A total of 18 saikosaponins have been discovered in the roots of Radix Bupleuri, including saikosaponins A-D (SSA-D), which primarily exist as isomers (33). Saikosaponins serve as the primary bioactive compound in Radix Bupleuri, exhibiting various pharmacological properties, including antitumor (25,26), anti-inflammatory (34), anti-oxidant (35), antifibrotic (36), antiviral (37,38) and immunomodulatory effects (39). The main saikosaponins with antitumor effects are SSA and SSD (40).

Polysaccharides are complex sugar chains composed of galactose, glucose, xylose, arabinose, rhamnose and other monosaccharides. These monosaccharides link to form a higher-order structure (41). Among these, glucose and arabinose are particularly notable monosaccharides that influence the activity of Radix Bupleuri. Polysaccharides extracted from Radix Bupleuri have been reported to enhance the functionality of macrophages and natural killer cells, boost the body's immune response and exhibit antioxidant, anti-inflammatory and immunomodulatory properties (42–45).

Flavonoids such as kaempferol, isorhamnetin and quercetin have been identified as possessing the ability to suppress the release and expression of certain pro-inflammatory factors, such as TNF-α, inducible nitric oxide synthase, IL-1β, IL-6 and IL-12p70 (46,47). Furthermore, flavonoids are capable of inhibiting the generation of free radicals in the body, scavenging free radicals, activating the body's anti-oxidant system (48) and regulating inflammation (49,50).

The eukaryotic cell growth and proliferation process encompasses replication and division, collectively referred to as the cell cycle, consisting of the G1, S, G2 and M phases. The advancement of the cell cycle is typically linked to an escalation in proliferative proteins, such as Cyclin, Cyclin-dependent kinase (CDK) and CDK inhibitor (56). Disturbances in any of the regulatory factors can result in abnormal cell cycle activity and uncontrolled cell proliferation, leading to tumor formation (57). SSD was shown to induce a G0/G1 phase blockade in doxorubicin (DOX)-resistant cells by decreasing the protein expression levels of Cyclin D1 and CDK4, while promoting DOX-induced apoptosis in vitro and in vivo by increasing cleaved Caspase 3 expression levels (58). p21 and p27 act as negative regulators of the cell cycle in coordination with cell cycle-associated kinases. Specifically, p21 halts cell cycle progression through the G/S phase and corrects abnormal centrosome replication, while p27 suppresses CDK expression, causing cell cycle arrest in the G0/G1 phase, inhibiting centrosome separation and impeding breast cancer cell proliferation (59). SSA induces apoptosis in the breast cancer cell lines, MDA-MB-231 and MCF-7, through p53/p21-independent and -dependent mechanisms (60). DNA synthesis during the S phase and mitosis in the G2/M phase are crucial for sustaining malignant tumor cell proliferation. Antitumor drugs primarily inhibit the progression of these cell cycle phases (61,62). The flavonoid, isorhamnetin, exhibits high anticancer activity, downregulating the protein expression levels of Cyclin B1 and CDK1 and inducing G2/M phase blockade, thus preventing the proliferation of MCF-7 cells (63).

Mitogen-activated protein kinase (MAPK) serves a crucial role in apoptosis induction. The downregulation of the p38 MAPK signaling pathway reduces the metastasis of breast cancer cells in the bone-tropic mouse mammary tumor virus-driven polyoma virus middle T oncoprotein transgenic (MMTV-PyMT) Bo1 mouse model (64–66). Additionally, SSD induces the apoptosis of MDA-MB-231 cells through the p38 MAPK signaling pathway by enhancing the p38its phosphorylation level and expression levels (67).

Excessive endoplasmic reticulum stress (ERS), or ER dysfunction, results in the persistent accumulation of unfolded and misfolded proteins in the ER, leading to an imbalance in cellular homeostasis, which can induce apoptosis (68). Calnexin monitors intracellular Ca²⁺ homeostasis by assisting in folding misfolded proteins and serving an essential role in ERS (69). SSD acts as an adenosine triphosphate (ATP)ase sarcoplasmic/ER Ca²⁺ transporting inhibitor in apoptosis-deficient cancer cells, such as MCF-7, and induces ERS and autophagic cell death by disrupting intracellular Ca²⁺ homeostasis via the Ca²⁺/calmodulin-dependent protein kinase 2/mammalian target of rapamycin (mTOR) pathway (70). Wnt/β-catenin signaling pathway-related genes are highly expressed in triple-negative breast cancer (TNBC) (71). After incubation with SSD, the protein and mRNA expression levels of β-catenin in the cytoplasm and nucleus of the TNBC cells, SUM-159, HCC1937, MDA-MB-468 and MDA-MB-231, were significantly decreased, as well as the expression of downstream target genes, such as c-myc and CyclinD1, indicating that SSD inhibits the proliferation of TNBC cells by significantly inhibiting β-catenin and its downstream target genes. SSD also induces Caspase-dependent apoptosis and inhibits the proliferation of SUM-159, HCC1937, MDA-MB-468 and MDA-MB-231 cells (72).

Therefore, saikosaponins and flavonoids inhibit the development of breast cancer cells by negatively regulating cell cycle-related proteins. Saikosaponins also regulate apoptosis-related genes, downregulate the Wnt/β-catenin and p38 MAPK signaling pathways, reduce the expression levels of calnexin, serve a vital role in inhibiting the proliferation and promoting the apoptosis and autophagy of breast cancer cells and exhibit a concentration and time dependent-effect compared with the clinical breast cancer drug, paclitaxel, in which saikosaponins have a higher potency (72).

Invasive metastatic recurrence is the leading cause of death in patients with breast cancer (73). Epithelial-mesenchymal transition (EMT) is an important mechanism for initiating the process of malignant phenotypic transformation and the development of metastasis in breast cancer (74). Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and promote the metastatic process of tumors, particularly MMP-2 and MMP-9 (75,76). SSA was shown to inhibit TNBC invasive metastasis by downregulating the MMP-9 and MMP-2 expression levels in SUM-149 and MDA-MB-231 cells (26). Phosphorylated signal transducers and activators of transcription 3 (p-STAT3) promotes the invasive metastasis of TNBC by mediating the EMT (77). Vasodilator stimulated phosphoprotein (VASP) can modulate actin polymerization and promote breast cancer development (78,79). SSB-2 suppresses the proliferation and migration of MCF-7 cells by inhibiting the STAT3 signaling pathway and reducing the expression levels of VASP, MMP-2 and MMP-9 (80).

The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mTOR pathway can regulate tumor cell growth, proliferation, survival, angiogenesis and other processes (81). The stromal cell-derived factor-1 (SDF-1)/chemokine receptor (CXCR4) axis is involved in the invasion and migration of MDA-MB-231 cells (82), and the crosstalk between the CXCR4/SDF-1 axis and the AKT/mTOR pathway occurs in circulating tumor cells (83). CXCR4 expression is associated with breast cancer growth, angiogenesis and distant metastasis and is an important indicator of infiltrative metastasis and poor prognosis (84,85). When compared with normal breast cancer cells, CXCR4 is highly expressed in TNBC cell lines (86). Results from an animal study showed that SSA downregulates the expression of CXCR4 in mouse lung high metastatic breast cancer luciferase cells (4T1-LUC cells) and exhibits anti-growth and anti-metastatic effects on TNBC through multiple signaling pathways, including PI3K/AKT/mTOR and MMPs (26).

Accordingly, different saikosaponins can inhibit the invasive and metastatic potential of breast cancer cells and prevent the occurrence and development of breast cancer through various signal transduction pathways such as the STAT3, VASP, MMPs, CXCR4/SDF-1, PI3K/Akt and Akt/mTOR pathways.

Solid tumor cells have the potential to proliferate indefinitely, and to support cell proliferation and survival, tumor cells rewire their metabolism to a glycolytic phenotype to satisfy their escalating bioenergetic demands, with TNBC being the breast cancer subtype most reliant on glycolysis for energy gain (87). Through the Warburg effect, the breakdown of glucose into lactic acid allows efficient access to the ATP required for unlimited proliferation of tumor cells, the raw material for the synthesis of tumor cell biomolecules and the precursors essential for other metabolic pathways, ultimately promoting tumor progression (88,89). SSA reduces the upregulation of p-STAT3 and p-AKT in MDA-MB-231 and MCF-7 cells and decreases lactate and ATP production and glucose uptake in tumor cells through downregulation of the AKT/STAT3 signaling pathway, inhibiting the proliferation of tumor cells (90).

Bioinformatics and network pharmacology analyses have shown that flavonoids and saikosaponins can regulate lipid metabolism-related genes and improve the depressive symptoms of patients with breast cancer (91,92). Saikosaponins can increase hepatic uptake of circulating fatty acids, promote mitochondrial respiration in fatty acid oxidation, repair imbalanced lipid metabolism and promote intracellular cholesterol efflux, high-density lipoprotein (HDL) remodeling and the clearance of low-density lipoprotein (LDL) particles and bile acid synthesis, significantly modulating cholesterol clearance (93,94). Certain commonly used drugs for the treatment of breast cancer, such as exemestane and chloroquine, are hepatotoxic and have major adverse effects (95,96). Saikosaponins have been reported to be effective liver protectants and Chaihu Liver Protection Tablets have been clinically used to combat oxidative stress and lipid peroxidation reactions associated with lipid overload (97).

The main component of the breast is adipose tissue and mammary glands, in which cancer-associated adipocytes can promote the proliferation and metastasis of breast cancer cells (98–100). SSA and SSD inhibit adipocyte 3T3-L1 production via the AMP-activated protein kinase (AMPK) and MAPK signaling pathways (101). Disorders of glucose and lipid metabolism predispose individuals to insulin resistance, abnormal glucose tolerance and altered lipid metabolism, all of which are risk factors for the development of breast cancer (102,103). It has been previously reported that SSA can be used to reduce the levels of blood glucose, triglyceride, free fatty acid, total cholesterol, LDL and HDL in mice, significantly reduce liver weight and fat accumulation, downregulate the expression of TNF-α and NF-κB, and upregulate the expression of fibroblast growth factor-21 and recombinant autophagy related protein 7, which stimulates the autophagy of cells and improves insulin resistance (104).

Therefore, saikosaponins can affect the glucose metabolism of breast cancer cells by regulating the production of aerobic enzymolysis products and inhibiting the activity of cytokines. Together with flavonoids, they can also serve a role in lipid regulation. Targeting the regulation of the AMPK and the MAPK signaling pathway and other aspects could enable treatments to target multiple breast tumors. The pharmacological effects of the active ingredients of Radix Bupleuri are shown in Table II.

The TME consists of cancer cells, stromal cells, cytokines, chemokines and other factors that serve a crucial role in breast cancer development, progression and drug resistance (105). Tumor-associated macrophages (TAMs) are the most abundant immune cell group in breast cancer, participate in every stage of cancer progression and are associated with tumor malignant progression (105,106). TAMs are typically divided into M1 and M2 types, with M1 TAMs contributing to the dormancy of metastatic breast cancer cells and M2 TAMs promoting tumor growth (105). Polysaccharides can upregulate the activity of macrophages, effectively inhibit the release of key factors of inflammatory response and immune regulation, exert anti-inflammatory activity, prevent tissue damage caused by excessive inflammation and may enhance the TME in breast cancer (43,44). SSD can significantly enhance the phagocytic ability of macrophages, increase their acid phosphatase levels and promote the expression of immune-related antigens on the cell surface (107).

Tumor infiltrating T cells are associated with improved clinical prognosis and survival in patients with breast cancer. As a crucial component of the TME, T cells serve a significant role in the immune response to cancer (108). The balance between T helper (Th)1 and Th2 cells is particularly important, as a shift from Th1 to Th2 promotes breast cancer progression (109). SSA activates the IL-12/STAT4 pathway, leading to a significant increase in the infiltration of CD8⁺ and CD4⁺ T cells in tumors. This activation occurs through the upregulation of IL-12, IL-12R and STAT4 gene expression, as well as the increased expression levels of IL-12, IL-12R and p-STAT4 proteins (25). CD8⁺ T cells exert tumor-killing activity by interacting with tumor antigens and releasing perforin, granzyme and cytokines, which directly or indirectly kill tumor cells. Additionally, this process promotes a shift in the Th1/Th2 balance towards a Th1 response, thereby inhibiting the development and progression of breast cancer (25).

Therefore, polysaccharides and saikosaponins jointly regulate the TME, which are important components of Radix Bupleuri for immune regulation.

The process of tumor angiogenesis includes endothelial cell activation, ECM degradation and endothelial cell migration, angiogenesis and extension into the tumor and is regulated by various cytokines in the TME (110). Vascular endothelial growth factor (VEGF) is an important angiogenesis factor and blocking VEGF is a potential strategy for the treatment of invasive breast cancer (111). The VEGF receptor 2 (VEGFR2) signaling pathway and its downstream proteins are an important signaling pathway that regulates endothelial cell function during angiogenesis (112). Clinically, patients with breast cancer who experience lymph node metastasis often harbor high expression levels of hypoxia inducible factor 1 subunit α (HIF-1α) and VEGF. The recurrence and metastasis of breast cancer may be related to the upregulation of HIF-1α and VEGF, promoting the angiogenesis of breast cancer and other related factors (113,114). SSA inhibits the phosphorylation of VEGFR2 and the activity of downstream protein kinases, phospholipase C-γ-1, focal adhesion kinase, Src and AKT, reducing tumor angiogenesis and subsequently inhibiting the growth of mouse breast cancer 4T1 cells (115). Thus, by downregulating the expression level or activity of angiogenesis-related proteins, saikosaponins inhibit tumor angiogenesis and limit the growth and metastasis of breast tumors.

At present, surgical resection is still the main treatment option for patients with breast cancer and postoperative radiotherapy or chemotherapy are often used to further improve patient prognosis (116). Radiotherapy and chemotherapy both non-selectively kill cancer cells and can cause toxic side effects (117). Traditional Chinese Medicine has been used for the prevention and treatment of breast cancer (118). Traditional Chinese Medicine has been reported to target tumors and can be combined with Western medicine to enhance the efficacy of these medicines and reduce potential toxicity (119).

During or after chemotherapy, the expression levels of multidrug resistance protein (MDR)1 and breast cancer resistance protein, ABCG2, can increase significantly (120). A mechanism of action of MDR is the upregulation of p-glycoprotein (P-gp), and the direct inhibition of P-gp upregulation by SSA and SSD in vitro reverses MDR in MCF-7 cells (121,122). Isorhamnetin mediates the inhibition of proliferation and the induction of apoptosis in MCF7/adriamycin (ADR) and MDAMB-231/DOX cells by enhancing the phosphorylation of AMPK, decreasing the phosphorylation levels of mTOR and p70S6K, inhibiting the expression levels of B-cell lymphoma-2 and increasing the cleavage of Caspase 3 (63). In addition, isorhamnetin also downregulates the expression level of P-gp, leading to increased intracellular DOX accumulation, increased toxicity, promotion of apoptosis and the restriction of tumor growth in vivo (63).

Moderate levels of reactive oxygen species (ROS) can activate various signaling pathways to promote tumor development (123,124). However, excessive ROS levels that cannot be effectively balanced by the antioxidant system can lead to oxidative stress, promoting apoptosis and the death of cancer cells. Drug-resistant cells produce higher levels of ROS compared with non-drug-resistant cells. To maintain redox homeostasis in an environment with high ROS levels, drug-resistant cancer cells enhance their antioxidant capacity by increasing the synthesis of reduced glutathione (GSH) and upregulating antioxidant enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1) (125). NQO1 is significantly upregulated in various types of solid tumors, suggesting its potential involvement in cellular defense during oncogenesis (126). NQO1 is reported to be a STAT1-regulated gene in MCF-7/DOX and MDA-MB-231/DOX-resistant strain cells, as STAT1 expression levels are positively correlated with NQO1 expression levels (58). Molecular docking has shown that SSD may interact with the active site of NQO1, forming two hydrogen bonds with the Leu103 and Tyr128 residues, suggesting that SSD has a strong binding affinity with the NQO1 protein (58). Subsequently, in vitro and in vivo experiments have been performed to confirm that SSD downregulates the STAT1/NQO1/peroxisome proliferator-activated receptor γcoactivator-1 α (PGC-1α) signaling pathway (58). After SSD inhibits NQO1, the consumption of nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH) decreases (58). The redox imbalance in the cell is in a high ROS state, and the antioxidants, GSH, NADPH and NADH, are consumed in large quantities in response to oxidative stress, which ultimately leads to a decrease in their levels (58), which leads to an increase in intracellular oxidative stress levels and ultimately induces MCF-7/DOX and MDA-MB-231/DOX apoptosis (58).

Patients with breast cancer treated with radiation and chemotherapy may have adverse reactions, such as insomnia, bone marrow suppression, leukopenia and depression (127). Radix Bupleuri, as the main ingredient of Xiao Chaihu Tang, Jia Wei Chaihu Gui Jiang Tang and other Traditional Chinese Medicine compound tonics, has been reported to clinically improve the postoperative adverse effects in patients with breast cancer, reduce or slow down the secretion of tumor markers and improve the efficacy of certain chemotherapeutic agents such as paclitaxel and cisplatin (27,128).

A variety of components of Radix Bupleuri can slow down the progression of breast cancer, reverse the MDR of tumor cells to chemotherapeutic drugs including ADR, vincristine and paclitaxel and alleviate postoperative adverse reactions (122).

The stability of subcellular organelles and metal ions is essential for various physiological and biochemical processes, such as homeostasis of the internal environment, regulation of cellular metabolism, substance synthesis, signal transmission and energy conversion (129). Staphylococcus aureus infection can cause an increase in inflammatory markers such as myeloperoxidase, TNF-α and IL-1 β in mice, as well as an accumulation of Fe²⁺; it significantly increases the expression levels of malondialdehyde (MDA) and reduces the expression levels of GSH, indicating that S. aureus induces ferroptosis in normal breast cells, leading to the development of mastitis. Additionally, S. aureus reduces the expression levels of sirtuin 1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1); however, SSA treatment increases the expression levels of SIRT1, Nrf2 and HO-1 in a dose-dependent manner, indicating that SSA inhibits S. aureus-induced mastitis by activating the SIRT1/Nrf2 signaling pathway (34).

Mitochondria are the metabolic centers and energy factories of cells (130). During respiration and oxidation, mitochondria store the generated energy as electrochemical potential energy in the inner mitochondrial membrane, causing an asymmetric distribution of protons and other ions on both sides of the inner membrane to form the mitochondrial membrane potential (ΔΨm) (130). Normal ΔΨm is a prerequisite for mitochondria to carry out oxidative phosphorylation and produce ATP, and ΔΨm instability promotes the development of cancer cells (131). With increasing SSD concentrations, the ΔΨm levels of MCF-7/DOX and MDA-MB-231/DOX cells decrease, the PGC-1α protein expression levels decrease and the protein expression levels of the double-stranded DNA breakage marker, γ-H2AX, increase, suggesting that SSD leads to the oxidative stress of drug-resistant cells by amplifying the ΔΨm loss, mitochondrial dysfunction and DNA damage to exert antitumor effects (58). Estrogen receptor 2 (ERβ) serves a key role in maintaining mitochondrial homeostasis in breast epithelial cells, but decreased expression levels of ERβ in some patients with breast cancer after surgical treatment leads to tumor recurrence and metastasis, suggesting that ERβ abnormalities are related to mitochondrial dysfunction. ERβ inhibits the invasive properties of EMT and TNBC cells and patients with ERβ deficiency are prone to tumorigenesis (132). SSD has weak estrogen-like effects and activates ERβ expression at high doses (133). The role of saikosaponins in inhibiting ferroptosis in normal breast cells in mastitis and in affecting the ΔΨm of drug-resistant cells provides a potential basis for the investigation of saikosaponin-induced ferroptosis in breast cancer and may suggest the feasibility of this approach for the future treatment of patients with this disease.

The study of signaling pathways has implications for the treatment of certain diseases and future biotechnological innovations. A number of relevant signaling pathways activated by Radix Bupleuri active ingredients cause anti-breast cancer effects (Fig. 2).