泌-骨骼系统的药效评价及机制研究；采用代谢组学技术研究内源性代谢物及代谢通路，采用血清药物化学结合分子对接技术，并采用细胞实验验证活性成分ER作用机制。网药研究发现QYF中共74 个化合物作用于围绝经期综合征 290 个靶点、主要富集在ER、PI3K-Akt等通路。各配伍组既有不同靶点，也有相同靶点作用于各个症状，协同发挥治疗作用。君药囊括方中216个靶点、40 个关键靶点和所有主要通路，依次增加臣、佐、使药后，靶点数量及各通路所涉及的靶点均有不同程度增加，提示君药肉苁蓉在方中的重要地位及其余药味对君药的辅佐补充。QYF各拆方组拮抗去卵巢大鼠生殖靶器官的萎缩及骨质疏松作用，作用机制与增加循环中的雌激素水平，上调靶器官ER蛋白表达相关。其中以君药组作用较强，依次增加臣、佐、使配伍在某些药效及机制指标发生一定的上调作用。代谢组学研究在负离子模式下，QYF检测出55个差异代谢物，主要涉及类固醇激素生物合成、不饱和脂肪酸的生物合成、初级胆汁酸生物合成、花生四烯酸代谢等多个途径；君药组检测出81个差异代谢物，依次增加臣、佐、使药配伍后差异代谢物及代谢通路均有不同程度的变化。血清药物化学检测出30种入血成分，其中9种为原型成分为水晶兰苷、车叶草苷、β-蜕皮甾酮等，原型成分与ERα与ERβ均具有较强的结合能分，可增加ER+阳性MCF-7 cell增殖，促进ER表达，是QYF在体内发挥雌激素样作用的活性成分及通过经典ER机制发挥作用。该研究从青盐方拆方的药效药理-内源性代谢产物及通路-入血活性成分及关联机制诠释青盐方“补肾益精”配伍规律的科学内涵，更好地指导临床遣方用药。