The Pharmacodynamic Effect Of ALO On Aconitine-induced Arrhythmia In Rats And Its Mechanism

Objective:To investigate the effects of aloperine（ALO）on aconitine-induced arrhythmias in rats and the effects of ALO on major ion channel currents（INa,Ito,ICa-L）and the gating kinetic characteristics on rat cardiomyocytes from an electrophysiological aspect,and to clarify its anti-arrhythmic mechanism.Methods:1.To observe the in vivo effects of ALO on aconitine-induced arrhythmias in rats by detecting changes in standard limb Ⅱ lead electrocardiograms（ECGs）.2.To detect the toxicity of ALO on neonatal rat cardiomyocytes by MTT method and to calculate the IC50 value.3.Single cardiomyocytes were isolated from adult SD rats by single enzymatic digestion using a modified Langendorff apparatus for reverse aortic perfusion of isolated hearts.4.Using whole-cell membrane clamp technique,the effects of ALO before and after administration on INa,Ito,and ICa-L on rat cardiomyocytes were recorded in voltage-clamp mode to observe their effects on the kinetic characteristics of various corresponding ion channel currents.Results:2.Effect of different concentrations of ALO on the ion channel current in rat cardiomyocyte（1）Effect of ALO on voltage-gated sodium channel current（INa）in rat cardiomyocyteTo explore whether ALO had an inhibitory effect on sodium channel in rat cardiomyocyte,we first tested 30 μM ALO and found that ALO had an inhibitory effect on INa.We then set a series of concentration gradients（1 μm,3 μM,30 μM,10μM,30 μM,100 μM）based on 30 μM.We found that ALO showed a concentration-dependent inhibitory effect on INa when the concentration was at 3 μM and above.In order to prevent the occurrence of arrhythmia caused by excessive inhibition of 100 μM ALO,3,10 and 30 μM ALO was next used for follow-up studies.In the experiments,we observed that 3,10 and 30 μM ALO made the inward current of INa decrease and shifted the current-voltage relationship（I-V）curve significantly upward,but did not affect the curve shape.When the concentration increased to 30μM,ALO could increase the activation threshold of Na channel,thereby increasing the difficulty of Na channel opening,and accelerating the inactivation process of Na channel in a concentration-dependent manner.Furthermore,10 and 30 μM ALO prolong the transition of sodium channel from the inactive state to the resting state.（2）Effect of ALO on transient outward potassium channel current（Ito）in rat cardiomyocyteTo explore whether ALO had an inhibitory effect on transient outward potassium channel in rat cardiomyocyte,we first tested 30 μM ALO,after observing that it had no effect on Ito,we reduced a concentration gradient to test 10 μM ALO,and found that ALO had a significant inhibitory effect on Ito.We then set a series of concentration gradients based on 10 μM,including 1 μM,3 μM,10 μM,30 μM,100μM.The results found that:3 and 10 μM ALO had an inhibitory effect on Ito,but when the dose was increased,displayed an activated effect,showing the bidirectional effect of ALO on Ito.Based on the consideration of maintaining consistency with the above doses,we selected ALO at 3 and 10 μM concentrations for follow-up studies.In the study of the Ⅰ-Ⅴ curves,we found that the 3 and 10 μM ALO shifted the Ⅰ-Ⅴcurve down,but the curve shape was unchanged,and the current density increased significantly with the raise of membrane potential.ALO could slow down the activation process,inhibit the opening degree of the transient outward potassium channel,reduce the threshold of inactivation,accelerate the inactivation process of transient outward potassium channel,and prolong the transition process from the inactivation state to the resting state.（3）Effect of ALO on L-type calcium channel current（Ica-L）in rat cardiomyocyteTo explore whether ALO had an inhibitory effect on the L-type calcium channel in rat cardiomyocyte,we first tested 30 μM ALO,and found that ALO had a significant inhibition effect on Ica-L.Based on the premise,a series of concentration gradients（1 μM,3 μM,10 μM,30 μM,100 μM）was set,and the concentration-dependent inhibitory effect of ALO on ICa-L was observed.Further study of the Ⅰ-Ⅴ curve found that ALO significantly shifted the Ⅰ-Ⅴ relationship curve of calcium channel,but did not change the shape of the curve.In gating kinetic experiments,ALO increased the activation threshold of calcium channels,slowing down the activation process,reduced the inactivation threshold,accelerating the inactivation process of transient outward potassium channels,and extended the recovery time after the inactivation,thus prolonging the time from inactive state to resting state.Conclusion:ALO can confront aconitine-induced ventricular arrhythmias,the mechanism may be related to the inhibition of INa,Ito,ICa-L and changes in its channel dynamics.