二仙汤有效组分配伍通过MAPKs和Nrf2/ARE通路对人卵巢颗粒细胞氧化应激保护作用机制的研究

Pathogenesis of Idiopathic Primary Ovarian Insufficiency is related with environmental、psychological and lifestyle factors closely .These factors can cause body oxidative stress state. MAPKs signaling pathways are involved in a variety of activities of the cells.JUK and P38MAPK,two signaling pathways of MAPKs,are closly related with apoptosis induced by oxidative stress.Nrf2/ARE signalpath is an important antioxidant stress pathway, so we put forward a hypothesis ,which is " Nrf2/ARE signaling pathway plays an important role in protection of ovarian function against oxidative stress injury ,and it has a close relationship with MAPKs pathway in the regulation of people ovarian granulosa cell apoptosis induced by oxidative stress." The hypothesis has not been confirmed so far. To test this hypothesis, we will set up people ovarian granulosa cell oxidation stress model for the first time, and using molecular biology technology, from the cellular level , observe influence of ovarian granulosa cell signaling pathways induced by oxidative stress .The results will provide new ideas and new targets for the prevention and control of ovarian aging. The basic pathogenesis of POI is kidney deficiency on the basis of traditional Chinese Medicine, and oxidative stress is one of the biological mechanism of kidney deficiency induced by aging.Therapy mechanisms of Erxian decoction pieces have been studied in treatment of many aging diseases,but decoction pieces compositions are complex, which can not provide good quality control standards for the innovation of traditional Chinese medicine research. This is the first optimal combination study of Erxian decoction active ingredient .We will clarify principle of the optimal combination, discuss mechanism of treating ovarian aging from oxidative stress signal pathways, in order to improve the quality of drug research and development innovation, provide scientific basis for traditional Chinese medicine.

特发性卵巢功能不全与环境心理生活因素有关，这些可造成体内氧化应激状态。氧化应激激活MAPKs信号通路促进细胞凋亡；作为重要的抗氧化应激通路,Nrf2/ARE有对抗细胞凋亡的作用。卵巢衰退的本质是颗粒细胞的凋亡，故我们提出"Nrf2/ARE信号通路在抑制氧化应激所致卵巢颗粒细胞凋亡方面发挥重要作用，与MAPKs家族之间可能存在密切的调控机制"的假说。为了证实这一假说，我们将建立人卵巢颗粒细胞的氧化应激模型，从细胞层面观察氧化应激对卵巢颗粒细胞的影响及其信号通路蛋白水平变化，为防治卵巢衰老提供新思路和新靶点。中医认为卵巢衰退基本病机是肾虚，氧化应激是肾虚衰老的生物机制之一，本研究探索抗衰老补肾名方- - 二仙汤有效组分最佳配伍在调控MAPKs和Nrf2/ARE信号通路发挥的抗氧化机制，为形成药效物质明确、质量可控、用于治疗卵巢功能不全的组分中药提供有价值的研究数据，为临床合理用药提供科学依据。

紧张和焦虑引发等社会环境因素引发的氧化应激状态影响女性的生殖系统健康，促使卵巢颗粒细胞凋亡。为了探究卵巢氧化应激机制以及评价二仙汤及有效组分抗卵巢氧化应激的作用。本研首先进行了观察性研究，采集行IVF或ICSI治疗的不孕患者的卵泡液，并从中提取颗粒细胞进行MDA、SOD检测及MAKP和Nrf2信号通路蛋白表达情况。结果提示P-JUK、P-P38、P-ERK1/2及Nrf2均有在颗粒细胞中表达，且不同年龄段的P-JUK蛋白表达具有显著差异。为进一步探索其作用机制，我们采用H2O2对人卵巢颗粒细胞株COV434进行诱导，用western blot、流式细胞仪及DAPI/TUNEL 的测定来检测H2O2作用在COV434后，细胞死亡的百分比。结果显示：H2O2作用在COV434细胞株，可造成细胞氧化应激状态，且 ROS的水平与H2O2作用的时间、浓度呈正相关性。WB结果显示：MAPKs的三个亚型均参与了H2O2诱导的人卵巢颗粒细胞氧化应激状态，Nrf2的蛋白表达与H2O2诱导人颗粒细胞氧化应激状态浓度和时间梯度呈明显正相关。并且线粒体细胞凋亡通路参与了ROS-JNK-p53在H2O2诱导人卵巢颗粒细胞氧化应激细胞凋亡机制。接下来我们通过MTT实验筛选了不同浓度的二仙汤六种有效单体及其拆方单体配伍对抗H2O2诱导COV434细胞的影响，结果均显示，与H2O2单用组相比较，中药单体各组或配伍组预处理并无降低H2O2对COV434细胞的抑制率的作用，提示了二仙汤发挥抗氧化应激作用的可能不是方中主要有效单体成分。而进一步研究发现，二仙汤的水提液在一定剂量范围内对H2O2诱导的COV434细胞损伤具有明显的保护作用，其保护效应可能与清除 ROS，减轻 DNA 氧化损伤，调控MAPK-Nrf2 信号通路以及其调控线粒体依赖的 Bax/Bcl-2 、Caspase-9家族参与了二仙汤水提液抑制H2O2诱导的氧化应激过程。本课题研究对指导临床用药具有一定指导意义。