基于膜片钳技术的丹红注射液对人诱导多能干细胞衍生心肌细胞电生理的影响研究

Effect of Danhong Injection on electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes using patch clamp technique

目的通过全细胞膜片钳实验,从心肌电生理层面探索丹红注射液(DHI)治疗乌头碱诱导心律失常的可能机制。方法基于人诱导多能干细胞衍生心肌细胞(hiPSC-CMs)膜片钳技术,即时给药并观察乌头碱不同浓度(3、9、27µmol·L^(−1))对钠通道、hERG钾通道的抑制效果,进行造模条件的筛选;设置对照组、模型组(乌头碱1µmol·L^(−1)长时间造模)、DHI(3、9、27µL·mL^(−1))组,乌头碱与DHI同时加药,记录动作电位幅度(APA)、放电频率、动作电位幅度下降50%时的时程(APD50)和动作电位幅度下降90%时的时程(APD90),并以对照组为标准,计算相对值;设置对照组、模型组、DHI(3µL·mL^(−1))组,乌头碱与DHI同时加药,观察3~5 min,记录钠离子、hERG钾离子、钙离子电流密度。结果给予即时药物处理,3、9、27µmol·L^(−1)的乌头碱对钠电流的抑制率分别为17.94%、31.07%、60.67%,对hERG通道的抑制率分别为5.02%、10.59%、28.59%;在短期内给乌头碱对hERG通道的抑制效果较为有限,而实际实验中,长时间的高浓度乌头碱孵育使钠电流被完全抑制,导致离子通道功能受损,因此选择乌头碱1µmol·L^(−1)长时间给药制备模型。与对照组比较,模型组hiPSC-CMs相对APA、相对APD50和相对APD90显著下降(P<0.01、0.001),相对放电频率显著升高(P<0.001);钠电流在一定时间内减小,抑制率为30.18%;hERG钾电流明显减小,抑制率为72.33%;钙电流明显增大,电流增大191.35%。与模型组比较,DHI 3、9、27µL·mL^(−1)组相对放电频率显著降低(P<0.05、0.001)、相对APD90显著升高(P<0.05、0.01),3µL·mL^(−1)组相对APD50显著升高(P<0.001);DHI组钠电流、hERG钾电流的减小及钙电流的增大均有一定缓解。结论DHI可能可以通过影响钠、钾、钙通道来缓解乌头碱导致的心律失常现象。

Objective To explore the possible mechanism of Danhong Injection(DHI)in the treatment of aconitine-induced arrhythmia from the perspective of cellular electrophysiology through whole-cell patch-clamp experiments.Method Based on the patch-clamp technique of human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs),the inhibition effects of aconitine at different concentrations(3,9,27μmol·L^(−1))on sodium channel and hERG potassium channel were observed,and the modeling conditions were screened.The control group,model group(1μmol·L^(−1) aconitine for a long time)and DHI(3,9,27μL·mL^(−1))groups were set up,and aconitine and DHI were added at the same time.The action potential amplitude(APA),discharge frequency,time-domain of 50%action potential decrease(APD50)and time-domain of 90%action potential decrease(APD90)were recorded,and the relative values were calculated using the control group as the standard.The control group,model group and DHI(3μL·mL^(−1))group were set up,and aconitine and DHI were added at the same time,observed for 3—5 min,and the current densities of sodium ion,hERG potassium ion,and calcium ion were recorded.Results The inhibition rates of aconitine at 3,9,and 27μmol·L^(−1) on odium current were 17.94%,31.07%,and 60.67%,respectively,and the inhibition rates of hERG channel were 5.02%,10.59%,and 28.59%,respectively.The inhibition effect of aconitine on hERG channel in a short period of time was limited,while in the actual experiment,the long-term incubation of high concentration of aconitine completely inhibited the sodium current,resulting in the damage of ion channel function,so the model was prepared by 1μmol·L^(−1) aconitine for a long time.Compared with the control group,the relative APA,relative APD50,and relative APD90 of hiPSC-CMs in the model group were significantly decreased(P<0.01,0.001),and the relative discharge frequency was significantly increased(P<0.001).The sodium current decreased within a certain time,with an inhibition rate of about 30.18%.The hERG potassium current decreased significantly,with an inhibition rate of about 72.33%.The calcium current increased significantly,with an increase of about 191.35%.Compared with model group,the relative discharge frequency of DHI 3,9,and 27μL·mL^(−1) groups was significantly decreased(P<0.05,0.001),and the relative APD90 was significantly increased(P<0.05,0.01),and the relative APD50 of 3μL·mL^(−1) group was significantly increased(P<0.001).The decrease of sodium current,the decrease of hERG potassium current,and the increase of calcium current in the DHI group were alleviated to some extent.Conclusion DHI may alleviate aconitine-induced arrhythmias by influencing sodium,potassium,and calcium channels.