We have developed an on-line detection method using acridine orange as the fluorescence probe and applied this method to rapidly identify active compounds in herbal medicines. This on-line method was equipped with a high-performance liquid chromatography tandem diode array detector, electrospray ionization-ion-trap time-of-flight mass spectrometry and DNA- acridine orange fluorescence detection （HPLC-DAD-MSn-DNA-AO-FLD）. A large amount of information could be simultaneously obtained during one run, which included HPLC fingerprint, ultraviolet spectra, total ion chromatograms, MSn data of high-resolution mass spectrometry and activity profile of each compound binding with DNA. The method also provided information on structureactivity relationships and mechanism of interaction. We used this on-line method to identify five DNA-binding activity components from Lithospermum erythrorhizon sample for the first time. The result showed that the parent nucleus of shikonin derivatives could bind with DNA. The structure-activity relationship showed that the parent nucleus of shikonin derivatives plays a major role in DNA binding, not the carboxyl group on the side chain. This simple, rapid, high precision and good stability on-line method should be useful for compound separation, structural identification and screening of DNA-binding compounds in herbal medicines.
目的:建立由于油酸大量摄入造成的HepG2细胞脂肪变性模型;发现HepG2细胞发生脂肪变性前、后转录因子DNA结合活性的差异。方法:将HepG2细胞在含2mmol/L油酸的培养基中培养24h,通过油红O染色鉴定细胞中的甘油三酯含量,并确定发生脂肪变性的程度。分别提取对照组和实验组的核蛋白,利用转录因子富集技术cat TFRE(a concatenated tandem array of the consensus transcription factor response element),同时运用深度覆盖的蛋白质组学手段,对HepG2细胞脂肪变性前、后的转录因子变化进行动态描绘和定量分析。最后,利用IPA(integrated pathway analysis)对DNA结合活性发生改变的转录因子进行功能分析。结果:对照组总共鉴定到170种转录因子,实验组总共鉴定到190种转录因子,两组总共鉴定到208种转录因子。HepG2细胞发生脂肪变性后,DNA结合活性发生变化的转录因子有101种(重复实验间差异小于2倍,不同处理间差异大于2倍),DNA结合活性加强的有67种,减弱的有34种。其中,调节糖代谢、脂代谢的关键转录因子MLX、MLXIPL的DNA结合活性减弱;NF-κB1的DNA结合活性增加,暗示了NF-κB介导的炎症反应的激活。IPA分析的结果表明,油酸激活了HepG2细胞内NRF2介导的氧化应激反应,促进了基因表达(gene expression)、细胞增殖(cell proliferation)、细胞分化(differentiation of cell)、细胞周期(cell cycle)及细胞存活(cell survival)的进程。结论:油酸诱导HepG2细胞脂肪变性会引起细胞内糖、脂代谢的紊乱,诱发炎症反应,并引起氧化损伤,但是在转录水平上,细胞通过加强脂类代谢基因的表达、减少糖类向脂肪酸的转化、促进细胞增殖及激活NRF2介导的氧化应激反应等,最终促进了HepG2细胞的存活。
