川芎丹参协同调控肾上腺素β2受体-G蛋白通路的活血行气作用生物学基础研究

How to heal the mechanism of blood circulation promoting with scientific language, is one of the core content of modern Chinese medicine. Our previous studies proved that the role of β2-adrenergic receptor activation mechanisms and signaling pathways related to blood circulation herbs. Meanwhile, the combination of Rhizoma Chuanxiong and Radix Salvia miltiorrhiza might show a synergy effect on β2-G protein pathway. Therefore, through the Bioactivity based UPLC-Q/TOF screening platform, to the aim of realize the efficacy of Chuanxiong and Salvia miltiorrhiza’s ingredients rapid identification. By Chou-Talalay combination index equation and phenotypic analysis of key receptor on platelet, to investigate the compatibility and matching principle of Chuanxiong and Salvia miltiorrhiza. By the blood stasis model, Vegfr2-luc transgenic mice, myocytes and smooth muscle cells, with the use of modern research methods included GPCR phenotype analysis, fluorescein tag living imaging technology and high-throughput real-time PCR chips, researches will be carried out around β2AR-G protein signaling pathway to clarify the material basis of its efficacy and molecular mechanism of action. While taking advantage of the classic prescription of blood circulation, we also try to investigate the integration of compatibility studies related signaling pathways regulating β2 receptor and to construct a ingredient-target-pathways network of coordinated regulation of Chuanxiong and Salvia miltiorrhiza. Based on the clarify of the biological basis of prescriptions of blood circulation, the scientific connotation on regulation of β2 adrenergic receptor – G protein pathway activation of blood circulation promoting and Qi activating treatment will be discovered.

活血化瘀中药治疗冠心病血瘀证的科学实质是中药现代化研究的核心内容之一。我们前期研究发现，血瘀证的病理变化与肾上腺素β2受体-G蛋白调控的效应器官生理功能密切相关，而活血化瘀中药的常见配伍方式-川芎丹参配伍具有潜在的协同调控β2受体-G蛋白信号通路的作用。鉴此，本课题拟通过Bioactivity based UPLC-Q/TOF筛选平台，实现对川芎及丹参β2受体活性成分的快速鉴定，并以Chou-Talalay中效原理为依据，借助血栓形成关键受体抗凝作用的三重定量分析明确配伍配比关系。利用冠心病血瘀证模型、Vegfr2-luc转基因鼠及心肌、平滑肌细胞，应用GPCR高内涵表型分析、活体成像、高通量PCR芯片等方法，围绕β2受体-G蛋白信号通路，阐明协同增效的分子作用机理，构建川芎丹参相须配伍治疗冠心病血瘀证的成分-靶点-通路多维网络，诠释基于β2受体-G蛋白通路的活血行气配伍的现代生物学基础。

活血化瘀中药治疗冠心病血瘀证的科学实质是中药现代化研究的核心内容之一。我们前期研究发现，血瘀证的病理变化与肾上腺素β2受体-G蛋白调控的效应器官生理功能密切相关，而活血化瘀中药的常见配伍方式-川芎丹参配伍具有潜在的协同调控β2受体-G蛋白信号通路的作用。鉴此，本课题共开展了四方面研究，围绕β2受体-G蛋白信号通路，阐明协同增效的分子作用机理，构建川芎丹参相须配伍治疗冠心病血瘀证的成分-靶点-通路多维网络，诠释基于β2受体-G蛋白通路的活血行气配伍的现代生物学基础。.本研究首先采用UPLC-Q/TOF对川芎及丹参的化学成分进行了定性分析，共鉴定出川芎中的21种化学成分，丹参中的23种化学成分。利用Bioactivity based UPLC-Q/TOF筛选平台对川芎及丹参β2受体活性成分进行，快速鉴定研究共得到川芎两种增强β2活性成分、丹参四种增强β2活性成分。以Chou-Talalay中效原理为依据，采用棋盘式表设计方法，利用高内涵检测方法评价川芎及丹参有效成分对血管作用及关键受体的配伍配比关系，丹参川芎配伍组、川芎组与模型相比可明显促进成环。经棋盘式表设计方法并计算CI值，发现丹参10-5mol/L与2*10-6至1*10-5mol/L川芎成分配伍效果显著。利用大鼠冠心病血瘀证模型及基于细胞层面开展川芎-丹参配伍有效成分调节β2-G通道下游机制研究，利用冠心病血瘀证模型进行配伍的药效学评价，采用基因沉默等技术对Vegfr2、β2受体等关键环节进行干预，明确药物通过β2受体激动作用产生的对血流动力学、微循环及血液流变性的改善作用。利用质谱成像技术考察配伍对药物成分在心脏组织的分布影响，证实丹参及川芎均有显著改善心肌损伤作用，且药效优于单用组，配伍可显著促进CD31和VEGF的表达，药效成分与单用药相比在心脏组织分布增多。利用G蛋白受体PCR芯片明确配伍在G蛋白混杂偶联中的作用，显示配伍可调节G蛋白偶联受体通路的相关基因，以激活HIF-1α信号通路调控下游的VEGF信号通路，上调VEGF蛋白表达和激活AKT、VEGFR2蛋白磷酸化，以促进心梗大鼠血管新生。利用IPA平台构建了川芎丹参相须配伍治疗冠心病血瘀证的成分-靶点-通路多维网络，诠释了基于β2受体-G蛋白通路的活血行气配伍的现代生物学基础。