益阴助阳活血法调控β肾上腺素受体经典及非经典信号通路抗心肌重塑的机理研究

In our previous work, the key pathogenesis of heart failure is considered as the deficiency of both Yin and Yang and the stagnation of the heart blood vessels. This project focuses on "blocking the cardiac remodeling" and suggests that activating Yang by nourishing Yin and promoting blood circulation will contributed to the treatment of heart failure. Under the guidance of the TCM theory, the in vivo and in vitro rat models of heart failure were established. Then, the examinations including cardiac hypertrophy, myocardial contractility, hemodynamic index, pathological change, angiotensin II, norepinephrine level were determined. Furthermore, the activities of matrix metalloproteinases, myocardial apoptosis, myocardial fibrosis, myocardial β1 receptor number, density and sensitivity were investigated. Moreover, the in vitro neonatal rat cardiac cell culture system induced by isoproterenol was established and the effects of the whole prescription and its active ingredients on myocardial energy metabolism, apoptosis, myocardial β1 receptor canonical signaling pathway (cAMP-PKA-CREB) as well as non-canonical signaling pathway (Ca2+/calmodulin kinase II) were explored. This study will provide scientific evidence of understanding activating Yang by nourishing Yin and promoting blood circulation for the treatment of heart failure.

我们前期的工作提示心衰的核心病机是阴阳两虚、心脉瘀滞，本项目拟围绕"阻断心肌重塑"这一心衰治疗的靶目标，提出益阴助阳活血为心衰治疗的有效法则。在该治法的指导下，拟于在体和离体大鼠心衰模型上，探讨益阴助阳活血方对宏观指标如心肌肥厚、心肌收缩力、血流动力学等以及微观指标如病理改变、血管紧张素Ⅱ、去甲肾上腺素水平等的影响；进而考察该方对心衰大鼠心脏MMP的活性、心肌细胞凋亡、心肌纤维化程度、心肌β1受体的数量、密度以及敏感性等的影响；进一步在体外乳鼠心肌细胞培养体系中，通过异丙肾上腺素诱导损伤，探讨全方及其活性成分对心肌细胞能量代谢、凋亡、心肌细胞经典的β1AR-cAMP-PKA-CREB信号通路以及非经典的β1AR-Ca2+/CaMKⅡ信号通路的影响，以此验证该方中主要活性成分的作用及其影响环节。本研究将有助于阐明益阴助阳活血法防治心衰的科学内涵，可进一步加深中医对于心衰证治规律的理解和认识。

我们前期的工作表明心衰的核心病机是阴阳两虚、心脉瘀滞，本项目围绕“阻断心肌重塑”这一心衰治疗的目标，提出益阴助阳活血为心衰治疗的有效法则。在该治法的指导下，我们分别在整体大鼠心衰模型和离体心肌细胞水平上，阐明益阴助阳活血方可显著改善心衰大鼠各种指标，包括宏观指标如心肌肥厚、心肌收缩力、血流动力学等以及多种微观指标如病理改变、血清中神经内分泌因子如血管紧张素Ⅱ、去甲肾上腺素水平等；进一步，我们发现益阴助阳活血方可显著下调心衰大鼠心脏MMP-2和MMP-9的活性、减轻心肌细胞凋亡、改善心肌纤维化程度；我们还构建了体外乳鼠心肌细胞培养体系，通过异丙肾上腺素诱导损伤，发现益阴助阳活血方含药血清可剂量依赖地增加心肌细胞损伤后的β1受体的表达水平以及PKA磷酸化活性，显著降低心肌细胞损伤后CREB的磷酸化。对于非经典β1AR-Ca2+/CaMK II信号通路，益阴助阳活血方含药血清则可显著降低心肌细胞损伤后CaMK II的表达。我们探讨了益阴助阳活血方中若干种活性成分作用于心肌重塑不同环节的特点和规律，发现黄芪甲苷和淫羊藿苷主要以增加心肌细胞β1受体表达和PKA活性为主，泽泻醇A以降低Bcl-2表达抵抗心肌细胞凋亡为主，而麦冬皂苷D则显著抑制心肌细胞损伤后CaMK II的表达。本研究将有助于阐明益阴助阳活血法防治心衰的科学内涵，可进一步加深中医对于心衰证治规律的理解和认识。本项目已发表了1篇SCI原著论文，2篇国内核心期刊论文。