【作者】 柳强妮；

【导师】 曾繁典；

【作者基本信息】 华中科技大学 ， 药理学， 2010， 博士

【摘要】 目的：蝙蝠葛碱(dauricine,Dau)和蝙蝠葛苏林碱(daurisoline,DS)是从中药蝙蝠葛(Menisperum dauricum,DC)根茎中提取的双苄基异喹林类生物碱。近年来,研究发现Dau和DS均具显著的抗实验性心律失常作用。长QT综合征(Long QT syndrome,LQTs)的基础是心肌细胞复极延缓。正常心肌细胞的动作电位复极时程和形态受外向电流和内向电流的影响。外向电流主要为快速激活延迟整流钾电流(rapidly activated delayed recitifying potassium current,IKr)和缓慢激活延迟整流钾电流(slowly activated delayed rectifying potassium current,IKs).内向电流主要为钠电流(sodium current,INa).L型钙通道电流(L-type calciumcurrent,ICa-L)。本研究采用膜片钳和Western blotting技术,研究Dau和DS对与获得性LQTs相关离子通道的影响规律,并在离子通道和蛋白水平探讨Dau和DS抗心律失常的作用机制。方法：1.采用酶消化法分离家兔左心室单个心肌细胞,用膜片钳技术,在电压钳制模式下记录单个细胞IK1、IKr、IKs和Ito,研究Dau和DS对心室肌细胞IK1、IKr、IKs和Ito的影响。2.用膜片钳技术记录在人胚肾细胞(HEK293)表达的hERG通道电流,研究Dau和DS对hERG通道电生理特性的影响,并与Quin和AM相比较。3.用Western blotting方法研究Dau和DS对hERG通道蛋白的影响。结果：1.以家兔单个心室肌细胞为研究对象,研究Dau和DS对IK1、IKr、IKs和Ito通道电流的影响(1)Dau：与给药前相比,Dau1,3,10,30,100μmol·L-1对IK1具有一定抑制作用,但差异均无统计学意义(P>0.05),对翻转电位也无影响(-40mV)。在-100 mV测试电压下,Dau 10,30,100μmol·L-1对IK1的抑制率分别为(5.8±1.8)%、(16.5±2.7)%和(39.3±8.1)%。在+60mV测试电压下,Dau 1,3,10,30μmol·L-1对Iκr的抑制率分别为(12.2±8.6)%、(30.4±7.1)%、(37.1±3.7)%和(64.1±5.8)%,半数抑制浓度(IC50)为14.0μmol·L-1。Dau 10μmol·L-1, 30μmol·L-1使IKr半激活电压由(-16.4±2.8)mV变为(-14.8±2.2)、(-17.8±4.2)mV变为(-20.7±1.7),曲线斜率由6.2±0.1变为6.8±0.4、6.2±0.2变为6.5±0.2,差异均无统计学意义(P>0.05)。在+60mV测试电压下,Dau 10,30μmol·L-1对IKs的抑制率分别为(28.7±3.3)%和(40.3±5.2)%。Dau 10,30μmol·L-1使IKs半激活电压由(14.1±0.7)mV变为(12.8±1.6)mV、(15.0±1.9)mV变为(10.5±1.0)mV,曲线斜率由6.2±0.2变为6.5±0.2、14.7±0.7变为15.5±1.7,差异均无统计学意义(P>0.05)。Dau 10,30,100μmol·L-1的对Ito电流均有抑制作用。在+30 mV测试电压下,Dau 30,100μmol·L-1对Ito的抑制率分别为(26.5±3.6)%和(38.7±0.7)%。Dau 30,100μmol·L-1使Ito半激活电压由(-12.4±4.6)mV变为(17.1±1.9)mV、(-12.5±4.6)mV变为(-14.0±3.2)mV,曲线斜率由19.9±1.4变为20.4±0.2、19.9±1.4变为19.8±0.7,差异均无统计学意义(P>0.05)。(2)DS：与给药前相比,DS 10,30,100μmol·L-1对IK1具有一定抑制作用,差异均无统计学意义(P>0.05),对翻转电位也无影响(-40 mV)。在-100 mV测试电压下,DS 10,30,100μmol·L-1对IK1的抑制率分别为(8.0±2.8)%、(16.0±3.4)%和(26.1±8.5)%。在+60 mV测试电压下,DS 1,3,10,30μmol·L-1对IKr的抑制率分别为(6.7±1.9)%、(26.1±6.0)%、(31.1±5.0)%和(55.4±6.9)%,IC50为19.9μmol·L-1。DS 10,30μmol·L-1使IKr半激活电压由(-22.5±1.1)mV变为(-25.2±1.5)mV、(-21.5±1.6)mV变为(-26.5±3.2)mV,曲线斜率由6.6±0.2变为5.6±0.4、6.7±0.4变为4.7±1.1,差异均无统计学意义(P>0.05)。在+60mV测试电压下,DS 10,30μmol·L-1对IKs的抑制率分别为(25.7±3.8)%和(38.0±3.8)%。DS 10,30μmol·L-1使Iκs半激活电压由(12.0±3.8)mV变为(11.5±1.7)mV、(12.2±3.0)mV变为(7.3±3.3)mV,曲线斜率由13.5±0.8变为12.3±1.3、13.9±0.9变为13.9±1.9,差异均无统计学意义(P>0.05)。DS 10,30μmol·L-1对Ito尾电流均有抑制作用。在+30 mV测试电压下,DS 10,30μmol·L-1对Ito的抑制率分别为(6.1±9.2)%和(36.5±11.4)%。DS 10,30μmol·L-1使Ito半激活电压由(-10.9±4.8)mV变为(-9.0±4.3)mV、(-19.9±1.9)mV变为(-15.7±1.5)mV,曲线斜率由17.0±1.5变为18.0±1.5、18.4±1.5变为17.6±1.8,差异均无统计学意义(P>0.05)。2.以HEK293细胞上表达的hERG通道为研究对象,研究Dau和DS对hERG通道动力学的影响(1)Dau：在20 mV时,Dau 1,3,10,30μmol·L-1对hERG去极化末电流的抑制率(16.1±5.5)%、(28.4±6.7)%(P<0.05,n=8)、(59.7±8.9)%(P<0.05,n=10)、(79.7±4.7)%(P<0.05,n=7),IC50为6.9μmol·L-1;在60mV时,对hERG尾电流的抑制率分别是(6.1±5.2)%、(26.5±3.6)%(P<0.05,n=4)、(57.6±8.8)%(P<0.05,n=9)、(85.3±2.2)%(P<0.05,n=6),IC50为8.4μmol·L-1。Dau1,3,10,30μmol·L-1分别使hERG的半激活电压V1/2从(4.5±3.0)mV变为(3.3±3.3)mV、(5.1±2.9)mV变为(2.6±4.0)mV、(0.7±2.8)mV变为(-6.2±5.8)mV、(1.1±2.7)mV变为(3.7±4.1)mV,差异均无显著性；激活曲线斜率分别从10.6±0.6变为11.0±0.7、10.7±0.6变为11.2±0.8、9.9±0.4变为13.6±1.6、9.6±0.4变为17.1±1.9(P<0.05)。Dau 10μmol·L-1分别使hERG的半失活电压V1/2从(-52.7±3.7)mV变为(-57.8±3.5)mV(P<0.05,n=11)；失活曲线斜率从-21.7±0.6变为-21.0±0.5(n=9,P>0.05)。单指数方程拟合稳态失活电流得到失活时间常数,在-120 mV和-110 mV电压下,Dau 10μmol·L-1使稳态失活时间常数明显下降,失活速率加快(P<0.05)。Dau对瞬时失活曲线和恢复时间常数无影响。Dau具有开放性阻断剂的特性。(2)DS：在20mV时,DS 1,3,10,30μmol·L-1对hERG去极化末电流的抑制率分别是(32.2±4.2)%(n=6,P<0.05)、(41.6±2.6)%(n=4,P<0.05)、(62.1±5.9)%(n=12,P<0.05)、(74.8±9.8)%(n=5,P<0.05),IC50为9.1μmol·L-1;在60 mV时,对hERG尾电流的抑制率分别是(16.7±5.8)%(n=6)、(31.1±4.5)%(n=7,P<0.05)、(55.1±11.2)%(n=13,P<0.05)、(81.2±7.0)%,IC50为9.6μmol·L-1。DS 1,3,10,30μmol·L-1分别使hERG的半激活电压V1/2从(8.2±2.0)mV变为(7.6±3.2)mV、(4.7±3.4)mV变为(6.5±3.1)mV、(2.8±3.3)mV变为(-2.6±4.2)mV、(0.4±4.5)mV变为(2.5±4.9)mV；激活曲线斜率分别从10.0±0.6变为11.1±0.8、10.5±0.7变为11.1±1.1、10.5±0.7变为13.2±2.2、10.0±0.7变为12.7±1.6,差异均无显著性。DS 10μmol·L-1分别使hERG的半失活电压V1/2从(-48.7±7.6)mV变为(-64.6±5.2)mV(P<0.05)；失活曲线斜率从-21.9±0.7变为-22.5±1.0,差异无显著性。在-120mV到-100mV电压下,DS 10μmol·L-1使稳态失活时间常数明显下降,失活速率加快(P<0.05)。DS 10μmol·L-1在-50 mV至+10 mV电压下对瞬时失活曲线有影响(左移),在-20 mV至+10 mV电压下瞬时失活时间常数缩短,差异有统计学意义。DS具有开放性阻断剂的特性。3.以HEK293细胞上表达的hERG通道为研究对象,采用Western blotting技术,研究Dau和DS对hERG通道蛋白表达水平的影响(1)Dau:HEK293-hERG细胞孵育24 h后,在大部分指令电压下Dau 10μmol·L-1可增加hERG尾电流(Itail),但无统计学差异。control组的膜电容为(32.4±4.4)pF(n=17),Dau 10μmol·L-1组膜电容为(35.9±4.7)pF(n=15),差异无统计学意义。分别用Dau 3,10,30μmol·L-1处理细胞24 h后进行Western blotting检测,结果发现Dau 30μmol·L-1可减少hERG通道蛋白的表达,统计学上有显著差异(P<0.05)。(2)DS:HEK293-hERG细胞孵育24 h后,DS 10μmol·L-1可抑制hERG去极化末电流(Istep),在-60 mV至0 mV差异有统计学意义(P<0.05)。在所有电压下对尾电流(Itail)均有抑制作用,差异有显著性(P<0.05)。control组的膜电容为(16.5±3.1)pF(n=12)，DS 10μmol·L-1组膜电容为(45.7±9.4)pF(n=6),差异有统计学意义(P<0.05)。分别用DS 3,10,30μmol·L-1处理细胞24h后进行Western blotting检测,结果发现DS 30μmol·L-1可减少hERG通道蛋白的表达,统计学上有显著差异(P<0.05)。结论：1.Dau和DS对IK1和Ito均有一定抑制作用,但作用较弱,100μmol·L-1时抑制率均未达到50%。Dau对IKr尾电流半数抑制浓度为14.0μmol·L-1,DS对IKr尾电流半数抑制浓度为19.9μmol·L-1,对IKr的激活曲线、半激活电压和激活曲线斜率的影响,均无统计学意义。Dau和DS对IKs尾电流的抑制作用较IKr弱,30μmol·L-1时抑制率均未达到50%。2.Dau、DS对hERG通道具有抑制作用,该作用具有电压依赖性,随着膜电位的去极化,抑制作用逐渐增强。但Dau和DS对通道的激活曲线几乎无影响。Dau.DS均能使hERG通道的半失活电压下降,失活时间常数减小,失活速率加快。Dau对瞬时失活曲线和恢复时间常数无影响。DS瞬时失活曲线左移,-20 mV-+10 mV电压下瞬时失活时间常数缩短,不影响恢复时间常数。Dau和DS对hERG通道的抑制作用具有开放性阻断剂的特征。3.hERG细胞在Dau 10μmol·L-1孵育24 h后,去极化末电流和尾电流都有增加的趋势,hERG通道蛋白表达减少,但均无统计学差异,Dau 30μmol·L-1时可显著减少hERG蛋白的表达。DS 10μmol·L-1孵育24 h后,在所有指令电压下尾电流都减小,有统计学差异,但DS 10μmol·L-1对hERG表达无显著影响,在30μmol·L-1时可显著减少hERG蛋白的表达。更多还原

【Abstract】 Objective:Daurincine(Dau) and daurinsoline(DS), the bisbenzyl isoquinoline alkaloids, are extracted from the root of Menispermum dauricum DC. Their antiarrhythmic effects have been demonstrated in various experimental models. The excessive prolonged repolarization of action potential duration (APD) is the electrophysiological basis of Long QT syndrome (LQTs).The APD and the morphology of action potential were controlled by the inward currents and outward currents. The main outward currents are the rapidly activated delayed rectifier potassium current(IKr) and the slowly activated delayed rectifier potassium current(IKs).The main outward currents are sodium current(INa) and L-type calcium current(ICa-L)。In this study, using patch-clamp technique and Western blotting, we studied the effects of Dau and DS on ion channels and their proteins which are related to the acquired LQTs, and explored their antiarrhythimic mechanisms.Methods:(1) Single left ventricular myocardial cells of rabbits were isolated by enzymic method. Using patch-clamp technique, we recorded the IK1, IKr, IKs and Ito of single cell and studied the effects of Dau and DS these potassium channels; (2) Using patch-clamp technique, we recorded the hERG current which stably expressed in HEK293 cells and studied the effects of Dau and DS on kinetics of hERG currents; (3) Using Western blotting techinique, we studied the effects of Dau and DS on hERG protein levels. Results:1. The effects of Dau and DS on IK1, IKr, IKs and Ito potassium current(1) Dau:Compared with control, Dau at 1,3,10,30,100μmol·L-1 could inhibit the IK1, but there were no statistical difference and Dau did not affect the reversal potetntials. The inhibiton rates of Dau at 10,30,100μmol·L-1 on IK1 were (5.8±1.8)%、(16.5±2.7)%v and (39.3±8.1)% at-100mV, respectively. The inhibiton rates of Dau at 1,3,10,30μmol·L-1 on IKr were (12.2±8.6)%, (30.4±7.1)%, (37.1±3.7)% and (64.1±5.8)% respectively at-60 mV and the IC50 was 14.0μmol·L-1。Dau 10,30μmol·L-1 made the half activation voltages of IKr change from(-16.4±2.8)mV to (-14.8±2.2) mV, from(-17.8±4.2) mV to(-20.7±1.7) mV, respectively and the slope of the activation curve change from 6.2±0.1 to 6.8±0.4, from 6.2±0.2 to 6.5±0.2, but there were all no statistical differences. The inhibition rates of Dau at 10,30μmol·L-1 on IKs at+60 mV were (28.7±3.3)% and (40.3±5.2)%, respectively。Dau at 10,30μmol·L-1 changed the half activation voltages from (14.1±0.7)mV to (12.8±1.6), from (15.0±1.9) mV to (10.5±1.0) mV, respectively and the slope of activation curve changed from 6.2±0.2 to 6.5±0.2, form 14.7±0.7 to 15.5±1.7, but there were all no statistical differences. Dau at 10,30,100μmol·L-1 inhibted the Ito current. The inhibition rate of Dau 30,100μmol·L-1 on Ito at+30 mV were (26.5±3.6)% and (38.7±0.7)%, respectively. Dau 30,100μmol·L-1 changed the half activation voltages of Ito from (-12.4±4.6) mV to (17.1±1.9) mV, from(-12.5±4.6) mV to (-14.0±3.2) mV, respectively and the activation slope changed from 19.9±1.4 to 20.4±0.2, from 19.9±1.4 to 19.8±0.7, but there were all no statistical differences.(2) DS:Compared with control, DS 10,30,100μmol·L-1 could inhibit the IK1, but there was no statistical difference and DS did not affect the reversal potetntials. The inhibiton rates of DS at 10,30,100μmol·L-1 on IK1 were (8.0±2.8)%、(16.0±3.4)% and (26.1±8.5)% at-100 mV, respectively. The inhibiton rate of DS at 1,3,10, 30μmol·L-1 on IKr were (6.7±1.9)%、(26.1±6.0)%、(31.1±5.0)% and (55.4±6.9)% at-60 mV and the IC50 was 19.9μmol·L-1. DS 10,30μmol·L-1 made the half activation voltages of IKr-change from(-22.5±1.1) mV to (-25.2±1.5) mV, from(-21.5±1.6) mV to (-26.5±3.2) mV, respectively and the slope of the activation curve change from 6.6±0.2 to 5.6±0.4, from 6.7±0.4 to 4.7±1.1, but there were all no statistical difference. The inhibition rates of DS at 10, 30μmol·L-1 on IKs at +60mV were (25.7±3.8)% and (38.0±3.8) %, respectively。DS at 10μmol·L-1, 30μmol·L-1changed the half activation voltages from (12.0±3.8) mV to (11.5±1.7) mV, from (12:2±3.0)mV to (7.3±3.3) mV, respectively and the slope of activation curve changed from 13.5±0.8 to 12.3±1.3, form 13.9±0.9 to 13.9±1.9, but there were all no statistical difference. DS 10, 30μmol·L-1 inhibted the Ito current. The inhibition rate of DS at 10, 30μmol·L-1 on Ito at +30 mV were (6.1±9.2)% and (36.5±11.4)%, respectively. DS 10, 30μmol·L-1 changed the half activation voltages of Ito from (-10.9±4.8) mV to (-9.0±4.3) mV, from(-19.9±1.9) mV to (-15.7±1.5) mV, respectively and the activation slope changed from 17.0±1.5 to 18.0±1.5, from 18.4±1.5 to 17.6±1.8, but there were all no statistical difference.2. The effects of Dau and DS on kinetics of hERG current, which stably expressed in HEK293 cells(1) Dau: At +20mV, the inhibition rates of Dau at 1, 3, 10, 30μmol·L-1 on hERG step current were (16.1±5.5)%, (28.4±6.7)% (P<0.05,n=8), (59.7±8.9)% (P<0.05,n=10), (79.7±4.7)% (P<0.05,n=7), respectively and the IC50 was 6.9μmol·L-1; At +60mV, the inhibition rates of Dau 1, 3, 10, 30μmol·L-1 on hERG tail current were (6.1±5.2)%, (26.5±3.6)% (P<0.05,n=4), (57.6±8.8)%(P<0.05,n=9), (85.3±2.2)% (P<0.05,n=6), respectively, and the IC50 was 8.4μmol·L-1. Dau 1,3, 10, 30μmol·L-1 changed the half activation voltages from (4.5±3.0)mV to (3.3±3.3) mV, from (5.1±2.9) mV to (2.6±4.0) mV, from (0.7±2.8) mV to (-6.2±5.8)mV, from (1.1±2.7) mV to (3.7±4.1) mV, respectively, but there were no statistical differences. The slopes of activation curve were changed from 10.6±0.6 to 11.0±0.7, from 10.7±0.6 to 11.2±0.8, from 9.9±0.4 to 13.6±1.6, from 9.6±0.4 to 17.1±1.9 (p<0.05) . Dau 10μmol·L-1 decreased the half inactivation voltage from -52.7±3.7 to -57.8±3.5 (P<0.05, n=11) and changed the slope of inactivation curve from -21.7±0.6 to -21.0+0.5(n=9)( P>0.05). Dau had no effects on steady-state inactivation curve, the onset of inactivation curve and the recovery time constant. Dau exerted the characteristics of an open channel blocker of hERG current. (2) DS:At+20 mV, the inhibition rates of DS at 1,3,10,30μmol·L-1 on hERG step current were (32.2±4.2)%(n=6, P<0.05), (41.6±2.6)%(n=4, P<0.05), (62.1±5.9)%(n=12,P<0.05), (74.8±9.8)%(n=5, P<0.05), respectively and the IC50 was 9.1μmol·L-1; At+60 mV, the inhibition rates of DS 1,3,10,30μmol·L-1 on hERG tail current were(16.7±5.8)%(n=6), (31.1±4.5)%(n=7, P<0.05), (55.1±11.2)% (n=13,P<0.05), (81.2±7.0) (P<0.05,n=6), respectively, and the IC50 was 9.6μmol·L-1. DS 1,3,10,30μmol·L-1 changed the half activation voltages of hERG current from (8.2±2.0) mV to (7.6±3.2) mV, from (4.7±3.4) mV to (6.5±3.1) mV, from (2.8±3.3) mV to (-2.6±4.2) mV, from (0.4±4.5) mV to (2.5±4.9) mV, respectively, but there were no statisitical differences. The slopes of activation curve were changed from 10.0±0.6 to 11.1±0.8, from 10.5±0.7 to 11.1±1.1, from 10.5±0.7 to 13.2±2.2, from 10.0±0.7 to 12.7±1.6, respectively, and there were no statistical differences. DS 10μmol·L-1 decreased the half inactivation voltages from (-48.7±7.6) mV to (-64.6±5.2) mV(P<0.05) and changed the slope of inactivation curve from-21.9±0.7 to-22.5±1.0 (P>0.05). DS 10μmol·L-1 increased the inactivation at the test potential from-120mV to-100 mV(P<0.05), shifted the onset of inactivation curve at the test potential from-50 mV to+10 mV(P<0.05), and decreased the time constant of the onset of inactivation at test potential from-20 mV to+10 mV(P<0.05).DS had no effect on the recovery time constant. DS exerted the characteristics of an open channel blocker of hERG current.3. The effects of Dau and DS on protein levels of hERG current, which stably expressed in HEK293 cells(1)Dau:After the HEK293-hERG cells were incubated for 24 hours with 10μmol·L-1 Dau, the step and tail hERG currents were increased(P>0.05). The capacitance of control and Dau were (32.4±4.4) pF(n=17) and (32.4±4.4) pF(n=17), respectively(P>0.05).The HEK293-hERG cells were incubated with Dau at 3,10,30μmol·L-1 for 24 hours and the protein were tested by using Western blotting technique. Dau 30μmol·L-1 could decrease the hERG protein expression(P<0.05).(1)DS:After the HEK293-hERG cells were incubated for 24 hours with 10 μmol·L-1 DS, the step and tail hERG currents were decreased at the test potential from-60 mV to 0 mV(P<0.05). The capacitance of control and DS were (16.5±3.1) pF(n=12) and (45.7±9.4)(n=6), respectively(P<0.05).The HEK293-hERG cells were incubated with DS at 3,10,30μmol·L-1 for 24 hours and the protein were tested by using Western blotting technique. DS 30μmol·L-1 could decrease the hERG protein expression(P<0.05).Conclusion:1. Dau and DS could inhibit IK1 and Ito, but the inhibiton rate did not reach 50% at 100μmol·L-1.The IC50 of Dau and DS on IKr tail current were 14.0μmol·L-1 and 19.9μmol·L-1, respectively. Dau and DS had no effects on the activation curves of IKr.The inhibitory effects of Dau and DS on IKs were weaker than their effects on IKr, the inhibiton rate did not reach 50% at 30μmol·L-1.2. Dau and DS had inhibitory effects on hERG currents and it was voltage-dependent. As the depolarization of membrane potential, the inhibitory effects became more and more strong. But Dau and DS had no effects on activaton curves of hERG current. Dau and DS could decrease the half inactivation voltage and the inactivation time constant of hERG current and could speed up the inactivaton. Dau did not affect the onset of inactivaton and the recovery time constant of hERG current. DS shifted the onset of inactivaton curve to left and markedly decreased the time constant of the onset of inactivation at test potential from-20mV to+10mV and had no effects on recovery time constant.3. After the HEK293-hERG cells were incubated with Dau 10μmol·L-1 for 24 hours, the step hERG currents were decreased, the tail hERG currents were increased and the hERG protein expression were decreased, but there were no significant difference. Dau 30μmol·L-1 couled markedly decreased the hERG protein expression. After the HEK293-hERG cells were incubated with DS 10μmol·L-1 for 24 hours, the step and tail hERG currents were significantly decreased, but DS 10μmol·L-1 had no effects on hERG protein expression. DS 30μmol·L-1 could markedly decrease the hERG protein expression.更多还原