金丹配伍干预心肌细胞多离子通道及调控心肌组织microRNA抗心律失常作用机制研究

Arrhythmia,a cardiovascular disease caused by myocardial cell electrophysiological abnormalities,may aggravate the condition of existing heart disease or even lead to sudden death.During former trials, we discovered that Hypericum attenuatum choisy has a significant effect of antiarrhythmic mechanism that helps regulate ICa-l and ATP-sensitive potassium related channels.Also, the Hypericum attenuatum choisy has an effect on INa channels.And we have found Hypericum attenuatum has a more significant anti-arrhythmic effect matched with Dan-shen Root.We speculate that there are anti-arrhythmic active substances in the pair of Hypericum attenuatum choisy with Dan-shen Root which react to multiple ion channels and they were related with microRNA during a stimulated model on arrhythmia.To test this hypothesis,we will copy rat model of arrhythmia and make use of a three level procedure- whole animal,cellular and molecules,which involving whole-cell patch-clamp technique， molecular biology and Realtime-PCR technique-to find out the effects of Hypericum attenuatum choisy match with Dan-shen Root on arrhythmia ion channels and microRNA expression.This research attempts to use a new way that on the ion channel and cellular and molecules level to explain the explicit function of Hypericum attenuatum choisy match with Dan-shen Root.Find out the rule of their work. Moreover,it aims to provide a technological platform for the development of ideal anti-arrhythmic medicine and to offer new idea and to the research of traditional Chinese medicine.

心律失常是心血管疾病引起的心肌细胞电生理异常，可加重原有心脏疾病，甚至导致猝死。研究者前期研究证明乌腺金丝桃抗心律失常机制与调节L型钙离子通道，ATP敏感钾通道有关，并观察到其对钠离子通道有影响，与丹参配伍后效应增强。我们推测乌腺金丝桃与丹参配伍后抗心律失常作用是其活性物质，其与对心律失常模型心肌细胞多个离子通道干预及对心肌组织microRNA调控作用有关。为验证这一假说，我们拟以大鼠心律失常模型，采用全细胞膜片钳、分子生物学、实时定量PCR（Realtime-PCR）等技术手段，从整体动物-细胞-分子，三个层次研究乌腺金丝桃和丹参配伍对于心律失常心肌离子通道及心肌组织microRNA表达的影响，从离子通道及分子生物水平解释金丹配伍抗心律失常的作用机制、阐明金丹配伍的组方原则，为开发理想抗心律失常方剂提供技术平台，为方剂配伍研究提供新思路。

本项目组前期进行了乌腺金丝桃抗心律失常的药效基础及作用机理的研究。. 完成乌腺金丝桃成分分离；研究乌腺金丝桃总黄酮对大鼠心肌细胞心律失常模型动作电位的影响，并探讨其离子通道机制；研究乌腺金丝桃与黄芪配伍对MIRI大鼠模型的影响，并探讨其作用机制；研究乌腺金丝桃与当归配伍对心肌缺血再灌注损伤大鼠心肌保护作用及其机制。.1.乌腺金丝桃与黄芪配伍1:1和1:2组与模型组相比均能明显降低MIRI大鼠模型血清CK含量、缩小心肌梗死面积。.2.乌腺金丝桃与黄芪配伍1:2组与模型组相比可明显改善MIRI大鼠模型心肌病理形态，明显减少心肌细胞凋亡。.3.乌腺金丝桃与黄芪配伍1:2组与模型组相比能明显降低MIRI大鼠模型血清MDA的含量，1:1组及1:2组均能增强模型大鼠血清中SOD的活力。.4.机理研究中，乌腺金丝桃与黄芪配伍1:2组与模型组相比能明显下调MIRI大鼠模型caspase-3蛋白及mRNA表达，下调TNF-α的蛋白及mRNA表达。.5.机理研究中，乌腺金丝桃与黄芪配伍1:2组与模型组相比可提高MIRI大鼠模型心肌组织HSP70蛋白的表达，并且减弱NF-κB p65活化，提高HSP70 mRNA表达水平，抑制NF-κB p65 mRNA表达水平。. 6.乌腺金丝桃与当归配伍2:1组可明显降低MIRI大鼠血清CK及LDH活性，说明2:1组为抗MIRI大鼠的最佳保护作用的配伍组合，可明显改善模型大鼠心肌病理形态，明显减少心肌细胞凋亡。.7.乌腺金丝桃与当归配伍2:1组可明显提高MIRI大鼠心肌组织中GSH-Px活力、Ca2+Mg2+-ATP与Na+K+-ATP 酶的活力，能明显上调模型大鼠心肌Bcl-2蛋白表达，下调Bax的蛋白表达。.8.乌腺金丝桃与当归配伍2:1组可明显提高MIRI大鼠心肌HSP70、Cx43蛋白的表达，并且减弱NF-κB p65蛋白活化，提高HSP70 mRNA、Cx43mRNA表达水平，抑制NF-κB p65 mRNA表达水平。. 本研究为李冀教授探讨“中医方药配伍的理论实质”研究系列之一，其起到承前启后的作用，本项目通过配伍已经清楚的乌腺金丝桃化学成分及丹参、黄芪、当归等有效组分进行探讨，为利用化学成分及有效组分配伍抗心律失常作用研究提供了一个技术平台，进而为揭示方剂的复杂药效物质基础、标准化科学化方剂理论打下坚实的理论基