Abstract Aim To verify the effects of modified Gengnianchun formula (MGNC), a traditional Chinese medicine, on a stressed diminished ovarian reserve (DOR) animal model and predict the underlying mechanisms through network pharmacology strategies. Methods Sexually mature female C57BL/6 mice were allocated to five groups, abbreviated as the control (C) group, stress manipulated model (M) group, stress with normal saline gavage (N) group, stress with low-dose MGNC gavage (L) group, and stress with high-dose MGNC gavage (H) group. Body weight and the estrous cycle were monitored during the stress and gavage process. Serum stress hormones and reproductive hormones were evaluated by ELISA. Ovarian follicle counts were calculated, and ovarian follicle-stimulating hormone receptor (FSHR) and anti-Müllerian hormone (AMH) expression were assessed by Western blotting and immunohistochemistry. Network pharmacology strategies included active compound screening, drug and disease target analysis, gene ontology analysis, pathway analysis, and visualization of results. Results MGNC treatment significantly decreased serum corticosterone (CORT) and follicle-stimulating hormone (FSH) levels and increased testosterone (T) levels in the H group compared with the M and N groups. Primordial and preantral follicle counts and ovarian AMH and FSHR expression were significantly increased in the H group compared to those in the M and N groups. Through pharmacokinetic screening, we found 244 active compounds in MGNC. A total of 186 candidate intersection target genes of disease and MGNC were further screened to construct the interaction network. Gene ontology and KEGG pathway enrichment analysis ultimately unveiled a series of key targets that mainly mediated the effects of MGNC on DOR induced by chronic stress. The PI3K-Akt pathway may serve as the critical pathway underlying this therapeutic mechanism. Conclusion MGNC is a promising formula to treat DOR induced by chronic stress, and the PI3K-Akt pathway may play an essential role in this effect. Keywords: modified Gengnianchun formula, diminished ovarian reserve, stress, follicle development, network pharmacology, target gene Introduction Diminished ovarian reserve (DOR) is defined by a decreased quantity or quality of ovarian follicles in women. It is a condition between normal reproductive physiology and premature ovarian insufficiency (POI). Although the criteria of DOR remain unresolved and are adopted differently in various regions,[35]1 it is basically characterized by an elevated serum follicle-stimulating hormone (FSH) level, decreased anti-Müllerian hormone (AMH) level and diminished antral follicle count on ultrasonic examination. According to US data, the prevalence of DOR increased from 19% to 26% from 2004 to 2011 among assisted reproductive treated patients.[36]2 Among various causes of DOR, psychological factors have emerged as an important one in recent years.[37]3–5 While acute or brief stressors only cause limited damage to women’s fertility, chronic stressors can be detrimental to ovarian reserve, leading to adverse fertility outcomes.[38]4,[39]6 Our previous animal study also indicated that chronic unpredictable stress (CUS) can induce a typical and long-lasting DOR phenotype in C57BL/6 mice.[40]7 The Western treatment of DOR is reproductive hormone-based therapy, which does not help to attenuate the pathology of DOR and stress conditions. There are also side effects that limit its clinical application.[41]8 Traditional Chinese medicine (TCM) has promising effects on treating gynecological endocrinology dysfunctions.[42]9–11 Gengnianchun (GNC) is a previously widely reported formula in the treatment of menopausal syndrome[43]12 and menopause-related diseases such as Alzheimer’s.[44]13 According to TCM theories, DOR shares a similar etiology with menopause, which is deficient in the kidney. With a significant effect of tonifying the kidney, GNC has also been used to treat patients with DOR and has shown a satisfactory effect in improving ovarian reserve, balancing reproductive endocrinology conditions, and improving the pregnancy rate and live birth rate. The modified Gengnianchun formula (MGNC), which constitutes three more herbs than GNC, was intended to better manage stress symptoms in patients with DOR.[45]14 Here, we plan to investigate the effects of MGNC in a stress-induced DOR animal model. Network pharmacology emerges as a novel method to integrate information from bioinformatics, systems biology, and polypharmacology.[46]15 With a “disease-gene-target-drug” network model, it evaluates the molecular mechanism of TCM formulas from a multidimensional perspective. This thinking coincides with the holistic and systemic characteristics of TCM.[47]16 By visualizing the multitarget, multigene, and multipathway interactions, the mechanisms of MGNC were expected to be unveiled and further explored. In this work, an in vivo experiment was carried out to observe the effects of MGNC on a stress-induced DOR mouse model. Serum corticosterone (CORT) was tested, and body weight was monitored to evaluate the effects of MGNC on stress. Serum FSH, luteinizing hormone (LH), testosterone (T), estradiol (E2), and AMH were analyzed; protein analysis of ovarian AMH and FSH receptor (FSHR) was undertaken to observe the endocrine regulation effects of MGNC. Furthermore, network pharmacology was applied to explore the active compounds and multitarget regulation of MGNC. Underlying mechanisms and signaling pathways were further suggested by GO and KEGG analysis to unveil the effects of MGNC against stress-induced DOR. Materials and Methods Animals and Treatment The animal experiment was based on a previously reported DOR mouse model induced by CUS.[48]7 All animal experimental procedures were approved by the Animal Ethics Committee of Fudan University. Seventy-five female C57BL/6 mice aged 6 to 8 weeks were randomly and equally divided into five groups. All mice were housed in the facility for 1 week before the experiment. Mice in the control (C) group were raised without intervention for 8 weeks. Mice in the model (M) group were subjected to 8 weeks of CUS. Mice in the other three groups were administered the 8-week CUS and oral administration of normal saline (N), a low-dose (2.23 g/kg body weight) of MGNC (L), and a high-dose (8.92 g/kg body weight) of MGNC (H). Mice were sacrificed after the 8-week manipulation. Sample collection was carried out from 8 am to 10 am when serum CORT was at the base level of daily fluctuation. The body weight of mice from each group was measured every week. The experiment was repeated three times. Preparation of MGNC Decoction The MGNC formula was prepared by mixing water extracts of 15 crude herbs purchased from Tianjiang Pharmaceutical Limited Company (Jiangyin, China) and dissolved in 100°C water to make the desired concentration for gavage. The products were manufactured with rigid quality control protocols following rigid specifications of the Pharmacopeia of China. The ingredients’ names and herbal information of MGNC are listed in [49]Table 1. The formula information of MGNC and the conversion of water extract weight and crude herbs provided by the manufacturer are listed in [50]Table 1. Table 1. Modified Gengnianchun Formula (MGNC) English Name (Chinese Name) Latin Name Plant Part Processing Method Crude Herb (g) Water Extract (g) Lot Number Radix Rehmanniae (Shengdi) Rehmannia glutinosa (Gaertn.) DC Root Dried 15 4.5 1,803,033 Epimedium Brevicornums (Yinyanghuo) Epimedium acuminatum Franch Root Dried 12 0.6 1,804,138 Radix Paeoniae Alba (Baishao) Paeonia lactiflora Pall Root Dried 12 1.2 1,804,029 Fructus Lycii (Gouqizi) Lycium barbarum L. Fruit Dried 12 4.8 1,804,052 Plastri Testudinis (Guiban) Carapax et plastrum Testudinis Carapax Stir-Baking with Vinegar 15 0.75 1,811,057 Rhizoma Anemarrhenae (Zhimu) Anemarrhena asphodeloides Bunge Root Dried 15 3.75 1,802,153 Semen Cuscutae (Tusizi) Cuscuta australis R.Br. Seed Dried 12 0.6 1,803,027 Moridae Officinalis (Bajitian) Morinda officinalis F.C.How Root Dried 12 3.6 1,803,168 Cistanche Salsa (Congrong) Cistanche deserticola Y.C.Ma Stem Dried 12 3.6 1,804,041 Cortex Phellodendri Amurensis (Huangbai) Phellodendron chinense Schneid Bark Dried 9 0.75 1,804,026 Rhizoma Coptidis (Huanglian) Coptis chinensis Franch. Rhizome Dried 3 0.5 1,803,165 Poria (Fuling) Poria cocos (Schw.) Wolf Sclerotium Dried 9 0.9 1,804,133 Radix Bupleuri (Chaihu) Bupleurum abchasicum Manden. Root Dried 9 1.5 1,803,134 Angelica Sinensis (Danggui) Angelica sinensis (Oliv.) Diels Root Dried 12 4.8 1,804,210 Ligusticum Wallichii (Chuanxiong) Conioselinum chinense (L.) Britton, Sterns & Poggenb Root Dried 9 3 1,804,003 [51]Open in a new tab High-Performance Liquid Chromatography (HPLC) A total of 34.85 g of granule mix was resolved in 336 mL of hot water to make a solution that equals 0.5 g of dry ingredients per milliliter. Samples were filtered through a 0.45-μm membrane filter before HPLC analysis. The HPLC machine was an Agilent S1200 system equipped with a Zorbax SB-C18 column (Agilent Technologies, Santa Clara, CA, USA). The detection wavelength was set at 210 nm, and the flow rate was 1.0 mL/min. The injection volume was 10 μL. The column temperature was maintained at 30°C. The mobile phases were acetonitrile (A) and water-phosphoric acid (B, 0.01%, v/v). The eluting conditions were as follows: 0–19 min, 10–18% (A); 19–25 min, 18% (A); 25–56 min, 18–35% (A). According to the Pharmacopeia of China, mangiferin, paeoniflorin, hyperin, and icariin are the most important therapeutic constituents of Zhimu, Baishao, Tusizi, and Yinyanghuo, respectively. Ferulic acid is the main therapeutic constituent of Danggui and Chuanxiong. These six ingredients also play a key role in regulating ovarian function and ameliorating emotional problems according to the traditional application of each ingredient. Therefore, for quality control, these five standard references were adopted to evaluate the quality and to