背景:开玉中玉方(KYZY)是中药(TCM)中的经典草药配方,已用于治疗与心理压力相关的不孕症已有三百多年的历史。
目的:心理压力对生育有重大影响,不同的结果取决于性质,力量,和压力的持续时间。压力可以直接扰乱排卵,卵母细胞质量,成熟,和胚胎发育。本研究旨在探讨KYZY在心理应激下提高卵母细胞发育潜能的分子机制。
方法:将4-5周龄的ICR雌性小鼠随机分为5组:对照组,在慢性不可预测压力模型(CUSM)中强调,并在38.2g/kg(KYZYH)的压力下加上KYZY处理,19.1g/kg(KYZYM),或9.6g/kg(KYZYL)。通过测量雌二醇(E2)的血清水平来评估卵巢功能,黄体生成素(LH),卵泡刺激素(FSH),和抗苗勒管激素(AMH)。根据活性氧(ROS)水平评估卵母细胞质量,凋亡DNA片段化,和线粒体分布。我们使用RNA测序(RNAseq)来鉴定组间的差异表达基因(DEGs),然后进一步分析DEGs的基因本体论(GO)术语富集和蛋白质-蛋白质相互作用。
结果:应激组小鼠血清E2、LH、和FSH以及增加的ROS水平,细胞凋亡增加,和干扰线粒体在卵母细胞中的分布。在所有三种剂量下用KYZY治疗逆转或改善了这些压力的负面影响。DEG分析确定了两个比较之间的187个常见基因(应激与控制和KYZYM与强调),其中33个注释有六个基因本体论(GO)的生物过程(BP)术语:细胞分化,凋亡,ATP合成,蛋白质同源寡聚化,神经元迁移,和肽酶活性的负调节。DEGs的蛋白质-蛋白质相互作用网络分析确定了关键枢纽基因。值得注意的是,Atp5o和Cyc1基因均参与ATP合成,在前三个hub基因中,这表明调节卵母细胞线粒体电子传递和ATP合成在应激反应中很重要,也是KYZY的可能作用机制。
结论:KYZY可有效改善压力对卵母细胞能力的不利影响,可能通过靶向线粒体呼吸链和ATP合酶。
BACKGROUND: The Kai Yu Zhong Yu recipe (KYZY) is a classic herbal formula in traditional Chinese medicine (TCM) that has been used to treat infertility associated with psychological stress for more than three hundred years.
OBJECTIVE: Psychological stress has major impacts on fertility, with variable outcomes depending on the nature, strength, and duration of the stress. Stress can directly disturb ovulation, oocyte quality, maturation, and embryo development. The aim of this
study is to investigate the molecular mechanism by which KYZY improves oocyte developmental potential under psychological stress.
METHODS: ICR female mice aged 4-5 weeks were randomly divided into five groups: control, stressed in the chronic unpredictable stress model (CUSM), and stressed plus KYZY treatment at 38.2 g/kg (KYZYH), 19.1 g/kg (KYZYM), or 9.6 g/kg (KYZYL). Ovary function was assessed by measuring serum levels of estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH). Oocyte quality was evaluated in terms of reactive oxygen species (ROS) levels, apoptotic DNA fragmentation, and mitochondria distribution. We used RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) between groups and then further analyzed the DEGs for gene ontology (GO) term enrichment and protein-protein interactions.
RESULTS: Mice in the stressed group had reduced serum E2, LH, and FSH as well as increased ROS levels, increased apoptosis, and disturbed mitochondria distribution in oocytes. Treatment with KYZY at all three doses reversed or ameliorated these negative effects of stress. DEG analysis identified 187 common genes between the two comparisons (stressed vs. control and KYZYM vs. stressed), 33 of which were annotated with six gene ontology (GO)\'s biological process (BP) terms: cell differentiation, apoptosis, ATP synthesis, protein homo-oligomerization, neuron migration, and negative regulation of peptidase activity. Protein-protein interaction network analysis of DEGs identified key hub genes. Notably, the genes Atp5o and Cyc1 were both involved in the ATP synthesis and among the top three hub genes, suggesting that regulation of oocyte mitochondrial electron transport and ATP synthesis is important in the response to stress and also is a possible mechanism of action for KYZY.
CONCLUSIONS: KYZY was effective in ameliorating the adverse effects of stress on oocyte competence, possibly by targeting the mitochondrial respiratory chain and ATP synthase.