参松养心胶囊抗心律失常机理研究
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摘要
背景：心律失常是临床上常见且又对患者的生活质量和生命安全影响极大的心血管疾病。长期以来，人们一直致力于抗心律失常治疗的研究。随着科学技术的进步，心律失常的非药物疗法，如除颤、起搏、射频消融、手术等在临床治疗中虽然起到越来越大的作用，但是许多类型的心律失常至今仍依赖于药物治疗，抗心律失常新药开发仍然是我国防治快速性心律失常的重要研究内容。
临床心律失常的种类繁杂，不能用一种机制来解释其发生的原因，从根本上说心肌细胞膜离子通道表达或功能异常将会影响动作电位的时程，进而引起心脏不同部位复极离散增大，是形成各种心律失常的最重要的病理生理基础。目前临床应用的抗心律失常西药主要包括钠通道阻断剂、β受体阻断剂、延长动作电位时程药(主要是钾离子通道阻断剂)和钙离子通道阻断剂。尽管药物分类不同，其作用机制有差异，归根到底是通过对各种心肌细胞离子通道功能的调节来起作用的。药物在发挥抗心律失常作用的同时，有潜在的致心律失常发生的危险，尤其是导致继发性QT间期延长和尖端扭转型室性心动过速，可增加患者死亡率。从90年代CAST和CASTII试验结果公布以来，人们开始重视抗心律失常药物治疗的收益与风险评估。重新评价临床应用的一些抗心律失常药物的安全性，以及开发广谱有效且副作用低的新型抗心律失常药物是目前研究的热点。能够改善离子通道紊乱并恢复其正常生理功能的药物，才能防治心律失常的发生并提高远期疗效。
同西药相比，抗心律失常作用的中药不仅对心脏离子通道功能有影响，而且中药往往是复合药物，具有多离子通道靶点的作用，因而毒性低副作用小。从中药中筛选出高效、低毒的抗心律失常药物是我国中药现代化的重要组成部分。用科学的方法来研究和评价中药的抗心律失常作用机理，对于指导临床用药和研发新药都有着重要的意义。
参松养心胶囊(SSYX)是一种复方制剂，方中多数药物均有较好的抗心律失常作用。动物实验证明，SSYX可以明显抑制药物诱发的心律失常；随机临床验显示，SSYX对治疗冠心病心律失常有明显疗效。关于SSYX抗心律失常的机制和对心肌细胞离子通道的影响至今未见报道。
目的：观察SSYX干粉提取溶液对单个心室肌细胞L型钙电流(I_(Ca)，L)，钠电流(I_(Na))，瞬时外向钾电流(I_to))，内向整流钾电流(I_(K1))，延迟整流钾电流慢成分(I_(Ks))的影响，以及对在HEK293细胞表达hERG通道电流(I_(Kr))的影响。探讨SSYX在离子通道水平的抗心律失常作用机制。
方法：急性分离豚鼠和大鼠心脏，用Langendoff灌流系统离体灌流，联合使用胶原酶Ⅱ和蛋白酶E，分离获得单个的心室肌细胞。培养HEK293细胞，用脂质体瞬时转染pcDNA3.1-hERG和pEGFP-C1质粒，于转染后24～48h在荧光显微镜下，对有绿色荧光表达的细胞进行电生理记录。用标准的全细胞膜片钳记录技术，记录豚鼠心室肌细胞的I_(Ca)，L，I_(Na)，I_(k)电流，大鼠心室肌细胞的I_(to)和I_(K1)电流，以及转基因阳性HEK293细胞的I_(Kr)电流。观察SSYX干粉提取溶液对各种离子通道电流的影响。
结果：通过酶解法得到的豚鼠和大鼠心室肌细胞能够记录到动作电位和各种离子通道电流，表明其具有良好的细胞电生理特性。大约40％～60％瞬时转染后HEK293细胞表达绿色荧光蛋白，同时可记录到I_(Kr)通道电流，表明细胞模型构建成功。
1．SSYX对动作电位的影响：在0.5％的药物使用前后，大鼠心室肌细胞的静息膜电位分别为-65.02±5.96mV和-62.67±7.59mV(n=6，P＞0.05)；动作电位超射幅度降低，超射值分别为101.31±8.78mV和84.84±5.47mV(n=6，P＜0.05)；复极时程延长，50％动作电位时程(APD_(50))分别为23.92±5.50ms和41.99±5.39ms(n=6，P＜0.05)；90％动作电位时程(APD_(90))分别为46.97±7.89ms和65.3±5.84ms(n=6，P＜0.05)。
2．SSYX对I_(Ca)，L通道的影响：记录稳态激活的I_(Ca)，L电流，观察到药物对I_(Ca)，L有抑制作用。以测试电压为+10 mV引出的I_(Ca-L)峰值电流密度作为统计指标，当药物浓度为1％，0.5％，0.25％时，分别对I_(Ca)，L的峰值电流密度的抑制率为50.69％±5.64％，44.82％±6.50％，和19.22％±1.10％，(n=5，P＜0.05)。药物可以使I_(Ca)，L的电流密度-电压(I-V)曲线上移，但不改变其激活电压、峰值电压和反转电位。分别用单指数和双指数方程拟合+10 mV引出电流的时间依赖性激活和失活曲线，药物浓度0.5％时，用药前后通道的激活时间常数(τ)分别为1.04±0.17ms和1.52±0.19ms(n=6，P＜0.05)，通道的快失活时间常数(τ_f)分别为13.00±3.72 ms和42.74±10.10ms(n=6，P＜0.05)，慢失活时间常数(τ_s)分别为97.75±20.79 ms和177.26±28.29ms(n=6，P＜0.05)。用Boltzmann方程分别拟合稳态激活和失活曲线。0.5％SSYX不改变I_(Ca-L)稳态激活曲线，可使I_(Ca-L)稳态失活曲线左移，用药前后的失活半电压(V_(1／2))分别为-15.38±2.4 mV和-20.44.2±5.01 mV(n=6，P＜0.05)，斜率因子(K)分别为5.25±0.61和5.33±0.51(n=6，P＞0.05)。记录通道的失活后恢复曲线，用单指数方程为最佳拟合，得到恢复时间常数(τ)，0.5％SSYX使τ从85.24±20.18ms增大到158.42±38.23 ms(n=5，P＜0.05)。
3．SSYX对I_(Na)通道的影响：记录稳态激活I_(Na)的I-V曲线，0.5％SSYX在每一指令电压水平上都减小I_(Na)电流密度，但不改变激活电压、峰值电压、反转电位和曲线的形状。当药物浓度为0.5％时，在测试电压为-20 mV的条件下，可以使I_(Na)峰值电流密度从27.21±5.35(pA／pF)降至14.88±2.75(pA／pF)，平均抑制率为44.84％±7.65％(n=5，P＜0.05)。用单指数方程拟合-20 mV条件下引出电流的时间依赖性激活和失活曲线，浓度0.5％药物使用前后，通道的激活τ分别为0.44±0.15ms和0.36±0.11 ms(n=5，P＞0.05)；失活τ分别为1.84±0.34ms和2.05±0.51 ms(n=5，P＞0.05)。0.5％药物使用前后，通道稳态激活V_(1／2)分别为-34.27±2.38mV和-31.40±1.63mV(n=5，P＜0.05)；稳态失活V_(1／2)分别为-73.68±4.80mV和-75.15±4.50mV(n=5，P＞0.05)。0.5％药物使用前后，通道的失活后恢复τ分别为19.03±1.13 ms和21.75±1.48 ms(n=5，P＞0.05)。
4．SSYX对I_(to)通道的影响：当药物浓度0.5％时，在60mV条件下使大鼠心室肌细胞I_(to)电流峰值密度从-19.82±7.10(pA／pF)降至-10.02±3.93(pA／pF)，平均抑制率为50.60±10.77％(n=6，P＜0.05)。用单指数方程拟合I_(to)通道的时间依赖性失活曲线，0.5％药物使用前后，失活的τ值分别为29.06±4.66ms和21.44±3.12ms(n=6，P＜0.05)；0.5％药物使I_(to)通道稳态失活曲线左移，用药前后的V_(1／2)分别为-15.67±2.52mV和-26.45±3.88mV，K分别为3.41±0.67和6.38±2.02(n=7，P＜0.05)。0.5％药物使用前后，I_(to)通道的失活后恢复减慢，τ分别为12.86±0.31ms和18.52±3.76ms(n=5，P＜0.05)。
5．SSYX对I_(K1)通道的影响：药物浓度0.5％时，可以使大鼠心肌细胞I_(k1)内向成分降低，以测试电压-100mV时引出的电流为统计指标，I_(k1)电流密度从-10.78±1.80(pA／pF)降至-7.18±2.05(pA／pF)，平均抑制率为33.10％±16.85％(n=11，P＜0.05)，但药物不改变电流的翻转电位及整流特性。
6．SSYX对I_(Ks)通道的影响：使用钾离子通道细胞内外液，维持电压-40mV，测试电压+50mV持续5s，可以在豚鼠心室肌细胞引出I_(Ks)电流。当SSYX药物浓度0.5％时，可以使豚鼠心肌细胞I_(Ks)最大电流密度从4.02±0.27(pA／pF)降低到1.39±0.30(pA／pF)，平均抑制率为65.21％±8.5％(n=5，P＜0.05)，同时对其尾电流的抑制率是30.77％±1.11％(n=5，P＜0.05)。
7．SSYX对I_(Kr)通道的影响：为了更好的观察SSYX对单纯的I_(Kr)电流的影响，我们用HEK293细胞瞬时表达hERG基因来研究药物对I_(Kr)的影响。在转染hERG基因阳性的HEK293细胞上记录到的I_(Kr)自—20mv激活，外向电流随去极化而增大，约在+20mV达到最大值，之后随进一步去极化电流幅值下降，具有内向整流特性。0.5％SSYX对I_(Kr)电流无显著影响。用药前后，在Vt为+20mV时，峰值电流密度分别为18.56±2.47 pA／pF和16.57±3.57 pA／pF(n=5，P＞0.05)。用标化的I_(Kr)尾电流对测试电压作图，得到I_(Kr)的稳态激活曲线，用Boltzmann方程拟合曲线，得到用药前激活V_(1／2)和K分别为-3.78±5.30 mV和8.14±1.26，0.5％药物使用后V_(1／2)和K分别为-5.65±5.29 mV和8.43±1.79(n=5，P＞0.05)。0.5％药物使用前后，在Vt-20mV时I_(Kr)尾电流的失活时间常数分别为16.85±3.78和14.76±1.34(n=5，P＞0.05)，I_(Kr)失活后恢复时间常数为6.29±0.84和5.94±0.50(n=5，P＞0.05)，差异均无显著性。
结论：参松养心胶囊干粉提取溶液对I_(Ca)，L、I_(Na)、I_(to)、I_(Ks)和I_(K1)多种心室肌离子通道均具有不同程度的阻滞作用。SSYX可以抑制豚鼠心室肌细胞I_(Ca)，L的峰值,电流，使通道的时间依赖性激活和失活减慢，并促进通道的电压依赖性失活，使稳态失活曲线的V_(1／2)左移。SSYX可以抑制豚鼠心室肌细胞I_(Na)的峰值电流，并使通道的稳态激活曲线右移。SSYX可以抑制大鼠心室肌细胞I_(to)峰值电流，加速通道时间依赖性失活，并促进通道的电压依赖性失活。SSYX可以抑制大鼠心室肌细胞I_(K1)的内向成分。SSYX可以抑制豚鼠心室肌细胞I_K电流，主要是作用于其慢激活成分I_(Ks)。SSYX对hERG通道的电流幅度，通道失活速度及通道失活后恢复速度无显著影响。SSYX对这些通道的综合作用可使得心肌细胞的除极幅度降低，动作电位时程和有效不应期延长，这可能是其产生抗心律失常作用的机制。同时这种多通道阻滞作用可能降低药物引发心律失常的风险，参松养心胶囊可能有广谱的抗心律失常作用。
Study on the mechanism of antiarrhythmic effects of ShenSongYangXin
Background: Cardiac arrhythmia is a kind of common cardiovascular diseases in the clinical practice, which have great effects on life quality and safety of the patients. People have been continuously devoted to investigation and development of antiarrhythmic drugs in the recent decades. Along with the progress in science and technology, non-pharmacotherapy (such as defibrillation, pacing, radio frequency ablation, surgery and so on) plays more and more roles in clinical treatment, but pharmacotherapy is still the essential way in control cardiac tachy-arrhythmia.
Cardiac arrhythmias result from abnormalities of impulse generation, conduction, or both. It is difficult, however, to illustrate the underlying mechanisms for all the clinical arrhythmias. At present, antiarrhythmic drugs have been classified as sodium channel blockers, [3-receptor blockers, action potential duration prolong agents (mainly potassium channel blockers) and calcium channel blockers. The abnormalities of expression and function of the membrane ion channels can impact on action potential duration, amplify dispersion of repolarization, induce triggered activity, and thus contribute to the development of cardiac arrhythmias. Although the classification of antiarrhythmic drugs are different, modulation the ion channels function is the basic mechanism. Besides the antiarrhythmic efforts, many antiarrhythmic agents have potential risk for proarrhythmia. The adverse side effects of drug may induce polymorphic ventricular tachycardia or torsade de pointes (TdP), which can increase mortality of the cardiovascular diseases. Since CAST and CASTⅡstudy published, it attached more attention to value the benefit risk ratio when antiarrhythmic drugs were applied. People begin to revalue the safety of antiarrhythmic drugs currently used in clinic and to develop new agents with fewer side effects.
Compared to Western medicine, some antiarrhythmic Chinese herbs with many components can multi-target modulate the disorder of ion channel function. It might exert antiarrhythmic efforts with lower incidence of adverse effects. Screening antiarrhythmic Chinese herbs might lead to a new trend in pharmacotherapy.
Shensong Yongxin (SSYX) is one of the compound recipe of Chinese materia medica including 12 ingredients such as Panax ginseng, dwarf lilyturf tuber, Nardostachys root, etc. Previous studies on animal model showed that SSYX significantly inhibited the arrhythmias induced by toxic chemical compounds or ischemia-reperfusion injury. Small random double-blind clinical trials also suggested that SSYX reduced the number of ventricular extra beats in patients with or without structure heart disease. However, the antiarrhythmic mechanisms of SSYX have not been studied.
Objectives: To determine the effects of ShenSongYangXin(SSYX) on L-type calcium channels( I_(Ca), L), sodium channels(I_(Na)), transient outward potassium current (I_(to)), delayed rectifier current (I_K), and inward rectifier potassium currents(I_(K1)) in isolated ventricular myocytes, and on hERG channel(I_(Kr)) in transfected HEK293 cells. To investigate the pharmacological mechanism of SSYX on the ion channels.
Methods: Single ventricular myocytes of guinea pigs and rats were obtained by enzymatic dissociation method. Cultured HEK293 cells were transfected transiently with pcDNA3.1-hERG by a lipofectamine method and green fluorescent protein cDNA (pEGFP-C1) was co-transfected to serve as an indicator. 24～48h after transfection, electrical studis were applied in cells with green fluorescence. Whole cell patch-clamp technique was used to record ion channel currents. I_(Ca,L,) I_(Na,) I_K were studied in ventricular myocytes of guinea pigs; I_(to,)I_(K1) were studied in ventricular myocytes of rats; and I_(Kr) were studied in transfected HEK293 cells.
Results: The action potential and ion currents could be recorded in the isolated ventricular myocytes of guinea pigs and rats, which indicated the myocytes had satisfactory electrophysiological properties. Approximate 40～60 percent transfected HEK293 cells took green fluorescence, in which I_(Kr) could be recorded.
1. The effect of SSYX on action potential. Before and after 0.5% drug application, the rest membrane potential of rat ventricular myocytes were -65.02±5.96mV and -62.67±7.59mV (n＝6,P＞0.05); the action potential amplitude were 101.31±8.78mV and 84.84±5.47mV(n=6, P＜0.05); 50% action potential duration(APD_(50)) were 23.92±5.50ms and 41.99±5.39ms(n=6, P＜0.05); 90% action potential duration(APD_(90)) were 46.97±7.89ms and 65.3±5.84ms(n=6, P＜0.05).
2. The effect of S SYX on I_(Ca,L.) At the test potential of＋10 mV, S SYX inhibited L-type calcium current in a dose-dependent manner. At 0.25%, 0.5%, 1% of concentration, the peak I_(Ca,L) was reduced by 19.22%±1.10 %, 44.82%±6.50 % and 50.69%±5.64%, respectively (n=5, all P＜0.05). In the presence of SSYX 0.5%, the current density-voltage curve was moved up and activation potential, the potential of peak current, and the shape of the I-V curve did not change. The avtivation time constant(τ) was measure as the monoexponential fit to the curret curve induced at＋10 mV, andτincreased from 1.04±0.17ms to 1.52±0.19ms(n=6, P＜0.05) after 0.5% drug application. The steady-state inactivation curve was fitted by a Boltzmann function. It was moved to more negative potential, the half inactivation potential (V_(1/2)) was -15.38±2.4 mV and -20.44.24±5.01 mV(n=6, P＜0.05) in control and SSYX 0.5% respectively. Monoexponential function was best fit the curve of recovery time from inactivation. After 0.5% drug use, the channel recovery time constant increased, from 85.24±20.18ms to 158.42±38.23 ms(n＝5, P＜0.05).
3. The effect of SSYX on I_(Na.) At the test potential of -20 mV, SSYX 0.5% decreased peak I_(Na) by 44.84%±7.65 % from 27.21±5.35 to 14.88±2.75 pA/pF (n=5, P＜0.05). SSYX up shifted the I-V curve of I_(Na) without changing the threshold, peak and reverse potentials. Vt -20mV, before and after SSYX 0.5% application, the time constant of activation were 0.444±0.15 ms and 0.364±0.11 ms (n=5, P＞0.05); the time constant of inactivation were 1.84±0.34ms and 2.054±0.51 ms (n=5, P＞0.05). Before and after SSYX 0.5% use, steady-state activation V_(1/2) were -34.27±2.38mV and -31.40±1.63mV (n=5, P＜0.05); steady-state inactivation V_(1/2) were -73.68±4.80mV and -75.15±4.50mV(n=5, P＞0.05); channel recovery time constant were 19.03±1.13 ms and 21.754±1.48 ms(n=5, P＞0.05).
4. The effect of SSYX on I_(to.) At 0.5% of concentration, the drug blocked the transient component of I_(to) by 50.60 % at membrane voltage of 60mV, from -19.82±7.10(pA/ pF) to -10.02±3.93 ( pA/ pF). SSYX 0.5% could accelerate the inactivation of I_(to,) the time constant were 29.06±4.66ms and 21.4 4±3.12ms (n=6, P＜0. 05). SSYX 0.5% negatively shifted the inactivation curve, the inactivation V_(1/2) were -15.67±2.52 mV and -26.45±3.88 mV (n=7, P＜0.05) respectively in control and in drug group. SSYX 0.5% delayed the channel recovery from inactivation. Before and after drug use, the recovery time constant were 12.86±0.31ms and 18.52±3.76ms (n=5, P＜0.05).
5. The effect of SSYX on I_(K1.) SSYX 0.5% inhibited the I_(K1) from -10.78±1.80 (pA/pF) to -7.18±2.05 (pA/pF)by 33.10 %±16.85 % (n＝11, P＜0.05) at the test potential of-100mV with little effect on reversal potential and the rectification property.
6. The effect of SSYX on/Ks. Holding potential at -40mV and test potential at +50mV last 5s could elicit time dependent increased I_(Ks) in isolated guinea pig ventricular cells. SSYX 0.5% inhibited I_(Ks) from 4.02±0.27(pA/pF) tol.39±0.30(pA/pF), by 65.21%±8.5%(n=5, P＜0.05), and inhibited I_(Ks,tail) by 30.77%±1.11%(n=5, P＜0.05).
7. The effect of SSYX on I_(Kr.) I_(Kr) was recorded in hERG-transfected HEK293 cells. During the depolarizing steps, the outward current was activated at voltages positive to -20mV, and the current amplitude was increased to reach a maximum at +20inV. With further depolarization, the current amplitude decreased progressively, because of the inward rectification. Test potential at +20mV, after 0.5% SSYX use the I_(Kr) amplitude changed from 18.56±2.47 pA/pF to16.57±3.57 pA/pF (n＝5, P＞0.05). Activation curve mearured with I_(Kr,tail) and fitted to a Boltzmann relationship. Before 0.5%SSYX use, the V_(1/2) and K were -3.78±5.30 mV and 8.14±1.26, and after drug use the V_(1/2) and K were -5.65±5.29 mV and 8.43±1.79 (n=5,P＞0.05). Test potential at -20mV, before and after 0.5% SSYX use, the I_(Kr,tail) inactivation time constant were 16.85±3.78 and 14.76±1.34 (n=5, P＞0.05); the recovery time constant from inactivation were 6.29±0.84 and 5.94±0.50 (n=5, P＞0.05).
Conclusions: It reveals that SSYX could block multiple ion channels includeI_(Ca,L) I_(Na,) I_(to,) I_(K1) and I_(Ks.) SSYX could inhibit I_(Ca,L) peak current, slow down time dependent activation and inactivation, and promote channel voltage dependent inactivation. SSYX could decrease I_(Na) peak current, and right shift the steady-state activation curve. SSYX could depress I_(to) peak current, promote channel time dependent and voltage dependent inactivation. SSYX could inhibit the inward current component of I_(K1.) SSYX could inhibit the slowly-activating component of I_K on guinea pig ventricular myocytes. SSYX had few effects on I_(Kr) current amplitude, time dependent inactivation and recovery from inactivation. The integrated effects of SSYX may change the action potential duration and contribute to some of its antiarrhythmic effects. Moreover, the block effect on multiple ion channels would be beneficial to reduce proarrhythmic side effects. SSYX may be expected to have a wild spectrum of antiarrhythmic effect with less proarrhythmic potential.
临床心律失常的种类繁杂，不能用一种机制来解释其发生的原因，从根本上说心肌细胞膜离子通道表达或功能异常将会影响动作电位的时程，进而引起心脏不同部位复极离散增大，是形成各种心律失常的最重要的病理生理基础。目前临床应用的抗心律失常西药主要包括钠通道阻断剂、β受体阻断剂、延长动作电位时程药(主要是钾离子通道阻断剂)和钙离子通道阻断剂。尽管药物分类不同，其作用机制有差异，归根到底是通过对各种心肌细胞离子通道功能的调节来起作用的。药物在发挥抗心律失常作用的同时，有潜在的致心律失常发生的危险，尤其是导致继发性QT间期延长和尖端扭转型室性心动过速，可增加患者死亡率。从90年代CAST和CASTII试验结果公布以来，人们开始重视抗心律失常药物治疗的收益与风险评估。重新评价临床应用的一些抗心律失常药物的安全性，以及开发广谱有效且副作用低的新型抗心律失常药物是目前研究的热点。能够改善离子通道紊乱并恢复其正常生理功能的药物，才能防治心律失常的发生并提高远期疗效。
同西药相比，抗心律失常作用的中药不仅对心脏离子通道功能有影响，而且中药往往是复合药物，具有多离子通道靶点的作用，因而毒性低副作用小。从中药中筛选出高效、低毒的抗心律失常药物是我国中药现代化的重要组成部分。用科学的方法来研究和评价中药的抗心律失常作用机理，对于指导临床用药和研发新药都有着重要的意义。
参松养心胶囊(SSYX)是一种复方制剂，方中多数药物均有较好的抗心律失常作用。动物实验证明，SSYX可以明显抑制药物诱发的心律失常；随机临床验显示，SSYX对治疗冠心病心律失常有明显疗效。关于SSYX抗心律失常的机制和对心肌细胞离子通道的影响至今未见报道。
目的：观察SSYX干粉提取溶液对单个心室肌细胞L型钙电流(I_(Ca)，L)，钠电流(I_(Na))，瞬时外向钾电流(I_to))，内向整流钾电流(I_(K1))，延迟整流钾电流慢成分(I_(Ks))的影响，以及对在HEK293细胞表达hERG通道电流(I_(Kr))的影响。探讨SSYX在离子通道水平的抗心律失常作用机制。
方法：急性分离豚鼠和大鼠心脏，用Langendoff灌流系统离体灌流，联合使用胶原酶Ⅱ和蛋白酶E，分离获得单个的心室肌细胞。培养HEK293细胞，用脂质体瞬时转染pcDNA3.1-hERG和pEGFP-C1质粒，于转染后24～48h在荧光显微镜下，对有绿色荧光表达的细胞进行电生理记录。用标准的全细胞膜片钳记录技术，记录豚鼠心室肌细胞的I_(Ca)，L，I_(Na)，I_(k)电流，大鼠心室肌细胞的I_(to)和I_(K1)电流，以及转基因阳性HEK293细胞的I_(Kr)电流。观察SSYX干粉提取溶液对各种离子通道电流的影响。
结果：通过酶解法得到的豚鼠和大鼠心室肌细胞能够记录到动作电位和各种离子通道电流，表明其具有良好的细胞电生理特性。大约40％～60％瞬时转染后HEK293细胞表达绿色荧光蛋白，同时可记录到I_(Kr)通道电流，表明细胞模型构建成功。
1．SSYX对动作电位的影响：在0.5％的药物使用前后，大鼠心室肌细胞的静息膜电位分别为-65.02±5.96mV和-62.67±7.59mV(n=6，P＞0.05)；动作电位超射幅度降低，超射值分别为101.31±8.78mV和84.84±5.47mV(n=6，P＜0.05)；复极时程延长，50％动作电位时程(APD_(50))分别为23.92±5.50ms和41.99±5.39ms(n=6，P＜0.05)；90％动作电位时程(APD_(90))分别为46.97±7.89ms和65.3±5.84ms(n=6，P＜0.05)。
2．SSYX对I_(Ca)，L通道的影响：记录稳态激活的I_(Ca)，L电流，观察到药物对I_(Ca)，L有抑制作用。以测试电压为+10 mV引出的I_(Ca-L)峰值电流密度作为统计指标，当药物浓度为1％，0.5％，0.25％时，分别对I_(Ca)，L的峰值电流密度的抑制率为50.69％±5.64％，44.82％±6.50％，和19.22％±1.10％，(n=5，P＜0.05)。药物可以使I_(Ca)，L的电流密度-电压(I-V)曲线上移，但不改变其激活电压、峰值电压和反转电位。分别用单指数和双指数方程拟合+10 mV引出电流的时间依赖性激活和失活曲线，药物浓度0.5％时，用药前后通道的激活时间常数(τ)分别为1.04±0.17ms和1.52±0.19ms(n=6，P＜0.05)，通道的快失活时间常数(τ_f)分别为13.00±3.72 ms和42.74±10.10ms(n=6，P＜0.05)，慢失活时间常数(τ_s)分别为97.75±20.79 ms和177.26±28.29ms(n=6，P＜0.05)。用Boltzmann方程分别拟合稳态激活和失活曲线。0.5％SSYX不改变I_(Ca-L)稳态激活曲线，可使I_(Ca-L)稳态失活曲线左移，用药前后的失活半电压(V_(1／2))分别为-15.38±2.4 mV和-20.44.2±5.01 mV(n=6，P＜0.05)，斜率因子(K)分别为5.25±0.61和5.33±0.51(n=6，P＞0.05)。记录通道的失活后恢复曲线，用单指数方程为最佳拟合，得到恢复时间常数(τ)，0.5％SSYX使τ从85.24±20.18ms增大到158.42±38.23 ms(n=5，P＜0.05)。
3．SSYX对I_(Na)通道的影响：记录稳态激活I_(Na)的I-V曲线，0.5％SSYX在每一指令电压水平上都减小I_(Na)电流密度，但不改变激活电压、峰值电压、反转电位和曲线的形状。当药物浓度为0.5％时，在测试电压为-20 mV的条件下，可以使I_(Na)峰值电流密度从27.21±5.35(pA／pF)降至14.88±2.75(pA／pF)，平均抑制率为44.84％±7.65％(n=5，P＜0.05)。用单指数方程拟合-20 mV条件下引出电流的时间依赖性激活和失活曲线，浓度0.5％药物使用前后，通道的激活τ分别为0.44±0.15ms和0.36±0.11 ms(n=5，P＞0.05)；失活τ分别为1.84±0.34ms和2.05±0.51 ms(n=5，P＞0.05)。0.5％药物使用前后，通道稳态激活V_(1／2)分别为-34.27±2.38mV和-31.40±1.63mV(n=5，P＜0.05)；稳态失活V_(1／2)分别为-73.68±4.80mV和-75.15±4.50mV(n=5，P＞0.05)。0.5％药物使用前后，通道的失活后恢复τ分别为19.03±1.13 ms和21.75±1.48 ms(n=5，P＞0.05)。
4．SSYX对I_(to)通道的影响：当药物浓度0.5％时，在60mV条件下使大鼠心室肌细胞I_(to)电流峰值密度从-19.82±7.10(pA／pF)降至-10.02±3.93(pA／pF)，平均抑制率为50.60±10.77％(n=6，P＜0.05)。用单指数方程拟合I_(to)通道的时间依赖性失活曲线，0.5％药物使用前后，失活的τ值分别为29.06±4.66ms和21.44±3.12ms(n=6，P＜0.05)；0.5％药物使I_(to)通道稳态失活曲线左移，用药前后的V_(1／2)分别为-15.67±2.52mV和-26.45±3.88mV，K分别为3.41±0.67和6.38±2.02(n=7，P＜0.05)。0.5％药物使用前后，I_(to)通道的失活后恢复减慢，τ分别为12.86±0.31ms和18.52±3.76ms(n=5，P＜0.05)。
5．SSYX对I_(K1)通道的影响：药物浓度0.5％时，可以使大鼠心肌细胞I_(k1)内向成分降低，以测试电压-100mV时引出的电流为统计指标，I_(k1)电流密度从-10.78±1.80(pA／pF)降至-7.18±2.05(pA／pF)，平均抑制率为33.10％±16.85％(n=11，P＜0.05)，但药物不改变电流的翻转电位及整流特性。
6．SSYX对I_(Ks)通道的影响：使用钾离子通道细胞内外液，维持电压-40mV，测试电压+50mV持续5s，可以在豚鼠心室肌细胞引出I_(Ks)电流。当SSYX药物浓度0.5％时，可以使豚鼠心肌细胞I_(Ks)最大电流密度从4.02±0.27(pA／pF)降低到1.39±0.30(pA／pF)，平均抑制率为65.21％±8.5％(n=5，P＜0.05)，同时对其尾电流的抑制率是30.77％±1.11％(n=5，P＜0.05)。
7．SSYX对I_(Kr)通道的影响：为了更好的观察SSYX对单纯的I_(Kr)电流的影响，我们用HEK293细胞瞬时表达hERG基因来研究药物对I_(Kr)的影响。在转染hERG基因阳性的HEK293细胞上记录到的I_(Kr)自—20mv激活，外向电流随去极化而增大，约在+20mV达到最大值，之后随进一步去极化电流幅值下降，具有内向整流特性。0.5％SSYX对I_(Kr)电流无显著影响。用药前后，在Vt为+20mV时，峰值电流密度分别为18.56±2.47 pA／pF和16.57±3.57 pA／pF(n=5，P＞0.05)。用标化的I_(Kr)尾电流对测试电压作图，得到I_(Kr)的稳态激活曲线，用Boltzmann方程拟合曲线，得到用药前激活V_(1／2)和K分别为-3.78±5.30 mV和8.14±1.26，0.5％药物使用后V_(1／2)和K分别为-5.65±5.29 mV和8.43±1.79(n=5，P＞0.05)。0.5％药物使用前后，在Vt-20mV时I_(Kr)尾电流的失活时间常数分别为16.85±3.78和14.76±1.34(n=5，P＞0.05)，I_(Kr)失活后恢复时间常数为6.29±0.84和5.94±0.50(n=5，P＞0.05)，差异均无显著性。
结论：参松养心胶囊干粉提取溶液对I_(Ca)，L、I_(Na)、I_(to)、I_(Ks)和I_(K1)多种心室肌离子通道均具有不同程度的阻滞作用。SSYX可以抑制豚鼠心室肌细胞I_(Ca)，L的峰值,电流，使通道的时间依赖性激活和失活减慢，并促进通道的电压依赖性失活，使稳态失活曲线的V_(1／2)左移。SSYX可以抑制豚鼠心室肌细胞I_(Na)的峰值电流，并使通道的稳态激活曲线右移。SSYX可以抑制大鼠心室肌细胞I_(to)峰值电流，加速通道时间依赖性失活，并促进通道的电压依赖性失活。SSYX可以抑制大鼠心室肌细胞I_(K1)的内向成分。SSYX可以抑制豚鼠心室肌细胞I_K电流，主要是作用于其慢激活成分I_(Ks)。SSYX对hERG通道的电流幅度，通道失活速度及通道失活后恢复速度无显著影响。SSYX对这些通道的综合作用可使得心肌细胞的除极幅度降低，动作电位时程和有效不应期延长，这可能是其产生抗心律失常作用的机制。同时这种多通道阻滞作用可能降低药物引发心律失常的风险，参松养心胶囊可能有广谱的抗心律失常作用。
Study on the mechanism of antiarrhythmic effects of ShenSongYangXin
Background: Cardiac arrhythmia is a kind of common cardiovascular diseases in the clinical practice, which have great effects on life quality and safety of the patients. People have been continuously devoted to investigation and development of antiarrhythmic drugs in the recent decades. Along with the progress in science and technology, non-pharmacotherapy (such as defibrillation, pacing, radio frequency ablation, surgery and so on) plays more and more roles in clinical treatment, but pharmacotherapy is still the essential way in control cardiac tachy-arrhythmia.
Cardiac arrhythmias result from abnormalities of impulse generation, conduction, or both. It is difficult, however, to illustrate the underlying mechanisms for all the clinical arrhythmias. At present, antiarrhythmic drugs have been classified as sodium channel blockers, [3-receptor blockers, action potential duration prolong agents (mainly potassium channel blockers) and calcium channel blockers. The abnormalities of expression and function of the membrane ion channels can impact on action potential duration, amplify dispersion of repolarization, induce triggered activity, and thus contribute to the development of cardiac arrhythmias. Although the classification of antiarrhythmic drugs are different, modulation the ion channels function is the basic mechanism. Besides the antiarrhythmic efforts, many antiarrhythmic agents have potential risk for proarrhythmia. The adverse side effects of drug may induce polymorphic ventricular tachycardia or torsade de pointes (TdP), which can increase mortality of the cardiovascular diseases. Since CAST and CASTⅡstudy published, it attached more attention to value the benefit risk ratio when antiarrhythmic drugs were applied. People begin to revalue the safety of antiarrhythmic drugs currently used in clinic and to develop new agents with fewer side effects.
Compared to Western medicine, some antiarrhythmic Chinese herbs with many components can multi-target modulate the disorder of ion channel function. It might exert antiarrhythmic efforts with lower incidence of adverse effects. Screening antiarrhythmic Chinese herbs might lead to a new trend in pharmacotherapy.
Shensong Yongxin (SSYX) is one of the compound recipe of Chinese materia medica including 12 ingredients such as Panax ginseng, dwarf lilyturf tuber, Nardostachys root, etc. Previous studies on animal model showed that SSYX significantly inhibited the arrhythmias induced by toxic chemical compounds or ischemia-reperfusion injury. Small random double-blind clinical trials also suggested that SSYX reduced the number of ventricular extra beats in patients with or without structure heart disease. However, the antiarrhythmic mechanisms of SSYX have not been studied.
Objectives: To determine the effects of ShenSongYangXin(SSYX) on L-type calcium channels( I_(Ca), L), sodium channels(I_(Na)), transient outward potassium current (I_(to)), delayed rectifier current (I_K), and inward rectifier potassium currents(I_(K1)) in isolated ventricular myocytes, and on hERG channel(I_(Kr)) in transfected HEK293 cells. To investigate the pharmacological mechanism of SSYX on the ion channels.
Methods: Single ventricular myocytes of guinea pigs and rats were obtained by enzymatic dissociation method. Cultured HEK293 cells were transfected transiently with pcDNA3.1-hERG by a lipofectamine method and green fluorescent protein cDNA (pEGFP-C1) was co-transfected to serve as an indicator. 24～48h after transfection, electrical studis were applied in cells with green fluorescence. Whole cell patch-clamp technique was used to record ion channel currents. I_(Ca,L,) I_(Na,) I_K were studied in ventricular myocytes of guinea pigs; I_(to,)I_(K1) were studied in ventricular myocytes of rats; and I_(Kr) were studied in transfected HEK293 cells.
Results: The action potential and ion currents could be recorded in the isolated ventricular myocytes of guinea pigs and rats, which indicated the myocytes had satisfactory electrophysiological properties. Approximate 40～60 percent transfected HEK293 cells took green fluorescence, in which I_(Kr) could be recorded.
1. The effect of SSYX on action potential. Before and after 0.5% drug application, the rest membrane potential of rat ventricular myocytes were -65.02±5.96mV and -62.67±7.59mV (n＝6,P＞0.05); the action potential amplitude were 101.31±8.78mV and 84.84±5.47mV(n=6, P＜0.05); 50% action potential duration(APD_(50)) were 23.92±5.50ms and 41.99±5.39ms(n=6, P＜0.05); 90% action potential duration(APD_(90)) were 46.97±7.89ms and 65.3±5.84ms(n=6, P＜0.05).
2. The effect of S SYX on I_(Ca,L.) At the test potential of＋10 mV, S SYX inhibited L-type calcium current in a dose-dependent manner. At 0.25%, 0.5%, 1% of concentration, the peak I_(Ca,L) was reduced by 19.22%±1.10 %, 44.82%±6.50 % and 50.69%±5.64%, respectively (n=5, all P＜0.05). In the presence of SSYX 0.5%, the current density-voltage curve was moved up and activation potential, the potential of peak current, and the shape of the I-V curve did not change. The avtivation time constant(τ) was measure as the monoexponential fit to the curret curve induced at＋10 mV, andτincreased from 1.04±0.17ms to 1.52±0.19ms(n=6, P＜0.05) after 0.5% drug application. The steady-state inactivation curve was fitted by a Boltzmann function. It was moved to more negative potential, the half inactivation potential (V_(1/2)) was -15.38±2.4 mV and -20.44.24±5.01 mV(n=6, P＜0.05) in control and SSYX 0.5% respectively. Monoexponential function was best fit the curve of recovery time from inactivation. After 0.5% drug use, the channel recovery time constant increased, from 85.24±20.18ms to 158.42±38.23 ms(n＝5, P＜0.05).
3. The effect of SSYX on I_(Na.) At the test potential of -20 mV, SSYX 0.5% decreased peak I_(Na) by 44.84%±7.65 % from 27.21±5.35 to 14.88±2.75 pA/pF (n=5, P＜0.05). SSYX up shifted the I-V curve of I_(Na) without changing the threshold, peak and reverse potentials. Vt -20mV, before and after SSYX 0.5% application, the time constant of activation were 0.444±0.15 ms and 0.364±0.11 ms (n=5, P＞0.05); the time constant of inactivation were 1.84±0.34ms and 2.054±0.51 ms (n=5, P＞0.05). Before and after SSYX 0.5% use, steady-state activation V_(1/2) were -34.27±2.38mV and -31.40±1.63mV (n=5, P＜0.05); steady-state inactivation V_(1/2) were -73.68±4.80mV and -75.15±4.50mV(n=5, P＞0.05); channel recovery time constant were 19.03±1.13 ms and 21.754±1.48 ms(n=5, P＞0.05).
4. The effect of SSYX on I_(to.) At 0.5% of concentration, the drug blocked the transient component of I_(to) by 50.60 % at membrane voltage of 60mV, from -19.82±7.10(pA/ pF) to -10.02±3.93 ( pA/ pF). SSYX 0.5% could accelerate the inactivation of I_(to,) the time constant were 29.06±4.66ms and 21.4 4±3.12ms (n=6, P＜0. 05). SSYX 0.5% negatively shifted the inactivation curve, the inactivation V_(1/2) were -15.67±2.52 mV and -26.45±3.88 mV (n=7, P＜0.05) respectively in control and in drug group. SSYX 0.5% delayed the channel recovery from inactivation. Before and after drug use, the recovery time constant were 12.86±0.31ms and 18.52±3.76ms (n=5, P＜0.05).
5. The effect of SSYX on I_(K1.) SSYX 0.5% inhibited the I_(K1) from -10.78±1.80 (pA/pF) to -7.18±2.05 (pA/pF)by 33.10 %±16.85 % (n＝11, P＜0.05) at the test potential of-100mV with little effect on reversal potential and the rectification property.
6. The effect of SSYX on/Ks. Holding potential at -40mV and test potential at +50mV last 5s could elicit time dependent increased I_(Ks) in isolated guinea pig ventricular cells. SSYX 0.5% inhibited I_(Ks) from 4.02±0.27(pA/pF) tol.39±0.30(pA/pF), by 65.21%±8.5%(n=5, P＜0.05), and inhibited I_(Ks,tail) by 30.77%±1.11%(n=5, P＜0.05).
7. The effect of SSYX on I_(Kr.) I_(Kr) was recorded in hERG-transfected HEK293 cells. During the depolarizing steps, the outward current was activated at voltages positive to -20mV, and the current amplitude was increased to reach a maximum at +20inV. With further depolarization, the current amplitude decreased progressively, because of the inward rectification. Test potential at +20mV, after 0.5% SSYX use the I_(Kr) amplitude changed from 18.56±2.47 pA/pF to16.57±3.57 pA/pF (n＝5, P＞0.05). Activation curve mearured with I_(Kr,tail) and fitted to a Boltzmann relationship. Before 0.5%SSYX use, the V_(1/2) and K were -3.78±5.30 mV and 8.14±1.26, and after drug use the V_(1/2) and K were -5.65±5.29 mV and 8.43±1.79 (n=5,P＞0.05). Test potential at -20mV, before and after 0.5% SSYX use, the I_(Kr,tail) inactivation time constant were 16.85±3.78 and 14.76±1.34 (n=5, P＞0.05); the recovery time constant from inactivation were 6.29±0.84 and 5.94±0.50 (n=5, P＞0.05).
Conclusions: It reveals that SSYX could block multiple ion channels includeI_(Ca,L) I_(Na,) I_(to,) I_(K1) and I_(Ks.) SSYX could inhibit I_(Ca,L) peak current, slow down time dependent activation and inactivation, and promote channel voltage dependent inactivation. SSYX could decrease I_(Na) peak current, and right shift the steady-state activation curve. SSYX could depress I_(to) peak current, promote channel time dependent and voltage dependent inactivation. SSYX could inhibit the inward current component of I_(K1.) SSYX could inhibit the slowly-activating component of I_K on guinea pig ventricular myocytes. SSYX had few effects on I_(Kr) current amplitude, time dependent inactivation and recovery from inactivation. The integrated effects of SSYX may change the action potential duration and contribute to some of its antiarrhythmic effects. Moreover, the block effect on multiple ion channels would be beneficial to reduce proarrhythmic side effects. SSYX may be expected to have a wild spectrum of antiarrhythmic effect with less proarrhythmic potential.
引文
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