Huang Yun, Zhang Qin, Shen Dan, Bao Xi
TCM Gynecology Department, Hangzhou Hospital of Traditional Chinese Medicine, NO.453 Ti Yuchang Road, Hangzhou, 310007, Zhejiang, China.
Geriatric Department, Hangzhou Hospital of Traditional Chinese Medicine, NO.453 Ti Yuchang Road, Hangzhou, 310007, Zhejiang, China.
J Ovarian Res. 2024 Dec 27;17(1):254. doi: 10.1186/s13048-024-01575-1.
He Shi Yu Lin Formula (HSYLF) is a clinically proven prescription for treating premature ovarian insufficiency (POI), and has shown a good curative effect. However, its molecular mechanisms are unclear. This study aimed to investigate the molecular mechanisms of HSYLF and clarify how network pharmacology analysis guides the design of animal experiments, including the selection of effective treatment doses and key targets, to ensure the relevance of the experimental results.
Network pharmacology, molecular docking, and animal experiments were utilized to investigate the effects of HSYLF. Key targets were identified by intersecting herb and disease targets to construct protein-protein interaction and "active components-intersection targets-disease" networks. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the clusterProfiler package in R. A total of 50 specific pathogen-free female mice of reproductive age were included in the animal experiments. They were divided into five groups: the positive control group, the high-dose HSYLF group, the low-dose HSYLF group, the model blank group, and the normal control group, to evaluate the serum anti-müllerian hormone levels, mitochondrial morphology in oocytes, the levels of reactive oxygen species (ROS), and mitochondrial membrane potential.
Network pharmacology identified 204 active components connecting 219 key therapeutic targets for POI. Gene Ontology enrichment analysis indicated that the anti-POI targets of HSYLF mainly regulated response to xenobiotic stimulus, cellular response to chemical stress, and response to oxidative stress; and the Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested the primary pathways, including lipid and atherosclerosis, advanced glycation end product-receptor for advanced glycation end product signaling pathway in diabetic complications, bladder cancer, tumor necrosis factor signaling pathway, and interleukin-17 signaling pathway. The low-dose (33 g/kg/d) HSYLF and high-dose (66 g/kg/d) HSYLF groups exhibited a marked elevation in serum anti-müllerian hormone levels (low-dose group: 2657.63 ± 354.82 PG/ml; high-dose group: 2823.73 ± 316.04 PG/ml) and mitochondrial membrane potential compared to the model blank group (P < 0.05 or P < 0.01), along with a significant decline in fluorescence intensity of 2',7'-dichlorofluorescein for the levels of ROS in oocytes (P < 0.05 or P < 0.01). Additionally, both groups showed varying degrees of improvement in the morphology, quantity, and distribution of mitochondria.
This study provides definite evidence for the molecular mechanism by which HSYLF treats POI by decreasing mitochondrial ROS, increasing membrane potential, and improving mitochondrial function. The results from active components of HSYLF and their related key targets also confirmed the characteristics of its multi-component, multi-target, multi-pathway, and overall regulatory effects on POI. Further research regarding the mechanisms is required to generalize these results, and the deeper clinical value of HSYLF also needs to be investigated in the future.
和时毓麟方（HSYLF）是经临床验证的治疗卵巢早衰（POI）的方剂，已显示出良好疗效。然而，其分子机制尚不清楚。本研究旨在探讨HSYLF的分子机制，并阐明网络药理学分析如何指导动物实验设计，包括有效治疗剂量和关键靶点的选择，以确保实验结果的相关性。
利用网络药理学、分子对接和动物实验研究HSYLF的作用。通过交叉草药和疾病靶点鉴定关键靶点，构建蛋白质-蛋白质相互作用和“活性成分-交叉靶点-疾病”网络。使用R中的clusterProfiler包进行基因本体论（Gene Ontology）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes）通路分析。动物实验共纳入50只特定病原体-free的育龄雌性小鼠。将它们分为五组：阳性对照组、HSYLF高剂量组、HSYLF低剂量组、模型空白组和正常对照组，以评估血清抗苗勒管激素水平、卵母细胞线粒体形态、活性氧（ROS）水平和线粒体膜电位。
网络药理学鉴定出204种活性成分，连接219个POI的关键治疗靶点。基因本体论富集分析表明，HSYLF的抗POI靶点主要调节对外源生物刺激的反应、细胞对化学应激的反应和对氧化应激的反应；京都基因与基因组百科全书通路分析提示主要通路包括脂质与动脉粥样硬化、糖尿病并发症中的晚期糖基化终产物-晚期糖基化终产物信号通路受体、膀胱癌、肿瘤坏死因子信号通路和白细胞介素-17信号通路。与模型空白组相比，低剂量（33 g/kg/d）HSYLF组和高剂量（66 g/kg/d）HSYLF组血清抗苗勒管激素水平显著升高（低剂量组：2657.63±354.82 PG/ml；高剂量组：2823.73±316.04 PG/ml）和线粒体膜电位升高（P<0.05或P<0.01），同时卵母细胞中ROS水平的2',7'-二氯荧光素荧光强度显著下降（P<0.05或P<0.01）。此外，两组线粒体的形态、数量和分布均有不同程度改善。
本研究为HSYLF通过降低线粒体ROS、增加膜电位和改善线粒体功能治疗POI的分子机制提供了确切证据。HSYLF活性成分及其相关关键靶点的结果也证实了其对POI的多成分、多靶点、多途径和整体调节作用的特点。需要进一步研究机制以推广这些结果，HSYLF更深层次的临床价值也有待未来研究。
