单核-巨噬细胞在"痰瘀互结"致动脉粥样硬化的作用及丹蒌片干预机制研究

Atherosclerosis(AS) is a serious disease hazards to humankind. Monocyte-macrophages play key roles on AS formation. Intermingled Phlegm and Blood Stasis are important etiological and pathological factors in Traditional Chinese Medicine theory, closely related with inflammation.Preliminary data showed that Dan-Lou Tablets took good performance in preventing and treating AS. However,the exact mechanisms are still unclear up to now. Our hypothesis is that Intermingled Phlegm and Blood Stasis can promote the abnormal differentiation of monocyte-macrophages and accelerate the formation of foam cells, resulting in AS.Also we hypothesize that Dan-Lou Tablets can prevent AS by regulating the abnormal differentiation of monocyte-macrophages and inhibit the formation of foam cells. The AS model of Intermingled Phlegm and Blood Stasis will be established by using ApoE -/- mice with carotid artery injury.The expressions and mechanisms of monocyte-macrophage subtypes will be systematically studied by taking flow cytometry and immunohistochemistry methods. Furthermore,we will explore the effects and mechanisms of Dan-Lou Tablets on the differentiation of monocyte-macrophages. Macrophages and foam cells will be cell-cultured, and the effects of Dan-Lou Tablets on the secretion of inflammatory factors and the macrophages foaming process will be studied. With this project going on, it will explore the secrets of how AS formed from Intermingled Phlegm and Blood Stasis, and will provide a novel theory and a potential pharmaceutical target in preventing and treating AS.

动脉粥样硬化（AS）是严重危害人类健康的重要疾病，单核-巨噬细胞在AS发生中发挥至关重要的作用，"痰瘀互结"是AS发生的重要病因病机，与炎症反应密切关系，前期研究发现，活血化痰中药丹蒌片具有良好的抗AS作用，但其确切机制尚未阐明。我们推测"痰瘀互结通过促使单核-巨噬细胞的异常分化，加速巨噬细胞泡沫化过程，导致AS的发生；丹蒌片通过抑制单核-巨噬细胞异常分化和巨噬细胞泡沫化过程，而发挥防治AS的作用"。为证实该假说，本研究以Apo-E基因敲除小鼠颈动脉损伤方法构建痰瘀互结型AS模型，通过流式细胞仪和细胞免疫组化技术，探讨单核-巨噬细胞亚型的表达规律及其作用机制，同时观察丹蒌片对AS模型中单核-巨噬细胞亚型表达的调节；构建巨噬细胞和泡沫细胞，观察观察丹蒌片对巨噬细胞炎症因子释放和泡沫化的影响及机制。本研究的开展将进一步揭示痰瘀互结致AS的发生机制，为其有效防治提供新思路和药物作用的新靶点。

背景：动脉粥样硬化（atheroscleosis, AS）是严重危害人类健康的重要疾病，单核-巨噬细胞在AS发生中发挥至关重要的作用，“痰瘀互结”是AS发生的重要病因病机，与炎症反应密切关系，前期研究发现，活血化痰中药丹蒌片具有良好的抗AS作用，但其确切机制尚未阐明。.目的：从整体和细胞、分子水平，阐明活血化痰中药丹蒌片通过调节单核—巨噬细胞异常分化，抑制巨噬细胞泡沫化过程，而发挥防治AS的作用，为AS的防治提供一种新思路和药物作用新靶点。.方法：本研究以ApoE基因敲除小鼠颈动脉损伤方法构建痰瘀互结型AS模型，通过流式细胞仪和细胞免疫组化技术，探讨单核-巨噬细胞亚型的表达规律及其作用机制，同时观察丹蒌片对AS模型中单核-巨噬细胞亚型表达的调节；构建巨噬细胞和泡沫细胞，观察丹蒌片对巨噬细胞炎症因子释放和泡沫化的影响及机制；构建小鼠缺血再灌注损伤模型，观察丹蒌片对缺血再灌注损伤小鼠模型的心肌保护作用。.结果：（1）丹蒌片能够减少ApoE-/-小鼠动脉粥样硬化模型的斑块形成，并降低血浆的LDL-C/TG/TC。丹蒌片各组及阿托伐他汀组小鼠的主动脉病变面积则逐步减轻，丹蒌片能减少巨噬细胞吞噬oxLDL,抑制巨噬细胞的泡沫化。丹蒌片能够下调巨噬细胞的清道夫受体CD36和血凝素样氧化低密度脂蛋白受体LOX-1的基因表达，上调ABCA1的基因表达。.（2）CD68免疫组化提示丹蒌片呈剂量依赖的减少斑块病变的巨噬细胞浸润；丹蒌片能降低AS模型小鼠脾细胞悬液中Th1-IFN-的百分比含量，与模型组相比，丹蒌片能下调炎症细胞因子IFN-和TNF-的mRNA的相对表达量，下调RAW264.7细胞泡沫化后TLR4的mRNA的相对表达量，并能减少泡沫细胞的炎症因子TNF-的蛋白量。.（3） 在小鼠心肌缺血再灌注损伤研究中发现，丹蒌片组和缺血预适应组的心肌梗死面积低于模型组（P<0.05），且丹蒌组心律失常发生率低于模型组。.结论：丹蒌片能减轻AS的斑块形成，降低血脂水平，抑制巨噬细胞泡沫化；能减少AS斑块中巨噬细胞的浸润，下调炎症细胞因子IFN-/TNF的表达及炎症受体TLR4的表达，并对小鼠心肌缺血再灌注损伤有明显的保护作用。