| Objective:To investigate the effects of Isovitexin(?)on the several major ion channel currents(Ito,INa,ICa-L)and their kinetic characteristics in rat ventricular myocytes.Methods:MTT assay was used to detect the effect of ? on cell viability.Retrograde perfusion of the aorta to separate adult rat cardiomyocytes by enzymatic hydrolysis.The whole-cell patch clamp technique was used to record and guide current.The changes of channel current and its kinetic characteristics before and after ? administration were observed by cumulative administration.Results:1.Effects of different concentrations of ? on the cardiomyocyte viability in neonatal ratsThe effect of ? on cell viability was measured by MTT assay.The results showed that at 24 h after administration,the low concentration of ?(1?3 ?M)had no significant effect on cell viability[the control group,(99.13±5.38)%;group IV,1 ?M(86.77±3.99)%,3 ?M(81.47±3.95)%(P>0.05,n=5)].When the concentration of ? was 10 ?M,the cell viability decreased gradually[(66.69±4.53)%(P<0.05,n=5)].However,high concentrations of IV(?30 ?M)significantly inhibited cell proliferation[30 ?M(48.62±2.44)%(P<0.01,n=5)].The results showed that IV had a significant effect on cell viability with the increase of dosing time and concentration[control,(99.93±1.64)%vs drug group,1?M(77.2±4.00)%,3 ?M(68.11±3.76)%,10 ?M(59.31±4.93)%and 30 ?M(43.11±2.73)%(P<0.01,n=5)].The IC50 of IV was 27.60?M and 22.43 ?M after 24 h and 48 h culture.2.Effects of different concentrations of IV on the current of ion channel(1)Effect of IV on transient outward potassium current(Ito)in rat ventricular myocytesThe whole-cell patch clamp technique was used to record and guide Ito,and to observe the effect of different doses of IV on the current.The results showed that the Ito was not significantly suppressed by ? in the low dose(?0.1 ?M).Whereas,with the increase of drug concentration(?0.3 ?M),the peak value of Ito decreased gradually.Which was reduced from(42.32±2.93)pA/pF to(31.83±4.30)pA/pF 1?M IV,(25.51±3.48)pA/pF 3 ?M IV and(16.35±2.50)pA/pF 10?MIV(P<0.01,n=6),and the inhibition was a concentration-dependent manner.And with the increase of IV concentration(?3 ?M),IV significantly shifts the ?-? curve of Ito.The activation curve showed that IV can affect the maximum half activation potential(V1/2)and move to the positive direction,and the V1/2 was increased from(19.59±1.60)mV to(22.81 ± 1.66)mV(P>0.05,n=6),(28.86±1.41)mV and(36.29±0.69)mV(P<0.01,n=6)at concentration of 1?M,3?M and 10 ?M.However,the maximum half inactivating potential(V1/2)of Ito had the opposite change,which was decreased from(-27.02±0.79)mV to(-44.71±0.71)mV 1 ?M,(-58.81±1.23)mV 3 ?M and(-70.11±1.13)mV 10?M(P<0.01,n=6).The inactivation recovery time of recovery curve after inactivation(?)was up-regulated significantly from(90.77±1.02)ms in control to(118.50±1.51)ms,(162.40±1.42)ms and(227.80±0.73)ms(P<0.01,n=6)at concentration of 1 ?M,3 ?M and 10 ?M.Meanwhile,the recovery from inactivation curve was shifted to the right,which suggested that it can prolong the recovery from inactivation time of the transient outward potassium channel.(2)Effect of IV on sodium channel current(INa)in rat ventricular myocytes With the increase of drug concentration,the inhibitory effect of IV on INa was gradually enhanced.The current density of INa changed from(-99.28±2.63)pA/pF to(-79.29±4.30)pA/pF,(-65.56±2.58)pA/pF and(-58.88±5.02)pA/pF(P<0.01,n=6),respectively,and the ?-? curve is obviously shifted up at concentration of 1 ?M,3?M and 10 ?M.Compared with pre-dose,the activation curve V1/2 was changed from(-15.6±0.42)mV to(-7.28±0.43)mV,(1.24±0.48)mV and(11.55±0.44)mV(P<0.01,n=6)at concentration of 1 ?M,3 ?M,and 10 ?M.The inactivation curve showed that with the increase of drug concentration,the inactivation curve shifted significantly to the left,and its V1/2 gradually migrated to the direction of hyperpolarization[control,(-74.98±0.52)mV;1V,(-81.15±0.34)mV 1 ?M(P>0.05,n=6),(-93.27±0.55)mV 3 ?M and(-102.6±0.26)mV 10 ?M(P<0.01,n=6)].At the same time,the recovery curve of INa was shifted to the right after administration.The recovery time after inactivation(?)was also significantly longer.[control,(8.16±0.45)ms,1 ?M(13.49±2.01)ms,3 ?M(17.65±0.89)ms and 10?M(22.27±0.52)ms(P<0.01,n=6)].(3)Effect of ? on L-type Ca2+ current(ICa-L)in rat ventricular myocytes Compared with the pre-dose current density(-23.69±2.34)pA/pF,the peak current density of ICa-L gradually decreased with the increase of drug concentration[1?M(-18.14±2.10)pA/pF,3?M(-12.81 ±1.97)pA/pF and 10 ?M(-6.61±1.30)pA/pF(P<0.01,n=6)].However,there was no significant difference in the current density of Ica-L after washout compared with control.For the activation curve of L-type calcium channel,different concentrations of IV increased V1/2 of ICa-L from(-15.18±0.30)mV before treatment to(-6.03±0.43)mV 1 ?M,(1.98±0.39)mV 3 ?M and(0.69±0.71)mV 10 ?M(P<0.01,n=6).Meanwhile,the inactivation curve of ICa-L shifted significantly to the left after administration.And moving in the negative direction of the membrane potential.The ? reduced the V1/2 of ICa-L from(-17.56±0.93)mV to(-28.03±0.72)mV 1 ?M,(-43.31 ± 0.82)mV 3 ?M,and(-53.10±0.63)mV 10 ?M(P<0.01,n=6).The recovery curve after inactivation of ICa-L increases with the increasing of drug concentration[control,?=(13.45±0.36)ms;1 ?M ? ?=(18.36±0.61)ms,3 ?M ? ?=(22.64±0.18)ms,10 ?M ? ?=(26.69±0.57)ms(P<0.01,n=6)].3.Effects of different solvents on INa in rat ventricular myocytesThe effect of different concentrations of solvents(methanol and DMSO)on INa was measured using the sodium current as a reference index.The results showed that when the content of methanol and DMSO was 0.1%,there was no obviously effect on INa.However,with the increase of methanol content in extracellular solution,it has inhibitory effect on INa.In addition,it was found that there was no obviously change in the experimental results when two different solvents(the content in the extracellular fluid was 0.1%)were used to dissolve the drugs.Conclusion:IV has different degrees of inhibition on several major ion channels(Ito,INa and ICa-L)on rat cardiomyocytes,and this effect may be related to the myocardial protective effect of IV. |
PDF Full Text Request