基于Ca2+/CaMKII通路研究附子甘草配伍的减毒强心作用与机制

Chronic heart failure is the serious stage of all heart diseases, with high morbidity. However, the traditional Chinese medicine which bases on the holistic view theory has obvious therapeutic effects on it, and also it can reduce the dangerous cardiac events. When the herbs are matched well, they would have the function of effect-enhancing and toxicity-reducing, together with the characteristics of many target spots. Fuzi and Gancao is classic traditional Chinese medicine compatibility. In our previous research, we found that the compatibility showed functions on improving myocardial hypertrophy, anti-inflammation and anti-apoptosis. Meantime, it can lower the intracellular concentration of Ca2+, and regulated the expressions of CaMKII and its related proteins. There have been studies revealed that the overload of Ca2+ could cause heart failure, and CaMKII was one crucial kinase who could adjust the balance of Ca2+ through RyR2 and SERCA2a channels. Accordingly, we speculated that Fuzi and Gancao compatibility could protect chronic heart failure through Ca2+/CaMKII signal pathway. Based on the previous research, this project plans to research by both in vitro and in vivo, build oxygen glucose deprivation model on H9c2 cells and chronic heart failure model on mice, and adopt the technologies of siRNA transfection, gene knockout and so on. Then, we shall survey the detoxification and cardiotonic effects of Fuzi and Gancao compatibility from cellular and integral level, and explore the role of Ca2+/CaMKII pathway in it. Therefore, this research on the one hand can enrich the basic theory of clinical treatments to compatibility of Fuzi and Gancao on chronic heart failure; on the other hand, it can provide vital scientific evidences for its clinical application.

慢性心衰是各种心脏病的严重阶段，发病率高。中医药从整体观的理论出发，对慢性心衰有确切疗效，可减少心血管终点事件。经方药配伍后可增效减毒，具有多靶点作用的特点。附子甘草配伍是临床经典的抗心衰中医药对。我们发现，该药对具有改善心肌肥厚、抗炎、抗凋亡等作用，并可降低钙离子浓度，调节CaMKII及其相关蛋白表达。研究表明，钙离子超载可诱发心衰，CaMKII是可通过RyR2、SERCA2a等通道调节钙离子平衡的重要激酶。故我们推测附子甘草配伍可能通过Ca2+/CaMKII通路产生抗心衰的作用。基于前期研究，本课题拟通过体外与体内实验相结合，建立H9c2心肌细胞缺氧缺糖及小鼠慢性心衰模型，采用siRNA转染及基因敲除等技术，从细胞和整体水平观察附子甘草配伍的减毒强心效果，研究Ca2+/CaMKII通路在其中的作用。因此，本项目不仅丰富了附子甘草配伍治疗心衰的理论基础，也为其临床应用提供重要的科学依据。

慢性心衰是各种心脏病的严重阶段，发病率高。中医药从整体观的理论出发，对慢性心衰有确切疗效，可减少心血管终点事件。经方药配伍后可增效减毒，具有多靶点作用的特点。附子甘草配伍是临床经典的抗心衰中医药对。我们发现，该药对具有改善心肌肥厚、抗炎、抗凋亡等作用，并可降低钙离子浓度，调节CaMKII及其相关蛋白表达。研究表明，钙离子超载可诱发心衰，CaMKII是可通过RyR2、SERCA2a等通道调节钙离子平衡的重要激酶。本课题通过体外与体内实验相结合，研究附子甘草单体配伍抗心衰的作用与代谢组学的相关性；采用系统网络药理学分析，应用QRT-PCR法及Western Blot法验证相关靶点基因的表达。采用小鼠非开胸式主动脉弓狭窄术构建心衰模型，通过血流彩色超声来判断心衰模型手术成功；利用心脏超声成像技术检测心衰对小鼠心肌损伤的影响；应用苏木精-伊红及天狼星红染色检测心衰小鼠心肌组织病理及纤维化情况；采用透射电镜技术观察小鼠心肌线粒体结构形态；用ELISA法检测小鼠血清中BNP、Na-K ATPase含量；应用Western Blot法检测心肌细胞Ca2+/CaMKII相关蛋白表达情况；发现附子甘草配伍对心衰小鼠心肌损伤具有保护作用。同时，以大鼠H9c2心肌细胞为研究对象，建立缺氧缺糖/复氧模型，通过CCK-8试剂显色反应确定附子甘草配伍最佳剂量；倒置显微镜下观察细胞形态；Hochest染色观察细胞凋亡情况；JC-1染色法观察线粒体膜电位的变化；ELISA法检测细胞上清液BNP、Na-K ATPase含量。发现高剂量附子甘草为最佳配伍给药剂量。接着应用Western Blot法检测心肌细胞Ca2+/CaMKII相关蛋白表达情况，发现附子甘草配伍可以有效降低Caspase-9、cleaved-Caspase-3、p-CaMKII的表达量，提高SERCA2a的表达量。表明附子甘草配伍组对心衰小鼠在Ca2+/CaMKII信号通路中发挥了保护作用。因此，本项目从细胞及动物方面丰富了附子甘草配伍治疗心衰的理论基础，也为其临床应用提供重要的科学依据。