龙牙楤木皂苷调控钙稳态失衡抑制内质网应激介导心肌保护的药效物质基础及分子机制研究

Disordered calcium homeostasis can lead to endoplasmic reticulum (ER) stress. Therefore, the attenuation of ER stress-induced apoptosis through modulating calcium homestasis may hold therapeutic promise for the treatment of MI/R injury. Aralia elata (Miq) Seem, which belongs to Araliaceae family, has the effects of promoting blood circulation and removing blood stasis. Continuous studies mainly on its cardioprotective effects have been carried out in our laboratory since 1980s. We found its novel use for the treatment of coronary heart disease, which has obtained Drug Clinical Trial Approval Document (Number 2003L01111) from the state Food and Drug Administration (SFDA), authorized 4 patents and published 12 papers. Recently, we for the first time found that total saponins of Aralia elata could increase the amplitude of E[Ca2+]i transients in a concentration-dependent manner, upregulate the expression of SERCA2a and PLB while decrease NCX expression, thereby reducing MIR-induced calcium overload. In this study, we aim to demonstrate that the cardioprotective activities of total saponins and its active components may be closely associated with modulating SERCA and other calcium signal transduction proteins, thereby inhibiting ER stress dependent apoptosis. Through elucidating the interaction between calcium and endoplasmic reticulum during IR injury, to confirm the signaling pathways and target molecules of Aralia elata saponins and explore the effective substance and molecular mechanism against MI/R injury.

钙稳态失衡是诱导内质网应激（ERS）发生的重要原因。通过调控钙稳态失衡进而抑制ERS介导的细胞凋亡成为药物抗MIRI的新途径。龙牙楤木系五加科楤木属植物，具有活血化瘀的功效。我们从80年代即开始了对龙牙楤木的研究，发现了其治疗冠心病、心绞痛的新用途，已获临床批件，授权了4项专利，发表12篇论文。近期我们首次发现龙牙楤木总皂苷可提高心肌细胞钙瞬变的幅度；提高SERCA活性，降低PLB 和 NCX蛋白表达。减少MIRI所致钙超载，抑制内质网应激诱导的凋亡。本课题拟在此基础上对龙牙楤木总皂苷及其主要活性成分进行基于调控钙稳态失衡抑制ERS介导MIRI保护机理研究，论证其心肌保护作用可能与直接干预SERCA等钙信号传导蛋白的表达抑制ERS，减少心肌细胞的凋亡，影响缺血再灌注时钙及内质网的交互作用，达到二者的稳态平衡有关，并确证其作用的信号通路与靶分子，探索其保护MIRI的药效物质基础及分子机制。

通过调控钙稳态失衡进而抑制内质网应激（ERS）介导的细胞凋亡成为药物抗MIRI的新途径。本项目研究发现龙牙楤木总皂苷心肌保护分子机制与调节Ca2+稳态失衡，抑制内质网应激，减少心肌细胞凋亡有关。进一步对龙牙楤木中的齐墩果烷类三萜皂苷抗MIRI进行活性筛选，发现了活性显著且收率较高的两个单体成分Araloside C和Calenduloside E，同时对其心肌缺血再灌注损伤的保护作用及分子机制进行了深入研究。结果表明，Araloside C能够改善由缺血再灌注损伤引起的心脏功能障碍、氧化应激及胞内钙超载。其抗心肌细胞收缩功能障碍及钙稳态失衡的保护作用与调控钙调蛋白 CaMKⅡ有关。进一步的分子机制发现，Araloside可以通过调节内质网应激信号通路PERK/eIF2α和ATF6信号通路，抑制内质网应激介导的凋亡途径，从而减轻缺氧/复氧所导致的心肌细胞损伤。HSP90的抑制剂17-AAG及siRNA干扰HSP90结果显示调控Hsp90-PERK-eIF2a内质网应激凋亡途径可能是Araloside C抗心肌缺血再灌的潜在靶点。另外，实验结果发现Calenduloside E抗心肌细胞收缩功能障碍及钙稳态失衡的保护作用与调控RyR等钙信号传导蛋白表达,维持心肌细胞功能有关。本项目的研究结果提示Araloside C和Calenduloside E具有防治心肌缺血再灌注损伤的潜在应用价值，同时也为龙牙楤木的临床应用及进一步开发提供了可靠的实验依据。本项目已发表相关学术论文5篇，其中包括SCI论文3篇，核心期刊论文2篇；授权国家发明专利1项；培养毕业博士研究生1名，毕业硕士研究生1名，在读博士研究生1名。