| Objective:To investigate the effects of orientin(Ori)on the main ion channel currents(INa,Ito,ICa-L)and dynamic characteristics of rat ventricular myocytes,and to explore the mechanism of its protective effect on rat arrhythmiaMethods:Separation of single,free rat ventricular myocytes by retrograde perfusion enzymatic hydrolysis in vitro;whole-cell patch-clamp technique was used to guide and record the current;the extracellular fluid drug perfusion was used to observe the effects of different dose groups of Ori before and after administration on the current and dynamic characteristics of each ion channel in rat ventricular myocytesResults:1.Effects of Ori on sodium channel current(INa)and its dynamic characteristics in rat ventricular myocytes(1)Effect of Ori on INaWe used successfully isolated rat ventricular myocytes to investigate whether Ori can affect INa on cells.The results showed that when we added Ori to the experimental system at a final concentration of 3 μM,INa-Peak changed from(-81.49±3.92)pA/pF before administration to(-69.07±3.72)pA/pF(P<0.01,n=6),which shows that Ori has a significant inhibitory effect on INa(2)Concentration dependence of Ori on INaCumulative dosing was used to observe the effects of Ori at 1,2,3,10 and 30 μM on INa in rat ventricular myocytes.The results showed that Ori at 1 and 2 μM caused the peak sodium current(INa-Peak)to change from(-81.49±3.92)pA/pF before administration to(-80.66±4.51)pA/pFand(-80.05±6.27)pA/pF(P>0.05,n=6),there is no statistically significant change;but as the Ori concentration increased to 3,10 and 30 μM,INa-Peak became(-69.07±3.72)pA/pF,(-63.05±2.85)pA/pF and(-55.35±3.24)pA/pF(P<0.01,n=6).It can be seen that the inhibitory effect of the drug on INa gradually increases with the increase of Ori concentration,showing a significant concentration dependence(3)Effects of Ori on the current density-voltage relationship(I-V)curve of INa3,10 and 30 μM of Ori can make the I-V curve of INa move up obviously in turn,but the trajectory change trend and inversion potential of the I-V curve have not changed.(4)Effects of Ori on the activation curve of INaOri shifts the activation curve of INa in the direction of depolarization(right).Among them,Ori at 3,10,and 30 μM changed the half activation potential from(-53.66±4.12)mV before administration to(-44.64±1.96)mV,(-38.95±1.73)mV,and(-30.21±1.55)mV(P<0.01,n=6),that is,Ori can significantly increase the activation difficulty of INa(5)Effects of Ori on the inactivation curve of INaOri shifts the inactivation curve of INa to the hyperpolarization direction(left).Ori at 3,10 and 30 μM reduced the half inactivation potential from(-51.68±0.76)mV before administration to(-60.1 7± 1.56)mV and(-68.51 ± 1.43)mV and(-75.22±1.37)mV(P<0.01,n=6),k changed from(5.26±0.58)to(7.99± 1.44),(10.33± 1.35)and(13.03±1.23)(P<0.01,n=6).Therefore,Ori can significantly accelerate the inactivation of INa.(6)Effects of Ori on recovery curve after inactivation of INaOri significantly shifted the recovery curve after INa inactivation to the right.Among them,Ori at 3,10,and 30 μM caused the recovery time(τ)value to change from(18.38±0.84)ms before administration to(24.53± 1.46)ms,(35.25±1.32)ms and(68.75±1.58)ms(P<0.01,n=6).This shows that with the increase of the dose of Ori,the drug gradually prolongs the time for INa to recover from the inactive state to the active state.2.Effects of Ori on transient outward potassium current(Ito)and its dynamic characteristics in rat ventricular myocytes(1)Effect of Ori on ItoWe used successfully isolated rat ventricular myocytes to investigate whether Ori can affect Ito on cells.The results showed that when we added Ori to the experimental system with a final concentration of 3 μM,Ito changed from(45.60±1.73)pA/pF before administration to(32.12±0.98)pA/pF(P<0.01,n=6),thus demonstrating:Ori has a significant inhibitory effect on Ito.(2)Concentration dependence of Ori on ItoCumulative dosing was used to observe the effects of Ori at 1,2,3,10 and 30 μM on ventricular myocyte Ito in rats.The results showed that Ori at 1 and 2 μM changed the peak Ito current from(45.60±1.73)pA/pF before administration to(43.79±3.76)pA/pF and(40.98±5.13)pA/pF(P>0.05,n=6).The amplitude was not statistically significant;but as the Ori concentration increased to 3,10,and 30 μM,the peak Ito current became(32.12±0.98)pA/pF,(25.84±2.14)pA/pF and(18.93± 1.57)pA/pF(P<0.01,n=6).It can be seen that the inhibitory effect of the drug on Ito gradually increases with the increase of Ori concentration,showing a significant concentration dependence(3)Effects of Ori on the current-voltage(Ⅰ-Ⅴ)curve of ItoAt 3,10 and 30 μM,the Ⅰ-Ⅴ curve of Ito can be shifted down significantly in order to suppress the Lto current density without changing the shape of the Ⅰ-Ⅴ curve(4)Effects of Ori on the activation curve of It.Ori shifts the activation curve of Ito towards the direction of depolarization(right).Among them,3,10 and 30 μM of Ori caused the half activation potential from(18.67±2.31)mV before administration to(23.11±3.46)mV,(28.84±2.86)mV and(39.88±3.17)mV(P<0.01,n=6),that is,Ori can significantly increase the difficulty of Ito activation.(5)Effects of Ori on the inactivation curve of ItoOri shifts the deactivation curve of Ito towards the direction of hyperpolarization(left)Among them,3,10 and 30 μM of Ori reduced the half inactivation potential from(-22.06±0.79)mV before administration to(-35.6 7±0.37)mV and(-52.12± 1.14)mV and(-63.65± 1.13)mV(P<0.01,n=6),k changed from(13.14±0.23)to(17.31 ±0.48),(19.46±0.21)and(20.73±0.79)(P<0.01,n=6).Therefore,Ori can significantly accelerate the inactivation of Ito(6)Effects of Ori on recovery curve after inactivation of It.Ori obviously shifted the recovery curve after inactivation of Ito.The Ori of 3,10,and 30μM caused the recovery time(τ)values to change from(77.93±3.76)ms before administration to(122.07±2.87)ms,(173.60± 1.99)ms,and(263.00±3.82)ms(P<0.01,n=6).This shows that with the increase of the dose of Ori,the drug gradually prolongs the time for Ito to recover from the inactive state to the active state.3.Effects of Ori on L-type calcium channel current(Ica-L)and its dynamic characteristics in rat ventricular myocytes(1)Effect of Ori on Ica-LWe used successfully isolated rat ventricular myocytes to investigate whether Ori can affect Ica-L on the cells.The results showed that when we added Ori to the experimental system at a final concentration of 10 μM,the peak Ica-L current changed from(-22.45±4.87)pA/pF before administration to(-15.74±2.37)pA/pF(P<0.01,n=6),This shows that Ori has a significant inhibitory effect on ICa-L(2)Concentration dependence of Ori on ICa-LCumulative dosing was used to observe the effects of Ori at 1,2,3,10,30 and 100μM on ICa-L in rat ventricular myocytes.The results showed that Ori at 1,2 and 3 μM caused Ica-L peak current to change from(-22.45±4.87)pA/pF before administration to(-21.14±3.49)pA/pF,(-20.66±4.73)pA/pF and(-19.87±4.56)pA/pF(P>0.05,n=6),the change amplitude was not statistically significant,but as the Ori concentration increased to 10,30,and 100 μM,the peak ICa-L current became(-15.74±2.37)pA/pF,(-13.16± 1.83)pA/pF and(-11.67±0.56)pA/pF(P<0.01,n=6).It can be seen that the inhibitory effect of the drug on ICa-L gradually increases with the increase of Ori concentration,showing a significant concentration dependence.(3)Effects of Ori on the current-voltage(Ⅰ-Ⅴ)curve of Ica-LOri at 10,30 and 100 μM can significantly shift the I-V curve of ICa-L.in turn,suppressing the current density of ICa-L without changing the shape of the I-V curve(4)Effects of Ori on the activation curve of ICa-LOri shifts the activation curve of ICa-L towards the direction of depolarization(right).Among them,Ori at 10,30,and 100 μM changed the half activation potential from(-16.50±2.37)mV to(-6.61 ±2.16)mV,(3.32±0.65)mV,and(11.86±3.28)mV(P<0.01,n=6),that is,Ori can change the dynamic characteristics of ICa-L.activation and delay Channel activation.(5)Effects of Ori on the inactivation curve of ICa-L.Ori shifts the deactivation curve of ICa-L towards the direction of hyperpolarization(left)Among them,Ori at 10,30,and 100 μM reduced the half-inactivation potential from(-11.22±1.92)mV to:(-30.12±2.31)mV,(-41.31 ±3.04)mV,and(-65.54±5.75)mV(P<0.01,n=6),that is,Ori can change the mechanical properties of ICa-L inactivation and accelerate the inactivation of calcium channels(6)Effects of Ori on recovery curve after inactivation of ICa-LOri significantly shifted the recovery curve after inactivation of ICa-L.Among them,Ori at 10,30,and 100 μM increased the recovery time τ from(16.48±0.76)ms before administration to(20.39±2.63)ms,(27.04±4.18)ms and(39.08±4.36)ms(P<0.01,n=6).,that is,Ori can change the recovery kinetic characteristics after ICa-L inactivation,and lengthen the transition time from inactivation state to activation state in a concentration-dependent manner.Conclusion:Ori has a concentration-dependent inhibitory effect on INa,Ito,and ICa-L on rat ventricular myocytes,and can change the channel dynamics.This result maybe related to Ori’s arrhythmia protection. |
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