蜂胶黄酮及其单体对巨噬细胞内质网应激凋亡途径的调控作用及机制

Endoplasmic reticulum stress (ERS)-mediated apoptosis in macrophages has been demonstrated to contribute to the instability of atherosclerotic plaques. We and other groups have found that propolis flavonoids and quercetin, a monomer of ?avonoids in propolis, protect macrophages and vascular endothelial cells from apoptosis stimuli, but the underlying mechanisms are not elucidated clearly. The studies from neuronal and intestinal epithelial cells have shown that propolis extract and quercetin inhibit apoptosis induced by ERS inducers. Therefore, we assume that propolis flavonoids and its monomers may regulate macrophage apoptosis and play an anti-atherosclerotic role through suppressing ERS-mediated apoptosis pathway. This study is designed to investigate the effects of propolis ?avonoids and its monomers, such as quercetin and pinocembrin, on the apoptosis in macrophages induced by oxidized low density lipoprotein (ox-LDL) or tunicamycin (a classical ERS inducer) and on the advanced atherosclerotic lesions of apoE-knockout mice. The ERS-mediated apoptosis pathways, which include C/EBP homologous protein (CHOP), c-Jun amino-terminal kinase (JNK) and caspase-12 pathways, are explored via Western blotting, immunohistochemistry, real-time PCR, and so on. This work will explain the diverse anti-atherosclerotic mechanisms of propolis and its active constituents, and shed a new light on the study of pharmacological mechanisms of flavonoids.

内质网应激(ERS)凋亡途径介导巨噬细胞凋亡并影响动脉粥样硬化(AS)斑块稳定性。我们既往研究和文献报道证实蜂胶黄酮及其单体槲皮素可抑制血管内皮和巨噬细胞凋亡，但其机制尚未完全阐明。来自神经和肠上皮细胞的研究表明蜂胶和槲皮素可抑制ERS诱导剂所诱导的细胞凋亡。因此我们假设蜂胶黄酮及其单体通过抑制ERS凋亡途径调控巨噬细胞凋亡，发挥其抗AS作用。本项目拟在氧化低密度脂蛋白和ERS诱导剂衣霉素诱导的巨噬源性泡沫细胞模型和ERS模型以及apoE-/-小鼠AS模型上，采用免疫印迹、免疫组化、实时定量PCR等方法，研究蜂胶黄酮及其单体槲皮素和乔松素对C/EBP同源蛋白(CHOP)、c-Jun氨基末端激酶(JNK)、caspase-12等ERS凋亡途径相关分子表达和活性的影响及其与巨噬细胞凋亡之间的关系，以进一步阐明蜂胶及其有效成分抗AS作用机制，并为黄酮类化合物药理作用机制研究提供新思路。

巨噬源性泡沫细胞和血管内皮细胞损伤和凋亡在动脉粥样硬化发生发展中起关键作用，氧化低密度脂蛋白(ox-LDL)是其主要原因。本课题在ox-LDL诱导的巨噬源性泡沫细胞模型、内皮细胞损伤模型和衣霉素（TM）诱导的RAW264.7细胞内质网应激（ERS）模型以及apoE-/-小鼠动脉粥样硬化模型上，探讨了内质网应激在动脉粥样硬化发病及防治机制中的作用。研究结果显示：（1）内质网应激信号途径介导巨噬源性泡沫细胞形成和凋亡；（2）蜂胶黄酮及其单体通过抑制内质网应激和氧化应激途径减轻巨噬源性泡沫细胞和血管内皮细胞凋亡；(3) 载脂蛋白AI模拟肽D4F通过抑制内质网应激和氧化应激途径减轻巨噬源性泡沫细胞和血管内皮细胞凋亡；（4）ox-LDL通过CD36介导的氧化应激诱导巨噬细胞自噬。上述研究结果表明内质网应激信号途径参与动脉粥样硬化的发生发展，而蜂胶黄酮及其单体和载脂蛋白AI模拟肽可通过抑制该信号途径减轻动脉粥样硬化进展，因此内质网应激有望成为动脉粥样硬化防治的新的干预靶点。