目的 应用双向电泳和质谱技术研究漆黄素对氧化低密度脂蛋白（ox-LDL）诱导的泡沫细胞蛋白质组的影响，分析漆黄素抑制泡沫细胞形成的分子机制。方法 采用MTT法检测ox-LDL对RAW264.7细胞及漆黄素对泡沫细胞活力的影响，化学法检测细胞内胆固醇酯（CE）的量，筛选合适的ox-LDL和漆黄素处理浓度。油红O染色法检测漆黄素对泡沫细胞内脂质累积情况的影响，并用双向电泳法建立漆黄素处理前后细胞蛋白质组图谱，质谱法鉴定差异表达的蛋白质；Western blotting法验证细胞色素b5（Cyt b5）的表达情况。结果 20μg/mL ox-LDL可以成功诱导泡沫细胞，100μg/mL漆黄素干预后，泡沫细胞内CE明显下降，脂质累积减少。蛋白质组学结果表明，漆黄素处理泡沫细胞后，Cyt b5、谷胱甘肽S-转移酶、NADH脱氢酶（泛醌）黄素蛋白2、Prelamin A/C、葡萄糖调节蛋白78、热休克蛋白60、Supervillin、胆固醇酰基转移酶表达量增加，而钙网蛋白、转录延伸因子B、蛋白核受体表达量下降。结论 漆黄素可能通过增强细胞抗氧化及抗炎能力，减弱细胞应激反应、降低细胞内胆固醇堆积、调节细胞凋亡及提高免疫能力等途径起到抗动脉粥样硬化的作用。

Objective To investigate the effect of fisetin(FIS) on foam cells by two-dimensional electrophoresis and mass spectrometry technology, and analyze the molecular mechanism of its inhibition. Methods MTT method was used to detect the effect of ox-LDL on viability of RAW264.7 cells and fisetin on foam cells separately as well as chemical method was used to detect intracellular cholesterol ester, screening appropriate ox-LDL, and FIS concentration. Oil red O staining displayed accumulation of lipids changed by FIS in the foam cells. Then established proteomic maps of foam cells before and after treatment with FIS by bi-directional electrophoresis, mass spectrometry was adopted to identify differences in the expression of proteins. The expression of Cyt b5 was verified by Western blotting. Results In our study, 20 μg/mL ox-LDL can successfully induce foam cells, as well as intervention of 100 μg/mL FIS would significantly decrease cholesterol ester and lipid accumulation within the foam cells. Proteomic experiment showed that foam cells treated by FIS had a lower expression of nuclear receptor, calreticulin and transcription elongation factor B, higher levels of glutathione S-transferase, cytochrome b5, Prelamin A/C, NADH dehydrogenase(ubiquinone) 2 flavin protein, lecithin-cholesterol acyltransferase, 78 kDa glucose regulation protein, supervillin, and heat shock protein 60. FIS can significantly improve the expression of Cyt b5 by WB. Conclusion These data suggest that FIS can significantly inhibit foam cells formation by enhancing the cellular antioxidant and anti-inflammatory capabilities, reducing cellular stress response, as well as decreasing accumulation of intracellular cholesterol, regulation of apoptosis and enhanced immunity to prevent atherosclerosis.

国家自然科学基金资助项目（81102841）