Acetylation reader BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS

Acetylation reader BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Acetylation reader BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS Yajing Weng, Yi Zhang, Wei Dong, Zhengquan Zhu, Zou Xiang, Guijun Yan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6321751/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by the developmental arrest and dysfunction of ovarian granulosa cells (GCs), serving as a major cause of infertility among women of reproductive age. Persistent activation of thioredoxin-interacting protein (TXNIP) due to aberrant histone acetylation modifications of transcription is a potential trigger; however, its precise upstream regulatory mechanism remains poorly understood. In this study, we found that TXNIP was aberrantly upregulated in both the dehydroepiandrosterone (DHEA)-induced PCOS-like rat model and the dihydrotestosterone (DHT)-induced primary GCs PCOS-like model in vitro. The TXNIP/NLRP3 inhibitor ruscogenin and the small interfering RNA (siRNA) targeting TXNIP remarkably inhibited NLRP3 inflammasome activation, subsequently reversing aberrant reproductive and metabolic phenotypes in PCOS-like models. Further bioinformatic analysis revealed that the promoter region of TXNIP contains binding motifs of bromodomain-containing protein 4 (BRD4) and androgen receptor (AR). BRD4 and AR exhibited inducible binding to the histone H3 acetylation-enriched TXNIP promoter, whereas intervention with the BRD4-selective inhibitor JQ1 and the AR-selective inhibitor attenuated this binding, leading to subsequent downregulation of TXNIP transcription that ultimately resulted in NLRP3 inflammasome suppression. Our data indicate that BRD4 upregulation and the resultant TXNIP transcriptional activation are crucial regulatory pathways for NLRP3 inflammasome activation, resulting in associated reproductive and metabolic abnormalities in ovarian GCs from PCOS. polycystic ovary syndrome bromodomain-containing protein 4 androgen receptor thioredoxin-interacting protein NLRP3 inflammasome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Polycystic ovary syndrome (PCOS), a prevalent endocrine-metabolic disorder affecting women during adolescence and reproductive years, demonstrates a global prevalence of 8–13%. PCOS is characterized by hyperandrogenism, blocked dominant follicle formation, and anovulation or oligoovulation. Granulosa cells (GCs), recognized as the most critical ovarian cell population, drive oocyte growth alongside maturation while facilitating both follicular development and formation. Oxidative stress coupled with NLRP3 inflammasome-driven pyroptosis in GCs serves as a key pathogenic mechanism underlying hormonal imbalance, subfertility, and PCOS-related ovarian dysfunction, manifesting through oocyte developmental arrest, follicular atresia, and anovulation [ 1 – 3 ]. Thioredoxin-interacting protein (TXNIP), a protein that interacts with thioredoxin (TRX), plays a role in the development and progression of diseases such as diabetes, neurodegenerative disorders, and chronic kidney diseases by regulating NLRP3 inflammasome activation and glucose/lipid metabolism [ 4 – 6 ]. Recent studies indicate that aberrant TXNIP upregulation is correlated with decreased insulin sensitivity in PCOS [ 7 – 10 ], but the precise upstream mechanisms underlying TXNIP/NLRP3 activation in PCOS remain unclear. TXNIP is regulated at multiple levels, including transcription, epigenetic modifications, mRNA stability, and protein stability [ 11 – 14 ]. MicroRNAs [ 15 , 16 ], circular RNAs [ 17 ], oncogenes [ 18 ], endoplasmic reticulum stress (ERs) [ 19 ], cytokines and growth factors [ 20 , 21 ] are associated with pathways/factors that affect TXNIP expression. Glucose-induced TXNIP levels are strongly linked to carbohydrate response elements and the associated transcription factors, such as the ChREBP/MondoA: Mlx complex [ 22 ], FOXO1 and MAPK [ 23 ]. In breast cancer, the mRNA decay factor ZFP36 targets TXNIP mRNA transcripts for degradation mediated by receptor tyrosine kinases [ 24 ]. Activated AMPK phosphorylates TXNIP, resulting in rapid degradation of its protein [ 25 ]. In addition, upregulation of TXNIP due to aberrant lysines 9/14/27 acetylation of histone H3 mediated via histone acetyltransferase p300 [ 26 – 29 ] may be the main cause of NLRP3 inflammasome activation. Although numerous studies have demonstrated the upstream regulatory mechanisms of TXNIP, the precise epigenetic regulatory mechanisms regulating TXNIP in PCOS remain to be fully elucidated. Histone acetylation, a frequently encountered form of epigenetic modification, promotes gene transcription activation by facilitating the dissociation of DNA from the histone octamer and relaxing the nucleosome structure. The process is primarily regulated by histone acetyltransferases (HATs), histone deacetylases (HDACs), and acetylation-reading proteins [ 30 ]. Bromodomain-containing protein 4 (BRD4) is an acetylation reader that binds to specific acetylated lysine residues on histone tails. Subsequently, it recruits various transcription factors and transcriptional coactivators to target gene promoters and enhancers to promote gene transcription [ 31 ]. Our previous research demonstrated that the aberrantly increased BRD4 and subsequent androgen receptor (AR) transcriptional activation are important triggers of ovarian fibrosis in PCOS [ 32 ]. However, whether BRD4 regulates TXNIP in PCOS is currently unknown. In this study, we investigated the epigenetic mechanism by which the acetylation reader BRD4 and the transcription factor AR promote TXNIP transcriptional activation, leading to ovarian pathology in PCOS. We provide strong evidence that upregulation of BRD4 and AR enhances TXNIP expression, subsequently triggering NLRP3 inflammasome activation and metabolic disturbances in PCOS. Our study aims to provide new epigenetic-based drug targets for PCOS and a theoretical foundation for developing new clinical therapies. 2. Materials and Methods 2.1. Animals and experimental protocols Three-week-old female Sprague-Dawley rats (Junke Biotechnology Corporation, China) were randomly divided into 4 groups (6 rats per group, n = 24, 55–65 g): (1) vehicle control (oil); (2) dehydroepiandrosterone (DHEA); (3) ruscogenin; (4) a combination of DHEA and ruscogenin treatment (D + R). All rats were housed in a specific pathogen-free (SPF) environment (Jiangsu Key Laboratory of Molecular Medicine), maintained at 24°C with a 12-hour light/dark cycle and humidity levels of 45%-65%. Free access to water and food was supplied. Rats in the DHEA treatment group received a subcutaneous DHEA (6 mg/100g body weight) injection daily for 35 consecutive days since postnatal day 23 to establish a PCOS-like rat model. The vehicle control rats received a hypodermic injection of experimental-grade soybean oil (Yuanye Biological Technology Corporation, China) with an identical volume. From the third week after DHEA or oil administration, the rats in the ruscogenin and D + R group were given ruscogenin (1 mg/kg, MCE, USA) by gavage once a day for 21 days. All the rats were euthanized to collect ovaries and blood at the end of the experiment. Unilateral ovaries were fixed with 4% paraformaldehyde (Servicebio, China) and embedded in paraffin for tissue sectioning. The remaining ovaries and serum were all preserved at -80℃ for further experiments. The experiments were carried out following the principles and guidelines for the use of laboratory animals and were approved by the institutional research animal committee of Nanjing University. 2.2. Estrous cycle analysis Vaginal cells were collected by lavage with saline and smeared from the indicated rats at 9:30AM for 10 consecutive days. The predominant cell types in the vaginal smears were determined under light microscopy using Giemsa staining. The stages of the estrous cycle were also determined. The proestrus stage was marked by predominantly nucleated cells; the estrus stage was represented by cornified squamous epithelial cells; the metestrus stage was typified by a combination of cornified cells and leukocytes; and the diestrus stage exhibited a predominance of leukocytes. 2.3. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) After a 12-hour fast, rats were injected with 20% glucose (2 g/kg; i.p.) or 0.75 U/kg insulin (i.p.). Blood samples were collected from the tail vein at time points of 0, 15, 45, 60 and 120 minutes after glucose or insulin injection to evaluate glucose concentrations. 2.4. Determination of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels Blood samples were taken from the inferior vena cava of rats. Next, serum was separated using centrifugation and stored at -80℃. Serum LH and FSH levels were determined using the enzyme-linked immunosorbent assay (ELISA) kits (Elabscience Biotechnology, China) following the manufacturer’s instructions. 2.5. Isolation of GCs Female Sprague-Dawley rats were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG, Sansheng Biological Technology Corporation, China) (20 IU) to promote follicular development, followed by euthanasia 48 hours later. The ovaries were isolated and the follicles were peeled off by manipulation with micro tweezers under a dissecting microscope. Next, the follicles were punctured to release GCs, and the cell suspension was filtered through a 70-µm cell strainer to remove residual cells and debris clumps. 2.6. Cell culture and treatment Primary rat GCs, a human granulosa-like tumor cell line (KGN cells) and human embryo kidney HEK-293T cells were cultured in DMEM-F12 or DMEM medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Gibco) at 37℃ with 5% CO 2 . Cells were treated with various concentrations of dihydrotestosterone (DHT; 0.5, 2, 5 µM; Meilun Biological Technology Corporation, China) for 48 hours to construct a PCOS-like model characterized by hyperandrogenism in vitro. Flutamide (20, 50 µM; Selleck, China) and JQ1 (1 µM; MCE) were added into cells for 24 hours with or without DHT. 2.7. Hematoxylin and eosin (H&E) Ovary tissues were processed for histopathology analysis following standard paraffin fixation, sectioning and H&E staining. The sections were viewed under an optical microscope (Leica Microsystems, Germany). 2.8. Immunohistochemistry Embedded ovaries were sectioned at 4 µm and stained with specific antibodies against TXNIP (1:100; HUABIO, China). Next, the sections were incubated with a secondary antibody goat anti-rabbit IgG (H + L)-HRP conjugate. Images were captured using an optical microscope (Leica Microsystems). 2.9. RNA isolation and real-time quantitative PCR (qRT-PCR) Total RNA from rat ovaries, GCs, and KGN cells was extracted using the TRIzol reagent (Beyotime, China), and cDNA was synthesized with a reverse transcription kit (Vazyme Biotech, China). qRT-PCR was performed with the ABI Viia 7 Real-Time PCR system (ABI, USA) using the SYBR Green PCR Master Mix (Vazyme Biotech), and the primers are shown in Tables 1 and 2 . The critical threshold cycle (Ct) value was determined for each reaction, which was transformed into relative quantification data using the 2 −ΔΔCt method. The housekeeping gene β-actin was used as an internal control. Table 1 Primer sequences for rats qRT-PCR Genes Forward Reverse β-actin 5’-TTCCTTCCTGGGTATGGAAT-3’ 5’-GAGGAGCAATGATCTTGATC-3’ TXNIP 5’-ATCATGGCGTGGCAAGAGTC-3’ 5’-TTTCTTGGAGCCAGGGACAC-3’ AR 5’-TCTGGTTGTCACTACG GAGC-3’ 5’-TGCAATCATTTCTGCTGGCAC-3’ NLRP3 5’-CAGCGATCAACAGGCGAGAC-3’ 5’-AGAGATATCCCAGCAAACCTATCCA-3’ GSDMD 5’-TTGAGTGTCTGGTGCTCGAC-3’ 5’-ATGGGGTGCTCTGTTCCAAG-3’ IL-1β 5’-CTACCTATGTCTTGCCCGTGGAG-3’ 5’-GGGAACATCACACACTAGCAGGTC-3’ ASC 5’-GGACCAACACAGGCAAGCACTC-3’ 5’-ACAAGTTCTTGCAGGTCAGGTTCC-3’ IL-18 5’-ACCTGTGGACTCTCAGACAAC-3’ 5’-AGCAGGCAGCTAGGATTACG-3’ Table 2 Primer sequences for human qRT-PCR Genes Forward Reverse β-actin 5’-CGTGGACATCCGCAAAGA-3’ 5’-GAAGGTGGACAGCGAGGC-3’ TXNIP 5’-CTGGCGTAAGCTTTTCAAGG-3’ 5’-AGTGCACAAAGGGGAAACAC-3’ 2.10. Western blot Ovaries, GCs, KGN cells, and HEK-293T cells were lysed using RIPA lysis buffer (Beyotime) containing 1 mM phosphatase inhibitor (MCE) and 1 mM protease inhibitor cocktail (MCE). Equal amounts of total proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the protein bands were transferred onto polyvinylidene difluoride membranes (Merck Millipore, USA). Target bands were incubated with corresponding primary antibodies against TXNIP (1:1000; HUABIO), NLRP3 (1:1000; CST, USA), GSDMD (1:1000; Proteintech, USA), IL-1β (1:1000; Abcam, UK), ASC (1:1000; Abcam), IL-18 (1:1000; HUABIO), AR (1:1000; Abcam), BRD4 (1:1000; CST), acetylated histone H3 (Ac-H3; 1:1000; Abcam) and β-actin (1:20000; Fudebio, China) overnight at 4℃, followed by the addition of HRP-labeled secondary antibodies (1:20000; Fudebio). The blots were visualized using chemiluminescent detection (Merck Millipore). The band intensity was quantified with Image J. 2.11. Immunofluorescence GCs were seeded in 24-well culture plates with wells containing cell climbing slices. Sections were fixed in 4% paraformaldehyde (Servicebio) for 30 minutes at 25°C, and then permeabilized with 0.3% Triton X-100 (Beyotime). After washing with PBS three times, the cells were blocked with 3% bovine serum albumin for 30 minutes at 25°C. Cells were incubated with antibodies against TXNIP (1:100), NLRP3 (1:100) and ASC (1:100) overnight at 4°C. After washing with phosphate buffered saline containing 0.5% of Tween-20 three times, cells were incubated with fluorescent secondary antibodies, including anti-rabbit Cy3 and anti-mouse Alexa 488 (Beyotime), at 25°C for 2 hours. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime) at a dilution of 1:2000 for 30 minutes and photographed using an Olympus laser scanning confocal microscope (FV3000, Japan). The fluorescence intensity was analyzed with Image J. 2.12. Lentiviral vector preparation and infection The TXNIP lentiviral particles for rats were purchased from GeneChem (China). Primary GCs were seeded in culture plates. At 70% confluence, cells were incubated with TXNIP lentiviral particles (MOI = 20) or control lentiviral particles along with HitransG P in DMEM-F12 medium. The transfection efficiency was determined 72 hours later by quantifying the intensity of green fluorescence and protein analysis using western blot. 2.13. RNA interference TXNIP knockdown in GCs and KGN cells was performed with small interfering RNA (siRNA). Scrambled RNA oligos of the rat TXNIP gene (sense oligo: 5’-GGACGUGAUUCCUGAAGAUTT-3’; antisense oligo: 5’-AUCUUCAGGAAUCACGUCCAT-3’) and human TXNIP gene (sense oligo: 5’-GAGAAUACAUGUUCCCGAA-3’; antisense oligo: 5’-UUCGGGAACAUGUAUUCUC-3’) were designed by Keygen Biotech (China). Cells were transfected with the siRNA using Lipofectamine 2000 (Invitrogen, USA) followed by incubation for 72 hours. Next, DHT (5 µM) was added and the plate was incubated for 48 hours. 2.14. Dual-luciferase reporter assay Wild type human TXNIP promoter (TXNIP WT), mutant human TXNIP promoter (TXNIP MUT), BRD4 negative control plasmid (NC-BRD4), and BRD4 overexpression plasmid (OE-BRD4) were designed by Tsingke Biotech (China). A Renilla luciferase reporter was used as an internal control. The mutant form of hTXNIPp-luc was constructed in which the BRD4 and AR responsive element CAGAACAGAGAGAAC was mutated to GTCTTGTCTCTCTTG. Plasmids were transfected into HEK-293T cells using GenJetTM Plus in Vitro DNA Transfection Reagent (SignaGen, USA) with a 48-hour incubation following the manufacturer’s instruction. The transfected cells were treated with DHT (5 µM) to activate AR, or treated with JQ1 (1 µM) to inhibit BRD4, or treated with flutamide (50 µM) to inhibit AR. Luciferase activities were determined with a GloMax Luminometer using a dual luciferase reporter assay kit (Vazyme Biotech). Luciferase activities were normalized to Renilla luciferase levels and expressed as relative fold changes. 2.15. Co-immunoprecipitation (Co-IP) The lysates of HEK-293T cells were first immunoprecipitated using antibodies to BRD4 (CST), AR (Abcam), or isotype-matched immunoglobulin followed by treatment with Protein A/G Magnetic Beads (Vazyme Biotech). Next, the immunoprecipitants were analyzed using western blot with antibodies to BRD4 (CST), AR (Abcam), or Ac-H3 (Abcam). 2.16. Chromatin immunoprecipitation (ChIP) ChIP assay was performed following the protocol of ChIP assay Kit (Beyotime). Ovarian tissues were processed into single-cell suspensions. Formaldehyde was added to the single-cell suspensions or HEK-293T cells for cross-linking the target proteins with genomic DNA. These samples were then treated with ultrasound to break the genomic DNA into 200–1000 bp fragments. The target proteins, including BRD4 (CST), AR (Abcam) or Ac-H3 (Abcam), and their bound DNA fragments were co-immunoprecipitated, purified, and amplified using PCR. The primer sequences for ChIP-PCR are provided in Table 3 . Table 3 Primer sequences for rats ChIP-PCR Gene Forward Reverse TXNIP 5’-TGGATGAGGTTCAGGGTCTCG-3’ 5’-TTGGCTACTTGGTCCTTGTTTA-3’ Table 4 Primer sequences for human ChIP-PCR Gene Forward Reverse TXNIP 5’-GAAGTGGGAGATAATGAGCG-3’ 5’-CGTGCCTGTGCTGTTCGTC-3’ 2.17. Statistics Statistical analyses were performed using GraphPad Prism 7.00 software. A two-tailed unpaired Student’s t test was used for comparing two groups. One-way analysis of variance (ANOVA) was used for comparing more than two groups, followed by the Bonferroni post hoc test. The Kruskal-Wallis test was performed for the comparisons of data with nonnormal distribution or heterogeneity of variance. The quantitative data are shown as the mean ± standard deviation (SD) with at least three independent experiments, with n indicating the number of replicates. A P -value ≤ 0.05 was considered statistically significant. 3. Results 3.1. TXNIP is abnormally increased in PCOS-like rats A PCOS-like model was established through daily subcutaneous injections of DHEA for 35 consecutive days. Compared with the oil-treated controls, DHEA-treated ovaries showed marked polycystic changes, lacked corpus luteum, and reduced ovarian size (Fig. 1 A). Immunohistochemical analysis of TXNIP confirmed dramatic upregulation in ovarian GCs of PCOS-like rats (Fig. 1 B). qRT-PCR and western blotting revealed a marked increase in TXNIP in PCOS ovaries (Fig. 1 C, D). Consistently, in a DHT-induced PCOS-like in vitro model using GCs and KGN cells, the expression of TXNIP were significantly elevated at mRNA and protein levels (Fig. 1 E-H). Both increased nuclear and cytoplasmic localization of TXNIP was determined using immunofluorescence (Fig. 1 I-J). Taken together, these results suggest that the aberrant TXNIP upregulation may contribute to ovarian pathological changes in PCOS. 3.2. TXNIP is a critical driver of NLRP3 inflammasome activation in PCOS To investigate the key role of TXNIP in NLRP3 inflammasome activation, we overexpressed TXNIP in GCs via lentiviral transduction. The overexpression efficiency of TXNIP was determined using western blot (Fig. 2 A), which consistently increased protein levels of NLRP3 and pyroptosis-related proteins, including GSDMD, IL-1β, ASC, and IL-18 (Fig. 2 B-D). Moreover, TXNIP was knocked down using siRNA in primary GCs and KGN cells separately. TXNIP silencing markedly reversed the elevated expression of NLRP3 inflammasome components (Fig. 2 E-J). These results indicate that aberrant TXNIP acts as a key regulator of NLRP3 inflammasome activation and pyroptosis in ovarian GCs of PCOS. 3.3. Inhibition of TXNIP/NLRP3 ameliorates metabolic abnormalities and ovarian pathology in PCOS Ruscogenin, a crucial steroidal sapogenin derivative, has been demonstrated to effectively suppress the activation of the TXNIP/NLRP3. Ruscogenin was used to treat PCOS-like rats and KGN cells, followed by an examination of relevant indicators. Our findings revealed that ruscogenin treatment reversed the upregulation of TXNIP/NLRP3 and pyroptosis-related proteins, including GSDMD, IL-1β, ASC, and IL-18 in both in vivo and in vitro models (Fig. 3 and Supplementary Fig. 1). We further evaluated the effect of TXNIP/NLRP3 inhibition on PCOS-associated metabolic abnormalities and ovarian dysfunction. The results showed that ruscogenin treatment did not effectively restore the reduced body weight in PCOS-like rats (Fig. 4 A), but it increased ovarian size (Fig. 4 B). Moreover, ruscogenin treatment improved glucose tolerance (Fig. 4 C), normalized serum levels of LH and FSH as well as the LH/FSH ratio (Fig. 4 D), restored ovarian health as evidenced by decreased preantral and cystic follicle counts with a concomitant increase in corpora lutea formation (Fig. 4 E), and ameliorated the disrupted estrous cyclicity (Fig. 4 F). Taken together, these results suggest that inhibition of TXNIP/NLRP3 markedly reversed metabolic disorders and ovarian dysfunction in PCOS-like rats. 3.4. AR-selective inhibition attenuates TXNIP and NLRP3 inflammasome activation Bioinformatic analysis of the NCBI Gene Expression Omnibus (GEO) database (GSE34526) showed that the TXNIP expression was profoundly elevated in women with PCOS compared with healthy women and was positively correlated with the AR expression (Fig. 5 A). Both DHEA and DHT profoundly increased AR activity in vivo and in vitro (Fig. 5 B, C). To further elucidate the upstream mechanisms responsible for the aberrant increase in TXNIP expression, we treated GCs and KGN cells with the AR inhibitor flutamide in vitro. Selective AR inhibition extensively reduced both mRNA and protein levels of TXNIP, NLRP3, and inflammasome activation-related factors in the DHT-induced in vitro PCOS-like model (Fig. 5 D-G and Supplementary Fig. 2). 3.5. AR-selective inhibition alleviates TXNIP transcription Bioinformatics analysis using the JASPAR database ( https://jaspar.elixir.no/ ) predicted high-affinity AR-binding motifs within the TXNIP promoter region, with prediction scores of 15.428571 (rat) and 13.350983 (human) (Fig. 6 A-D). To validate AR as a transcription factor of TXNIP, we performed ChIP assays to confirm AR binding to the TXNIP promoter regions containing AR-binding motifs in the DHEA-treated ovaries and HEK-293T cells, and this interaction was abolished by flutamide (Fig. 6 E, F). TXNIP promoter-luciferase reporter plasmid was transfected into HEK-293T cells, and we found that flutamide attenuated the aberrant TXNIP-LUC transactivation induced by the AR agonist DHT (Fig. 6 G). Taken together, these results indicate that AR directly promotes TXNIP transcriptional activation and subsequent NLRP3 inflammasome activation, both of which are effectively inhibited by flutamide. 3.6. BRD4 activates TXNIP via AR and acetylated histone-associated transcriptional activation BRD4 is a well-known acetylation reader that facilitates gene transcriptional activation through the recruitment of transcription factors and transcriptional coactivators. Building on our prior demonstration that AR is a transcription factor for TXNIP, we hypothesized that BRD4 binds the TXNIP promoter and recruits AR to promote TXNIP transcription. ChIP assays showed that BRD4 was inducibly bound to the AR motif-containing TXNIP promoter enriched with Ac-H3 in DHT-induced PCOS-like model in vitro, which was inhibited by JQ1 (Fig. 7 A, B). In addition, we constructed a TXNIP promoter-luciferase reporter TXNIP-LUC and a mutant reporter muTXNIP-LUC, in which the BRD4 motif TCAGAACAGAGAGAAC was replaced by GTCTTGTCTCTCTTG. The indicated plasmids were transfected into HEK-293T cells and then the cells were treated with plasmids for BRD4 overexpression or a negative control, along with the BRD4-selective inhibitor JQ1. We found that BRD4 overexpression increased the transactivation of TXNIP-LUC, but not muTXNIP-LUC, and this effect was significantly blocked by JQ1 (Fig. 7 C). We further treated HEK-293T cells with the AR agonist DHT followed by JQ1. The results showed that DHT treatment increased the transactivation of TXNIP-LUC, but not muTXNIP-LUC, which was blocked by JQ1 (Fig. 7 D). Furthermore, we confirmed that BRD4 was inducibly associated with AR and Ac-H3 in DHT-treated HEK-293T cells, which was inhibited by JQ1 (Fig. 7 E, F). To summarize, these results strongly support that BRD4 promotes TXNIP transcriptional activation in PCOS through inducible interaction with the AR and Ac-H3, culminating in its chromatin enrichment at the TXNIP promoter. 4. Discussion In this study, we demonstrated that histone acetylation-dependent TXNIP transcriptional activation is a critical determinant in driving NLRP3 inflammasome activation and ovarian dysfunction in PCOS ovarian GCs. We found that the TXNIP/NLRP3 inhibitor ruscogenin effectively reversed NLRP3 inflammasome activation and metabolic dysfunction in PCOS-like rats. Furthermore, BRD4 drives TXNIP transcriptional activation via inducible interaction with the AR and Ac-H3, coupled with chromatin recruitment to the TXNIP promoter. Notably, this regulatory axis is suppressed by pharmacological inhibition of BRD4 or AR. Therefore, aberrant TXNIP activation mediated by histone acetylation is an important epigenetic mechanism for NLRP3 inflammasome activation and ovarian pathophysiology in PCOS. As a key α-arrestin family member, TXNIP promotes apoptotic signaling through TRX inhibition and subsequent ASK-1 activation [ 33 ], while simultaneously driving NLRP3 inflammasome assembly via redox-dependent mechanisms [ 34 ], contributing to neurodegenerative disorders [ 35 ], diabetes/complications [ 36 , 37 ], and cardiovascular diseases [ 38 ]. Previous studies have demonstrated that high glucose promotes TXNIP overexpression, leading to β-cell apoptosis, disrupted glucose homeostasis, and impaired glucose uptake in skeletal muscle, liver, and adipose tissues [ 36 , 39 ]. Additionally, increased TXNIP induces G0/G1 arrest by inhibiting cyclin A [ 40 ]. In this study, we discovered that TXNIP/NLRP3 inhibition ameliorated ovarian histopathological alterations and endocrine-metabolic abnormalities, suggesting aberrant TXNIP/NLRP3 is a crucial element of ovarian dysfunction in PCOS. Demonstration of AR-activated TXNIP transcription in promoting aberrant TXNIP overexpression and GCs pyroptosis in PCOS is a crucial discovery of this study. Our previous research has shown that sustained AR activation is an important driver of various pathologies in DHEA-induced PCOS-like rats, including ovarian fibrosis and anovulation [ 32 ]. Complementarily, AR knockout reverses DHT-induced reproductive and metabolic abnormalities in mice [ 41 ]. We analyzed the TXNIP promoter using JASPAR and found the AR binding motifs, which are located very close to the transcription start site with high prediction score (rat: 15.428571; human: 13.350983). We showed that AR can directly bind to the TXNIP promoter region, and TXNIP transcription was blocked by AR inhibition and the mutation of AR binding motif on the TXNIP promoter. It is noteworthy that our data does not exclude the possibility of other transcription factors contributing to TXNIP activation in PCOS, but establishes a basis for future research. The bromodomain and extra-terminal domain (BET) family, comprised of BRD2, BRD3, BRD4, and bromodomain testis-specific protein (BRDT), orchestrates genomic regulation and cellular homeostasis through dynamic recognition of acetylated chromatin substrates. These epigenetic readers bind acetylated histones and non-histone protein, while exhibiting DNA/RNA-binding capacity to coordinate transcriptional activation, DNA damage repair, and cell fate determination via chromatin remodeling [ 42 , 43 ]. BRD4, the most studied member, is involved in the pathology of cancer, inflammatory diseases, viral infections, and neurological disorders [ 44 ]. BRD4 also interacts with non-acetylated transcription factors like c-Jun, AP2, Myc, YYA, V/EBPβ, and p53 [ 45 ]. The availability of JQ1, a specific inhibitor of BRD4, offers a valuable tool for scientific investigation. In this study, we observed that BRD4 recruits AR to the TXNIP promoter region containing Ac-H3, thereby facilitating TXNIP transcription, which is suppressed by JQ1. These data demonstrate that beyond targeted therapies, epigenetic modulation via histone acetylation may represent a novel therapeutic strategy to mitigate ovarian GC developmental disorders and protect ovarian function in PCOS. 5. Conclusion In conclusion, this study reveals BRD4 recognizes and inducibly binds to Ac-H3 at the TXNIP promoter, recruits AR to drive TXNIP transcription, which ultimately activating NLRP3 inflammasome with concomitant metabolic derangements and ovarian functional impairment in PCOS-like rats. Our study reveals a crucial epigenetic mechanism of TXNIP activation, and provides a novel target for the treatment of PCOS with clinical implications. Abbreviations Ac-H3 acetylated histone H3 ANOVA one-way analysis of variance AR androgen receptor BET bromodomain and extra-terminal structural domain BRDT bromodomain testis-specific protein BRD4 bromodomain-containing protein 4 ChIP chromatin immunoprecipitation Co-IP co-immunoprecipitation Ct critical threshold cycle DAPI 4’,6-diamidino-2-phenylindole DHEA dehydroepiandrosterone DHT dihydrotestosterone D + R a combination of DHEA and ruscogenin treatment ELISA enzyme-linked immunosorbent assay ERs endoplasmic reticulum stress FBS fetal bovine serum FSH follicle stimulating hormone GCs granulosa cells GEO Gene Expression Omnibus HATs histone acetyltransferases HDACs histone deacetylases H&E hematoxylin and eosin IPGTT intraperitoneal glucose tolerance test ITT insulin tolerance test KGN cells human granulosa-like tumor cell line LH luteinizing hormone NC-BRD4 BRD4 negative control plasmid OE-BRD4 BRD4 overexpression plasmid PCOS polycystic ovary syndrome PMSG pregnant mare serum gonadotropin qRT-PCR real-time quantitative PCR SD means ± standard deviation siRNA small interfering RNA SPF specific pathogen-free TRX thioredoxin TXNIP thioredoxin-interacting protein TXNIP MUT mutant human TXNIP promoter TXNIP WT wild type human TXNIP promoter Declarations Ethics approval and consent to participate All animal experiments were conducted according to the principles and guidelines of the Institutional Animal Care and approved by the Institutional Research Animal Committee of Nanjing University. Informed consent was obtained from all individual participants included in the study. Consent for publication The manuscript is approved by all authors for publication. Availability of data and material Data and material will be made available on request. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was supported by the Jiangsu Provincial Department of Science and Technology (BE2022755), Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases (Obstetrics and Gynecology Hospital, Fudan University, 20DZ2271300) and National Natural Science Foundation of China (82201795 and 81971346). Authors’ contributions Yajing Weng: Writing-review & editing, Writing-original draft, Methodology, Investigation, Conceptualization. Yi Zhang: Methodology, Investigation. Wei Dong: Software, Conceptualization. Zhengquan Zhu: Methodology, Software. Zou Xiang: Writing-review & editing, Supervision. Guijun Yan: Resources. Hongwei Wang: Visualization, Data curation. Yanting Wen: Visualization, Project administration. Min Liu: Visualization, Funding acquisition. Daojuan Wang: Writing-review & editing, Supervision. Yong Wang: Project administration, Funding acquisition, Conceptualization. Acknowledgements Thanks to Professor Yong Wang and everyone in the laboratory for their help. References Fan X, Bialecka M, Moustakas I, Lam E, Torrens-Juaneda V, Borggreven NV, Trouw L, Louwe LA, Pilgram GSK, Mei H, van der Westerlaken L (2019) Chuva de Sousa Lopes SM. Single-cell reconstruction of follicular remodeling in the human adult ovary. Nat Commun 10(1):3164 Wang D, Wang T, Wang R, Zhang X, Wang L, Xiang Z, Zhuang L, Shen S, Wang H, Gao Q, Wang Y (2020) Suppression of p66Shc prevents hyperandrogenism-induced ovarian oxidative stress and fibrosis. 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Diabetes 57(4):938–944 Han SH, Jeon JH, Ju HR, Jung U, Kim KY, Yoo HS, Lee YH, Song KS, Hwang HM, Na YS, Yang Y, Lee KN, Choi I (2003) VDUP1 upregulated by TGF-beta1 and 1,25-dihydorxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression. Oncogene 22(26):4035–4046 Ma Y, Andrisse S, Chen Y, Childress S, Xue P, Wang Z, Jones D, Ko C, Divall S, Wu S (2017) Androgen Receptor in the Ovary Theca Cells Plays a Critical Role in Androgen-Induced Reproductive Dysfunction. Endocrinology 158(1):98–108 Ali HA, Li Y, Bilal AHM, Qin T, Yuan Z, Zhao W (2022) A Comprehensive Review of BET Protein Biochemistry, Physiology, and Pathological Roles. Front Pharmacol 13:818891 Wu SY, Chiang CM (2007) The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem 282(18):13141–13145 Liang Y, Tian J, Wu T (2021) BRD4 in physiology and pathology: ''BET'' on its partners. BioEssays 43(12):e2100180 Wu SY, Nin DS, Lee AY, Simanski S, Kodadek T, Chiang CM (2016) BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression. Cell Rep 16(6):1733–1748 Supplementary Files SupplementaryFigure1.jpg Supplementary Fig. 1: Inhibition of TXNIP decreases the GSDMD level in PCOS-like rat ovaries. Rats received DHEA or combined with ruscogenin treatment. (A) GSDMD expression in ovaries was analyzed by immunohistochemical staining. DHEA, dehydroepiandrosterone; RUS, ruscogenin; D+R, DHEA combined with ruscogenin. SupplementaryFigure2.jpg Supplementary Fig. 2: AR-selective inhibition attenuates NLRP3 inflammasome activation. GCs were treated with DHT alone or together with flutamide. (A) The mRNA levels of AR and NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in GCs were assessed using qRT-PCR. (B) The protein levels of NLRP3 inflammasome-related factors (GSDMD, IL-1β, ASC, and IL-18) in GCs were assessed using western blot (left panel). Band intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group. DHT, dihydrotestosterone; Flu, flutamide. Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 02 May, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 28 Mar, 2025 First submitted to journal 27 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6321751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436555623,"identity":"009d0400-670d-4928-ab71-abea0b071d3b","order_by":0,"name":"Yajing Weng","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital: Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yajing","middleName":"","lastName":"Weng","suffix":""},{"id":436555624,"identity":"2f07f6d3-63a2-48ed-893e-aef65649a353","order_by":1,"name":"Yi 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15:14:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6321751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6321751/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05925-0","type":"published","date":"2025-11-26T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81032431,"identity":"f794e8fe-cbb1-49b2-8374-d68d55dc674c","added_by":"auto","created_at":"2025-04-21 11:33:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXNIP is abnormally increased in PCOS-like rats.\u003c/strong\u003e Rats received DHEA for induction of PCOS. Primary GCs and KGN cells were treated with DHT. (A) Ovarian and follicular morphology were assessed using H\u0026amp;E staining. (B) TXNIP expression in ovaries was analyzed using immunohistochemical staining. (C) The mRNA levels of TXNIP in ovaries were assessed using qRT-PCR. (D) The protein levels of TXNIP in ovaries were assessed using western blot (left panel). Band intensities were quantified (right panel). (E and F) The mRNA levels of TXNIP in GCs (E) and KGN cells (F) were assessed using qRT-PCR. (G and H) The protein levels of TXNIP in GCs (G) and KGN cells (H) were assessed using western blot (upper panel). Band intensities were quantified (lower panel). (I and J) Levels of TXNIP in GCs (I) and KGN cells (J) were analyzed using immunofluorescence staining (60×) (left panel). Fluorescence intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eDHEA, dehydroepiandrosterone; TXNIP, thioredoxin-interacting protein; DHT, dihydrotestosterone; Ctrl, control.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/5a1bf8c6ce7027932892096f.jpeg"},{"id":81033348,"identity":"5b9437cd-8296-4a61-9c69-b56f0e65b4f2","added_by":"auto","created_at":"2025-04-21 11:49:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXNIP is a critical driver of NLRP3 inflammasome activation in PCOS. \u003c/strong\u003ePrimary GCs were transfected with a lentivirus overexpressing TXNIP. Primary GCs and KGN cells were treated with DHT, or TXNIP siRNA followed by treatment with DHT. (A) The protein levels of TXNIP in GCs were assessed using western blot. (B) The expression of NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in GCs was assessed using western blot. (C) Band intensities were quantified (A and B). (D) Levels of NLRP3 and ASC in GCs were analyzed using immunofluorescence staining (60×) (left panel). Fluorescence intensities were quantified (right panel). (E and F) The mRNA levels of TXNIP in GCs (E) and KGN cells (F) were analyzed using qRT-PCR. (G and H) The protein levels of TXNIP in GCs (G) and KGN cells (H) were assessed using western blot (upper panel). Band intensities were quantified (lower panel). (I and J) The expression of NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in GCs (I) and KGN cells (J) was assessed using western blot (left panel). Band intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eOE, overexpression; TXNIP, thioredoxin-interacting protein; NC, negative control; DHT, dihydrotestosterone; siRNA, small interfering RNA.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/d3b57e533e823742855b85c5.jpeg"},{"id":81032434,"identity":"5160ad02-4f44-460f-a292-32b17740e00e","added_by":"auto","created_at":"2025-04-21 11:33:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRuscogenin inhibits TXNIP/NLRP3 in PCOS. \u003c/strong\u003eRats received DHEA alone or together with ruscogenin. KGN cells were treated with DHT alone or together with ruscogenin. (A) The protein levels of TXNIP in ovaries were assessed using western blot (upper panel). Band intensities were quantified (lower panel). (B) The expression of NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in ovaries was assessed using western blot (left panel). Band intensities were quantified (right panel). (C) The protein levels of TXNIP in KGN cells were assessed using western blot (upper panel). Band intensities were quantified (lower panel). (D) The expression of NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in KGN cells was assessed using western blot (left panel). Band intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eDHEA, dehydroepiandrosterone; RUS, ruscogenin; TXNIP, thioredoxin-interacting protein; D+R, a combination of DHEA and ruscogenin treatment; DHT, dihydrotestosterone; N.S, not significant.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/ff749f21d7f9f339c4d0134a.jpeg"},{"id":81032446,"identity":"b4bf0fd8-2025-4922-ad9e-f195d98b47f5","added_by":"auto","created_at":"2025-04-21 11:33:28","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of TXNIP/NLRP3 ameliorates metabolic abnormalities and ovarian pathology in PCOS.\u003c/strong\u003e Rats received DHEA in the presence or absence of ruscogenin. (A) Average body weights of rats were recorded (left panel). Body weights at the end of the treatment (right panel). (B) Intraperitoneal glucose tolerance test and insulin tolerance test were carried out. (C) Ovary weights were recorded. (D) Serum LH and FSH levels were analyzed using ELISA. (E) Ovarian and follicular morphology was assessed using H\u0026amp;E staining (left panel). The features of follicles were analyzed (right panel). (F) The estrous cycle was analyzed (left panel). The percentages of each period of the estrous cycle were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control.\u003c/p\u003e\n\u003cp\u003eDHEA, dehydroepiandrosterone; RUS, ruscogenin; D+R, a combination of DHEA and ruscogenin treatment; LH, luteinizing hormone; FSH, follicle-stimulating hormone; PAF, preantral and early antral follicle; AF, antral follicle; CF, cystic follicle; CL, corpus luteum; M, metestrus; D, diestrus; P, proestrus; E, estrus; N.S, not significant.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/461794cc01a8893fe969ee83.jpeg"},{"id":81032445,"identity":"f4dcdee6-270c-498b-95bc-9af33c969a5a","added_by":"auto","created_at":"2025-04-21 11:33:28","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR-selective inhibition attenuates TXNIP and NLRP3 inflammasome activation.\u003c/strong\u003e Gene expression data were downloaded from the NCBI Gene Expression Omnibus (GEO) database. Rats received DHEA for induction of PCOS. GCs and KGN cells were treated with DHT alone or together with flutamide. (A) TXNIP and AR gene expression analysis was performed using the GEO data set (GSE34526). (B) The protein levels of AR in ovaries, GCs and KGN cells were assessed using western blot. (C) Band intensities were quantified (B). (D and E) The mRNA levels of TXNIP in GCs (D) and KGN cells (E) were analyzed using qRT-PCR. (F and G) The protein levels of AR, TXNIP, and NLRP3 in GCs (F) and KGN cells (G) were assessed using western blot (left panel). Band intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eGEO, Gene Expression Omnibus; PCOS, polycystic ovary syndrome; TXNIP, thioredoxin-interacting protein; AR, androgen receptor; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; GCs, granulosa cells; Flu, flutamide; N.S, not significant.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/bdc0e4464c81bbc95236949e.jpeg"},{"id":81032457,"identity":"69ec7be9-2db1-4e20-a343-9cdf52a3ce4f","added_by":"auto","created_at":"2025-04-21 11:33:28","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR-selective inhibition alleviates TXNIP transcription.\u003c/strong\u003e Rats received DHEA for induction of PCOS. HEK-293T cells were treated with DHT alone or together with flutamide. (A and B) The binding sites of AR in the rat (A) and human (B) TXNIP promoter regions were predicted using bioinformatics analysis. (C and D) Rat (C) and human (D) AR-binding motifs are shown. (E and F) ChIP assays were carried out. The ovary tissues (E) or HEK-293T cells (F) as indicated were immunoprecipitated with antibody to AR or an isotype-matched immunoglobulin (▽). The genomic DNA (input) and the antibody-bound DNA fragments on the TXNIP promoter were PCR amplified. The PCR products were revealed on an agarose gel (left panel). Band intensities were quantified (right panel). (G) A luciferase assay was carried out. HEK-293T cells were transfected with the human TXNIP promoter reporter plus a Renilla luciferase plasmid alone or together with DHT and flutamide. The luciferase activity was measured and normalized with the Renilla luciferase activity. Data are shown as the mean± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eAR, androgen receptor; TXNIP, thioredoxin-interacting protein; DHEA, dehydroepiandrosterone; IgG, Immunoglobulin G; DHT, dihydrotestosterone; Flu, flutamide.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/3476e212be80d6c31c2f18d0.jpeg"},{"id":81033082,"identity":"b5cbdb48-5021-4a13-9cb8-95e808b8b1b1","added_by":"auto","created_at":"2025-04-21 11:41:28","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRD4 activates TXNIP via AR and acetylated histone-associated transcriptional activation.\u003c/strong\u003e (A and B) ChIP assays were carried out. HEK-293T cells were treated with DHT with or without JQ1. The HEK-293T cells were immunoprecipitated with antibody to BRD4 (A) or Ac-H3 (B). Isotype-matched immunoglobulin was used as specificity control (▽), Next, the genomic DNA (input) and the antibody-bound DNA fragments on the TXNIP promoter were PCR-amplified. The PCR products were revealed on an agarose gel (left panel). Band intensities were quantified (right panel). (C) HEK-293T cells were transfected with the wild type human TXNIP promoter reporter (TXNIP WT) or the mutant human TXNIP promoter reporter (TXNIP MUT) plus a Renilla luciferase plasmid and BRD4 overexpression plasmid (OE-BRD4) or negative control (NC-BRD4), followed by treatment with JQ1 for 24 hours. The luciferase activity was measured and normalized against Renilla luciferase activity. (D) HEK-293T cells were transfected with the TXNIP WT reporter or the TXNIP MUT reporter plus a Renilla luciferase plasmid alone or together with DHT and JQ1. The luciferase activity was measured and normalized against the Renilla luciferase activity. (E) A Co-IP assay was carried out. The expression levels of AR, Ac-H3, and BRD4 proteins in the HEK-293T cells were measured for (input). The same tissue lysates were immunoprecipitated with isoform-matched immunoglobulin or antibody to BRD4 or AR, followed by analysis of AR, Ac-H3 or BRD4 levels in the immunoprecipitants using western blot. (F) Band intensities were quantified (E). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eDHT, dihydrotestosterone; BRD4, bromodomain-containing protein 4; IgG, Immunoglobulin G; TXNIP, thioredoxin-interacting protein; NC-BRD4, BRD4 negative control plasmid; OE-BRD4, BRD4 overexpression plasmid; TXNIP WT, wild type human TXNIP promoter; TXNIP MUT, mutant human TXNIP promoter; N.S, not significant.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/d04a9df4b91ee766766ee402.jpeg"},{"id":81032458,"identity":"6b3ed05a-c9c8-4021-bb62-c650fca09bd8","added_by":"auto","created_at":"2025-04-21 11:33:28","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":139444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA schematic diagram showing how BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS.\u003c/strong\u003e Hyperandrogenism upregulates BRD4 and AR. BRD4 recognizes the acetylated histone H3 of TXNIP and recruits AR to modulate TXNIP transcriptional enhancement, which leads to NLRP3 inflammasome activation, and ultimately contributes to both metabolic abnormalities and ovarian pathology in PCOS-like rats.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/85716b612e068d16121df302.jpeg"},{"id":97178294,"identity":"2b42c692-3bb2-4565-822b-448c421aa2b4","added_by":"auto","created_at":"2025-12-01 16:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2565072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/17bf0fa1-dccc-45fe-9aff-16f76924eb63.pdf"},{"id":81032436,"identity":"69191235-e81b-4cb9-a875-e211e0dedfa7","added_by":"auto","created_at":"2025-04-21 11:33:27","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3429532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1: Inhibition of TXNIP decreases the GSDMD level in PCOS-like rat ovaries. \u003c/strong\u003eRats received DHEA or combined with ruscogenin treatment. (A) GSDMD expression in ovaries was analyzed by immunohistochemical staining.\u003c/p\u003e\n\u003cp\u003eDHEA, dehydroepiandrosterone; RUS, ruscogenin; D+R, DHEA combined with ruscogenin.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/76d46e101065612a2cf2697e.jpg"},{"id":81032433,"identity":"d150173f-e607-446f-b7bc-e155b1ac9f03","added_by":"auto","created_at":"2025-04-21 11:33:27","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1955903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2: AR-selective inhibition attenuates NLRP3 inflammasome activation. \u003c/strong\u003eGCs were treated with DHT alone or together with flutamide. (A) The mRNA levels of AR and NLRP3 inflammasome-related factors (NLRP3, GSDMD, IL-1β, ASC, and IL-18) in GCs were assessed using qRT-PCR. (B) The protein levels of NLRP3 inflammasome-related factors (GSDMD, IL-1β, ASC, and IL-18) in GCs were assessed using western blot (left panel). Band intensities were quantified (right panel). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Each treatment group was compared with the control group.\u003c/p\u003e\n\u003cp\u003eDHT, dihydrotestosterone; Flu, flutamide.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6321751/v1/8fa79dd0f73345acb7ff5702.jpg"}],"financialInterests":"","formattedTitle":"Acetylation reader BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS), a prevalent endocrine-metabolic disorder affecting women during adolescence and reproductive years, demonstrates a global prevalence of 8\u0026ndash;13%. PCOS is characterized by hyperandrogenism, blocked dominant follicle formation, and anovulation or oligoovulation. Granulosa cells (GCs), recognized as the most critical ovarian cell population, drive oocyte growth alongside maturation while facilitating both follicular development and formation. Oxidative stress coupled with NLRP3 inflammasome-driven pyroptosis in GCs serves as a key pathogenic mechanism underlying hormonal imbalance, subfertility, and PCOS-related ovarian dysfunction, manifesting through oocyte developmental arrest, follicular atresia, and anovulation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thioredoxin-interacting protein (TXNIP), a protein that interacts with thioredoxin (TRX), plays a role in the development and progression of diseases such as diabetes, neurodegenerative disorders, and chronic kidney diseases by regulating NLRP3 inflammasome activation and glucose/lipid metabolism [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies indicate that aberrant TXNIP upregulation is correlated with decreased insulin sensitivity in PCOS [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], but the precise upstream mechanisms underlying TXNIP/NLRP3 activation in PCOS remain unclear.\u003c/p\u003e \u003cp\u003eTXNIP is regulated at multiple levels, including transcription, epigenetic modifications, mRNA stability, and protein stability [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. MicroRNAs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], circular RNAs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], oncogenes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], endoplasmic reticulum stress (ERs) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], cytokines and growth factors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] are associated with pathways/factors that affect TXNIP expression. Glucose-induced TXNIP levels are strongly linked to carbohydrate response elements and the associated transcription factors, such as the ChREBP/MondoA: Mlx complex [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], FOXO1 and MAPK [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In breast cancer, the mRNA decay factor ZFP36 targets TXNIP mRNA transcripts for degradation mediated by receptor tyrosine kinases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Activated AMPK phosphorylates TXNIP, resulting in rapid degradation of its protein [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, upregulation of TXNIP due to aberrant lysines 9/14/27 acetylation of histone H3 mediated via histone acetyltransferase p300 [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] may be the main cause of NLRP3 inflammasome activation. Although numerous studies have demonstrated the upstream regulatory mechanisms of TXNIP, the precise epigenetic regulatory mechanisms regulating TXNIP in PCOS remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eHistone acetylation, a frequently encountered form of epigenetic modification, promotes gene transcription activation by facilitating the dissociation of DNA from the histone octamer and relaxing the nucleosome structure. The process is primarily regulated by histone acetyltransferases (HATs), histone deacetylases (HDACs), and acetylation-reading proteins [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Bromodomain-containing protein 4 (BRD4) is an acetylation reader that binds to specific acetylated lysine residues on histone tails. Subsequently, it recruits various transcription factors and transcriptional coactivators to target gene promoters and enhancers to promote gene transcription [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our previous research demonstrated that the aberrantly increased BRD4 and subsequent androgen receptor (AR) transcriptional activation are important triggers of ovarian fibrosis in PCOS [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, whether BRD4 regulates TXNIP in PCOS is currently unknown.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the epigenetic mechanism by which the acetylation reader BRD4 and the transcription factor AR promote TXNIP transcriptional activation, leading to ovarian pathology in PCOS. We provide strong evidence that upregulation of BRD4 and AR enhances TXNIP expression, subsequently triggering NLRP3 inflammasome activation and metabolic disturbances in PCOS. Our study aims to provide new epigenetic-based drug targets for PCOS and a theoretical foundation for developing new clinical therapies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and experimental protocols\u003c/h2\u003e \u003cp\u003eThree-week-old female Sprague-Dawley rats (Junke Biotechnology Corporation, China) were randomly divided into 4 groups (6 rats per group, n\u0026thinsp;=\u0026thinsp;24, 55\u0026ndash;65 g): (1) vehicle control (oil); (2) dehydroepiandrosterone (DHEA); (3) ruscogenin; (4) a combination of DHEA and ruscogenin treatment (D\u0026thinsp;+\u0026thinsp;R). All rats were housed in a specific pathogen-free (SPF) environment (Jiangsu Key Laboratory of Molecular Medicine), maintained at 24\u0026deg;C with a 12-hour light/dark cycle and humidity levels of 45%-65%. Free access to water and food was supplied. Rats in the DHEA treatment group received a subcutaneous DHEA (6 mg/100g body weight) injection daily for 35 consecutive days since postnatal day 23 to establish a PCOS-like rat model. The vehicle control rats received a hypodermic injection of experimental-grade soybean oil (Yuanye Biological Technology Corporation, China) with an identical volume. From the third week after DHEA or oil administration, the rats in the ruscogenin and D\u0026thinsp;+\u0026thinsp;R group were given ruscogenin (1 mg/kg, MCE, USA) by gavage once a day for 21 days. All the rats were euthanized to collect ovaries and blood at the end of the experiment. Unilateral ovaries were fixed with 4% paraformaldehyde (Servicebio, China) and embedded in paraffin for tissue sectioning. The remaining ovaries and serum were all preserved at -80℃ for further experiments. The experiments were carried out following the principles and guidelines for the use of laboratory animals and were approved by the institutional research animal committee of Nanjing University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Estrous cycle analysis\u003c/h2\u003e \u003cp\u003eVaginal cells were collected by lavage with saline and smeared from the indicated rats at 9:30AM for 10 consecutive days. The predominant cell types in the vaginal smears were determined under light microscopy using Giemsa staining. The stages of the estrous cycle were also determined. The proestrus stage was marked by predominantly nucleated cells; the estrus stage was represented by cornified squamous epithelial cells; the metestrus stage was typified by a combination of cornified cells and leukocytes; and the diestrus stage exhibited a predominance of leukocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT)\u003c/h2\u003e \u003cp\u003eAfter a 12-hour fast, rats were injected with 20% glucose (2 g/kg; i.p.) or 0.75 U/kg insulin (i.p.). Blood samples were collected from the tail vein at time points of 0, 15, 45, 60 and 120 minutes after glucose or insulin injection to evaluate glucose concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Determination of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels\u003c/h2\u003e \u003cp\u003eBlood samples were taken from the inferior vena cava of rats. Next, serum was separated using centrifugation and stored at -80℃. Serum LH and FSH levels were determined using the enzyme-linked immunosorbent assay (ELISA) kits (Elabscience Biotechnology, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Isolation of GCs\u003c/h2\u003e \u003cp\u003e Female Sprague-Dawley rats were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG, Sansheng Biological Technology Corporation, China) (20 IU) to promote follicular development, followed by euthanasia 48 hours later. The ovaries were isolated and the follicles were peeled off by manipulation with micro tweezers under a dissecting microscope. Next, the follicles were punctured to release GCs, and the cell suspension was filtered through a 70-\u0026micro;m cell strainer to remove residual cells and debris clumps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell culture and treatment\u003c/h2\u003e \u003cp\u003ePrimary rat GCs, a human granulosa-like tumor cell line (KGN cells) and human embryo kidney HEK-293T cells were cultured in DMEM-F12 or DMEM medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Gibco) at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were treated with various concentrations of dihydrotestosterone (DHT; 0.5, 2, 5 \u0026micro;M; Meilun Biological Technology Corporation, China) for 48 hours to construct a PCOS-like model characterized by hyperandrogenism in vitro. Flutamide (20, 50 \u0026micro;M; Selleck, China) and JQ1 (1 \u0026micro;M; MCE) were added into cells for 24 hours with or without DHT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Hematoxylin and eosin (H\u0026amp;E)\u003c/h2\u003e \u003cp\u003eOvary tissues were processed for histopathology analysis following standard paraffin fixation, sectioning and H\u0026amp;E staining. The sections were viewed under an optical microscope (Leica Microsystems, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eEmbedded ovaries were sectioned at 4 \u0026micro;m and stained with specific antibodies against TXNIP (1:100; HUABIO, China). Next, the sections were incubated with a secondary antibody goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP conjugate. Images were captured using an optical microscope (Leica Microsystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. RNA isolation and real-time quantitative PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from rat ovaries, GCs, and KGN cells was extracted using the TRIzol reagent (Beyotime, China), and cDNA was synthesized with a reverse transcription kit (Vazyme Biotech, China). qRT-PCR was performed with the ABI Viia 7 Real-Time PCR system (ABI, USA) using the SYBR Green PCR Master Mix (Vazyme Biotech), and the primers are shown in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The critical threshold cycle (Ct) value was determined for each reaction, which was transformed into relative quantification data using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The housekeeping gene β-actin was used as an internal control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences for rats qRT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-TTCCTTCCTGGGTATGGAAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GAGGAGCAATGATCTTGATC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTXNIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-ATCATGGCGTGGCAAGAGTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TTTCTTGGAGCCAGGGACAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-TCTGGTTGTCACTACG GAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TGCAATCATTTCTGCTGGCAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CAGCGATCAACAGGCGAGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGAGATATCCCAGCAAACCTATCCA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-TTGAGTGTCTGGTGCTCGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATGGGGTGCTCTGTTCCAAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CTACCTATGTCTTGCCCGTGGAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GGGAACATCACACACTAGCAGGTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-GGACCAACACAGGCAAGCACTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ACAAGTTCTTGCAGGTCAGGTTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-ACCTGTGGACTCTCAGACAAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGCAGGCAGCTAGGATTACG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences for human qRT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CGTGGACATCCGCAAAGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GAAGGTGGACAGCGAGGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTXNIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CTGGCGTAAGCTTTTCAAGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGTGCACAAAGGGGAAACAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Western blot\u003c/h2\u003e \u003cp\u003eOvaries, GCs, KGN cells, and HEK-293T cells were lysed using RIPA lysis buffer (Beyotime) containing 1 mM phosphatase inhibitor (MCE) and 1 mM protease inhibitor cocktail (MCE). Equal amounts of total proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the protein bands were transferred onto polyvinylidene difluoride membranes (Merck Millipore, USA). Target bands were incubated with corresponding primary antibodies against TXNIP (1:1000; HUABIO), NLRP3 (1:1000; CST, USA), GSDMD (1:1000; Proteintech, USA), IL-1β (1:1000; Abcam, UK), ASC (1:1000; Abcam), IL-18 (1:1000; HUABIO), AR (1:1000; Abcam), BRD4 (1:1000; CST), acetylated histone H3 (Ac-H3; 1:1000; Abcam) and β-actin (1:20000; Fudebio, China) overnight at 4℃, followed by the addition of HRP-labeled secondary antibodies (1:20000; Fudebio). The blots were visualized using chemiluminescent detection (Merck Millipore). The band intensity was quantified with Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Immunofluorescence\u003c/h2\u003e \u003cp\u003eGCs were seeded in 24-well culture plates with wells containing cell climbing slices. Sections were fixed in 4% paraformaldehyde (Servicebio) for 30 minutes at 25\u0026deg;C, and then permeabilized with 0.3% Triton X-100 (Beyotime). After washing with PBS three times, the cells were blocked with 3% bovine serum albumin for 30 minutes at 25\u0026deg;C. Cells were incubated with antibodies against TXNIP (1:100), NLRP3 (1:100) and ASC (1:100) overnight at 4\u0026deg;C. After washing with phosphate buffered saline containing 0.5% of Tween-20 three times, cells were incubated with fluorescent secondary antibodies, including anti-rabbit Cy3 and anti-mouse Alexa 488 (Beyotime), at 25\u0026deg;C for 2 hours. Nuclei were counterstained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI, Beyotime) at a dilution of 1:2000 for 30 minutes and photographed using an Olympus laser scanning confocal microscope (FV3000, Japan). The fluorescence intensity was analyzed with Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Lentiviral vector preparation and infection\u003c/h2\u003e \u003cp\u003eThe TXNIP lentiviral particles for rats were purchased from GeneChem (China). Primary GCs were seeded in culture plates. At 70% confluence, cells were incubated with TXNIP lentiviral particles (MOI\u0026thinsp;=\u0026thinsp;20) or control lentiviral particles along with HitransG P in DMEM-F12 medium. The transfection efficiency was determined 72 hours later by quantifying the intensity of green fluorescence and protein analysis using western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. RNA interference\u003c/h2\u003e \u003cp\u003eTXNIP knockdown in GCs and KGN cells was performed with small interfering RNA (siRNA). Scrambled RNA oligos of the rat TXNIP gene (sense oligo: 5\u0026rsquo;-GGACGUGAUUCCUGAAGAUTT-3\u0026rsquo;; antisense oligo: 5\u0026rsquo;-AUCUUCAGGAAUCACGUCCAT-3\u0026rsquo;) and human TXNIP gene (sense oligo: 5\u0026rsquo;-GAGAAUACAUGUUCCCGAA-3\u0026rsquo;; antisense oligo: 5\u0026rsquo;-UUCGGGAACAUGUAUUCUC-3\u0026rsquo;) were designed by Keygen Biotech (China). Cells were transfected with the siRNA using Lipofectamine 2000 (Invitrogen, USA) followed by incubation for 72 hours. Next, DHT (5 \u0026micro;M) was added and the plate was incubated for 48 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Dual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eWild type human TXNIP promoter (TXNIP WT), mutant human TXNIP promoter (TXNIP MUT), BRD4 negative control plasmid (NC-BRD4), and BRD4 overexpression plasmid (OE-BRD4) were designed by Tsingke Biotech (China). A Renilla luciferase reporter was used as an internal control. The mutant form of hTXNIPp-luc was constructed in which the BRD4 and AR responsive element CAGAACAGAGAGAAC was mutated to GTCTTGTCTCTCTTG. Plasmids were transfected into HEK-293T cells using GenJetTM Plus in Vitro DNA Transfection Reagent (SignaGen, USA) with a 48-hour incubation following the manufacturer\u0026rsquo;s instruction. The transfected cells were treated with DHT (5 \u0026micro;M) to activate AR, or treated with JQ1 (1 \u0026micro;M) to inhibit BRD4, or treated with flutamide (50 \u0026micro;M) to inhibit AR. Luciferase activities were determined with a GloMax Luminometer using a dual luciferase reporter assay kit (Vazyme Biotech). Luciferase activities were normalized to Renilla luciferase levels and expressed as relative fold changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Co-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eThe lysates of HEK-293T cells were first immunoprecipitated using antibodies to BRD4 (CST), AR (Abcam), or isotype-matched immunoglobulin followed by treatment with Protein A/G Magnetic Beads (Vazyme Biotech). Next, the immunoprecipitants were analyzed using western blot with antibodies to BRD4 (CST), AR (Abcam), or Ac-H3 (Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Chromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eChIP assay was performed following the protocol of ChIP assay Kit (Beyotime). Ovarian tissues were processed into single-cell suspensions. Formaldehyde was added to the single-cell suspensions or HEK-293T cells for cross-linking the target proteins with genomic DNA. These samples were then treated with ultrasound to break the genomic DNA into 200\u0026ndash;1000 bp fragments. The target proteins, including BRD4 (CST), AR (Abcam) or Ac-H3 (Abcam), and their bound DNA fragments were co-immunoprecipitated, purified, and amplified using PCR. The primer sequences for ChIP-PCR are provided in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences for rats ChIP-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTXNIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-TGGATGAGGTTCAGGGTCTCG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TTGGCTACTTGGTCCTTGTTTA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences for human ChIP-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTXNIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-GAAGTGGGAGATAATGAGCG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CGTGCCTGTGCTGTTCGTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Statistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 7.00 software. A two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was used for comparing two groups. One-way analysis of variance (ANOVA) was used for comparing more than two groups, followed by the Bonferroni post hoc test. The Kruskal-Wallis test was performed for the comparisons of data with nonnormal distribution or heterogeneity of variance. The quantitative data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) with at least three independent experiments, with n indicating the number of replicates. A \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. TXNIP is abnormally increased in PCOS-like rats\u003c/h2\u003e \u003cp\u003eA PCOS-like model was established through daily subcutaneous injections of DHEA for 35 consecutive days. Compared with the oil-treated controls, DHEA-treated ovaries showed marked polycystic changes, lacked corpus luteum, and reduced ovarian size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunohistochemical analysis of TXNIP confirmed dramatic upregulation in ovarian GCs of PCOS-like rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). qRT-PCR and western blotting revealed a marked increase in TXNIP in PCOS ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Consistently, in a DHT-induced PCOS-like in vitro model using GCs and KGN cells, the expression of TXNIP were significantly elevated at mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H). Both increased nuclear and cytoplasmic localization of TXNIP was determined using immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Taken together, these results suggest that the aberrant TXNIP upregulation may contribute to ovarian pathological changes in PCOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. TXNIP is a critical driver of NLRP3 inflammasome activation in PCOS\u003c/h2\u003e \u003cp\u003eTo investigate the key role of TXNIP in NLRP3 inflammasome activation, we overexpressed TXNIP in GCs via lentiviral transduction. The overexpression efficiency of TXNIP was determined using western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which consistently increased protein levels of NLRP3 and pyroptosis-related proteins, including GSDMD, IL-1β, ASC, and IL-18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Moreover, TXNIP was knocked down using siRNA in primary GCs and KGN cells separately. TXNIP silencing markedly reversed the elevated expression of NLRP3 inflammasome components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-J). These results indicate that aberrant TXNIP acts as a key regulator of NLRP3 inflammasome activation and pyroptosis in ovarian GCs of PCOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Inhibition of TXNIP/NLRP3 ameliorates metabolic abnormalities and ovarian pathology in PCOS\u003c/h2\u003e \u003cp\u003eRuscogenin, a crucial steroidal sapogenin derivative, has been demonstrated to effectively suppress the activation of the TXNIP/NLRP3. Ruscogenin was used to treat PCOS-like rats and KGN cells, followed by an examination of relevant indicators. Our findings revealed that ruscogenin treatment reversed the upregulation of TXNIP/NLRP3 and pyroptosis-related proteins, including GSDMD, IL-1β, ASC, and IL-18 in both in vivo and in vitro models (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further evaluated the effect of TXNIP/NLRP3 inhibition on PCOS-associated metabolic abnormalities and ovarian dysfunction. The results showed that ruscogenin treatment did not effectively restore the reduced body weight in PCOS-like rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), but it increased ovarian size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, ruscogenin treatment improved glucose tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), normalized serum levels of LH and FSH as well as the LH/FSH ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), restored ovarian health as evidenced by decreased preantral and cystic follicle counts with a concomitant increase in corpora lutea formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and ameliorated the disrupted estrous cyclicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Taken together, these results suggest that inhibition of TXNIP/NLRP3 markedly reversed metabolic disorders and ovarian dysfunction in PCOS-like rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. AR-selective inhibition attenuates TXNIP and NLRP3 inflammasome activation\u003c/h2\u003e \u003cp\u003eBioinformatic analysis of the NCBI Gene Expression Omnibus (GEO) database (GSE34526) showed that the TXNIP expression was profoundly elevated in women with PCOS compared with healthy women and was positively correlated with the AR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Both DHEA and DHT profoundly increased AR activity in vivo and in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). To further elucidate the upstream mechanisms responsible for the aberrant increase in TXNIP expression, we treated GCs and KGN cells with the AR inhibitor flutamide in vitro. Selective AR inhibition extensively reduced both mRNA and protein levels of TXNIP, NLRP3, and inflammasome activation-related factors in the DHT-induced in vitro PCOS-like model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G and Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. AR-selective inhibition alleviates TXNIP transcription\u003c/h2\u003e \u003cp\u003eBioinformatics analysis using the JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.elixir.no/\u003c/span\u003e\u003cspan address=\"https://jaspar.elixir.no/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicted high-affinity AR-binding motifs within the TXNIP promoter region, with prediction scores of 15.428571 (rat) and 13.350983 (human) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). To validate AR as a transcription factor of TXNIP, we performed ChIP assays to confirm AR binding to the TXNIP promoter regions containing AR-binding motifs in the DHEA-treated ovaries and HEK-293T cells, and this interaction was abolished by flutamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). TXNIP promoter-luciferase reporter plasmid was transfected into HEK-293T cells, and we found that flutamide attenuated the aberrant TXNIP-LUC transactivation induced by the AR agonist DHT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Taken together, these results indicate that AR directly promotes TXNIP transcriptional activation and subsequent NLRP3 inflammasome activation, both of which are effectively inhibited by flutamide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. BRD4 activates TXNIP via AR and acetylated histone-associated transcriptional activation\u003c/h2\u003e \u003cp\u003eBRD4 is a well-known acetylation reader that facilitates gene transcriptional activation through the recruitment of transcription factors and transcriptional coactivators. Building on our prior demonstration that AR is a transcription factor for TXNIP, we hypothesized that BRD4 binds the TXNIP promoter and recruits AR to promote TXNIP transcription. ChIP assays showed that BRD4 was inducibly bound to the AR motif-containing TXNIP promoter enriched with Ac-H3 in DHT-induced PCOS-like model in vitro, which was inhibited by JQ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). In addition, we constructed a TXNIP promoter-luciferase reporter TXNIP-LUC and a mutant reporter muTXNIP-LUC, in which the BRD4 motif TCAGAACAGAGAGAAC was replaced by GTCTTGTCTCTCTTG. The indicated plasmids were transfected into HEK-293T cells and then the cells were treated with plasmids for BRD4 overexpression or a negative control, along with the BRD4-selective inhibitor JQ1. We found that BRD4 overexpression increased the transactivation of TXNIP-LUC, but not muTXNIP-LUC, and this effect was significantly blocked by JQ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further treated HEK-293T cells with the AR agonist DHT followed by JQ1. The results showed that DHT treatment increased the transactivation of TXNIP-LUC, but not muTXNIP-LUC, which was blocked by JQ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, we confirmed that BRD4 was inducibly associated with AR and Ac-H3 in DHT-treated HEK-293T cells, which was inhibited by JQ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). To summarize, these results strongly support that BRD4 promotes TXNIP transcriptional activation in PCOS through inducible interaction with the AR and Ac-H3, culminating in its chromatin enrichment at the TXNIP promoter.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we demonstrated that histone acetylation-dependent TXNIP transcriptional activation is a critical determinant in driving NLRP3 inflammasome activation and ovarian dysfunction in PCOS ovarian GCs. We found that the TXNIP/NLRP3 inhibitor ruscogenin effectively reversed NLRP3 inflammasome activation and metabolic dysfunction in PCOS-like rats. Furthermore, BRD4 drives TXNIP transcriptional activation via inducible interaction with the AR and Ac-H3, coupled with chromatin recruitment to the TXNIP promoter. Notably, this regulatory axis is suppressed by pharmacological inhibition of BRD4 or AR. Therefore, aberrant TXNIP activation mediated by histone acetylation is an important epigenetic mechanism for NLRP3 inflammasome activation and ovarian pathophysiology in PCOS.\u003c/p\u003e \u003cp\u003eAs a key α-arrestin family member, TXNIP promotes apoptotic signaling through TRX inhibition and subsequent ASK-1 activation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while simultaneously driving NLRP3 inflammasome assembly via redox-dependent mechanisms [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], contributing to neurodegenerative disorders [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], diabetes/complications [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and cardiovascular diseases [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Previous studies have demonstrated that high glucose promotes TXNIP overexpression, leading to β-cell apoptosis, disrupted glucose homeostasis, and impaired glucose uptake in skeletal muscle, liver, and adipose tissues [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, increased TXNIP induces G0/G1 arrest by inhibiting cyclin A [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we discovered that TXNIP/NLRP3 inhibition ameliorated ovarian histopathological alterations and endocrine-metabolic abnormalities, suggesting aberrant TXNIP/NLRP3 is a crucial element of ovarian dysfunction in PCOS.\u003c/p\u003e \u003cp\u003eDemonstration of AR-activated TXNIP transcription in promoting aberrant TXNIP overexpression and GCs pyroptosis in PCOS is a crucial discovery of this study. Our previous research has shown that sustained AR activation is an important driver of various pathologies in DHEA-induced PCOS-like rats, including ovarian fibrosis and anovulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Complementarily, AR knockout reverses DHT-induced reproductive and metabolic abnormalities in mice [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We analyzed the TXNIP promoter using JASPAR and found the AR binding motifs, which are located very close to the transcription start site with high prediction score (rat: 15.428571; human: 13.350983). We showed that AR can directly bind to the TXNIP promoter region, and TXNIP transcription was blocked by AR inhibition and the mutation of AR binding motif on the TXNIP promoter. It is noteworthy that our data does not exclude the possibility of other transcription factors contributing to TXNIP activation in PCOS, but establishes a basis for future research.\u003c/p\u003e \u003cp\u003eThe bromodomain and extra-terminal domain (BET) family, comprised of BRD2, BRD3, BRD4, and bromodomain testis-specific protein (BRDT), orchestrates genomic regulation and cellular homeostasis through dynamic recognition of acetylated chromatin substrates. These epigenetic readers bind acetylated histones and non-histone protein, while exhibiting DNA/RNA-binding capacity to coordinate transcriptional activation, DNA damage repair, and cell fate determination via chromatin remodeling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. BRD4, the most studied member, is involved in the pathology of cancer, inflammatory diseases, viral infections, and neurological disorders [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. BRD4 also interacts with non-acetylated transcription factors like c-Jun, AP2, Myc, YYA, V/EBPβ, and p53 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The availability of JQ1, a specific inhibitor of BRD4, offers a valuable tool for scientific investigation. In this study, we observed that BRD4 recruits AR to the TXNIP promoter region containing Ac-H3, thereby facilitating TXNIP transcription, which is suppressed by JQ1. These data demonstrate that beyond targeted therapies, epigenetic modulation via histone acetylation may represent a novel therapeutic strategy to mitigate ovarian GC developmental disorders and protect ovarian function in PCOS.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study reveals BRD4 recognizes and inducibly binds to Ac-H3 at the TXNIP promoter, recruits AR to drive TXNIP transcription, which ultimately activating NLRP3 inflammasome with concomitant metabolic derangements and ovarian functional impairment in PCOS-like rats. Our study reveals a crucial epigenetic mechanism of TXNIP activation, and provides a novel target for the treatment of PCOS with clinical implications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAc-H3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacetylated histone H3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eone-way analysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eandrogen receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebromodomain and extra-terminal structural domain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBRDT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebromodomain testis-specific protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBRD4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebromodomain-containing protein 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echromatin immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCo-IP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eco-immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecritical threshold cycle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4\u0026rsquo;,6-diamidino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edehydroepiandrosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edihydrotestosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD\u0026thinsp;+\u0026thinsp;R\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ea combination of DHEA and ruscogenin treatment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendoplasmic reticulum stress\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efollicle stimulating hormone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egranulosa cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGEO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Expression Omnibus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHATs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehistone acetyltransferases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDACs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehistone deacetylases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPGTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneal glucose tolerance test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eITT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einsulin tolerance test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKGN cells\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman granulosa-like tumor cell line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eluteinizing hormone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNC-BRD4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBRD4 negative control plasmid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOE-BRD4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBRD4 overexpression plasmid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolycystic ovary syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epregnant mare serum gonadotropin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereal-time quantitative PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emeans\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmall interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003especific pathogen-free\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethioredoxin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTXNIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethioredoxin-interacting protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTXNIP MUT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emutant human TXNIP promoter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTXNIP WT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild type human TXNIP promoter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted according to the principles and guidelines of the Institutional Animal Care and approved by the Institutional Research Animal Committee of Nanjing University. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript is approved by all authors for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and material will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Jiangsu Provincial Department of Science and Technology (BE2022755), Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases (Obstetrics and Gynecology Hospital, Fudan University, 20DZ2271300) and National Natural Science Foundation of China (82201795 and 81971346).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYajing Weng: Writing-review \u0026amp; editing, Writing-original draft, Methodology, Investigation, Conceptualization. Yi Zhang: Methodology, Investigation. Wei Dong: Software, Conceptualization. Zhengquan Zhu: Methodology, Software. Zou Xiang: Writing-review \u0026amp; editing, Supervision. Guijun Yan: Resources. Hongwei Wang: Visualization, Data curation. Yanting Wen: Visualization, Project administration. Min Liu: Visualization, Funding acquisition. Daojuan Wang: Writing-review \u0026amp; editing, Supervision. Yong Wang: Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to Professor Yong Wang and everyone in the laboratory for their help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFan X, Bialecka M, Moustakas I, Lam E, Torrens-Juaneda V, Borggreven NV, Trouw L, Louwe LA, Pilgram GSK, Mei H, van der Westerlaken L (2019) Chuva de Sousa Lopes SM. Single-cell reconstruction of follicular remodeling in the human adult ovary. 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BioEssays 43(12):e2100180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu SY, Nin DS, Lee AY, Simanski S, Kodadek T, Chiang CM (2016) BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression. Cell Rep 16(6):1733\u0026ndash;1748\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"polycystic ovary syndrome, bromodomain-containing protein 4, androgen receptor, thioredoxin-interacting protein, NLRP3 inflammasome","lastPublishedDoi":"10.21203/rs.3.rs-6321751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6321751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycystic ovary syndrome (PCOS) is an endocrine disorder characterized by the developmental arrest and dysfunction of ovarian granulosa cells (GCs), serving as a major cause of infertility among women of reproductive age. Persistent activation of thioredoxin-interacting protein (TXNIP) due to aberrant histone acetylation modifications of transcription is a potential trigger; however, its precise upstream regulatory mechanism remains poorly understood. In this study, we found that TXNIP was aberrantly upregulated in both the dehydroepiandrosterone (DHEA)-induced PCOS-like rat model and the dihydrotestosterone (DHT)-induced primary GCs PCOS-like model in vitro. The TXNIP/NLRP3 inhibitor ruscogenin and the small interfering RNA (siRNA) targeting TXNIP remarkably inhibited NLRP3 inflammasome activation, subsequently reversing aberrant reproductive and metabolic phenotypes in PCOS-like models. Further bioinformatic analysis revealed that the promoter region of TXNIP contains binding motifs of bromodomain-containing protein 4 (BRD4) and androgen receptor (AR). BRD4 and AR exhibited inducible binding to the histone H3 acetylation-enriched TXNIP promoter, whereas intervention with the BRD4-selective inhibitor JQ1 and the AR-selective inhibitor attenuated this binding, leading to subsequent downregulation of TXNIP transcription that ultimately resulted in NLRP3 inflammasome suppression. Our data indicate that BRD4 upregulation and the resultant TXNIP transcriptional activation are crucial regulatory pathways for NLRP3 inflammasome activation, resulting in associated reproductive and metabolic abnormalities in ovarian GCs from PCOS.\u003c/p\u003e","manuscriptTitle":"Acetylation reader BRD4-driven TXNIP transcription enhances NLRP3 inflammasome activation in PCOS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 11:33:22","doi":"10.21203/rs.3.rs-6321751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-05-02T22:15:23+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-01T15:14:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T22:38:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-28T04:54:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-03-27T11:11:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6bc0bc6-39b1-464c-a897-6429eee13f31","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:00:31+00:00","versionOfRecord":{"articleIdentity":"rs-6321751","link":"https://doi.org/10.1007/s00018-025-05925-0","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-11-26 15:57:16","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-04-21 11:33:22","video":"","vorDoi":"10.1007/s00018-025-05925-0","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05925-0","workflowStages":[]},"version":"v1","identity":"rs-6321751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6321751","identity":"rs-6321751","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}