雷公藤甲素影响线粒体通透性转化孔的调控致心肌细胞毒性的机制研究

Triptolide (TP), a major active ingredient of the Chinese herb Tripterygium wilfordii Hook f. (TWHF), which is widely used in Traditional Chinese Medicine for centuries, has been shown to possess multiple biological activities. However, the clinical applications of TP are limited by its narrow therapeutic window and severe toxicity on heart, liver and kidney. The mechanism of TP-induced cardiotoxicity has not been fully elucidated. Our previous studies demonstrated that TP exerted toxic action on rat and Beagle dog (2013), and recent results also indicated that TP induced mitochondrial dependent cell death in H9c2 cardiomyocytes. These preliminary results indicate H9c2 cells that knockdown GSK-3β which can translocate to the mitochondria and binds to mPTP subunit proteins ANT-VDAC, or p53 which can translocate to the mitochondria and binds to an mPTP subunit protein CypD by small interfering RNA, and GSK-3β-/- or p53-/-mice will be used to determine the cardiotoxicity of TP. Levels of mitochondrial membrane potential depolarization, mitochondrial CytC release, and the key proteins Caspase-3 for apoptosis and RIP3 for necrosis expression were assessed as the toxic indices for TP. This research would suggest that TP modulates the mPTP opening by inactivating GSK-3β and p53, and then the opened mPTP triggers the cell death. Prevention of the mPTP opening may underlie the mechanism against TP-induced cardiotoxicity. The research could clarify the mechanisms of TP-induced toxicity in cardiomyocytess, and provide a rational basis for seeking antidotes to TP-induced cardiomyopathy in clinical application, further can be applied to the similar Traditional Chinese Medicine.

中药单体雷公藤甲素(TP)药理作用明确，临床应用广泛，然而毒副作用明显、治疗安全窗狭窄成为限制其应用的主要原因。目前TP心脏毒性的作用机制尚未彻底阐明。在我们前期工作观察到TP能引起动物毒性反应(2013)及近期预实验发现TP对H9c2心肌细胞系有明显毒性作用的基础上，本课题拟在细胞和动物水平上，干扰与线粒体通透性转化孔(mPTP)重要组成亚基ANT、VDAC和CypD相互作用而开放mPTP的基因GSK-3β和p53，以线粒体膜电势、细胞色素C释放及死亡关键蛋白Caspase-3和RIP3表达水平的变化为主要指标，观察基因沉默GSK-3β和p53前后，TP对心肌细胞和心脏组织的毒性作用。以论证TP是通过影响mPTP开放的调控因子GSK-3β和p53来诱导心肌细胞死亡，产生心脏毒性之假说，详析TP致心脏毒性的作用机制，为其临床应用的减毒提供思路，进而为同类中药的使用推广提供理论依据。

中药单体雷公藤甲素(Triptolide，TP)药理作用明确，临床应用广泛，然而毒副作用明显、治疗安全窗狭窄成为限制其应用的主要原因。本研究采用体外细胞及体内动物模型，选取心肌细胞重要细胞器线粒体为对象，围绕雷公藤甲素引起心脏毒性反应及损伤的类型、线粒体通透性转化孔（mPTP）开放是否参与TP诱导心脏毒性、GSK-3β与p53如何调节mPTP开放等科学问题，进一步阐明TP的心脏毒性机理。我们的研究结果得出：TP诱导的氧化应激，可损伤Nrf2抗氧化系统，抑制自噬，导致心肌细胞损伤；通过GSK-3β活化线粒体mPTP亚单位Cyp-D而介导mPTP的开放；通过上调p53蛋白，可招募Bax蛋白诱导线粒体mPTP开放，也可抑制IKKβ-NF-κB通路介导的GLUTs表达，抑制糖代谢与能量生成，最终导致心肌细胞凋亡。研究结果为TP临床应用的减毒策略提供理论与实验依据。