Integrative network pharmacology and experimental validation to decipher the anti-angiogenic mechanisms of Xuandan Sanjie decotion (玄丹散结汤) in adenomyosis

Abstract

OBJECTIVE:

To investigate the mechanisms by which Xuandan Sanjie decoction (玄丹散结汤, XSD) inhibits angiogenesis in adenomyosis (AM), using both AM-derived primary endometrial stromal cells (ESCs) and a mouse model.

METHODS:

Core components and targets of XSD for AM treatment were predicted using multiple databases. Gene ontology and kyoto encyclopedia of genes and genomes pathway analyses were used to elucidate the potential mechanisms. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was applied to identify chemical constituents of XSD. A cobalt (II) chloride (CoCl₂)-induced hypoxia-mimicking ESC model and an AM mouse model were established to experimentally validate the anti-angiogenic effects of XSD.

RESULTS:

According to bioinformatics analysis, hypoxia signaling and angiogenesis are crucial for AM progression. Twenty chemical constituents of the 106 compounds identified from the Traditional Chinese Medicine systems pharmacology database and analysis platform database were validated by UHPLC-MS/MS. Network pharmacology identified protein kinase B alpha, epidermal growth factor receptor, hypoxia inducible factor-1α (HIF-1α), and estrogen receptor 1 (ESR1) as core targets. According to in vitro and in vivo experiments, XSD exhibits anti-angiogenic properties through ESR1/G protein-coupled estrogen receptor (GPER)-mediated HIF-1α signaling.

CONCLUSION:

Our study confirms that XSD inhibits angiogenesis in AM viaESR1/GPER-mediated HIF-1α signaling. These findings elucidate the molecular mechanisms underlying XSD's therapeutic effects for abnormal uterine bleeding associated with adenomyosis, offering new therapeutic strategies for this condition.

1. INTRODUCTION

Abnormal uterine bleeding (AUB), a representative symptom of adenomyosis (AM), significantly impacts females' reproductive health and life quality.¹ Menorrhagia, the most common bleeding pattern in AUB associated with AM (AUB-A), can cause severe anemia and hypercoagulability, increasing thrombosis risk.² Hormonal therapies for AM can reduce menorrhagia but often lead to menstrual irregularities, hypoestrogenism, and a higher risk of thrombosis,³ highlighting the need for safer and more effective treatments for AUB-A.⁴

Recent studies indicate that excessive angiogenesis increases vascular permeability and fragility, thereby contributing to AUB-A. Thus, targeting abnormal angiogenesis has emerged as a valuable treatment strategy.^5,6 Traditional Chinese Medicine (TCM), with its multi-component and multi-target characteristics, has shown good efficacy and few side effects in treating AUB.⁷ Xuandan Sanjie decoction (玄丹散结汤, XSD), developed by Prof. YANG Guiyun and patented (ZL201610482871.0), significantly relieves dysmenorrhea, menorrhagia, and other AM symptoms by activating blood circulation, resolving stasis, and dispersing masses. However, whether its therapeutic effects involve angiogenesis regulation remains unknown, providing an opportunity to explore XSD’s mechanism in AUB-A.

A network pharmacology strategy was employed herein to elucidate interactions among angiogenesis, relevant signaling pathways, and therapeutic targets in AM. In vitro and in vivo experiments using primary endometrial stromal cells (ESCs) and a mouse AM model further clarify XSD's mechanism in treating AUB-A.

2. MATERIALS AND METHODS

2.1. XSD active compound screening and target prediction

We retrieved active compounds and target proteins of XSD from the Traditional Chinese Medicine systems pharmacology database and analysis platform (TCMSP) Database.⁸ Compounds were screened using absorption, distribution, metabolism and eliminationparameters: oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, and half-life (HL) ≥ 4 h. Target proteins were converted to Homo sapiens gene symbols via UniProt.

2.2. Potential target screening for AM

Potential targets associated with AM were identified from GeneCards and national center for biotechnology information. After eliminating duplicate entries, AM-related targets were obtained.

2.3. Protein-protein interaction (PPI) network construction

A PPI network was constructed using Cytoscape software (Boston, MA, USA), based on the overlapping targets and their corresponding compounds. First, overlapping targets were subjected to PPI analysis in the search tool for the tetrieval of interaction gene/proteins (STRING) platform, restricted to Homo sapiens.⁹ This interaction network was used to identify core regulatory genes. The degree of each node was calculated using STRING after removing unconnected nodes. The final PPI network was visualized in Cytoscape software (Boston, MA, USA), where nodes with higher degrees were considered key targets.¹⁰

2.4. Functional enrichment analyses

To elucidate the potential signaling pathways underlying XSD’s therapeutic effects on AM, kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed using Metascape, while gene ontology (GO) analysis was conducted using the database for annotation, visualization and integrated discovery. The species was set to "human". Results were ranked in ascending order of P-values, and the top 10 GO terms and KEGG pathways were selected for further investigation.

2.5. Experimental drugs and preparation

XSD is composed of seven herbs, i.e., Danshen (Radix Salviae Miltiorrhizae) 15 g, Yanhusuo (Rhizoma Corydalis Yanhusuo) 10 g, Danggui (Radix Angelicae Sinensis) 10 g, Wuyao (Radix Linderae Aggregatae) 10 g, Yujin (Radix Curcumae Wenyujin) 10 g, Shijianchuan (Herba Salviae Chinensis) 15 g, Guijianyu (Ramulus Euonymi) 10 g. These Chinese herbs were obtained from Jiangsu Province Hospital of Integrated Traditional and Western Medicine (Nanjing, China). Herbs (80 g total) were mixed with 10 × (w/v) distilled water, boiled at 100 ℃ for 40 min, and strained. The residue was decocted again. The combined decoctions were centrifuged at 4 ℃ for 10 min, concentrated using a rotary evaporator (80 ℃), and filtered through a 0.22 μm filter for cellular use.

2.6. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis

Thawed XSD extract was vortexed (30 s) and centrifuged (12 000 rpm, 15 min, 4 ℃). Supernatant (300 μL) was mixed with 1000 μL extraction solution (methanol:water = 4∶1, v/v) containing 10 μg/mL internal standard, vortexed, ultrasonicated (ice-water bath, 5 min), incubated at －40 ℃ for 1 h, and centrifuged again (12 000 rpm, 15 min, 4 ℃). The supernatant (500 μL) was filtered (0.22 μm) for analysis.

UHPLC-MS/MS was performed on an Agilent 1290 UHPLC coupled with an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Both positive and negative ion modes were used, with data-dependent acquisition mode for MS1/MS2 (scan range m/z 100-1500). Separation used an an Aquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) (Waters, Milford, MA, USA) at 40 ℃, flow rate 0.3 mL/min, injection 5 μL. Data were processed with XCMS for feature detection, retention time correction, and alignment, and compounds were identified by matching fragment ions against a secondary MS database provided by Shanghai BIOTREE biotech Co., Ltd. (Shanghai, China).

2.7. Chemicals

A 200 mM stock solution was prepared by dissolving cobalt (II) chloride (CoCl₂) (Sigma, St. Louis, MO, USA) in dulbecco's modified eagle medium/nutrient mixture F-12 (1∶1). Hypoxia was induced in cells by culturing them for 24 h in media supplemented with 200 μM CoCl₂ to induce hypoxia. 17β-Estradiol (E2) (Sigma, St Louis, MO, USA) was dissolved in 10 μM ethanol (stock concentration) and stored at －80 ℃. Preparation of G15 (MedChemExpress, Shanghai, China; HY-103449) stock solutions was achieved through dissolving each compound in dimethyl sulfoxide (DMSO) at 100 μM. Giredestrant (a selective estrogen receptor degrader and full antagonist) was dissolved in DMSO and diluted to a final concentration of 10 nM in culture medium. Calcein-AM powder (Yeasen, Shanghai, China) was dissolved in DMSO to 6.25 mg/mL (1 mg in 160 μL DMSO) and stored at −20 ℃. Mifepristone (25 mg/tablet, Resources Zizhu, Beijing, China) was dissolved in DMSO to 1×10^-2 M, then diluted to 1 × 10^-5 M (final DMSO < 0.5%) for use.

2.8. Cell culture

Primary ESCs were isolated from eutopic endometrial tissues of AM patients as previously described in detail.¹¹ ESCs were cultured in dulbecco's modified eagle medium/nutrient mixture F-12 (1∶1) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), and maintained at 37 ℃ in 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell medium (ScienCell, CA, Carlsbad, USA). To obtain conditioned medium (CM), ESCs were cultured for 48 h, and CM was centrifuged (3000 × g, 10 min) and stored at －80 ℃.

2.9. Cell treatment and drug intervention

To verify the regulatory effects of quercetin and kaempferol on the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway, a hypoxia-mimicking model was established at the cellular level. AM lesions are typically exposed to a chronic hypoxic microenvironment, in which hypoxia stabilizes HIF-1α and promotes VEGF expression, thereby inducing angiogenesis and cellular invasion while providing a supportive milieu for lesion growth. Chemical hypoxia induction using CoCl₂ is one of the classical in vitro methods. Its mechanism involves Co²⁺-mediated inhibition of prolyl hydroxylase activity, which prevents the degradation of HIF-1α protein under normoxic conditions, leading to HIF-1α stabilization and activation of downstream signaling pathways.^12,13 According to multiple studies,^14-16 treatment with 200 μM CoCl₂ for 24 h effectively induces activation of the HIF-1α/VEGF pathway without compromising cell viability. Therefore, this condition was employed in the present study to establish a stable and reliable hypoxia-mimicking model for subsequent intervention experiments involving monomeric compounds.

Subsequently, two representative flavonoid constituents of XSD-quercetin (Sigma-Aldrich, St Louis, MO, USA; Q4951) and kaempferol (Sigma-Aldrich, St Louis, MO, USA; 60010)-were selected for intervention. According to the literature,¹⁷ both compounds can modulate multiple cellular signaling pathways at concentrations of 5-20 μM without causing notable cytotoxicity. In this study, concentrations of 5, 10, and 20 μM were applied for 24 h following CoCl₂ induction. The control group received an equal volume of DMSO (final concentration < 0.1%). All experiments were performed in triplicate as independent biological replicates.

2.10. Cell viability assay [cell counting kit-8 (CCK-8) method]

ESC viability was assessed using CCK-8 (Dojindo, Tokyo, Japan; CK04-500T). Cells were seeded in 96-well plates, treated for 24 h, then 10 μL CCK-8 solution was added and incubated for 2 h at 37 ℃. OD was measured at 450 nm. Cell viability (%) = (OD of the treatment group-OD of the blank group) / (OD of the control group-OD of the blank group) × 100%. Each group had three technical replicates and three independent repeats.

2.11. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis

After treatment, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; 15596026). The purity was assessed by measuring the A260/A280 ratio, which was maintained between 1.8 and 2.0. Using 1 μg of RNA as the template, complementary DNA (cDNA) was synthesized with the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China; RR047A). Quantitative PCR was performed with PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA; A25742). The reaction volume was 20 μL, and cycling conditions were as follows: pre-denaturation at 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 30 s. Amplification specificity was confirmed by melt-curve analysis. Primer sequences are listed in supplementary Table 1.

Relative expression levels were calculated using the 2^-ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase as the internal reference and normalization to the control group (set to 1). Each sample was analyzed in triplicate, and all experiments were independently repeated three times.

2.12. Preparation of the XSD-mediated serum

XSD herbs were obtained from the Jiangsu Province Hospital for the Integration of Chinese and Western Medicine (Nanjing, China). Twenty Sprague-Dawley rats were randomly assigned to XSD (n = 10) or vehicle (n = 10) groups. XSD group received intragastric XSD aqueous extract (7.2 g/kg) twice daily for 5 d; vehicle group received sterilized water. One hour after the last administration, blood was collected from the abdominal aorta under anesthesia. Serum was obtained by centrifugation (3000 × g, 10 min, 4 ℃), filtered (0.22 μm), heat-inactivated (56 ℃, 30 min), and stored at －80 ℃.

2.13. Enzyme-linked immunosorbent assay (ELISA)

The collected culture supernatants were centrifuged and filtered. The resulting supernatants were maintained at －80 ℃. Mouse uterine tissues were homogenized in tissue lysis buffer (Beyotime, Shanghai, China) to obtain protein lysates. Specific ELISA kits (Elabscience, Wuhan, China) were used to measure the secreted VEGF and matrix metalloproteinase-9 (MMP9) levels in culture supernatants and protein lysates.

2.14. Western blot

Phosphatase and protease inhibitor-containing radioimmuno precipitation assay lysis buffer was used to extract total protein, and protein quantification was performed using the pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). After 8%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation, an equal volume of protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). PVDF membranes underwent 1 h of blockage in 5% nonfat milk, and one night of incubation at 4 ℃ using en receptor (GPER) (ABclonal, Wuhan, China; A10217), phosphatidy-linositol 3-kinase (PI3K) (Proteintech, Wuhan, China; 60225-1-Ig), phospho-PI3K (p-PI3K) (bs-3332R, Bioss, China), protein kinase B (AKT) (Proteintech, Wuhan, China; 10176-2-AP), phospho-AKT (p-AKT) (Proteintech, Wuhan, China; 66444-1-Ig), extracellular signal-regulated kinase (ERK) (16443-1-AP, Proteintech), phospho-ERK (p-ERK) (Proteintech, Wuhan, China; 28733-1-AP), HIF-1α (Proteintech, Wuhan, China; 20960-1-AP), estrogen receptor 1 (ESR1) (Proteintech, Wuhan, China; 21244-1-AP). After the phosphate buffered saline with tween-20 wash, membranes were incubated with the secondary antibody for 1 h at room temperature. After 3 additional washes, High-Sig ECL Western Blot Substrate (Tanon, Shanghai, China) was added. Bands were detected using X-ray film cassette (Yuehua, Shantou, China) in a darkroom.

2.15. Tube formation assay

We assessed HUVECs' pro-angiogenic activity using a capillary tube formation assay on a matrigel (Corning, Bedford, MA, USA; 356231)-precoated μ-slide Angiogenesis ibiTreat plate (ibidi, Martinsried, Germany). Briefly, each chamber well was added with 10 μL of Matrigel and incubated for half an hour of incubation at 37 ℃ for gelation. HUVECs were suspended in preconditioned medium (2 × 10⁵ cells/mL), and each well was added with 50 μL of the cell suspension. After 6 h incubation at 37 ℃, Calcein-AM was dissolved in serum-free medium and added for cell staining. After incubation for 30 min, cells were washed 3 times with PBS. We utilized an inverted fluorescence microscope (Olympus IX71, Shanghai, China) to capture the fluorescence images. Vessel area and total vessel length were quantified using AngioTool software (National Cancer Institute, Bethesda, MD, USA).

2.16. Migration and invasion assay

Transwell chambers (24-well, 8.0 μm pore polyester membrane, Corning, Bedford, MA, USA) were used to assess cell migration and invasion according to the manufacturer's protocol. The migration assay was conducted, seeding 5 × 10⁴ cells/well into the upper chamber using 100 μL serum-free medium, while adding 750 μL of complete medium to the lower chamber to serve as a chemoattractant. Cells were incubated for 24 h at 37 ℃, after which cells remaining on the upper surface were removed with cotton swabs. Migrated cells on the lower surface were fixed with 4% paraformaldehyde (PFA), stained with 0.1% crystal violet, and imaged under an inverted fluorescence microscope. For the invasion assay, the upper chamber was pre-coated with 100 μL of Matrigel (1:5 dilution in Dulbecco's modified eagle medium) (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 30 min at 37 ℃ before cell seeding. Cells were incubated for 48 h, and the same staining and imaging procedures were applied as in the migration assay.

2.17. Establishment and treatment of a mouse model of AM

Animal procedures herein obtained the approval of the Institutional Review Board of the Jiangsu Province Academy of Traditional Chinese Medicine. Neonatal Institute of Cancer Research mice (Day 1 after birth) and their mothers came from Shanghai SLAC Laboratory Animal (Shanghai, China). Mice were housed in individual cages under controlled conditions. The AM mouse model was induced by continuous tamoxifen administration, as previously described.¹⁸ Briefly, tamoxifen (MedChemExpress, Shanghai, China; HY-13757A) was suspended in a mixture of peanut oil, lecithin and condensed milk (2:0.2:3, v/v) and administered to female neonatal mice at 2.7 μmol/kg body weight from Days 2-5 post-birth. Control mice (n = 6) received the same vehicle without tamoxifen. At 3 weeks of age, weaned pups were separated. At 3 months post-birth, 10 tamoxifen-treated and 10 control mice were euthanized, followed by hematoxylin and eosin (HE) staining to ascertain the AM incidence. The remaining tamoxifen-treated mice fell into six groups (n = 8): Control: No treatment; Model: 0.2 mL/d saline; Vehicle: 0.2 mL/d distilled water; Positive (Mifepristone): 3.25 mg/kg body weight (bw) mifepristone; XSD-L: 5.2 g/kg bw XSD (low dose); XSD-H: 10.4 g/kg bw XSD (high dose), with all groups except the control group receiving intragastric administration. After 30 d of treatment, all mice were sacrificed. After fixation in 4% PFA and paraffin embedding, uterine tissues were prepared for histopathological analysis.

2.18. HE and immunohistochemistry (IHC) analysis

AM lesions in the AM mouse model were rinsed with PBS and then underwent 24 h of fixation in 4% PFA at 4 ℃, dehydration, and paraffin-embedding. 3 μm-thick slices were subjected to HE staining (Solarbio, Beijing, China). Uterine morphological structures were assessed as described by previous research.¹⁹ Following the previous description,²⁰ pathology scores ranged from 0 (regressed lesion accompanied by stromal fibrosis, hemosiderin deposition, and absence of glandular structures) to 3 (active lesion accompanied by fresh blood, extensive stromal cell infiltration, and fine glandular structures).

IHC staining was conducted using an IHC staining kit (ZCGB-BIO, Beijing, China). Dewaxed and dehydrated sections underwent one night of incubation with primary antibodies targeting: GPER (1∶200; ABclonal, Wuhan, China; A10217), ESR1 (21244-1-AP, 1∶500, Proteintech, Wuhan, China), HIF-1α (20960-1-AP, 1∶200, Proteintech, Wuhan, China), and CD31 (1∶100; Bioss, Beijing, China; bsm-10825M). After 30 min of incubation with biotinylated secondary antibodies at room temperature, sections were stained with DAB (ZCGB-BIO, Beijing, China). Sections were counterstained with hematoxylin (3 min) and eosin (20 s). IHC staining intensity was analyzed semi-quantitatively using the H-score as previously described by our group.²¹ Digital images were captured using a scanning biomicroscope (Olympus SLIDEVIEW VS200, Tokyo, Japan).

2.19. Statistical analysis

All experiments were independently repeated at least three times to ensure the reliability of the statistical analysis. The sample size for the animal experiments was n = 8 per group. The intergroup effect sizes (Cohen's d) for key indicators — including ESR1, GPER, HIF-1α, and CD31 — ranged from 3.88 to 12.02, with corresponding statistical power values approaching or equal to 1.000 (supplementary Table 2), indicating strong statistical robustness of the study. Data analysis relied on SPSS 23.0 (IBM Corp., Armonk, NY, USA) together with GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). The presentation of measurement data followed the format of mean ± standard deviation. An independent Student's t-test was used to compare two groups, and a one-way analysis of variance was used to compare multiple groups. P < 0.05 denoted statistical significance.

3. RESULTS

3.1. Bioinformatic analysis for AM-related gene targets retrieved from the database

To explore signaling pathways involved in abnormal angiogenesis in AM, bioinformatics analysis was performed using public databases. A total of 90 common AM related gene targets were retrieved from GeneCards and NCBI. A PPI network was constructed with 90 nodes and 406 edges (supplementary Figure 1A). Degree centrality ranking identified the top ten hub genes (supplementary Table 3). GO enrichment analysis revealed significant enrichment in vasculature development, angiogenesis, and response to hypoxia, indicating that hypoxia driven angiogenesis critically affects AM progression (supplementary Figure 1B). KEGG analysis showed remarkable enrichment of the PI3K/AKT and HIF 1α signaling pathways (supplementary Figure 1C). A Sankey diagram was then constructed to illustrate the relationships among the three enriched GO terms and their associated gene targets, suggesting a collaborative regulatory mechanism (supplementary Figure 1D). HIF 1α and vascular endothelial growth factor A (VEGFA) were found to be significantly enriched in angiogenesis, vasculature development, and hypoxia response. Finally, KEGG enrichment analysis further confirmed that angiogenesis related genes, particularly HIF 1α and VEGF, are primarily modulated by the PI3K/AKT and HIF 1α signaling pathways (supplementary Figure 1E). Collectively, hypoxia and angiogenesis, especially the HIF 1α pathway, play pivotal roles in AM pathogenesis and progression.

3.2. Network pharmacology analysis for the theoretical active ingredients of XSD against AM

Given the significant enrichment of hypoxia- and angiogenesis-related pathways in AM, we further employed a network pharmacology approach to predict the potential active components and intervention targets of XSD. We retrieved 116 chemical components of XSD from the TCMSP database (supplementary Table 4). Using the Swiss Target Prediction platform, we identified 941 drug targets and obtained 355 disease-related targets from the GeneCards and NCBI databases. A Venn diagram identified 68 common targets shared between AM and XSD using the Venny 2.1 online analytical tool (supplementary Figure 2A; supplementary Table 5). These 68 common targets were considered potential therapeutic targets for XSD in AM-related angiogenesis. To visualize the interactions between XSD ingredients and AM targets, a XSD-ingredients-targets-AM network diagram was generated using Cytoscape, comprising 192 nodes and 909 edges (supplementary Figure 2B). Network analysis suggested that XSD exerted its pharmacological effects on AM through a multi-component, multi-target mechanism.

To further explore XSD's potential mechanisms of treating AM, a PPI network of 68 common targets was built (supplementary Figure 2C), encompassing 68 nodes and 1,086 unique edges. The top 10 hub genes by connectivity degrees within the network were identified as the core targets, namely, protein kinase B alpha (AKT1), ESR1, VEGFA, signal transducer and activator of transcription 3, interleukin-1 beta, Cyclin D1, Epidermal Growth Factor Receptor, prostaglandin-endoperoxide synthase 2, hypoxia inducible factor 1 subunit alpha and MMP9 (supplementary Table 6), suggesting their roles as key targets in XSD-mediated AM treatment. Functional analysis using the Enrich platform revealed significant GO term enrichment in steroid hormone response, hypoxia response, and angiogenesis regulation (supplementary Figure 2D). According to KEGG enrichment analysis, XSD-targeted genes showed a significant relevance to the PI3K/AKT, HIF-1α, and Estrogen signaling pathways (supplementary Figure 2E). To enhance the visual association between key components and functional pathways, we constructed a compound-target-pathway (CTP) network based on 116 components, 68 shared targets, and the enriched pathway results (supplementary Figure 2F). As shown, several representative components exhibited high connectivity with critical targets such as ESR1, G-protein coupled estrogen receptor 1, phosphate-dylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, AKT1, and hypoxia inducible factor 1 alpha which were primarily enriched in the PI3K-Akt, HIF-1α, Estrogen, and VEGF signaling modules. These findings suggest that XSD exerts its regulatory effects through a coordinated "multi-component-multi-target-multi-pathway" mechanism, closely aligning with key signaling axes implicated in aberrant angiogenesis in AM.

3.3. Chemical constituent identification in XSD by UHPLC-MS/MS

UHPLC-MS/MS analysis of XSD aqueous extract provided total ion chromatograms in positive and negative modes (Figure 1A). Table 1 lists the 20 identified compounds (10 in positive, 10 in negative mode). Kaempferol, coumaric acid, and sclareol were the three most abundant ingredients. Metabolite class composition showed flavonoids (30%), pheny-lpropanoids (20%), and alkaloids (20%) (Figure 1B). A Sankey diagram indicated that flavonoids are the primary components in Danshen (Radix Salviae Miltiorrhizae), Yanhusuo (Rhizoma Corydalis Yanhusuo), Danggui (Radix Angelicae Sinensis), Wuyao (Radix Linderae Aggregatae), Yujin (Radix Curcumae Wenyujin), and Guijianyu (Ramulus Euonymi) (Figure 1C).

Figure 1. Identification of chemical constituents in XSD by UHPLC-MS/MS.

A: A1: XSD in ESI positive mode; A2: XSD in ESI negative mode. The red numbers in the mass spectrum represent 20 identified compounds. 1: kaempferol; 2: coumaric acid; 3: sclareol; 4: nicotinic acid; 5: palmatine; 6: allocryptopine; 7: acetophenone; 8: boldine; 9: camphor; 10: linderane; 11: apigenin; 12: kaempferol; 13: azelaic acid; 14: naringenin; 15: isoimperatorin; 16: coumaric acid; 17: caffeic acid; 18: cirsimaritin; 19: vanillic acid; 20: quercetin; B: metabolite class composition ring diagram; C: sankey diagram illustrating relationships between the ingredients and source herbs in XSD. XSD: Xuandan Sanjie decoction; UHPLC-MS/MS: ultra-high performance liquid chromatography coupled with tandem mass spectrometry; ESI: electrospray ionization.

Table 1.

Characterization of compounds of XSD by UPLC-MS/MS

No.	Compound	Formula	mzmed	ms2Adduct	Relative content	Class
1	Kaempferol	C15H10O6	287.055	[M+H]+	3860556.051	Flavonoids
2	Coumaric acid	C9H8O3	147.043	[M+H-H2O]+	42418783.33	Phenylpropanoids
3	sclareol	C20H36O2	326.305	[M+NH4]+	31763629.87	Terpenoids
4	Nicotinic acid	C6H5NO2	124.039	[M+H]	87096072.94	Alkaloid
5	Palmatine	C21H22NO4	352.153	[M]+	1414855.134	Alkaloid
6	Allocryptopine	C21H23NO5	370.165	[M+H]	20554792.16	Alkaloid
7	Acetophenone	C8H8O	121.064	[M+H]	4027023.846	Phenols
8	Boldine	C19H21NO4	328.153	[M+H]+	4970057868	Alkaloid
9	Camphor	C10H16O	153.127	[M+H]	1193613.001	Terpenoids
10	Linderane	C15H16O4	261.111	[M+H]	103593915.9	Terpenoids
11	Apigenin	C15H10O5	269.045	[M-H]-	2469278.777	Flavonoids
12	Kaempferol	C15H10O6	285.042	[M-H]-	3370501.518	Flavonoids
13	Azelaic acid	C9H16O4	187.097	[M-H]-	6112865.346	Aliphatic acyl
14	Naringenin	C15H12O5	271.060	[M-H]	7626711.301	Flavonoids
15	isoimperatorin	C16H14O4	269.082	[M-H]-	11272505.7	Phenylpropanoids
16	Coumaric acid	C9H8O3	163.039	[M-H]-	12214940.45	Phenylpropanoids
17	Caffeic acid	C9H8O4	179.034	[M-H]-	42289183.19	Phenylpropanoids
18	cirsimaritin	C17H14O6	313.071	[M-H]-	55540661.17	Flavonoids
19	Vanillic acid	C8H8O4	167.034	[M-H]-	67417507.56	Cinnamic acid derivatives
20	Quercetin	C15H10O7	301.037	[M-H]-	3860556.051	Flavonoids

Notes: XSD: Xuandan Sanjie decoction; UHPLC-MS/MS: ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

3.4. XSD inhibits angiogenesis by modulating the secretion of VEGF and MMP9 in vitro

Hypoxia promotes blood vessel growth via HIF-1α activation leading to VEGF and MMP9 production. To evaluate the effects of XSD on estrogen-mediated pro-angiogenic activity in ESCs under hypoxia, we measured VEGF and MMP9 secretion in ESC supernatants by ELISA. ESCs were treated with control, E2 (10 nM), Saline-mediated serum (vehicle), various concentrations of XSD-mediated serum, or mifepristone. Results showed that estrogen significantly upregulated VEGF and MMP9 secretion in ESCs under hypoxia, while 10% XSD-mediated serum markedly reduced these effects (P < 0.001) (Figures 2A, 2B). A Matrigel-based tube formation assay showed that E2 increased HUVEC tube formation, which was significantly suppressed by 10% XSD serum (Figure 2C, supplementary Figures 3A, 3B). Migration and invasion assays showed that E2-induced enhancement was also inhibited by 10% XSD serum (Figures 2D, 2E, supplementary Figures 3C, 3D). These results suggest that XSD effectively attenuates strogen-mediated pro-angiogenic activity in hypoxic ESCs, prompting investigation of the ESR1/GPER-HIF-1α axis.

Figure 2. XSD inhibits angiogenesis by modulating VEGF and MMP9 secretion in vitro.

A: VEGF levels in ESC supernatants after 24 h of treatment; B: MMP9 levels in ESC supernatants after 24 h of treatment; C: C1-C7: tube formation assays of HUVECs treated with conditioned media from each group (× 40); C8-C14: representative images of endothelial tube formation analyzed by AngioTool; D: migration assays of HUVECs treated with conditioned media (× 100); E: invasion assays of HUVECs treated with conditioned media (× 100). C1, C8, D1, E1: Control group; C2, C9, D2, E2: E2 group; C3, C10, D3, E3: Vehicle group; C4, C11, D4, E4: Low dose XSD group; C5, C12, D5, E5: Middle dose XSD group; C6, C13, D6, E6: High dose XSD group; C7, C14, D7, E7: Mifepristone group. Control: ESCs treated with 200 μM CoCl₂. E2: ESCs treated with 10 nM E2. Vehicle: ESCs treated with blank. XSD-L: ESCs treated with 2.5% (v/v) XSD-containing serum. XSD-M: ESCs treated with 5% (v/v) XSD-containing serum. XSD-H: ESCs treated with 10% (v/v) XSD-containing serum. Mifepristone: ESCs treated with 10 μM mifepristone. VEGF: vascular endothelial growth factor; MMP9: matrix metalloproteinase-9; ESCs: endometrial stromal cells; E2: Estrogen; XSD: Xuandan Sanjie decoction; ANOVA: analysis of variance. ANOVA tests were used among these groups. Data were presented as mean ± standard deviation (n = 3). Compared with the Control group, ^aP < 0.05; compared with the Vehicle group, ^bP > 0.05, ^cP < 0.05, ^dP < 0.01.

3.5. XSD regulates VEGF and MMP9 expression through the modulation of ESR1/GPER-HIF-1α pathways

Increasing evidence indicates that hypoxia activates HIF-1α and its downstream targets, promoting angiogenesis, aggressiveness, and therapy resistance. Additionally, estrogen exerts its effects via ESR1 and the non-canonical receptor GPER.²² To dissect the molecular mechanism of XSD against HIF-1α under estrogen signaling and hypoxia, we examined ESR1 and GPER expression in E2-stimulated ESCs following XSD-mediated serum treatment. E2 significantly upregulated HIF-1α expression, which was reversed by the ESR1 antagonist Giredestrant, the GPER-specific inhibitor G15, or XSD. qRT-PCR showed that E2 elevated HIF-1α and VEGF mRNA levels, and this was reduced by Giredestrant and XSD (Figures 3A, 3B). Western blot confirmed ESR1 regulation of HIF-1α protein (Figure 3C). Similarly, XSD or G15 reduced E2-induced HIF-1α and VEGF mRNA (Figures 3E, 3F). The PI3K/AKT/ERK pathway is a rapid early response cascade.²³ Since our own data confirmed that its activation reaches a steady state by 1 h post-stimulation, this time point was selected for validation. Western blot showed that E2-induced hypoxia increased GPER, phosphorylated PI3K/AKT/ERK, and HIF-1α protein levels, and these effects were significantly suppressed by XSD or G15 (P < 0.01) (Figure 3G, supplementary Figure 4). ELISA confirmed that XSD, especially combined with Giredestrant or G15, inhibited E2-induced VEGF secretion under hypoxia (P < 0.05) (Figures 3D, 3H). Thus, XSD functions as a dual estrogen receptor modulator, inhibiting both ESR1 and GPER signaling.

Figure 3. XSD regulates VEGF and MMP9 expression via ESR1/GPER-HIF-1α pathways.

A: qPCR analysis of HIF-1α mRNA in ESCs after 24 h; B: qPCR analysis of VEGF mRNA after 24 h; C: Western blot of ESR1 and HIF-1α at 24 h; D: VEGF levels in ESC supernatants (24 h); E: early (1 h) expression of HIF-1α mRNA; F: early (1 h) expression of VEGF mRNA; G: Western blot of GPER and HIF-1α at 1 h post-stimulation; H: VEGF secretion after 1 h assessed by ELISA. Group definitions: Control: 200 μM CoCl₂; E2: 10 nM E2 under hypoxia; Vehicle: blank serum under hypoxia + E2; XSD: 10% (v/v) XSD serum under hypoxia + E2; Giredestrant: 10 nM Giredestrant under hypoxia + E2; G15: 100 nM G15 under hypoxia + E2. qPCR: quantitative reverse transcription polymerase chain reaction; ESCs: endometrial stromal cells; VEGF: vascular endothelial growth factor; MMP9: matrix metalloproteinase-9; CoCl₂: cobalt (II) chloride; ESR1: estrogen receptor 1; HIF-1α: hypoxia inducible factor-1α; ELISA: enzyme-linked immunosorbent assay; GPER: G protein-coupled estrogen receptor; PI3K: phosphatidylinositol 3-kinase; p-PI3K: phospho-PI3K; AKT: protein kinase B; p-AKT: phospho-AKT; ERK: extracellular signal-regulated kinase; p-ERK: phospho-ERK; XSD: Xuandan Sanjie decoction; ANOVA: analysis of variance. ANOVA tests were used among these groups. Data were presented as mean ± standard deviation (n = 3). Compared with the Control group, ^aP < 0.05; compared with the Vehicle group, ^bP < 0.05; compared with the XSD group, ^cP > 0.05.

3.6. XSD suppresses angiogenesis in AM by modulating ESR1/GPER-HIF-1α pathways in vivo

Having confirmed in vitro that XSD modulates the ESR1/GPER-HIF-1α pathway, we further verified this mechanism in vitro using a tamoxifen-induced AM mouse model. ELISA analysis of ectopic lesions revealed that the model and vehicle groups showed significantly higher VEGF and MMP9 levels than the control group. However, these levels exhibited an obvious reduction in the positive and XSD-H groups (P < 0.05) (Figures 4A, 4B). Increased uterine index and AM nodules were observed in the model and vehicle group, but these effects diminished following positive and XSD-H treatment versus the control group (P < 0.05) (Figures 4C, 4D). Pathological scoring confirmed significant lesion regression in model mice treated with XSD-H, alongside the restoration of endometrial stromal architecture (Figures 4E, 4F). Furthermore, IHC staining revealed that ectopic lesions from the model and vehicle groups had considerably elevated ESR1, GPER, and HIF-1α expression, while these expressions showed an obvious reduction in the positive and XSD-H groups (P < 0.05) (Figures 4G-4J). CD31 staining showed that ectopic tissues in AM model mice exhibited increased vascularization compared with the control group, whereas positive and XSD-H significantly decreased vascular density (Figure 4G, supplementary Figure 5). These results confirm that XSD inhibits lesion-associated angiogenesis and AM progression via the ESR1/GPER-HIF-1α axis.

Figure 4. XSD inhibits angiogenesis in vivo in AM through ESR1/GPER-HIF-1α signaling.

A: VEGF levels in ectopic lesions from all groups (ELISA); B: MMP9 levels in ectopic lesions from all groups (ELISA); C: uterus index comparison; D: number of AM nodules; E: HE staining of uterine tissues (× 10); F: pathology scoring; G: immunohistochemistry staining of GPER, ESR1, HIF-1α, and CD31 (× 200); E1, G1, G7, G13, G19: Control group; E2, G2, G8, G14, G20: Model group; E3, G3, G9, G15, G21: Vehicle group; E4, G4, G10, G16, G22: Positive group; E5, G5, G11, G17, G23: XSD-L group; E6, G6, G12, G18, G24: XSD-H group. H: H-score quantification of GPER. I: H-score quantification of ESR1. J: H-score quantification of HIF-1α. Group definitions: Control: untreated mice; Model: saline (0.2 mL/d); Vehicle: distilled water (0.2 mL/d); Positive control (Mifepristone): 3.25 mg/kg bw; XSD-L: low dose (5.2 g/kg bw) XSD; XSD-H: high dose (10.4 g/kg bw) XSD. ESR1: estrogen receptor 1; GPER: G protein-coupled estrogen receptor; HIF-1α: hypoxia inducible factor-1α; VEGF: vascular endothelial growth factor; MMP9: matrix metalloproteinase-9; HE: hematoxylin and eosin; ELISA: enzyme-linked immunosorbent assay; AM: adenomyosis; XSD: Xuandan Sanjie decoction; ANOVA: analysis of variance. Statistical analysis was carried out using ANOVA tests among these groups. Data were presented as mean ± standard deviation (n = 8). Compared with the Control group, ^aP < 0.05; compared with the Model group, ^bP > 0.05, ^cP < 0.01, ^dP < 0.05.

3.7. Quercetin and kaempferol reverse hypoxia-mimicking activation of the HIF-1α/VEGF signaling pathway

To validate the specific roles of quercetin and kaempferol in the HIF-1α/VEGF axis, we established a chemical hypoxia model using 200 μM CoCl₂ for 24 h. Primary ESCs were treated with quercetin or kaempferol at 5, 10, and 20 μM. CCK-8 assays showed that after 24 h, ESC morphology remained intact, with no evident apoptosis or detachment. Relative cell viability in all concentration groups was ≥ 85%, with no significant difference from the control group (P > 0.05) (supplementary Figure 6A), indicating no cytotoxicity within this concentration range.

CoCl₂ induction markedly upregulated HIF-1α and VEGF mRNA levels (P < 0.001), confirming the hypoxia model. Both quercetin and kaempferol dose-dependently downregulated the transcriptional levels of HIF-1α and VEGF (supplementary Figures 6B-6E). Specifically, at 20 μM, quercetin reduced HIF-1α expression by ~65% and VEGF by ~55%; kaempferol reduced them by ~60% and ~50%, respectively. These results indicate that quercetin and kaempferol suppress CoCl₂-induced activation of the HIF-1α/VEGF pathway at the transcriptional level (supplementary Figure 6F), thereby reversing hypoxia-mediated signaling abnormalities and validating the "compound-target-effect" chain predicted by network pharmacology.

4. DISCUSSION

This study is the first to elucidate that XSD suppresses aberrant angiogenesis in AM by targeting the ESR1/GPER-HIF-1α axis. XSD inhibited estrogen- and hypoxia-driven angiogenesis in vivo and in vitro, downregulated VEGF/MMP9, and blocked PI3K/AKT/ ERK signaling. These findings reveal a multi-layer regulatory mechanism and support XSD as a multi-target, non-hormonal strategy for AM.

AM is an estrogen-dependent disorder where dysregulated estrogen signaling drives angiogenesis and AUB-A.¹¹ Hypoxic stabilization of HIF-1α and VEGF activation are central to vascular abnormalities in AM.^24-26 This study further confirms that estrogen concurrently activates ESR1 and GPER, leading to rapid upregulation of HIF-1α and downstream signaling pathways such as PI3K/AKT and ERK, ultimately promoting the expression of VEGF and MMP9. Notably, XSD significantly inhibits estrogen-induced ESR1/GPER activation and attenuates hypoxia-related signaling, offering a new perspective on the increased vascular fragility observed in AM. Collectively, these results identify the ESR1/GPER-HIF-1α axis as a pivotal regulator of angiogenesis in AM and a critical therapeutic target of XSD.

As a TCM compound, XSD exhibits prominent multi-target and multi-pathway synergistic regulatory properties, aligning closely with the complex pathophysiology of AM. In this study, network pharmacology analysis identified 68 potential targets, among which ESR1, EGFR, AKT1, and HIF-1α emerged as critical hub nodes implicated in multiple pathological processes. Combined with UHPLC-MS/MS component profiling, we found that the representative constituents of XSD predominantly belong to the flavonoid class — a group of compounds widely reported to possess anti-angiogenic and anti-inflammatory properties.^27,28 Furthermore, monomeric compound experiments confirmed that both quercetin and kaempferol suppress CoCl₂-induced expression of HIF-1α and VEGF in a dose-dependent manner, providing direct phytochemical evidence supporting the synergistic mechanism of XSD. Notably, UHPLC-MS/MS also identified other abundant constituents such as p-coumaric acid and sclareol. In vitro studies have demonstrated that p-coumaric acid significantly inhibits the migration and tube formation of human endothelial cells and suppresses endothelial sprouting in rat aortic ring assays, indicating clear anti-angiogenic activity.²⁹ In addition, a recent review summarized the pharmacological effects of sclareol across various disease models, including anti-tumor, anti-inflammatory, antidiabetic, and antihypertensive activities, along with involvement in several key signaling pathways.³⁰ These findings suggest that both p-coumaric acid and sclareol may contribute to the multi-target pharmacological actions of XSD, particularly in modulating oxidative stress, inflammation, and angiogenesis. Although their specific functions were not directly validated in this study, their documented bioactivities make them compelling candidates for further mechanistic exploration. Importantly, the inhibition of ESR1 and GPER by XSD was not transient but was accompanied by a rapid downregulation of non-genomic pathways such as PI3K/AKT and ERK. This observation suggests that XSD may exert coordinated regulation at multiple levels, from receptor engagement to intracellular signaling cascades and transcriptional control. This multi-node, multi-layered mode of intervention distinguishes XSD from single-pathway inhibitors and reflects the integrative regulatory landscape described by network pharmacology. Owing to this coordinated multi-pathway mechanism, our study observed a clear dose-dependent inhibitory effect of XSD on VEGF and MMP9 expression, with the most pronounced effect observed in the 10% drug-containing serum group. In summary, XSD functions as a biologically potent multi-target modulator, whose inhibition of the ESR1/GPER-HIF-1α axis is not solely attributable to individual active compounds, but rather to the synergistic interplay of its composite ingredients.

From a clinical perspective, although progestins remain the primary treatment for AM, they are associated with significant side effects and high relapse rates after discontinuation.³¹ Thus, safer and more sustained therapies are needed. XSD’s modulation of the ESR1/GPER-HIF-1α axis provides mechanistic support for alleviating menorrhagia and dysmenorrhea in AM patients. Compared to conventional anti-VEGF/VEGFR agents, XSD’s multi-target profile may avoid safety issues such as hypertension and proteinuria.³² Moreover, because anti-VEGF therapies often encounter resistance via compensatory pathway activation,³³ XSD’s simultaneous inhibition of ESR1, GPER, and downstream effectors (PI3K/AKT/ERK) may more effectively block such “escape routes”.³⁴ Based on the findings of this study, XSD holds promise as an adjunct or alternative therapy for mild to moderate AM and may also serve as a complementary treatment to hormonal therapy, potentially reducing dose-dependent adverse effects. In the future, the use of molecular markers such as HIF-1α and VEGF for efficacy prediction or patient stratification could further enhance XSD's clinical value. This research not only establishes a clear mechanistic foundation for the rational clinical use of XSD but also offers a novel framework for multi-target precision therapy in AM.

Despite a series of mechanistic findings, this study has several limitations. First, the animal sample size (n = 8 per group), though based on effect size and power analysis, remains modest; larger cohorts are needed. Second, although UHPLC-MS/MS and network pharmacology identified various flavonoids and phenylpropanoids as potential active compounds, their anti-angiogenic effects have not been independently validated. Causal relationships for the ESR1/GPER-HIF-1α pathway require genetic approaches (e.g., siRNA). Third, the current endpoint-only design lacks dynamic observations across multiple time points, limiting understanding of molecular progression. Fourth, species differences between human ESCs and the murine uterine microenvironment, the exclusive use of female mice without sex-based pharmacokinetic analysis, and the absence of clinical sample validation constrain translational value. Finally, key monomers such as quercetin and kaempferol need individual validation. Future studies will address these limitations by expanding sample size, incorporating temporal analyses, and integrating clinical specimens.

In conclusion, XSD exerts significant anti-angiogenic activity in AM by modulating the ESR1/GPER-mediated HIF-1α signaling pathway. This study not only elucidates the molecular mechanism of XSD but also offers a novel multi-target intervention strategy for chronic gynecological conditions such as AUB-A, bridging traditional medicine with modern molecular targeting.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://www.journaltcm.com.