高血压肝阳上亢证模型大鼠代谢节律稳态及钩藤调控机制研究

Hypertension wth liver yang hyperactivity syndrome is the most common clinical hypertension TCM syndromes, and has the syndrome characteristics of integrity and dynamics. The dynamics from rhythm analysis and integrity from metabolomics is deeply integrated in this subject and introduce the conception of " Metabolic rhythm homeostasis". Around the metabolic rhythm homeostasis imbalance and reconstruction, twelve time nodes in 24 hours are acquired to analysis the rhythm of central and peripheral clock gene, metabolic key enzymes, proteins and metabolites. The hythmic oscillation of metabolic process including upstream, downstream and the rate-limiting enzyme would be realized and the imbalance of metabolic rhythm homeostasis would be claified. The chinese medicine, Uncaria rhynchophylla will be used to disproof the rhythm imbalance of hypertension wth liver yang hyperactivity syndrome rats and explore the intervention mechanism on the level of metabolic rhythm homeostasis reconstruction. Studying "metabolic rhythm homeostasis imbalance and reconstruction" systematically and deeply could explain the intrinsic nature of TCM syndromes and the intervention mechanism of traditional Chinese medicine in the three-dimensional, networking, systematic and dynamic level. If so, the Chinese medicine conception of “cure hypertension is not only lower blood pressure and focus on rhythm adjustment” could be acquired, while the universal research model containing originality theoretical characteristics of TCM syndrome would be constructed.

高血压肝阳上亢证是临床最常见的高血压中医证候表型，具备动态性和整体性的证候特点，本课题深度融合节律分析的动态性和代谢组学的整体性于一体，引入“代谢节律稳态”的概念，围绕代谢节律稳态失衡与重构，以十二时辰为时间节点，开展高血压病肝阳上亢证模型大鼠中枢和外周生物钟基因、代谢关键酶及蛋白、终端代谢物的十二时辰数据的节律分析，发现代谢过程中节律波动的上游、下游以及限速酶环节，厘清高血压肝阳上亢证大鼠生物钟基因代谢节律稳态失衡的过程，进而以药测证，以钩藤干预病证模型大鼠，反证代谢节律稳态失衡的同时从代谢节律稳态重构的层面探明钩藤的干预机制。遵循“天人合一”、“因时制宜”中医理论，系统深入的研究“代谢节律稳态的失衡与重构”将有助于在立体化、网络化、系统化和动态化的层面阐述中医证候的内在本质和中药的干预机制，实现“治血压不惟降血压，重在调整节律”，从而构建蕴含中医证候理论特征的普适性研究模式。

本研究遵循“天人合一”、“因时制宜”等中医理论，采用吻合这一理论特征的“代谢节律稳态”的概念，从代谢节律稳态失衡和重构入手，纳入中医十二时辰时间节点，耦联代谢生物钟和代谢组学，以代谢组学、RT-PCR 以及Western blot 分析生物钟基因、节律标志物和代谢酶及其蛋白的水平，进而开展节律分析描述其动态，描述高血压肝阳上亢证大鼠节律的上游、下游和代谢酶及其蛋白节律失衡状态，从代谢的“上游-限速酶-下游”的节律失衡状态探寻该疾病的发生机制和过程；以钩藤反证高血压肝阳上亢证大鼠的代谢节律稳态失衡，从代谢节律稳态重构的层面阐明钩藤干预高血压肝阳上亢证大鼠模型的机制。研究结果如下：①通过非靶向代谢组学技术，发现了高血压肝阳上亢证大鼠节律失衡的标志物及代谢通路；②分别针对氨基酸、神经递质及脂质类 3类节律标志物进行靶向代谢组学分析，并通过余弦分析建立了高血压肝阳上亢证大鼠代谢节律特征模型，揭示了高血压肝阳上亢证大鼠代谢节律稳态失衡的“下游”；③采用q-PCR技术，测定了高血压肝阳上亢证大鼠中枢/外周生物钟基因clock、bmall、per2、cry1表达水平的节律性变化特征，揭示了高血压肝阳上亢证大鼠代谢节律稳态失衡的“上游”；④采用q-PCR、Western Blot分子生物学技术，测定了高血压肝阳上亢证大鼠节律失衡代谢通路的关键mRNA 表达、核心蛋白表达，结合生物钟基因和节律标志物分析，共同揭示了高血压肝阳上亢证大鼠节律稳态失衡状态的代谢上游、下游和靶点通路，厘清了代谢节律稳态失衡与高血压大鼠疾病发生的完整作用环节；⑤分别测定了中药钩藤干预后高血压肝阳上亢证大鼠下丘脑、肝脏和血液的中枢/外周生物钟的基因表达、节律标志物表达以及生物钟失衡代谢通路中关键蛋白表达，分析对比干预前后代谢节律模型震荡和动力学特征的改变，从调控高血压节律紊乱（振幅、相位等余弦模型）角度建立了钩藤整体降压药效作用评价的新模式；⑥开展了钩藤中主要降压成分-异钩藤碱干预高血压肝阳上亢证大鼠生物种基因与节律失衡代谢通路的分子生物学机制研究，发现了其上调节律蛋白Bmal1表达从而调控脂质和多巴胺节律代谢紊乱，阐明了其通过调控生物钟负反馈环回调节律代谢通路，抑制RAAS激活和交感神经兴奋，进而发挥抗高血压的作用。