Synergistic RU486 and olaparib therapy enhances apoptosis in endometriosis by simultaneously targeting hormonal signalling and DNA repair

Background and Purpose Endometriosis is a chronic, hormone-dependent disorder characterized by ectopic implantation of endometrial tissue, often accompanied by pain and infertility. Although the progesterone receptor modulator RU486 is effective for pain relief, its impact on lesion regression is limited, possibly due to apoptosis resistance and overexpression of PARP1 in endometriotic cells. This study aimed to evaluate the synergistic therapeutic efficacy of combining RU486 with the PARP inhibitor olaparib, focusing on reactivating p53-dependent apoptosis in endometriotic lesions. Experimental Approach Primary ectopic endometrial stromal cells (EESCs) were used to assess the effects of RU486 and olaparib on cytotoxicity, apoptosis, DNA damage, mitochondrial membrane potential, and cell migration. Mechanistic investigations included flow cytometry, western blotting, RT-qPCR, immunofluorescence, and comet assays. A murine endometriosis model was established to evaluate in vivo lesion suppression, pain behaviour, hormone levels, and treatment safety following combination therapy. Key Results RU486 and olaparib exhibited strong synergy at a 1:4 ratio, significantly enhancing apoptosis in EESCs by coactivating p53 through distinct mechanisms—transcriptional up-regulation by RU486 and posttranslational stabilization by olaparib. This synergy intensified Bax/Bcl-2-mediated mitochondrial dysfunction, caspase-3 activation, and PARP1 cleavage. In vivo, the combination therapy achieved a 77.5% lesion weight regression, significantly exceeding monotherapies, while maintaining robust analgesic effects. Histological and molecular analyses confirmed DNA damage and enhanced apoptosis in lesions.

and Implications The combination medication enhanced apoptotic sensitivity in endometriosis by restoring p53 activity through targeting hormonal signalling and DNA repair pathways. This strategy offered strong potential for clinical translation. Graphical Abstract Abbreviations | | | |---|---| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | What is already known - RU486 relieves endometriosis-related pain clinically, but its ability to induce lesion apoptosis is limited. - PARP1 is highly expressed in endometriosis and impairs DNA damage responses. What does this study add - RU486 and olaparib synergistically restored p53 activity, enhancing apoptosis, targeting hormonal signalling and DNA repair. - The combination treatment achieved stronger lesion reduction and pain relief than either drug alone. What is the clinical significance - The combination therapy provided a potential new therapeutic option for clinical management of endometriosis. 1 INTRODUCTION Endometriosis is a chronic, hormone-dependent inflammatory disorder characterized by the ectopic implantation of endometrial-like glands and stroma that undergo cyclical activity outside the uterine cavity (Home & Missmer, 2022; Saunders & Horne, 2021; H. S. Taylor et al., 2021). It typically manifests as progressively worsening dysmenorrhoea, menorrhagia, and infertility, significantly impairing both physical and mental health (Nelsen et al., 2018). Globally, approximately 10% of women of reproductive age are affected by the disease (Home & Missmer, 2022; Saunders & Horne, 2021; H. S. Taylor et al., 2021), and its associated alterations in the peritoneal microenvironment, along with disruptions in fertilization or embryo development, contribute to infertility rates spanning 30%–50% (La Marca et al., 2025). Current clinical management primarily involves surgical intervention and pharmacological therapy; nevertheless, surgery often carries risks of damage to the myometrium and pelvic structures and is associated with high recurrence rates of up to 40%–50% (Chapron et al., 2019; Guo et al., 2009; Moses et al., 2021), limiting its suitability for women seeking to preserve fertility. In this context, pharmacological therapy, as a noninvasive approach, has emerged as a promising strategy for long-term disease management. Mifepristone (RU486) as a selective progesterone receptor modulator (SPRM) has demonstrated pronounced efficacy across a range of hormone-dependent disorders (Chang et al., 2021; L. Chen et al., 2025). Extensive clinical studies have shown that RU486 effectively alleviated pain symptoms in patients with endometriosis by antagonizing progesterone receptors (PRs). Clinically, although RU486 provides substantial pain relief, its efficacy in reducing lesion size is limited, and symptoms frequently recur following discontinuation of the drug (Li et al., 2018; Song et al., 2018; Zheng et al., 2023). Our recent studies showed that RU486, whether administered as a free drug or in a nanotechnology-based formulation, achieved lesion inhibition rates of only 25% and 37.84%, respectively, highlighting the restricted therapeutic efficacy of RU486 monotherapy (Yan et al., 2025; M. Zhang, Ye, et al., 2024). In the search for more effective treatment strategies (Moses et al., 2021; Volpini et al., 2023), numerous studies have investigated the biological characteristics of endometriosis. Notably, its tumour-like behaviour characterized by resistance to apoptosis and overexpression of PARP1 leads to an aberrant DNA damage response that closely mirrors the molecular features of ovarian cancer (Barreta et al., 2019; Belmonte et al., 2024; Gupta et al., 2025). Regarding apoptosis resistance, many studies have demonstrated that dysregulation of p53-mediated apoptotic pathways may drive the progression of endometriosis (Allavena et al., 2015; Arimoto et al., 2003; Kobayashi et al., 2024). Gene expression profiling conducted by Arimoto et al. identified a significant down regulation of p53 and other apoptosis-related genes in ovarian endometrioma tissues, suggesting a reduction in apoptotic activity (Arimoto et al., 2003). This mechanistic insight was further supported by the findings of Azam et al., who reported dual phenotypic alterations characterized by reduced apoptosis and simultaneous activation of anti-apoptotic pathways (Azam et al., 2022). The limited therapeutic efficacy of RU486 at lesion sites may be attributed to insufficient activation of the p53-mediated apoptotic pathway. The second characteristic of endometriosis—overexpression of PARP1, provides us with another potential therapeutic target. PARP1 is a DNA repair enzyme that plays a key role in the base excision repair pathway by detecting DNA strand breaks and facilitating repair through poly ADP-ribosylation of target proteins. PARP inhibitors block this activity, leading to the accumulation of DNA damage and cell death, particularly in cells deficient in homologous recombination repair. Clinically, PARP inhibitors are primarily used in the treatment of BRCA (Breast Cancer Susceptibility Gene)-mutated ovarian, breast, prostate, and pancreatic cancers. Notably, PARP inhibitor-induced single-strand DNA breaks (SSBs) can be converted into double-strand breaks (DSBs), amplifying the DNA damage response and activating the p53 pathway (Lu et al., 2024; M. J. Taylor et al., 2022), which is expected to potentiate the proapoptotic effects of RU486. Activation of p53 induces mitochondrial release of caspase-3, thereby creating a positive feedback loop that further enhances the therapeutic efficacy of PARP inhibitors. For example, studies on PARP1-p53 coregulation have demonstrated that an intact p53 signalling pathway enhanced PARP inhibitor-induced apoptosis (Zaza et al., 2024). Based on these distinct yet potentially complementary mechanisms of action, we proposed that the combination of PARPi with RU486 might produce synergistic effects in the treatment of endometriosis. Based on this hypothesis, this study was designed to assess the therapeutic effect of combining RU486 and olaparib in endometriosis and explore their potential synergistic mechanism. The primary ectopic endometrial stromal cells (EESCs) were used to assess the effects of treatment on key pathological processes, including cytotoxicity, cell migration, apoptosis, mitochondrial dysfunction, cell cycle arrest, and DNA damage. Specifically, a comprehensive analysis of apoptotic signalling was conducted to elucidate the mechanism by which RU486 and olaparib synergistically enhance apoptosis. In parallel, a murine model of endometriosis was employed in vivo to assess lesion suppression, histopathological changes, and treatment safety. To date, the combination of RU486 and olaparib for endometriosis treatment has been rarely reported, highlighting the novelty of this therapeutic strategy and its potential for clinical translation. 2 METHODS 2.1 Materials RU486, olaparib, and dimethyl sulfoxide were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Hydroxypropyl-β-cyclodextrin was obtained from Qingdao Youshunfa Biotechnology Co., Ltd. (Qingdao, China). β-estradiol was acquired from MilliporeSigma (Darmstadt, Germany). Sterile phosphate-buffered saline (PBS), SDS-PAGE running buffer powder, and RT-qPCR primers for caspase-3, Bax, and p53 were obtained from Servicebio Technology Co., Ltd. (Wuhan, China). DMEM/F-12 (Dulbecco's Modified Eagle Medium/F12 Nutrient Mixture) cell culture medium, fetal bovine serum (FBS), Collagenase Type I, and MitoProbe™ JC-1 Assay Kit were purchased from Thermo Fisher Scientific Inc. (Eugene, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Melonepharma Biotechnology Co., Ltd. (Dalian, China). PARP1 monoclonal antibody (Proteintech Cat# CL650-66520, RRID:AB_2920479), Cleaved PARP (Abcam Cat# ab32064, RRID:AB_777102), cleaved caspase-3 monoclonal antibody (Proteintech Cat# 68773-1-Ig, RRID:AB_3665444), γ-H2AX polyclonal antibody (Proteintech Cat# 10856-1-AP, RRID:AB_2114985), GAPDH monoclonal antibody (Proteintech Cat# 60004-1-Ig, RRID:AB_2107436), prestained protein marker, Bax monoclonal antibody (Proteintech Cat# CL594-60267, RRID:AB_2919907), Bcl-2 monoclonal antibody (Proteintech Cat# 68103-1-Ig, RRID:AB_2923635), and cocktail protease inhibitor were obtained from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, China). The p53 monoclonal antibody (Cell Signaling Technology Cat# 2524, RRID:AB_331743) was obtained from Cell Signaling Technology, Inc. (Danvers, USA). RIPA lysis buffer, BCA microplate protein assay kit, phosphatase inhibitor cocktail, 0.05% trypsin, Cell Cycle and Apoptosis Analysis Kit, Annexin V-FITC Apoptosis Detection Kit, Comet Assay Kit, and DNA Damage Assay Kit (γ-H2AX immunofluorescence) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Ultra-sensitive chemiluminescence (ECL) reagent and SDS-PAGE gels were obtained from Yeasen Biotechnology Co., Ltd. (Shanghai, China). The rapid transfer membrane solution and antibody dilution buffer were obtained from New Cell & Molecular Biotech Co., Ltd. (Suzhou, China). Total RNA extraction reagent was purchased from Biosharp Biotechnology Co., Ltd. (Beijing, China). 2.2 Cells and animals The enzymatic isolation of EESCs from surgically excised ectopic lesions was performed according to our previously established methods (Yan et al., 2025). EESCs were enzymatically isolated from surgically excised ectopic lesions obtained from patients with endometriosis. All specimens were acquired following informed consent, and the study protocol received ethical approval from the Ethics Committee of the Women's Hospital, Zhejiang University School of Medicine (Approval No. IRB-20240104-R). Ectopic endometrial tissues were rinsed with PBS to remove blood and debris. The cyst wall surface was carefully scraped under sterile conditions using a scalpel, and the obtained tissue was minced into ~1 mm3 pieces. These fragments were then digested in 3 ml of 0.1% collagenase I solution at 37°C with gentle shaking for 50 min. Following enzymatic digestion, the resulting cell suspension was centrifuged at 100 × g for 5 min to collect the cells. The pellet was resuspended in DMEM/F-12 complete medium supplemented with 12% FBS and seeded into culture flasks. Incubation was carried out at 37°C in a 5% CO2-supplemented humidified atmosphere. Following the initial 24 h period, the culture medium was changed to remove floating cells and tissue debris. Ongoing observations of cell morphology, adhesion characteristics, and proliferation were conducted using an inverted microscope, with media refreshed at regular intervals. Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). Female BALB/c mice (aged 3–5 weeks) were obtained from Shanghai BK Laboratory Animal Co., Ltd. and housed under standard conditions in the animal facility of Zhejiang Chinese Medical University. All experimental protocols involving animals were reviewed and approved by the Laboratory Animal Ethics Committee of Zhejiang Chinese Medical University (IACUC Approval No. 20240506-02). Mice were housed at a 12:12 h light/dark cycle, a temperature of 22–24°C and a relative humidity of 55%–60%. Standard conditions were maintained, allowing the mice free access to both food and water. The mice were randomly divided into four groups (11 mice per group), different groups were allocated in a randomized manner, and investigators were blinded to the allocation of different groups when carrying out surgeries and outcome evaluations. After the experiment, all animals were humanely killed using CO2. To confirm death, permanent cessation of circulation was verified. 2.3 Cytotoxicity and combination index (CI) Cytotoxicity was assessed using a cell viability assay, as described by previous studies (Shao et al., 2024; M. Zhang, Ye, et al., 2024). EESCs were plated in 96-well plates at a density of 5000 cells per well. To identify the most effective combinatorial ratio of RU486 and olaparib, a series of concentration-dependent experiments were conducted, and then data were analysed using Synergy Finder 2.0 by employing Zero Interaction Potency (ZIP) models to assess potential synergistic, additive, or antagonistic effects between RU486 and olaparib. To investigate the CI under the optimal drug ratio, the experiment was designed to include blank, control, and treatment groups. A range of RU486 (0–25 μg·ml−1) and olaparib (0–100 μg·ml−1) concentrations were applied to the treatment groups, with each dose tested in six parallel replicates. Following 24 h of drug exposure, the medium was discarded and 100 μl fresh medium containing 10% CCK-8 reagent was added to each well. After a 2 h incubation period, absorbance was recorded using a microplate reader (Thermo Fisher Scientific, Waltham, USA) to assess cell viability. The half-maximal inhibitory concentration (IC50) values for each treatment were subsequently determined, and the CI values were analysed using CompuSyn software. 2.4 Transwell migration assay The Transwell migration assay was performed as previously described (Cai et al., 2024; Justus et al., 2023). Transwell chambers with 8 μm pores were inserted into 24-well plates. The lower compartment was filled with 1.2 ml of complete culture medium containing 10% FBS to serve as a chemoattractant. In the upper compartment, 300 μl of a cell suspension (3 × 104 cells) was carefully added, cells were either untreated or pretreated with RU486 (15 μg·ml−1), olaparib (60 μg·ml−1), or their combination (RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1) prior to seeding. After 24 h of incubation, cells were fixed with 4% paraformaldehyde, rinsed with PBS, and subsequently stained with 0.1% crystal violet. The migrated cells were visualized under a DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany) and quantified in ImageJ software by an investigator blinded to group allocation. 2.5 Scratch assay The wound-healing assay was performed as described in previous reports (X. X. Chen et al., 2021; W. Zhang, Zhang, et al., 2024). To assess the migration capacity of EESCs, a wound-healing assay was performed. Cells were plated in six-well plates and grown to approximately 90% confluence. A uniform linear scratch was made through the monolayer using a sterile 1 ml pipette tip, subsequently, each well was supplemented with drug-free or drug-containing culture medium (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or the combination of RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1) containing 1% FBS to suppress cell proliferation while maintaining cell viability. At 0, 6, 12, and 24 h post-scratch, the wound sites were imaged using a phase-contrast microscope (Olympus, Tokyo, Japan). The closure of the scratch was quantitatively assessed in ImageJ software by an investigator blinded to group allocation. 2.6 Apoptosis assay The apoptosis assay was conducted as previously described (Qiao et al., 2024; Wang et al., 2024). EESCs were seeded into a 12-well plate with a density of 1 × 105 per well. After overnight incubation, the cells were assigned to four groups: control, RU486 (15 μg·ml−1), olaparib (60 μg·ml−1), and their combination (RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1), with six replicates per group. Then cells were harvested 24 h after drug administration and washed with cold PBS. Apoptotic events were detected using Annexin V-FITC/PI double staining as per the manufacturer's guidelines. Quantitative analysis was conducted via FACSVerse™ flow cytometry (BD Biosciences, San Jose, USA) by an operator blinded to group allocation, with apoptotic subpopulations categorized accordingly. 2.7 Detection of mitochondrial membrane potential The JC-1 assay for mitochondrial membrane potential was performed following the protocol described in previous studies (Peng et al., 2025). To evaluate drug-induced alterations in mitochondrial membrane potential (MMP), EESCs were divided into four groups: control (untreated), RU486 (15 μg·ml−1), olaparib (60 μg·ml−1), and their combination (RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1). Cells were treated for 24 h and the JC-1 assay was conducted with both confocal microscopy and flow cytometry. For confocal imaging, the JC-1 staining solution was prepared as per the manufacturer's protocol, and 5 μl was added to each confocal culture dish. After a 30-min incubation, mitochondrial fluorescence was imaged using an FV1200 confocal laser scanning microscope (Olympus, Japan), with red JC-1 aggregates indicating high membrane potential and green monomers indicating depolarized mitochondria. For flow cytometric analysis, 2 × 105 cells were seeded per well in six-well plates. After 24 h of drug treatment as described above, cells were harvested, resuspended in a JC-1 working solution, and incubated for 30 min in a humidified incubator. Subsequently, cells were rinsed with PBS and subjected to analysis using the NovoCyte flow cytometer (Agilent Technologies, Santa Clara, USA) by an operator blinded to group allocation. JC-1 monomers and aggregates were detected by recording fluorescence signals through the FITC (Fluorescein Isothiocyanate) and EYFP (Enhanced Yellow Fluorescent Protein) channels, respectively. 2.8 Immunofluorescence detection of DNA double-strand breaks (DSBs) via γ-H2AX staining The detection of DNA DSBs was performed as described in previous studies (Q. J. Zhang et al., 2023). To detect the DSBs of DNA following drug exposure (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or their combination of RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1), γ-H2AX was visualized using immunofluorescence staining with a standard detection kit. Cells were cultured in confocal imaging dishes at a density of 1 × 105 per dish and treated for 24 h. Following treatment, the culture medium was discarded, and the cells were gently rinsed once with PBS before being fixed at room temperature for around 15 min. Non-specific binding was blocked with a blocking buffer for 10–20 min, after which cells were incubated with a γ-H2AX primary antibody for 1 h at room temperature. Following three washes with PBS, the cells were incubated for 1 h with a secondary antibody conjugated to a fluorescent dye. Subsequently, DAPI was used to counterstain the nuclei for 5 min, and fluorescence imaging was performed on an FV1200 confocal laser scanning microscope under blinded conditions to evaluate DNA damage. 2.9 Comet assay The comet assay was conducted following the method described by previous studies (Collins et al., 2023). To gain additional insight into DNA damage, comet detection was conducted utilizing a commercially available comet assay kit. Following treatment (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or their combination of RU486 at 15 μg·ml−1 and olaparib at 60 μg·ml−1), with untreated as control, cells were collected and prepared as a single-cell suspension at a density of 1 × 104–1 × 106 cells·ml−1. For slide preparation, cells were encapsulated within a triple-layer agarose matrix comprising a base layer of 1% normal melting point agarose, a central layer containing 0.7% low melting point agarose mixed with the cell suspension, and a protective top layer of 0.7% low melting point agarose. Once solidified, the slides were immersed in lysis buffer and incubated at 4°C for 2 h. Following PBS washing, the slides were incubated in alkaline electrophoresis buffer at room temperature for 30 min to allow DNA unwinding, and electrophoresis was then performed at 25 V and 300 mA for 30 min. The slides were subsequently neutralized with Tris–HCl buffer and stained with ethidium bromide in the dark for 20 min. DNA damage was assessed by capturing and analysing comet images with a DMi8 inverted fluorescence microscope. 2.10 Cell cycle assay The cell cycle assay was performed following the procedure described in previous studies (Huang et al., 2023; Ramos et al., 2023). For cell cycle profiling, 2 × 105 cells were seeded into six-well plates and cultured until reaching approximately 80% confluency. Following 24 h of drug exposure (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or their combination of RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1), cells were harvested by trypsinization and centrifugation, then fixed with prechilled 70% ethanol at 4°C for 2 h to permeabilize cellular membranes and preserve nuclear DNA. After fixation, ethanol was removed by centrifugation, and cells were rinsed once with PBS. The resulting pellet was then resuspended in a propidium iodide (PI) staining solution containing RNase A and incubated at 37°C for 30 min in the dark. DNA content was measured using a NovoCyte flow cytometer, and cell cycle profiles were analysed with NovoExpress software (Agilent Technologies, Santa Clara, USA) by a blinded assessor. 2.11 Western blot analysis Western blotting was performed as described in previous studies (Cao et al., 2021). After 24 h of drug treatment (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or their combination of RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1), cellular proteins were isolated using lysis buffer, followed by incubation on ice and centrifugation to remove debris. The protein content was quantified using the BCA assay. Protein samples (20 μg per lane) were combined with SDS sample buffer, heated at 100°C for 10 min to denature, and subsequently resolved by SDS–PAGE. Gel concentrations were selected based on the molecular weights of the target proteins: 7.5% gels for PARP1 and cleaved PARP1, and 12% gels for cleaved caspase-3, γ-H2AX, p53, Bax, and Bcl-2. GAPDH was selected as the loading control. Proteins were transferred onto preactivated PVDF (Polyvinylidene Difluoride membrane) membranes and blocked using a rapid blocking buffer for 5 min at ambient temperature. All primary antibodies were diluted 1:1000, except for GAPDH, which was diluted 1:5000. The anti-mouse (Proteintech Cat# SA00001-1, RRID:AB_2722565) and anti-rabbit (Proteintech Cat# SA00001-2, RRID:AB_2722564) secondary antibody (Sanying Biotechnology, Wuhan, China) were diluted 1:5000. All diluted antibodies were stored at 4°C and discarded after three uses. The membranes were incubated with primary antibodies overnight at 4°C, and subsequently exposed to HRP (Horseradish Peroxidase)-linked secondary antibodies for 1 h at room temperature. After washing, the membranes were incubated using an ECL reagent. Protein levels were semiquantified using ImageJ in a blinded manner to avoid bias and the immuno-related procedures comply with the recommendations of the British Journal of Pharmacology (Alexander et al., 2018). 2.12 Real-time quantitative reverse transcription PCR analysis RT-qPCR analysis was performed following the previous studies (W. C. Wei et al., 2025). RT-qPCR analysis was employed to measure mRNA levels of key apoptotic markers (caspase-3, Bax, and p53) in response to drug treatment. After 24 h of drug treatment (RU486 at 15 μg·ml−1, olaparib at 60 μg·ml−1, or their combination of RU486 at 15 μg·ml−1 + olaparib at 60 μg·ml−1), total RNA was extracted from cultured cells using a conventional isolation procedure and subsequently reverse transcribed into cDNA following the manufacturer's guidelines. RT-qPCR was conducted using SYBR Green dye on a CFX Connect™ detection system (Bio-Rad, Hercules, USA), applying the following thermal profile: initial denaturation at 95°C for 30 s, followed by 40 amplification cycles of 95°C for 15 s and 60°C for 30 s. All RT-qPCR reactions were conducted in triplicate. Relative mRNA expression was calculated by the 2−ΔΔCt method, with both data processing and quality control conducted by a blinded assessor. Primer information is available in Table S1. 2.13 Construction of the endometriosis mouse model The establishment of the endometriosis mouse model was conducted using methods previously described (N. Wu et al., 2025; M. Zhang, Ye, et al., 2024). Female BALB/c mice were used to establish the endometriosis model. Prior to model construction, each mouse was subcutaneously injected with three doses of β-estradiol (150 μg·kg−1 per 3 days) to synchronize the oestrous cycle. Donor mice were randomly selected and humanely euthanized using CO2 inhalation, permanent cessation of circulation was verified, then their uteri were collected and cut into approximately 1 mm3 segments. One hour before the surgery, recipient mice were orally administered Meloxicam at a dose of 5 mg·kg−1, as recommended in previous studies for analgesia (Furumoto et al., 2021; Oh & Narver, 2024). Following this, anaesthesia was induced with 3% isoflurane and maintained with 2% isoflurane. Under sterile conditions, the abdominal cavity was opened. Four uterine segments were implanted into the peritoneal cavity of each recipient mice, where the fragments were allowed to freely disperse without suturing. This approach facilitated random deposition, adhesion, and vascularization of the ectopic endometrial tissue, providing a more accurate simulation of the disease pathology. Before closing the abdomen, penicillin was administered subcutaneously at a recommended dose of 90,000 IU per mouse to prevent postoperative infection (Ding et al., 2025; Shen et al., 2023). The inner abdominal wall was sutured using surgical needles, and the outer skin was closed with mouse-specific wound clips to complete the surgical procedure. Postoperatively, recipient mice were given Meloxicam (5 mg·kg−1 per 12 h) for a total of six doses to manage pain during the recovery period. Additionally, to maintain the estrogen-dependent growth of ectopic endometrial tissue, mice were subcutaneously injected three doses of β-estradiol (150 μg·kg−1 per 3 days). Twenty days after model establishment, ultrasound imaging was performed to confirm lesion formation, followed by subsequent pharmacodynamic evaluations. The modelling success rate was 100%. 2.14 In vivo treatment of endometriosis To assess the in vivo efficacy of RU486 and olaparib, endometriosis model mice received oral gavage administration of RU486 (10 mg·kg−1) and olaparib (40 mg·kg−1) every 3 days for a total of seven treatments. A comprehensive evaluation was performed using behavioural, biochemical, and histological indicators. To evaluate nociceptive sensitivity during drug administration, the hot plate test was conducted weekly. Mice were exposed to a thermal stimulus at 50°C, and the latency to pain response behaviours (including paw licking, lifting, or jumping) was documented. Each mouse was allowed a 3 min acclimatization period before testing. Changes in response latency were used to assess the analgesic effect of the treatment on endometriosis-associated pain sensitivity. At the end of treatment, ectopic endometrial lesions were collected from the peritoneal cavity. The number, volume, and weight of ectopic nodules were recorded to assess lesion regression. Serum estradiol and progesterone levels were measured via ELISA following blood collection, providing insight into systemic endocrine changes. To further assess cellular proliferation and DNA damage in ectopic lesions, immunohistochemical staining for Ki67 (ServiceBio Cat# GB121141, RRID:AB_3083641) and γ-H2AX was performed. In addition, TUNEL immunofluorescence analysis was performed to visualize apoptosis in ectopic tissue sections. Outcome data were collected and analysed by investigators unaware of group assignments. 2.15 In vivo biosafety evaluation To assess the systemic safety of RU486 and olaparib treatment, serum biochemical analysis and histopathological examination of major organs were performed at the end of the experiment. Blood samples were collected and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were measured to evaluate potential hepatotoxicity and nephrotoxicity. Representative organs (heart, liver, spleen, lungs, kidneys, and uterus) were excised and weighed to compute organ-to-body weight ratios (organ weight/body weight × 100%), then subjected to haematoxylin and eosin (H&E) staining for microscopic pathology evaluation. Pathological features such as inflammation, cellular degeneration, and tissue damage were examined in order to comprehensively assess the in vivo safety profile of the drug treatment. All safety assessments were conducted under blinded conditions, with coded specimen IDs and assessors/pathologists unaware of treatment allocation. 2.16 Statistical analysis Data and statistical analysis complied with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2025). Data were analysed using GraphPad Prism 10.1.2 and are presented as mean ± standard error of the mean. Statistical analysis was undertaken only for studies where each group size was at least n = 5. IC50 values were determined by nonlinear regression using the dose–response inhibition model. Western blot band intensities were quantified with ImageJ software. Drug combination effects were assessed using CompuSyn 1.2, and flow cytometry data were analysed with NovoExpress. The comparison of the test groups was determined by a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test for parametric data. Post-hoc tests were run only if F achieved P < 0.05, and there was no significant variance inhomogeneity. P values < 0.05 were considered significantly different. 2.17 Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander, Cidlowski, et al., 2023; Alexander, Fabbro, et al., 2023; Alexander, Kelly, et al., 2023). 3 RESULTS 3.1 In vitro cytotoxicity Given the clinical limitations of RU486 monotherapy in treating endometriosis, particularly its inadequate therapeutic impact on lesion regression, exploring effective combination strategies has become essential to improve therapeutic outcomes. Following enzymatic digestion and overnight culture of ectopic endometrial tissues, the EESCs were successfully isolated as shown in Figure S1. The cells exhibited typical spindle-shaped morphology and a swirling growth pattern under bright-field microscopy. According to the ZIP synergy model, a RU486:olaparib ratio of approximately 1:4 resulted in multiple concentration points consistently located within the synergistic region (Figure 1a), suggesting a robust synergistic interaction between the two drugs. Subsequently, the cytotoxicity of the drugs at the 1:4 ratio was shown in Figure 1b, the IC50 values of RU486 and olaparib were respectively 18.15 and 88.68 μg·ml−1. In the combination group, 1.41 μg·ml−1 of RU486 and 5.64 μg·ml−1 of olaparib could achieve the half inhibition effect, which highlighted a synergistic enhancement of cytotoxic efficacy upon coadministration. Figure 1c illustrates the CI analysis, with all of the values falling below 1, consistent with a strong synergistic effect between RU486 and olaparib. Co-administration with olaparib markedly enhanced cellular sensitivity to RU486, allowing comparable cytotoxic effects to be achieved at a lower drug dose. Similar findings had been reported in hormone-driven malignancies, where PARP inhibition was shown to increase responsiveness to hormonal therapies (de Bono et al., 2020). 3.2 Cell migration assay Endometriosis exhibited tumour-like behaviours in clinical settings, including local invasion, recurrence, and distant implantation. These aggressive features were largely attributed to the enhanced migratory capacity of EESCs (Hung et al., 2021; Wu et al., 2020). In clinical practice, the invasive and migratory nature of ectopic endometrial cells leads to widespread lesion distribution, often involving the ovaries, pelvic peritoneum, bowel, and even distant organs. This poses significant challenges for timely diagnosis and effective treatment. Given the confirmed synergistic inhibitory effect of RU486 and olaparib on cell proliferation, the subsequent study evaluated their impact on cellular motility through transwell migration and wound healing assays. As shown in Figure 1d,e, in the wound healing assay at 12 h, all treatment groups exhibited markedly reduced migration rates compared with the control group, indicating effective suppression of cell migration. However, no significant differences were observed among the RU486, olaparib, and combination groups. By 24 h, both RU486 (26.08 ± 5.86%) and olaparib (33.21 ± 5.48%) monotherapies significantly reduced the migration area compared with the control group (83.07 ± 4.94%). Notably, the combination treatment (14.45 ± 4.828%) resulted in the most substantial inhibition, with a significantly greater reduction than either monotherapy alone. These findings indicated a time-dependent synergistic effect of RU486 and olaparib on inhibiting cell migration. In the transwell migration assay, the number of migrated cells was substantially reduced following RU486 (152.5 ± 30.27) and olaparib (139.67 ± 24.91) treatment compared with the control group (Figure 1f,g). Notably, the combination treatment exhibited the lowest number of migrated cells (61.67 ± 16.10), indicating a markedly enhanced suppression of cell migration relative to either monotherapy. These findings demonstrated that both RU486 and olaparib possessed inhibitory effects on the invasion and migration of EESCs. Given that the combination of these two agents significantly reduced the proliferative activity of EESCs, their synergistic use exhibited a markedly enhanced inhibitory effect on invasion and migration compared with monotherapy. The subsequent study investigated the underlying mechanisms of this synergistic effect. 3.3 In vitro apoptosis study Flow cytometry analysis revealed that the apoptotic rates in each group were as follows: 2.95 ± 0.74% in the control group, 29.87 ± 2.75% in the RU486 group, 15.71 ± 2.15% in the olaparib group, and 48.855 ± 5.03% in the combination treatment group (Figure 2a,b). The low apoptosis rate observed in EESCs is consistent with previous studies, indicating that the apoptotic signalling in EESCs is inherently weak (Azam et al., 2022; Ni et al., 2022). Compared with the control, both RU486 and olaparib monotherapies considerably increased the proportion of apoptotic cells. Notably, the apoptotic rate in the combination group was significantly higher than that observed in either monotherapy group. These findings suggested that the combination of RU486 and olaparib significantly enhanced apoptotic responses in EESCs, further supporting the presence of a synergistic interaction at the cellular level. 3.4 In vitro mitochondrial membrane potential analysis In light of the vital role of mitochondrial dysfunction in intrinsic apoptosis, this study evaluated MMP using JC-1 staining through both flow cytometry and confocal imaging. With drug treatment, Flow cytometry results demonstrated a progressive increase in the proportion of JC-1 monomers, which serve as an early indicator of apoptosis (Figure 2c,d). The proportion rose from 9.42 ± 2.36% in the control group to 33.98 ± 2.65% with RU486 treatment and 24.60 ± 2.77% with olaparib treatment. The combination treatment further elevated the apoptosis proportion to 45.82 ± 4.87%. Compared with the control, both RU486 and olaparib greatly induced MMP loss. Moreover, the combination treatment led to a greater accumulation of JC-1 monomers than either monotherapy, suggesting its more pronounced mitochondrial dysfunction effect. In addition, confocal microscopy further confirmed these findings: drug-treated cells exhibited markedly decreased red fluorescence (JC-1 aggregates) and increased green fluorescence (JC-1 monomers), with the most prominent shift observed in the combination group (Figure 2e). This shift from JC-1 aggregates to monomers reflects a loss of MMP, a hallmark of early mitochondrial dysfunction. Under physiological conditions, healthy mitochondria maintain a high membrane potential, enabling JC-1 to form red fluorescent aggregates. Upon mitochondrial depolarization, JC-1 remains in its monomeric form, emitting green fluorescence. Thus, the observed increase in JC-1 monomers and decrease in aggregates demonstrated that the combination treatment markedly compromises mitochondrial integrity, potentially activating intrinsic apoptotic pathways. Given that mitochondrial dysfunction is increasingly recognized as a key contributor to energy imbalance and oxidative stress in endometriosis (Kobayashi et al., 2023), our findings suggested that olaparib enhances RU486-induced mitochondrial damage, exerting a synergistic proapoptotic effect. 3.5 In vitro DNA damage analysis In this study, the effects of RU486 and olaparib on DNA damage were assessed using comet assays and γ-H2AX immunofluorescence staining. Comet assay results showed that both RU486 and olaparib monotherapies significantly increased DNA tail length compared with the control group, indicating their ability to induce DNA strand breaks (Figure 3a,b). The DNA damage induced by olaparib is attributed to its inhibition of PARP activity, whereas RU486 may exert its effect through activation of the p53 pathway (Delenko et al., 2024; Duan et al., 2020; X. Ma et al., 2022). As shown in Figure 3b, olaparib induced slightly more pronounced DNA damage than RU486. The combination treatment group exhibited a further increase in tail length, with a highly pronounced difference compared with the control. Consistently, γ-H2AX immunofluorescence staining (Figure 3c) showed increased γ-H2AX signals in the RU486-treated group compared with the control group. Cells treated with olaparib exhibited even stronger γ-H2AX signals than those treated with RU486. Among all groups, the combination treatment induced the most intense and widespread γ-H2AX foci, indicating a significantly elevated level of DNA DSBs under cotreatment conditions. Through this study, we proposed that RU486 induced DNA damage and apoptosis in EESCs via mitochondrial dysfunction. Endometriotic cells were known to have impaired DNA repair capacity, rendering them more vulnerable to genomic instability (Chen et al., 2018). When combined with olaparib, the inhibition of PARP enzymatic activity further compromises DNA damage repair, thereby exacerbating DNA damage (Javle et al., 2021; Wilson et al., 2022) and ultimately leading to enhanced apoptosis induction and proliferation inhibition. 3.6 In vitro cell cycle analysis Given the close interplay between DNA damage and cell cycle regulation, particularly during DNA replication and checkpoint transitions, this study next examined whether RU486 and olaparib treatments alter the cell cycle distribution of EESCs. The results of cell cycle analysis are presented in Figure 3d,e. In the control group, the proportions of cells in the G1, S, and G2 phases were 62.56 ± 0.74%, 23.06 ± 0.78%, and 14.73 ± 0.54%, respectively. RU486 treatment significantly increased the proportion of cells in the G1 phase, suggesting G1/S checkpoint arrest as a potential antiproliferative mechanism. This finding is consistent with previous studies in ovarian cancer cells that share biological characteristics with endometriosis-like lesions where RU486 induced G1 phase arrest and impaired DNA synthesis (Goyeneche et al., 2005). In contrast, olaparib treatment markedly increased the proportions of cells in the S phase (32.91 ± 1.62%) and G2 phase (21.10 ± 1.47%), indicative of replication stress and activation of the DNA damage response. This effect is likely due to the trapping of PARP1 at sites of DNA damage, leading to replication fork destabilization and the accumulation of DNA DSBs (Simoneau et al., 2021). Notably, the combination treatment group showed a decreased S phase population compared with olaparib alone, along with partial restoration of G1 phase levels. These results suggested that RU486 primarily induced G1-phase arrest, while olaparib contributed to S-phase accumulation when used in combination. This complementary modulation of the cell cycle, by simultaneously impeding G1 entry and S-phase progression, may enhance antiproliferative efficacy through dual checkpoint interference. 3.7 Effects on expression of apoptosis-related proteins and genes To further investigate the mechanism underlying the synergistic effect of RU486 and olaparib on cellular damage and apoptotic signalling pathways, western blot and RT-qPCR analyses were conducted. The protein bands were shown in Figure 4a,b. As a selective progesterone receptor modulator (SPRM), RU486 alleviates PR and glucocorticoid receptor (GR)-mediated repression of the p53 pathway. Accordingly, p53 expression was markedly up-regulated at both the mRNA and protein levels, initiating a transcriptional cascade associated with apoptotic signalling (Figure 4c,d). Building on this p53 activation, downstream mitochondrial apoptotic regulators were also significantly altered. Bax protein and mRNA levels were increased (Figure 4e,f), while Bcl-2 protein was markedly reduced (Figure 4g), leading to a pronounced elevation in the Bax/Bcl-2 ratio (Figure 4h). An increased Bax/Bcl-2 ratio is widely recognized as a molecular hallmark of cells undergoing apoptosis (W. Zhang, Zhang, et al., 2024), and is frequently used as a key parameter to evaluate apoptotic activity in various disease models (Gitego et al., 2023; Han et al., 2024). This strong effect was consistent with the observations in the study of Zipponi et al., which highlighted the central role of the p53/Bax pathway in the regulation of apoptosis in EESCs and its potential as a therapeutic target (Zipponi et al., 2025). The accumulation of Bax is known to compromise mitochondrial membrane potential, thereby facilitating apoptotic progression. Consistently, RU486 robustly activated caspase-3, as evidenced by significantly increased levels of cleaved caspase-3 protein (Figure 4i) and mRNA (Figure 4j), indicating that apoptosis was initiated at both the transcriptional and functional levels. These findings suggest that RU486 induces apoptosis in EESCs via the PR/GR–p53–Bax–mitochondrial damage–caspase-3 signalling axis. However, it was noteworthy that the ability of RU486 to up-regulate Bax expression through p53 activation appeared to be limited, as no significant difference in Bax protein or mRNA levels was observed compared with the control group. Consequently, the downstream apoptotic cascade might not have been fully activated. We speculated that this limited induction of Bax may underlie the suboptimal efficacy of RU486 in controlling lesion progression. Western blot analysis showed a high level of PARP1 expression in EESCs, which is consistent with previous reports (Barreta et al., 2019; Belmonte et al., 2024; Gupta et al., 2025). This finding suggests that EESCs possess a robust DNA damage repair capacity, thereby providing a theoretical rationale for the application of PARP inhibitors in their treatment. Olaparib, a potent PARP inhibitor, blocks the repair of SSBs, promoting their conversion into DSBs during replication. This accumulation of unresolved DNA lesions was reflected by a notable increase in γ-H2AX protein levels. As displayed in Figure 4k, the γ-H2AX protein levels of EESCs increased significantly after olaparib treatment. Notably, this study observed a significant increase in p53 protein levels in EESCs following olaparib treatment, while its mRNA expression remained unchanged. It was speculated that this discrepancy is due to the ability of olaparib-induced DSBs to stabilize the p53 protein by reducing its degradation. DNA damage signalling may inhibit the interaction between p53 and MDM2, similar to the mechanism of action of MDM2 antagonists (Fallatah et al., 2023; Marcellino et al., 2023), or regulate other E3 ubiquitin ligases such as DCAF13 to decrease p53 ubiquitination and degradation (S. Wei et al., 2024). These mechanisms may explain the observed phenomenon. Further investigations demonstrated that olaparib-mediated stabilization of p53 increased the expression of the proapoptotic protein Bax (Figure 4e), reduced the expression of the anti-apoptotic protein Bcl-2 (Figure 4g), and significantly elevated the Bax/Bcl-2 ratio (Figure 4h). These findings suggested that olaparib could promote apoptosis by modulating the p53-dependent apoptotic signalling pathway. Importantly, this study seems to identify a potential mechanism underlying the enhanced efficacy of the combination therapy. Specifically, RU486 was found to promote the transcriptional activation of p53, while olaparib stabilized the p53 protein by preventing its degradation. In the combination treatment group, p53 expression was significantly higher at both the protein and mRNA levels compared with either monotherapy group. Moreover, the Bax/Bcl-2 ratio was also markedly elevated in the combination group relative to both single-drug groups. The increased Bax expression induced by the combination treatment facilitates its translocation from the nucleus to the cytoplasm, leading to mitochondrial dysfunction and subsequent activation of caspase-3. Activated caspase-3, in turn, mediates the proteolytic cleavage of PARP1, a hallmark event in the execution phase of apoptosis (Q. Chen et al., 2022; Mashimo et al., 2021). As shown in Figure 4l,m, total PARP1 expression was significantly lower in the combination group than in the other three groups, whereas cleaved PARP1 expression was significantly higher. Since PARP1 cleavage enhances the therapeutic efficacy of olaparib, these findings suggest that the combination therapy exerts a more potent proapoptotic effect than either agent alone through multiple converging mechanisms. In general, this part of the study clarifies the synergistic mechanism of the combination of two drugs (Figure 4n). Briefly, RU486 antagonized PR/GR signalling, thereby relieving transcriptional repression of the p53 pathway and promoting the expression of proapoptotic genes such as Bax and caspase-3, sensitizing cells to apoptosis. In parallel, olaparib inhibited PARP activity, blocking the repair of SSBs, which were subsequently converted into more deleterious DSBs during replication. This accumulation of DSBs induced γ-H2AX formation and activated the p53 axis, thereby amplifying DNA damage signalling and driving cell death. Critically, the combination therapy achieved synergy through the convergence of these distinct mechanisms on the central p53 hub. RU486 initiated transcriptional activation of the p53 pathway and its apoptotic targets, while olaparib induced DNA damage stress leading to p53 protein stabilization and further activation. This mutual reinforcement at the p53 node intensified cellular stress, ultimately resulting in mitochondrial destabilization and robust programmed cell death. 3.8 In vivo pain relief research To evaluate the therapeutic effects of RU486 and olaparib on endometriosis in mice, a comprehensive analysis was conducted involving multiple parameters, including nociceptive behaviour, ectopic lesion burdens, serum sex hormone levels, histological morphology, and molecular marker expression. To enhance the representativeness of the study data, more than 10 endometriosis model animals were included in each experimental group. The modelling and treatment workflow are illustrated in Figure 5a,b. Using our established protocol, the modelling success rate reached 100%. Pain is one of the most prominent and debilitating symptoms of endometriosis. It significantly impairs the quality of life in affected women, contributing to chronic pelvic pain, dysmenorrhoea, dyspareunia, and infertility, and is often associated with emotional and psychological distress such as anxiety and depression. In this study, pain sensitivity was assessed using the hot plate test (Figure 5c). Baseline latencies were comparable across groups. After treating 1 week, all drug-treated groups exhibited prolonged response times compared with control (22.58 ± 3.49 s), with RU486: 39.74 ± 6.50 s, olaparib: 33.66 ± 5.35 s, and combination: 44.35 ± 4.55 s. This effect was also sustained through Weeks 2 and 3. Our results demonstrated that RU486 monotherapy exerted a robust and durable analgesic effect, which was in agreement with prior clinical and experimental reports (Li et al., 2018; Y. X. Zhang, 2016). In contrast, although olaparib could alleviate pain via inhibition of oxidative DNA damage and microglial activation (Senguttuvan et al., 2023), its combination with RU486 did not enhance analgesia, this lack of synergism may be attributed to the ability of RU486 alone to sufficiently engage the predominant nociceptive pathways, thereby masking the olaparib's pharmacological contribution. Given the critical role of sex hormones in promoting pain sensitization (Bulun et al., 2023; Yang et al., 2024), we subsequently quantified serum levels of progesterone and oestrogen to assess their potential contributions to these processes. All treatment groups distinctly increased serum progesterone levels compared with the control group (3.35 ± 0.70 ng·ml−1), with RU486: 4.22 ± 0.58 ng·ml−1, olaparib: 4.20 ± 0.35 ng·ml−1, and combination: 4.92 ± 0.59 ng·ml−1 (Figure 5d). Notably, both groups containing RU486 showed statistically significant differences compared with control. Similarly, only the RU486 group (8.51 ± 2.76 pg·ml−1) and the combination group (6.86 ± 1.45 pg·ml−1) significantly reduced serum oestrogen levels relative to the control group (13.13 ± 4.80 pg·ml−1), whereas olaparib monotherapy (12.32 ± 2.87 pg·ml−1) showed no significant effect (Figure 5e). Overall, in the analgesic study, both RU486 and the combination treatment exhibited significant pain-relieving effects, primarily through antagonism of PR/GR receptors. However, the combination therapy did not show a significantly greater analgesic effect compared with RU486 monotherapy. 3.9 In vivo lesion suppression analysis Regarding lesion characteristics, RU486 monotherapy demonstrated a moderate ability to suppress the number, volume, and weight of endometriotic lesions (number: 4.00 ± 1.18; volume: 0.3072 ± 0.1170 cm3; weight: 274.55 ± 101.52 mg) (Figure 6a–d). However, these differences were not statistically significant compared with the control group (number: 4.55 ± 2.02; volume: 0.3891 ± 0.1074 cm3; weight: 338.73 ± 68.31 mg). This finding aligns with previous reports indicating that although RU486 exerts significant analgesic effects, its ability to suppress lesion progression remains limited (Yan et al., 2025; M. Zhang, Ye, et al., 2024). In contrast, olaparib monotherapy resulted in significant reductions in lesion volume and weight. The results demonstrated that olaparib was more effective than RU486 in reducing lesion volume and weight. Among all treatment groups, the combination therapy exhibited the most pronounced therapeutic efficacy, with lesions showing the lowest number (2.08 ± 0.90), volume (0.0953 ± 0.0609 cm3), and weight (91.92 ± 61.37 mg) (Figure 6a–d). Compared with the olaparib group, all three lesion parameters were slightly reduced in the combination group, and compared with the RU486 group, the combination group achieved superior control of lesion weight and volume. Taken together, these findings highlight the synergistic therapeutic effect of olaparib in suppressing ectopic lesions and underscore its strong therapeutic potential in the treatment of endometriosis. Immunohistochemical staining showed elevated γ-H2AX expression in both the olaparib and combination groups (Figure 6e), indicating enhanced DNA damage. This result was consistent with the findings from cellular experiments, in which olaparib effectively up-regulated γ-H2AX expression. In clinical samples, the Ki-67 positivity rate is considered a predictive marker of apoptosis resistance, with lower Ki-67 expression associated with reduced resistance to cell death (M. Chen et al., 2016). In our study, Ki-67 expression was lowest in the combination group, suggesting that the dual-drug treatment effectively suppressed cell proliferation (Figure 6f) and enhanced the sensitivity of EESCs to apoptotic signals. TUNEL immunofluorescence further confirmed increased apoptosis in all treatment groups, with fluorescence intensity significantly elevated in both the RU486 and olaparib groups. Notably, compared with either monotherapy, the combination group showed a further enhancement in apoptotic signalling within ectopic lesions (Figure 6g). In conclusion, both RU486 and olaparib independently inhibited the growth of ectopic lesions and induced apoptosis to varying degrees. RU486 interacted with hormone receptors and exerted its effects in two primary ways: firstly, by adjusting the expression of oestrogen and progesterone, it mitigated the lesion microenvironment, effectively alleviating pain; secondly, it eliminated the suppression of p53, activating the apoptotic pathway. However, the intrinsic apoptotic resistance of ectopic cells (Azam et al., 2022), driven by non-hormone-dependent survival mechanisms, restricted the therapeutic efficacy of RU486 when used in isolation, preventing it from significantly reducing the number, volume, and weight of ectopic lesions. Olaparib, functioning as a PARP inhibitor, instigated SSBs and further intensified DSBs. Subsequent to this DNA damage, there was an increased activation and stabilization of p53. Serving as a tumour suppressor, p53 elevated the expression of Bax, which amplified the permeability of the mitochondrial outer membrane. This action subsequently activated caspase-3, initiating the endogenous apoptosis pathway. This sequence significantly augmented apoptosis, thereby effectively inhibiting lesion growth. The combination therapy maximized the distinct mechanisms of both drugs: RU486 functioned as the initial trigger, activating p53 expression and supplying the primary energy required to launch the apoptosis machinery, enabling a gradual onset of the process, thus reducing pain and offering preliminary lesion suppression. In contrast, olaparib acted as the accelerant, propelling p53 activation with great force, guiding the machinery swiftly and accurately, thereby rapidly progressing the apoptosis process and markedly inhibiting lesion expansion. 3.10 In vivo biosafety As shown in Figure 7a–c, serum biochemical analysis showed no significant differences in ALT, AST, and BUN levels among the treatment groups, indicating that the combination of RU486 and olaparib did not induce apparent liver or kidney dysfunction. Consistently, analysis of organ indices for the heart, liver, spleen, lung, and kidney (Figure S2) showed no significant differences across groups, further supporting the overall systemic safety of the treatment regimen. Despite the overall favourable safety profile of the drugs, H&E staining demonstrated slight toxicity in several critical organs (Figure 7d). Following RU486 or olaparib monotherapy, cardiac tissue exhibited mild myocardial cell atrophy and reduced cell diameter; hepatic tissue showed mild oedema and cytoplasmic vacuolization; and in the spleen, both treatments caused follicular atrophy, decreased lymphocyte density, and notable hemosiderin deposition. In the lungs, both drugs led to pronounced tissue atrophy, epithelial hyperplasia, and alveolar fusion. In the kidneys, mild oedema was observed, along with partial loosening of renal tubular epithelial cells. To further enhance the safety and therapeutic efficacy of this regimen, future studies should consider optimizing the drug delivery approach. For example, improving drug formulations may help reduce localized toxicity while enhancing bioavailability and targeting specificity. In recent years, nanocarrier-based hormone delivery systems have demonstrated significant advantages in both oncology and gynaecological applications, not only improving therapeutic potency but also enhancing tissue selectivity and systemic safety (J. Ma et al., 2025; Wu et al., 2025). Accordingly, the development of RU486 and olaparib into nanoparticle formulations may offer a more effective and safer approach to endometriosis treatment (Anthis et al., 2024; Liu et al., 2024). Moreover, our previous studies have shown that some drugs have the first-pass effect in the uterus after vaginal administration, which means that their specific distribution in the uterine tissue is increased and their distribution in the whole body is reduced (Xin et al., 2025). Thus, subsequent studies can prepare RU486 and olaparib into vaginal gels to reduce adverse drug reactions if it is clear that they have a first-pass effect in the uterus. 4 DISCUSSION Given the limited clinical efficacy of RU486 in eliminating endometriotic lesions, this study established a dual-pronged therapeutic strategy in which RU486 and olaparib converge mechanistically on p53 activation. RU486 initiated transcriptional awakening of p53 through nuclear receptors, while olaparib promoted functional hyperactivation via PARP inhibition-induced DNA damage, stabilizing phosphorylated p53 to orchestrate Bax-mediated mitochondrial apoptosis. This synergistic interaction established a self-amplifying circuit in which RU486-mediated transcriptional activation and olaparib-induced posttranslational modification of p53 cooperatively enhanced caspase-3 activation, leading to PARP1 cleavage. The subsequent inactivation of PARP1 further potentiated the therapeutic efficacy of olaparib, thereby reinforcing the apoptotic cascade. In vitro, this combination potently suppressed cell viability, induced apoptosis, disrupted mitochondrial membrane potential, and inhibited cell migration. In vivo, combination therapy achieved a 77.5% reduction in lesion mass while maintaining sustained analgesic effects. These findings highlight the combination of RU486 and PARP inhibitors as a promising and innovative therapeutic strategy for the treatment of endometriosis. Building on this p53-centric pharmacology, future research should prioritize developing targeted delivery systems to minimize systemic toxicity, or explore other effective adjunctive p53 stabilizers to further enhance apoptotic priming, and implementing biomarker-guided patient stratification to identify individuals with intact p53 signalling for personalized therapy. Combination therapy is a well-established approach to overcome the limitations of single-agent treatments. Among them, olaparib, a clinically approved PARP inhibitor, has become a pivotal component in various oncological combination regimens due to its ability to amplify DNA damage responses and potentiate apoptosis. Recent studies have confirmed the feasibility of such synergistic strategies across different cancers. For instance, combining olaparib with abiraterone significantly improved progression-free survival in metastatic castration-resistant prostate cancer (Saad et al., 2025); gallic acid restored olaparib sensitivity in osteosarcoma by enhancing apoptotic signalling (Erdogan & Usca, 2025); and olaparib with the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 achieved robust antitumor effects in temozolomide-resistant glioblastoma (Sha et al., 2025). Given the tumour-like behaviours of endometriosis, characterized by aberrant proliferation, invasion, angiogenesis, and apoptotic resistance, integrating PARP inhibition into its treatment represents a rational and feasible strategy. The RU486–olaparib combination thus offers a promising approach to overcome monotherapy limitations and achieve enhanced therapeutic synergy. According to the latest European Society of Human Reproduction and Embryology (ESHRE) and American College of Obstetricians and Gynecologists (ACOG) clinical guidelines, current therapeutic approaches for endometriosis primarily rely on hormonal suppression or surgical excision. Although progestins such as RU486 have shown efficacy in alleviating pain and suppressing lesion activity, their long-term use is often limited by hormone-related adverse effects, including menstrual irregularities, adrenal suppression, and metabolic disturbances. The present study introduces a mechanistically distinct combination strategy in which RU486 and the PARP inhibitor olaparib converge on the activation of the p53 pathway, integrating hormone receptor modulation with DNA damage–induced apoptosis. This dual-targeted approach not only enhances therapeutic efficacy but also allows a significant reduction in RU486 dosage, thereby minimizing endocrine-related side effects while maintaining potent lesion inhibition. Importantly, both RU486 and olaparib are clinically approved agents with well-characterized pharmacokinetics and safety profiles, supporting their translational potential for rapid clinical adaptation. Furthermore, this strategy aligns with the future directions emphasized in current international guidelines, which advocate the development of non–hormone-dependent, mechanism-based, and precision therapies for endometriosis management. By focusing on the central p53 signalling hub, the RU486–olaparib combination exemplifies this forward-looking therapeutic paradigm. In addition, given the shared molecular features of apoptotic resistance and DNA repair dysregulation in related gynaecological disorders such as adenomyosis, endometrial hyperplasia, and ovarian endometrioid carcinoma, the mechanistic framework established here may also offer translational insight beyond endometriosis itself. Collectively, these findings support a clinically feasible, low-dose, and mechanism-guided therapeutic approach that not only meets current management priorities but also paves the way for broader applications across p53-associated gynaecological diseases. AUTHOR CONTRIBUTIONS Yujie Peng: Investigation (lead); methodology (supporting); writing—original draft (lead); writing—review and editing (supporting). Meng Zhang: Data curation (supporting); investigation (lead); methodology (lead); writing—original draft (supporting). Libo Zhu: Data curation (supporting); formal analysis (supporting); resources (lead). Wenqiang Qian: Investigation (supporting); software (supporting); writing—original draft (supporting); writing—review and editing (supporting). Jingjing Yan: Investigation (supporting); writing—review and editing (supporting). Huidi Jiang: Resources (equal); writing—review and editing (equal). Yu Xin: Investigation (supporting); writing—review and editing (lead). Ying Zhang: Investigation (supporting); software (supporting); writing—original draft (supporting). Dongli Sun: Investigation (supporting); methodology (equal); writing—review and editing (supporting). Weidong Fei: Conceptualization (lead); funding acquisition (lead); project administration (equal); writing—review and editing (equal). Mengdan Zhao: Conceptualization (equal); funding acquisition (lead); project administration (equal); writing—review and editing (equal).

This study was financially supported by the National Natural Science Foundation of China (82071616 and 82401930), the Natural Science Foundation of Zhejiang Province (LY19H040011), and the Zhejiang Pharmaceutical Association Hospital Pharmacy Scientific Research Program (2022ZYYL08). CONFLICT OF INTEREST STATEMENT The authors declared that no competing interest exists. DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation and as recommended by funding agencies, publishers, and other organizations engaged with supporting research. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.