{"paper_id":"72d7ceaf-49fb-4dac-982a-b0759562151e","body_text":"RESEARCH Open Access\n© The Author(s) 2025. Open Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 \nInternational License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you \ngive appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the \nlicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or \nexceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit  h t t p  : / /  c r e a  t i  \nv e c  o m m  o n s .  o r  g / l  i c e  n s e s  / b  y - n c - n d / 4 . 0 /.\nGao et al. Journal of Ovarian Research           (2025) 18:87 \nhttps://doi.org/10.1186/s13048-025-01679-2\nJournal of Ovarian Research\n†Ranran Gao and Yeke Wu contributed equally to this work and \nshould be considered co-first authors.\n*Correspondence:\nBaojun Guo\nhnsrmyygbj@zzu.edu.cn\n1Department of TCM, Henan Provincial People’s Hospital, Zhengzhou \nUniversity, No. 7 Weiwu Road, Jinshui District, Zhengzhou, Henan  \n450003, China\n2Department of Stomatology, Hospital of Chengdu University of \nTraditional Chinese Medicine, Chengdu, Sichuan 610072, China\n3Department of Clinical Bioinformatics Experimental Center, Henan \nProvincial People’s Hospital, Zhengzhou University, Zhengzhou,  \nHenan 450003, China\n4Department of Gynaecology, Hospital of Chengdu University of \nTraditional Chinese Medicine, Chengdu, Sichuan 610072, China\nAbstract\nBackground Premature ovarian failure (POF) is defined as amenorrhea that occurs before the age of 40 when the \novaries weaken or even fail. This disease seriously affects a woman’s future health and fertility.\nMethods Potential targets of Yijing Decoction (YJD) and POF were predicted by web-based pharmacology-related \ndatabases. The POF rat models and human ovarian granulosa cells injury models were induced by triptolide. In \naddition, the estrous cycle of the rats was monitored by vaginal smear and the ovarian tissue morphology was \nstained by HE staining. Immunohistochemistry, qRT-PCR and Western blotting were used to evaluate the levels of \nreproductive and angiogenesis related factors. Moreover, serum levels of the sex hormones and the oxidative stress \nindicators were measured by ELISA.\nResults YJD treatment resulted in the improvement of triptolide-induced abnormal ovarian function by restoring \nnormal estrous cycle, maintaining nearly normal ovarian size, reducing follicular atresia and increasing vascularization. \nAdditionally, YJD treatment normalized the serum levels of P , E2, FSH, LH, AMH, MDA and SOD, while activating the \nVEGF/VEGFR-2/FAK pathway. However, the VEGF/VEGFR-2/FAK pathway inhibitors reversed these pharmacological \neffects that YJD exhibited in POF rats. Furthermore, YJD increased the pregnancy rate and the number of live births in \nPOF rats.\nConclusion YJD reduced oxidative stress level, promoted angiogenesis and improved ovarian function in POF rats by \nactivating VEGF/VEGFR-2/FAK pathway. Moreover, YJD improved the intrauterine microenvironment for implantation \nin POF rats, thereby improving fertility.\nClinical trial number Not applicable.\nKeywords Yijing Decoction, Premature ovarian failure, Oxidative stress, Angiogenesis\nYijing Decoction improves premature ovarian \nfailure in rats by activating VEGF/VEGFR-2/FAK \npathway\nRanran Gao1†, Yeke Wu2†, Yuqin Tang3, Keming Wu4 and Baojun Guo1*\n\nPage 2 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nIntroduction\nPremature ovarian failure (POF) is defined as the ces -\nsation of menstruation for a period of at least 3 months \nbefore the age of 40, accompanied by elevated levels of \nfollicle stimulating hormone (FSH) exceeding 40 IU·L-1 \nor menopausal levels, as well as estradiol (E2) levels \nbelow 50 pg·mL− 1  on two separate occasions one month \napart. This condition is typically characterized by a defi -\nciency of mature follicles, diminished levels of FSH, and \nreduced ovarian reserve [ 1]. Common clinical mani -\nfestations among affected individuals include irregular \nmenstruation, amenorrhea, and infertility [ 2]. As qual -\nity of life improves and societal factors evolve, the inci -\ndence of POF is gradually younger and the incidence \nrate is increasing year by year, which has attracted more \nand more attention. The primary treatment options for \nPOF include hormone replacement therapy and assisted \nreproductive technology, with varying degrees of effec -\ntiveness and no definitive cure. These treatments may \nalso lead to adverse reactions such as osteoporosis and \ncardiovascular diseases [3]. Traditional Chinese medicine \noffers a promising alternative with its diverse therapeu -\ntic applications and potential to mitigate the side effects \nassociated with Western medicine. In recent years, Chi -\nnese medicine has gradually taken the advantage in the \ntreatment of POF, thus receiving the attention of the \nmajority of researchers and scholars [4].\nThe efficacy of Yijing Decoction (YJD) in treating sec -\nondary amenorrhea, infertility, and menopausal syn -\ndrome has been validated through modern clinical \napplication. Therefore, the renowned herbal medicine \nYJD was chosen for inclusion in this study. YJD is com -\nprised of eleven herbs, including Radix Rehmanniae \nPraeparata, Atractylodes macrocephala Koidz , Yam, \nRadix Angelica sinensis , Jujube seed , Radix adenopho -\nrae, cortex moutan, Ginseng Radix, Radix Paeoniae Alba, \nBupleurum, and Radix Eucommia ulmoides. The formula \nconsists of various herbs that have specific functions in \nnourishing and tonifying different organs in the body. \nRadix Rehmanniae Praeparata  nourishes kidney and \nis accompanied by Radix Eucommia ulmoides  to tonify \nkidney [ 5]. Radix Angelica sinensis  and Radix Paeoniae \nAlba nourish blood and smooth the liver [ 6, 7]. Bupleu-\nrum and cortex moutan  are included in the formula to \ndetoxify the liver [ 8]. Additionally, Yam and Atracty-\nlodes macrocephala Koidz  are utilized to strengthen the \nspleen and nourish blood [ 9]. Ginseng Radix  and Radix \nadenophorae help to unblock the liver [ 10], while Jujube \nseed nourishes the heart and calms the mind [ 11]. Over-\nall, this formula is beneficial for kidney health, spleen \nstrength, heart nourishment, liver regulation, and pro -\nmotion of blood circulation and menstruation. Clinical \ntrials have demonstrated that the combination of modi -\nfied YJD and artificial periodic therapy enhances ovarian \nfunction in individuals with ovarian reserve dysfunction, \nrenal insufficiency, and liver depletion [ 12]. YJD posi -\ntively impacts sex hormone levels, endometrial thickness, \nand Chinese medicine symptomatology, ultimately lead -\ning to improved quality of life for patients. The observed \nclinical efficacy of this treatment approach is noteworthy.\nOvarian angiogenesis plays a crucial role in follicular \ndevelopment, with vascular endothelial growth factor \n(VEGF) serving as a key regulator of vascular growth and \ndevelopment [13]. VEGF is primarily produced by granu-\nlosa cells, membrane cells, and luteal cells within ovarian \ntissues. Both VEGF and its receptor VEGFR-2 play sig -\nnificant roles in follicular development, ovulation, and \nluteal formation [ 14]. Activation of the VEGF/VEGFR-2 \nsignaling pathway leads to the activation of downstream \neffectors, including focal adhesion kinase (FAK), which \npromotes the survival, permeability, and proliferation of \novarian vascular endothelial cells, ultimately aiding in the \nrestoration of ovarian function [ 15]. Research has dem -\nonstrated the significant impact of oxidative stress on \novulation and its role in the progressive accumulation of \noxidative damage, which is a key factor in ovarian aging \n[16]. Patients with POF exhibit elevated levels of reactive \noxygen species (ROS), resulting in an imbalance of oxida-\ntive processes within the body [ 17]. Additionally, studies \nhave indicated that heightened expression of VEGF can \nmitigate oxidative stress within cells [ 18, 19]. Conse -\nquently, investigating the influence of VEGF on ovarian \nangiogenesis and oxidative stress is essential for enhanc -\ning clinical interventions aimed at improving ovarian \nfunction.\nThis study aims to explore the target of action and \npotential molecular mechanism of YJD in treating POF \nthrough network pharmacology analysis. Given the wide-\nspread clinical efficacy of YJD, the objective of this inves -\ntigation is to establish a theoretical framework for the \nclinical utilization of YJD in POF treatment. By delving \ninto the action mechanism of YJD, this research endeav -\nors to pave the way for future investigations and offer \ninsights to advance the field.\nMaterials and methods\nScreening of potential targets for YJD and POF\nYJD compounds were gathered from the Traditional \nChinese Medicine Systematic Pharmacology (TCMSP) \ndatabase and the Encyclopedia of Traditional Chinese \nMedicine (ETCM), followed by the identification of \nYJD-related targets from the TCMSP and Swiss Target \nPrediction databases. A target dataset for POF proteins \nwas established by utilizing the Online Mendelian Inheri-\ntance in Humans (OMIM), Gene Cards (GC), and Gene \nExpression Omnibus (GEO) databases.\n\nPage 3 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nConstruction and analysis of protein-protein interaction \n(PPI) network\nThe common targets of drugs and diseases interactions \nwere entered into the String database for the construc -\ntion and analysis of PPI networks. The biological species \nwas set as human, and the PPI network was obtained and \nplotted using Cytoscape 3.7.2 software.\nEnrichment analysis of gene ontology (GO) and Kyoto \nencyclopedia of genes and genomes (KEGG)\nGO and KEGG data were enriched and analyzed using \nFunRich software and the ClusterProfiler software \npackage.\nPreparation of YJD\n30 g each of Radix Rehmanniae Praeparata and Atracty-\nlodes macrocephala Koidz , 15 g each of Yam and Radix \nAngelica sinensis, 9 g each of Jujube seed and Radix ade-\nnophorae, 6 g each of cortex moutan and Ginseng Radix, \nand 3 g each of Radix Paeoniae Alba , Bupleurum and \nRadix Eucommia ulmoides were combined and decocted \ntogether for 1 h with water. The resulting filtrate was then \nextracted and concentrated to create high dose (2.36 g/\nmL) (H-YJD), medium dose (1.18 g/mL) (M-YJD), and \nlow dose (0.59 g/mL) (L-YJD) based on the amount of \nraw drug. The solution was refrigerated at 4 °C.\nAnimals and treatment\nSpecific pathogen free (SPF) healthy Sprague Dawley \n(SD) female and male rats (weighing 200–250 g and aged \n7 weeks) were purchased from Beijing HFK bioscience \nCO., Ltd. (Beijing, China). All protocols were authorized \nby the Ethics Committee of Henan Academy of Tradi -\ntional Chinese Medicine Animal Experiment Center \n(SYXK (Yu) 2022-0007). After one week of acclimatiza -\ntion feeding, 60 female rats with normal estrous cycle \nwere selected and randomly divided into six groups. \nThe control group received daily oral gavage of 1 mL/kg \nphysiological saline, while the remaining five groups were \nadministered 400 µg/kg triptolide (TP) via oral gavage \ndaily for a duration of 8 weeks. Following 2 weeks of con -\ntinuous TP administration, rats in the treatment group \nrespectively received low (5.9 g/kg), medium (11.8 g/kg) \nand high (23.6 g/kg) doses of YJD via oral gavage based \non the amount of raw drug, and divided into TP + L-YJD \ngroup, TP + M-YJD group and TP + H-YJD group [ 20]. \nThe rats in TP + DHEA group were given a daily oral \ngavage dehydroepiandrosterone (DHEA) at dose of 13.5 \nmg/kg for 8 weeks [ 21]. Saline (1 mL/kg per day) was \nadministered via oral gavage to rats in both the TP and \ncontrol groups, and the first day of drug administration \nwas recorded as D1. In addition, rats were continuously \nmonitored for vaginal smears, ovaries, and body weight \nfor 56 days from D1 onwards. All the diestrus females \nwere sacrificed after the model was established, and the \nserum and ovaries of the diestrus females were obtained \nfor studying the mechanism of action of YJD on POF rats \nin vivo.\nFemale rats in estrus after modeling were utilized to \nevaluate the effect of YJD on female fertility. Male rats \nwere individually housed in cages for mating purposes. \nFemale rats that exhibited estrus, as determined by \ncounting estrous cycles in the preceding experimental \nphase, were paired with males in a 1:1 ratio. The presence \nof a vaginal plug was an indicator of successful mating, \nwith the day of plug detection recorded as the first day \nof pregnancy (P1). At P10, three female rats from each \ngroup were euthanized to assess the number of implanted \nembryos, as well as to collect blood and ovaries for sex \nhormone level analysis during pregnancy. The remaining \npregnant female rats in each group were kept until natu -\nral delivery and the number of pups born alive/dead and \nlitter weight were recorded. The pregnancy rate, average \nnumber of embryos implanted, number of live births and \nneonatal weight of rats in each group were counted.\nHematoxylin-eosin staining (HE)\nThe tissue was dehydrated with ethanol and xylene, fol -\nlowed by paraffin embedding. After the tissue was cut \ninto wax slices with a thickness of 5 μm, the sections were \ndried, deparaffinized and rinsed with distilled water. Sub-\nsequently, the sections were stained with hematoxylin, \nrinsed with flowing water, and then stained with eosin. \nAfter being dehydrated and sealed, the prepared sections \nwere examined under a light microscope. The histomor -\nphology of the rat ovary was observed and the number \nof follicular cells at all levels (primordial follicle, primary \nfollicle, secondary follicle, sinus follicle, atretic follicle) \nwas statistically analyzed. Hematoxylin and eosin were \npurchased from Wuhan Xavier Biotechnology Co., LTD \n(Wuhan, Hubei, China).\nImmunohistochemistry (IHC)\nThe sections were rapidly cooled with cold water after \nimmersion in an antigen retrieval solution, followed by \nwashing twice with phosphate-buffered saline (PBS). The \nwashed sections were then treated with an endogenous \nperoxidase blocking agent and incubated in darkness at \nroom temperature. Subsequently, primary antibodies \nwere applied to the sections at 37 ℃ for 30 min, followed \nby incubation with enzyme-labeled goat anti-mouse/\nrabbit IgG secondary antibody (1:100, PV-6000; Bei -\njing Zhongshan Jinqiao Biotechnology Co. LTD, Bei -\njing, China) at 37 ℃ for 20 min. Color development was \nachieved using 3,3’-diaminobenzidine (DAB) (Wuhan \nXavier Biotechnology Co., LTD, Wuhan, Hubei, China), \nfollowed by staining with hematoxylin and final dehy -\ndration to seal the sections. The prepared sections were \n\nPage 4 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nplaced under a light microscope and the images were \ncaptured. Primary antibodies were used as follows: anti-\nPlatelet endothelial cell adhesion molecule-1 (CD31) \nantibody (1:200, A01513-3), anti-α-Smooth muscle actin \n(α-SMA) antibody (1:200, BM3902), anti-VEGF antibody \n(1:200, BA0407), anti-VEGFR-2 antibody (1:200, A00901-\n3), anti-FAK antibody (1:200, BM4303). These primary \nantibodies were purchased from Boster Biological Tech -\nnology (Pleasanton, CA, USA).\nImmunofluorescence (IF)\nThe deparaffinization and antigen retrieval of tissue \nsections were performed as described in immunohis -\ntochemical procedures. The antigen-repaired sections \nwere subjected to incubation with primary antibodies \nagainst mouse vasa homologue (MVH) (1:200, BA2882; \nBoster Biological Technology, Pleasanton, CA, USA) and \noctamer-binding transcription factor 4 (Oct4) (1:200, \nA00174; Boster Biological Technology, Pleasanton, CA, \nUSA) overnight at 4 °C. Following primary antibody \nincubation, the tissues underwent incubation with Cy3-\nlabeled goat anti-rabbit IgG secondary antibody (1:100, \nGB21303; Wuhan Xavier Biotechnology Co., LTD, \nWuhan, Hubei, China) for 1 h at room temperature in the \nabsence of light. Subsequently, the sections were washed \nwith PBS and incubated with DAPI for 15 min. An anti-\nfluorescence quencher was then applied, and the slices \nwere sealed and dried. All procedures were performed in \ndarkness after the introduction of secondary antibodies. \nThe tissue sections were placed under an inverted confo -\ncal microscope for tissue scanning, and the original data \nimages were reconstructed and simulated in three dimen-\nsions by using Imaris software and Image J software.\nEnzyme-linked immunosorbent assay (ELISA)\nSerum levels of the hormones progesterone (P), E2, FSH, \nluteinizing hormone (LH), and anti-mullerian hormone \n(AMH), and the oxidative stress indicators malondial -\ndehyde (MDA) and superoxide dismutase (SOD) were \nmeasured by using ELISA kits. And the experiments were \nperformed according to the instructions for the kits. Rat \nP ELISA kit, rat E2 ELISA kit, rat FSH ELISA kit, rat LH \nELISA kit, rat AMH ELISA kit, and rat MDA ELISA kit \nwere purchased from Elabscience Biotechnology Co., \nLTD (Wuhan, Hubei, China). The rat SOD ELISA kit was \npurchased from Jianglai Biotechnology Co., LTD (Shang -\nhai, China).\nQuantitative real-time PCR (qRT-PCR)\nTotal RNA was extracted by adding TRIzol reagent (Invi-\ntrogen, Thermo Fisher Scientific Inc., Waltham, MA, \nUSA) in each group. Subsequently, the extracted total \nRNA was reversely transcribed into cDNA according to \nthe instructions of PrimeScript RT reagent Kit (Takara, \nTokyo, Japan), and then the cDNA was amplified. The \nprimers were designed by Primer-BLAST software and \nsynthesized by Thermo Fisher Scientific (Waltham, MA, \nUSA), and the primer sequences and product lengths \nare shown in Table 1. The relative expression of VEGF, \nVEGFR-2, and FAK mRNA was calculated using the \n2−ΔΔCT  method with β-actin as an internal reference.\nWestern blotting (WB)\nThe total protein extracted was quantified for protein \nconcentration utilizing the bicinchoninic acid (BCA) kit \n(Biosharp, Guangzhou, Guangdong, China). Following \na 5-minute heating and denaturation process, a portion \nof the denatured protein samples underwent electro -\nphoresis on sodium dodecyl sulfate (SDS) polyacryl -\namide gel. Subsequently, the proteins were transferred \nfrom the gel to polyvinylidene fluoride (PVDF) mem -\nbrane and blocked with 5% skimmed milk powder for a \nduration of 1 h at ambient temperature. The membrane \nwas then subjected to incubation with primary antibod -\nies for VEGF(1:1,000, BA0407; Boster, Pleasanton, CA, \nUSA), VEGFR-2 (1:1,000, BM4256; Boster, Pleasanton, \nCA, USA), FAK(1:1,000, PB0662; Boster, Pleasanton, CA, \nUSA), p-VEGFR-2(1:1,000, AP0382; ABclonal Technol -\nogy Co., LTD, Wuhan, Hubei, China), p-FAK(1:1,000, \nAF1960; Beyotime Biotechnology, Shanghai, China), \nB-cell lymphoma-2 (Bcl-2) (1:1,000, ab182858; Abcam, \nCambridge, MA, USA), pro-Caspase-3(1:1,000, ab32499; \nAbcam, Cambridge, MA, USA), Caspase-3 (1:1,000, \nab32351; Abcam, Cambridge, MA, USA), apoptosis-\ninducing factor (AIF) (1:1,000, ab137725; Abcam, Cam -\nbridge, MA, USA), and β-actin (1:1,000, ab8227; Abcam, \nTable 1 List of primers used in the study\nNumber Gene Primer sequence Primer length\n1 β-actin Forward 5’-CTGAGAGGGAAATCGTGCGT-3’\nReverse 5’-CCACAGGATCCATACCCAAGA-3’ 150 bp\n2 VEGF Forward 5’-GAGCGTTCACTGTGAGCCTTGT-3’\nReverse 5’-TTAACTCAAGCTGCCTCGCCT-3’ 122 bp\n3 VEGFR-2 Forward 5’-TTGGCAAATACAACCCTTCAGAT-3’\nReverse 5’-GCAGAAGATACTGTCACCACCG-3’ 132 bp\n4 FAK Forward 5’-CAACCACCTGGGCCAGTATTATC-3’\nReverse 5’-CCATAGCAGGCCACATGCTTTA-3’ 138 bp\n\nPage 5 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nCambridge, MA, USA) overnight at 4 °C. After washing \nthree times with TBST buffer, the horseradish peroxidase \n(HRP)-labeled goat anti-rabbit IgG secondary antibody \n(1:5000, BM3894; Boster, Pleasanton, CA, USA) was \nintroduced and the membrane was incubated for 1 h at \nroom temperature. The relative expression of the target \nprotein was analyzed by Image J v1.8.0 software.\nCell culture and treatment\nHuman ovarian granulosa (KGN) cells were purchased \nfrom the Procell life science & technology Co., LTD \n(Wuhan, Hubei, China). The cells were cultured in Dul -\nbecco’s Modified Eagle Medium/Nutrient Mixture F-12 \n(DMEM/F12) medium (Gibco, Thermo Fisher Scientific \nInc., Waltham, MA, USA) supplemented with 10% fetal \nbovine serum (Gibco, Thermo Fisher Scientific Inc., \nWaltham, MA, USA) and 1% penicillin-streptomycin \n(Sangon-Biotech, Shanghai, China). The culture con -\ndition was 37 ℃ and 5% CO 2. After the KGN cells were \nadhered to the plate for 24 h, the supernatant was dis -\ncarded. In order to establish a cell model of ovarian \ndamage, KGN cells were treated with 200 µL of TP at \na concentration of 100 nM and incubated for 12 h. The \nKGN cells were exposed to medicated serum at varying \nconcentrations (0%, 5%, 10%, 15%, and 20%) to determine \nthe optimal concentration of YJD in subsequent experi -\nments. Subsequently, the effect of YJD on the cell model \nwas further investigated by treating TP-induced KGN \ncells with 5%, 10%, and 15% medicated serum. Addition -\nally, the potential involvement of the VEGF/VEGFR-2/\nFAK signaling pathway in the therapeutic effects of YJD \non POF was explored by adding VEGF inhibitor (Avas -\ntin), VEGFR-2 inhibitor (SU5408) and FAK inhibitor \n(Y15) to TP-induced KGN cells supplemented with 15% \nmedicated serum. After drug treatment, the culture was \ncontinued for 24 h.\nCell counting kit-8 (CCK-8) assay\nA medicated serum was generated through continu -\nous gavage of SD rats with M-YJD. Various concentra -\ntions (0%, 5%, 10%, 15%, and 20%) of medicated serum \nwere applied to KGN cells, and their inhibition ratio was \nassessed using CCK-8 kit (Yeasen Biotechnology Co., \nLTD, Shanghai, China). Subsequently, 5%, 10%, and 15% \nmedicated serum were employed to treat TP-induced \nKGN cells for cell viability assessment and subsequent \nexperiments.\nAnnexin V-fluorescein isothiocyanate (Annexin V-FITC) \napoptosis assay\nCell samples from each experimental group were col -\nlected and processed to achieve a concentration of 10^5 \ncells per milliliter in a suspension. The cells were subse -\nquently washed with phosphate-buffered saline (PBS), \ncentrifuged, and the resulting supernatant was removed \nbefore resuspending the cells. Following this, the cells \nwere treated with Annexin V-FITC/propidium iodide (PI) \nkit (Invitrogen, Thermo Fisher Scientific Inc., Waltham, \nMA, USA) according to the instructions of the kit, and \nthen allowed to incubate at room temperature for 15 min. \nFinally, the samples were analyzed using flow cytometry.\nStatistical analysis\nEach assay was performed for 3 times. Data were ana -\nlyzed by GraphPad Prism 8.0 (La Jolla, CA, USA) and \nexpressed as mean ± standard deviation. Two-tailed \nStudent’s t test were used for comparing two variables. \nOne-way ANOVA test was used for multiple variable \ncomparison. P < 0.05 was considered as a significant \ndifference.\nResults\nAngiogenesis and oxidative stress might be involved in \ntreatment of YJD on POF by bioinformatics analysis\nThe targets corresponding to the active ingredients of \nYJD were obtained from TCMSP , which yielded over -\nlapping 171 targets upon intersecting with POF disease \ntargets from Gene card (Fig. 1A). VEGFA was observed \nin a network map of the relationships between these 171 \ntargets and the active ingredients of 11 herbs in YJD (Fig. \n1B). Additionally, VEGFA and VEGFR were overlapping \ntarget genes of POF and YJD and had relatively strong \ninteraction with other target genes by the PPI network \ndiagram (Fig. 2A). The biological processes (BP), cell \ncomponents (CC) and molecular functions (MF) involved \nin overlapping target genes were analyzed by GO enrich -\nment analysis (Fig. 2B). Among them, overlapping target \ngenes enrichment was observed in response to oxidative \nstress, response to decreased oxygen levels and response \nto ROS in the top 10 BP entries. Furthermore, KEGG \nenrichment analysis was utilized to cluster the pathway \nfunctions on the overlapping target genes. Chemical car -\ncinogenic ROS and VEGF signaling pathways existed in \nthe top 20 pathways enriched by overlapping target genes \nof YJD and POF (Fig. 2C). Therefore, these findings indi-\ncated that oxidative stress and angiogenesis might be \ninvolved in treatment of YJD on POF.\nYJD reduced oxidative stress, enhanced angiogenesis and \nimproved ovarian function in POF rats\nVaginal cell smears in rats showed different cell compo -\nsitions during different stages of the estrous cycle: pro -\nestrus had mostly nuclear epithelial cells, estrus had \npatches of keratinized cells, metoestrus had equal pro -\nportions of keratinized cells and leukocytes, and dies -\ntrus had mostly leukocytes with some mucus (Fig. 3A). \nThe results of vaginal cell smears implied that the estrous \ncycle of the TP model group was disturbed, which \n\nPage 6 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nreturned to normal after YJD or DHEA treatment (Fig. \n3B-C). Additionally, YJD or DHEA therapy restored the \nweight loss induced by TP-induced POF in rats (Fig. 3D). \nThe decrease in ovarian wet weight and ovarian index \nin the POF rat models was improved by YJD or DHEA \ntreatment (Fig. 3E-F). The smaller ovaries with fewer fol -\nlicles and disorganized granulosa cells were observed in \nTP group by HE staining. YJD or DHEA caused normal -\nized ovarian volume and an increased number of follicles \n(Fig. 3G). Quantification of follicles at various develop -\nmental stages through HE staining indicated that the \nmodel group had fewer follicles at all levels compared \nto the normal group, with an increase in atretic follicles. \nAddition of YJD or DHEA resulted in the normalization \nof follicle counts across all stages, with the most pro -\nnounced therapeutic effect observed with M-YJD treat -\nment (Fig. 3H-L). Thus, YJD exhibited improvement in \novarian function in POF rats, among which M-YJD had \nthe strongest effect.\nThe results of IHC assay presented that decreased \nmicrovessel density were found in TP model group. \nTreatment with either YJD or DHEA led to a marked \nincrease in CD31 and α-SMA positive staining in the fol -\nlicles and corpus luteum and microvessel density of POF \nrats (Fig. 4A-C). P , E2, and AMH levels in the TP model \ngroup decreased while FSH and LH levels increased com-\npared to the control group by ELISA assays. After treat -\nment with YJD or DHEA, an increase of P , E2, and AMH \nFig. 2 Functional and pathway enrichment analysis of the overlapping target genes of YJD and POF. (A) PPI network analysis was utilized to observed the \ninteraction between these overlapping targets. (B) Bar charts of GO enrichment analysis of YJD and POF shared targets of action. (C) Bubble plots of KEGG \nenrichment analysis of YJD and POF shared targets of action\n \nFig. 1 Bioinformatics analysis of the correlation between YJD and POF. (A) Venn diagram of POF target genes established by Gene Card and YJD active \ningredients target genes obtained from TCMSP . (B) The network map of the overlapping target genes and the 11 herbs active components of YJD\n \n\nPage 7 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nFig. 3 YJD affected ovarian function in POF rats. ( A) Vaginal exfoliated cell smears were used to observe the morphological changes of cells at various \nstages of the estrous cycle of rats; Red arrows point to nucleated epithelial cells; Blue arrows point to keratinized cells; Green arrows point to white blood \ncells; (Note: The estrous cycle of rats is 4 ~ 5 days. The morphological changes in vaginal cytological smears of the rat estrous cycle were categorized as \nproestrus, estrus, metoestrus, and diestrus. After rats were modeled with TP , L-YJD, M-YJD and H-YJD as well as DHEA positive controls were added. (B-C) \nFrom the beginning of drug administration (D1), the estrous cycle of each rat was examined daily by vaginal smear, and the estrous cycles of the different \ngroups were counted. (D) Since the beginning of drug administration (D1), the dynamic changes in body weight of rats in each group were counted. \n(E) Bilateral intact ovary weights were weighed. ( F) Ovarian index was calculated (ovarian index = bilateral ovarian wet weight (mg)/body weight (g) × \n100%). (G) Ovarian tissue morphology was observed by HE staining and light microscopy; Black arrows point to the follicle. (H–L) The number of follicles \nat each level in HE staining was counted. (Primordial follicle: oocyte surrounded by a layer of flattened granulosa cells; Primary follicle: oocyte surrounded \nby a layer of cuboidal granulosa cells; Secondary follicle: oocyte with two or more layers of cuboidal granulosa cells, and without follicular sinus; Sinus \nfollicle: follicular sinus cavity is large, with a pronounced cumulus; Atretic follicle: follicular wall collapsed with damage to the structure of the oocyte, and \nthe loss of hyaline zone.) *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. TP + M-YJD\n \n\nPage 8 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nlevels and a decrease of FSH and LH levels were observed \n(Fig. 4D-H). MDA and SOD levels also normalized after \ntreatment with YJD or DHEA (Fig. 4I-J).\nAs a result, YJD demonstrated efficacy in improving \nTP-induced ovarian dysfunction by restoring a normal \nestrous cycle, leading to near-normal ovarian size, and \ndecreasing follicular atresia. Additionally, YJD treat -\nment caused an increase in ovarian angiogenesis and a \ndecrease in oxidative stress in POF rats.\nYJD activated VEGF/VEGFR-2/FAK signaling pathway in \nPOF rats\nIF analysis of germ cell-specific markers MVH and Oct4 \ndemonstrated a significant decrease in expression levels \nin the TP group compared to the control group. Con -\nversely, expression levels of both MVH and Oct4 were \nsignificantly elevated in the L-YJD, M-YJD, and H-YJD \ngroups compared to the TP group, with the best thera -\npeutic effect in M-YJD treatment. Furthermore, the \nFig. 4 YJD affected angiogenesis and oxidative stress in POF rats. (A) IHC was performed to detect the expression of CD31 and α-SMA, which is located \naround growing follicles in the ovaries; Black arrows point to areas of positive staining. (B) Average optical density of CD31. (C) The average optical density \nof α-SMA. (D–H) Serum levels of the hormones P , E2, FSH, LH, and AMH were examined by ELISA. (I–J) Serum levels of indicators of oxidative stress, MDA \nand SOD, were assessed by ELISA. ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. TP + M-YJD\n \n\nPage 9 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nDHEA group also exhibited a significant increase in \nexpression levels (Fig. 5A-B). Additionally, the qRT-PCR \nand IHC results indicated that YJD led to a significant \nincrease in the expression of VEGF, VEGFR-2, and FAK \nin POF rats (Fig. 5C-D). The results of WB assay further \nrevealed that the expression of p-VEGFR-2 and p-FAK \nalso was increased after YJD treatment in TP-induced \nPOF rats (Fig. 5E-F). Hence, YJD resulted in the activa -\ntion of the VEGF/VEGFR-2/FAK signaling pathway in \nrats with POF.\nYJD improved TP-induced KGN cell injury and activated \nVEGF/VEGFR-2/FAK pathway in vitro\nCCK-8 method was employed to screen out 5%, 10% \nand 15% YJD medicated serum for the treatment on \nTP-induced KGN cells in subsequent experiments by \nassessing the cell inhibition ratio of various concentra -\ntions of YJD medicated serum on KGN cells (Fig. 6A). \nCells viability decreased in TP-induced KGN cells but \nincreased with the increase of YJD concentration (Fig. \n6B). Levels of P , E2, and AMH were significantly reduced \nin the TP group, while FSH and LH levels were elevated. \nThe addition of YJD gradually restored all sex hormone \nlevels to normal by ELISA detection (Fig. 6C-G). In addi-\ntion, qRT-PCR results exhibited decreased expression of \nVEGF, VEGFR-2, and FAK in TP group, but increased \ntheir expression with YJD (Fig. 6H). WB confirmed that \nphosphorylation levels of angiogenesis markers were \ngradually increased with the increase of YJD concentra -\ntion (Fig. 6I-J). Moreover, the results of Annexin V-FITC \nflow cytometry showed that TP-induced apoptosis of \nKGN cells was gradually reduced with the increase of \nYJD treatment concentration (Fig. 6K). With the increase \nof YJD treatment concentration, the expression of pro-\nCaspase-3, Caspase-3 and AIF was gradually decreased, \nwhile the expression of anti-apoptotic factor Bcl-2 was \nincreased as confirmed by WB (Fig. 6L). Therefore, YJD \ninduced the activation of VEGF/VEGFR-2/FAK pathway \nand the inhibition of apoptosis in TP-induced KGN cells.\nFig. 5 YJD affected the VEGF/VEGFR-2/FAK pathway in vivo. ( A–B) Germ cell markers MVH and Oct4 were detected by IF. ( C) The expression of VEGF, \nVEGFR-2, and FAK was detected by qRT-PCR. (D) VEGF, VEGFR-2, FAK expression in the ovaries was examined by IHC; Black arrows point to areas of positive \nstaining. (E-F) The expression of VEGF, VEGFR-2, FAK, p-VEGFR-2 and p-FAK were assessed by WB. ** P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, \n###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01 vs. TP + M-YJD\n \n\nPage 10 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nYJD improved POF and reduced oxidative stress by \nactivating VEGF/VEGFR-2/FAK signaling pathway in vitro\nTo explore whether YJD improved POF by regulating \nVEGF/VEGFR-2/FAK signaling pathway, TP-induced \nKGN cells treated with 15% medicated serum were sup -\nplemented with VEGF inhibitor, VEGFR-2 inhibitor, \nand FAK inhibitor, respectively. Compared with the TP \n+ 15% medicated serum treatment group, the addition \nof angiogenesis marker inhibitors caused a reduction \nin the levels of P , E2 and AMH and an increase in the \nlevels of FSH and LH, as well as an abnormality in the \nlevels of oxidative stress markers MDA and SOD by \nELISA (Fig. 7A-G). Furthermore, the results of qRT-\nPCR and WB revealed that the increased expression of \nVEGF, VEGFR-2, FAK, p-VEGFR-2 and p-FAK induced \nby YJD in TP-induced KGN cells was reversed by the \naddition of Avastin, SU5408 or Y15 (Fig. 7H-L). Simi -\nlarly, the inhibited apoptosis and reduced expression of \nFig. 6 YJD affected the VEGF/VEGFR-2/FAK pathway and KGN cell injury in vitro. ( A) Screening of YJD for medicated serum concentrations utilized the \nCCK-8 assay. (B) The cell viability of each group was measured by CCK-8. ( C–G) Serum levels of the hormones P , E2, FSH, LH, and AMH were examined \nby ELISA. (H) The expression of VEGF, VEGFR-2, and FAK was detected by qRT-PCR. (I-J) The expression of VEGF, VEGFR-2, FAK, p-VEGFR-2 and p-FAK were \nassessed by WB. (K) Apoptosis index was detected by Annexin V-FITC flow cytometry. (L) The expression of Bcl-2, pro-Caspase-3, Caspase-3 and AIF were \nassessed by WB. **P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TP\n \n\nPage 11 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nFig. 7 YJD affected POF by regulating the VEGF/VEGFR-2/FAK signaling pathway. ( A–E) Serum levels of the hormones P , E2, FSH, LH, and AMH were \nexamined by ELISA. Control and TP groups were set up, and the remaining groups were co-treated with TP and 15% medicated serum, along with VEGF \ninhibitor (Avastin), VEGFR-2 inhibitor (SU5408), or FAK inhibitor (Y15). (F-G) Serum levels of indicators of oxidative stress, MDA and SOD were assessed by \nELISA. (H-J) The expression of VEGF, VEGFR-2, and FAK was detected by qRT-PCR. (K-L) The expression of VEGF, VEGFR-2, FAK, p-VEGFR-2 and p-FAK were as-\nsessed by WB. (M) Apoptosis rate was detected by Annexin V-FITC flow cytometry. (N) The expression of Bcl-2, pro-Caspase-3, Caspase-3 and AIF were as-\nsessed by western blotting. **P < 0.01, ***P < 0.001 vs. Control; ##P < 0.01, ###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. TP + 15% medicated serum\n \n\nPage 12 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \napoptotic factors in TP-induced KGN cells by YJD were \nalso altered by the addition of Avastin, SU5408 or Y15 \nvia flow cytometry and WB (Fig. 7M-N). These findings \npresented above indicated that YJD induced normaliza -\ntion of sex hormone and oxidative stress levels, as well \nas promoted angiogenesis in TP-induced KGN cell mod -\nels. However, the effect of YJD on TP-induced KGN cell \nmodels was reversed by angiogenesis inhibitors. There -\nfore, YJD improved TP-induced KGN cell function and \nreduced oxidative stress levels through the activation of \nthe VEGF/VEGFR-2/FAK signaling pathway.\nYJD improved the fertility of pregnant POF rats\nIn order to investigate the impact of YJD on the fertil -\nity of POF rats, an analysis was conducted on the preg -\nnancy rate, number of implanted embryos, number of \nlive births, and weight of newborn rats in each group, \nas detailed in Table 2. Both YJD and DHEA treatments \nresulted in an increase in the pregnancy rate, number \nof implanted embryos, number of live births, and body \nweight of newborn rats in POF rats. Furthermore, the \nELISA assays revealed a significant decrease in the levels \nof P , E2, and AMH in pregnant rats with POF, along with \na significant increase in FSH and LH. Additionally, treat -\nment with YJD was found to improve the sex hormone \nlevels in pregnant POF rats, with the best effect in M-YJD \ntreatment (Fig. 8A-E). Moreover, the uterine embryo \nlanding plots of different groups of pregnant mice were \nshown in Fig. 8F. YJD and DHEA group embryos were \nuniformly sized, fully developed, and had no blood in the \nuterus, while model group embryos were unevenly dis -\ntributed, small, and irregularly shaped with some blood \nin the uterus. In summary, YJD improved sex hormone \nlevels and ovarian function in pregnant POF rats and \nimproved their reproductive capacity.\nDiscussion\nPOF is one of the most serious diseases affecting women’s \nreproductive health in the current era. Although there \nare various causes of POF, such as genetic abnormalities, \nautoimmune factors, medical factors, infectious factors, \ntoxins, and environmental factors, in most cases, the \ncause of POF cannot be identified after a comprehensive \nevaluation [22]. With the increasing incidence of cancer, \nradiotherapy and chemotherapy are gradually becoming \nthe main cause of POF in young women [ 23]. This not \nonly affects women’s fertility, but also seriously influences \ntheir mental health and quality of life [ 24]. This study \nconfirmed that YJD improved POF symptoms by amelio -\nrating oxidative stress damage and promoting angiogen -\nesis, which provided a direction for clinical treatment to \nimprove ovarian function.\nTable 2 Pregnancy rate, number of implanted embryos, number of live births, and body weight of newborn rats\nGroup Pregnancy rate (%) Average number of implantation sites Number of live births Body weight of newborn rats (g)\nControl 90.00% (9/10) 16.67 ± 0.58 15.67 ± 1.22 7.62 ± 0.14\nTP 42.86% (3/7) 9.33 ± 1.53*** 9.33 ± 1.53*** 6.87 ± 0.25***\nTP + L-YJD 50.00% (4/8) 12.33 ± 0.58 12.00 ± 0.41### 7.00 ± 0.17##\nTP + M-YJD 62.50% (5/8) 14.00 ± 0.00## 12.40 ± 1.14### 7.31 ± 0.16###\nTP + H-YJD 57.14% (4/7) 12.33 ± 0.58# 11.75 ± 0.96## 7.01 ± 0.25#\nTP + DHEA 77.78% (7/9) 15.00 ± 1.00### 14.71 ± 1.38###& 7.41 ± 0.20###&&\n*P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01, &&& P < 0.001 vs. TP + M-YJD\nFig. 8 Effect of YJD on fertility in rats with POF. (A–E) The levels of serum hormones P , E2, FSH, LH and AMH in rats were assessed by ELISA. After rats were \nmodeled with TP , L-YJD, M-YJD and H-YJD as well as DHEA positive controls were administrated to POF rats. ( F) Representative images of the uterus of \npregnant rats. ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TP; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. TP + M-YJD\n \n\nPage 13 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nTP is an active compound extracted from the Chinese \nherb Lei Gong Teng [ 25]. It has been shown to possess \nanti-inflammatory, immunosuppressive and anticancer \nactivities. However, the use and development of TP has \nbeen extremely limited because it causes serious damage \nto the liver, kidneys and reproductive system [26]. Oxida-\ntive stress is the predominant mechanism of TP-induced \ninjury [27]. TP and its toxic metabolites interfere with the \nintracellular antioxidant system and impair its detoxifi -\ncation. This may eventually lead to oocyte degeneration, \ngranulosa cell apoptosis and impaired hormone secre -\ntion [28]. Furthermore, it has also been shown that exces-\nsive oxidative stress damage has a detrimental effect on \nangiogenesis. SOD is an important enzyme in the antiox -\nidant defense process and plays an important role in pro -\ntecting cells and tissues [ 29]. MDA is a major metabolite \nof lipid peroxidation and an important marker of oxida -\ntive stress damage [ 30]. The present study utilized TP to \ninduce POF models in rats and KGN cells. The observed \ndecrease in SOD levels and increase in MDA accumula -\ntion in the TP-induced model resulted in localized oxida-\ntive damage within the ovary, aligning with findings from \nprevious research. Moreover, treatment with YJD dem -\nonstrated an ameliorative effect on TP-induced oxidative \nstress levels.\nThe rodent estrous cycle is characterized by morpho -\nlogical changes in the ovaries, uterus, and vagina, making \nit a valuable metric for monitoring reproductive perfor -\nmance [31]. The estrous cycle in female rats typically lasts \nfrom 4 to 5 days [ 32]. Although there might be poten -\ntial delays or advances in specific phases of the estrous \ncycle in the rat models in the course of this study, result -\ning in irregularity of the estrous cycle. However, it had \nno remarkable impact on the results of the study. These \nfindings confirmed that YJD improved ovarian dysfunc -\ntion in TP-induced POF rats by restoring the normal \nestrous cycle and facilitating ovarian size normalization. \nMoreover, MVH and Oct4 are often used as molecular \nmarkers of germ cells [ 33, 34]. In the POF rat models, \ntreatment with YJD restored the TP-induced reduction \nin MVH and Oct4 expression. On the other hand, sex \nhormone levels are closely related to ovarian function. \nAMH inhibits the development of male mullerian ducts \nand regulates the development of reproductive cells and \ngonads in both sexes [35, 36]. It can be used as an indica -\ntor to evaluate the ovarian reserve function. In addition, \nE2 is a naturally occurring estrogen that is integral to the \npreservation of female secondary sexual characteristics. \nThe expression of P and E2 has been linked to follicular \ngrowth [37]. FSH is primarily produced by the basophilic \ncells of the pituitary gland. Working in conjunction with \nLH, FSH facilitates the maturation of follicles, leading to \nthe secretion of P and estrogen [ 38, 39]. Our experimen-\ntal data showed that YJD increased E2, P , and AMH levels \nas well as reduced FSH and LH levels, improving ovar -\nian function and fertility in POF rats during diestrus and \npregnancy.\nThe process of angiogenesis in the ovary is intricate, \ninvolving both neovascularization and vascular matu -\nration [ 40]. Key cell types implicated in this process \nare vascular endothelial cells and pericytes [ 41]. Vascu-\nlar endothelial cells proliferate and migrate to establish \nneovascularization, while pericytes play a crucial role \nin ensuring vascular integrity and stabilization through \ntheir recruitment [ 42]. The collaboration between vas -\ncular endothelial cells and pericytes is vital for microvas -\ncular remodeling and stabilization [ 43]. In the present \ninvestigation, vascular endothelial cells and pericytes \nwere identified using CD31 and α-SMA markers, respec -\ntively. Findings revealed a notable elevation in CD31 and \nα-SMA positive staining within the follicle and corpus \nluteum following YJD treatment as opposed to the TP-\ninduced model group. These results indicated that YJD \nmitigated vascular damage in the ovaries of POF rats by \nenhancing angiogenesis and promoting vascular stabil -\nity. Furthermore, the angiogenic process necessitates \nthe collaborative involvement of multiple essential fac -\ntors. VEGF is a major factor involved in cell proliferation, \nmigration, survival and vascular permeability of vascu -\nlar endothelial cells [ 44]. The pro-angiogenic effect of \nVEGF is mediated by binding to VEGFR-2, which trig -\ngers a series of intracellular signaling channels. FAK is a \ndownstream signal effector of VEGF/ VEGFR-2 pathway, \nwhich is closely related to cell adhesion, diffusion, pro -\nliferation, migration and apoptosis [ 45]. In both POF rat \nand KGN cell models, YJD demonstrated a significant \nenhancement in the expression levels of VEGF, VEGFR-2, \nand FAK, ultimately facilitating ovarian angiogenesis. In \naddition, VEGF/VEGFR-2/FAK inhibitors were found to \nreverse the effect of YJD in TP-induced KGN cells. Con -\nsequently, the modulation of the VEGF/VEGFR-2/FAK \nsignaling pathway by YJD appeared to be a promising \ntherapeutic approach for improving POF.\nCaspase-3 is at the center of the caspase cascade reac -\ntion and is a common pathway for all apoptotic signal -\ning pathway [46]. Bcl-2 and Caspase-3 interact with each \nother to regulate the process of apoptosis. In this work, \nthe treatment of YJD significantly decreased the expres -\nsion of Caspase-3 and AIF, while the expression of Bcl-2 \nwas significantly increased. The effect of YJD on apopto -\nsis in the treatment of POF deserved further exploration.\nSeveral recent studies have indicated that maternal \nexposure to TP to conception may impact pregnancy \noutcomes and result in long-term negative consequences \nfor future generations [ 47]. Our study revealed notable \ndecrease in pregnancy rates, reduction in the number \nof implanted embryos and severe uterine hemorrhaging \nin the TP-induced model group. Conversely, the group \n\nPage 14 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \ntreated with YJD did not exhibit uterine hemorrhaging \nand displayed a more uniform distribution of embryos. \nThe findings of this study indicated that M-YJD had a \nsignificant impact on both the pregnancy rate and the \nnumber of embryos implanted, suggesting its potential \nto enhance the intrauterine microenvironment for suc -\ncessful implantation and subsequent live births. Further \ncomprehensive research is needed to fully understand \nthe mechanisms by which YJD may influence in utero \nembryo development. In the present research, the ther -\napeutic effects of YJD at the selected dose gradients on \nPOF rats did not exhibit a dose-dependent response. This \nphenomenon may be attributed to the complexity of the \nherbal formula components, the multi-target synergistic \neffects, and the feedback regulation mechanisms of the \nbody [ 48]. The pharmacological effects of the various \ncomponents in the herbal formula may be either syner -\ngistic or antagonistic [ 49]. Additionally, the current dose \ngradient of YJD used in this work may not fully cover \nthe critical nodes of the “dose-response” curve, which \ncould explain the absence of a dose-dependent effect. In \nfuture studies, it would be beneficial to further explore \nthe optimal dose range of YJD by incorporating pharma -\ncokinetic analyses and to comprehensively evaluate the \noverall effects of the herbal formula using multi-omics \napproaches, which will enhance the potential of YJD in \nthe treatment of POF.\nConclusion\nIn conclusion, the administration of YJD resulted in a \nsignificant improvement in sex hormone levels and ovar -\nian function in rats with POF. Furthermore, YJD demon -\nstrated a protective effect on ovarian tissue by alleviating \noxidative damage through enhancing SOD levels and \nreducing MDA accumulation, as well as promot -\ning angiogenesis through the regulation of the VEGF/\nVEGFR-2/FAK signaling pathway. Additionally, YJD was \nfound to enhance the intrauterine implantation microen-\nvironment, leading to an increase in pregnancy rates and \nlive birth numbers.\nAbbreviations\nAMH  Anti-mullerian hormone\nCD31  Platelet endothelial cell adhesion molecule-1\nDHEA  Dehydroepiandrosterone\nE2  Estradiol\nELISA  Enzyme-linked immunosorbent assay\nETCM  Encyclopedia of traditional Chinese medicine\nFITC  Fluorescein isothiocyanate\nFSH  Follicle stimulating hormone\nGEO  Gene expression omnibus\nGO  Gene ontology\nHE  Staining of hematoxylin and eosin\nKEGG  Kyoto encyclopedia of genes and genomes\nLH  Luteinizing hormone\nMDA  Malondialdehyde\nOMIM  Online mendelian inheritance in humans\nP  Progesterone\nPBS  Phosphate buffer solution\nPI  Propidium iodide\nPOF  Premature varian failure\nPPI  Protein-protein interaction\nPMSG  Pregnant Mare Serum Gonadotropin\nqRT-PCR  Quantitative real-time PCR\nROS  Reactive oxygen species\nSD  Sprague Dawley\nSOD  Superoxide dismutase\nTCM  Traditional Chinese medicine\nTCMSP  Traditional Chinese medicine systematic pharmacology\nTP  Triptolide\nYJD  Yijing decoction\nα-SMA  α-Smooth muscle actin\nSupplementary Information\nThe online version contains supplementary material available at  h t t p s :   /  / d o  i .  o r  \ng  /  1 0  . 1 1   8 6  / s 1 3  0 4 8 -  0 2 5 - 0  1 6 7 9 - 2.\nSupplementary Material 1\nAcknowledgements\nThe authors thank all colleagues and support from Henan Provincial People’s \nHospital.\nAuthor contributions\nRG: Conceptualization, Data curation, Formal analysis, Investigation, Writing-\noriginal draft, Writing-review & editing. YW: Conceptualization, Data curation, \nFormal analysis, Investigation, Writing-original draft, Writing-review & editing. \nYT: Formal analysis, Software, Methodology, Writing-original draft, Writing-\nreview & editing. KW: Software, Methodology, Writing-review & editing. BG: \nConceptualization, Funding acquisition, Project administration, Resources, \nSupervision, Writing-review & editing.\nFunding\nThe present study was supported by the National Natural Science \nFoundation of China (Grant No. 81873334 and 81973684), the Natural Science \nFoundation of Sichuan Province (Grant No. 2023NSFSC1760) and Henan \nProvince Postdoctoral Research Program (Grant No. HN2024079), and Health \nCommission of Chengdu and Chengdu University of Traditional Chinese \nMedicine Joint Innovation Fund in 2024 (Grant No. WXLH202402019).\nData availability\nThe datasets generated and/or analysed during the current study are not \npublicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available \nfrom the corresponding author on reasonable request.\nDeclarations\nEthics approval and consent to participate\nAll animal experimental procedures were performed in accordance with the \nInstitutional Animal Care and Use Committee of Henan Provincial People’s \nHospital, which was approved by the Institutional Animal Care and Use \nCommittee of Henan Academy of Traditional Chinese Medicine Animal \nExperiment Center (SYXK (Yu) 2022-0007).\nPatient consent for publication\nNot applicable.\nCompeting interests\nThe authors declare no competing interests.\nReceived: 19 November 2024 / Accepted: 21 April 2025\n\n\nPage 15 of 15\nGao et al. Journal of Ovarian Research            (2025) 18:87 \nReferences\n1. Ghahremani-Nasab M, et al. Premature ovarian failure and tissue engineering. \nJ Cell Physiol. 2020;235(5):4217–26.\n2. Liu M, et al. MicroRNA-144-3p protects against chemotherapy-induced \napoptosis of ovarian granulosa cells and activation of primordial follicles by \ntargeting MAP3K9. Eur J Med Res. 2023;28(1):264.\n3. Deady J. Clinical monograph: hormone replacement therapy. J Managed \nCare Pharmacy: JMCP . 2004;10(1):33–47.\n4. Wang Y, Teng X, Liu J. Research progress on the effect of traditional Chinese \nmedicine on signal pathway related to premature ovarian insufficiency. \nEvidence-based Complement Altern Medicine: eCAM. 2022;2022:p7012978.\n5. Yan J, et al. [Effects of Rehmanniae Radix and Rehmanniae Radix Praeparata \non proteomics and autophagy in mice with type 2 diabetes mellitus induced \nby high-fat diet coupled with streptozotocin]. Zhongguo Zhong yao za zhi = \nZhongguo zhongyao zazhi = China. J Chin Materia Med. 2023;48(6):1535–45.\n6. Zhou W, et al. Comprehensive quality evaluation of two different geogra-\nphy originated Angelica sinensis Radix based on potential production area \ndevelopment and resource protection. Volume 201. Plant physiology and \nbiochemistry: PPB; 2023. p. 107878.\n7. Chen C, et al. Radix paeoniae Alba attenuates Radix Bupleuri-induced \nhepatotoxicity by modulating gut microbiota to alleviate the Inhibition of \nsaikosaponins on glutathione synthetase. J Pharm Anal. 2023;13(6):640–59.\n8. Jeong J, et al. Triple herbal extract DA-9805 exerts a neuroprotective effect \nvia amelioration of mitochondrial damage in experimental models of Parkin-\nson’s disease. Sci Rep. 2018;8(1):15953.\n9. Hu P , et al. An UPLC-MS/MS method for targeted analysis of microbial and \nhost Tryptophan metabolism after administration of polysaccharides from \natractylodes macrocephala Koidz. In ulcerative colitis mice. J Pharm Biomed \nAnal. 2023;235:115585.\n10. Zhang X, et al. First report of root rot caused by fusarium Armeniacum on \nAmerican ginseng in China. Plant Dis. 2020;105(4):1223.\n11. Ji Q, et al. Comparative transcriptome profiling analysis provides insight into \nthe mechanisms for sugar change in Chinese jujube (Ziziphus Jujuba Mill.) \nunder rain-proof cultivation. Plant Genome. 2023;16(2):e20341.\n12. Xue-Han LJ-JHG-RL. Clinical study of modified Yijing Decoction combined \nwith artificial periodic therapy for the treatment of diminished ovarian reser-\nvation. J Guangzhou Univ Traditional Chin Med. 2022;39(9):2021–7.\n13. Shimizu T. Molecular and cellular mechanisms for the regulation of ovarian \nfollicular function in cows. J Reprod Dev. 2016;62(4):323–9.\n14. Qiao J, Feng H. Extra- and intra-ovarian factors in polycystic ovary syndrome: \nimpact on oocyte maturation and embryo developmental competence. \nHum Reprod Update. 2011;17(1):17–33.\n15. Robinson R, et al. Angiogenesis and vascular function in the ovary. Reprod \n(Cambridge England). 2009;138(6):869–81.\n16. Nie X, et al. Establishment of a mouse model of premature ovarian failure \nusing consecutive superovulation. Cell Physiol Biochemistry: Int J Experimen-\ntal Cell Physiol Biochem Pharmacol. 2018;51(5):2341–58.\n17. Moldogazieva N, et al. Oxidative stress and advanced lipoxidation and \nglycation end products (ALEs and AGEs) in aging and Age-Related diseases. \nOxidative Med Cell Longev. 2019;2019:p3085756.\n18. Schallreuter K, et al. In vivo and in vitro evidence for hydrogen peroxide \n(H2O2) accumulation in the epidermis of patients with vitiligo and its suc-\ncessful removal by a UVB-activated pseudocatalase. J Invest Dermatology \nSymp Proc. 1999;4(1):91–6.\n19. Kashino G, et al. VEGF affects mitochondrial ROS generation in glioma \ncells and acts as a radioresistance factor. Radiat Environ Biophys. \n2023;62(2):213–20.\n20. Limei L, et al. Effect of modified Yijing Decoction on sex hormones and \novarian histomorphology in rats with ovarian hypofunction. Chin Med Rev. \n2021;27(02):24–6.\n21. Zhou F, et al. Si-Wu-Tang facilitates ovarian function through improving \novarian microenvironment and angiogenesis in a mouse model of premature \novarian failure. J Ethnopharmacol. 2021;280:114431.\n22. Ebrahimi M, Akbari F, Asbagh. Pathogenesis and causes of premature ovarian \nfailure: an update. Int J Fertility Steril. 2011;5(2):54–65.\n23. Rodriguez-Wallberg K, Oktay K. Options on fertility preservation in female \ncancer patients. Cancer Treat Rev. 2012;38(5):354–61.\n24. Howard-Anderson J, et al. Quality of life, fertility concerns, and behavioral \nhealth outcomes in younger breast cancer survivors: a systematic review. J \nNatl Cancer Inst. 2012;104(5):386–405.\n25. Gao H, et al. Triptolide induces autophagy and apoptosis through ERK activa-\ntion in human breast cancer MCF-7 cells. Experimental Therapeutic Med. \n2018;15(4):3413–9.\n26. Deng Y, et al. Triptolide sensitizes breast cancer cells to doxorubicin through \nthe DNA damage response Inhibition. Mol Carcinog. 2018;57(6):807–14.\n27. Zhao F, et al. Triptolide induces protective autophagy through activation of \nthe CaMKKβ-AMPK signaling pathway in prostate cancer cells. Oncotarget. \n2016;7(5):5366–82.\n28. Vliegenthart A, et al. Characterization of Triptolide-Induced hepatotoxic-\nity by imaging and transcriptomics in a novel zebrafish model. Toxicol Sci. \n2017;159(2):380–91.\n29. Fu K, et al. Xanthotoxin induced photoactivated toxicity, oxidative stress \nand cellular apoptosis in Caenorhabditis elegans under ultraviolet A. Comp \nBiochem Physiol C Toxicol Pharmacol. 2022;251:109217.\n30. Ramachandran A. H. Jaeschke 2019 Acetaminophen hepatotoxicity: A mito-\nchondrial perspective. Adv Pharmacol (San Diego Calif ) 85 195–219.\n31. Goldman JM, Murr AS, Cooper RL. The rodent estrous cycle: characterization \nof vaginal cytology and its utility in toxicological studies. Birth Defects Res B \nDev Reprod Toxicol. 2007;80(2):84–97.\n32. Cora MC, Kooistra L, Travlos G. Vaginal cytology of the laboratory rat and \nmouse: review and criteria for the staging of the estrous cycle using stained \nvaginal smears. Toxicol Pathol. 2015;43(6):776–93.\n33. Nagamori I, Cruickshank V, Sassone-Corsi P . Regulation of an RNA granule \nduring spermatogenesis: acetylation of MVH in the chromatoid body of germ \ncells. J Cell Sci. 2011;124:4346–55.\n34. Morrison G, Brickman J. Conserved roles for Oct4 homologues in maintain-\ning multipotency during early vertebrate development. Development. \n2006;133(10):2011–22.\n35. Boucret L, et al. Relationship between diminished ovarian reserve and \nmitochondrial biogenesis in cumulus cells. Hum Reprod (Oxford England). \n2015;30(7):1653–64.\n36. Lunding S, et al. AMH as predictor of premature ovarian insufficiency: A \nlongitudinal study of 120 Turner syndrome patients. J Clin Endocrinol Metab. \n2015;100(7):E1030–8.\n37. Komatsu K, Masubuchi S. The concentration-dependent effect of progester-\none on follicle growth in the mouse ovary. J Reprod Dev. 2017;63(3):271–7.\n38. Bosch E, et al. Reduced FSH and LH action: implications for medically assisted \nreproduction. Hum Reprod (Oxford England). 2021;36(6):1469–80.\n39. Moolhuijsen L, Visser J. Anti-Müllerian hormone and ovarian reserve: \nupdate on assessing ovarian function. J Clin Endocrinol Metab. \n2020;105(11):3361–73.\n40. Gaengel K, et al. Endothelial-mural cell signaling in vascular development \nand angiogenesis. Arterioscler Thromb Vasc Biol. 2009;29(5):630–8.\n41. Herbert S, Stainier D. Molecular control of endothelial cell behaviour during \nblood vessel morphogenesis. Nat Rev Mol Cell Biol. 2011;12(9):551–64.\n42. Sugino N, et al. Angiogenesis in the human corpus luteum. Reproductive \nMed Biology. 2008;7(2):91–103.\n43. Davis GE, Norden PR, Bowers SL. Molecular control of capillary morphogene-\nsis and maturation by recognition and remodeling of the extracellular matrix: \nfunctional roles of endothelial cells and pericytes in health and disease. \nConnect Tissue Res. 2015;56(5):392–402.\n44. Apte R, Chen D, Ferrara N. VEGF in signaling and disease: beyond discovery \nand development. Cell. 2019;176(6):1248–64.\n45. Claesson-Welsh L, Welsh M. VEGFA and tumour angiogenesis. J Intern Med. \n2013;273(2):114–27.\n46. Hadji A, et al. Caspase-3 triggers a TPCK-sensitive protease pathway leading \nto degradation of the BH3-only protein puma. Apoptosis: Int J Program Cell \nDeath. 2010;15(12):1529–39.\n47. Wen X, et al. A systematic review and Meta-Analysis of the inhibitory effects \nof Plant-Derived sterilants on rodent population abundance. Toxins (Basel). \n2022;14(7).\n48. Li C, et al. Traditional Chinese medicine in depression treatment: from mol-\necules to systems. Front Pharmacol. 2020;11:586.\n49. Ung CY, et al. Are herb-pairs of traditional Chinese medicine distinguishable \nfrom others? Pattern analysis and artificial intelligence classification study of \ntraditionally defined herbal properties. J Ethnopharmacol. 2007;111(2):371–7.\nPublisher’s note\nSpringer Nature remains neutral with regard to jurisdictional claims in \npublished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}