温阳消饮法调控cAMP-PKA信号通路影响水通道蛋白表达治疗水饮病的分子机制研究

In the previous National Natural Science Foundation, we have discovered that the treatment of warming yang and resolving fluid-retention can significantly inhibit the expression of Aquaporin1 of the mesothelial cells of parietal pleura, but the regulation mechanism is unclear. As is known, AQP1 is regulated by cAMP-PKA pathway of multiple links, and the pathway of fluid reabsorption mediated by antidiuretic hormone is a branch of cAMP-PKA pathway,which can control the formation and excretion of urine. So the hypothesis was put forward that "cAMP-PKA pathway is comprehensive controled of multiple links by the treatment of warming yang and resolving fluid-retention, and then finally regulate the VWR to effect the secretion and formation of urine in order to resolving the fluid".In our study, Xua-yin(rat model of inflammatory pleural effusion) and Zhi-yin(rat model of congestive heart failure induced by drug) will be taken as the vector of fluid-retention, the decoction of Ling-gui-zhu-gan and Ting-li-da-zao-xie-fei decoction will be used as representative prescriptions of warming yang and resolving fluid-retention. Gene expression profile chip and other technologies will be applied to uncover the regulation of treatment to cAMP-PKA pathway on different parts (pleura, lung, kidney) through AQPs, which will elucidate the scientific connotation of the treatment, provide a scientific basis for expanding the clinical applications and a new target of clinical medicine research for the fluid-retention

前自然基金研究发现温阳消饮法显著抑制胸膜间皮细胞水通道蛋白AQP1及AQP1mRNA表达消除悬饮，但调节机制不清。AQP1受cAMP-PKA信号通路多个环节调控，其中抗利尿激素介导的水重吸收通路（VWR）是cAMP信号通路的分支，可控制尿液生成与排泄。故提出"温阳消饮法能多环节综合调控cAMP-PKA信号通路并最终调节VWR影响尿液生成以消退不同部位水饮"的假说，以悬饮（炎性胸腔积液大鼠模型）、支饮（充血性心力衰竭大鼠模型）为水饮病载体，以苓桂术甘汤合葶苈大枣泻肺汤为温阳消饮法代表方，采用cAMP-PKA信号通路表达谱芯片、RT-PCR、Western杂交、免疫组化等技术，以AQPs为切入点，分析温阳消饮法治疗悬饮、支饮在心、肺、肾三脏cAMP-PKA信号通路上多环节调控的异同，诠释"温药和之"法的科学内涵，为拓展温阳消饮法的临床应用提供科学依据，也为水饮病临床方药研发提供新的靶向。

温阳消饮法是治疗水饮病的基本治法，中医的水饮病包括了西医学多系统的多种疾病，对温阳消饮法的研究具有临床应用价值。本研究首次选择类似中医水饮病悬饮的胸腔积液与类似水饮病支饮的慢性充血性心衰的两种动物模型，从宏观功能学、微观分子学及基因表达层面切入，研究结果证实温阳消饮法在悬饮（胸腔积液）模型中能减少模型的胸液量，降低胸液生成相关因子的含量或活性，改善病变造成的胸膜、小肠、肾组织细胞异常变化。其温阳消饮效果可能是通过影响Mcpt8I3基因调控肾素血管紧张素系统通路，影响Ugt2b3基因调控甾类激素生物合成通路，并上调肾水通道蛋白2、小肠水通道蛋白4，以达到调节全身水液代谢而发挥其治疗效应。在支饮（慢性心衰）模型中，能改善阿霉素所致CHF模型部分心脏内分泌功能（如降低血清BNP、血浆AngⅡ含量）与血流动力学（如提高动脉舒张压，降低左心室舒张期末压）、心肌组织细胞超微结构的异常变化；该法能上调此模型大鼠肾脏AQP2、肺脏AQP5表达水平；从CHF大鼠肾髓质中发现了异常表达的Ryr2基因，提示肾脏在心衰发生的心肌兴奋-收缩耦联过程中亦发挥着调控作用；该法可能通过调控MAPK信号转导通路、NOD样受体信号通路，参与抑制肾脏细胞的凋亡，发挥保护肾功能的作用。本研究科学阐释了临床常用治法——温阳消饮法多环节、多靶点发挥其治疗胸腔积液与慢性充血性心衰的作用机理，为拓展该法的临床运用或进行深入研究提供了实验研究依据,有益于拓展该治法的应用范围，也为深入研究该治法发现了新的切入靶点。由此可见，该课题研究成果研究具有较高的学术价值和临床应用价值。