Apelin/APJ受体在脂肪乳剂解救布比卡因心脏毒性中的作用及机制研究

Long-acting amides local anesthetics, particularly bupivacaine, are widely applied in anesthesia. Due to their significant cardiac toxicity, local anesthetics can lead to severe circulatory collapse and cardiac arrest if absorbed into bloodstream in larger dosage. And standard resuscitation does not work effectively. Our previous studies showed that lipid resuscitation of local anesthetic-induced cardiac toxicity was partially associated with "Lipid Sink", but could not be fully explained by "Lipid Sink". Similar to lipid emulsion, Apelin/APJ receptor has cardiac inotropic effect as well as myocardial protection through inhibiting the opening of mPTP and reducing ROS. However, the role of Apelin/APJ receptor on lipid resuscitation of bupivacaine-induced cardiac toxicity has not been studied or reported. This study will investigate the mechanism of Apelin/APJ receptor on lipid resuscitation and the effects of exogenous Apelin on lipid resuscitation by applying patch clamp technique, Western Blot analysis, RT-PCR, gene transfection, etc. The goal of this study is to illustrate the effects and mechanism of Apelin/APJ receptor on lipid resuscitation of bupivacaine-induced cardiac toxicity, and provide experimental basis and new methods for the treatment of local anesthetic-induced cardiac toxicity.

以布比卡因为代表的长效酰胺类局麻药在麻醉领域应用广泛，但心脏毒性突出，一旦入血量偏大，可造成严重循环衰竭，甚至心脏停搏，常规复苏非常困难。我们在前期研究中发现，脂肪乳剂对局麻药心脏毒性的救治与其脂质包裹（Lipid Sink）有关，但不能完全以"Lipid Sink"解释。Apelin/APJ受体与脂肪乳剂均具有正性肌力作用，均可通过减少ROS释放和mPTP开放实现心肌保护，但Apelin/APJ受体在脂肪乳剂解救布比卡因心脏毒性中的作用及机制国内外未见相关报道。本课题拟通过膜片钳、Western Blot、RT-PCR、RNA干扰等技术研究Apelin/APJ受体在脂肪乳剂救治中发挥作用的具体机制及外源性Apelin对脂肪乳剂救治效果的影响。通过本项目的研究，阐明Apelin/APJ受体在脂肪乳剂解救布比卡因心脏毒性中发挥的作用及机制，为局麻药心脏毒性的救治提供实验依据和新思路。

研究背景：Apelin作为七次跨膜G蛋白偶联受体APJ的内源性配体，主要调节心血管系统和液体的平衡。Apelin在心血管疾病如动脉粥样硬化、心力衰竭、高血压、心房颤动等的发生发展中发挥着重要作用，同时也是继瘦素和脂肪细胞因子后另一调节能量代谢的脂肪因子。但是Apelin/APJ系统在布比卡因中毒致心脏骤停的心肺复苏中的作用尚未见研究报道。.研究内容: 我们采用原代大鼠心肌细胞布比卡因中毒模型和大鼠布比卡因中毒心脏骤停模型，从在体、细胞和分子水平，探讨Apelin/APJ系统在布比卡因致大鼠心脏毒性中的表达变化及外源性[pyr1]Apelin-13对大鼠布比卡因心脏中毒的复苏作用及可能机制。.重要结果和关键数据：布比卡因作用于H9C2心肌细胞后，APJ和Apelin表达量都显著低于对照组（P<0.05）。外源性(Pyr1)apelin-13可提高布比卡因所致的SD大鼠心脏骤停的复苏率（BA组vs B组=100% vs 50%），APJ拮抗剂可消除外源性apelin的解救作用（BA组vs FBA=100% vs 25%）。B、BA组首次心跳时间显著短于FBA组（P<0.05）。对大鼠心脏进行CG-RNA seq发现apelin能明显改善线粒体生物合成相关蛋白。大鼠心脏BA组PGC-1α、ACC、AMPK mRNA表达显著高于S、B组，BA组AMPK、 ACC蛋白表达显著高于S、B组。布比卡因可抑制乳鼠心肌细胞搏动次数，而外源性(Pyr1)apelin-13可拮抗这种抑制，增加60min时的pAMPK/AMPK和pACC/ACC表达量，改善线粒体超微结构，调节OCR，用腺病毒抑制APJ表达后apelin的保护作用消失。Apelin-13可逆转布比卡因导致的大鼠心功能抑制；Apelin可逆转布比卡因导致的大鼠原代心室肌细胞钠通道抑制。.科学意义：动物实验结果表明(Pyr1)Apelin-13能解救大鼠布比卡因所致的心脏骤停和逆转布比卡因对乳鼠心肌细胞搏动。机制包括逆转大鼠心肌细胞钠通道抑制，通过APJ-活化AMPK/ACC-调节OCR，改善线粒体形态和功能，为心肌细胞的跳动提供能量。