大黄素介导的声动力治疗通过裂解巨噬细胞微管诱导细胞凋亡从而稳定动脉粥样硬化斑块

One of the mechanisms of atherosclerosis is inflammation. The infiltration of macrophages will lead to vulnerable plaque, which can trigger acute coronary syndrome. It was discovered that sonodynamic therapy mediated by emodin(emodin-SDT) could stabilize atherosclerotic plaque; there was a synergistic action of emodin and ultrasound exposure to induce the cleavage of cytoskeleton and the apoptosis of macrophages; emodin scattered in cytoplasm and produced reactive oxygen species(ROS) under ultrasound exposure. Why does emodin-SDT can lead to the cleavage of cytoskeleton and induce the apoptosis of macrophages? The subunits of tubulin fold and form hydrophobic regions. As a hydrophobe, emodin might insert into the hydrophobic regions to oxidize binding sites under ultrasound exposure, and the oxidation of amino acids would change the space conformation of tubulin, then the tubulin couldn't polymerize to form microtubules; at the same time, the dysfunction of mitochondrial respiratory chain caused by ROS would lead to the decrease in ATP, and the tubulin couldn't polymerize for lack of energy, and moreover, the cleavage of microtubules and the decrease in ATP would promote each other; consequently, cell apoptosis took place through the mitochondrial pathway. The research works will check these hypothesis with cells and animal models by immunohistochemistry, immunofluorescence, western blot, and so on. The research will contribute to the mechanism of emodin-SDT stabilizing atherosclerotic plaque, thus to bring about a new selection for sonosensitizers.

动脉粥样硬化发病机制之一是炎症，巨噬细胞在斑块大量浸润会致其不稳定，引发心血管急性事件。前期工作发现大黄素介导的声动力治疗可以稳定兔动脉粥样硬化斑块；大黄素与超声波协同作用于巨噬细胞，引发细胞骨架断裂，诱导细胞凋亡；大黄素分布在细胞微管中，在超声辐照下产生活性氧。大黄素介导的声动力治疗为何能够引发巨噬细胞骨架断裂、诱导细胞凋亡都尚未得知。假设大黄素嵌入微管蛋白亚基折叠后形成的疏水区中，在超声辐照下氧化结合位点，氨基酸氧化引发微管蛋白空间构象改变，后者不能多聚化形成微管；或活性氧直接干扰线粒体呼吸链，导致ATP不足，微管蛋白多聚化受阻，且微管解聚与ATP不足相互促进；进而激活线粒体通路，细胞凋亡。拟利用细胞及动物模型，采取免疫组化、免疫荧光、蛋白质免疫印迹等方法验证上述假设。该研究有助于揭示大黄素-声动力稳定动脉粥样硬化斑块的机制，且为声敏剂的选择提供新的对象。

项目背景：动脉粥样硬化由于斑块的不稳定会发生一系列恶性事件，包括恶性心律失常、心脏骤停、甚至猝死，目前医学界致力于稳定斑块的各种医疗方法。声动力治疗正是在这一背景下的新尝试。针对各种声敏剂的探索也在进行中。前期工作证实大黄素具有声敏剂效应，体内实验证实其介导的声动力治疗稳定家兔及apoE-/-小鼠动脉粥样硬化斑块，体外实验发现大黄素-声动力治疗裂解巨噬细胞微管、诱导巨噬细胞凋亡。大黄素在超声辐照下可以产生活性氧，进而产生一系列反应。自活性氧产生到微管发生裂解，细胞凋亡，这中间发生了哪些变化是此次立项的目的，旨在研究微管裂解之前的细胞内变化。.主要研究内容：⑴动物实验检验预实验中的发现：大黄素-声动力稳定动脉粥样硬化斑块；⑵细胞实验对大黄素-声动力诱导巨噬细胞凋亡的机制进行探索：①巨噬细胞微管氧化；②ATP合成不足；③微管解聚与ATP不足互相促进；④通过线粒体通路诱导细胞凋亡。.重要结果.⑴动物实验.①大黄素-声动力缩小家兔动脉粥样硬化斑块，减少巨噬细胞负荷，诱导巨噬细胞凋亡；.②大黄素-声动力抑制apoE-/-小鼠动脉粥样硬化斑块中的炎症反应；.⑵细胞实验.大黄素-声动力通过诱导微管蛋白氧化导致微管裂解，通过线粒体途径诱导细胞凋亡。.关键数据.⑴动物实验：大黄素-声动力稳定家兔动脉粥样硬化斑块，减少巨噬细胞负荷，诱导细胞凋亡；apoE-/-小鼠动脉粥样硬化斑块炎症因子表达减少；.⑵细胞实验：①大黄素90%分布于细胞微管中；②大黄素-声动力诱导活性氧生成增多；③大黄素-声动力引发微管氧化；④大黄素-声动力使细胞ATP合成不足；⑤大黄素-声动力引发线粒体膜电位下降。.科学意义：有关声敏剂的探索一直没有终止，大黄素是传统中药，用于临床已经多年，易于获得，其安全性可靠，其声敏效应的开发基于原有药理学、毒理学基础之上，探索其作用机制有利于其作为新型声敏剂今早广泛应用于临床研究。