| Objective:To investigate the effect of Nobiletin(NOB)on aconitine-induced ventricular arrhythmias in rats,and to explore the effect of NOB on the kinetic characteristics of the main ion channel currents(INa,Ito,ICa-L)on rat cardiomyocytes from the perspective of electrophysiological as well as molecular docking,and to elucidate its anti-arrhythmic mechanism.Methods:1.A rat ventricular arrhythmia model was established by tail vein injection of aconitine,and the effect of NOB on the arrhythmia of this model was evaluated.2.The toxicity of NOB to mammary rat cardiomyocytes was measured using the MTT method to determine the safe range.3.Aortic reverse perfusion was performed in isolated rat hearts using the langendorff apparatus,and type Ⅱ collagenase with gradient calcium replication was used to isolate calcium-resistant individual cardiomyocytes.4.Whole-cell patch clamp techniques were used to study changes in the current of each ion channel before and after drug administration and to assess the effect of NOB on the kinetic characteristics of each channel.5.Using molecular docking techniques,the effect of NOB on the structure of Nav1.5 channel proteins(including the inactivated state)was calculated and predicted to explain the structural biological basis of drug effects.Results:1.Effect of NOB on aconitine-induced arrhythmias in SD ratsWe successfully established a ventricular arrhythmia model in rats by tail vein injection of aconitine to determine whether the different doses of NOB have anti-ventricular arrhythmic effects by the prophylactic administration.The results showed that NOB pretreatment at 5,10 and 20 mg/kg delayed the onset of aconitine-induced ventricular premature from(6.53±0.60)min to(9.57±0.59)min,(10.46±0.81)min and(11.58±0.55)min(n=10,p<0.05),respectively;ventricular tachycardia was sequentially delayed from(7.74±0.72)min to(18.55±1.25)min,(20.31±1.84)min,(21.46±1.53)min(n=10,p<0.01),before drug administration;the incidence of fatal ventricular fibrillation was reduced from 90%to 20%,10%and 10%(n=10,p<0.01)before drug administration.It is clear that NOB has an anti-aconitine-induced arrhythmia effect,and this effect is similar to that of the positive control group(amiodarone treatment).2.Effect of different concentrations of NOB on cardiac cell viability in SD mammary ratsThe effect of relevant concentration gradients of NOB on the activity of primary cultured SD mammary rat cardiomyocytes was examined by MTT method.The results indicated that the administration of 5,10,25,50,100 and 200 μmol/L of NOB to the culture medium of breast rat cardiomyocytes cultured for 24 h decreased the cell activity by(0.99±3.17)%,(5.43±5.38)%,(6.91±5.38)%(9.63±3.64)%,(16.30±1.36)%(n=6,p>0.05),(17.04±3.80)%(n=6,p<0.05).It can be concluded that ambient NOB concentrations below 200 μmol/L had no significant effect on cardiomyocyte viability.Therefore,the concentration of NOB for subsequent patch clamp experiments could be within 200 μmol/L.3.Effects of different concentrations of NOB on various ion channels in rat ventricular myocytes(1)Effect of NOB on voltage-gated sodium channels in rat ventricular myocytesThe voltage-gated sodium channel current INa was guided and recorded via using a whole-cell patch clamp technique,and the changes in this current at each dose of NOB were observed by the self-control method before and after administration.The results revealed that 25,50 and 100 μmol/L of NOB decreased the peak sodium current from(-87.79±4.21)pA/pF before administration to(-61.87±2.54)pA/pF,(-50.88±5.41)pA/pF and(-38.90±3.21)pA/pF,respectively(n=10,p<0.01).The inhibition rates were(29.53±2.90)%,(42.04±6.16)%and(55.69±3.66)%(n=10,p<0.01),respectively,with a significant concentration-dependent inhibition.The current-voltage(I-V)relationship curve of Ira was affected by different concentrations of NOB,and the curve shifted significantly upwards close to the baseline as the concentration administered increased,while the trend of the trace and the peak potential were largely unaffected;NOB at 25,50 and 100 μmol/L shifted the activation fitted curves significantly in the direction of depolarization along the positive axis,and the half-activation voltage(V1/2-ac)changed sequentially from(-62.00±0.53)mV before administration to(-56.17±0.44)mV,(-53.77±0.29)mV,and(49.89±0.18)mV(n=7,p<0.01);25,50,and 100 μmol/L NOB shifted the inactivation fitted curves in the hyperpolarization direction,and the half inactivation voltage(V 1/2-in)changed sequentially from(-75.00±0.57)mV before administration to(-79.59±0.44)mV,(-82.72±0.64)mV and(-87.94±0.60)mV(n=7 p<0.05),and the slope factor κ changed sequentially from(8.03±0.53)before administration to(8.55 ±0.56),(9.23±0.47),and(9.39±0.39)(n=7,p<0.05);Moreover,NOB at 25,50,and 100 μmol/L shifted the recovery curve along the time axis to the right after sodium channel inactivation,which prolonged the recovery time τ from(21.51±0.95)ms before administration to(26.75±0.94)ms,(29.16±0.34)ms,(32.04±0.78)ms(n=7,p<0.01).This suggests that NOB blocks voltage-gated sodium channels from the inactivated state into the resting state,raising the threshold for channel opening and prolonging the inactivated state of the channel.(2)Effect of NOB on the transient outward potassium channel current(Ito)and its kinetic characteristics in rat ventricular myocytesThe voltage clamp mode with the corresponding stimulation protocol was used to guide and record the changes in Ito at each dose of NOB.The results showed that NOB at 10,25,50 and 100μmol/L decreased the peak current density of Ito in rat ventricular myocytes from(66.12±5.88)pA/pF before administration to(61.03±5.13)pA/pF,(54.60±4.92)pA/pF(n=6,p>0.05),(52.17±2.00)pA/pF(n=6,p<0.05),(33.18±5.99)pA/pF(n=6,p<0.01),the inhibition effect showed concentration dependence.In subsequent experiments,both 50 and 100μmol/L affected the current-voltage(I-V)relationship curves of Ito,and the curves shifted significantly downward close to the baseline with increasing dosing administration,while the trend of trace and the peak potential were largely unaffected;NOB at 50 and 100μmol/L shifted the activation fitting curve of Ito to the right in the positive direction of the voltage axis,and the half-activation voltage(V1/2-ac)changed sequentially from(4.94±0.45)mV before administration to(10.61±0.63)mV and(27.64±0.67)mV(n=6,p<0.01);50 and 100 μmol/L of NOB shifted the inactivation fit curve significantly to the left,with the half deactivation voltage(V1/2-in)decreasing sequentially from(-25.02±1.02)mV before administration to(-29.80±0.61)mV,(-35.47±0.54)mV(n=6,p<0.01),and the slope factor κ changing sequentially from(4.50±0.60)before administration to(4.73±0.84),(5.34±0.47)before administration(n=6,p>0.05);50 and 100μmol/L NOB also shifted the post-inactivation recovery curve of Ito to the right along the time axis,prolonging the recovery time τ from(12.75±2.81)ms before administration to(16.58±6.63)ms,(25.77±2.56)ms(n=6,p<0.01).This suggests that NOB blocked the transient outward potassium channel from the inactivated state into the resting state,prolonged the inactivation of the channel and increased the ease of opening the transient outward potassium channel.(3)Effect of NOB on L-type calcium channel(ICa-L)and their kinetic characteristics in rat ventricular myocytesThe changes in ICa-L were recorded at each dose of NOB using the voltage clamp mode with the corresponding stimulation protocol.10,25,50,and 100 μmol/L of NOB sequentially decreased the peak current density of ICa-L from(29.76±5.41)pA/pF before administration to(-27.03±4.87)pA/pF,(-25.53±4.35)pA/pF,(-25.19±3.39)pA/pF(n=6,p>0.05),and(-21.80±4.01)pA/pF(n=6,p<0.05).NOB had an inhibitory effect on ICa-L,and this effect showed concentration dependence.NOB at 50 and 100 μmol/L affects the current-voltage(I-V)relationship curve of ICa-L and shifts significantly upward with the curve close to the baseline,while the trend of the trace and the peak potential are largely unaffected;50,100 μmol/L NOB shifted the activation fitted curve of ICa-L in the positive direction of the voltage axis,with the half activation voltage(V 1/2-ac)changing sequentially from(-31.19±2.07)mV before administration to(-21.50±2.64)mV,(-7.28±2.72)mV(n=6,p<0.01);NOB at 50 and 100 μmol/L shifted the inactivation fitting curve in the direction of hyperpolarization of the voltage axis,and the half inactivation voltage(V 1/2-in)changed sequentially from(-20.90±0.80)mV before administration to(-34.00±1.50)mV,(-47.90±1.28)mV(n=6,p<0.01),and the slope factor κ changed from(9.27±1.14)sequentially to(9.66±0.71),(10.55±1.51)(n=6,p>0.05);In addition,NOB at 50 and 100 μmol/L shifted the post-inactivation recovery curve of ICa-L to the right along the time axis and prolonged the recovery time τ from(18.86±0.85)ms to(33.21±2.06)ms and(42.56±3.92)ms(n=6,p<0.01)before administration,respectively.The results indicate that NOB blocked L-type calcium channels from the inactivated state into the resting state,increased the threshold for channel opening and prolonged the inactivated state of the channels.4.Results of NOB and Nav1.5 molecular docking calculationsThe results of molecular docking showed that the binding energies of NOB to rNav1.5,Nav1.5(QQQ)and hNav1.5 were-6.655,-6.562 and-5.693 kcal/mol,respectively,and the binding energies of MM-GBSA were-36.44,-29.32 and-51.71 kcal/mol,showing that NOB has good binding potential to the voltage-gated sodium channel-associated protein Nav1.5.The protein-ligand interaction profile showed that NOB binds to a phenylalanine residue at position 1762(Phe-1762)with a π-π stacking and to a serine at position 1712 with a hydrogen bond.Conclusion:NOB pretreatment had an anti-aconitine-induced effect on the development of ventricular arrhythmias in rats,which was associated with inhibition of INa,Ito,ICa-L and alteration of their channel kinetic properties. |
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