基于傅氏“嫉妒不孕”理论研究肝郁-心理应激对小鼠卵母细胞ROS-线粒体凋亡通路的影响

Modern research has found that psychosocial factors are one important pathogenesis of infertility. The TCM theory of “Infertility due to Jealousy” holds that infertility could be caused by liver Qi stagnation, but its mechanism of actions was not clearly identified. In light of this theory, we hypothesized that “liver Qi stagnation-psychological stress induces ROS over-production by ovaries, which damaged the structure and function of mitochondria in oocyte, activated its apoptosis pathway, and reduced the oocyte’s fertilization ability and the embryo’s developmental potential, thus leading to oocyte apoptosis via endogenous pathway and eventually result in infertility.” In this study, this hypothesis will be verified using technologies such as the DCFH-fluorescence method, the TBA method, Mito-Tracker Green, TUNEL and RT-PCR to measure ROS, SOD, MDA, the structure and function of mitochondria and the apoptosis rate of oocyte in Psychological Stress Mouse Models under different stimulus intensity. Based on the method that TCM syndrome could be determined by investigating the effects of the “syndrome-specific” drug on the subject, we will observe the effects of Fu Qingzhu’s prescription Kaiyu Zhongyu Tang on the fertilization ability of oocyte and the developmental potential of embryo. The study holds promise to reveal the mechanism of liver Qi stagnation-psychological stress to cause infertility, and will provide insights into clinical treatment of infertility, and also provide scientific evidence for the promotion and application of TCM in modern medical practice.

现代研究表明社会心理因素是导致不孕症的重要因素。中医学早已提出肝郁所致“嫉妒不孕”的理论，但其具体机制尚未明确。基于此，本研究提出“肝郁—心理应激可导致卵巢内产生过量ROS，破坏卵母细胞线粒体结构及功能，激活线粒体凋亡通路，降低卵母细胞受精能力及胚胎发育潜能，通过内源性途径诱发卵母细胞凋亡而导致不孕症发生”的假说。通过建立不同刺激强度心理应激小鼠模型，采用DCFH-荧光法、TBA法、Mito-Tracker Green、TUNEL、RT-PCR等技术，检测ROS、SOD、MDA、线粒体结构及功能、卵母细胞凋亡率等来验证该假说。并运用“以药测证”的方法，观察傅氏开郁种玉汤对卵母细胞受精能力及胚胎发育潜能的影响。预期通过该项目揭示肝郁-心理应激导致不孕症的机制，为临床治疗不孕症提供新思路，并为中医药在现代医学模式下的推广应用提供科学依据。

社会心理因素可导致不孕症的发生。中医学提出“嫉妒不孕”理论，但其具体机制尚未明确。基于此，本研究提出“肝郁-心理应激可导致卵巢内产生过量ROS，破坏卵母细胞线粒体结构及功能，激活线粒体凋亡通路，卵母细胞凋亡增加，卵母细胞受精能力及胚胎发育潜能下降，进而导致不孕症”的假说。通过建立不同刺激强度心理应激小鼠模型，采用DCFH-荧光法、Mito-Tracker Green、RT-PCR等技术，检测氧化应激相关指标、线粒体结构及功能、卵母细胞凋亡率等来验证该假说。在本项目中，首先通过动物实验证实了不同程度肝郁-心理应激刺激强度下，小鼠体内氧化应激水平增加，抗氧化酶水平减退，血清性激素水平下降，卵巢储备功能下降程度与刺激强度呈正相关；其次，通过肝郁-心理应激造模后获取小鼠卵母细胞，进一步观察到随着肝郁-心理应激刺激程度的增强，卵母细胞内氧化应激水平增加，抗氧化酶活性下降，线粒体分布不均，线粒体mtDNA拷贝数减少，卵母细胞内促凋亡因子表达水平增加，卵母细胞凋亡增加，胚胎发育潜能下降；最后，在造模基础上添加中药开郁种玉汤及原花青素作为干预措施后，发现小鼠卵母细胞内氧化应激水平下降，线粒体形态、分布及mtDNA拷贝数均有所改善，促凋亡因子表达水平下降，卵母细胞凋亡减少，与模型组相比，差异均有统计学意义，说明中药开郁种玉汤与抗氧化剂均可有效改善肝郁-心理应激导致的小鼠卵巢储备功能减退及卵母细胞质量下降的问题。当今社会工作生活压力剧增，慢性心理应激在女性不孕症中扮演的角色愈发得到重视，本研究证实了心理应激可导致卵巢储备功能减退及卵母细胞质量下降及其具体机制，为中医学“嫉妒不孕”理论在临床中的应用提供了科学数据，为中药复方开郁种玉汤治疗临床因心理应激所致不孕症提供了理论基础，亦为中医药现代化及临床推广应用奠定了基础。