脂肪乳调控PI3K/Akt/GSK-3β信号通路逆转布比卡因心脏毒性的作用机制

Lipid emultion (LE) is an effective rescue therapy for the cardiotoxicity induced by bupivacaine (BPV), but the positive mechanisms are still unclear. It is proved that the cardiotoxicity caused by BPV is associated with uncoupling the mitochondrial oxidative phosphorylation, inhibiting respiratory chain complexes, enhancing ROS productions and inhibiting of mPTP opening. Recent studies demonstrated that rescue of BPV-induced cardiotoxicity by LE is associated with inhibition of mPTP, but lacking of positive mechanisms. The prelimary experiment we did showed that LE can ameliorate the myocardial cell apoptosis, and LE induced cardiprotection is linked with increased phosphorylation levels of Akt and GSK-3β. This study bases on the hypothesis that LE reverses BPV-induced cardiotoxicity via PI3K/Akt/GSK-3β signal pathways.The aim of our study is using the PI3K/Akt and GSK-3β specific inhibitor combined with the GSK-3β RNAi technology to confirm how LE inhibiting mPTP opening and then reversing the myocardium mitochondial indury caused by BPV via the signal pathways of PI3K/Akt/GSK-3β, to assure the impact of LE acts on the cardioprotection induced by BPV, then the exact relationship between GSK-3β and the main components of mPTP is need to be investigated，the final goal is to verify the hypothesis mentioned above in different levels, find out the exact effect target and direction of LE in reversing the cardiotoxicity caused by BPV.

脂肪乳（LE）是逆转布比卡因（BPV）心脏毒性极具前景的治疗药物，但确切机制不清。已证实BPV心脏毒性与线粒体氧化磷酸化解偶联，呼吸链复合酶抑制，ROS生成增加及mPTP开放有关。最新研究发现LE可通过抑制mPTP的开放而减轻BPV心脏毒性，但机制未明。我们预实验发现LE能够抑制BPV致心肌细胞凋亡，且磷酸化的Akt和GSK3β在LE处理后明显增高。因此我们推测，LE通过调控PI3K/Akt/GSK3β信号通路逆转BPV心肌细胞线粒体凋亡。本课题旨在通过给予PI3K/Akt及GSK3β特异性阻滞剂联合GSK3β基因沉默技术确定PI3K/Akt/GSK3β信号通路在LE抑制BPV致mPTP开放从而减轻心肌线粒体损伤中的作用，并进一步明确GSK3β与mPTP各组分之间的确切作用关系，从在体、离体、细胞及分子水平上阐明LE逆转BPV心肌毒性的作用及机制，为BPV心脏毒性的防治提供新的靶点和方向。

临床上布比卡因（BPV）常用作外周神经阻滞、硬脊膜外阻滞和蛛网膜下腔阻滞，如果误入静脉或剂量过大，则可导致严重的心血管毒性反应，有报道称脂肪乳（LE）可逆转BPV的心脏毒性，但就LE逆转BPV心脏毒性的确切机制并不清楚。所以阐明LE逆转BPV所致心脏毒性的机制，寻求有效的治疗手段，是医学领域的重要课题之一。故本项目在动物实验整体上观察脂肪乳（LE）对布比卡因（BPV）所致大鼠心肌线粒体损伤以及能量代谢的影响，进而研究BPV所致大鼠离体心脏毒性后给予LE后对心肌细胞线粒体的影响，明确PI3K/Akt/GSK-3β信号通路分子在LE救治BPV致大鼠心肌细胞凋亡模型中的表达，运用RNAi技术明确了GSK-3β在LE逆转BPV致大鼠心肌细胞毒性中的作用。实验结果显示：脂肪乳逆转布比卡因大鼠心肌毒性作用的机制与抑制线粒体通透性转换孔（mPTP）的开放有关；脂肪乳减轻布比卡因诱发大鼠心肌毒性的机制可能与改善线粒体能量代谢有关；脂肪乳通过抑制mPTP的开放逆转布比卡因所致H9C2心肌细胞毒性时能量生成的减少以及细胞凋亡率的增加； 脂肪乳逆转布比卡因致大鼠心肌细胞mPTP的开放是通过PI3K/Akt/GSK-3β信号通路来实现的。