【作者】 刘毅；

【导师】 金满文；

【作者基本信息】 华中科技大学 ， 药理学， 2012， 博士

【摘要】 第一部分三甲基芹菜素对多种心脏钾离子通道的阻断作用背景和目的：天然黄酮和多甲氧基黄酮被报道具有心血管保护作用,而这类化合物对心脏离子通道的潜在影响少有报道。我们使用膜片钳技术,在全细胞模式下研究槲皮素,芹菜素与其甲基化衍生物3,7,3’,4’-四甲基槲皮素,3,5,7,3’,4’-五甲基槲皮素,7,4’-二甲基芹菜素和5,7,4’-三甲基芹菜素对稳定表达hKv1.5通道的HEK293细胞IKur电流的影响。我们发现三甲基芹菜素在六个化合物中对hKv1.5的抑制作用最强,因此,我们针对三甲基芹菜素深入研究其阻断hKv1.5的作用特点以及其对其他重要心脏钾离子通道的影响。结果：实验结果表明,三甲基芹菜素对hKv1.5通道的阻断作用表现为浓度与频率依赖性。三甲基芹菜素对hKv1.5阻断的IC50为6.4 gM,对急性分离人心房肌细胞超速激活延迟整流钾电流(IKur)阻断的ICs0为8.0μM。三甲基芹菜素是hKv1.5的开放性阻断剂,表现随时间延长加速通道失活速率,三甲基芹菜素3μM与10μM干预后药物敏感电流曲线的失活时间常数分别为126.1±15.4 ms和59.6±4.7 ms。三甲基芹菜素对hKv1.5的抑制作用随刺激频率的升高而增强,其在1,2和5 Hz下的抑制率分别为6.2,5.1和4.2μM。此外,三甲基芹菜素对乙酰胆碱激活钾电流的抑制作用也较强(IC50=6.8μM),而对人心房瞬时外向钾电流Ito的抑制ICs0为19.3μM,对hKv4.3的抑制强度与人心房Ito抑制强度接近。然而,三甲基芹菜素对hERG的抑制相比IKur及IKACh较弱(IC50=18-31μM)并且对大鼠心室肌IK1的影响更小(IC50>30μM)。结论：以上研究结果证明三甲基芹菜素可显著抑制心房特异表达钾电流IKur及IKACh,同时对hERG及IK1的影响较小,提示三甲基芹菜素可能具有潜在心房选择性,有可能具有抗房颤作用。第二部分三甲基芹菜素增加人心房肌细胞及稳定表达于HEK293细胞Nav1.5通道迟钠电流的作用研究背景和目的：心肌细胞上的钠通道开放是动作电位0相去极化的最重要成分,也是多种抗心律失常药物的作用靶点。三甲基芹菜素已经被证明具有多种钾离子通道阻断作用并对心房特异表达的钾通道抑制作用更强,但其对钠通道是否有影响未见报道。我们利用急性分离人心房肌细胞与稳定表达Nav1.5通道的HEK293细胞研究三甲基芹菜素对钠通道的潜在影响。结果：我们发现三甲基芹菜素(1.10μM)对人心房肌钠电流峰值没有影响,但能够浓度依赖性显著减缓钠电流“失活”过程,表现为在等电压下,三甲基芹菜素随浓度增加而使钠电流回复到基线时间明显延长,并且使钠电流“失活”过程由单指数方程拟合转变为双指数方程拟合。同时,这种减慢失活作用在刺激频率1,2,5与10Hz下没有差异,说明其具有非频率依赖特点。三甲基芹菜素10μM能使电压-失活曲线左移,半数失活电压由-94.3±0.27 mV左移至-99.3±0.2 mV,但不改变失活曲线斜率参数,此外其半数激活电压与激活虚线斜率也不受影响。三甲基芹菜素能显著延长人心房钠通道从失活状态恢复时间,通过间隔递增双刺激实验证明其恢复时间常数由8.2±0.12 ms增加至13.9±0.26 ms。三甲基芹菜素对克隆Nav1.5通道的影响与原代人心房肌细胞基本一致,表现为不影响钠电流峰值但显著增加钠总内流电量,减缓钠电流“失活”过程,使双指数方程拟合获得的时间常数tau1与tau2增大,正常状态下tau1与tau2分别为6.69±0.17ms,1.58±0.02 ms,三甲基芹菜素10μM干预后分别为11.39±0.16 ms与1.75±0.03 ms。同时,三甲基芹菜素可明显左移失活曲线并降低半数失活电压,对激活曲线则没有显著影响,并且同人心房肌类似,干预后其失活恢复时间常数由13.6±0.50 ms增加至25.2±1.25 ms。结论：以上结果证明三甲基芹菜素能在不影响钠电流激活峰值的情况下显著减缓钠电流失活过程,并且在人原代心房肌细胞与克隆Nav1.5通道上获得的结果趋势一致,提示三甲基芹菜素可能是通过非孔道阻塞性作用于α亚基引起通道失活减缓。这种不改变峰值但最终造成钠内流增加的结果与海葵毒素ATX-Ⅱ的作用极为相像,而在黄酮类化合物中则是首次发现。更多还原

【Abstract】 PartⅠEffects of the natural flavone trimethylapigenin on cardiac potassium currentsBackground and purpose:The natural flavones and polymethylflavone have been reported to have cardiovascular protective effects. In the present study, we determined whether quecertin, apigenin and their methylated compounds (3,7,3’,4’-tetramethylquecertin,3,5,7,3’,4’-pentamethylquecertin,7,4’-dimethylapigenin, and 5,7,4’-trimethylapigenin) would block the atrial specific potassium channel hKv1.5 using a whole-cell patch voltage-clamp technique.Results:We found that among the six flavonoids, trimethylapigenin showed the strongest inhibitory effect on hKv1.5 channel current. This compound suppressed hKv1.5 current in HEK 293 cell line (IC50=6.4μM), and the ultra-rapid delayed rectify K+current IKur in human atrial myocytes (IC50=8.0μM) by binding to the open channels. The mean value of blocking time constant was 126.1±15.4 ms for 3μM trimethylapigenin, and 59.6±4.7 ms for 10μM trimethylapigenin. The blockade showed a use-and frequency-dependent manner, the inhibition potent was increased with the elevation of the frequency, the IC50 on hKv1.5 was 6.2,5.1, and 4.2μM at 1,2, and 5 Hz, respectively. In addition, trimethylapigenin decreased transient outward potassium current (Ito) in human atrial myocytes, inhibited acetylcholine-activated K+current (IC50=6.8μM) in rat atrial myocytes. Interestingly, trimethylapigenin had a weak inhibition of hERG channel current (IC50=18-31μM) and rat ventricle IK1(IC50>30μM).Conclusion and implication:Our results indicate that trimethyapigenin significantly inhibits the atrial potassium currents hKv1.5/IKur and IKACh, which suggests that trimethylapigenin may be a potential candidate for anti-atrial fibrillation. Part II Trimethylapigenin increases inward sodium current on human atrial myocyte and cloned Navl.5 channelBackground and Purpose:Sodium channel is responsible for forming the 0 phase of cardiac action potential. It is also the target of Class I anti-arrythmia drugs. Trimethylapigenin has been proved to be functional inhibiting multipule potassium channels, especially those specially expressed only in atrial, but the effect of trimethylapigenin on sodium channe has not been well studied. We employed human atrial myocytes and HEK293 cell which stably expressed Navl.5 gene to study the potential influence.Results:We found that 1-10μM trimethylapigenin did not show much influence on the peak amplitude of sodium current on human atrial myocyte, interestingly, it could concentration dependently delay the "inactivation" procedure of sodium current:With the concentration elevated, it took much more time for the sodium current return to the basal line, meanwhile, the mono-exponential fitting for the inactivation was poor after trimethylapigenin was given but it fit much better to a bi-exponential function. Besides, this "delay" of inactivation showed no difference under 1,2,5 and 10 Hz stimulation, indicating the frequency was irrelevant. Trimethylapigenin 10μM could left shift the voltage-dependent available curve, the V0.5 was decresed from-94.3±0.27 mV to-99.3±0.2 mV but the slope was not affected. The time constant of recovery from inactivation was increased from 8.2±0.12 ms to 13.9±0.26 ms.The influence of trimethylapigenin of cloned Navl.5 was close to what we found on human atrial. Trimethylapigenin did not change the peak amplitude but obviously increase the total inward quantity of electricity and delay the inactivation procedure. The fitting parameter obtained from bi-exponential function taul and tau2 were both increased from 6.69±0.17 ms,1.58±0.02 ms to 11.39±0.16 ms,1.75±0.03 ms, respectively. Meanwhile, trimethylapigenin left shifted the voltage-dependent avalible curve and showed no influence on the voltage-dependent activation. Similar as human atrial, the recovery time constant from inactivation was prolonged from 13.6±0.50 ms to 25.2±1.25 ms.Conclusions:These results demonstrate that trimethylapigenin delay the inactivation procedure of INa without affecting the peak amplitude. The channel opening properties were similar between human atrial myocytes and cloned Navl.5 which indicated that the a subunit probable be the target of trimethylapigenin and the delay was not by directly blocking the pore of sodium channel. This effect was very alike the influence of ATX-II (a sea anemones toxin) and it is the first time that a flavonoid was proved to have a similar function.更多还原