Na/K 泵α1亚基介导哇巴因心肌毒性的分子机制研究

Cardiac glycosides (CGs) have the disadvantage of a relatively low therapeutic index, which increases the likelihood for toxic manifestations following their administration, representing a major limitation to their use in heart failure. Na/K pump is the pharmacological receptor for CGs. The mechanism whereby CGs cause a positive inotropic effect and cardiotoxicity is still not completely clear. Recent experiments have shown that the cardiotoxicity effect of ouabain is mediatde by α1 isoform of Na/K pump, and the first and second transmembrane region (H1-H2 and H3-H4) is currently considered as an important specific binding site for ouabain. Our previous study suggest that SSA78, which can recognize and detect the Hl-H2 domain of Na/K pump α1 isoform, may have a protective effect in ouabain-induced cardiotoxicity contraction. In this study, the specific binding peptide of Na/K pump α1 subunit H3-H4 extramembrane domain was screened from phage display random peptide libraries, which can block the combination of ouabain and Na/K pump with that SSA78. We will investigate the role and molecular mechanism of on ouabain-induced cardiotoxicity by measurement of Na/K pump activity, the rise of [Ca2+]i and contraction of the cardiomyocytes induced by ouabain, and SR-leak was also detected, which lay the foundation for the further exploring the possible interaction mechanism of Na/K pump with ouabain and give light to the discovery and development of new high-efficiency and low toxicity preparation.

强心苷类药物(CGs)治疗心衰因其治疗窗窄使临床应用受到很大限制。心肌Na/K泵是强心苷作用的靶点，然而强心苷与Na/K泵相互作用的机制仍有待精确阐明。目前认为Na/K泵的α1亚基与哇巴因的心脏毒性有关，且Na/K泵第一个膜外区H1-H2和第二个膜外区H3-H4是哇巴因可能的结合位点。我们前期研究发现利用制备的Na/K泵α1亚基H1-H2膜外区抗体(SSA78)可以减少哇巴因引起的心肌细胞收缩力毒性。因此，本申请拟应用噬菌体随机肽库技术筛选特异性Na/K泵H3-H4膜外区结合肽，与抗体SSA78同时封闭上述两个结合位点，有效拮抗哇巴因对Na/K泵的抑制作用，通过比较封闭前后哇巴因对心肌细胞Na/K泵活性影响、细胞收缩力、细胞内钙、肌浆网钙泄漏等以及整体动物的变化，更深入地揭示Na/K泵与哇巴因的可能作用机制，为临床研制高效低毒的抗心衰药物提供理论和实验依据。

强心苷类药物(CGs)临床治疗心衰因其治疗窗窄使应用受到很大限制。心肌钠泵是强心苷作用的靶点，然而强心苷与钠泵相互作用的机制仍有待精确阐明。目前认为钠泵的H1-H2及H3-H4膜外区是哇巴因（Ouabain，OUA）可能的结合位点。首先利用作用在心肌钠泵α1 亚基H1-H2膜外区抗体SSA78及以钠泵α1亚基H3-H4膜外区氨基酸序列作为靶蛋白，从噬菌体随机肽库中筛选能与之特异性结合的多肽为工具，通过比较封闭前后哇巴因对心肌细胞钠泵活性的影响，结果表明没有完全取消高浓度哇巴因对钠泵的抑制，说明尚有其他结构域参与哇巴因与钠泵的结合。其次利用作用在心肌钠泵α2亚基H1-H2膜外区结构域的多克隆抗体WJS为工具，封闭该结合位点，有效拮抗低浓度哇巴因对Na/K泵的抑制作用，通过比较封闭前后哇巴因对心肌细胞钠泵活性影响、细胞收缩力、细胞内钙的变化，结果表明钠泵α2亚基抗体取消高亲和力钠泵电流和低浓度OUA所致的心肌细胞收缩力增强作用，表明高亲和力钠泵α2亚基介导OUA的治疗作用。最后，通过给予不同信号转导抑制剂检测究竟是哪些信号转导通路参与治疗浓度哇巴因的正性肌力作用。我们的实验结果表明钠泵/Src/EGFR/Ras/ROS信号途径参与了低浓度哇巴因的正性肌力作用。说明强心苷的正性肌力作用中，既有钠泵的离子泵功能参与，也有其信号转导功能参与，而低浓度（nM）强心苷的正性肌力作用与钠泵的离子泵功能无关，可能仅涉及了钠泵的信号传导功能。总之，本实验利用钠泵膜外区特异性抗体，从细胞、分子水平比较封闭前后哇巴因对钠泵功能的影响及可能作用的机制。明确该膜外区参与哇巴因正性肌力作用的机制，为临床治疗心衰采用新的治疗手段提供了新途径，同时也为研发高效低毒的药物提供理论和实验依据。

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