Zeng Pengfei, Zhou Hang, Guo Pei, Han Nana, Zhang Xuan, Yin Zhixing, Xia Wanting, Huang Jinzhu, Zeng Qian
Department of Gynecology, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China.
School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Heliyon. 2023 Sep 13;9(9):e20104. doi: 10.1016/j.heliyon.2023.e20104. eCollection 2023 Sep.
This study aimed to explore the mechanism of the Bushen Huoxue Formula (BHF) in treating diminished ovarian reserve (DOR) through the use of metabolomics and integrated network pharmacology.
The study involved 24 non-pregnant female Sprague-Dawley rats, divided into four groups of six rats each: control, model, BHF, and DHEA (n = 6 per group). The model group was induced with DOR by administering Tripterygium glycosides orally [50 mg (kg·d)] for 14 days. Subsequently, BHF and Dehydroepiandrosterone (DHEA) treatments were given to the respective groups. Ovarian reserve function was assessed by measuring anti-Müllerian hormone (AMH), estradiol (E), and follicle-stimulating hormone (FSH) levels and conducting hematoxylin-eosin staining. In addition, UHPLC-QTOF-MS analysis was performed to identify differential metabolites and pathways in DOR rats treated with BHF. In this study, LC-MS was utilized to identify the active ingredients of BHF, while network pharmacology was employed to investigate the correlations between BHF-related genes and DOR-related genes. An integrated analysis of metabonomics and network pharmacology was conducted to elucidate the mechanisms underlying the efficacy of BHF in treating DOR.
The model group exhibited a poor general condition and a significant decrease in the number of primordial, primary, and secondary follicles ( < 0.05) when compared to the control group. However, BHF intervention resulted in an increase in the number of primordial, primary, and secondary follicles ( < 0.05), along with elevated levels of AMH and E ( < 0.05), and a decrease in FSH levels ( < 0.05) in DOR rats. The modeling process identified eleven classes of metabolites, including cholesterol esters (CE), diacylglycerols (DAG), hexosylceramides (HCER), lysophosphatidylcholines (LPC), phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomyelins (SM), ceramides (CER), free fatty acids (FFA), triacylglycerols (TAG), and lysophosphatidylethanolamines (LPE). The study found that PC, CE, DAG, and TAG are important metabolites in the treatment of DOR with BHF. LC-MS analysis showed that there were 183 active ingredients in ESI(+) mode and 51 in ESI(-) mode. Network pharmacology analysis identified 285 potential genes associated with BHF treatment for DOR in ESI(+) mode and 177 in ESI(-) mode. The combined analysis indicated that linoleic acid metabolism is the primary pathway in treating DOR with BHF.
BHF was found to improve ovarian function in rats with DOR induced by Tripterygium glycosides. The study identified key metabolites such as phosphatidylcholine (PC), cholesteryl ester (CE), diacylglycerol (DAG), triacylglycerol (TAG), and the linoleic acid metabolism pathway, which were crucial in improving ovarian function in DOR rats treated with BHF.
本研究旨在通过代谢组学和整合网络药理学方法，探讨补肾活血方（BHF）治疗卵巢储备功能下降（DOR）的机制。
本研究选用24只未孕雌性Sprague-Dawley大鼠，分为四组，每组6只：对照组、模型组、BHF组和脱氢表雄酮（DHEA）组（每组n = 6）。模型组通过口服雷公藤多苷[50 mg/(kg·d)]诱导14天建立DOR模型。随后，分别对BHF组和脱氢表雄酮（DHEA）组进行相应处理。通过检测抗苗勒管激素（AMH）、雌二醇（E）和卵泡刺激素（FSH）水平以及苏木精-伊红染色评估卵巢储备功能。此外，采用超高效液相色谱-四极杆飞行时间质谱（UHPLC-QTOF-MS）分析，鉴定BHF治疗DOR大鼠的差异代谢物和代谢途径。本研究利用液相色谱-质谱联用（LC-MS）鉴定BHF的活性成分，同时运用网络药理学研究BHF相关基因与DOR相关基因之间的相关性。进行代谢组学和网络药理学的整合分析，以阐明BHF治疗DOR的疗效机制。
与对照组相比，模型组大鼠一般状况较差，原始卵泡、初级卵泡和次级卵泡数量显著减少（P < 0.05）。然而，BHF干预可使DOR大鼠的原始卵泡、初级卵泡和次级卵泡数量增加（P < 0.05），同时AMH和E水平升高（P < 0.05），FSH水平降低（P < 0.05）。建模过程鉴定出11类代谢物，包括胆固醇酯（CE）、二酰基甘油（DAG）、己糖神经酰胺（HCER）、溶血磷脂酰胆碱（LPC）、磷脂酰胆碱（PC）、磷脂酰乙醇胺（PE）、鞘磷脂（SM）、神经酰胺（CER）、游离脂肪酸（FFA）、三酰基甘油（TAG）和溶血磷脂酰乙醇胺（LPE）。研究发现，PC、CE、DAG和TAG是BHF治疗DOR的重要代谢物。LC-MS分析显示，电喷雾电离正离子（ESI(+)）模式下有183种活性成分，电喷雾电离负离子（ESI(-)）模式下有51种。网络药理学分析在ESI(+)模式下鉴定出285个与BHF治疗DOR相关的潜在基因，ESI(-)模式下有177个。综合分析表明，亚油酸代谢是BHF治疗DOR的主要途径。
发现BHF可改善雷公藤多苷诱导的DOR大鼠的卵巢功能。本研究鉴定出关键代谢物，如磷脂酰胆碱（PC）、胆固醇酯（CE）、二酰基甘油（DAG）、三酰基甘油（TAG）以及亚油酸代谢途径，这些对于改善BHF治疗的DOR大鼠的卵巢功能至关重要。
