基于五子衍宗丸中药物盐炙前后分别组成五子衍宗丸来研究“盐炙入肾-肾主生殖”的机理

Processing and compound are two important characteristics of clinical medication in TCM. In recent years, considerable researches are on the processing of the single drug-oriented while study on Chinese herb compound is less. And the study which combine the processing theory of “stir-frying with salt-water into the kidney, kidney governing reproduction” with the viscera manifestation doctrine of TCM is even less. This subject is based on pervious study “stir-frying with salt-water into the kidney” which was under the financial aid of National natural funding. At first, the subject forms differert Wuzi Yanzong Wan respectively with raw drugs and salt-processed drugs. Then this subject takes it as break through point, combines the Wuzi Yanzong Wan’s functions of treating Spermatorrhea premature ejaculation impotence and sterility and uses animals model on syndrome in Chinese medicine of kidney essence deficiency to screen the best combinations of Wuzi Yanzong Wan; then use the comprehensive study mode “pharmacodynamics - fingerprint spectrum of active fractio - spectrum activity relationship - serum fingerprint - effective compositions PK and PD” to study the material basis of salt-processing drugs and its change regulation in body; and employ a series of techniques such as correlation spectrum efficiency, LC-MS/MS, real-time fluorescence quantitative and protein imprinting to research its mechanism from multi-angle and multi-level. The subject is aimed to clarifying scientific connotation of “stir-frying with salt-water into the kidney , kidney governing reproduction” which fully reflects the polypharmacy characteristic of TCM, enhances the practicability of research, highlights the guidance of the basic theory of TCM and promote and develop the theory of traditional processing and TCM.

炮制和复方是中医用药的两大特色，但目前单味药的炮制研究较多，少有组成复方研究，更缺乏将“盐炙入肾-肾主生殖” 的中药炮制理论和中医藏象学说的结合研究。本课题在前期三个自然基金资助“盐炙入肾”研究基础上，将“五子衍宗丸”中药物盐制前后分别组成“五子衍宗丸”为切入点，紧密结合“五子衍宗丸”治疗遗精早泄、阳痿不育的主治功能，利用“肾精亏虚”的中医证侯动物模型，对“五子衍宗丸”中药物炮制品最优组合进行筛选；再采用“药效学-有效部位指纹图谱-谱效关系-血清指纹图谱-药效成分PK-PD”的综合研究模式，对“五子衍宗丸”中药物盐炙物质基础及体内变化规律进行研究；并引入谱效关联、LC-MS联用、实时荧光定量、蛋白印迹法等技术，从多角度、多层次展开其机制研究。旨在阐明“盐炙入肾-肾主生殖”的科学内涵，充分体现中医复方用药特点，增强研究的实用性，又突出中医药基础理论的指导，来推动和发展中药炮制和中医药理论。

“盐制入肾”是中药炮制经典理论之一，中医藏象学说有“肾主生殖”。炮制和复方是中医用药两大特色，但目前单味药的炮制研究较多，少有组成复方研究，更缺乏将“盐制入肾-肾主生殖”的中药炮制理论和中医藏象学说的结合研究。本项目在“盐制入肾”和“肾主生殖”理论指导下，通过五子衍宗丸中单味药炮制品入选依据和不同组合方式疗效对比研究，筛选五子衍宗丸中最优炮制品；通过有效部位、谱效关系、血清指纹图谱及主要成分代谢等研究，揭示盐制对五子衍宗丸物质基础及体内变化规律影响；通过观察盐制前后组成五子衍宗丸对模型大鼠脏器形态、肾功能、免疫功能、抗氧化能力、“下丘脑-垂体-性腺轴”、睾丸TGF-β1/Smads信号转导通路、生精细胞凋亡等影响，阐明“盐制入肾-肾主生殖”科学内涵。结果如下：.（1）通过对枸杞子、覆盆子和五味子盐制前后分别对肾阴虚、肾阳虚多尿和肾精亏虚模型的药效比较研究，发现盐覆盆子与盐五味子效果均优于生品；结合生精障碍型大鼠模型，盐制后对大鼠精子密度、活率及其活力改善效果最佳。.（2）通过有效部位、谱效关系和含量测定等研究，发现盐制对绿原酸、鞣花酸、金丝桃苷、毛蕊花糖苷、异槲皮苷、紫云英苷、山柰酚、五味子醇甲等主要成分含量有影响。血清指纹图谱研究发现盐制前后入血成分差别不大，但部分峰峰面积有所改变。体内药动及代谢组学研究表明，盐制通过影响化学成分体内代谢，从而改变机体氨基酸代谢、脂质代谢和核苷酸代谢等，以对氨基酸代谢影响最为显著。.（3）药物盐制后组成的五子衍宗丸主要从修复肾脏和睾丸病理损伤，改善精子质量、肾功能和免疫功能，增强肾及附睾的抗氧化能力等方面增强“补肾”作用，阐明了“盐制入肾”的作用机理。.（4）药物盐制后组成的五子衍宗丸主要通过调节“下丘脑-垂体-性腺轴”分泌的性激素、降低睾丸组织中TGF-β1，Smad2、Smad4 mRNA和蛋白的表达，调控Bcl-2、Bax，共同改善生殖功能。从生殖内分泌及睾丸TGF-β1/Smads信号转导通路的角度阐明“肾主生殖”的作用机理。