异牡荆素对大鼠心室肌细胞瞬时外向钾电流的影响

Effects of isovitexin on transient outward potassium current of ventricular myocytes in rats

探究异牡荆素对大鼠心室肌细胞瞬时外向钾通道电流的影响。该实验采用MTT检测法探究异牡荆素的安全范围,结果显示该药物的IC5010~30μmol·L^(-1),而膜片钳实验所选用的药物浓度1~3μmol·L^(-1)均在该安全范围之内。此外,采用主动脉逆行灌流单酶解法得到单个心室肌细胞,应用全细胞膜片钳技术引导、记录大鼠心室肌细胞瞬时外向钾电流(Ito)并分析在给予异牡荆素(IV)前后该电流特征的变化。当IV终浓度低于0.1μmol·L^(-1)时,其对Ito无明显影响,但随着浓度的升高(≥0.3μmol·L^(-1)),Ito峰值逐渐降低[由给药前的(32.32±2.9)p A/p F分别降为(25.83±4.3)p A/p F,1μmol·L^(-1)IV和(19.51±3.5)p A/p F,3μmol·L^(-1)IV],呈现出浓度依赖性抑制现象。在1~3μmol·L^(-1)浓度内,IV使Ito的I-V曲线明显下移。激活曲线结果显示,IV可使最大半数激活电位(V1/2)向正值方向移动[给药前V1/2为(19.59±1.6)m V,给药后V1/2分别升高(22.81±1.7)m V,1μmol·L^(-1)IV;(28.86±1.4)m V,3μmol·L^(-1)IV],同时激活曲线右移。Ito稳态失活曲线的最大半数失活电位(V1/2):给药组(-61.81±1.3)m V,1μmol·L^(-1)和(-71.50±1.4)m V,3μmol·L^(-1)较给药前(-51.43±0.99)m V显著降低。而就失活后恢复曲线的失活时间常数(τ)而言,给药后的τ较给药前明显升高[给药前(94.89±0.73)ms;给药后(118.5±1.5)ms,1μmol·L^(-1);(162.4±1.4)ms,3μmol·L^(-1)],IV使Ito的失活后恢复曲线右移,提示其能延长瞬时外向钾通道的失活后恢复时间。异牡荆素对大鼠心室肌细胞的Ito具有明显的抑制作用。

To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC50 of the drug was in the range of 10-30 μmol·L^-1, and the drug concentration of 1-3 μmol·L^-1 for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current （Ito） of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied. When the concentration of IV was less than 0.1 μmol·L^-1, there was no significant effect on Ito. However, with the increase in the concentration of IV （≥ 0.3 μmol·L^-1）, the peak of Ito was decreased gradually, from （32.32±2.9） pA/pF to （25.83±4.3） pA/pF, 1 μmol·L^-1 IV and （19.51±3.5） pA/pF, 3 μmol·L^-1 IV respectively, with an inhibition effect in a concentration-dependent manner. In the range of 1-3 μmol·L^-1, IV down-regulated the I-V curve of Ito significantly. The activation curve showed that IV can enable the maximum half activation potential （V1/2） to move to the positive direction, and the V1/2 was increased from （19.59±1.6） mV to （22.81±1.7） mV and （28.86±1.4） mV at concentration of 1, 3 μmol·L^-1, meanwhile the activation curve moved to the right. However, the maximum half inactivating potential （V1/2） of the steady-state inactivation curve of Ito was significantly decreased from （-51.43±0.99） mV to （-61.81±1.3） mV with concentration of 1 μmol·L^-1 and （-71.50±1.4） mV with concentration of 3 μmol·L^-1. The inactivation time constant of recovery from inactivation （τ） was up-regulated significantly from （94.89±0.73） ms to （118.5±1.5） ms and （162.4±1.4） ms at concentration of 1, 3 μmol·L^-1respectively. Meanwhile IV could enable the inactivation recovery curve to move to the right, which suggested that it can prolong the recovery time from inactivation of the transient outward potassium channel. In conclusion, isovitexin had a high inhibitory effect on Ito in rat ventricular myocytes.