基于甾体激素受体核转录调控途径白术黄酮苷AMFG-5改善高雄诱导的卵泡发育障碍的作用机制研究

As the most common ovarian follicle development disorder, polycystic ovary syndrome (PCOS) negatively affects 10% women of childbearing age worldwide. In the previous work, the applicant has conducted various studies and successfully isolated and identified an active ingredient (Atractylodes macrocephala Koidz flavonoid glycoside-5, AMFG-5) from Atractylodes macrocephala Koidz ( a Chinese medicinal herb), which can significantly improve ovarian follicle development disorders. Based above, the present study will further confirm the effects of AMFG-5 on the synthesis and metabolism of steroid hormones, transport of water and glucose uptake in the ovarian granulosa cells on the basis of rat models with PCOS and human ovarian granulosa cells--KGN. The effects of AMFG-5 on the key functional genes in the human ovarian granulosa cells will be demonstrated through RNAi, plasmids and other methods. The relationship between regulatory region sequences with the response element will be verified with bioinformatics techniques. The key binding sites for the response elements will be screened with verification of functions using a luciferase reporter system after base point mutation of DNA sequences of the response elements. The nuclear transcription regulatory pathways of steroid hormone receptors underlying AMFG-5 improving the ovarian follicle development disorder induced by hyperandrogenism will be finally demonstrated. The outcomes of the present study will propose a new theory of the pathway for Atractylodes macrocephala Koidz flavonoid glycosides and provide a scientific basis for the researches on treating PCOS with Chinese medicinal herbs. It will also provide a scientific basis for the development of new drugs with an activity of improving follicular developmental disorders.

多囊卵巢综合征（PCOS）是最常见的卵泡发育障碍性疾病，影响着10%育龄期女性的生殖健康。申请人前期对中药白术进行了多方面研究，并分离、鉴定出一种能够改善卵泡发育障碍的活性成分-白术黄酮苷（AMFG-5）。本项目在此基础上，将通过PCOS大鼠模型和人卵巢颗粒细胞系KGN研究AMFG-5对卵巢颗粒细胞甾体激素合成代谢、水转运及葡萄糖摄取的影响；利用RNAi，质粒构建等技术明确AMFG-5对颗粒细胞中关键功能基因的调节作用；利用生物信息学等技术验证调控区域序列与反应元件之间的结合关系；将寻找出的反应元件DNA序列进行逐个碱基点突变后构入荧光素酶报告系统进行功能验证，筛选出关键结合位点；最终从甾体激素受体核转录调控角度，阐明AMFG-5改善高雄诱导的卵泡发育障碍的作用机制。研究成果将提出白术黄酮苷作用通路的新理论，为中药治疗PCOS的研究提供借鉴，也为开发改善卵泡发育障碍的新药提供科学依据。

白术是一种极具开发价值的中草药。项目组从浙江天台产白术中分离并鉴定出一种能够改善卵巢卵泡发育障碍的活性成分-白术黄酮苷AMFG-5。在此基础上，本项目通过大鼠模型和人卵巢颗粒细胞系KGN进一步验证了白术黄酮苷AMFG-5对卵巢颗粒细胞的甾体激素合成代谢、水转运及葡萄糖摄取的影响；通过RNAi技术、质粒构建技术、生物信息学技术验证了反应元件中关键位点，从甾体核受体调控下游卵巢功能关键目的基因的转录活性层面，明确了白术黄酮苷AMFG-5改善卵巢卵泡发育障碍的作用机制。研究成果为开发具有改善卵泡发育障碍活性的功能食品提供科学依据，同时提出了白术黄酮苷作用通路的新理论。