植物性功能食品原料的内分泌干扰效应及其机制

发布时间：2018-04-24 20:39

本文选题：植物性功能食品 + 雌激素受体　；参考：《南京医科大学》2013年硕士论文

【摘要】：随着社会的发展和人们保健意识的提高，一些传统的食用食物和中草药被广泛的应用于植物性功能食品中，例如凉茶，其主要原料就有甘草、布渣叶、金银花、菊花、仙草、鸡蛋花、夏枯草等。这些植物性功能食品的发展极大地扩大了人群对各种植物化学物质的暴露范围。植物中含有丰富的化学物质，包括植物性来源的雌激素和雄激素，这些是植物体内天然的激素物质，具有一定的内分泌干涉效应。部分植物性功能食品利用植物激素的内分泌干预作用，获得相应的疾病预防作用，如大豆异黄酮预防肿瘤、骨质疏松和心血管疾病。但是对于特定的人群，例如正在生长发育的儿童、更年期妇女、乳腺癌或者前列腺癌人群来说，摄入了这些植物类激素就有可能产生潜在的风险。目前，甘草和当归就被报道具有雌激素效应，能够促进乳腺癌细胞的增长，而对其他大部分植物性功能食品（饮料）的原料的内分泌干扰效应尚缺乏了解，尤其是雄激素效应方面。所以，为了更安全的食用这些植物性功能食品，就有必要对它们是否具有内分泌干扰效应及其机制进行研究。目的：本研究采用雌激素依赖性乳腺癌细胞（MCF-7）和雄激素依赖性前列腺癌细胞(22RV1)为模型，研究植物性功能食品的原料是否具有内分泌干扰效应并探讨其机制。方法：采用MTS实验检测植物性功能食品原料提取物对细胞活性的影响；采用RT-PCR检测提取物对雌激素受体（ER）和雄激素受体(AR)靶基因的影响；通过Western blot检测提取物对ERα、ERK/1/2蛋白表达的影响和ERα及ERK1/2蛋白的分别在浆核的表达情况；应用免疫荧光方法检测ERα及ERK1/2的浆核定位；采用免疫沉淀实验检测ERα和ERK1/2蛋白是否相互结合。结果：对八种植物提取物（当归、布渣叶、甘草、金银花、菊花、仙草、鸡蛋花、夏枯草）进行筛检，在雌激素依赖性乳腺癌细胞MCF-7中，MTS实验结果显示，与对照相比布渣叶、当归提取物在一定浓度下能够增加MCF-7细胞的活性，且这种活性能够被ERα的拮抗剂ICI182，780（ICI）和MEK1/2抑制剂U0126所抑制，这些结果表明ERα和ERK1/2信号通路参与了提取物介导的细胞活性的增加；Western blot显示当归和布渣叶在MCF-7细胞中能够下调ERα蛋白的水平，且能够介导ER的靶基因pS2表达的增加，加入ICI或者U0126之后，提取物介导的pS2水平明显下降；当归和布渣叶能诱导延迟的ERK1/2激活，并呈现时间效应，当加了U0126后，这种诱导作用被阻断，而ICI对提取物介导的ERK1/2的激活没有影响，表明ERK1/2激活位于ERα介导的信号通路的上游；当归和布渣叶能够促进ERα的核转位，诱导磷酸化的ERK1/2和ERα蛋白的结合。在雄激素依赖型前列腺癌细胞22RV1中，对布渣叶、甘草和当归三种植物提取物进行筛检，布渣叶提取物与对照相比，能够促进22RV1细胞的活性，并且能够诱导雄激素受体靶基因PSA的表达，当加入雄激素受体抑制剂后，细胞活性和雄激素受体靶基因水平均明显下降，当归和甘草提取物与对照相比并没有明显改变；当加入ERK1/2的抑制剂U0126后，同样抑制了布渣叶提取物介导的细胞活性和PSA的表达。结论：当归、布渣叶具有雌激素效应，布渣叶同时还具有雄激素效应；这些提取物介导的内分泌效应的发生是通过ERα或AR介导；且延迟的ERK1/2的激活是当归、甘草和布渣叶介导的雌激素效应所需要的。
[Abstract]:With the development of society and the improvement of people's awareness of health care, some traditional food and Chinese herbal medicine are widely used in plant functional food, such as herbal tea. The main materials are Glycyrrhiza, dregs leaves, honeysuckle, chrysanthemum, fairy grass, egg flower, and Prunella vulgaris. The development of these plant functional food has greatly expanded the population to the population. The exposure range of various plant chemicals. Plants contain abundant chemicals, including phytoestrogens and androgens, which are natural hormone substances in plants and have certain endocrine interference effects. Some plant functional foods use the endocrine intervention of plant hormones to obtain corresponding diseases. Anti - effects, such as soy isoflavones, prevent cancer, osteoporosis, and cardiovascular disease. But for a particular population, such as growing children, menopause women, breast or prostate cancers, the intake of these plant hormones may lead to potential risks. Hormone effects can promote the growth of breast cancer cells, but the endocrine disrupting effects on the raw materials of most other plant functional foods (beverages) are still lacking, especially in the androgenic effect. Therefore, it is necessary to have endocrine disrupting effects and whether it is necessary to eat these plant functional foods more safely. The mechanism is studied.
Objective: in this study, estrogen dependent breast cancer cells (MCF-7) and androgen dependent prostate cancer cells (22RV1) were used as a model to study the endocrine disrupting effects of plant functional food materials and to explore its mechanism.
Methods: the effects of extracts from plant functional food materials on cell activity were detected by MTS, and the effects of extracts on the target genes of estrogen receptor (ER) and androgen receptor (AR) were detected by RT-PCR, and the effects of extracts on the expression of ER A and ERK/1/2 protein were detected by Western blot, and the ER alpha and ERK1/2 proteins were in the pulp nucleus respectively. The expression of ER and ERK1/2 was detected by immunofluorescence. The localization of ER and ERK1/2 protein was detected by immunoprecipitation assay.
Results: eight plant extracts (Angelica, dregs leaf, licorice, honeysuckle, chrysanthemum, chrysanthemum, chrysanthemum, egg flower, Xia Kucao) were screened, and in estrogen dependent breast cancer cell MCF-7, the results of MTS experiment showed that the extract of Angelica sinensis could increase the activity of MCF-7 cells at a fixed concentration, and the activity could be ER Inhibition of alpha antagonist ICI182780 (ICI) and MEK1/2 inhibitor U0126, these results suggest that ER alpha and ERK1/2 signaling pathway participates in the increase in activity of extracts mediated by extracts; Western blot shows that Angelica sinensis and cloth residue leaves can reduce the level of ER alpha in MCF-7 cells, and can mediate the increase of ER target gene pS2 expression. Or after U0126, the pS2 level of the extract was obviously decreased; Angelica and dregs leaves could induce delayed ERK1/2 activation and have a time effect. When U0126 was added, the induction was blocked, and ICI had no effect on the activation of ERK1/2, which indicated that ERK1/ 2 activated the upstream of the signal pathway mediated by ER a; Angelica sinensis. And cloth residue leaves can promote the nuclear transposition of ER A and induce the binding of phosphorylated ERK1/2 and ER alpha protein. In the androgen dependent prostate cancer cell 22RV1, the extracts of three kinds of plant extracts from the leaf, the licorice and Angelica sinensis were screened. The residue leaf extract of the cloth can promote the activity of 22RV1 cells and can induce the androgen receptor. When the target gene PSA was expressed, the level of cell activity and androgen receptor target gene decreased obviously when the androgen receptor inhibitor was added, and the extract of Angelica and Glycyrrhiza Glycyrrhiza did not significantly change compared with the control. When the inhibitor U0126 was added to ERK1/2, the cell activity and the expression of PSA were also inhibited by the leaf extract of the dregs.
Conclusion: Angelica, the leaf of dregs has estrogen effect, and the leaf of the cloth also has androgen effect; the endocrine effect mediated by these extracts is mediated by ER alpha or AR; and the delayed activation of ERK1/2 is necessary for the estrogen effect of angelica, licorice and cloth residue leaves.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R173

【参考文献】