基于中医水液代谢理论从AQPs探讨慢性心衰大鼠水平衡紊乱的分子机制

Disorders of water balance is an significant pathophysiological mechanism of Chronic Heart Failure(CHF). Aquaporins(AQPs) have been found as a water transport protein, relating to water balance regulation.However,the mechanism of AQPs in heart failure is unknown. Our previous research indicates that the increase of AQPs level and blood stasis and edema are closely related. Combining with the Chinese medicine theory and the Traditional Chinese Medicine(TCM) pathogenesis of heart failure, we propose that heart failure which is caused by deficiency of Qi (Yang) and blood stasis, is followed by the increasing release of active substance such as angiotensin Ⅱ（AngⅡ）and TNF-a, resulting in the abnormal expression of AQPs in lung,kidney and other tissues and then affecting fluid exchange". The purpose of this study is to explore the expression and regulatory mechanism of AQPs subtypes on lung and kidney, as well as the influence of AQPs on the formaiton and development of edema in the process of heart failure, through the changes of angiotensin Ⅱ type l receptor、TNF-α and AQPs on different organs in rats at different stages of CHF , combining vitro experiment.Then we are going to verify the scientificalness of the hypothesis about"YiQiHuoXue-affacting mediators of neuroendocrine and inflammation - regulating the expression of AQPs - adjusting the function of internal organs - improving the metabolism of water". From a new persepctive-AQPs,the molecular mechanism of disorders of water metabolism in CHF and the scientific meaning of TCM about water metabolism will be explained. This study are important for guiding the treatment of CHF.

水平衡紊乱是慢性心衰(CHF)形成的重要病理生理机制，水通道蛋白（AQPs）是新近发现的参与水平衡调节的水转运蛋白，然而其在心衰形成中的作用尚不清楚。我们前期工作提示AQPs表达增高与血瘀、水肿密切相关，结合中医水液代谢理论及心衰病机特点，我们提出"心衰时气（阳）虚、血瘀伴随AngⅡ和TNF-a等活性物质释放增加,导致肺、肾等组织AQPs表达异常，进而影响液体交换而病水"的假说，拟通过CHF大鼠不同组织AngⅡ受体、TNF-α和AQPs等的表达变化，并以肾AQP2为切入点，结合体外实验探讨AQPs亚型在肺和肾脏的表达规律、调控及其在心源性水肿发生、发展中的作用；继之以经典方药验证"益气活血-影响神经内分泌炎症介质-调控AQPs-调整脏腑功能-改善水代谢"的科学性。以期从AQPs这一全新角度阐释CHF水代谢异常的分子机制及中医水液代谢理论的科学内涵，这对于指导CHF治疗具有重要意义。

本研究通过结扎冠脉前降支，建立大鼠急性心肌梗死（模型，观察益气、活血代表方保元汤联合桃红四物汤对心梗后心衰大鼠尿量及心脏功能影响，ELISA方法检测血浆AVP和AngII水平，免疫组化检测肾髓AQP2、pS256-AQP2，肺组织α-SMA、TNF-α以及心肌组织α-SMA、TNF-α和LC3蛋白表达，Western blotting方法检测肾组织AQP2、pS256-AQP2及V2R、AT1R、Epac1、ERKl/2、TNF-α、IL-6等神经内分泌、炎性因子表达，检测肺组织AQP1及α-SMA、collagen I、TGF-β1、Smad3、TNF-α、TLR4和NF-κBp65等纤维化、炎性因子表达，以及心脏α-SMA、collagen I、TGF-β1、Smad3、TNF-α、TLR4、NF-κBp65和IL-6表达；体外采用dDAVP刺激小鼠肾集合管细胞（IMCD3）细胞，益气活血方药液作用于IMCD3 24h，ELISA法检测cAMP水平，Western blotting检测AQP2、Epac1和ERKl/2蛋白的表达情况。结果：益气活血方治疗后大鼠尿量明显增加，肺重量减低，左室射血分数明显增加，血浆AVP 和AngII水平降低，肾髓质AQP2、pS256-AQP2表达降低，肾组织V2R、AT1R、Epac1、ERK1/2、TNF-α和IL-6等神经内分泌、炎性因子表达降低，肺组织AQP1表达升高，肺和心脏α-SMA、collagen I、TGF-β1、Smad3、TNF-α、TLR4和NF-κBp65等炎症、纤维化指标均明显降低；dDAVP诱导的IMCD3细胞AQP2 、Epac1和ERKl/2表达增加，cAMP水平升高，益气活血方治疗后AQP2 、Epac1和ERKl/2表达减低，cAMP水平下降。结论：益气活血方通过调控肾AQP2和肺AQP1蛋白表达，改善心梗后心衰大鼠肾和肺水代谢，其机制与益气活血方影响神经内分泌、炎性因子表达，调控AVP-V2R，AngII-AT1R、TNF-α-AT1R及Epac1/ERKl/2等相关信号通路有关。我们的研究结果从AQPs这一全新角度阐释了慢性心衰水代谢异常的分子机制及中医水液代谢理论的科学内涵，揭示了益气活血法“化瘀利水”的作用靶点和分子机制。