基于钙超载理论对“痰瘀互结”致心律失常的作用及丹蒌片干预机制研究

Arrhythmia is a serious risk to human health . " Phlegm and blood stasis ", play an important role in the occurrence of cardiac arrhythmias . Our previous studies have confirmed Danlou Tablet can reduce arrhythmia caused by myocardial ischemia, but its exact mechanism has not been elucidated . It was reported that calcium overload played an important role in arrhythmias. We speculate " Phlegm and blood stasis" increased calcium overload by promoting myocardial dysfunction of L-type calcium channel(ICaL) and sodium calcium exchanger(NCX), leading to arrhythmias; Danlou Tablet inhibits channel dysfunction, reduce calcium overload, so as to suppress arrhythmias.To confirm this hypothesis , we'll construct arrhythmia model of phlegm and blood stasis by ligating coronary artery of Apo-E knockout mice, monitoring arrhythmia by electrocardiogram,detecting calcium transient by optical mapping and confocal,meausuring ICaL and Incx currents by patch clamp, detecting the protein expression of CaV1.2 and NCX. Next, observing the effect of Danlou Tablet on this model.Thirdly,using in vitro experiments and computer simulation,observing the effect of Danlou Tablets on arrhythmia. This research will further provide the new targets and experimental evidence for the effective prevention of Tanlou tablet on clinical application .

心律失常,严重危险人类健康."痰瘀互结、心血瘀阻"是心律失常的重要病机。我们的前期研究发现化痰祛瘀中药丹蒌片能改善心肌缺血所导致的心律失常，但其确切机制尚未阐明.目前研究认为,钙超载在心律失常中起到重要作用.我们推测"痰瘀互结"，促进心肌细胞L型钙离子通道(ICaL)与钠钙泵(NCX)功能紊乱，加重钙超载，导致心律失常的发生；丹蒌片通过抑制ICaL与NCX，减轻钙超载，而抑制心律失常。为证实该假说，本研究以Apo-E基因敲除小鼠冠状动脉结扎方法构建痰瘀互结型心律失常模型，通过心电图检测心律失常,光学标测结合激光共聚焦检测心肌组织与细胞钙流,膜片钳结检测ICaL、NCX电流,免疫印迹检测NCX、CaV1.2蛋白表达，并且观察丹蒌片对痰瘀互结型心律失常的影响,进一步行体外试验与计算机模拟试验，观察丹蒌片对钙超载的作用机制。本研究的开展为丹蒌片治疗心律失常的临床应用提供新的作用靶点和试验依据。

心肌缺血所导致的恶性心律失常,严重危险人类健康.“痰瘀互结”是其重要病机。我们通过Apo-E基因敲除小鼠冠状动脉结扎方法构建痰瘀互结型心律失常，观察丹蒌片对痰瘀互结模型的影响,发现丹蒌片可减少心律失常的发生，减少心肌梗死面积和cTnI, 进一步行体外试验构建H9c2心肌细胞缺血模型，观察丹蒌片对心肌缺血损伤及心肌细胞钙超载的影响，通过外源性给予丹蒌片含药血清，采用细胞形态学观察法，MTT法检测细胞活性，激光共聚焦法检测各组心肌细胞内钙流浓度，蛋白免疫印迹技术检测钙超载相关蛋白p-CaMKⅡ及CaMKⅡ的表达。通过以上方法确定采用5mM Na2S204缺氧处理2h以建立稳定的心肌细胞缺氧损伤模型。与对照组相比，模型组心肌细胞内游离Ca2+水平显著升高，P-CaMKⅡ表达增加；与模型相比，抑制剂组及丹蒌片组心肌细胞内游离Ca2+水平均显著降低，P-CaMKⅡ表达降低。计算机仿真试验提示Ca2+、CaMKII在钙超载致心律失常中发挥重要作用，丹蒌片对心肌缺血损伤的保护作用与减少外钙内流、降低内钙释放，调节P-CaMKⅡ表达有关。本研究的开展为丹蒌片治疗心律失常的临床应用提供新的作用靶点和试验依据。