AS斑块铁沉积与槲皮素干预：巨噬细胞Hepcidin自分泌信号调控

"Iron hypothesis" is still highly questioned in spite of common iron deposition in atherosclerosis (AS) plaque. Based on the new finding of hepcidin autocrine from macrophages, our project is designed to explore systematically the pathophysiological significance for AS progression and potential signaling pathways of hepcidin autocrine following a deep characteristic analysis on iron deposition in the presence of AS, limited research on autocrine signaling pathway of macrophages induced by bacterial infection but emerging studies and recent development on the regulatory pathways implicated in hepatic hepcidin expression. The potential signaling pathways, including BMPs/SMAD, endoplasmic reticulum stress, toll-like receptors, hypoxia inducible factor, and etc will be studied and verified in vivo and in vitro by using (local) gene transfection or silence, signaling activation or inhibition, double fluorescent staining for cellular compartmentalization through laser scanning co-focal technique, chromatinImmunoprecipitation, electrophoretic mobility shift assay and so on. In addition, naturally occurring quercetin is selected for AS intervention due to its target accumulation into plaque macrophages, beneficial iron-chelating and anti-AS activities. The effect and potential target of quercetin on hepcidin autocrine from plaque macrophages will be discussed to elucidate the molecular mechanisms of quercetin against AS from a new perspective. Thus, the project will not only help to further clarify the underlying reason and pathological consequence of focal iron deposition in the presence of AS and enrich "iron hypothesis", will also provide a new target for the development and application of phytochemicals and nutritional intervention for AS.

尽管动脉粥样硬化（AS）斑块铁沉积普遍，但"铁假说"仍备受置疑。本项目拟以新近发现的巨噬细胞Hepcidin自分泌为切入点，结合肝脏Hepcidin调控通路的新近进展，以ApoE-/-高脂AS模型和ox-LDL诱导活化的巨噬细胞为对象，借助基因转染与沉默、通路激动与阻断、转录因子/启动子结合活性分析等方法，对斑块巨噬细胞Hepcidin自分泌及BMPs/SMAD、ERS、TLRs、HIF等潜在通路层层剖析和验证，阐明其病理生理学意义与信号调控通路。在此基础上，选择可天然靶向富集于AS斑块巨噬细胞且具有良好铁螯合和抗AS效应的槲皮素进行干预，分析其对巨噬细胞Hepcidin自分泌的调控及潜在的作用靶点，从全新的角度深入揭示其拮抗AS的分子机制。本项目的实施，不仅有助于进一步阐释AS局灶性铁沉积特征和病理学意义、丰富AS"铁假说"，还将为AS的营养干预及植物化学物的开发应用提供新的靶点。

动脉粥样硬化（AS）斑块铁沉积普遍，但“铁假说”仍备受质疑。本项目以新近发现的巨噬细胞Hepcidin自分泌为切入点，以斑块巨噬细胞泡沫化为中心，利用高脂膳食喂养ApoE-/-小鼠建立AS模型，借助基因沉默、信号通路激活与阻断等分析方法，结合细胞水平分子实验系统全面分析和验证，阐明巨噬细胞Hepcidin自分泌在AS发生发展中的病理生理学意义与信号调控通路。在此基础上，我们利用高铁膳食喂养建立铁过载模型，在ApoE-/-小鼠中对“铁假说”进行了验证，借助肝脏定量蛋白组学研究，通过对差异表达蛋白相关生物学过程及信号通路的分析及验证，结合各项生化指标检测，阐明了不同于AS斑块处局灶性铁沉积加速斑块形成的有害作用，系统性铁过载通过损害肝脏脂代谢而表现出AS斑块形成减少的可能分子机制。经过4年的努力，取得的主要研究成果有：1、氧化型低密度脂蛋白通过激活TLR4/NF-κB信号通路诱导人和小鼠AS斑块巨噬细胞Hepcidin自分泌，并引发巨噬细胞铁沉积及泡沫化；2、巨噬细胞铁沉积可反向激活TLR4/NF-κB信号通路，并由此通过Hepcidin自分泌形成铁沉积与TLR4/NF-κB信号通路激活之间的恶性循环，进一步加剧铁沉积和脂质蓄积；3、巨噬Hepcidin自分泌诱导的细胞内铁沉积可引发CYP27A1/27-羟固醇轴调节障碍而导致胆固醇稳态失衡，从而加速细胞泡沫化。4、与对照组相比，高铁膳食喂养建立的铁过载ApoE-/-小鼠血脂降低，未见明显主动脉斑块沉积，表明在此模型中“铁假说”并不成立。5、肝脏定量蛋白组学研究结果表明系统性铁过载通过抑制肝脏脂肪酸摄取及转运相关蛋白表达，诱发严重肝脏脂代谢异常。6、槲皮素通过抑制衰老细胞的形成发挥AS保护作用。. 本研究的开展，不仅深入探讨了AS斑块巨噬细胞Hepcidin自分泌发生的分子机制及其在巨噬细胞铁沉积及泡沫化发生发展中的作用机制，同时发现并阐明了系统性铁过载通过损害ApoE-/-小鼠肝脏脂代谢而“缓解”AS的相关分子机制，在极大程度上丰富了“铁假说”。进一步发现并证实槲皮素有效抑制衰老细胞形成的作用，为AS的预防和治疗提供了充分的理论依据并开辟了新的路径。