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Tanshinone IIA alleviates tri-ortho-cresyl phosphate-induced ovarian damage through Hippo signaling pathway activation in mice

Journal of Ovarian Research volume 18, Article number: 85 (2025) Cite this article

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.

Introduction

Worldwide, infertility affects 15–20% of couples, with 18.6 million individuals impacted by 2024, making it the third most prevalent disease globally [1]. In China, infertility rates have surged from 12 to 18% within a decade, now affecting ~ 50 million people [2]. Environmental toxicants, particularly plasticizers such as triphenyl phosphate (TOCP), are increasingly implicated in female reproductive dysfunction due to their endocrine-disrupting properties [3,4,5].

Tri-ortho-cresyl phosphate (TOCP), the most toxic isomer of tricresyl phosphate, is widely used in plastics, lubricants, and flame retardants [6, 7]. Human exposure occurs via inhalation, ingestion, and dermal absorption, with higher urinary TOCP levels detected in women than men [8,9,10]. TOCP exerts multi-organ toxicity, including reproductive damage: in males, it disrupts spermatogenesis [11, 12], while in females, it impairs granulosa cell function, induces oocyte mitochondrial dysfunction, and disrupts cytoskeletal organization, leading to follicular atresia and hormonal imbalance [13,14,15]. 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 [16,17,18]. 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 [19,20,21]. 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) [22, 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 [24, 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 [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 [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 [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 [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. 1): Group 1: Control (The normal control group received an equivalent dose of corn oil, according to previous studies [28, 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
figure 1

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

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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 [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, http://lsp.nwu.edu.cn/tcmsp.php), (ii) the Pharm Mapper Server (https://lilab-ecust.cn/pharmmapper/index.html), and (iii) the Swiss Target Prediction (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 (https://www.genecards.org/), Online Mendelian Inheritance in Man (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 (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 (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 (https://www.bioinformatics.com.cn/) and David database (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 × 103 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. 2). The morphology of the ovarian tissue was significantly improved compared to the TOCP-exposed group (Fig. 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. 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. 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. 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. 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. 2E). Particularly, the number of amenorrhea decreased significantly in the high-concentration tanshinone IIA treated group (p < 0.05) (Fig. 2E). These results indicate that tanshinone IIA exhibits a favorable effect on the recovery of ovarian follicle developmental damage induced by TOCP.

Fig. 2
figure 2

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

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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. 3B, p < 0.001) and FSH (Fig. 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. 3A–B). For E2, an opposite trend was observed: the TOCP group had significantly elevated E2 levels (Fig. 3C, p < 0.001), while tanshinone IIA-treated groups demonstrated restored E2 levels (p < 0.05, Fig. 3C).Immunohistochemistry (Fig. 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. 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
figure 3

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

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

CCK- 8 analysis (Fig. 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. 4B). EDU assays (Fig. 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. 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
figure 4

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

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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. 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. 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. 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
figure 5

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

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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. 6A). Additionally, the apoptosis rate increased in the TOCP-exposed group (Fig. 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. 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. 6B-C). These results indicated that exposure to TOCP induces ovarian apoptosis, whereas tanshinone IIA inhibits this process.

Fig. 6
figure 6

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

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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. 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. 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. 7C–F). However, after the mice were treated with tanshinone IIA, CCN2 (p < 0.05) levels increased in a dose-dependent manner (Fig. 7D-E); however, CCN1 levels remained unchanged (Fig. 7C-D). Figure 7E-F demonstrates a striking dose–response relationship in CCN2 expression rescue with tanshinone IIA.

Fig. 7
figure 7

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

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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. 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. 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
figure 8

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

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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. 8G).

Discussion

TOCP has been well-documented to induce neurotoxicity, hepatotoxicity, and reproductive toxicity [31,32,33,34]. Our prior research [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 [34,35,36]. 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 [13]. Tanshinone IIA has been reported to inhibit ovarian granulosa cell apoptosis [24] and cervical cancer cell progression via the Hippo pathway [37], suggesting a potential regulatory role in apoptosis-related pathways. Given the typical murine dosage range of tanshinone IIA (20–50 mg/kg/d) [24, 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 [39, 40], making it a focal point for experimental validation.

Mechanistically, the Hippo pathway is central to TOCP-induced follicular dysfunction [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 [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 [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 [27]. However, the PI3 K/AKT, ERK, and P38 signaling pathways play crucial roles in regulating and recovering from tanshinone IIA [42, 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 [44,45,46]. 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. 9).

Fig. 9
figure 9

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

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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 [37, 47,48,49], 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.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Cimadomo D, Garolla A, Vitagliano A. P4 Reproductive Medicine: Prediction, Prevention, Personalization, and Participation in Infertility Care. J Clin Med. 2024;13(19):5860. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/jcm13195860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhou Z, Zheng D, Wu H, Li R, Xu S, Kang Y, Cao Y, Chen X, Zhu Y, Xu S, Chen ZJ, Mol BW, Qiao J. Epidemiology of infertility in China: a population-based study. BJOG. 2018;125(4):432–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1471-0528.14966.

    Article  CAS  PubMed  Google Scholar 

  3. Segal TR, Giudice LC. Before the beginning: environmental exposures and reproductive and obstetrical outcomes. Fertil Steril. 2019;112(4):613–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.fertnstert.2019.08.001.

    Article  CAS  PubMed  Google Scholar 

  4. Caserta D, Bordi G, Ciardo F, Marci R, La Rocca C, Tait S, Bergamasco B, Stecca L, Mantovani A, Guerranti C, Fanello EL, Perra G, Borghini F, Focardi SE, Moscarini M. The influence of endocrine disruptors in a selected population of infertile women. Gynecol Endocrinol. 2013;29(5):444–7. https://doiorg.publicaciones.saludcastillayleon.es/10.3109/09513590.2012.758702.

    Article  CAS  PubMed  Google Scholar 

  5. Mok-Lin E, Ehrlich S, Williams PL, Petrozza J, Wright DL, Calafat AM, Ye X, Hauser R. Urinary bisphenol A concentrations and ovarian response among women undergoing IVF. Int J Androl. 2010;33(2):385–93. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1365-2605.2009.01014.x.

    Article  CAS  PubMed  Google Scholar 

  6. Craig PH, Barth ML. Evaluation of the hazards of industrial exposure to tricresyl phosphate: a review and interpretation of the literature. J Toxicol Environ Health B Crit Rev. 1999;2(4):281–300. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/109374099281142.

    Article  CAS  PubMed  Google Scholar 

  7. Fowler MJ, Flaskos J, McLean WG, Hargreaves AJ. Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells. J Neurochem. 2001;76(3):671–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1046/j.1471-4159.2001.00020.x.

    Article  CAS  PubMed  Google Scholar 

  8. Morgan JP. The Jamaica ginger paralysis. JAMA. 1982Oct 15;248(15):1864–7 (PMID: 6750161).

    Article  CAS  PubMed  Google Scholar 

  9. Ding Q, Fang S, Chen X, Wang Y, Li J, Tian F, Xu X, Attali B, Xie X, Gao Z. TRPA1 channel mediates organophosphate-induced delayed neuropathy. Cell Discov. 2017;3:17024. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/celldisc.2017.24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li J, Dong Z, Wang Y, Bao J, Yan Y, Jin J. Different organophosphate flame retardant and metabolite concentrations in urine from male and female university students in Beijing and an assessment of exposure via indoor dust. Environ Toxicol Chem. 2019;38(4):760–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/etc.4365.

    Article  CAS  PubMed  Google Scholar 

  11. Liu ML, Wang JL, Wei J, Xu LL, Yu M, Liu XM, Ruan WL, Chen JX. Tri-ortho-cresyl phosphate induces autophagy of rat spermatogonial stem cells. Reproduction. 2015;149(2):163–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1530/REP-14-0446.

    Article  CAS  PubMed  Google Scholar 

  12. Liu X, Xu L, Shen J, Wang J, Ruan W, Yu M, Chen J. Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro. Reprod Biol Endocrinol. 2016;14(1):30. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12958-016-0165-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hu L, Peng T, Huang J, Su T, Luo R, Zheng Y, Zhong Z, Yu P, Nie K, Zheng L. Tri-ortho-cresyl phosphate (TOCP) induced ovarian failure in mice is related to the Hippo signaling pathway disruption. Reprod Toxicol. 2019;83:21–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.reprotox.2018.10.007.

    Article  CAS  PubMed  Google Scholar 

  14. Wang J, Ruan W, Huang B, Shao S, Yang D, Liu M, Zeng L, Wei J, Chen J. Tri-ortho-cresyl phosphate induces autophagy of mouse ovarian granulosa cells. Reproduction. 2019;158(1):61–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1530/REP-18-0456.

    Article  CAS  PubMed  Google Scholar 

  15. Meng F, Zhang Y, Du J, Li N, Qiao X, Yao Y, Zhao T, Wu D, Peng F, Wang D, Yang S, Shi J, Liu R, Zhou W, Hao A. Nicotinamide mononucleotide maintains cytoskeletal stability and fortifies mitochondrial function to mitigate oocyte damage induced by Triocresyl phosphate. Ecotoxicol Environ Saf. 2024;15(275): 116264. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ecoenv.2024.116264.

    Article  CAS  Google Scholar 

  16. Zeng L, Zhou C, Xu W, Huang Y, Wang W, Ma Z, Huang J, Li J, Hu L, Xue Y, Luo T, Zheng L. The ovarian-related effects of polystyrene nanoplastics on human ovarian granulosa cells and female mice. Ecotoxicol Environ Saf. 2023;257: 114941. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ecoenv.2023.114941.

    Article  CAS  PubMed  Google Scholar 

  17. Lin JY, Ke YM, Lai JS, Ho TF. Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells. Phytomedicine. 2015;22(10):929–38. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.phymed.2015.06.012.

    Article  CAS  PubMed  Google Scholar 

  18. Jin Z, Chenghao Y, Cheng P. Anticancer Effect of Tanshinones on Female Breast Cancer and Gynecological Cancer. Front Pharmacol. 2022;12: 824531. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fphar.2021.824531.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang R, Liu Y, You J, Ge B. Tanshinone IIA inhibits ischemia-reperfusion-induced inflammation, ferroptosis and apoptosis through activation of the PI3K/Akt/mTOR pathway. Hum Exp Toxicol. 2023;42:9603271231180864. https://doiorg.publicaciones.saludcastillayleon.es/10.1177/09603271231180864.

    Article  CAS  PubMed  Google Scholar 

  20. Xu L, He D, Wu Y, Shen L, Wang Y, Xu Y. Tanshinone IIA inhibits cardiomyocyte apoptosis and rescues cardiac function during doxorubicin-induced cardiotoxicity by activating the DAXX/MEK/ERK1/2 pathway. Phytomedicine. 2022;107: 154471. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.phymed.2022.154471.

    Article  CAS  PubMed  Google Scholar 

  21. Yang Y, Wang B, Jiang Y, Fu W. Tanshinone IIA mitigates postoperative cognitive dysfunction in aged rats by inhibiting hippocampal inflammation and ferroptosis: Role of Nrf2/SLC7A11/GPX4 axis activation. Neurotoxicology. 2025;107:62–73. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neuro.2025.02.003.

    Article  CAS  PubMed  Google Scholar 

  22. Li Y, Deng X, Zhuang W, Li Y, Xue H, Lv X, Zhu S. Tanshinone IIA down-regulates -transforming growth factor beta 1 to relieve renal tubular epithelial cell inflammation and pyroptosis caused by high glucose. Bioengineered. 2022;13(5):12224–36. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/21655979.2022.2074619.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang G, Ma L, Wang B, Gao F, Li J, Cai H, Wang J, Zhang T, Guo H, Xie P, Li Y. Tanshinone IIA Accomplished Protection against Radiation-Induced Cardiomyocyte Injury by Regulating the p38/p53 Pathway. Mediators Inflamm. 2022;2022:1478181. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2022/1478181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bai L, He G, Gao C, Yang H, Li M, Huang Y, Moussa M, Xu C. Tanshinone IIA enhances the ovarian reserve and attenuates ovarian oxidative stress in aged mice. Vet Med Sci. 2022;8(4):1617–25. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/vms3.811.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Huang J, Zeng F, Xu Q, Ma J. Cryptotanshinone decreases granulosa cell apoptosis and restores ovarian function in mice with premature ovarian failure. Gen Physiol Biophys. 2020;39(3):277–83. https://doiorg.publicaciones.saludcastillayleon.es/10.4149/gpb_2019059.

    Article  CAS  PubMed  Google Scholar 

  26. Gershon E, Dekel N. Newly Identified Regulators of Ovarian Folliculogenesis and Ovulation. Int J Mol Sci. 2020;21(12):4565. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms21124565.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Qin J, Shi H, Xu Y, Zhao F, Wang Q. Tanshinone IIA inhibits cervix carcinoma stem cells migration and invasion via inhibiting YAP transcriptional activity. Biomed Pharmacother. 2018;105:758–65. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biopha.2018.06.028.

    Article  CAS  PubMed  Google Scholar 

  28. Xin X, Zeng T, Dou DD, Zhao S, Du JY, Pei JJ, Xie KQ, Zhao XL. Changes of mitochondrial ultrastructures and function in central nervous tissue of hens treated with tri-ortho-cresyl phosphate (TOCP). Hum Exp Toxicol. 2011;30(8):1062–72. https://doiorg.publicaciones.saludcastillayleon.es/10.1177/0960327110386815.

    Article  CAS  PubMed  Google Scholar 

  29. Carlton BD, Basaran AH, Mezza LE, Smith MK. Examination of the reproductive effects of tricresyl phosphate administered to Long-Evans rats. Toxicology. 1987;46(3):321–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0300-483x(87)90212-5.

    Article  CAS  PubMed  Google Scholar 

  30. Faire M, Skillern A, Arora R, Nguyen DH, Wang J, Chamberlain C, German MS, Fung JC, Laird DJ. Follicle dynamics and global organization in the intact mouse ovary. Dev Biol. 2015;403(1):69–79. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ydbio.2015.04.006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Read DJ, Langford L, Barbour HR, Forshaw PJ, Glynn P. Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells. Toxicol Appl Pharmacol. 2007;219(2–3):190–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.taap.2006.08.002.

    Article  CAS  PubMed  Google Scholar 

  32. Xu LL, Long CY, Wang JL, Yu M, Chen JX. Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced liver injury in male mice. Hum Exp Toxicol. 2016;35(10):1093–101. https://doiorg.publicaciones.saludcastillayleon.es/10.1177/0960327115621363.

    Article  CAS  PubMed  Google Scholar 

  33. Chen JX, Xu LL, Mei JH, Yu XB, Kuang HB, Liu HY, Wu YJ, Wang JL. Involvement of neuropathy target esterase in tri-ortho-cresyl phosphate-induced testicular spermatogenesis failure and growth inhibition of spermatogonial stem cells in mice. Toxicol Lett. 2012;211(1):54–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.toxlet.2012.03.004.

    Article  CAS  PubMed  Google Scholar 

  34. Yang X, Zhang Y, Wu X, Bae CS, Hou L, Kuang H, Wang Y, Stener-Victorin E. Cryptotanshinone reverses reproductive and metabolic disturbances in prenatally androgenized rats via regulation of ovarian signaling mechanisms and androgen synthesis. Am J Physiol Regul Integr Comp Physiol. 2011;300(4):R869–75. https://doiorg.publicaciones.saludcastillayleon.es/10.1152/ajpregu.00334.2010.

    Article  CAS  PubMed  Google Scholar 

  35. Roth A, Zhao P, Soukup ST, Guigas C, Stärke J, Kulling SE, Diel P. Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems. Toxicol Lett. 2023;15(375):21–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.toxlet.2022.12.002.

    Article  CAS  Google Scholar 

  36. Fang J, Little PJ, Xu S. Atheroprotective Effects and Molecular Targets of Tanshinones Derived From Herbal Medicine Danshen. Med Res Rev. 2018;38(1):201–28. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/med.21438.

    Article  CAS  PubMed  Google Scholar 

  37. Zhu X, Shan Y, Yu M, Shi J, Tang L, Cao H, Sheng M. Tetramethylpyrazine Ameliorates Peritoneal Angiogenesis by Regulating VEGF/Hippo/YAP Signaling. Front Pharmacol. 2021;12: 649581. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fphar.2021.649581.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ding B, Lin C, Liu Q, He Y, Ruganzu JB, Jin H, Peng X, Ji S, Ma Y, Yang W. Tanshinone IIA attenuates neuroinflammation via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro. J Neuroinflammation. 2020;17(1):302. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12974-020-01981-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ye X, Lin Y, Ying Y, Shen X, Ni F, Wang F, Chen J, Zhao W, Yu X, Zhang D, Liu Y. Human Amniotic Epithelial Stem Cells Alleviate Autoimmune Premature Ovarian Insufficiency in Mice by Targeting Granulosa Cells via AKT/ERK Pathways. Stem Cell Rev Rep. 2024 Jun 4. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12015-024-10745-z. Epub ahead of print.

  40. Song A, Zhang S, Zhao X, Wu S, Qi X, Gao S, Qi J, Li P, Tan J. Exosomes derived from menstrual blood stromal cells ameliorated premature ovarian insufficiency and granulosa cell apoptosis by regulating SMAD3/AKT/MDM2/P53 pathway via delivery of thrombospondin-1. Biomed Pharmacother. 2023;166: 115319. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biopha.2023.115319.

    Article  CAS  PubMed  Google Scholar 

  41. Nagashima T, Kim J, Li Q, Lydon JP, DeMayo FJ, Lyons KM, Matzuk MM. Connective tissue growth factor is required for normal follicle development and ovulation. Mol Endocrinol. 2011;25(10):1740–59. https://doiorg.publicaciones.saludcastillayleon.es/10.1210/me.2011-1045.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Chi MC, Lin ZC, Lee CW, Huang CC, Peng KT, Lin CM, Lee HC, Fang ML, Chiang YC. Tanshinone IIA suppresses burning incense-induced oxidative stress and inflammatory pathways in astrocytes. Ecotoxicol Environ Saf. 2023;258: 114987. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ecoenv.2023.114987.

    Article  CAS  PubMed  Google Scholar 

  43. Dai JP, Zhu DX, Sheng JT, Chen XX, Li WZ, Wang GF, Li KS, Su Y. Inhibition of Tanshinone IIA, salvianolic acid A and salvianolic acid B on Areca nut extract-induced oral submucous fibrosis in vitro. Molecules. 2015;20(4):6794–807. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/molecules20046794.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang W, Han W, Qin A, Wang Z, Xu J, Qian Y. The emerging role of Hippo signaling pathway in regulating osteoclast formation. J Cell Physiol. 2018;233(6):4606–17. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jcp.26372.

    Article  CAS  PubMed  Google Scholar 

  45. Wu Z, Zhou C, Yuan Q, Zhang D, Xie J, Zou S. CTGF facilitates cell-cell communication in chondrocytes via PI3K/Akt signalling pathway. Cell Prolif. 2021;54(3): e13001. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cpr.13001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu SC, Hsu CJ, Chen HT, Tsou HK, Chuang SM, Tang CH. CTGF increases IL-6 expression in human synovial fibroblasts through integrin-dependent signaling pathway. PLoS ONE. 2012;7(12): e51097. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0051097.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Feng J, Liu L, Yao F, Zhou D, He Y, Wang J. The protective effect of tanshinone IIa on endothelial cells: a generalist among clinical therapeutics. Expert Rev Clin Pharmacol. 2021;14(2):239–48. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/17512433.2021.1878877.

    Article  CAS  PubMed  Google Scholar 

  48. Ma X, Zhang L, Gao F, Jia W, Li C. Salvia miltiorrhiza and Tanshinone IIA reduce endothelial inflammation and atherosclerotic plaque formation through inhibiting COX-2. Biomed Pharmacother. 2023;167: 115501. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biopha.2023.115501.

    Article  CAS  PubMed  Google Scholar 

  49. Wen J, Chang Y, Huo S, Li W, Huang H, Gao Y, Lin H, Zhang J, Zhang Y, Zuo Y, Cao X, Zhong F. Tanshinone IIA attenuates atherosclerosis via inhibiting NLRP3 inflammasome activation. Aging (Albany NY). 2020;13(1):910–32. https://doiorg.publicaciones.saludcastillayleon.es/10.18632/aging.202202.

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank Home for Researchers editorial team (www.home-for-researchers.com) for language editing service.3

Clinical trial number

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers [82160288] and [82160284]); the Natural Science Foundation of Jiangxi Province (grant number [20212BAB216044]); the Research Project of Traditional Chinese Medicine in Jiangxi Province (grant number [2021B722] and [2021Z019]).

Author information

Author notes

  1. Zhangqiang Ma and Na Hu contributed equally to this work.

Authors and Affiliations

  1. Jiangxi Province Key Laboratory of Immunology and Inflammation, Jiangxi Provincial Clinical Research Center for Laboratory Medicine, Department of Clinical Laboratory, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, No. 566 Xuefu Road, Honggutan District, Nanchang, Jiangxi, 330036, China

    Jianlin Yu & Liaoliao Hu

  2. School of Public Health, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China

    Zhangqiang Ma, Na Hu, Liping Zheng, Wencan Wang, Xiu Cheng, Jianlin Yu & Liaoliao Hu

  3. School of Basic Medical Sciences, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China

    Liping Zheng, Yue Xue & Chong Zhou

  4. Institute of Biomedical Innovation, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China

    Tao Luo

  5. Jiangxi Provincial Key Laboratory of Disease Prevention and Public Health, Nanchang University, Nanchang, Jiangxi, China

    Zhangqiang Ma, Na Hu, Liping Zheng, Yue Xue, Chong Zhou, Wencan Wang, Xiu Cheng & Tao Luo

Authors
  1. Zhangqiang Ma
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  2. Na Hu
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  3. Liping Zheng
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  4. Yue Xue
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  5. Chong Zhou
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  6. Wencan Wang
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  7. Xiu Cheng
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  8. Tao Luo
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  9. Jianlin Yu
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  10. Liaoliao Hu
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Contributions

Liaoliao Hu and Zhangqiang Ma wrote the main manuscript text. Zhangqiang Ma, Na Hu and Yue Xue analyzed the data. Liping Zheng and Tao Luo administrated the project. Chong Zhou and Wencan Wang prepared Figs. 1–3. Na Hu and Xiu Cheng prepared Figs. 4–7. Jianlin Yu prepared Fig. 8 and additional files. All authors reviewed the manuscript.

Corresponding author

Correspondence to Liaoliao Hu.

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Ethics approval and consent to participate

The Institutional Animal Care and Use Committee of Nanchang University (Protocol number: NCULAE- 20221031138) approved all experimental procedures and all animal experiments were conducted in accordance with the National Institute of Health and the National Research Council for the care and use of laboratory animals’ guidelines.

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The authors declare no competing interests.

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Ma, Z., Hu, N., Zheng, L. et al. Tanshinone IIA alleviates tri-ortho-cresyl phosphate-induced ovarian damage through Hippo signaling pathway activation in mice. J Ovarian Res 18, 85 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13048-025-01671-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13048-025-01671-w

Keywords

Journal of Ovarian Research

ISSN: 1757-2215

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