祛痰化瘀方剂调控巨噬细胞内氧化应激与促炎因子产生稳定易损斑块的机制

Inflammation and oxidative stress are the crucial mechanism that involve in vulnerable plaques. Macrophages in plaques produce a large number of reactive oxygen species and proinflammatory factors. Recently research shows that the PKC/cPLA2/AA signaling pathway plays the key role in network control of oxidative stress and proinflammatory factor production. Arachidonic acid (AA) can induce the production of NADPH oxidase and, as an inflammatory medium, series of proinflammatory factor, thereby the mentioned pathway is an important intervention target. The reducing phlegm and removing dampness method can improve clinical symptoms in patients with atherosclerosis, inhibiting inflammatory reaction and stable plaques. Previous studies showed the reducing phlegm and removing dampness method can reduce AA production in macrophages. In this basis, we propose a hypothesis that the reducing phlegm and removing dampness method can regulate macrophage oxidative stress and proinflammatory factor production through the PKC/cPLA2/AA signaling pathways in order to achieve stable plaques. This research intends to use the reducing phlegm and removing dampness method intervention, on the basis of related animal and cell models, to analysis of the action mechanism of the reducing phlegm and removing dampness method in the process of reactive oxygen species and proinflammatory factor and the related material production from PKC/cPLA2/AA signaling pathways, in order to clarify the drug targets, provide new therapies for clinical treatment.

炎症与氧化应激是动脉粥样硬化斑块易损的关键机制，斑块内巨噬细胞（Mф）是促炎因子与氧化应激的主要来源。近期研究发现氧化应激与促炎因子的产生受PKC/cPLA2/AA信号通路共同调控。花生四烯酸（AA）不仅是调控氧化应激关键酶--NADPH氧化酶活性的重要因素，其代谢产物又是炎性介质，可放大炎症反应，因此上述通路为重要的干预靶点。祛痰化瘀方可通过抑制炎症反应、抗氧化减缓动脉粥样硬化进程并稳定斑块，且前期工作显示祛痰化瘀方能下调Mф内AA的水平。据此我们提出假设：祛痰化瘀方可通过PKC/cPLA2/AA信号通路调控Mф氧化应激与促炎因子的产生达到稳定斑块的作用。本研究拟以祛痰化瘀方干预apoE-/- 小鼠动脉粥样硬化模型和Mф源性泡沫细胞模型，基于PKC/cPLA2/AA信号通路观察活性氧与促炎因子产生过程中相关物质的变化，揭示祛痰化瘀方稳定斑块作用的分子机制及具体靶点，为临床治疗另辟新径。

动脉粥样硬化是以富含脂质的巨噬细胞在动脉内膜蓄积为特点的一种慢性炎症性疾病。蓄积的巨噬细胞发挥多种功能促进动脉粥样硬化病变发展，因此，巨噬细胞被视为抗动脉粥样硬化的主要靶点。丹蒌片是一种长期应用于临床治疗心血管疾病的中成药。本研究利用ApoE基因敲除小鼠模型和巨噬细胞源性泡沫细胞模型探究丹蒌片醇提物治疗动脉粥样硬化的机制。采用网络药理学分析预测丹蒌片与动脉粥样硬化相关的潜在靶点，筛选出了93个相关基因，设计了对应的miRNA芯片。高脂干预APOE-/-小鼠并采用丹蒌片干预，分组如下：对照组（C57BL/6小鼠，正常饮食）、模型组（ApoE-/-小鼠，高脂饮食饲养20周）、丹蒌片组（ApoE-/-小鼠，高脂饮食饲养20周，其中后12周灌胃丹蒌片治疗）；利用mRNA芯片和油红O染色研究主动脉斑块内AS相关指标表达情况及脂质含量；采用mRNA芯片、炎症因子芯片检测 RAW264.7细胞中炎症因子和胆固醇流出情况，并探索潜在机制。结果显示，丹蒌片通过抑制NF-kB通路减少炎症因子表达，通过激活PPARα/ABCA1通路促进胆固醇流出。此外，激活NF-κB通路和抑制PPARα/ABCA1通路后，丹蒌片抗炎作用及促胆固醇流出作用消失。综上所述，本研究表明丹蒌片在巨噬细胞内通过抑制NF-κB通路抗炎和激活PPARα/ABCA1通路促胆固醇流出发挥抗动脉粥样硬化作用。