Intestinal homeostasis is essential for systemic health and longevity, and its disruption contributes to colitis and age-related gut dysfunction. N-acetylneuraminic acid (Neu5Ac), a major form of sialic acid enriched in bird's nest and human milk, exhibits immunomodulatory and antioxidant properties, yet its physiological role in intestinal integrity remains unclear. Here, we demonstrate that oral Neu5Ac supplementation preserves intestinal homeostasis in both natural aging and dextran sulfate sodium (DSS)-induced colitis models. Neu5Ac enhanced epithelial barrier integrity, increased tight-junction proteins, and maintained mucosal architecture. It alleviated systemic and local inflammation by suppressing macrophage infiltration and polarization toward the pro-inflammatory phenotype while maintaining tissue-reparative macrophages. Neu5Ac also selectively enriched butyrate-producing bacterial taxa, including Butyricimonas synergistica and Parabacteroides goldsteinii, thereby increasing fecal butyrate levels, without globally altering microbial diversity. Mechanistically, transcriptomic profiling implicated the HIF-1 signaling pathway in mediating the anti-inflammatory effects of Neu5Ac. Consistently, Neu5Ac reduced colonic HIF-1α protein signals, predominantly localized to inflammatory cells, and suppressed HIF-1α expression in LPS-stimulated macrophages. Neu5Ac promoted epithelial regeneration and mitigated senescence-associated p53 activation, thereby restoring gut homeostasis. Importantly, Neu5Ac exhibited excellent biosafety in vivo. Together, these findings identify Neu5Ac as a bioactive nutritional molecule that sustains intestinal homeostasis through coordinated epithelial, immune, and microbial modulation, offering a promising preventive strategy against aging- and inflammation-driven intestinal disorders.

Objective This trial investigates the efficacy of neoadjuvant therapy using rezvilutamide combined with androgen deprivation therapy (ADT), with or without docetaxel, in treating oligometastatic hormone-sensitive prostate cancer (omHSPC).Methods and analysis This prospective, open-label, multicentre trial aims to enrol 100 patients newly diagnosed with omHSPC (defined as ≤5 bone or lymph node metastases confirmed by conventional imaging, without visceral metastasis) who must express a desire to undergo surgery. All patients undergo a prostate-specific membrane antigen positron emission tomography/CT (PSMA-PET/CT) scan at enrolment or within 4 weeks before enrolment to assess and confirm the number of metastases at baseline. Scans should be performed before initiating ADT to avoid compromising test sensitivity. Then patients will be allocated into groups in parallel according to their own preferences: one group will receive an LHRH agonist or antagonist for 24 weeks to maintain continuous ADT or have undergone bilateral orchiectomy. Treatment with rezvilutamide will be maintained daily. The other group will be scheduled to complete up to six cycles of docetaxel within 24 weeks, with maintenance of continuous ADT and rezvilutamide for 24 weeks. Both groups will receive a conventional imaging evaluation at the 12th week. After neoadjuvant therapy, patients will undergo conventional imaging and a second PSMA-PET/CT assessment, followed by cytoreductive radical prostatectomy within the subsequent 6 weeks. After surgery, patients may choose to continue with ADT or rezvilutamide at their own discretion, until disease progression. The primary endpoint is pathological complete response, defined as the absence of residual viable tumour cells in the tumour bed on pathological evaluation of the postoperative specimen. Secondary endpoints include 1 year biochemical progression-free survival, overall survival, radiographic progression-free survival, time to prostate-specific antigen progression, quality of life scores (total and subscale) assessed using the Functional Assessment of Cancer Therapy-Prostate questionnaire, time to symptomatic progression, time to deterioration in Eastern Cooperative Oncology Group performance status, the interval from enrolment to an increase in score from baseline, the proportion of patients with a ≥30% reduction in prostate volume on imaging before cytoreductive surgery compared with pre-neoadjuvant therapy, minimal residual disease and major pathological response. The study plans to enrol a total of 100 patients. Patient recruitment for this study is scheduled to begin in May 2025.Ethics and dissemination This has been approved by the Ethics Committee in Clinical Research of the First Affiliated Hospital of Wenzhou Medical University (number KY2024-231). Results will be published in peer-reviewed publications.Discussion This study is expected to provide prospective evidence on the feasibility and potential clinical value of rezvilutamide combined with ADT, with or without docetaxel, as neoadjuvant treatment for newly diagnosed omHSPC.Trial registration number Chinese Clinical Trial Registry (ChiCTR2400093262).

Astrocyte polarization into a complement component 3 positive (C3+) neurotoxic A1 phenotype is a hallmark of the inflammatory cascade following spinal cord injury (SCI), where it plays a pivotal role in exacerbating neuronal senescence, inhibiting axonal regeneration, and limiting functional recovery. Although cyclic adenosine monophosphate (cAMP) signaling has been implicated in modulating astrocyte activation, the precise molecular pathways regulating C3+ astrocyte differentiation and their downstream effects on neuroinflammation remain insufficiently understood. In this study, we identified cAMP response element binding protein (CREB) as a critical regulator of astrocyte polarization. Activation of CREB using dibutyryl-cAMP (db-cAMP), a cell-permeable cAMP analog, led to a 43% reduction in the number of C3+ astrocytes and a 15% decrease in C3 protein expression within 24 h in vitro. Functionally, the suppression of neurotoxic astrocytes alleviated neuronal senescence and reshaped the immune microenvironment by reducing classically activated macrophage (M1 macrophage) polarization, enhancing alternatively activated macrophage (M2 macrophage) responses, and promoting efferocytosis. Our transcriptomic profiling and pathway enrichment analyses further revealed that the CREB/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling axis mediates the anti-inflammatory and neuroprotective reprogramming of astrocytes. We further engineered an adhesive microneedle patch (db-cAMP@MP) capable of sustained, targeted delivery of db-cAMP into the injured spinal cord lesion site to achieve localized therapeutic modulation of astrocyte phenotypes in vivo. The application of db-cAMP@MP following SCI effectively activated CREB signaling in spinal astrocytes, resulting in reduced C3+ astrocyte accumulation, decreased neuronal senescence marker expression, and a 1.4-fold increase in motor evoked potential (MEP) amplitude and motor functional recovery, with a Basso Mouse Scale (BMS) score of 3.8 ± 0.83 in the SCI model. Our findings establish CREB as a central molecular switch in astrocyte-driven neuroinflammation and identify db-cAMP@MP as a promising, precision-targeted therapeutic platform to modulate astrocyte phenotypes and promote functional repair after SCI.

Background: Accumulating evidence indicates that long non-coding RNA (lncRNA) miR17HG plays important roles in tumorigenesis in various cancers. However, its underlying effect on bladder cancer (BC) remains to be explored. Objective: This study aims to elucidate the function and molecular mechanisms of lncRNA miR17HG in the proliferation and metastasis of BC. Methods: BC tissues and matched para-cancerous tissues were collected from our hospital. Oncology functional assays, quantitative reverse-transcription polymerase chain reaction, and western blotting were conducted to confirm the role of lncRNA miR17HG in BC development. The interations among lncRNA miR17HG, miR-144-3p and AXIN2 were verified via luciferase reporter assay. Nude mouse subcutaneous tumor models were established to investigate the effect of lncRNA miR17HG on BC tumor growth. Results: LncRNA miR17HG and miR-144-3p were significantly upregulated, while AXIN2 was downregulated in BC tissues. Knockdown of lncRNA miR17HG or inhibition of miR-144-3p suppressed cell proliferation, migration and invasion, whereas miR-144-3p mimic exerted the opposite effects. Mechanistically, lncRNA miR17HG sponged miR-144-3p to regulate the expression of AXIN2. Furthermore, lncRNA miR17HG knockdown effectively suppressed tumor xenograft growth in mice. Conclusion: In summary, this study identifies a novel lncRNA miR17HG/miR-144-3p/AXIN2 pathway in BC and demonstrate that this regulatory axis may serve as a promising therapeutic target for BC.

CONTEXT:Dipeptidyl peptidase-4 inhibitors (DPP-4i) serve as an incretin-based hypoglycemic class for the treatment of type 2 diabetes (T2D). DPP-4i have been reported to produce a pleiotropic effect on lipid profiles in addition to regulation of glucose homeostasis. OBJECTIVE:The aim of this systematic review and meta-analysis was to quantitatively evaluate the impact of DPP-4i on lipid parameters in patients with T2D. DATA SOURCES:PubMed, Embase, and The Cochrane Library were systematically searched for randomized controlled trials. DATA EXTRACTION:Trials were identified if changes in lipid parameters, including low-density-lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglycerides (TG), high-density-lipoprotein cholesterol (HDL-C), non-HDL-C, and apolipoprotein B (ApoB) were reported. DATA ANALYSIS:A total of 95 publications were identified. DPP-4i significantly reduced levels of LDL-C (-3.48 mg/dL; 95% CI, -4.77 to -2.20; I2 = 70%, P < .00001), TC (-2.59 mg/dL; 95% CI, -3.88 to -1.29; I2 = 73%, P < .0001), TG (-5.39 mg/dL; 95% CI, -8.04 to -2.75; I2 = 77%, P < .0001), and non-HDL-C (-6.27 mg/dL; 95% CI, -10.94 to -1.60; I2 = 53%, P = .008). No significant effect was found on HDL-C (-0.32 mg/dL; 95% CI, -1.19 to 0.55; I2 = 97%, P = .47) and ApoB (-0.88 mg/dL; 95% CI, -3.36 to 1.60; I2 = 36%, P = .49) during DPP-4i treatment. CONCLUSION:DDP-4i significantly improved lipid parameters including LDL-C, TC, TG, and non-HDL-C in patients with T2D. This underscores the potential cardiovascular benefits of DPP-4i and their role in improving diabetes-related outcomes. SYSTEMATIC REVIEW REGISTRATION:PROSPERO registration no. CRD42020175999.

Curcumin is a natural polyphenolic compound extracted from the rhizomes of Curcuma longa, exhibiting diverse biological activities, including anti-inflammatory, antioxidant, antitumor, antibacterial, antiviral, and neuroprotective effects. However, its clinical application is severely limited by poor oral absorption and low bioavailability. To address these challenges, this study synthesized a novel folate-targeted curcumin-loaded material-CU-FRα/PEG-2-CHO. This delivery system significantly enhances tumor cell uptake efficiency through folate receptor (FRα)-mediated active targeting (FRα denotes the folate receptor alpha subtype). Additionally, polyethylene glycol (PEG) modification and aldehyde (-CHO) functionalization further improve its water solubility and stability. Experimental results demonstrate that this material not only substantially increases curcumin's absorption rate and bioavailability but also enhances its antitumor activity via targeted delivery mechanisms. This study developed a new type (significantly improving various performances) folate receptor-targeted curcumin nano-delivery system (CU-FRα/PEG-2-CHO) and systematically compared the antitumor effects of two curcumin nanomaterials (CU-FRα/PEG-1-CHO and CU-FRα/PEG-2-CHO), demonstrating that CU-FRα/PEG-2-CHO significantly inhibited the growth of A2780 and HO8910PM ovarian cancer cell lines. After targeting tumor cells, CU-FRα/PEG-2-CHO released curcumin, which was mechanistically shown to specifically target Cdc42-binding kinase β (MRCKβ, IC50 = 2.12 μM), with its binding characteristics confirmed by surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and cellular thermal shift assay (CESTA). Molecular docking analysis revealed that MRCKβ precisely binds to the ATP-binding pocket of curcumin. Further transcriptomic analysis demonstrated that MRCKβ silencing significantly affected the TGF-β-SMAD4-LAG-3 signaling pathway, and CU-FRα/PEG-2-CHO exerted its therapeutic effects by regulating the MRCKβ-APC-SMAD4-CTLA-4 signaling axis to suppress tumor cell proliferation and immune evasion. Animal experiments confirmed that CU-FRα/PEG-2-CHO administration at a dose of 1 mg/kg every two days significantly inhibited tumor growth with a dose-dependent effect. Notably, although cisplatin (5 mg/kg every two days) and αPD-L1 (5 mg/kg every two days) also showed certain therapeutic effects, their tumor suppression efficacy was significantly lower than that of CU-FRα/PEG-2-CHO. These findings not only elucidate the mechanism of a novel folate receptor-targeted curcumin nanoliposome but also provide important evidence for developing MRCKβ-targeted therapeutic strategies against ovarian cancer.

Introduction:Bladder cancer is a highly recurrent malignancy and frequently shows drug resistance. Although gemcitabine initially works well for most patients, the majority of treated patients progressively generate resistance after multiple rounds of therapy, eventually leading to tumor recurrence. Recent studies suggest that a small subpopulation of cancer cells with stem cell-like properties - cancer stem cells - may be responsible for chemoresistance and tumor recurrence. Circular RNAs (circRNAs) are novel non-coding RNAs with great potential as cancer therapy. Material and methods:The levels of circRNA_103809 in bladder cancer cells and a normal urothelial cell line were measured by quantitative polymerase chain reaction (qPCR). Bladder cancer cells were depleted of circRNA_103809, then in vitro and in vivo cell growth, migration, invasion, sphere formation ability, and resistance to gemcitabine were analyzed. The biomarkers of cell migration, invasion, and stemness were detected by western blotting assay. The interaction between miR-516a with circRNA_103809 or FBXL18 3'UTR was detected by luciferase reporter gene assay and an RNA pulldown experiment. Results:Depletion of circRNA_103809 significantly suppressed the viability of bladder cancer cells, reduced cell migration and invasion, increased sensitivity to gemcitabine, and repressed cancer cell stemness. Further investigation of the molecular mechanism revealed that circRNA_103809 interacted with miR-516a to modulate the expression of FBXL18 in bladder cancer cells. Conclusions:CircRNA_103809 acts as a potential promoter of bladder cancer through sponging miR-516a to upregulate FBXL18. Our findings identify circRNA_103809 as a potential target for bladder cancer therapy.

Spinal cord injury (SCI) causes permanent sensory and motor function loss below the injury site, with limited treatment options. Conductive hydrogels have shown promise for SCI repair due to their electrical and mechanical properties, while neurotrophic factors and extracellular vesicles exhibit anti-inflammatory and neurorestorative effects. This study developed a dual-loaded conductive hydrogel (Exo-N/NT3@ICH) containing both neurotrophic factors and extracellular vesicles and evaluated its efficacy combined with electroacupuncture (EA) for SCI treatment. The hydrogel was synthesized through Schiff base reactions using oxidized hyaluronic acid and aniline trimer, creating a physically crosslinked, injectable conductive matrix. Assessments examined the hydrogel's morphology, mechanical and electrical properties, swelling, degradation, drug release, and electrochemical behaviour. In vitro and in vivo studies further investigated its biocompatibility, anti-inflammatory effects, and pro-angiogenic potential. Results showed that Exo-N/NT3@ICH enhanced cell proliferation and differentiation through its conductivity, controlled release, and antioxidant properties. In a rat SCI model, the hydrogel improved functional outcomes, attributed to its neurotrophic and neuroregenerative effects. This study highlights Exo-N/NT3@ICH, when combined with EA, as a potential injectable therapeutic system to promote neurogenesis and tissue regeneration after SCI.

Curcumin has been shown to have antitumor properties, but its low potency and bioavailability has limited its clinical application. We designed a novel curcuminoid, [1-propyl-3,5-bis(2-bromobenzylidene)-4-piperidinone] (PBPD), which has higher antitumor strength and improves bioavailability. Cell counting kit-8 was used to detect cell activity. Transwell assay was used to detect cell invasion and migration ability. Western blot and quantitative polymerase chain reaction were used to detect protein levels and their messenger RNA expression. Immunofluorescence was used to detect the protein location. PBPD significantly inhibited the proliferation of cervical cancer cells, with an IC50 value of 4.16 μM for Hela cells and 3.78 μM for SiHa cells, leading to the induction of cuproptosis. Transcriptome sequencing analysis revealed that PBPD significantly inhibited the Notch1/Recombination Signal Binding Protein for Immunoglobulin kappa J Region (RBP-J) and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathways while upregulating ferredoxin 1 (FDX1) expression. Knockdown of Notch1 or RBP-J significantly inhibited NRF2 expression and upregulated FDX1 expression, leading to the inhibition of nicotinamide adenine dinucleotide phosphate activity and the induction of oxidative stress, which in turn activated endoplasmic reticulum stress and induced cell death. The overexpression of Notch1 or RBP-J resulted in the enrichment of RBP-J within the NRF2 promoter region, thereby stimulating NRF2 transcription. NRF2 knockdown resulted in increase in FDX1 expression, leading to cuproptosis. In addition, PBPD inhibited the acidification of tumor niche and reduced cell metabolism to inhibit cervical cancer cell invasion and migration. In conclusion, PBPD significantly inhibits the proliferation, invasion, and migration of cervical cancer cells and may be a novel potential drug candidate for treatment of cervical cancer.

Chlorinated bisphenol A (BPA) derivatives are formed during chlorination process of drinking water, whereas bisphenol S (BPS) and brominated BPA and BPS (TBBPA and TBBPS) were synthesized for many industrial uses such as fire retardants. However, the effect of halogenated BPA and BPS derivatives on glucocorticoid metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) remains unclear. The inhibitory effects of 6 BPA derivatives in the inhibition of human and rat 11β-HSD1 were investigated. The potencies for inhibition on human 11β-HSD1 were TBBPA (IC50, 3.87 μM) = monochloro BPA (MCBPA, 4.08 μM) = trichloro BPA (TrCBPA, 4.41 μM) > tetrachloro BPA (TCBPA, 9.75 μM) > TBBPS (>100 μM) = BPS (>100 μM), and those for rat 11β-HSD1 were TrCBPA (IC50, 2.76 μM) = MCBPA (3.75 μM) > TBBPA (39.58 μM) > TCBPA = TBBPS = BPS. All these BPA derivatives are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that MCBPA, TrCBPA, TCBPA, and TBBPA all bind to the active site of human 11β-HSD1, forming hydrogen bonds with catalytic residue Ser170 except TCBPA. Regression of the lowest binding energy with IC50 values revealed a significant inverse linear regression. In conclusion, halogenated BPA derivatives are mostly potent inhibitors of human and rat 11β-HSD1, and there is structure-dependent inhibition.

Single-cell multiomic and exosome analyses are potent tools in various fields, such as cancer research, immunology, neuroscience, microbiology, and drug development. They facilitate the in-depth exploration of biological systems, providing insights into disease mechanisms and aiding in treatment. Single-cell isolation, which is crucial for single-cell analysis, ensures reliable cell isolation and quality control for further downstream analyses. Microfluidic chips are small lightweight systems that facilitate efficient and high-throughput single-cell isolation and real-time single-cell analysis on- or off-chip. Therefore, most current single-cell isolation and analysis technologies are based on the single-cell microfluidic technology. This review offers comprehensive guidance to researchers across different fields on the selection of appropriate microfluidic chip technologies for single-cell isolation and analysis. This review describes the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips available for single-cell isolation. Moreover, this review highlights the implications of using this technology for subsequent analyses, including single-cell multiomic and exosome analyses. Finally, the current challenges and future prospects of microfluidic chip technology are outlined for multiplex single-cell isolation and multiomic and exosome analyses.

Medical image registration is an essential topic in medical image analysis. In this paper, we propose a method for medical image registration using a pretrained large language model. We find that using the pretrained large language model to encode deep features of the medical images in the registration model can effectively improve image registration accuracy, indicating the great potential of the large language model in medical image registration tasks. We use dual encoders to perform deep feature extraction on image pairs and then input the features into the pretrained large language model. To adapt the large language model to our registration task, the weights of the large language model are frozen in the registration model, and an adapter is utilized to fine-tune the large language model, which aims at (a) mapping the visual tokens to the language space before the large language model computing, (b) project the modeled language tokens output from the large language model to the visual space. Our method combines output features from the fine-tuned large language model with the features output from each encoder layer to gradually generate the deformation fields required for registration in the decoder. To demonstrate the effectiveness of the large prediction model in registration tasks, we conducted experiments on knee and brain MRI and achieved state-of-the-art results.

Bisphenol A (BPA) is a widely used plastic material and its potential endocrine disrupting effect has restricted its use and increasing use of BPA alternatives has raised health concerns. However, the effect of bisphenol alternatives on steroidogenesis remains unclear. The objective of this study was to compare inhibitory potencies of 10 BPA alternatives in the inhibition of gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) in three species (human, rat and mouse). The inhibitory potency for human 3β-HSD2, rat 3β-HSD1, and mouse 3β-HSD6 ranged from bisphenol FL (IC50, 3.32 μM for human, 5.19 μM for rat, and 3.26 μM for mouse) to bisphenol E, F, and thiodiphenol (ineffective at 100 μM). Most BPA alternatives were mixed inhibitors of gonadal 3β-HSD and they dose-dependently inhibited progesterone formation in KGN cells. Molecular docking analysis showed that all BPA analogs bind to steroid and NAD+ active sites. Lipophilicity of BPA alternatives was inversely correlated with IC50 values. In conclusion, BPA alternatives mostly can inhibit gonadal 3β-HSDs and lipophilicity determines their inhibitory strength.

Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 and regulatory transcription factor Nr3c2 at 100–200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 μM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1, which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis.

AIMS:Curcumin is a natural compound and has good antitumor properties, but its clinical use is limited by its low bioavailability. We constructed the derivative CP41 (3,5-bis(2-chlorobenzylidene)-1-piperidin-4-one) by enhancing the bioavailability of curcumin while retaining its antitumor properties.MAIN METHODS:CCK-8 (Cell Counting Kit-8) was used to detect the effect of CP41 on cell proliferation; Western blotting, immunofluorescence, immunoprecipitation, quantitative PCR and enzyme-linked immunosorbent assay were used to evaluate the expression of subcutaneous tumor-related molecules in cells and mice.KEY FINDINGS:Our results showed that CP41 inhibited the proliferation of endometrial cancer cells by suppressing the proliferation of AN3CA and HEC-1-B cells. We found that CP41 significantly increased H3F3A and inhibited proteasome activity, which activated MAPK signaling and led to apoptosis. Further experiments showed that H3F3A is a potential target of CP41. Correlation analysis showed that H3F3A was positively correlated with the sensitivity to chemotherapeutic agents in endometrial cancer. CP41 significantly induced reactive oxygen species (ROS) levels and activated endoplasmic reticulum stress, which led to apoptosis. The safety profile of CP41 was also evaluated, and CP41 did not cause significant drug toxicity in mice.SIGNIFICANCE:CP41 showed stronger antitumor potency than curcumin, and its antitumor activity may be achieved by inducing ROS and activating H3F3A-mediated apoptosis.

Tetrachlorobisphenol A (TCBPA), a halogenated flame retardant and endocrine disruptor, has been detected in human urine and serum. While previous research has shown its impact on the reproductive system, investigations into its mechanisms during puberty remain limited. This study aims to explore the effects of TCBPA on Leydig cells in adolescent mice and potential underlying mechanisms. Male C57 mice of age 28 days were gavaged with 50, 100, and 200 mg/kg/day for 28 days. TCBPA did not alter body weight and testis weight but lowered testosterone levels at 100 and 200 mg/kg and reduced sperm count in the epididymis at 200 mg/kg. TCBPA lowered Leydig cell number at 200 mg/kg while it downregulated key Leydig cell gene (Lhcgr, Scarb1, Cyp11a1, Cyp17a1, Hsd3b6, Hsd17b3 and Insl3) as low as 50 mg/kg. Further study indicated that TCBPA induced reactive oxygen species and caused endoplasmic reticulum stress. In vitro study in TM3 mouse Leydig cells showed that TCBPA indeed induced reactive oxygen species and caused endoplasmic reticulum stress at 75 μM and inhibited testosterone production at this concentration and addition of antioxidant tocopherol can reverse it. These discoveries provide new insights and references for a deeper understanding of the toxic mechanisms of TCBPA on Leydig cells during puberty.

Benzophenone (BP) UV-filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3β-hydroxysteroid dehydrogenases (3b-HSD) catalyse conversion of pregnenolone to progesterone. This study explored effect of 12 BPs on human, rat, and mouse 3b-HSD isoforms and analysed the structure-activity relationship (SAR) and underlying mechanisms. The potency of BP-1 (IC50, 5.66±0.95 mM)>BP-2 (5.84±2.22 mM)>BP-6 (>100 mM) >BP3-BP12 in inhibition of human KGN cell 3β-HSD2, or BP-2 (5.90±1.02 mM)>BP-1 (7.55±1.26 mM) > BP3-B12 for rat testicular 3b-HSD1, or BP-1 (15.04±5.20)>BP-2 (22.64±11.81)>BP-6=BP-7 (~100 mM)>other BPs for mouse testicular 3b-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3b-HSD, and BP-2 is a mixed inhibitor of human and rat 3b-HSD and a noncompetitive inhibitor of mouse 3b-HSD. 4-Hydroxy substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, mouse gonadal 3b-HSD. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at >10 mM. Docking analysis revealed that 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bond with residue Ser123 of human 3b-HSD2 and residue Asp127 of rat 3b-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, mouse gonadal 3β-HSD and that there is a significant SAR difference.

Bisphenol A (BPA) is a widely used plastic material, and halogenated BPA derivatives are formed either by synthesis or environmental processes. However, the effect of halogenated bisphenols on steroidogenesis remains unclear. The aim of this study was to compare inhibition of 6 BPA derivatives on gonadal 3p-hydroxysteroid dehydrogenases (3p-HSDs) in three species (human, rat, and mouse). The inhibition on human 3p-HSD2 was tetrabromo BPA (TBBPA, IC50, 1.01 mu M)>trichloro BPA (TrCBPA, 3.95 mu M)>tetrachloro BPA (TCBPA, 4.14 mu M)> monochloro BPA (MCBPA, 4.74 mu M)>others with TrCBPA of competitive, TBBPA of noncompetitive and MCBPA/TCBPA of mixed inhibition. The inhibition on rat 3p-HSD1 was TCBPA (1.68 mu M)>TrCBPA (1.72 mu M)> MCBPA (2.80 mu M)>BPA>others with mixed inhibition. The inhibition on mouse 3p-HSD6 was TrCBPA (1.59 mu M) >MCBPA (3.36 mu M)>TCBPA (3.72 mu M)>others with mixed inhibition. Molecular docking analysis showed that TBBPA, TrCBPA, and TCBPA bind to steroid active sites, contacting with catalytic residue Tyr154 of human 3pHSD2. MCBPA, TrCBPA, and TCBPA bind to steroid active site of rat 3p-HSD1. MCBPA and TrCBPA bind to active site of mouse 3p-HSD6. Regression of lowest binding energy values with Ki values revealed a significant negative linear regression (P < 0.05). In conclusion, halogenated BPA derivatives are more potent inhibitors of three 3pHSDs than BPA and there is structure-dependent inhibition. Synopsis: Chlorinated bisphenol derivatives after water chlorination process and other halogenated bisphenols effectively inhibit human and rat 3p-HSD activity, thereby leading to steroid hormone deficiency.

Bisphenol A (BPA) is a chemical used in the production of certain plastics and resins. Recent research has found that BPA can inhibit the activity of 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerases (3β-HSDs). Whether benzene ring BPA substitutes can inhibit human, rat, and mouse gonadal 3β-HSDs, the structure-activity relationship and the underlying mechanism remain unclear. In this study, we compared 6 benzene ring BPA substitutes to BPA in the inhibition of human, rat, and mouse gonadal 3β-HSDs and conducted structure-activity relationship and in silico docking analysis. The inhibitory activity (IC50) of human 3β-HSD2 in KGN cells ranged from about 0.02 μM for bisphenol H to 8.75 μM for BPA, that of rat 3β-HSD1 in testicular microsomes ranged from 0.099 μM for bisphenol H to 31.32 μM for BPA, and that of mouse 3β-HSD6 ranged from 0.021 μM for BPH to ineffectiveness for 100 μM BPA. These compounds acted as mixed inhibitors with LogP inversely correlated with IC50 and ΔG positively correlated with IC50 value. Docking analysis showed that these compounds bind to the steroid active site of the 3β-HSD enzymes. In conclusion, some benzene ring BPA substitutes potently inhibit gonadal 3β-HSD in various species, and lipophilicity and binding affinity determine their inhibitory strength.

Azole fungicides are widely used in the agricultural industry to control fungal infections in crops. However, recent studies have shown that some azole fungicides inhibit the activity of 3β-hydroxysteroid dehydrogenases (3β-HSDs) in the gonads. Out of the 16 azole fungicides tested, 8 were found to inhibit human KGN cell 3β-HSD2 with IC50 values of less than 100 μM. The strongest inhibitor was difenoconazole, with an IC50 value of 1.88 μM. In contrast, only 3 of the azole fungicides inhibited rat testicular 3β-HSD1, which was less sensitive to inhibition. Azole fungicides potently inhibited progesterone secretion by KGN cells under basal and forskolin stimulated conditions at ≥ 5 μM. The inhibitory strength of azole fungicides was determined by their lipophilicity (LogP), molecular weight, pKa, and binding energy. A pharmacophore analysis revealed that the hydrogen bond acceptor-lipid group was a critical feature required for inhibition. Overall, these findings suggest that the use of azole fungicides have unintended consequences on reproductive health due to their inhibition of gonadal 3β-HSDs. Key words: Azole fungicides; steroid hormones; 3β-hydroxysteroid dehydrogenase; docking analysis; lipophilicity.