Network pharmacology approach and experimental verification of Danzhi Xiaoyao Powder for breast cancer treatment based on UHPLC-Q-Orbitrap HRMS

Introduction

Breast cancer (BC) is a common malignancy worldwide, accounting for approximately 25% of all cancers diagnosed in women, and approximately 12% of all cancers overall (1-3). BC comprises multiple subtypes, of which hormone receptor-positive tumors predominate. These are characterized by high estrogen receptor (ER) and progesterone receptor expression, and represent approximately 80% of all BC cases (4).

Endocrine therapy (ET), aimed at inhibiting ER signaling, remains the primary treatment strategy for ER-positive patients without human epidermal growth factor receptor 2 overexpression (5,6). For postmenopausal women diagnosed with ER-positive BC, aromatase inhibitors, which block androgen-to-estrogen conversion, and selective ER modulators, which antagonize ER signaling by disrupting coactivator binding, are standard therapeutic choices. These drugs are commonly used as adjuvant therapies for durations of up to 10 years (7,8). Although ET can reduce mortality in primary BC by up to 40%, resistance often occurs, leading to recurrence and metastasis (9-11). In postmenopausal patients with ER-positive metastatic BC, ET combined with targeted inhibitors is the standard treatment. Despite various therapeutic combinations, resistance remains a significant issue and eventually leads to death. The PI3K/AKT/mTOR signaling axis is one of the most frequently activated pathways in BC, contributing to cell proliferation, survival, and metastasis. Aberrant activation of this pathway has been linked to endocrine therapy resistance and poor prognosis in BC patients. Growing evidence indicates that natural products derived from traditional Chinese medicine (TCM) can exert anti-cancer effects by modulating this pathway, making it a promising therapeutic target for BC intervention. Therefore, understanding the mechanisms underlying therapeutic resistance in metastatic BC progression is essential (12,13).

Following advances in cellular biotechnology, Chinese herbal medicine has made significant breakthroughs in anti-cancer research. In Asia, TCM is widely recognized as a complementary and alternative cancer therapy (14-17). Danzhi Xiaoyao Powder (DZXYP), also known as Jiawei Xiaoyao Powder, originated from the Abstract of Internal Medicine, and is derived from Xiaoyao Powder (recorded in The Taiping Huimin Heji Jufang), supplemented with moutan peel and Gardenia jasminoides. Numerous clinical studies have demonstrated the widespread use of this formulation in the treatment of psychiatric disorders (18,19).

Apart from anti-anxiety and antidepressant effects, DZXYP exhibits multiple benefits, including liver protection, sedation, and hypnosis. DZXYP notably soothes the liver, strengthens the spleen, alleviates depression, nourishes Yin, and clears heat, making it particularly important in the treatment of depression (19-21). Clinical research has also demonstrated that DZXYP effectively treats depression while mitigating liver injury (22). Studies indicate DZXYP exhibits therapeutic effects on BC and precancerous lesions, particularly in ER-positive BC (22,23). Thus, DZXYP possesses potential as a therapeutic agent for BC. However, its active constituents, specific targets, and molecular mechanisms remain unclear.

The therapeutic effects of TCM are achieved through a multi-targeted approach aimed at restoring homeostasis in physiological systems (24-26). Network pharmacology, a novel methodology grounded in biological network analyses, is increasingly being employed to explore complex interactions between drugs, active constituents, biological targets, and diseases (27). Integrating TCM with network pharmacology thus provides an innovative means to identify disease-associated genes, clarify drug-disease interactions, and further determine the regulatory mechanisms underlying TCM efficacy. In this study, network pharmacology, metabolomics, and cellular experiments were used in combination to identify key bioactive compounds, relevant molecular targets, and the potential mechanisms by which DZXYP exerts anti-BC activities. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0431/rc).

Methods

Identification of BC-related targets

The Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and DrugBank databases were searched using the keyword “breast cancer”. Overlapping targets from these databases were then selected.

Analysis of active ingredients and targets of DZXYP

Oral bioavailability (OB) and drug likeness (DL) play essential roles in determining a drug’s absorption capacity across the gastrointestinal epithelium. Thus, to screen active constituents from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), the herbs “Danpi”, “Zhizi”, “Pachia”, “Atractylodes”, “Dangguo”, “Bupleuri”, “Peony”, and “Licorice” were employed as search terms, selecting only components with an OB ≥30% and a DL ≥18%. Subsequently, candidate molecular targets of these selected active components were predicted using databases such as Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM), Search Tool for Interactions of Chemicals (STITCH), and Swiss Target Prediction.

Construction of the protein-protein interaction (PPI) network

Overlapping predicted target genes were subjected to PPI analysis using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Genes exhibiting interaction scores <0.7, indicative of weak or absent interactions, were excluded. Consequently, a refined PPI network of closely interacting genes was established.

Enrichment analysis of active ingredients and targets

To identify the genes that were correlated with active substances, functional annotation of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using the Metascape platform. The GO terms were categorized into three distinct groups: cellular components, molecular functions, and biological processes.

Cell culture

Chinese Academy of Sciences’ Shanghai Institute for Biological Sciences provided the MCF-7 and MDA-MB-231 BC cell lines. Dulbecco’s Modified Eagle Medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) was used to culture the MCF-7 cells, while Roswell Park Memorial Institute 1640 medium (Gibco, CA, USA) was used to culture the MDA-MB-231 cells, which were likewise enhanced with 10% FBS. The two cell cultures were maintained at 37 ℃ in 5% carbon dioxide.

Cell viability assays

The MCF-7 cells were seeded into 96-well plates. A range of DZXYP concentrations (10%, 15%, and 20%) was subsequently applied to the cells for 72 hours. After treatment, Cell Counting Kit 8 (CCK-8) reagent (Biosharp, Beijing, China) was added to each well, and the cells were incubated for 2 hours at 37 ℃. Absorbance at 450 nm was then measured using a multifunctional microplate reader (BioTek, Vermont, USA). The selection of 10%, 15%, and 20% DZXYP-containing serum was based on preliminary experiments in which MCF-7 cells were treated with a range of concentrations (5%, 10%, 15%, 20%, and 25%). Concentrations below 10% showed negligible inhibitory effects, while concentrations above 20% reduced cell viability by more than 70% and induced significantly morphological changes. Therefore, 10%, 15%, and 20% were chosen for subsequent experiments.

Cell migration and invasion assays

To examine their migration ability, the cells were mixed with serum-free medium and then seeded into the upper chambers of Transwell devices, while the lower chambers were filled with complete medium. Fixation, staining, and microscopic counting were performed on migrating cells after 48 hours of incubation. To assess invasion, the migration assay was employed using membranes of the upper chamber coated with diluted Matrigel (BD, New Jersey, USA), which was allowed to solidify.

Western blot (WB)

Using RIPA buffer that was treated with PMSF, proteins were extracted from the grown cells. The concentration of the extracted protein was measured using the BCA Protein Assay Kit. Next, proteins were separated by SDS-PAGE and deposited onto polyvinylidene fluoride membranes. The membranes were then blocked in a 5% skim milk solution, after which they were incubated at 4 ℃ overnight with primary antibodies targeting Bcl-2 (1:1,000, Cat#15071, CST), Ki67 (1:2,000, Cat# 9449, CST), MMP2 (1:1,000, Cat# 40994, CST), E-cadherin (1:2,000, Cat# 14472, CST), Vimentin (1:2,000, Cat# 5741, CST), and GAPDH (1:5,000, Cat# 5174, CST). After rinsing, the membranes were treated with secondary antibodies for 2 hours at 37 ℃. An improved chemiluminescence detection kit (Thermo, Massachusetts, USA) was used to visualize the protein bands.

Preparation of serum containing DZXYP

The DZXYP formulation comprised eight medicinal herbs (Danpi, Gardenia, Poria, Atractylodes, Angelica sinensis, Radix Bupleuri, Peony, and Licorice) plus 3 g of mint and 3 g of ginger (Table 1). All the herbs were sourced from the Traditional Chinese Medicine Hospital in Jiangsu Province. First, 126 g of these herbs were soaked in distilled water (10× volume) at room temperature for 1 hour. After the mixture was brought to a boil, it was simmered over low heat for an additional 40 min.

The resulting herbal concoction was administered to the rats orally once daily for seven days. Two hours after the final administration, the rats were anesthetized with 3% pentobarbital, and blood samples were collected from the abdominal aorta. The serum samples were allowed to coagulate at room temperature for 1 hour, and then centrifuged at 3,000 rpm for 15 min at 4 ℃. The serum were subsequently heat-inactivated at 57 ℃ for 30 min, filtered through a 0.22-µm membrane, and stored at −80 ℃ awaiting testing. The total herbal weight of 126 g corresponds to the standard adult daily dose of DZXYP as recorded in Taiping Huimin Heji Jufang. According to body surface area conversion (human‑to‑rat factor of 6.3), this dose is equivalent to approximately 12.6 g/kg per day for rats.

Chromatographic conditions

The chromatographic analysis was performed using a Vanquish Flex UHPLC system equipped with an Acquity UPLC HSS T3 column. The column temperature was maintained at 40 ℃ with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (phase A) and acetonitrile (phase B). The injection volume was 6.0 µL.

Mass spectrometry (MS) conditions

MS was performed using a Q Exactive hybrid quadrupole Orbitrap mass spectrometer equipped with a HESI-II ionization probe. Nitrogen was used as the sheath gas, auxiliary gas, and collision gas at a pressure of 1.5 mTorr. The data were acquired in the data-dependent mass spectrometry squared (dd-MS²) mode. The complete-scan settings included a resolution of 70,000, an automatic gain control (ACG) target of 1×10⁶, and a maximum injection time of 50 ms. The dd-MS² parameters were set as follows: resolution: 17,500, AGC target: 1×10⁵, maximum injection time: 50 ms, top 10 data-dependent precursor selection, isolation width: m/z², stepped collision energies: 10, 30, and 60 eV, and intensity threshold: 1 × 10⁵.

In vivo tumorigenesis assays

The animal study was reviewed and approved by the Ethics review committee of Affiliated Hospital of Nantong University (No. S20220305-066), in compliance with institutional guidelines for the care and use of animals were followed. The mice were purchased from the Animal Center of Nantong University. Twelve male BALB/c nude mice (5–6 weeks old, 19–23 g) were randomly assigned to two groups of six mice per each. The mice were subcutaneously injected with the MCF-7 cells (5×10⁶ cells in 100 µL of serum‑free medium per mouse) that had been pretreated with either saline or serum containing DZXYP. Tumor size was measured every five days, and after 21 days, the rats were euthanized. During the 21 days treatment period, no mortality, significant body weight loss, reduced activity, or abnormal behavior was observed in either group. The excised tumors were weighed, embedded in paraffin, and subjected to histological examination using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC).

Statistical analysis

Data are presented as the mean ± standard deviation. Statistical analyses involving multiple-group comparisons were conducted using one-way analysis of variance followed by the Student’s t-test using GraphPad Prism 7.0 software. Statistical significance was defined as a P value <0.05.

Results

Identification and screening of components in DZXYP

The molecular mechanisms underlying the therapeutic effects of DZXYP against BC were investigated by analyzing the chemical components of the formulation and serum samples from the treated rats. The analysis was performed using high-performance liquid chromatography—quadrupole orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). By comparison with reference and theoretical compound databases, as well as manual verification, 46 compounds were identified (Figure 1 and Table 2).

Figure 1 The total ion chromatograms of DZXYP aqueous extract (A,B), blank serum (C,D), and dosed serum samples (E,F) in positive and negative modes, respectively. DZXYP, Danzhi Xiaoyao Powder.

Prediction of targets and bioinformatics analysis of DZXYP in BC

Initially, the Genecards, OMIM, TTD, and DrugBank databases were searched using the keyword “breast cancer”, and 5,518 potential target genes were identified after merging and removing duplicates. Next, the active components of DZXYP were identified from the TCMSP database using specified screening thresholds (an OB ≥30% and a DL ≥18%), and employing the keywords “Danpi, Gardenia, Poria, Atractylodes, Angelica, Bupleurum, Peony, and Licorice”. The potential molecular targets associated with these identified components were predicted through multiple databases (i.e., BATMAN-TCM, STITCH, and Swiss Target Prediction), resulting in 477 putative targets. Cross-referencing these 477 targets with a set of 5,518 known BC-associated targets revealed 288 overlapping genes (Figure 2A).

Figure 2 Construction of DZXYP component-BC target network. (A) Venn diagram of common targets of DZXYP and BC. (B) Construction of PPI network between components of DZXYP and BC targets. (C,D) Enrichment analysis of GO and KEGG pathways of key target genes. BC, breast cancer; DZXYP, Danzhi Xiaoyao Powder; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

To further explore the mechanisms underlying DZXYP’s anti-BC action, the interactions between the DZXYP components and these common targets were visualized using Cytoscape software (version 3.8.0). PPIs among these overlapping targets were assessed by STRING analysis, discarding those with interaction scores <0.7 to focus on the most significant relationships (Figure 2B).

Further, enrichment analyses of the GO terms and KEGG pathways for these 288 overlapping targets were carried out using RStudio software. The top 10 enriched terms were visualized in graphs, highlighting associations primarily with steroid metabolic processes, nuclear receptor activities, and membrane raft structures (Figure 2C). The KEGG pathway enrichment analysis indicated that DZXYP potentially inhibits BC progression via multiple signaling pathways, including the PI3K-AKT signaling cascade, receptor activation pathways, lipid metabolism and atherosclerosis pathways, and reactive oxygen species (ROS)-related pathways (Figure 2D).

Construction of the DZXYP component-BC target network

In recent years, there has been growing interest in the metabolomic characterization of bioactive constituents derived from TCM. In the present study, decoctions of the eight primary herbal components of DZXYP were administered orally to experimental rats via gastric gavage once daily for one week. As illustrated in Figure 3A, DZXYP decoction was orally administered to rats once daily for seven consecutive days. Two hours after the final administration, blood samples were collected from the abdominal aorta. The serum was separated by centrifugation, heat-inactivated at 57 ℃ for 30 min, and then filtered through a sterile 0.22-µm membrane. These processed serum samples, referred to as DZXYP-containing serum, were subsequently analyzed by UHPLC-Q-Orbitrap HRMS to identify the absorbed bioactive components. The anticipated drug components and their related targets were used to develop a comprehensive drug component-target-pathway-disease network. Nodes in this network represent drug components, target genes, and associated signaling pathways.

Subsequently, Cytoscape’s Network Clustering Algorithm was employed to identify 26 core targets (Figure 3B-3D), for which further GO and KEGG analyses were conducted. The results indicated that the active components of DZXYP were primarily involved in responses to endogenous stimuli and oxygen-containing compounds, as well as the regulation of cell death. These components regulate BC through pathways related to lipid and atherosclerosis, mTOR signaling, and cancer-associated pathways. In total, 46 major active components and 26 key genes were identified in the drug-containing serum. Most of the key genes were involved in the mTOR signaling pathway (Figure 3E,3F).

Figure 3 Identification of the active ingredients involved in DZXYP in the treatment of BC. (A) Schematics of preparation of the DZXYP-containing serum. (B-D) Active ingredient-target-pathway network. (E,F) GO and KEGG analyses of the common genes associated with DZXYP and BC. BC, breast cancer; DZXYP, Danzhi Xiaoyao Powder; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

DZXYP inhibits the malignant progression of BC cells in vitro

To assess the inhibitory effects of the DZXYP serum metabolites on BC cell growth, the MCF-7 and MDA-MB-231 cell lines were treated with serum containing different concentrations of DZXYP (10%, 15%, or 20%) over a period of 72 hours. Cellular viability was measured at 24-hour intervals using the CCK-8 assay. Among the tested concentrations, 20% serum demonstrated the most pronounced suppression of BC cell proliferation (Figure 4A), and was thus used in subsequent assays.

Figure 4 DZXYP inhibits the malignant progression of BC cells in vitro. (A) CCK8 assays were used to evaluate the inhibitory effects of different concentrations of the active ingredients of DZXYP on the growth of BC cells. (B,C) Transwell assays, Magnification: 20×. (D) ROS assays, Magnification: 20×. (E) Scratch-wound assays, Magnification: 20×. (F) Immunofluorescence staining, Magnification: 20×. *, P<0.05; **, P<0.01; ***, P<0.001. BC, breast cancer; CCK8, cell counting kit-8; DZXYP, Danzhi Xiaoyao Powder; ROS, reactive oxygen species.

A series of in vitro experiments was conducted to further elucidate the effects of DZXYP-containing serum on the malignant biological behaviors of BC cells. Transwell assays revealed notable reductions in the migratory and invasive capabilities of the cells after DZXYP treatment (Figure 4B,4C).

The pathway enrichment analysis revealed that ROS, lipid metabolic pathways, and the mTOR signaling cascade were significantly modulated by the DZXYP treatment. Measurements of intracellular ROS levels revealed a significant increase following exposure to DZXYP-containing serum, suggesting that DZXYP constituents may attenuate BC cell proliferation by modulating intracellular ROS through mTOR signaling (Figure 4D).

Additionally, scratch-wound assays provided further evidence of the decreased proliferative potential of the BC cells treated with DZXYP (Figure 4E). Immunofluorescence staining showed reduced expression of the mesenchymal protein marker Vimentin, coupled with elevated expression of the epithelial marker E-cadherin, upon treatment with DZXYP (Figure 4F). Collectively, these findings strongly indicate that the serum metabolites of DZXYP effectively inhibit the malignant phenotypes of BC cells.

DZXYP inhibits the malignant progression of BC cells in vivo

For the in vivo evaluation of DZXYP metabolite efficacy against tumor growth, BC cells previously incubated with DZXYP-containing serum were subcutaneously injected into the nude mice. Tumor size was subsequently measured every five days to construct tumor growth curves. The DZXYP-treated group exhibited significantly slower tumor growth compared to the control group (Figure 5A-5C). After 21 days, tumors in the DZXYP group were smaller and weighed significantly less. Histological (H&E and IHC) staining showed significantly lower percentages of cells expressing Bcl-2, Ki67, MMP2, and Vimentin, and increased E-cadherin expression in the DZXYP group compared with the control group. These findings were supported by the WB results (Figure 5D,5E). Thus, the xenograft experiments showed that DZXYP effectively inhibits the proliferation and metastasis of BC cells in vivo.

Figure 5 DZXYP inhibits the malignant progression of BC cells in vivo. (A) Images of nude mice and transplanted tumors after 21 days of treatment with the control or DZXYP. (B) Tumor size. (C) Tumor weight. (D) Western blotting was used to determine the protein expression of Bcl2, Ki67, MMP2, E-Cad, and Vimentin. (E) Representative H&E staining images and immunohistochemical images of Bcl2, Ki67, MMP2, E-Cad, and Vimentin, Magnification: 10×. **, P<0.01; ****, P<0.0001. BC, breast cancer; DZXYP, Danzhi Xiaoyao Powder; H&E, hematoxylin and eosin.

DZXYP inhibited the PI3K/AKT/mTOR signal pathway

We then investigated the main pathophysiological mechanism by which DZXYP regulates the malignant progression of BC. The maximal clique centrality, maximum neighborhood component, and degree algorithms were used to identify the top 20 candidate hub genes from the 288 interacting genes. The genes associated with the BC pathway were intersected, resulting in the identification of two key hub genes (Figure 6A). Using The Cancer Genome Atlas (TCGA) database, we found that AKT1 was significantly upregulated while PIK3R1 was downregulated in BC (Figure 6B).

Figure 6 DZXYP inhibits the PI3K/AKT/mTOR signaling pathway. (A) Venn diagram of the common targets of key hub genes. (B) AKT1 and PIK3R1 expression in TCGA. (C,D) Molecular docking results of the DZXYP active ingredients and hub targets (AKT1, PIK3R1, and mTOR). (E) Western blotting was used to detect the effect of DZXYP on AKT1, PIK3R1, and mTOR. **, P<0.01; ****, P<0.0001. DZXYP, Danzhi Xiaoyao Powder; MCC, maximal clique centrality; MNC, maximum neighborhood component; TCGA, The Cancer Genome Atlas.

To investigate the affinity of the active components of DZXYP with the key targets of potential pathways, a molecular docking (MD) analysis was performed. The binding positions and interactions between candidate metabolites and PIK3R1, AKT1, and mTOR were obtained using Autodock Vina version 1.2.2 software, and the corresponding binding energies of the interactions were calculated (Figure 6C,6D). The results revealed that the candidate metabolites formed hydrogen bonds and exhibited strong electrostatic interactions with key proteins of the PI3K/AKT/mTOR signal pathway.

Further, the WB analysis showed that treatment with the DZXYP-containing serum decreased the AKT1 and mTOR levels, while increasing the PIK3R1 levels in the BC cells. These results suggest that the DZXYP active components increase the level of PIK3R1, thereby inhibiting the continuous activation of the AKT1/mTOR signaling pathway, leading to the accumulation of ROS in the BC cells (Figure 6E).

Discussion

BC is a common malignancy, accounting for approximately 25% of all cancer cases in women worldwide. TCM has emerged as an influential complementary strategy in cancer therapeutics. Recently, research has focused on identifying and extracting active metabolic components from TCM for potential application in cancer treatments (28,29). It is among the most popular adjuvant therapies after radical cancer resection in East Asia. Due to its unique advantages, TCM has received increasing global attention. In addition to its widely recognized anti-depressant and liver-protective effects, DZXYP has attracted considerable attention for its anti-cancer properties (20,30,31). This study applied network pharmacology and metabolomics methodologies to comprehensively analyze potential molecular mechanisms and identify therapeutic targets associated with DZXYP-derived bioactive compounds in modulating the malignant progression of BC.

In this study, key targets related to DZXYP and BC were identified using public databases. The GO analysis suggested that these targets were primarily involved in steroid and lipid metabolism. The KEGG pathway enrichment analyses suggested that the bioactive components in DZXYP primarily influence BC progression via the modulation of the PI3K/AKT and mTOR pathways. Extensive research indicates that the PI3K/AKT signaling axis significantly contributes to cellular proliferation, survival, and metastatic processes in cancer, and is closely associated with the pathogenesis of BC (32). Moreover, the suppression of the PI3K/AKT signaling axis can significantly enhance therapeutic efficacy in the ET of BC. Further, the overactivation of this pathway contributes to drug resistance, metastasis, and other malignant behaviors in BC (33).

Jiang et al. developed a nanosheet exhibiting oxidase and peroxidase activities that significantly increase tamoxifen sensitivity in BC cells via PI3K-related proteins (34). Chi et al. demonstrated that the increased expression of CapG significantly elevated paclitaxel resistance in BC cells, and this resistance was positively associated with the enhanced activation of the PI3K/AKT network. Further mechanistic exploration revealed that CapG interacts with the transcriptional coactivator p300/CBP, leading to the recruitment of CapG to the PIK3R1 promoter. This event facilitated the transcriptional activation of PIK3R1/P50 by augmenting the histone H3K27 acetylation levels (35). Moreover, Wu et al. demonstrated that SIRT1 reduces doxorubicin-triggered senescence in MCF-7 cells through the negative modulation of the PI3K/AKT/mTOR pathway (36). Notably, MS and bioinformatics analyses in this study revealed that key active components in DZXYP-containing serum are involved in the malignant progression of BC through the modulation of the mTOR pathway.

It is important to note that MCF-7 cells used in this study are ER-positive, and ER signaling plays a dominant role in their growth. Although our network pharmacology and KEGG analyses did not identify ER as a direct core target of DZXYP, and we did not experimentally measure ER expression or activity, the possibility of an indirect interaction between DZXYP and ER signaling cannot be excluded. The PI3K/AKT/mTOR pathway is known to engage in extensive cross‑talk with ER signaling, and inhibition of this pathway can modulate ER transcriptional activity. Therefore, the anti-proliferative effects of DZXYP observed in MCF-7 cells might be mediated, at least in part, through PI3K/AKT/mTOR-dependent modulation of ER function. Alternatively, DZXYP may suppress MCF-7 cell growth through ER-independent mechanisms, such as the elevation of ROS levels and regulation of lipid metabolism, as demonstrated in this study. Future studies are warranted to directly examine whether DZXYP components bind to ER or alter its expression and transcriptional activity.

Conclusions

To investigate the potential mechanisms underlying DZXYP treatment in BC, this study integrated network pharmacology, metabolomics, and both in vitro and in vivo experiments (Figure 7). UHPLC-Q-Orbitrap HRMS analysis identified 46 absorbed bioactive components in DZXYP-containing serum. In vitro experiments demonstrated that DZXYP-containing serum, particularly at the concentration of 20%, significantly inhibited BC cell proliferation, migration, and invasion, elevated ROS levels, and suppressed the PI3K/AKT/mTOR pathway as confirmed by WB. In vivo xenograft assays further confirmed the anti-tumor efficacy of DZXYP. MD revealed strong binding affinities between DZXYP metabolites and key proteins (PIK3R1, AKT1, mTOR). Collectively, these findings support the conclusion that DZXYP exerts anti-BC effects through multi-component regulation of the PI3K/AKT/mTOR signaling pathway.

Figure 7 Schematic diagram of the mechanism by which DZXYP regulates the PI3K/AKT/mTOR signaling pathway, leading to the accumulation of ROS in BC, thereby inhibiting the malignant progression of BC. (The figure is created via Adobe Illustrator). BC, breast cancer; DZXYP, Danzhi Xiaoyao Powder; ROS, reactive oxygen species.

Acknowledgments

We thank all those who participated in this study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0431/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0431/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0431/prf

Funding: This work was financially supported by the Nantong Social Livelihood Science and Technology Project (No. MS2023037), the Natural Science Foundation of Jiangsu Province (No. BK20211018), the Nanjing Medical Science and Technique Development Foundation (No. YKK21163) and the Nantong City Natural Science Foundation (No. JC2024013).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0431/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The animal study was reviewed and approved by the Ethics review committee of Affiliated Hospital of Nantong University (No. S20220305-066), in compliance with institutional guidelines for the care and use of animals were followed.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)