基于血清药物化学与ERK、Wnt信号通路对骨质疏松肾阳虚证与肾阴虚证"同病异治"的研究

Osteoporosis is a systemic bone metabolic disease, divided into two kinds: kidney-yang deficiency and kidney-yin deficiency in Chinese medicine clinical mainly. This project is to provide the basis for treatment based on syndrome of osteoporosis in the signal transduction levels through studying on the osteoblast growth curve, activity of alkaline phosphatase, degree of mineralization, expression of Runx2, ERK and Wnt mRNA and protein in rat osteoblasts, which are from normal, osteoporosis, osteoporosis with kidney-yang deficiency, osteoporosis with kidney-yin deficiency rats, and finging out the difference. Each group of animals are administered Zuogui pill and Yougui pill respectively which are the classic clinical decoction for osteoporosis with kidney-yang deficiency or kidney-yin deficiency, then explore the serum to find out the difference in the blood components, at the same time, the osteoblasts are intervened by the serum containing medicine ,which aims at studying the influence of compositions into blood of Zuogui pill and Yougui pill on indexes such as expression of ERK, Wnt signal pathways and so on of rat osteoblasts from different syndrome rats. The project is to research the rationality of treatment based on syndrome differentiation and different treatments for the same disease of traditional Chinese medical science in knowledge of osteoporosis (kidney-yang deficiency and kidney-yin deficiency) from two aspects: serum pharmacochemistry and molecular biology signaling pathways, and provide the reference for the similar study.

骨质疏松是全身性骨代谢性疾病，中医临床主要分为肾阳虚证和肾阴虚证。本项目拟通过检测正常、骨质疏松、骨质疏松肾阳虚证、骨质疏松肾阴虚证大鼠成骨细胞的生长曲线、碱性磷酸酶活性、矿化结节程度、Runx2 、ERK、β-catenin mRNA及蛋白表达情况，分析比较并发现其差异，为骨质疏松的"辨证论治"提供信号通路层面的实验依据。选取治疗骨质疏松补肾阳、滋肾阴的代表方：右归丸、左归丸，分别灌胃给予各组动物模型，分析每组的含药血清成分，探究不同证候下入血成分的差异；同时将右归丸、左归丸的含药血清干预各组成骨细胞，研究不同证候下左归丸、右归丸的入血成分对各证候成骨细胞ERK、Wnt信号通路等指标的影响，最终从血清药物化学和分子生物学信号通路两个方面探究骨质疏松肾阳虚证、肾阴虚证"辨证论治"、"同病异治"的合理性，为同类相关研究提供借鉴和参考。

本项目采用手术去卵巢叠加氢化可的松/甲状腺素法建立骨质疏松（OP）、OP肾阳虚、OP肾阴虚模型，分别给予左归丸、右归丸，首次研究OP不同证型左、右归丸含药血清对成骨细胞及ERK、Wnt通路相关因子mRNA及蛋白表达是否存在差异；OP不同证型左、右归丸的入血成分及体内变化是否存在差异；结果发现：OP不同证型左、右归丸含药血清对成骨细胞及ERK、Wnt/β-catenin通路相关因子基因、蛋白的表达存在明显差异，OP肾阳虚右归丸含药血清对成骨细胞增殖、ALP表达、β-catenin、ERK1、ERK2 mRNA及蛋白表达的促进作用最强，而OP肾阴虚左归丸含药血清对成骨细胞增殖、ALP表达、β-catenin、ERK1、ERK2 mRNA及蛋白表达的促进作用最强。4种证型左、右归丸的入血成分基本一致，但不同证型入血成分的体内动态变化存在明显差异。右归丸含药血浆均分析鉴定20个主要入血成分，12个主要成分药动学表明，右归丸补肾阳君药附子和佐药杜仲的主要成分—乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、松脂醇二葡萄苷在OP肾阳虚状态下吸收较多，作用时间较长，初步推测这些成分可能是右归丸补肾阳的功效成分；而滋肾阴臣药熟地、山茱萸的主要成分—桃叶珊瑚苷、莫诺苷、马钱苷在OP肾阴虚状态下吸收、分布较大，可能是右归丸滋肾阴的主要功效成分。左归丸含药血浆均分析鉴定10个主要入血成分，5个主要成分的药动学表明，左归丸滋肾阴君药熟地和臣药山茱萸、枸杞的主要成分桃叶珊瑚苷、莫诺苷、马钱苷、原儿茶醛在OP肾阴虚状态下吸收较多，作用时间较长，初步推测这些成分可能是左归丸滋肾阴的功效成分。这与OP不同证型左、右归丸含药血清对成骨细胞的作用相一致，也与“右归丸补肾阳、左归丸滋肾阴”的中医理论相吻合。首次从细胞信号通路和化学层面诠释中医治疗骨质疏松肾阳虚、肾阴虚证的“辨证论治”、“同病异治”的科学内涵。