代代花活性成分对3T3-L1前脂肪细胞分化和凋亡分子调控的影响

Obesity is the main inducing factors for type II diabetes, hypertension, atherosclerosis and other metabolic diseases. And the differentiation and apoptosis of preadipocyte and its disorder in metabolic pathways are closely related to obesity. Flower of Citrus aurantium L. var. amara Engl, an edicine edible species, has been proved to have good anti-obesity and health care effects after a long practice. Two monomeric compound, 5-hydroxy-6,7,3', 4'-tetramethoxy flavones (HTF) and limonexic acid (LA) that can obviously inhibit the proliferation of pre-adipocytes and tumor cells, has been isolated and identified from C. aurantium var. amara. in our previous work. This project will further use HTF and LA to intervene the differentiation and apoptosis of 3T3-L1 preadipocyte of mouse embryos, to investigate their influence on 3T3-L1 preadipocyte apoptosis, and at the same time, to analysis the preadipocyte's differentiation, apoptosis and the gene expression levels of the key enzymes and proteins and other regulators in different metabolic pathways, to determine the effect of the two compounds against the preadipocyte energy metabolism, cell apoptosis, fatty acid oxidation and esters degradation, etc., so as to reveal the main pathways and regulators that HTF and LA would act on. This project will have the obvious originality in the research on anti-obesity active substance from C. aurantium var. amara, laying the foundation for the study on the metabolic pathways and targets of the HTF and LA, and will show inspiration and demonstration significance in the development of Chinese medicine edible species and their new functional factors.

肥胖是II型糖尿病、高血压、动脉硬化等代谢疾病的主要诱因，而前脂肪细胞的分化、凋亡及其代谢途径紊乱与肥胖症密切相关，药食同源品种代代花经过长期实践证明具有良好的减肥保健功效。课题组前期从代代花中获得的两个单体化合物5-羟基-6,7,3',4'-四甲氧基黄酮（HTF）和Limonexic acid（LA）对前脂肪细胞和肿瘤细胞增殖有明显抑制作用。本项目进一步用HTF和LA对小鼠3T3-L1前脂肪细胞的分化和凋亡进行干预，考察两者对前脂肪细胞凋亡的影响，同时分析前脂肪细胞分化、凋亡和代谢途径的关键酶和蛋白等调控因子的基因表达水平，确定两个化合物对前脂肪细胞能量代谢、细胞凋亡、脂肪酸氧化和酯类降解等途径的作用效果，揭示HTF和LA作用的主要途径和调控因子。本项目在代代花减肥活性物质研究方面有原创性，为研究两个化合物作用的路径和靶点奠定基础，对我国药食同源品种及其新型功能因子的开发有示范借鉴意义。

代代（Citrus aurantium L. var. amara Engl）系芸香科（Rutaceae）柑橘属（Citrus）酸橙（Citrus aurantium L.）的变种之一。代代花是代代的干燥花蕾，有关代代花化学成分和药理作用的研究报道很少，对其化学成分及其活性的研究，为其在更多领域应用提供一定的理论依据，有利于其资源的进一步开发。. 本项目采用有机溶剂萃取法对其进行分级萃取，获得代代花乙醇提取浸膏，经过大型硅胶柱，Sephadex LH-20 葡聚糖凝胶柱，分析型柱层析，再结合溶剂重结晶等方法，对其进行分离纯化，得到的化合物，利用理化性质，质谱、核磁解析如1H-NMR和13C-NMR等技术，鉴定其中2个首次从该植物中分离得到的化合物为：5-羟基-6，7，3’，4’-四甲氧基黄酮（5-Hydroxy-6,7,3',4'-tetramethoxyflavone）和Limonexic acid. 并对这两种化学物进行了抗氧化、抗癌、抗肿瘤、抗炎、抗菌和抗病毒的活性研究。. 本项目研究了代代花总黄酮抑制3T3-L1细胞增殖及诱导其凋亡的作用。不同浓度的代代花总黄酮作用于3T3-L1细胞24h之后，采用MTT法检测代代花总黄酮对细胞增殖的抑制作用；使用倒置显微镜观察细胞形态学的变化；Annexin V-EGFP/PI 标记检测细胞凋亡率；PI标记法检测了代代花总黄酮对细胞周期的影响；活性氧（ROS）试剂盒检测细胞内ROS水平；荧光定量PCR检测了凋亡相关基因的mRNA水平表达。结果表明，高浓度（300μg/mL，400μg/mL)的代代花总黄酮可显著抑制3T3-L1细胞增殖，且细胞形态发生了明显的变化，并显著升高了细胞内ROS的浓度。细胞凋亡实验结果显示，代代花总黄酮可诱导3T3-L1细胞早期凋亡和晚期凋亡。而细胞周期实验则表明，处理后的3T3-L1细胞G0/G1期细胞数比例从下降，而相应的S期细胞数比例呈小幅度的增加，G2/M期细胞数比例变化不明显。凋亡相关基因p21及p53的mRNA表达明显升高及促凋亡基因bax与抗凋亡基因bcl-2比例的升高使3T3-L1进入细胞凋亡程序。