从细胞内钙稳态角度探索liguzinediol正性肌力作用机制

The positive inotropic drugs in the market are divided into cardiac glycosides non-cardiac glycosides, as well as a new positive inotropic drug levosimendan. But they all have different degrees of adverse reactions. Liguzinediol is a compound of the series of derivatives, which were modified from tetramethylpyrazine. The earlier study was showed that its positive inotropic effect is strong, and has a very good effect on acute heart failure animals and chronic heart failure animals. It has no arrhythmia side effects which are commonly seen in current positive inotropic drugs. But its mechanisms are unknown. The results of previous mechanism research were that its target is not the α1 receptors , β- receptor , the D1 receptor and H1 receptor. Its mechanism was different from milrinone. Liguzinediol can increase the concentration of liguzinediol intracellular calcium, so we hypothesized: the positive inotropic mechanism of liguzinediol is achieved by changing the intracellular calcium ion concentration. We focus on the calcium to find its positive inotropic mechanism from three areas, the extracellular calcium influx , intracellular calcium release and excretion of calcium. The cAMP-PKA signaling pathway and PLC-IP3-Ca2+ signaling pathway are affected the release of calcium, we will detect the relationship between Liguzinediol and these two pathways, and will prove the positive inotropic effect pathway with appropriate blocker.

目前已上市的正性肌力药物包括强心苷类和非强心苷类以及新型正性肌力药左西孟坦。但其都有不同程度的不良反应。Liguzinediol是川芎嗪经结构修饰所得的一种化合物，前期研究结果表明其正性肌力作用强，对急慢性心衰动物均有非常好的疗效，且无常见的心律失常等副作用，但其作用机制尚不清楚。前期机理初探发现其作用机制与现有的强心药米力农不同，作用的靶点也不是α1受体、β受体、D1受体、H1受体，而Liguzinediol能增加细胞内钙离子，故我们提出假设：liguzinediol正性肌力作用是通过调节细胞内钙离子浓度实现的。围绕钙离子从细胞外钙内流、细胞内钙释放及细胞膜上钙的排出三方面来寻找其强心机制。影响Ca2+释放的信号转导系统有cAMP-PKA信号通路和IP3- Ca2+信号通路。因此我们检测Liguzinediol与这两通路间的关系，加入不同的阻断剂来证明其通过哪条途径来实现其正性肌力作用。

Liguzinediol是川芎嗪经结构修饰所得系列衍生物中的一个化合物，前期研究结果表明其正性肌力作用强，对急慢性心衰动物均有非常好的疗效，且无已有正性肌力药物常见的心律失常等副作用。前期机理初探发现其作用机制与现有的强心药米力农和地高辛都不同，作用的靶点也不是α1受体、β受体、D1受体、H1受体，而Liguzinediol能增加细胞内钙离子，因此我们围绕钙离子从细胞外钙内流、细胞内钙释放及细胞膜上钙的排出三方面来寻找其强心机制。我们利用钙成像系统检测Liguzinediol 对细胞外钙内流的影响，结果显示证实Liguzinediol对细胞外钙内流这个环节影响不大。内钙释放的实验用阿霉素复制心肌损伤模型，结果提示Liguzinediol可增加心肌细胞内肌浆网上RyR2的蛋白和RyR2 mRNA表达，降低p- RyR2的蛋白表达，增加心肌细胞内肌浆网上IP3R2的蛋白表达和FKBP12.6的蛋白表达，促进RyR2和FKBP12.6之间的结合，缓解舒张期Ca2+渗漏。Liguzinediol还可以通过增强肌浆网钙泵的功能来调控细胞内钙离子的浓度。在线粒体部分实验结果表明Liguzinediol明显下调MCU、fis1、Fundc1、VDAC1、tricA通道和drp1的表达，上调MFN1和MFN2蛋白表达增加，说明Liguzinediol可以通过线粒体来缓冲心肌细胞内钙离子浓度，维持细胞内钙稳态。在内钙外流方面实验结果显示Liguzinediol可增强细胞膜上Ca2+-ATP和降低NCX蛋白和 mRNA的表达，说明Liguzinediol能够改变心肌细胞内钙离子的浓度与减弱细胞膜上的钠钙交换体的功能和增强细胞膜上钙泵有关。在信号通路方面研究结果表明Liguzinediol可降低细胞内cAMP含量和下调PKA的蛋白表达，可下调PLC的蛋白表达和基因表达，PLB的蛋白表达，PP1的表达，CaMKII 、CaN的表达，说明 Liguzinediol可以同时通过cAMP-PKA 信号通路和 PLC-IP3- Ca2+信号通路实现对细胞内钙离子浓度的调控。因此Liguzinediol可以同时通过cAMP-PKA 信号通路和 PLC-IP3- Ca2+信号通路影响细胞内钙释放和内钙外排两个环节实现其正性肌力作用。