基于β2受体通路探讨去甲乌药碱的强心、抗心肌细胞凋亡及诱导生理性肥大作用

In our previous study, we found Chinese herb "Aconite root" also named "fuzi" and Chinese medical prescriptions that characterized by using "fuzi" as monarch, can improve the cardiac function and remodeling in human and rat. Higenamine , as the main inotropic functional compound of "fuzi", was demonstrated recently in vitro by our group to attenuate apoptosis through beta2/Gi/PI3K/AKT pathway. Interestingly, we also found Higenamine induced hypertrophy in primary cultured adult mouse ventricular myocytes. Based on preliminary data, we hypothesize that: [1]. Higenamine plays inotropic role in heart via exciting beta2/Gs and membrane- specific cAMP/PKA pathway, which leads to cardiac inotropic. [2]. By activating beta2/Gi/PI3k/AKT pathway, Higenamine attenuates cardiac apoptosis in vivo and in vitro. [3]. Also through this signal pathway, Higenamine triggers physiological cardial hypertrophy, which may be complementary benefit in later heart failure. To test our hypothesis, first, ex-vivo Langendorff heart perfusion system and fluorescence resonance energy ransfer(FRET) will be utilized to measure beta2 AR/Gs, membrane specific AC/cAMP/PKA pathway and inotropic function of Higenamine. Secondly, ischemia/reperfusion mouse model and myocardial infarction model in vivo, siRNA technology to knock down target gene in vitro will be performed to confirm the anti-apoptotic function of Higenamine. Finally, mouse transverse aortic constriction (TAC) model in vivo, pharmacological inhibitor and RNAi knock down in vitro will be done to support that: Higenamine cause physiological instead of pathological cardiac hypertrophy through beta2/Gi/PI3K/AKT pathway. This findings will provide strong evidence of inotropic, anti-apoptotic and physiological hypertrophy inducible function of Higenamine in later stage of heart failure, and Higenamine may have potential to develop new candidate for therapy of heart failure.

本课题组以往临床和实验研究显示附子及附子为主的经验方改善心功能和心室重构，前期对其水溶性强心单体去甲乌药碱（Higenamine）的体外研究发现其通过β2/Gi/PI3K/AKT途径抑制新生大鼠、成年小鼠心肌细胞氧化应激引起的凋亡，同时发现该药物同样导致成年小鼠心肌细胞肥大。在此基础上，我们拟采用体外灌流小鼠心脏、单个心肌细胞荧光共振能量转移（FRET）技术证明Higenamine通过β2兴奋细胞膜局部cAMP/PKA活性发挥强心作用；采用缺血再灌注和心梗后心衰小鼠模型及体外RNA干扰技术证明其通过β2/Gi/PI3K/AKT通路在体内及体外抗心肌细胞凋亡，保护心功能；采用小鼠主动脉缩窄（TAC）模型及体外RNA干扰、药物抑制的方法证明其诱导的心肌细胞肥大是有良性代偿意义的生理性肥大而非病理性肥大，其通路也是β2/Gi/PI3K/AKT。完善附子的现代分子生物学研究，以期开发心衰治疗新药。

心肌细胞凋亡加重心肌缺血损伤，并加剧心力衰竭的进展。生理性心肌细胞肥大可以适应机体生理性供血需要，可能对晚期心力衰竭保持心肌收缩力有良性的代偿作用。数千年来，中医将附子用于治疗有心衰症状的疾病，去甲乌药碱作为附子的主要水溶性强心成份，有研究表明其在某些细胞有抗凋亡作用，如心肌细胞和神经元细胞。但是对于心肌细胞，去甲乌药碱的药理学靶点和分子机制尚不十分明确。在此，我们发现去甲乌药碱保护心肌细胞凋亡和缺血再灌注损伤是通过选择性激活β2肾上腺素受体。而且，我们阐明了在小鼠体内，去甲乌药碱明显减少了由缺血再灌注导致的心肌梗塞面积。无论在新生大鼠还是成年小鼠心肌细胞，我们不仅证明了去甲乌药碱可以抑制细胞凋亡，还证明了它可以降低凋亡生物标志物的表达，比如caspase3和caspase9。更重要的是，我们证明了在心肌细胞中，去甲乌药碱的作用可以被β2-AR阻滞剂抑制，而不能被β1-AR阻滞剂抑制。同时，我们明确了在小鼠心脏中，去甲乌药碱减少了因缺血再灌注而导致的心肌损伤，并且减少了依赖β2-AR通路的凋亡蛋白。在心肌细胞中，去甲乌药碱是通过活化磷酸化的AKT和激活PI3K而发挥抗凋亡的作用。这些发现共同证明了去甲乌药碱是通过调节β2-AR/PI3K/AKT通路发挥抗凋亡和保护心肌的作用。我们采用FRET技术观察去甲乌药碱对心肌细胞细胞膜局部cAMP、PKA的激活作用，证明其仅激活细胞膜附近的cAMP pool发挥强心作用，而无β1受体激活全细胞cAMP/PKA导致的心肌细胞肥大和凋亡的副作用。我们采用小鼠主动脉缩窄心脏肥厚模型，发现单独应用去甲乌药碱组的心肌细胞面积增大，但是心功能及心衰的生物标志物均正常，提示其诱导了心肌细胞的生理性肥大；在中晚期TAC模型组中，发现去甲乌药碱可以抑制病理性心肌肥厚，表现在心肌细胞面积减小，心功能及心衰的生物标志物均恢复正常。本课题研究结果完善了强心药附子的有效成分去甲乌药碱的现代分子生物学基础，而且有力地支持了附子在心力衰竭方面的治疗地位，有可能作为先导化合物开发新型抗心力衰竭药物。