月见草油靶向FOXO1基因改善PCOS胰岛素抵抗的作用机制

Objective To observe the effect of the treatment of evening primrose oil on insulin resistance of polycystic ovary syndrome. To study the related mechanism of the forkhead box O1 on insulin resistance of polycystic ovary syndrome. Previous findings confirmed IRS-1/PI3K signaling pathway was inhibited in project approved by National Science Foundation，and regulatory mechanism was closely related with insulin resistance between Akt/ FOXO1 and STIR1/ FOXO1 signaling pathway from the literature reports. The latest experimental results showed phosphorylation level of FOXO1 protein was increased and the serum inflammatory cytokines level were increased in successful model rats.But whether prostaglandin E,which is the active ingredient of Evening primrose oil,targeted FOXO1 factor to improve the symptoms of IR has not been reported, To explore mechanism of Improving insulin resistance, IR model rats and IR model cells were treatmented with evening primrose oil. Research Group use gene transfection, Western-blot, EMSA, Chip-Seq, C1 Single-Cell Auto capture technology and luciferase reporter to detect the expression of inflammatory cytokine and to measure the level of oxidative stress and to Improve the blood supply and vascular endothelium function.It revealed FOXO1 signaling plays an important role to insulin resistance in PCOS model rats treatmented with evening primrose oil.And it proved evening primrose oil has a good effect on treatment of insulin resistance.It provided theoretical basis and new ideas for clinical traditional Chinese medicine in the treatment of PCOS.

多囊卵巢综合征（PCOS）胰岛素抵抗（IR）是胰岛素敏感性的问题，抑制叉头转录因子1（FOXO1）可提高胰岛素敏感性，因此抑制FOXO1基因的表达对于改善IR具有重要意义。最近研究表明黄素化颗粒细胞IRS-1/PI3K通路受损，IR发生与Akt/FOXO1及SIRT1/FOXO1相关，月见草油能够改善IR症状。前期工作证实PCOS IR模型大鼠卵巢FOXO1磷酸化及炎性因子表达升高，氧化应激失衡。然而月见草油是否会抑制FOXO1因子改善PCOS IR的症状未见报道。因此课题组以IR细胞模型、IR大鼠模型和Cre/loxP大鼠为研究对象，应用转染、Western-blot、EMSA、Chip-Seq、单细胞测序及荧光素酶等技术，从抗氧化，抗炎及保护血管方面研究月见草油抑制FOXO1因子改善IR，甚至逆转IR的作用机制，揭示FOXO1及其下游分子如何调节见草油对PCOS胰岛素敏感性的保护机制。

多囊卵巢综合征（PCOS）胰岛素抵抗（IR）是胰岛素敏感性的问题，IR发生与Akt/FOXO1及SIRT1/FOXO1相关，月见草油能够改善IR症状。前期工作证实PCOS IR模型大鼠卵巢FOXO1磷酸化及炎性因子表达升高，氧化应激失衡。因此试验中以月见草油组和 PGE1 组为实验组，通过与胰岛素抵抗 PCOS 大鼠模型及正常对照组、二甲双胍西药阳性对照组(剂量同上)及高脂诱导 IR ApoE-/-大鼠组（普通饲料脂肪 7.39%，高脂饲料脂肪 21.28%） 进行比较分析，结果证实（1）月见草油通过胰岛素下游通路 IRS-1/Akt 发挥改善血管内皮的作用，明确月见草油通过激活 Akt/FOXO1 改善大鼠模型卵巢血液供应、抑制糖异生关键酶基因表达的机制，揭示其可能促进卵泡发育、成熟、排卵的作用机制。（2）月见草油对PCOS模型大鼠生殖内分泌和糖代谢水平、细胞凋亡及卵巢组织形态变化的影响，确定了月见草油对NLRP3炎症体及相关蛋白表达的影响，分析月见草油对PCOS大鼠治疗前后NLRP3炎症体的作用，明确了月见草油通过NLRP3炎症体作用改善PCOS疾病的发病机制。（3）月见草油和二甲双胍的单用和联合应用可通过PINK/Parkin通路介导的线粒体自噬来改善来曲唑诱导的PCOS模型大鼠雄激素过多以及改善发情周期和卵巢病理变化。（4）取分离纯化的IVF-ET卵巢颗粒细胞，activin A和Bimagrumab继续培养24小时，结果证实activin A能够增加Act RⅡ、p-Smad2/Smad2 和 CYP19A1 的表达水平，且activin A促进颗粒细胞p-Smad2/Smad2和CYP19A1表达与Act RⅡ信号通路相关。（5）月见草油活性成分β-谷甾醇培养KGN细胞，采用CCK-8测定增殖，流式细胞检测细胞凋亡及周期，Western blot 检测PI3K/AKT/mTOR 通路，添加20μmol·L-1浓度的β-谷甾醇可通过对细胞蛋白表达水平的影响，促进颗粒细胞的增殖同时可抑制凋亡，其机制与 PI3K/Akt信号通路。以上研究确定了月见草油对PCOS模型大鼠内分泌和代谢水平的干预效果，以及对卵巢NLRP3炎症体通路表达的影响，探讨月见草油干预PCOS慢性炎症的可能作用机制，为月见草油的开发、应用和PCOS的临床治疗提供科学依据。