基于心肌细胞钙稳态研究益气活血药对舒张性心功能不全的干预机制

Diastolic dysfunction is not only an important pathological types but also an important part of the development of the heart failure, but the treatment has no significant improvement during the last decades. Therefore diastolic dysfunction has become the hot point of heart failure. Calcium homeostasis abnormalities are important mechanisms that lead to the development of heart failure, there are still no effective treatment.TCM believes that qi deficiency and blood stasis syndrome is basic pathogenesis of heart failure. Using the herbs for nourishing qi and activating blood early internention can be significanly improved the syndromes and diastolic function in patients with heart failure, delay heart failure progression. The preliminary research of our group suggests that using the herbs for nourishing qi and activating blood early internention can be significanly improved the syndromes and diastolic function in rat model with heart failure, delay heart failure progression. Another experiment pointed out that the herbs for nourishing qi and activating blood can regulated Calcium transients.The current, lack of a system research of calcium homeostasis as a major energy dissipation mechanism in diastolic myocardial cells for diastolic dysfunction on Yiqihuoxue Therapy.Accordingly proposed: Regulating calcium homeostasis maybe one of the mechanism of Yiqihuoxue Therapy guide diastolic dysfunction treatment.We make the model of Cardiac hypertrophy- - heart failure by narrowing the abdominal aorta. We intend to use animal echocardiography to monitor left ventricular systolic and diastolic function, and P-V Loop catheter to evaluate hemodynamics, and IonOptix system simultaneous to detect calcium transient and sarcomete motion in single cardiomyoctye, and molecular biotechnolog to detect calcium regulatory protein. We explore the microscopic mechanism and the role of Yiqihuoxue Therapy guide diastolic dysfunction treatment; Further explain the essence of qi deficiency and blood stasis syndrome of early heart failure diastolic dysfunction, and provide a scientific basis for reasonable compatibility of in herbs for nourishing qi and activating blood heart failure at different stages.

舒张性心功能不全是心衰重要的病理类型，也是心衰发生发展的重要环节，钙稳态异常是导致心衰发生发展的重要机制和潜在可干预靶点，目前仍缺乏有效的治疗药物。益气活血法是心衰治疗的基本法则。课题组前期研究提示益气活血方药可以改善心衰模型大鼠心脏舒缩功能，并观察到其对心肌细胞钙瞬变有一定调节作用。据此提出维持心肌细胞钙稳态，可能是益气活血方药改善舒张性心功能不全的机制之一的假说。建立缩窄主动脉模型，模拟心肌肥厚-心衰的演变过程。采用益气药（党参、黄芪），活血药（丹参、三七）及其配伍进行干预。运用小动物超声心动监测左室舒缩功能、P-V Loop导管评价血流动力学；并通过单细胞收缩-钙成像系统检测钙瞬变和肌小节运动、分子生物学方法检测钙调控相关蛋白，从整体、细胞、分子三个层面探讨益气活血法对舒张性心功能不全的干预作用及其作用的微观机制；并为心衰不同阶段益气药与活血药合理配伍应用提供科学依据。

舒张性心功能不全HFpEF是心血管学科面临的难点，心肌细胞钙稳态是调节心肌细胞舒缩功能的关键环节。气虚血瘀被认为是HFpEF的基本病机。我们前期研究发现益气活血方药可改善大鼠左室舒张功能及肥大心肌细胞的舒缩功能，并对钙转运产生良性调节作用。但对HFpEF灌注不足，益气活血药物之间不同配伍及剂量关系仍需进一步验证。据此提出”维持心肌细胞钙稳态，可能是益气活血方药改善舒张性心功能不全的机制之一，并且益气活血药物不同配伍之间存在疗效差异”的假说。拟复制改良缩窄腹主动脉术致压力负荷HFpEF大鼠模型，分别使用益气药（黄芪+党参）、活血药（丹参+三七）及益气活血药1：1，2:1比例不同配伍进行干预，以美托洛尔为西药对照组，运用小动物超声、左心室悬浮导管、激光共聚焦技术、Western blot等技术，从整体、组织、细胞及分子水平进行验证。重要研究结果：1）缩窄腹主动脉后12周，模型大鼠左室二尖瓣血流E/e值增高，左室舒张末期压力升高，心肌组织出现明显的间质纤维化及血管周围纤维化，左室室壁增厚，益气组、1:1、2:1组均可改善上述病理改变，美托洛尔组仅可减轻室壁厚度，2：1组改善作用明显优于1:1组，而活血组、美托洛尔组并不能改善心室舒张功能；2）模型大鼠钙瞬变50%时间、钙回落50%时间、舒张期钙消除时间常数显著增加，心肌细胞舒张速率减慢，舒张50%时间延长，益气组、1:1组、2:1组可同时改善模型大鼠心肌细胞钙瞬变、钙回摄及舒张功能，以2:1组作用最优，活血组、美托洛尔组仅可缩短心肌舒张时间；3）模型大鼠术后12周CaMK、PKA、RyR2水平升高，PLB(S16)、PLB(T17)、SERCA2a、NCX1、N/S比值表达水平下降，仅2:1组对各蛋白异常表达均有改善作用。研究结论：1）益气活血配伍组防治舒张性心功能不全疗效优于单纯的益气、活血和美托洛尔组；2）重用益气药物的益气活血配伍组防治舒张性心功能不全疗效优于常规益气活血配伍组。本研究对益气活血药临床防治HFpEF具有重要指导意义，为进一步阐释气血理论科学内涵提供了重要科学依据。