抗心律失常复方中药稳心颗粒的心脏安全性再评价

Cardiac safety reevaluation of anti-arrhythmic Chinese medicine Wenxin Keli

目的整合在体、离体、细胞、分子多功能指标,观察抗心律失常复方中药稳心颗粒(WXKL)对大鼠心脏和豚鼠离体心脏心电图、心肌细胞表型及hERG离子通道电流作用,对心脏安全性再评价。方法 1大鼠在体实验:WXKL72.9 g·kg^(-1)剂量ig给予,连续7 d,然后进行Ⅱ导联心电图记录。2豚鼠离体心脏实验:按照正常灌流液10 min,WXKL 3×10^(-2) g·L^(-1) 10 min顺序灌流,记录心电图。3高内涵细胞表型实验:H9C2心肌细胞加入WXKL 1×10^(-2)、1×10^(-3) g·L^(-1) 24 h后,换成0.1μmol·L^(-1)Hoechst 33342、Mito Tracker Deep Red FM 2种荧光分子探针作用20 min,再加入Rhod2 AM荧光分子探针作用30 min,进行活细胞成像。4全自动膜片钳Ionworks系统记录hERG通道电流实验:加入含有(1×10^(-2)、1×10^(-2.5)、1×10^(-3)、1×10^(-3.4) g·L^(-1))WXKL的细胞外液,记录hERG通道电流,分析hERG通道活性I_x。结果 1大鼠在体实验:与空白对照组比,WXKL72.9 g·kg^(-1)及阳性药VER 3.6×10^(-2)g·kg^(-1)对P-R间期、QRS间期、QTs间期、心率差异无统计学意义。2豚鼠离体实验:与空白对照组相比,WXKL 3×10^(-2) g·L^(-1)组P-R间期、QRS间期、Q-T间期、心率并没有显著性差异,阳性药奎尼丁3.25×10^(-3) g·L^(-1)延长QRS间期、Q-T间期,减慢心率(P<0.01),而P-R间期差异无统计学意义。3高内涵细胞表型实验:与空白对照组相比,各浓度WXKL对H9C2心肌细胞的数量、线粒体质量及细胞内钙流强度未显著性改变,而Ⅲ类抗心律失常阳性药胺碘酮在6.82×10^(-3) g·L^(-1)剂量下则对H9C2心肌细胞有显著性改变损害。4全自动膜片钳Ionworks系统记录hERG通道电流实验:与空白对照组相比,各浓度的WXKL未显著性改变hERG通道电流,hERG通道活性没有差异,阳性药奎尼丁3.25×10^(-4)g·L^(-1)阻断hERG通道,抑制hERG通道活性(P<0.01)。结论根据本研究首次建立的多指标综合评价方式,WXKL是一种对心脏较安全的抗心律失常复方中药。

Objective To reevaluate cardiac safety of Wenxin Keli（WXKL） using an integrated in vivo and in vitro assays. Methods 1 In vivo ECG recordings were made to analyze effects WXKL on rat hearts. 2 In vitro ECG recordings were made to analyze effects of WXKL on P-R, QRS, Q-T intervals and heart rates in isolated hearts of guinea pig. 3 High-content screening assay： following WXKL treatment for 24 h, H9C2 cells were stained with Hoechst 33342, Mito Tracker Deep Red FM and Rhod2 AM. After PBS wash, cardiomyocyte images were scanned by Operetta HCA system. 4 Automated patch clamp technique was used to record the effects of WXKL on h ERG channel currents, a gold standard for cardiac safety evaluation. Results 1 WXKL at 72.9 g·kg-1 or Verapamil at 3.6×10- 2 g·kg- 1 in vivo did not change P-R, QRS, QTc intervals and heart rates, compared with Control group. 2 Compared with Control group, WXKL at 3×10- 2 g·L- 1 in vitro did not change P-R, QRS, QT intervals and heart rates, whereas Quinidine at 3.25×10- 3 g·L- 1 slowed heart rates and prolonged QRS and QT but not PR intervals. 3 Compared with Control group, WXKL at 1×10- 2, 1×10- 3 g·L- 1 did not affect H9C2 cell phenotypes whereas positive drug. Amiodarone at 6.82×10- 3g·L- 1 caused significant toxicity shown by reduced cell number, increased mitochondrial mass, and increased calcium overload. 4 Quinidine at 3.25×10- 4 g·L- 1 can significantly altered h ERG channel activity but WXKL at 1×10- 2, 1×10- 2.5, 1×10- 3, 1×10- 3.4 g·L- 1 had no effect. Conclusion Wenxin Keli is a safe antiarrhythmic compound Chinese medicine by multiple integrated analyses.