Abstract

Background

   Tri-ortho-cresyl phosphate (TOCP), a widely used plasticizer, has been
   shown to impair ovarian function. While tanshinone IIA exhibits ovarian
   protective effects in aging models, its potential to counteract
   TOCP-induced ovarian damage and associated signaling mechanisms remains
   unexplored. This study investigates the therapeutic effects of
   tanshinone IIA on TOCP-damaged ovaries in mice, with focus on Hippo,
   AKT, and MAPK pathways.

Results

   TOCP exposure (200 mg/kg/d for 28 days) significantly reduced ovarian
   follicle counts (primordial, preovulatory, and mature follicles) and
   disrupted hormone levels (elevated Estrogen(E2), decreased Follicle
   stimulating hormone(FSH)/ Anti-Mueller tube hormone(AMH)) in mice.
   Treatment with high-dose tanshinone IIA restored ovarian structure and
   function: growing follicle counts increased significantly (p < 0.001),
   FSH (p < 0.001) and AMH (p < 0.001) levels surged to marked degrees,
   while E2 (p < 0.001) levels decreased significantly. All changes were
   statistically significant. Immunohistochemistry and Western blot
   analysis revealed that tanshinone IIA restored ovarian AMH and
   Follicle-Stimulating Hormone Receptor (FSHR) protein expression, which
   were suppressed by TOCP. In vitro experiments further demonstrated that
   TOCP dose-dependently inhibited granulosa cell viability (p < 0.001)
   and proliferation (p < 0.001). Co-treatment with tanshinone IIA
   (0.01 mM) rescued cell viability (p < 0.01) and proliferation
   (p < 0.05). Mechanistically, tanshinone IIA suppressed ovarian
   apoptosis (p < 0.01) and modulated multiple signaling pathways: it
   attenuated Hippo signaling (p < 0.05) and reactivated PI3K/AKT
   (p < 0.05), p38 (p < 0.05), and ERK1/2 (p < 0.01) pathways.

Conclusions

   Tanshinone IIA alleviates TOCP-induced ovarian dysfunction primarily
   through coordinated modulation of Hippo signaling and AKT/MAPK pathway
   activities, offering a potential therapeutic strategy for
   chemical-induced ovarian injury.

   Keywords: Hippo signaling pathway, Apoptosis, Tanshinone IIA,
   Tri-ortho-cresyl phosphate, Granulosa cells

Introduction

   Worldwide, infertility affects 15–20% of couples, with 18.6 million
   individuals impacted by 2024, making it the third most prevalent
   disease globally [[42]1]. In China, infertility rates have surged from
   12 to 18% within a decade, now affecting ~ 50 million people [[43]2].
   Environmental toxicants, particularly plasticizers such as triphenyl
   phosphate (TOCP), are increasingly implicated in female reproductive
   dysfunction due to their endocrine-disrupting properties [[44]3–[45]5].

   Tri-ortho-cresyl phosphate (TOCP), the most toxic isomer of tricresyl
   phosphate, is widely used in plastics, lubricants, and flame retardants
   [[46]6, [47]7]. Human exposure occurs via inhalation, ingestion, and
   dermal absorption, with higher urinary TOCP levels detected in women
   than men [[48]8–[49]10]. TOCP exerts multi-organ toxicity, including
   reproductive damage: in males, it disrupts spermatogenesis [[50]11,
   [51]12], while in females, it impairs granulosa cell function, induces
   oocyte mitochondrial dysfunction, and disrupts cytoskeletal
   organization, leading to follicular atresia and hormonal imbalance
   [[52]13–[53]15]. Despite these risks, no approved therapies exist to
   counteract TOCP-induced ovarian injury, underscoring the urgency to
   identify protective agents.

   Traditional Chinese medicine (TCM) offers promise for multi-target
   interventions in ovarian protection. Tanshinone IIA, a bioactive
   diterpenoid from Salvia miltiorrhiza, demonstrates pleiotropic effects
   in gynecological disorders, including antioxidant, anti-inflammatory,
   and anti-apoptotic activities [[54]16–[55]18]. Preclinical studies
   highlight its ability to restore ovarian reserve in aging and premature
   ovarian failure models by modulating hormone levels (FSH, AMH, E2) and
   suppressing oxidative stress via pathways such as Nrf2/SLC7 A11/GPX4
   [[56]19–[57]21]. In gynecological therapeutics, tanshinone IIA protects
   ovarian function by activating antioxidant pathways (e.g., Nrf2/SLC7
   A11/GPX4) to mitigate oxidative damage and inflammatory cytokines
   (e.g., IL- 6, TNF-α) [[58]22, [59]23], while enhancing ovarian reserve,
   reducing granulosa cell apoptosis, and restoring hormone balance (LH,
   FSH, AMH, E2) in aging and Premature ovarian failure (POF) mouse models
   [[60]24, [61]25].

   The Hippo pathway plays a pivotal role in developing and functioning
   the ovary by regulating the activation and survival of follicles, as
   well as the proliferation of ovarian cells [[62]26]. Our previous
   research findings suggested that after ovarian loss caused by TOCP
   exposure, the level of follicle apoptosis increased and that the levels
   of sex hormones in mice were abnormal, which may be associated with the
   Hippo signaling pathway [[63]13]. Additionally, it has been reported
   that tanshinone IIA can suppress the formation, migration, and invasion
   of cancer stem cells through the Hippo signaling pathway [[64]27].
   Consequently, we put forward the hypothesis that tanshinone IIA could
   potentially mitigate TOCP-induced damage to ovarian function by
   activating the Hippo signaling pathway, which is closely linked to
   ovarian function regulation and regulates cell proliferation and
   apoptosis. Specifically, we aim to (i) assess the impact of Tanshinone
   IIA on follicle survival and sex hormone levels in TOCP-exposed mice,
   (ii) determine the role of the Hippo pathway in mediating these
   effects, and (iii) explore the molecular mechanisms by which Tanshinone
   IIA regulates ovarian cell proliferation and apoptosis.

Materials and methods

Animals and experimental design

   Female balb/c mice (6–8 weeks old, 20–25 g, n = 32) were purchased from
   the Center for Experimental Animals at Nanchang University. A total of
   mice were housed under controlled conditions (12-h light/dark cycles,
   24 ± 2 ℃ ambient temperature and 50–60% humidity) with ad libitum
   access to food and water. All animal procedures were approved by the
   Institutional Animal Care and Use Committee of Nanchang University, in
   compliance with ethical guidelines for animal research.

   Based on our previous studies, which demonstrated that TOCP induces
   ovarian dysfunction in a dose-dependent manner [[65]13]. To investigate
   the effects of tanshinone IIA on the recovery from TOCP-induced
   poisoning, we chose the lowest dose (200 mg/kg/d) that can cause
   significant toxicity for TOCP exposure model. The mice were randomly
   assigned into four groups (n = 8 in each group) (Fig. [66]1): Group 1:
   Control (The normal control group received an equivalent dose of corn
   oil, according to previous studies [[67]28, [68]29]); Group 2: 200
   mg/kg/d TOCP; Group 3: 200 mg/kg/d TOCP + Tanshinone IIA 25 mg/kg/d;
   Group 4: 200 mg/kg/d TOCP + Tanshinone IIA 50 mg/kg/d.

Fig. 1.

   [69]Fig. 1
   [70]Open in a new tab

   Schematic diagram of mice grouping. The mice were randomly assigned
   into four groups (n = 8 in each group): Mice in Group 1 were treated
   with corn oil for six weeks as control. Mice in Group 2 were exposed to
   200 mg/kg/d TOCP for four weeks and then received corn oil for two
   weeks. Mice in Group 3 were exposed to 200 mg/kg/d TOCP for four weeks
   and then received 25 mg/kg/d tanshinone IIA for two weeks. Mice in
   Group 4 were exposed to 200 mg/kg/d TOCP for four weeks and then
   received 50 mg/kg/d tanshinone IIA for two weeks

Morphological analysis and follicular classification

   The ovaries were collected and fixed in 4% paraformaldehyde overnight.
   After dehydration in ethanol and embedding in paraffin, serial sections
   were obtained, and hematoxylin and eosin staining was performed
   according to standard procedures. The images were processed using the
   same parameters, and the follicles were classified according to
   previous descriptions [[71]30].

Determination of serum levels of estradiol (E2), anti-mullerian hormone
(AMH), and follicle-stimulating hormone (FSH)

   Blood samples were collected, and serum was then separated by
   centrifugation and stored at –80 ℃ for further analyses. The levels of
   E2 (ml063198), AMH (ml037597), and FSH (ml063198) in mice plasma
   weremeasured using commercial ELISA kits (Shanghai Enzyme-linked
   Biotechnology Co., Ltd, Shanghai, China). Measurements were conducted
   according to the manufacturer's rotocol.

Acquisition of Tanshinone IIA targets

   To collect as many potential targets for Tanshinone IIA as possible, we
   utilized the following four databases: (i) the TCM Systems Pharmacology
   Database and Analysis Platform (TCMSP,
   [72]http://lsp.nwu.edu.cn/tcmsp.php), (ii) the Pharm Mapper Server
   ([73]https://lilab-ecust.cn/pharmmapper/index.html), and (iii) the
   Swiss Target Prediction ([74]https://www.swisstargetprediction.ch/).

TOCP damage related target retrieval (Search for Related Targets of
TOCP-Induced Injury)

   The keyword'Tri-ortho-cresyl phosphate damage'as tilized to conduct a
   search for targets related to the TOCP damage in the Genecards
   ([75]https://www.genecards.org/), Online Mendelian Inheritance in Man
   ([76]https://www.omim.org/) and The Comparative Toxicogenomics Database
   (ctdbase.org) databases. The final list of targets related to TOCP
   damage was generated after removing false positives.The Uniprot
   database was utilized to standardize the target list.

Protein–protein interaction network analysis

   Using the Bioinformatics online platform
   ([77]https://www.bioinformatics.com.cn/), we constructed a Venn diagram
   of the intersection targets between tanshinone IIA and TOCP-induced
   damage. We also identified the intersection targets and imported them
   into the STRING database ([78]https://string-db.org/) for
   Protein–Protein Interaction (PPI) network analysis. To do so, we
   specified the species as"Homo Sapiens"and set the"Minimum Required
   Interaction Score"to 0.4. We then constructed a PPI network using the
   STRING online database. The PPI network diagram was then exported as
   TSV format and uploaded to Cytoscape 3.10.2 software to generate a PPI
   protein interaction network map.

GO and KEGG enrichment analysis

   GO and KEGG enrichment analyses were conducted using the online
   platform ([79]https://www.bioinformatics.com.cn/) and David database
   ([80]https://david.ncifcrf.gov/).

Molecular docking

   PyMol 2.6 was utilized to produce the receptor proteins and visualize
   the results of the docking process. AutoDock 1.5.7 was employed for
   docking, with he docking conformation with the highest output score
   deemed the most suitable binding conformation. The docking energy
   represented the energy absorbed or released during molecular binding,
   and was used to assess the affinity and stability of the molecular
   docking. The visualization of the results was achieved using PyMol and
   Discovery studio.

Terminal deoxynucleotidyl transferase-mediated UTP ick-end labeling (TUNEL)
assay

   To detect DNA fragmentation in the ovary, paraffin-embedded ovary
   tissues were sectioned into 5 μm sections and assayed using a TUNEL kit
   (KGA7035, KeyGEN Biotechnology, European, China) according to the
   manufacturer's protocol. The stained sections were imaged using a
   fluorescence microscope equipped with an Olympus camera (Olympus IX73P2
   F). The apoptotic signal was recorded as positive when dUTP stained the
   nuclei green.

Protein extraction and western blot analysis

   Total protein was extracted from the ovaries of each group using RIPA
   reagent (Beyotime, P0013 F) for western blot analysis. Proteins were
   transferred from the SDS-PAGE gel to a PVDF membrane (Millipore,
   Billerica, USA) as a routine procedure. After transfer, the membrane
   was incubated for 1 h at room temperature in 5% BSA and then incubated
   overnight at 4 ℃ with the following primary antibodies: anti-BAX
   (Proteintech 0599–2), anti-BCL- 2 (Proteintech 68,103–1), anti-YAP
   (Cell Signaling Technology, 4912S), anti-P-YAP (Cell Signaling
   Technology, 13008S), anti-CCN1 (Proteintech 26,689–1), anti-CCN2,
   anti-AKT (Proteintech 60,203–2), anti-P-AKT (Proteintech 80,455–1),
   anti-ERK 1/2 (Proteintech 1257–1), anti-P-ERK 1/2 (Proteintech
   80,031–1), anti-P38 (Proteintech 14,064–1), anti-P-P38 (Proteintech
   28,796), anti-CYP19 A1 (Proteintech 16,554–1-AP), anti-AMH (Proteintech
   14,461–1-AP), anti-FSHR (Proteintech 22,665–1-AP), and anti-STAR
   (Proteintech 12,225–1-AP). The membrane was washed with TBST and then
   incubated with a suitable secondary antibody at room temperature for
   1 h. β-actin (Proteintech 66,009–1) serves as a loading control.
   Proteins were imaged using a Gel Imaging System (Bio-Radical Chemical
   Document™ XRS +) and analyzed using the Gelscan software (Image Lab
   5.2.1).

Immunohistochemistry (IHC)

   Ovarian tissues fixed in 4% paraformaldehyde were embedded in paraffin
   and sectioned at 5 μm thickness. After deparaffinization and
   rehydration, antigen retrieval was performed using citrate buffer (pH
   6.0) at 95℃ for 20 min. Endogenous peroxidase activity was blocked for
   15 min. Sections were incubated overnight at 4 °C with primary
   antibodies against FSHR (Proteintech 22,665–1-AP) or AMH (Proteintech
   14,461–1-AP), followed by HRP-conjugated secondary antibody (Beyotime
   A0208) incubation for 1 h at 37℃. DAB (Beyotime P0203) was used for
   chromogenic detection, and nuclei were counterstained with hematoxylin.
   Images were captured using an Olympus BX53 microscope and analyzed with
   ImageJ software (v1.53 t).

Isolation of Mouse Granulosa Cells (Mechanical Disruption Method)

   Euthanize female balb/c mice (6–8 weeks old) and excise ovaries. Remove
   surrounding adipose and connective tissues in ice-cold PBS. Place
   ovaries in a 35 mm dish containing 1 mL PBS. Mechanically dissociate
   granulosa cells by gently puncturing the ovaries 50–60 times with a 1
   mL syringe (26G needle) for 5 min, releasing follicular contents.
   Transfer the cell suspension through a 70 μm cell strainer to remove
   ovarian stroma and debris. Rinse the dish with 2 mL PBS and filter the
   rinse through the same strainer. Centrifuge the combined filtrate at
   300 × g for 5 min at 4℃. Discard the supernatant and resuspend the
   pellet in 1 mL complete medium. Seed cells into 6-well plates at a
   density of 2 × 105 cells per well and incubate at 37℃ in a 5% CO₂
   atmosphere.

CCK- 8 cell viability assay

   Granulosa cells were seeded in 96-well plates at 5 × 10^3 cells/well
   and treated with TOCP (0–200 μM) or tanshinone IIA (10–50 μM) for 24
   h,48 h and 72 h. CCK- 8 reagent (Beyotime C0038, 10 μL/well) was added
   and incubated for 2 h. Absorbance was measured at 450 nm using a
   microplate reader (BioTek Synergy H1). Cell viability (%) = (OD
   [treatment]/OD [control]) × 100.

EdU proliferation assay

   Cell proliferation was assessed using the BeyoClick™ EdU Kit (Beyotime
   C0075S). Granulosa cells were treated with tanshinone IIA (25–50 μM)
   for 24 h, then incubated with 10 μM EdU for 2 h. Cells were fixed with
   4% paraformaldehyde, permeabilized with 0.3% Triton X- 100, and stained
   with Click Reaction Mix (Azide 594, 30 min). Nuclei were counterstained
   with Hoechst 33,342. Images were captured using a fluorescence
   microscope (Olympus IX73P2 F) and EdU-positive cells were quantified
   with ImageJ.

Statistical analysis

   Statistical analyses were performed using GraphPad Prism 5.0 software.
   Data were analyzed by one-way ANOVA (single-factor variance analysis)
   followed by Tukey’s post hoc multiple comparison test. Results are
   expressed as mean ± SEM with n = 3 biological replicates per
   experimental group. Significance levels were defined as p < 0.05 (*),
   p < 0.01 (**), and p < 0.001 (***).

Results

Tanshinone IIA restores follicular development damage caused by TOCP

   In the control group, the ovarian tissue structure was complete, and
   the morphology of various types of follicles was normal. In the
   TOCP-exposed group, the ovarian tissue exhibited significant structural
   abnormalities with extensive structural changes (Fig. [81]2). The
   morphology of the ovarian tissue was significantly improved compared to
   the TOCP-exposed group (Fig. [82]2C-D). A low concentration of
   tanshinone IIA resulted in a dense ovarian tissue structure; however,
   the recovery of various types of follicular morphology was
   unsatisfactory (Fig. [83]2C). In the high-concentration recovery group,
   not only did the ovarian tissue remain complete, but also the various
   types of follicular morphology were normal (Fig. [84]2D). Furthermore,
   compared to the control group, the TOCP-exposed group exhibited a
   significant decrease in the number of primordial (p < 0.001),
   preovulatory (p < 0.001), and mature follicles (p < 0.05)
   (Fig. [85]2E). Conversely, the TOCP-exposed group exhibited a
   significant increase in the number of amenorrhea cases compared with
   the control group (p < 0.01) (Fig. [86]2E). Surprisingly, the number of
   primordial (p < 0.01), preovulation (p < 0.01), and mature follicles
   (p < 0.05) increased significantly in the tanshinone IIA-treated group
   compared to the TOCP-exposed group (Fig. [87]2E). Particularly, the
   number of amenorrhea decreased significantly in the high-concentration
   tanshinone IIA treated group (p < 0.05) (Fig. [88]2E). These results
   indicate that tanshinone IIA exhibits a favorable effect on the
   recovery of ovarian follicle developmental damage induced by TOCP.

Fig. 2.

   [89]Fig. 2
   [90]Open in a new tab

   Tanshinone IIA can mitigate the impact of TOCP on the development of
   the ovaries. Morphological changes of ovaries treated with a vehicle
   control (A), 200 mg/kg/d TOCP (B), 200 mg/kg/d TOCP + 25 mg/kg/d
   tanshinone IIA (C) or 200 mg/kg/d TOCP + 50 mg/kg/d tanshinone IIA (D)
   (n = 3). Scale bars represent 100 μm. (E) Quantification of ovarian
   follicle types in mice ovaries after treatment with a vehicle control,
   200 mg/kg/d TOCP, 200 mg/kg/d TOCP + 25 mg/kg/d tanshinone IIA or 200
   mg/kg/d TOCP + 50 mg/kg/d tanshinone IIA (n = 3). The data are
   presented as mean ± SEM from at least three independent experiments.
   *p < 0.05, **p < 0.01, ***p < 0.001

Tanshinone IIA restores TOCP-induced sex hormone disorders and related
protein expression in female mice

   ELISA was employed to measure serum FSH, AMH, and E2 levels. The
   TOCP-exposed group (TOCP 200 mg/kg/d) exhibited significant reductions
   in AMH (Fig. [91]3B, p < 0.001) and FSH (Fig. [92]3A, p < 0.001) levels
   compared with the control group. In tanshinone IIA-treated groups, AMH
   (p < 0.05) and FSH (p < 0.01) levels were notably higher than those in
   the TOCP group. Remarkably, the 50 mg/kg/d tanshinone IIA-treated group
   showed hormone levels approaching those of the control group
   (Fig. [93]3A–B). For E2, an opposite trend was observed: the TOCP group
   had significantly elevated E2 levels (Fig. [94]3C, p < 0.001), while
   tanshinone IIA-treated groups demonstrated restored E2 levels (p <
   0.05, Fig. [95]3C).Immunohistochemistry (Fig. [96]3D) revealed weakened
   positive staining for AMH and FSHR in ovarian tissues of the TOCP
   group, which was enhanced in tanshinone IIA-treated groups—especially
   the 50 mg/kg/d group, nearing the control level. Western blot analysis
   (Fig. [97]3E–F) further confirmed that AMH and FSHR protein levels were
   significantly lower in the TOCP group than in the control group.
   Conversely, tanshinone IIA treatment significantly upregulated AMH and
   FSHR expression. Collectively, these results indicate that TOCP
   exposure disrupts sex hormone levels and related protein expression in
   female mice, while tanshinone IIA effectively restores these disorders.

Fig. 3.

   [98]Fig. 3
   [99]Open in a new tab

   Tanshinone IIA restores TOCP-induced sex hormone disorders and related
   protein expression in female mice. Female mice were allocated into four
   groups: Control, TOCP 200 mg/kg/d, Tanshinone IIA 25 mg/kg/d + TOCP 200
   mg/kg/d, and Tanshinone IIA 50 mg/kg/d + TOCP 200 mg/kg/d.(A–C) Serum
   levels of follicle-stimulating hormone (FSH), anti-Müllerian hormone
   (AMH), and estradiol (E2) were quantified via ELISA (n = 3). (D)
   Immunohistochemical analysis of AMH and FSHR (follicle-stimulating
   hormone receptor) expression in ovarian tissues (n = 3). (E–F) Western
   blot detection of AMH and FSHR protein levels, with β-actin as the
   loading control. F presents the quantitative analysis of AMH and FSHR,
   expressed as fold change relative to the control group (n = 3). The
   data are presented as mean ± SEM from at least three independent
   experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Tanshinone IIA Ameliorates TOCP-Induced Impairment of Granulosa Cell
Viability, Proliferation, and Steroidogenic Enzymes

   CCK- 8 analysis (Fig. [100]4A) revealed that TOCP inhibited granulosa
   cell viability in a concentration- and time-dependent manner. Treatment
   with 0.125 mM, 0.25 mM, or 0.5 mM TOCP significantly reduced cell
   viability compared to the control group over 24 h, 48 h, and 72 h, with
   maximal inhibition observed at 0.5 mM TOCP for 72 h (p < 0.05).
   Tanshinone IIA alleviated TOCP-induced cytotoxicity: co-treatment with
   0.01 mM tanshinone restored cell viability at 72 h (p < 0.01,
   Fig. [101]4B). EDU assays (Fig. [102]4C–D) further demonstrated that
   TOCP suppressed cell proliferation (p < 0.001), an effect reversed by
   tanshinone IIA (p < 0.05). Notably, TOCP exposure upregulated STAR
   protein levels (p < 0.01, Fig. [103]4F), indicating dysregulated
   steroidogenesis. Tanshinone IIA treatment dose-dependently reduced STAR
   expression (25–50 mg/kg/d), restoring progesterone/estrogen balance.
   These findings suggest TOCP disrupts ovarian steroidogenesis by
   regulating STAR/CYP19 A1, while tanshinone IIA counteracts this
   imbalance through targeted modulation of steroidogenic enzymes.

Fig. 4.

   [104]Fig. 4
   [105]Open in a new tab

   Tanshinone IIA Ameliorates TOCP-Induced Impairment of Granulosa Cell
   Viability, Proliferation, and Steroidogenic Enzymes. A. CCK- 8 assay
   showing cell viability in control, 0.125 mM, 0.25 mM, and 0.5 mM
   TOCP-treated groups at 24 h, 48 h, and 72 h. B. CCK- 8 analysis of cell
   viability in control, 0.5 mM TOCP, and 0.5 mM TOCP + tanshinone (0.001
   mM, 0.01 mM) groups at 72 h. C-D. EDU staining quantification (C) and
   representative images (D) showing cell proliferation in control, 0.5 mM
   TOCP, and 0.5 mM TOCP + tanshinone IIA (0.001 mM, 0.01 mM) groups. E.
   Western blot analysis of CYP19 A1 and STAR protein expression
   normalized to β-actin in control, TOCP (0.5 mM), and tanshinone IIA
   (0.01 mM,0.001 mM) groups. F. Quantification of STAR protein levels in
   control, TOCP (0.5 mM), and tanshinone IIA (0.01 mM,0.001 mM) groups.
   The data are presented as mean ± SEM from at least three independent
   experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Network pharmacology-based strategy for predicting potential targets of
Tanshinone IIA for TOCP

   Through the TCMSP, PharmMapper, and Swiss Target Prediction databases,
   a total of 489 targets for Tanshinone IIA were retrieved after
   deduplication (Additional file 1). The data obtained through querying
   various databases such as the Geencards, OMIM, and CTD was filtered to
   remove duplicate targets, resulting in a total of 380 disease targets
   related to TOCP-induced damage (Additional file 2). The targets
   associated with Tanshinone IIA were identified using the online
   microbioinformatics platform, and were mapped to the targets related to
   the TOCP. A Venn diagram was drawn, indicating 23 intersectional target
   points (Fig. [106]5A). In the STRING database, we imported the
   intersecting targets and download the stringinteractions.csv file.
   Then, we visualized the protein–protein interactions (PPI) graph using
   Cytoscape (Fig. [107]5B). Finally, we analyzed the network topology
   parameters and obtained the top-ranked targets such as TP53, CASP3,
   GPT, and BCL2. We utilized DAVID and microbeSTORM platforms to perform
   GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes)
   analysis on the targets that were identified. The results revealed that
   the Tanshinone IIA targets primarily overlapped in various biological
   processes, including EGFR tyrosine kinase inhibitor resistance, P53
   signaling pathway, endocrine resistance, apoptosis, MAPK signaling
   pathway, and PI3 K-AKT signaling pathway (Fig. [108]5C-D). These
   pathways were all implicated in the damage caused by TOCP. In
   particular, the apoptosis, MAPK signaling pathway, and PI3 K-AKT
   signaling pathway have been identified as classic pathways that are
   closely linked to ovarian function. Consequently, the targets related
   to the MAPK signaling pathway and PI3 K-AKT signaling pathway were
   selected as candidate targets for Tanshinone IIA to recover the damage
   caused by TOCP.

Fig. 5.

   [109]Fig. 5
   [110]Open in a new tab

   Targets related to TOCP and active ingredient-targets of Tanshinone
   IIA. (A) Venn diagram of TOCP targets and Tanshinone IIA targets. (B)
   Construction of PPI target network. (C) GO enrichment analysis of
   potential therapeutic targets. (D) KEGG pathway enrichment analysis of
   potential therapeutic targets

Tanshinone IIA inhibits follicular apoptosis induced by TOCP

   To evaluate the extent of apoptosis in the ovaries of each group of
   mice, ovarian tissues were stained with the TUNEL Assay Kit. The
   results demonstrated a higher number of TUNEL-positive follicles in the
   ovaries of the TOCP-exposed group (Fig. [111]6A). Additionally, the
   apoptosis rate increased in the TOCP-exposed group (Fig. [112]6A).
   TUNEL-positive follicles were not observed in the ovaries of the mice
   in the tanshinone IIA treated group. Meanwhile, the ratio of BAX to
   BCL- 2 is frequently employed as a marker for determining
   susceptibility to apoptosis. BAX is linked to apoptosis and promotes
   blockage, whereas BCL- 2 inhibits this process and promotes ovarian
   survival. Western blotting was performed to evaluate the expression
   levels of BAX and BCL- 2 proteins in the ovaries of each group. As
   expected, the BAX level increased, while the BCL- 2 (p < 0.01) level
   decreased significantly in the exposed group compared with the control
   group (Fig. [113]6B-C). Additionally, the level of BAX decreased, and
   the level of BCL- 2 (p < 0.01) increased significantly in the
   tanshinone IIA high-concentration-treated group compared with the
   infected group (Fig. [114]6B-C). These results indicated that exposure
   to TOCP induces ovarian apoptosis, whereas tanshinone IIA inhibits this
   process.

Fig. 6.

   [115]Fig. 6
   [116]Open in a new tab

   Tanshinone IIA inhibits TOCP-induced apoptosis. (A) The apoptosis level
   of ovaries treated with a vehicle control, 200 mg/kg/d TOCP, 200
   mg/kg/d TOCP + 25 mg/kg/d tanshinone IIA or 200 mg/kg/d TOCP + 50
   mg/kg/d tanshinone IIA was determined using TUNEL staining(n = 3).
   Scale bars represent 100 μm. (B) The protein expression levels of BAX
   and BCL- 2 were examined using western blot analysis(n = 3). (C)
   Quantification of the BAX/BCL- 2 ratio using western blot analysis(n
   = 3). The data are presented as mean ± SEM from at least three
   independent experiments. *p < 0.05, **p < 0.01

Hippo pathway plays a role in tanshinone IIA’s therapeutic effect on
TOCP-induced ovarian dysfunction

   To investigate the mechanism by which tanshinone IIA rescued ovarian
   damage caused by exposure to TOCP, we examined the expression levels of
   P-YAP and YAP and found that the ratio of P-YAP/YAP (p < 0.01) in the
   TOCP-exposed group increased significantly compared to the control
   group (Fig. [117]7A-B). However, the ratio of P-YAP/YAP (p < 0.05) in
   the high-concentration tanshinone IIA-treated group decreased
   significantly, whereas the P-YAP/YAP ratio in the low-concentration
   tanshinone IIA-treated group increased slightly but did not reach
   statistical significance (Fig. [118]7B). In addition, we examined the
   expression levels of downstream targets of the Hippo pathway,
   specifically CCN1 and CCN2. The findings displayed that the levels of
   CCN1 (p < 0.01) and CCN2 (p < 0.05) were decreased in the TOCP-exposed
   group (Fig. [119]7C–F). However, after the mice were treated with
   tanshinone IIA, CCN2 (p < 0.05) levels increased in a dose-dependent
   manner (Fig. [120]7D-E); however, CCN1 levels remained unchanged
   (Fig. [121]7C-D). Figure [122]7E-F demonstrates a striking
   dose–response relationship in CCN2 expression rescue with tanshinone
   IIA.

Fig. 7.

   [123]Fig. 7
   [124]Open in a new tab

   Tanshinone IIA activates the Hippo signaling pathway, which is
   inhibited by TOCP in the ovary. Female mice were administered with a
   vehicle control, 200 mg/kg/d TOCP, 200 mg/kg/d TOCP + 25 mg/kg/d
   tanshinone IIA, or 200 mg/kg/d TOCP + 50 mg/kg/d tanshinone IIA. (A)
   The protein expression levels of YAP, P-YAP, CCN1, and CCN2 were
   examined using western blot analysis (n = 3). (B) Quantification of the
   P-YAP/YAP ratio using western blot analysis (n = 3). Quantification of
   (C) CCN1 and (D) CCN2 expression levels (n = 3). The data are presented
   as mean ± SEM from at least three independent experiments. *p < 0.05,
   **p < 0.01, ***p < 0.001

Tanshinone IIA reverses the inhibition of the AKT, P38, and ERK1/2 signaling
pathways induced by TOCP

   To acquire a deeper insight into the mechanism by which tanshinone IIA
   can counteract TOCP-caused ovarian dysfunction, we conducted a thorough
   analysis of the expression levels of associated proteins. Our findings
   revealed that the expression levels of P-AKT/AKT (p < 0.01), P-P38/P38
   (p < 0.001), and P-ERK1/2/ERK1/2 (p < 0.05) significantly decreased in
   the TOCP-exposed group (Fig. [125]8). However, tanshinone IIA treatment
   reversed these changes, resulting in significantly higher ratios of
   P-P38/P38 (p < 0.05), P-AKT/AKT (p < 0.05), and P-ERK1/2/ERK1/2 (p <
   0.05) compared to the TOCP poisoning group (Fig. [126]8). These
   findings suggest that tanshinone IIA may alleviate TOCP-caused ovarian
   damage through the PI3 K/AKT, ERK, and P38 MAPK signaling pathways.

Fig. 8.

   [127]Fig. 8
   [128]Open in a new tab

   Tanshinone IIA induces the activation of the AKT, P38, and ERK1/2
   signaling pathways. Western blot analysis for AKT and P-AKT expression
   levels in ovaries were treated with a vehicle control, 200 mg/kg/d
   TOCP, 200 mg/kg/d TOCP + 25 mg/kg/d tanshinone IIA or 200 mg/kg/d TOCP
   + 50 mg/kg/d tanshinone IIA. (A) Western blot analysis for P-AKT and
   AKT expression levels. (B) Quantification of P-AKT/AKT expression level
   using western blot analysis (n = 3).(C)Western blot analysis for P38
   and P-P38 expression levels. (D) Quantification of P-P38/P38 expression
   level using western blot analysis (n = 3). (E) Western blot analysis
   for ERK1/2 and P-ERK1/2 expression levels (n = 3). (F) Quantification
   of P-ERK1/2/ERK1/2 expression level using western blot analysis (n =
   3). (G) Molecular docking site and three-dimensional force analysis
   diagram of tanshinone IIA and ERK, P38, AKT (n = 3). The data are
   presented as mean ± SEM from at least three independent experiments.
   *p < 0.05, **p < 0.01, ***p < 0.001

   Next, we employed molecular docking technology to investigate whether
   tanshinone IIA can directly or indirectly bind to AKT, ERK, and p38,
   and thereby exert a functional effect. The results showed that the
   docking binding energy of tanshinone IIA with AKT was − 7.62 kcal/mol,
   with three binding sites being ARG- 41, GLU- 40, and GLN- 47. The
   binding energy with p38 was − 7.69 kcal/mol, with the binding site
   being ASP- 88. The binding energy with ERK was − 7.44 kcal/mol, and the
   binding sites were VAL- 304 and HIS- 141. All the binding energies were
   less than − 5 kcal/mol, indicating a strong binding ability. Similar
   results were also obtained from the two-dimensional force analysis
   (Fig. [129]8G).

Discussion

   TOCP has been well-documented to induce neurotoxicity, hepatotoxicity,
   and reproductive toxicity [[130]31–[131]34]. Our prior research
   [[132]13] demonstrated that TOCP exposure reduces primordial, anterior
   sinus, and mature follicle counts in ovaries, accompanied by disrupted
   serum hormone levels—elevated E2 alongside decreased FSH and AMH,
   consistent with existing laboratory findings. Traditional Chinese
   medicine, with its resource abundance and cost-effectiveness, offers
   dual roles in disease treatment and prevention. Among them,
   tanshinones, particularly Tanshinone IIA (a key active component of
   Salvia miltiorrhiza), have shown efficacy in ameliorating polycystic
   ovary syndrome (PCOS)-related reproductive and metabolic dysregulation
   in rats, alongside non-classical estrogenic activity [[133]34–[134]36].
   This prompted our exploration into whether tanshinone IIA could rectify
   ovarian dysfunction in TOCP-exposed mice.

   Notably, TOCP exposure elevates ovarian apoptosis via the Hippo
   signaling pathway, a process linked to ovarian failure [[135]13].
   Tanshinone IIA has been reported to inhibit ovarian granulosa cell
   apoptosis [[136]24] and cervical cancer cell progression via the Hippo
   pathway [[137]37], suggesting a potential regulatory role in
   apoptosis-related pathways. Given the typical murine dosage range of
   tanshinone IIA (20–50 mg/kg/d) [[138]24, [139]38], we selected 25 and
   50 mg/kg/d for TOCP-model mice, hypothesizing its restorative effects
   on ovarian structure and hormone balance.

   Our network pharmacology analysis revealed 23 shared targets between
   tanshinone IIA and TOCP. Key targets like TP53, CASP3, and BCL2,
   alongside enriched pathways (P53, MAPK, PI3 K-AKT), highlighted
   potential therapeutic mechanisms. The PI3 K-AKT pathway, critical in
   ovarian diseases, regulates cell proliferation and metabolism [[140]39,
   [141]40], making it a focal point for experimental validation.

   Mechanistically, the Hippo pathway is central to TOCP-induced
   follicular dysfunction [[142]13]. TOCP alters Hippo pathway
   phosphorylation (reduced P-MST/MST, increased P-YAP/YAP), affecting
   nuclear gene expression. Previous studies showed YAP overexpression
   mitigates TOCP-induced ovarian injury [[143]13]. In this study, TOCP
   exposure decreased YAP levels and elevated P-YAP/YAP, aligning with
   prior findings. As YAP target genes, CCN1 and CCN2—critical in
   proliferation and apoptosis—were investigated. TOCP exposure reduced
   CCN1/2 expression, while tanshinone IIA dose-dependently restored CCN2,
   consistent with CCN2’s role in ovarian function regulation [[144]41].

   The processes of damage and recovery from tanshinone are complex.
   Research has revealed that tanshinone IIA can hinder the migration and
   invasion of cervical cancer stem cells through the Hippo signaling
   pathway [[145]27]. However, the PI3 K/AKT, ERK, and P38 signaling
   pathways play crucial roles in regulating and recovering from
   tanshinone IIA [[146]42, [147]43]. Besides, CCN2 is frequently involved
   in anti-apoptotic processes through the PI3 K/AKT, ERK, and P38
   signaling pathways as a downstream target gene of the Hippo pathway
   [[148]44–[149]46]. This study utilized molecular docking technology for
   verification, conducting molecular docking between key target proteins
   in PI3 K and MAPK pathways and tanshinone IIA. It was found that both
   exhibited strong affinity, explaining at the molecular level that
   tanshinone IIA can restore ovarian damage caused by TOCP through AKT,
   P38, and ERK targets. To further investigate the pathway network, we
   examined the ratios of P-AKT/AKT, P-ERK1/2/ERK1/2, and P-P38/P38. We
   observed a significant decrease in these ratios in the TOCP poisoning
   group compared to those in the control group. However, in the
   tanshinone IIA treated group, these ratios gradually recovered, with a
   more pronounced increase observed in the high-dose tanshinone IIA
   treated group. Consequently, tanshinone IIA may exert its effect by
   forming a regulatory network through the downstream targets of CCN2 in
   the Hippo pathway and the PI3 K/AKT, ERK, and P38 pathways  (Fig.
   [150]9).

Fig. 9.

   Fig. 9
   [151]Open in a new tab

   Molecular mechanism schematic diagram illustrating the recovery effect
   of tanshinone IIA on TOCPinduced ovarian dysfunction. TOCP induces
   ovarian apoptosis mediated by inhibiting the YAP phosphorylation,
   ultimately leading to ovarian dysfunction. Tanshinone IIA stimulates
   the YAP phosphorylation, causing its retention in the cytoplasm.
   Unphosphorylated YAP translocates to the nucleus and enhances the
   expression of downstream target genes CCN1 and CCN2. Upregulation
   of CCN2 promotes the phosphorylation of AKT, ERK1/2, and P38 signaling
   pathways, ultimately inhibiting ovarian apoptosis and reversing
   TOCP-induced ovarian dysfunction

   Our study unveils three pivotal advances in combating TOCP-induced
   ovotoxicity: First therapeutic evidence: We provide the first
   demonstration that tanshinone IIA effectively rescues TOCP-damaged
   ovarian function by restoring folliculogenesis and rebalancing sex
   hormones addressing a critical gap in environmental toxicant antidote
   development. Multi-pathway synergy: Mechanistically, tanshinone IIA
   orchestrates Hippo-AKT/MAPK crosstalk—suppressing Hippo hyper
   activation while reactivating PI3 K/AKT and MAPK signaling,
   establishing a novel multi-target paradigm against plasticizer
   toxicity. Clinical translation road map: Despite known effects on
   cardiovascular systems [[152]37, [153]47–[154]49], our findings
   position tanshinone IIA as a compelling candidate for localized ovarian
   therapy (e.g., intraovarian delivery). Future work will: Optimize
   herb-drug combinations (e.g., with astragaloside IV for mitochondrial
   protection) to enhance efficacy while minimizing systemic exposure;
   Develop nanoparticle-encapsulated formulations to achieve
   ovary-specific targeting, leveraging its lipophilic properties;
   Validate safety thresholds through organ-on-chip models simulating
   multi-tissue interactions. This work not only deciphers the molecular
   interplay of TOCP-induced ovarian damage but also pioneers a
   phytochemical-based strategy for occupational/environmental
   reproductive toxicology, bridging traditional medicine with precision
   toxicant countermeasures.

Conclusion

   This study provides the first evidence that Tanshinone IIA counteracts
   plasticizer-induced ovarian toxicity through a tripartite mechanism:
   (i) Restoring follicular development and sex hormone balance
   (FSH/AMH/E2). (ii) Coordinated regulation of the Hippo-PI3 K/AKT-MAPK
   signaling axis to suppress excessive apoptosis. (iii) Breakthrough
   reversal of abnormal Hippo pathway activation (reducing P-YAP/YAP
   ratio). Its multi-target properties establish a new paradigm for
   treating environmental ovarian injury. Future research should focus on
   developing ovary-specific drug delivery systems (e.g.,
   nanoparticle-based carriers) and synergistic herbal formulations to
   enhance efficacy, bridging traditional medicinal components with
   precision reproductive toxicology.

Acknowledgements