靶向hERG通道评价羟基吴茱萸次碱的心脏安全性

Evaluation of cardiac safety of hydroxyrutaecarpine,hERG channel inhibitor

药物诱发长QT间期综合征(long QT syndrome,LQTS)已成为临床上重要的研究课题,其中获得性长QT间期综合征(acLQTS)的发生主要是药物抑制人类ether-α-go-go相关基因(the human ether-α-go-go related gene,hERG)通道所致。hERG基因编码快速激活延迟整流钾离子通道(rapid component of the delayed rectifier potassium current,Ikr)的α亚基,其在动作电位3期复极过程中发挥重要作用,也是大多数抗心律失常药物作用的靶点。本文旨在探讨羟基吴茱萸次碱(hydroxyrutaecarpine,HRU)对hERG通道的影响,评估其心脏安全性。利用全细胞膜片钳技术记录HRU对hERG通道电流及动力学的影响,并验证与hERG通道的结合位点。运用PCR技术测定HRU对hERG mRNA表达水平的影响。利用Western blot技术检测HRU对hERG蛋白和转录因子Sp1(specificity protein 1)表达的影响。采用免疫荧光技术证实HRU对hERG蛋白和转录因子Sp1的定位和表达的影响。研究显示,HRU瞬时给药后对hERG电流具有抑制作用,降低hERG通道的失活电流,缩小失活时间常数,作用位点是S6片段的两个芳香族氨基酸即第656位的苯丙氨酸F656和第652位的酪氨酸Y652。HRU孵育给药能够减少hERG蛋白表达量,并抑制hERG电流,降低hERG mRNA的水平,降低细胞核内转录因子Sp1和细胞浆内hERG蛋白表达水平。激光扫描共聚焦实验也显示细胞核内转录因子Sp1和细胞浆内hERG蛋白表达都减少,说明HRU抑制Sp1表达是导致hERG mRNA表达减少的原因。以上结果表明,HRU瞬时给药抑制hERG电流的作用是通过结合hERG通道内F656和Y652位点,缩小失活时间常数,加快通道失活,从而抑制hERG通道功能。此外,HRU还抑制hERG蛋白表达,主要是通过抑制转录因子Sp1的表达,使hERG通道蛋白的转录功能下调,最终导致hERG蛋白减少。

Drug-induced long QT syndrome(LQTS)has become an important clinical research topic,and the occurrence of acquired long QT syndrome(acLQTS)is mainly caused by drug inhibition of the human ether-α-go-go related gene(hERG)channel.The hERG gene encodes theαsubunit of the fast-activating delayed rectifying potassium ion channel(Ikr),which plays an important role in the process of action potential phase 3 repolarization and is also the target of most antiarrhythmic drugs.The purpose of this study was to investigate the effect of hydroxyrutaecarpine(HRU)on the hERG channel and to evaluate its cardiotoxicity.The whole cell patch clamp technique was used to detect the effects of HRU on the current and kinetics of the hERG channel,and to confirm the binding site on the hERG channel.PCR was used to determine the effect of HRU on hERG mRNA expression.Western blotting was used to detect the effects of HRU on the expression of hERG protein and transcription factor Sp1.Immunofluorescence was used to confirm the effects of HRU on localization and expression of hERG protein and transcription factor Sp1.Studies have shown that transient HRU can inhibit hERG current and shorten the inactivation time constant.Its binding sites to the hERG channel are F656 and Y652.After incubation for 24 h,HRU can reduce the expression of hERG protein,inhibit the hERG current,reduce the level of hERG mRNA,and reduce the expression of transcription factor Sp1 in the nucleus and hERG protein in the cytoplasm.Immunofluorescence experiments also showed the same results suggesting that the inhibition of Sp1 expression by HRU is the cause of the decreased expression of hERG mRNA.In conclusion,the acute inhibition of HRU accelerates the channel inactivation process and reduces the inactivation time constant by binding to the F656 and Y652 sites in the hERG channel,thus reducing the hERG current.In addition,HRU also inhibits the expression of hERG protein,mainly by inhibiting the expression of transcription factor Sp1,the transcription function of hERG channel protein is down-regulated,so that the hERG protein is reduced.