Specnuezhenide attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice

Specnuezhenide attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice | 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 Article Specnuezhenide attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice Xuesong Bai, Bo Y. Jiang, Weijie Du, Beijia Wang, He Sui, Songnan Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6338624/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Bisphenol A (BPA), a xenoestrogenic compound, disrupts spermatogenesis by inducing oxidative stress (OS) through iron ion-dependent mechanisms, ultimately contributing to male infertility. Specnuezhenide (SPN), a secoiridoid derived from Fructus Ligustri Lucidi (FLL) , exhibits notable antioxidant and anti-inflammatory properties. However, whether SPN can protect against BPA-induced OS and its detrimental effects on spermatogenesis remains unclear. Furthermore, the underlying mechanisms by which SPN alleviates BPA-induced male reproductive toxicity are poorly understood. Purpose: To demonstrate the efficacy of SPN in mitigating BPA-provoked testicular damage. Study Design : Specnuezhenide was verified to attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice. Methods: The male ICR mice have been divided into five groups to investigate these questions, including: the control group, the BPA group (50 mg/kg [bw], orally for 28 days), and three SPN+BPA groups receiving BPA (50 mg/kg [bw], orally for 28 days) along with SPN (30 mg/kg [bw], orally for 21, 28, and 35 days, respectively). The extent od testicular damage was evaluated by basic parameters of body weight, sperm quality, hormonal levels and hematoxylin-eosin (H&E) staining. The mRNA and protein levels of ferroptosis and apoptosis pathways in testes were evaluated by qPCR amplification, western blotting and immunofluorescence analysis. Results: BPA exposure significantly impaired sperm quality, induced OS, caused iron accumulation, and led to mitochondrial damage; restored serum hormone levels, including testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH), while increasing estradiol (E2) levels; reduced the activities of antioxidant enzyme, such as superoxide dismutase, catalase, and glutathione peroxidase; and sharply elevates in the expressions of NCOA4 ( a marker of ferritinophagy), GPX4 and SLC7A11 (markers of ferroptosis), cysteine- dependent aspartate-specific protease-3 (Caspase-3), cysteine- dependent aspartate-specific protease-9 (Caspase-9) and BCL2-associated X protein (Bax) (markers of apoptosis). Conversely, SPN supplementation considerably mitigated BPA-induced testicular damage by inhibiting iron accumulation and OS, thereby downregulating ferroptosis and apoptosis pathways. Conclusions: These findings underscore potential of SPN as a therapeutic agent and highlight the necessity for in-depth investigation into the detailed mechanisms underlying BPA-induced toxicity. Biological sciences/Molecular biology Earth and environmental sciences/Environmental sciences Health sciences/Endocrinology Health sciences/Pathogenesis Biological sciences/Physiology/Metabolism Biological sciences/Physiology/Reproductive biology Bisphenol A Specnuezhenide ferroptosis testicular toxicity spermatogenesis apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bisphenol A (BPA) is a prevalent environmental pollutant arising from its extensive production and widespread application[ 1 ]. It is frequently detected in various consumer goods, such as food packaging materials, baby bottles, reusable water bottles, PVC films, paper products, cardboard, and the epoxy resin linings of food cans[ 2 ]. Additionally, BPA is extensively utilized in the manufacturing of dental sealants and thermal paper[ 3 ].Human exposure to BPA happens through diverse pathways, including ingestion, inhalation, and dermal absorption[ 4 ], with dietary intake considered the primary route[ 5 ]. BPA was reported to impair male fertility, at the initial research, by mimicking endogenous estrogen or interacting with androgen receptors[ 6 ]. However, subsequent studies have further demonstrated that BPA also affects male fertility by causing mitochondrial dysfunction, driven by excessive accumulation of iron ion-dependent ROS, which triggers apoptosis and ferroptosis in testicular cells[ 7 ]. Natural substances are being investigated for their potential protective and therapeutic properties to mitigate the harmful effects of BPA. For instance, Cuscuta chinensis flavonoids (CCF) significantly reduced testicular cell apoptosis in male offspring when administered to pregnant mice exposed to BPA on postnatal days 21 and 56[ 8 ]. Similarly, crocin (Cr) alleviated BPA-induced apoptosis in adult rats by downregulating Caspase-3 expression[ 9 ]. Melatonin has been shown to mitigate testicular damage by reducing oxidative stress (OS)[ 10 ], while vitamin E improved apoptosis in reproductive cells caused by BPA exposure[ 11 ]. These findings underscore the growing interest in natural compounds as low-toxicity, multifunctional agents with strong antioxidative properties, offering promising potential to address BPA-induced reproductive damage. The traditional Chinese medicinal herb, Fructus Ligustri Lucidi (FLL), which widely incorporated in tonic formulas, was first documented in the Shennong Materia Medica [ 12 ]. Specnuezhenide (SPN), a key bioactive compound in FLL, is recognized as a phytochemical marker for assessing the herb’s quality, as described in the current edition of the Chinese Pharmacopoeia[ 13 ]. SPN has demonstrated significant antioxidative properties, notably its ability to mitigate OS caused by free radicals[ 14 ], but its potential to alleviate BPA-induced testicular cell death remains unclear. Previous studies have shown that SPN can attenuate CCl 4 -induced liver injury in mice by counteracting OS through activation of the NRF2 signaling pathway and suppressing apoptosis through downregulation of the BCL-2 pathway[ 15 ]. These mechanisms suggest a potential parallel in the ability of SPN to counteract BPA-induced testicular OS and apoptosis. Thus, we speculate that SPN has the potential to alleviate BPA-induced testicular injury. In this review, we investigate how SPN can ameliorate BPA-induced testicular damage, with a focus on elucidating its role in modulating key cell death pathways, such as apoptosis and ferroptosis. By integrating findings from in vivo studies, we aim to provide a comprehensive understanding of the underlying mechanisms through which SPN exerts its protective effects. This analysis not only highlights SPN’s therapeutic potential but also offers valuable scientific insights and references for strategies to safeguard and enhance reproductive health. Materials and methods 2.1. Chemical and Reagents BPA (purity ≥ 99%) was purchased from Sigma (USA) and stored at 4°C. Specnuezhenide (SPN, HPLC > 98%) was sourced from Nanjing Jingzhu Biotechnology Co., Ltd. (Jiangsu, China), with its content and composition verified by HPLC; the corresponding results are provided in Supplementary Fig. S1 . Corn oil was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China) to use as a solvent. Biochemical kits for superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), nitric oxide (NO), nitric oxide synthase (NOS), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) were sourced from Nanjing Jiancheng Institute of Biotechnology Co., Ltd. (Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kits for hormones of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH), while increasing estradiol (E2), cytokines of interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) were supplied by Jingmei Biotechnology Co., Ltd. (Jiangsu, China). Additionally, cysteine- dependent aspartate-specific protease-3 (Caspase-3) and cysteine- dependent aspartate-specific protease-9 (Caspase-9) assay kits, immunofluorescence staining reagents (anti-rabbit Alexa Fluor 488), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 2.2. Animals and treatments The experiments were conducted with male ICR mice weighting 20 ± 2 g, supplied by the Changchun Yisi Laboratory Animal Technology Co. Ltd. (License number: SCXK(Ji)2018-0007, Jilin, China. No.01021685682051708). The animals were housed under controlled conditions, with a temperature of 25 ± 2°C, humidity of 50 ± 10%, and water with 12-hour light-dark cycle. They were fed a laboratory diet (SCXK(liao) 2022-0001). Sixty mice were randomly divided into three groups, each receiving treatments detailed in Table S1 . Mice in BPA and BPA + SPN groups were intragastric administration with BPA for 28 days. Then, mice in BPA + SPN groups were intragastric administration with SPN for 21-, 28-, and 35-days, respectively. At each experimental endpoint (21, 28, and 35 days), mice were anesthetized and euthanized to collect testes and serum for subsequent analyses. The body weight and testis weight of each mouse were recorded, and the relative organ weight was determined by the ratio of testis weight to body weight. For consistency, samples from 15 mice in either the control or model group were pooled for experimental analysis and data calculation, as per the study design. The experimental protocol was approved by the Ethics Committee of Jilin Medical University, Jilin, China (2023-LW015). All procedures were performed by qualified technicians with appropriate laboratory animal handling certifications, strictly adhering to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals regarding rodent disposal standards. The reporting complies with the ARRIVE guidelines. The entire experiment followed the "3R principles" (Reduction, Refinement, Replacement). After a 6-hour preoperative fast, the animals were deeply anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg), with the disappearance of corneal reflexes and absence of response to painful stimuli serving as the criteria for effective anesthesia. For euthanasia, cervical dislocation was performed on mice after confirming a state of anesthesia. 2.3. Epididymal sperm analysis Epididymal tails from each group of mice were dissected into small pieces and subsequent incubated in the 0.9% NaCl solution (0.5 mL) that prewarmed to 37°C for 30 mins. The resulting suspension was used to isolate epididymal sperm. A 20 µL aliquot of the sperm suspension was loaded onto a Makler counting chamber and then using a computer-assisted sperm analysis system (SCA, Microptic, Spain) to analyze them. For each sample, ten fields of view were selected to assess sperm concentration and viability. 2.4. Analysis of serum hormone and inflammatory cytokine levels The serum samples were analyzed quantitatively using ELISA kits to measure the hormones of T, LH, FSH, and E2, pro-inflammatory cytokines of IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokines of IL-4 and IL-10 levels in accordance with the manufacturer’s instructions (Jiangsu Jingmei Biotechnology Co., Ltd.). 2.5. Determination of ferritin contents in serum and Fe 2+ levels in testicular tissues Serum ferritin levels were quantified using a mouse ferritin ELISA kit (Jiangsu Jingmei Biotechnology Co., Ltd.), under the guidance of the manufacturer’s protocol. The absorbance was measured at the wavelength of 450 nm using an Epoch plate reader (Epoch, BioTek, America), and the ferritin concentration was subsequently calculated. Iron concentrations in testicular tissues were assessed by means of Iron Assay Kit (Nanjing Jiancheng Institute of Biotechnology Co., Ltd.). Testicular cell lysates were treated with an iron reducer to convert ferric iron (Fe³⁺) into ferrous iron (Fe²⁺), followed by incubation in a water bath at 100°C, with 5 mins, allowing the formation of a colored complex. The absorbance measurement was performed at the wavelength of 520 nm, and Fe²⁺ levels were determined according to the provided protocol. 2.6. Mitochondrial function analysis Testicular homogenates were collected at 3000 g, 4°C for 20 mins at to obtain supernatants for subsequent analyses. The electron transport chain (ETC) complexes (I, II, and V) activities in testicular homogenates were measured by means of ELISA kits (Beijing Solarbio Science & Technology Co., Ltd., China) in accordance with the protocols, with absorbance values recorded on an Epoch microplate reader (Epoch, BioTek, America). All experiments were performed in triplicate. Adenosine triphosphate (ATP) levels in testicular homogenates were measured employing an ATP assay kit (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China). Tissue samples were lysed with an ATP-releasing reagent, and subsequently, lysates were mixed with ATP detection solution. ATP concentrations were normalized to protein content, which was determined using an enhanced BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). The NAD⁺/NADH and NADPH/NADP⁺ ratios in testicular homogenates were measured by means of assay kits (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China). The absorbance measurement was performed at 570 nm with a microplate reader, and the ratios were determined according to the absorbance values as specified in the kit instructions. 2.7. Oxidative stress assay ROS levels in testicular homogenates of mice were measured using a ROS assay kit (Jingmei Biotechnology Co., Ltd., Jiangsu, China) under the guidance of the manufacturer’s instructions. Briefly, 50 µL of testicular homogenate was added to an ELISA plate in duplicate, incubated at 37°C for 30 mins, and subsequently washing. After a second incubation at same conditions with 50 µL of enzyme-labeled reagents, followed by washing, a colorimetric reaction was initiated. Then using an Epoch microplate reader (Epoch, BioTek, America) to measure the values of optical density (OD) at 450 nm. Enzymatic activities of SOD, NOS, CAT, and GSH-Px, along with the concentrations of MDA, GSH, and NO, were assessed in serum and testicular supernatants by means of commercial kits (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China) in accordance with the manufacturer’s instructions. The supernatants protein concentrations were determined by means of Bradford Protein Assay Kits (BCA, Beyotime Biotechnology Co., Ltd., Shanghai, China). All measurements were conducted in triplicate to ensure accuracy. For further analysis the anti-oxidative effect of SPN, the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl ( · OH) radical scavenging capacity and iron reducing power were also measured in vitro (Detailed procedures were shown in Fig. S2 and S8)[ 17 , 18 ]. 2.8. Histopathological analysis Testicular tissues were fixed and preserved in 4% paraformaldehyde, dehydrated, and subsequently embedded in paraffin. Sections, 5 µm thick, were prepared by means of a rotary microtome (RM2245, Leica, Wetzlar, Germany) and subsequently stained with hematoxylin and eosin (H&E). Light microscope (BX53, Olympus, Tokyo, Japan) was use to examine the histopathological changes. To ensure consistency, three mice were analyzed, with three sections evaluated per mouse. 2.9. Immunofluorescence analysis The subcellular localization of target proteins was assessed using immunofluorescence, following previously established protocols[ 15 ]. Paraffin-embedded testicular sections were successively deparaffinized, rehydrated, permeabilized, and blocked. The slides were subsequently reacted with primary antibodies (details provided in Table S2) and then exposed to an Alexa Fluor 488-conjugated secondary antibody using an immunofluorescence staining kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Cellular nuclei were stained with DAPI as a counterstain, and the slides were mounted with anti-fluorescence quenching solution and sealed. Fluorescence signals were observed by means of a fluorescence microscope (BX53, Olympus, Tokyo, Japan). 2.10. Caspase-3 and − 9 measurement Caspase-3 and − 9 activities in testicular homogenates were measured using commercial assay kits (Beyotime Biotechnology Co., Ltd., Shanghai, China). The absorbance measurement was determined at a wavelength of 405 nm. 2.11. Western blotting Total protein from testes were extracted, separated by means of 10–12% SDS-PAGE, and then transferred onto the PVDF membranes (Millipore, Merck, Germany). Those samples were exposed to 5% BSA in TBST buffer (0.1% Triton X-100 in PBS), for 1 h with pH of 7.4, at room temperature. After subsequent blocking, they were exposed to the specified primary antibodies (Table S3) at 4°C overnight, followed by the corresponding secondary antibodies at 37°C for 1 h. After three washes with TBST, protein bands were detected using a chemiluminescence reagent (Beijing Solarbio Science & Technology Co., Ltd., China). The levels of relative protein were normalized to β-actin, which served as the loading control. Band intensities were quantified using a Bio-Rad GelDoc XR + system and analyzed with Image-Pro Plus software (Bio-Rad Laboratories, Inc.). 2.12. qPCR amplification Total RNA was isolated from testicular tissues by means of Trizol reagent (Invitrogen, Thermo Fisher Scientific, USA) in accordance with the manufacturer’s protocols. The first strand cDNAs were synthesized by means of the Start Script First-Strand cDNA Synthesis Kit (GenStar, Co., Ltd., China). Quantitative PCR was conducted employing the RealStar Green Fast Kit (GenStar, Co., Ltd., China) in an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA). Primer sequences for target genes (listed in Table S4) were synthesized and provided by Sangon Biotech Co., Ltd. (Shanghai, China). The qPCR protocol can separate in two main steps: an initial denaturation at 95°C for 2 mins, and a second step of successively 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Each experimental group included samples from five individual mice, with three technical replicates per sample. GAPDH served as the internal control, and the expression levels of relative mRNA were calculated using the 2 −ΔΔCT method. 2.13. Statistical analysis All data are presented as means ± standard error of the mean (SEM). Differences between groups were evaluated using one-way ANOVA, with a statistical significance of P < 0.05. Fig.s and visualizations were created using GraphPad Prism software to illustrate the results. Results 3.1. SPN alleviates BPA-induced sperm quality reduction Body weight changes were monitored throughout the experimental period. After BPA treatment, the body weight was deceased compared to control group ( p < 0.01), whereas there was no remarkable difference between control and BPA groups at the beginning of the experiments (Table S5). After SPN treatment, the body weights of BPA + SPN groups were increased relative to the BPA group from day 0 to day 35 ( p < 0.05, p < 0.01, Table S6). On day 21, the body weight of BPA + SPN (35) were higher compared with BPA group ( p < 0.05). On day 28, the body weights of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) were increased relative to BPA group ( p < 0.01). On day 35, the body weights of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) showed an increase as compared to BPA group ( p < 0.05, p < 0.01). The body weight gain was dependent on SPN dose increase. The effects of BPA and SPN treatments on testicular and sperm parameters were further analyzed (Fig. 1 a-d). BPA exposure resulted in significant reductions in testicular weight, testicular organ coefficient, sperm concentration, and sperm motility relative to the control group ( p < 0.01). SPN supplementation effectively counteracted these adverse effects. The testicular weight, testicular organ coefficient, sperm concentration and sperm motility of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) increased significantly compared with that of BPA group ( p < 0.01). Notably, the testicular weight in the BPA + SPN (28) and BPA + SPN (35) groups was statistically higher than that in the BPA + SPN (21) group ( p < 0.05). Similarly, sperm concentration in the BPA + SPN (28) group showed a prominent increase relative to the BPA + SPN (21) group ( p < 0.05). 3.2. SPN alleviates BPA-induced hormonal disorder and testicular tissue damage Hormonal analysis revealed significant alterations in plasma hormone levels following BPA exposure (Fig. 2 a and 2 b). T, FSH and LH levels were dramatically reduced in the BPA group when compared with the results of control group ( p < 0.01), while E2 levels showed a considerable increase ( p < 0.01). SPN treatment significantly restored T, FSH, LH, and E2 levels in all BPA + SPN groups relative to the BPA group ( p < 0.01). Notably, T, FSH, and E2 levels displayed statistically remarkable differences among the BPA + SPN treatment groups ( p < 0.05, p < 0.01). LH levels were meaningfully higher in the BPA + SPN (35) group relative to the BPA + SPN (21) and BPA + SPN (28) groups ( p < 0.01). H&E staining showed well-ordered testicular cells with regular structure in control group (Fig. 2 c). Seminiferous tubules were enlarged with disordered testicular cells in model group. Granular degeneration, nuclear condensation, interstitial proliferation and inflammatory cell infiltration were also pronounced in model group. BPA + SPN (35) group showed apparently alleviated pathological lesions in testis with slight enlargement of seminiferous tubules. SPN treatment effectively protected testes structures against BPA damage and reduced the loss of spermatophores. 3.3. SPN alleviates BPA-induced testicular mitochondrial dysfunction The BPA-treated mice exhibited a remarkable increment in the ROS level in comparison with the control group ( p < 0.01, Fig. 3 a). Concurrently, the relative ratios of NAD⁺/NADH and NADPH/NADP⁺ in testicular tissues were reduced significantly in the BPA group versus the control group ( p < 0.01, Fig. 3 b and 3 c). SPN administration decreased the ROS levels and increased the testicular NAD + /NADH and NADPH/NADP + relative ratios ( p < 0.01, Fig. 3 a- 3 c). This situation was developed time-dependently (Fig. 3 a- 3 c). The effects of BPA and SPN on ATP levels and the activity of ETC complexes (I, II, and V) were also examined. BPA exposure resulted in significant reductions in ATP content and the activities of ETC complexes ( p < 0.01, Fig. 3 d and 3 e), while SPN administration markedly improved ATP levels and ETC complex activities in SPN-treated mice ( p < 0.05, p < 0.01, Fig. 3 d and 3 e). 3.4. SPN alleviates BPA-induced oxidative damage in mice Both in vitro and in vivo experiments were performed to confirm the anti-oxidant activity of SPN. In vitro, anti-oxidant activity was calculated based on clearance rates, with lower IC-50 values indicating higher activity. The IC-50 values of the SPN (Fig. S2a and S2b), calculated using DPPH and hydroxyl radical scavenging capacity, demonstrated that the removal of different kinds of free radicals may be related to the different biological activities of SPN. In addition, the IC-50 of the SPN (DPPH IC-50 = 271.5 µg/mL, hydroxyl radical IC-50 = 572.98 µg/mL) is lower compared to the VC (DPPH IC-50 = 44.69 µg/mL, hydroxyl radical IC-50 = 375.80 µg/mL) (Fig. S2c). In vivo experiments, the antioxidant oxidase activity and the markers of oxidative damage were also determined by a mice model. Compared with control group, BPA exposure led to significant reductions in SOD and GSH-Px activities in testicular tissues and serum, accompanied by increased MDA, NOS, and NO levels, while CAT activity in testicular tissue also decreased significantly ( p < 0.01, Fig. 4 a- 4 f). However, SPN supplementation restored the SOD, GSH-Px, CAT and NOS activities, the MDA and NO contents in BPA + SPN (28) and BPA + SPN (35) groups when compared with BPA-treated group ( p < 0.01, Fig. 4 a- 4 f). Moreover, SPN supplementation significantly restored the SOD and CAT activities, MDA and NO contents of serum and testicular tissues, NOS activity of serum in BPA + SPN (21) group relative to the BPA group ( p < 0.01, Fig. 4 a- 4 f). SPN treatment for 28 and 35 days showed better resistance to BPA- induced oxidative damage than SPN treatment for 21 days. SPN treatment for 35 days restored the MDA content in serum and testicular tissues, SOD activity in testes, NOS activity and NO content in serum when compared to SPN treatment for 28 days ( p < 0.05, p 0.05, Fig. 4 c and 4 d). Moreover, the NAD(P)H dehydrogenase, quinone 1 (NQO1) expression level in BPA group was higher compared with the control group ( p < 0.01, Fig. 4 g and 4 h), while SPN supplementation significantly reduced NQO1 expression levels in SPN-treated mice ( p < 0.05, p 0.05, Fig. 4 g and 4 h). The heme oxygenase-1 (HO-1) expression in the BPA + SPN (35) group was meaningfully lower than that of other groups ( p < 0.05, p < 0.01, Fig. 4 g and 4 h). Immunofluorescence analysis confirmed that BPA exposure markedly increased neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression levels, and decreased CAT, superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) expression levels in comparison with the control group ( p < 0.01, Fig. S3-S7), while it was reversed by SPN administration in a time-dependent manner ( p < 0.05, p < 0.01). GSH content in testicular tissues was notably lower in the BPA group when compared with the control group ( p < 0.01, Fig. 4 i). SPN supplementation significantly enhanced GSH contents in all BPA + SPN groups ( p < 0.01), with BPA + SPN (28) and BPA + SPN (35) groups showing higher GSH levels than the BPA + SPN (21) group ( p 0.05). 3.5. SPN alleviates BPA-induced inflammation in testes The mRNA and protein levels of IL-6, IL-1β, and TNF-α in BPA group have a significantly increase when compared to the control group due to the BPA exposure ( p < 0.01, Fig. 5 a and 5 b). However, SPN supplementation significantly reduced the mRNA and protein expressions of IL-6 and IL-1β in BPA + SPN groups relative to the BPA-treated group ( p < 0.05, p 0.05). Changes in IL-6 mRNA and TNF-α protein levels were significant among BPA + SPN groups ( p < 0.01), with TNF-α mRNA levels notably lower in BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group ( p < 0.05). The levels of IL-4 and IL-10 mRNA and protein have a marked decrease in the BPA group when in comparison with the results of control group due to the BPA exposure ( p < 0.01, Fig. 5 c and 5 d). SPN supplementation significantly increased IL-4 and IL-10 expressions in BPA + SPN (28) and BPA + SPN (35) groups relative to the BPA group ( p < 0.05, p < 0.01). IL-10 mRNA levels in BPA + SPN (28) and BPA + SPN (35) groups were higher than those in BPA + SPN (21) group ( p < 0.05, p < 0.01), while IL-4 mRNA levels in BPA + SPN (35) group were higher than in BPA + SPN (21) and BPA + SPN (28) groups ( p < 0.01). Protein expressions of IL-4 and IL-10 in the BPA + SPN (35) group were also markedly elevated as compared to the BPA + SPN (21) group due to the SPN treatment ( p < 0.05). 3.6. Effects of SPN on iron metabolism pathways To assess whether BPA exposure increased iron uptake in testicular cells, we investigated the mRNA and protein expressions of major extracellular ferric iron importers, including transferrin receptor1 (TFR1, CD71), solute carrier family 39 (zinc transporter), member 8 (ZIP8, SLC39A8) and member 14 (ZIP14, SLC39A14). RT-PCR examination shown that the levels of mRNA of Tfr1 and Zip14 have significant changes ( p < 0.01), whereas SPN treatment mitigated these effects, reducing iron absorption ( p < 0.01, Fig. 6 a). Notably, the expression of Tfr1 mRNA in the BPA + SPN (35) group was higher versus with BPA + SPN (21) and BPA + SPN (28) groups ( p < 0.01, Fig. 6 a). Conversely, Zip14 mRNA expression reduced in BPA + SPN (28) and BPA + SPN (35) groups relative to BPA + SPN (21) ( p < 0.05, Fig. 6 a). At the protein level, TFR1 expression was elevated in the BPA group compared to the control ( p < 0.05, Fig. 6 b), but SPN treatment significantly reduced TFR1 levels in BPA + SPN (21) and BPA + SPN (28) groups compared to the BPA group ( p < 0.01, Fig. 6 b). TFR1 protein levels further declined in BPA + SPN (28) group compared to BPA + SPN (21) group (p < 0.05), with an increase in the BPA + SPN (35) group relative to BPA + SPN (28) group ( p 0.05, Fig. 6 b). Similarly, Zip8 mRNA and protein expressions did not show substantial differences between the BPA and control groups ( p > 0.05, Fig. 6 a and 6 b). However, Zip8 mRNA was lower in BPA + SPN (28) compared to BPA + SPN (21) ( p < 0.05, Fig. 6 a), and the level of Zip8 mRNA or ZIP8 protein displayed higher in BPA + SPN (35) compared to the BPA + SPN (28) ( p < 0.05, p < 0.01, Fig. 6 a and 6 b). Since increased intracellular ferrous iron may result from impaired export function, we examined the expression of the primary iron exporter, ferroportin1 (FPN1, SLC40A1). ANOVA analysis shown that BPA exposure substantially increased the expression of Fpn1 mRNA compared to the control group ( p < 0.01, Fig. 6 c). SPN treatment considerably decreased Fpn1 mRNA expression in the BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group ( p < 0.05, p 0.05, Fig. 6 d). BPA exposure also significantly upregulated mRNA levels of ferritin heavy chain (Fth) and ferritin light chain (Ftl), key components of iron storage ( p < 0.01, Fig. 6 c). SPN supplementation caused significant differences in Ftl mRNA expression between the BPA and BPA + SPN groups ( p 0.05, Fig. 6 d). Similarly, no meaningful differences in FTH protein expression were observed between the BPA and BPA + SPN groups ( p > 0.05, Fig. 6 d). Notably, Fth mRNA expression changes were significantly difference among BPA + SPN groups ( p < 0.01, Fig. 6 c). After entering the endosome, imported intracellular iron is reduced to ferrous iron by the metalloreductase six-transmembrane epithelial antigen of the prostate 3 (STEAP3) and subsequently released into the cytoplasmic labile iron pool via divalent metal transporter 1 (DMT1) for cellular utilization or storage in ferritin. ANOVA analysis revealed that BPA exposure markedly affected Steap3 mRNA expression, which was reversed by SPN treatment ( p < 0.01, Fig. 6 e). Steap3 mRNA expression was lower in the BPA + SPN (28) and BPA + SPN (35) groups relative to the BPA + SPN (21) group ( p 0.05, Fig. 6 f), and while STEAP3 protein levels tended to decrease in the BPA + SPN groups, the differences were not significant in comparison with the BPA group ( p > 0.05, Fig. 6 f). Similarly, DMT1(SLC11A2) protein levels not displayed significantly differences between BPA, SPN, and control groups ( p > 0.05, Fig. 6 f). However, BPA exposure significantly increased Dmt1 mRNA expression compared with the control group ( p < 0.01, Fig. 6 e). SPN supplementation reduced Dmt1 mRNA levels in all BPA + SPN groups compared to the BPA group ( p < 0.01, Fig. 6 e). Furthermore, Dmt1 mRNA levels were lower in the BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group, but it was reversed in protein levels ( p < 0.05, Fig. 6 e). Acting as an autophagic cargo receptor, NCOA4 facilitates the degradation of ferritin, resulting in elevated levels of free iron. To determine whether the observed iron accumulation in our model was associated with ferritinophagy, we analysed the protein and mRNA expression levels of NCOA4 (Fig. 6 e and 6 f). The results indicated no noticeable differences in NCOA4 protein expression among the groups ( p > 0.05, Fig. 6 f). However, the levels of Ncoa4 mRNA in the BPA group were higher when compared to the control group ( p < 0.01, Fig. 6 e), and were lower in the BPA + SPN groups relative to the BPA group ( p < 0.05, p < 0.01, Fig. 6 e). Additionally, the levels of Ncoa4 mRNA in BPA + SPN (28) group were decreased in comparison with BPA + SPN (21) ( p < 0.01), but increased in the BPA + SPN (35) group in comparison with BPA + SPN (28) ( p < 0.01, Fig. 6 e). 3.7. SPN alleviates BPA-induced testicular ferroptosis To assess the role of iron in SPN-mediated protection against oxidative damage, the iron-reducing power of SPN was evaluated in vitro. As shown in Fig. S8, SPN demonstrated strong iron-reducing ability. In vivo results further indicated that BPA exposure increased serum ferritin levels and testicular ferrous iron content compared with the control group ( p < 0.01, Fig. 7 a and 7 b). These effects were reversed in BPA + SPN groups ( p < 0.01, Fig. 7 a and 7 b). Western blotting assay showed BPA dramatically decreased the expression of glutathione peroxidase 4 (GPX4) in testicular tissues ( p < 0.01, Fig. 7 c) and these effects were reversed in BPA + SPN groups ( p < 0.05, p 0.05, Fig. 7 c), and it was reversed by SPN ( p > 0.05). Addition, the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) in BPA group were decreased in comparison with control group ( p 0.05). These results were further supported by immunofluorescence staining, which revealed enhanced expression of GPX4, SLC7A11, and ACSL4 after SPN treatment ( p < 0.05, p < 0.01, Fig. 7 d and S9 - S11 ). Moreover, SPN upregulated the mRNA expression of Gpx4 and Acsl4 in testes exposed to BPA ( p < 0.05, p < 0.01, Fig. S12). 3.8. SPN alleviates BPA-induced testicular apoptosis No significant differences in BCL2-associated X protein (BAX) and B-cell CLL/lymphoma 2 (BCL-2) protein expression were observed between the BPA and BPA + SPN groups ( p > 0.05, Fig. 8 a and 8 b). Immunofluorescence staining showed apparent changes in BAX and BCL-2 antibodies between BPA + SPN(35) and BPA groups ( p < 0.01, Fig. 8 c, S13 and S14 ). At the mRNA level, BPA exposure upregulated the expression of the pro-apoptotic marker Bax and downregulated the anti-apoptotic marker Bcl-2 ( p < 0.01, Fig. 8 d). SPN treatment reversed these changes, normalizing the mRNA levels of Bax and Bcl-2 ( p < 0.05, p < 0.01, Fig. 8 d). Furthermore, BPA exposure increased the protein and mRNA expression of Caspase-3 and Caspase-9 ( p < 0.01, Fig. 8 d and 8 e). SPN treatment suppressed the expression of these apoptosis-related markers at both the protein and mRNA levels. Discussion Some studies demonstrated that BPA exposure induces OS, iron deposition, and mitochondrial structural abnormalities, while also altering the expression of proteins related to ferritinophagy, ferroptosis, and apoptosis, suggesting that BPA triggers OS, promotes iron accumulation, and leads to cell death in mouse testes[ 7 ]. SPN exhibits diverse pharmacological activities, which may stem from its strong free radical scavenging ability, as observed in this study [ 14 , 15 ]. Based on these properties, we hypothesized that SPN could mitigate testicular cell death-induced by BPA through modulating OS and iron accumulation pathways. To investigate the therapeutic effects of SPN on BPA-induced testicular damage, we conducted comprehensive analyses of dosage optimization for both compounds. The establishment of an effective mouse model required careful determination of BPA dosage. Our BPA exposure regimen (50 mg/kg BW/day) was selected based on the previously reported NOAEL (No Observable Adverse Effect Level) for reproductive and developmental toxicity[ 16 ]. Subsequent evaluation of SPN's therapeutic potential revealed dose-dependent effects on reproductive system toxicity (Fig. S15). Sperm motility improvements showed progressive enhancement across the 30–60 mg/kg SPN range. Hormonal analyses demonstrated concomitant dose-dependent increases in T, FSH, and LH levels, along with a reduction in E2 concentrations. Comparative analysis between 15 mg/kg and 30 mg/kg SPN doses revealed comparable therapeutic efficacy against BPA-induced testicular toxicity. We selected 30 mg/kg as the optimal therapeutic dose, balancing efficacy with safety considerations while avoiding potential toxicity associated with higher doses. Treatment duration analysis revealed temporal patterns in therapeutic response (Fig. S16). Biochemical parameters including serum hormone levels (T, FSH, LH, E2) and testicular oxidative stress markers (SOD, MDA) demonstrated significantly enhanced recovery in the 21–35 days treatment groups compared to shorter durations (7–14 days). Based on these findings, we focused subsequent investigations on 21-, 28-, and 35-day treatment periods to identify the optimal therapeutic window. To elucidate the molecular mechanisms underlying SPN's protective effects, we systematically investigated its capacity to mitigate BPA-induced iron accumulation and associated pathological processes in testicular tissue. This iron dysregulation, if unaddressed, could lead to chronic cellular toxicity and programmed cell death. In testicular tissue, BPA exhibits antiandrogenic and estrogen-like effects, causing further damage to testicular cells[ 6 ]. As a potent endocrine disruptor, it impairs male gonads and disrupts the redox balance in testes and sperm[ 19 , 20 ]. Studies have shown that BPA negatively affects sperm parameters, including reduced testicular organ coefficients, sperm motility, and concentration[ 20 ]. Consistent with these findings, the results in Fig. 1 support this hypothesis in this work. Furthermore, our results also demonstrate that SPN supplementation effectively reversed these changes. Prolonged SPN treatment led to increased testicular weight, organ coefficients, sperm concentration, and motility. As noted, BPA disrupted the endocrine system in treated mice. Recent epidemiological studies report that BPA exposure decreases LH, FSH, and T levels while increasing progesterone and E2 levels[ 21 ]. Similarly, prior studies have shown reduced LH, FSH and T levels in rats treated with BPA at 50 mg/kg/day for 30 days [ 22 ]. These findings align closely with our laboratory results. Moreover, in the present study, hormone levels in BPA + SPN groups were higher compared to the BPA group, suggesting that SPN facilitates the restoration and/or enhancement of antioxidant capacity, which supports spermatogenesis (Fig. 2 ). The protective role of SPN against BPA-induced reproductive toxicity was further confirmed through H&E staining results. Our results provide evidence that BPA exposure significantly increased ROS in the testes, suggesting BPA heightened OS (Fig. 3 ). According to our findings, SPN is capable of scavenge and neutralize free radicals in vitro and it can significantly mitigated BPA-induced ROS production, similarly to other researchers' findings[ 14 , 15 , 23 ]. NADPH plays a vital role in converting oxidized glutathione (GSSG) into reduced GSH[ 24 ], with NADPH, GSH, and SOD serving as key endogenous antioxidants [ 24 – 28 ]. In this study, BPA exposure markedly decreased the NADPH/NADP⁺ ratio, GSH content, and SOD activity in testicular tissues, indicating reduced antioxidant capacity (Fig. 4 ). SPN treatment not only restored the NADPH/NADP⁺ ratio but also increased GSH content and SOD activity after BPA treated, confirming its antioxidant properties. In the cytoplasm, lactate dehydrogenase reduces NAD⁺ to NADH [ 29 ] while the oxidative phosphorylation ETC complex I (NADH dehydrogenase) oxidizes NADH back to NAD⁺, facilitating the reduction of oxygen to water and ATP production[ 30 ]. Electron leakage from the ETC and reduced ATP synthesis can elevate mitochondrial ROS levels, triggering apoptosis[ 31 – 33 ]. In this study, BPA exposure decreased the NAD⁺/NADH ratio, which was restored by SPN supplementation. Similarly, BPA reduced ATP content and inhibited ETC complex (I, II, and V) activities, leading to elevated ROS levels. Expectedly, SPN reverse the decrease of ATP synthesis and ETC complexes (I, Ⅱ, and V) levels in our results. It is implied that SPN can improve the function of mitochondria. The mitochondrial antioxidant enzyme defense system plays a crucial role in the normal process of spermatogenesis. Key antioxidant enzymes such as SOD, CAT and GSH-Px help eliminate ROS and maintain redox balance[ 7 ]. BPA exposure has been shown to increase NO, nitric oxide synthase 2A (NOS2A), and ROS levels while reducing NADPH levels[ 34 ]. Notably, BPA-induced NO production is largely driven by inducible nitric oxide synthase (iNOS) in cells[ 35 ]. In this study, SPN treatment effectively prevented testicular oxidative damage in BPA-exposed mice by enhancing antioxidant levels, including SOD, CAT, GSH-Px and GSH in serum and testicular tissues (Fig. 4 ). SPN also mitigated BPA-induced toxicity by reducing NOS activity, MDA, and NO levels in serum and testes. In line with the results of NOS, the increase of nNOS and iNOS, decrease of CAT, SOD1 and SOD2 expressions-treated by BPA were also confirmed by immunofluorescence and reversed by SPN treatment (Fig. S3-S7). Downregulation nNOS and iNOS (markers of RNS), upregulation of SOD1 and SOD2 (markers of ROS) levels would be an effective way to alleviate BPA-induced testicular destroy in mice. Our study provides evidence that SPN alleviates oxidative damage induced by BPA. BPA also promotes the accumulation of proteins associated with OS signaling[ 36 ]. The NRF2/HO-1/NQO1 signaling pathway has garnered attention for its critical role in regulating cellular OS and offering protection against BPA-induced reproductive toxicity[ 37 ]. Additionally, the NRF2/HO-1 pathway exhibits anti-inflammatory effects by suppressing inflammatory proteins such as TNF-α and IL-6[ 38 ]. Contrary to the previous findings, our study observed increased NRF2, HO-1, and NQO1 protein levels following BPA exposure. This elevation is likely a compensatory response to BPA-induced testicular injury and was reversed by SPN treatment in BPA-exposed mice. Furthermore, SPN reduced the expression of IL-6, IL-1β and TNF-α significantly, while enhancing IL-4 and IL-10 at both protein and gene levels (Fig. 5 ). Similar effects of IL-4 and IL-10 have been reported in BPA-exposed mice in previous studies[ 39 ]. Overall, SPN appears to mitigate BPA-induced damage by activating antioxidant and anti-inflammatory pathways at both the expression of protein and gene. Research indicates that ferrous iron accumulation is mainly attributed to disturbances in iron homeostasis, which are intrinsically connected to iron metabolism. Under normal conditions, circulating ferric iron is taken up by cells through TFR1[ 40 ]. Extracellular iron can also be imported via metal transporters such as ZIP8 and ZIP14. Once inside the cell, ferric iron is reduced to ferrous iron by STEAP3 in the endosome and subsequently released into the cytoplasmic labile iron pool by DMT1[ 41 , 42 ]. Ferrous iron is then used for physiological reactions or stored in FTH, while excess iron is exported by FPN1[ 42 , 43 ]. Disruptions in these pathways can trigger ferroptosis. In this study, BPA exposure decreased DMT1, FPN1, FTH and FTL protein levels, while increasing the TFR1, ZIP8, ZIP14, and STEAP3 protein expressions. However, compared to the control group, BPA exposure did not significantly alter the protein expressions of DMT1, FPN1, ZIP8, ZIP14, FTH, or FTL, suggesting that iron import via ZIP8 and ZIP14, export by FPN1, and storage by FTH and FTL were not evidently affected (Fig. 6 ). SPN treatment reversed these proteins expression changes, notably downregulating TFR1, indicating a decrease in intracellular iron transport demand. Additionally, ferritinophagy mediated by NCOA4 has been reported as a pathway for ferroptosis induction[ 44 ]. While silencing NCOA4 has been shown to reverse BPA-induced FTH degradation and MDA accumulation[ 45 ], our results revealed no remarkable differences in NCOA4 protein expression between the BPA and control groups. However, NCOA4 expression was lower in the SPN-treated groups compared to the BPA group, although this difference lacked statistical significance. These results indicate that BPA-induced iron accumulation may involve ferritinophagy, and SPN could partially reverse this process. Overall, BPA-induced iron accumulation appears to result from enhanced iron absorption and transport combined with reduced storage and export. SPN alleviates these effects by regulating iron metabolism and inhibiting ferritinophagy, a pathway associated with autophagy-dependent ferroptosis. As a potential negative regulator of ferritinophagy in testicular cells, SPN may contribute to maintaining iron homeostasis. Notably, some mRNA results were inconsistent with protein findings, suggesting that SPN's regulation of iron homeostasis may extend beyond the pathways examined here, warranting further investigation into the underlying mechanisms. Iron metabolism in testicular tissues must be tightly regulated to prevent iron overload. Ferritin, a highly conserved protein composed of FTH and FTL polypeptide chains, plays a key role in maintaining iron homeostasis. FTH catalyzes Fe²⁺ oxidation, while FTL facilitates ferric iron (Fe³⁺) storage[ 23 ]. Ferroptosis regulation involves GPX4, an antioxidant enzyme in mitochondria, SLC7A11, a cysteine transporter crucial for GSH synthesis, and ACSL4, which influences lipid composition[ 23 , 46 ]. GPX4 converts lipid hydroperoxides to lipid alcohols, preventing Fe²⁺-dependent toxic lipid ROS formation [ 23 ]. BPA has been reported to induce Fe²⁺ accumulation in testes, along with decreased GPX4 expression and increased ferritin levels, contributing to ferroptosis in testicular cells[ 23 , 36 ]. In this study, BPA treatment significantly elevated Fe²⁺ levels in testes and ferritin levels in serum while reducing GPX4 and SLC7A11 expression in testicular tissue, consistent with previous findings, suggesting ferroptosis as a key mechanism in BPA-induced testicular injury (Fig. 7 ). Meanwhile, SPN-mediated ferroptosis resistance was achieved via decrease Fe 2+ and ferritin contents, upregulation of the GPX4 and SLC7A11 expression levels, suggesting that SPN can also inhibit BPA-induced ferroptosis in testes. Besides, expressions of GPX4 and SLC7A11 were also assessed by immunofluorescence. Immunofluorescence analysis further confirmed that SPN significantly suppressed BPA-induced NRF2 overexpression while restoring GPX4, GPX4, and SLC7A11 levels. These results suggest that SPN activates NRF2 downstream pathways involving GSH, SLC7A11, and GPX4, thereby protecting against BPA-induced testicular ferroptosis. ACSL4 is a key isozyme involved in the metabolism of polyunsaturated fatty acids (PUFAs), playing a crucial role in determining ferroptosis sensitivity[ 42 ]. Ferroptosis is a controlled cell death mechanism driven by iron-dependent lipid peroxidation reaching toxic levels[ 7 , 36 ]. While the role of ACSL4 in BPA-induced lipid peroxidation and ferroptosis remains unclear, increased ACSL4 levels have been observed in BPA-induced lipid accumulation in the liver[ 47 ]. To investigate the protective effects of ACSL4, western blotting and immunofluorescence analyses were conducted on testicular tissue undergoing BPA-induced ferroptosis (Fig. 8 ). BPA significantly downregulated ACSL4 expression in the testes, which was notably ameliorated by SPN treatment. These findings suggest that ACSL4 is a critical determinant of ferroptosis sensitivity and that SPN may exert protective effects by modulating ACSL4 expression and maintaining cellular homeostasis. BPA-induced testicular damage through OS can occur via both ferroptosis and apoptosis[ 7 , 48 ]. To explore how SPN mitigates BPA-induced testicular apoptosis, we investigated the expression of apoptosis-related proteins. Previous studies have reported that BPA exposure increases the expression of Caspase-3, Caspase-9, and Bax, while reducing Bcl-2 expression[ 7 , 48 ]. Our mRNA and protein analyses corroborated these findings, showing similar changes in apoptosis-related markers. Notably, SPN treatment reversed these alterations, suggesting that SPN alleviates testicular cell death by modulating the mitochondria-dependent apoptotic pathway. It is intriguing that in this study, SPN rescued BPA-induced damage do not seem to be at a time-dependent manner, the results in SPN + BPA (28) group were apparently superior to the other two groups. This nonlinear relationship may be due to the fact that although SPN is an antioxidant, high dose SPN produced toxic side effects and enhanced the toxicity of BPA. Conclusions This study is the first to demonstrate that SPN has an observable testicular protective effect on BPA exposure mice by increasing its ability to suppress hormonal imbalance, OS, iron accumulation, ferroptosis, and apoptosis. It is also proposed for the first time that the regulation of NAD + /NADH, NADPH/NADP + ratios and ACSL4 expression are involved in the protective effects of SPN against BPA-induced reproductive system damage. These findings provide a foundation for the potential prevention and treatment of BPA-induced testicular toxicity. Abbreviations BPA OS SPN FLL H&E T LH FSH E2 Gpx4,GPX4 SLC7A11 Caspase-3 Caspase-9 Bax, BAX Bcl-2, BCL-2 CCF Cr SOD MDA CAT NO NOS GSH-Px GSH ELISA IL-1β IL-4 IL-6 IL-10 TNF-α ROS DAPI ETC ATP OD BCA DPPH · OH NQO1 HO-1 NRF2 nNOS iNOS SOD1 SOD2 TFR1,CD71 Zip8,ZIP8 Zip14,ZIP14 Fpn1,FPN1 Fth,FTH Ftl,FTL Steap3, STEAP3 Dmt1, DMT1 Acsl4, ACSL4 Bisphenol A oxidative stress specnuezhenide Fructus Ligustri Lucidi hematoxylin-eosin testosterone luteinizing hormone follicle stimulating hormone estradiol glutathione peroxidase 4 solute carrier family 7 member 11 cysteine- dependent aspartate-specific protease-3 cysteine- dependent aspartate-specific protease-9 BCL2-associated X protein B-cell CLL/lymphoma 2 Cuscuta chinensis flavonoids Crocin superoxide dismutase malondialdehyde catalase nitric oxide nitric oxide synthase glutathione peroxidase glutathione enzyme-linked immunosorbent assay interleukin-1β interleukin-4 interleukin-6 interleukin-10 tumor necrosis factor-α reactive oxygen species 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride electron transport chain Adenosine triphosphate optical density Bradford Protein Assay 1,1-diphenyl-2-picrylhydrazyl Hydroxyl NAD(P)H dehydrogenase, quinone 1 heme oxygenase-1 NF-E2-related factor 2 neuronal nitric oxide synthase inducible nitric oxide synthase superoxide dismutase 1 superoxide dismutase 2 transferrin receptor1 solute carrier family 39 (zinc transporter), member 8 solute carrier family 39 (zinc transporter), member14 ferroportin1 ferritin heavy chain ferritin light chain metalloreductase six-transmembrane epithelial antigen of the prostate 3 divalent metal transporter 1 acyl-CoA synthetase long-chain family member 4 Declarations CRediT authorship contribution statement Conceptualization: X.B. and Y.X.; Data Curation: B.J., W.D., B.W., H.S., and S.Y.; Writing-review and editing: X.B. and Y.X.; Funding acquisition: Y.X.; All authors have read and agreed to the published version of the manuscript. Declaration of Competing Interest The authors declared no competing interest. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Acknowledgments This research was funded by Key Research and Development project of Science and Technology Department of Jilin Province (NOS.20220204035 YY), Innovation and Entrepreneurship Training project for College Students (NOS. 2021013). The author(s) would like to thank the Research and Experiment Center of Chronic Disease Prevention, Jilin Medical University, Jilin, China. Conflict of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The study was approved by the Animal Care Committee of Jilin Medical University, China (2023-LW015). There are no human subjects in this article and informed consent is not applicable. 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Kaur, S., Saluja, M., Aniqa, A., Sadwal, S., 2021. Selenium attenuates bisphenol A incurred damage and apoptosis in mice testes by regulating mitogen-activated protein kinase signalling. Andrologia. 53(3):e13975. Additional Declarations No competing interests reported. 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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-6338624","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":454165575,"identity":"c1b105d0-ea68-4d07-8966-b7e305fbec18","order_by":0,"name":"Xuesong Bai","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuesong","middleName":"","lastName":"Bai","suffix":""},{"id":454165576,"identity":"a9ceb22a-4b5c-4f62-bb91-0cd1852bb4f6","order_by":1,"name":"Bo Y. Jiang","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"Y.","lastName":"Jiang","suffix":""},{"id":454165577,"identity":"dbe637e4-021d-41ed-959e-155588814cee","order_by":2,"name":"Weijie Du","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Du","suffix":""},{"id":454165578,"identity":"d173c02c-fb00-47bf-a215-116e85bd754b","order_by":3,"name":"Beijia Wang","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Beijia","middleName":"","lastName":"Wang","suffix":""},{"id":454165579,"identity":"01c44611-eb47-4b6b-9af5-bd6ad15a390b","order_by":4,"name":"He Sui","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Sui","suffix":""},{"id":454165580,"identity":"3b186965-a090-401c-946f-4ed128ec4b5f","order_by":5,"name":"Songnan Yu","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Songnan","middleName":"","lastName":"Yu","suffix":""},{"id":454165582,"identity":"3c5fcbef-10c8-4f18-b997-70379ba50bf3","order_by":6,"name":"Yanli Xi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3PsYoCMRCA4RkGYhO1sPGw8BWyCIvFss8iBFJZWFquBNzmYFsfwzcwOqiNYGthIQjWHtdcIeJee4VJeUW+KgnzkwQgiv4htFQ4mD7zerG+3FWWByRYJwenqRQ6WUyMDrmnAJw7LCuZduR9U289qMQZT8SZmhZSypQjaPB26XsYL+St9WHBXMfq3AJpzMmbyC5RYmE/GKsbQVemAYkiXDHOe0PFWIQlI8aZJdGD8MQZjVZQ8qmMFr6/JBXzd/OZ5Vgdvy4/jyxvN3j3Pin+noh347/6voEoiqIIXsdBTFb5xUJGAAAAAElFTkSuQmCC","orcid":"","institution":"Jilin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Xi","suffix":""}],"badges":[],"createdAt":"2025-03-30 13:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6338624/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6338624/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82622310,"identity":"7b7539e5-bab9-469d-8e43-b91cd5fdb81c","added_by":"auto","created_at":"2025-05-13 12:27:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":290516,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPN on testis weight, organ coefficient and sperm quality reduction. Comparisons of testicular weight (a), testicular organ coefficient (b), sperm concentration (c), and sperm motility (d) among the groups. Data are shown as means ± SEM.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA+SPN (21) group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/9e712ee51c936d5e090ed388.png"},{"id":82622312,"identity":"f584c6c1-813c-46eb-a109-a119bbedad51","added_by":"auto","created_at":"2025-05-13 12:27:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1287220,"visible":true,"origin":"","legend":"\u003cp\u003eSPN alleviates BPA-induced hormonal disorder and testicular tissue damage. Comparisons of hormones (a,b) among the groups. Representative photomicrographs of H\u0026amp;E staining sections (c) of testes (scale bar is 100 µm) from each group. Data are presented as means ± SEM.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA+SPN (21) group, \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05,\u003csup\u003e ΔΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA+SPN (28) group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/e6fb7dc506e65452eadf5c84.png"},{"id":82622315,"identity":"fb40b2b6-8eb2-4298-9fd7-493d69911fca","added_by":"auto","created_at":"2025-05-13 12:27:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287577,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of BPA and SPN on testicular mitochondrial function. (a) ROS levels, (b) NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio, (c) NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratio, (d) ATP contents and (e) ETC complexes levels were measured by kits. Data are shown as means ± SEM.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (21) group, \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ΔΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (28) group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/2d757b7996200c5d55a578b8.png"},{"id":82622314,"identity":"4b0bee8b-dc87-4e00-8a9a-8534508d137d","added_by":"auto","created_at":"2025-05-13 12:27:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":669887,"visible":true,"origin":"","legend":"\u003cp\u003eSPN attenuates BPA-induced oxidative stress and regulates antioxidant enzyme activity. Activities of SOD and levels of MDA in serum (a) and testicular tissues (b), CAT and GSH-Px activities in serum (c) and testicular tissues (d), along with the NOS activity and NO levels in serum (e) and testicular tissues (f) were evaluated, with data are shown as means ± SEM. (g) Western blot analysis of NRF2, HO-1, and NQO1 expression in each group. (h) The gray values of western blot bands were quantified using Image-Pro Plus 7.0 software, with data shown as means ± SEM from at least three independent experiments. (i) The contents of GSH in testes were detected. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN(21) group, \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ΔΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN(28) group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/942359af6c3db9285019b61a.png"},{"id":82622328,"identity":"666d0f31-a65b-41e7-8466-76e2287cc05e","added_by":"auto","created_at":"2025-05-13 12:27:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":468239,"visible":true,"origin":"","legend":"\u003cp\u003eModulatory effects of SPN on BPA-induced testicular inflammatory reactions in all groups. (a) mRNA and (b) protein expressions of IL-6, IL-1β and TNF-α, and mRNA (c)and (d) protein expressions of IL-4 and IL-10 were evaluated. Data are shown as means ± SEM.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (21) group, \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ΔΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (28) group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/73a74211c7f5ce1f0e1192c3.png"},{"id":82622653,"identity":"0e18f42a-c07e-4e3d-b4a5-bc5f83c86cce","added_by":"auto","created_at":"2025-05-13 12:35:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":805451,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of BPA and SPN on iron import, storage, and export in testes. The data of gene was expressed as the relative levels of GAPDH, data are presented as means ± SEM. Western blotting were normalized to β-actin, and the gray values of western blotting bands were calculated by Image-Pro Plus 7.0 software, with data displayed means ± SEM. (a) Tfr1, Zip8 and Zip14 mRNA expressions. (b) The protein expression levels of TFR1, ZIP8 and ZIP14 in testes.(c) Fpn1, Fth and Ftl mRNA expressions. (d) The protein expression levels of FPN1, FTH and FTL in testes. (e) Dmt1, Steap3 and Ncoa4 mRNA expressions. (f) The protein expression levels of DMT1, STEAP3 and NCOA3 in testes. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05 in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 in comparison with the BPA+SPN (21) group;\u003csup\u003e Δ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ΔΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (28) group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/6293d599280853749b2d27eb.png"},{"id":82622649,"identity":"79f913d7-2ca0-4946-a66c-1263eae247ce","added_by":"auto","created_at":"2025-05-13 12:35:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2696706,"visible":true,"origin":"","legend":"\u003cp\u003eSPN alleviates BPA-induced testicular ferroptosis. (a) Ferritin contents in serum were measured using an ELISA kit. (b) Ferrous iron in testes was analyzed using a commercial kit. Data are presented as means ± SEM. (c) The protein expression of GPX4, SLC7A11, and ACSL4, and the gray values of western blotting bands were calculated by Image-Pro Plus 7.0 software, with data displayed means ± SEM. (d) Representative photographs of testes stained by immunofluorescence technique with GPX4, SLC7A11, and ACSL4 antibodies, scale bar=100 µm. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN(21) group;\u003csup\u003e Δ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e \u003c/sup\u003ein comparison with the BPA+SPN(28) group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/04655ce8ea17e3a4f7f8d502.png"},{"id":82622648,"identity":"1130be3a-bf28-4463-8bb4-90d62d9cc8e6","added_by":"auto","created_at":"2025-05-13 12:35:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2136091,"visible":true,"origin":"","legend":"\u003cp\u003eSPN alleviates BPA-induced testicular apoptosis. The protein expression of BAX (a)and BCL-2(b), with data are presented as means ± SEM. (c) The representative photographs of testes stained by immunofluorescence technique with BAX and BCL-2 antibodies, scale bar is 100 µm. (d) The mRNA expressions of Bax, Bcl-2, Caspase-3 and Caspase-9 relative to that of GAPDH. (e) Caspase-3 and Caspase-9 protein levels were also analyzed by kits. Data are presented as means ± SEM. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt;0.01, in comparison with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, in comparison with the BPA group; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, in comparison with the BPA+SPN (21) group.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/c691b26ed1c5fcfa7614b5f4.png"},{"id":82624695,"identity":"955bda17-84a1-4f9f-b128-20c0ed676ede","added_by":"auto","created_at":"2025-05-13 12:51:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9701890,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/80da2707-2cf7-45fc-b920-d88e4bddcd36.pdf"},{"id":82622342,"identity":"8991971a-fde8-4609-83c0-63e1c10ae488","added_by":"auto","created_at":"2025-05-13 12:27:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14405217,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfiguresandTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/f404a202d095f79f99368955.docx"},{"id":82622647,"identity":"1eaf6381-15c5-4755-b764-54c3dc973687","added_by":"auto","created_at":"2025-05-13 12:35:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":58478,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6338624/v1/f2fdefd87fa2585eb6e51eff.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Specnuezhenide attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBisphenol A (BPA) is a prevalent environmental pollutant arising from its extensive production and widespread application[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is frequently detected in various consumer goods, such as food packaging materials, baby bottles, reusable water bottles, PVC films, paper products, cardboard, and the epoxy resin linings of food cans[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Additionally, BPA is extensively utilized in the manufacturing of dental sealants and thermal paper[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Human exposure to BPA happens through diverse pathways, including ingestion, inhalation, and dermal absorption[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], with dietary intake considered the primary route[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. BPA was reported to impair male fertility, at the initial research, by mimicking endogenous estrogen or interacting with androgen receptors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, subsequent studies have further demonstrated that BPA also affects male fertility by causing mitochondrial dysfunction, driven by excessive accumulation of iron ion-dependent ROS, which triggers apoptosis and ferroptosis in testicular cells[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNatural substances are being investigated for their potential protective and therapeutic properties to mitigate the harmful effects of BPA. For instance, Cuscuta chinensis flavonoids (CCF) significantly reduced testicular cell apoptosis in male offspring when administered to pregnant mice exposed to BPA on postnatal days 21 and 56[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, crocin (Cr) alleviated BPA-induced apoptosis in adult rats by downregulating Caspase-3 expression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Melatonin has been shown to mitigate testicular damage by reducing oxidative stress (OS)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while vitamin E improved apoptosis in reproductive cells caused by BPA exposure[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings underscore the growing interest in natural compounds as low-toxicity, multifunctional agents with strong antioxidative properties, offering promising potential to address BPA-induced reproductive damage.\u003c/p\u003e \u003cp\u003eThe traditional Chinese medicinal herb, \u003cem\u003eFructus Ligustri Lucidi\u003c/em\u003e (FLL), which widely incorporated in tonic formulas, was first documented in the \u003cem\u003eShennong Materia Medica\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Specnuezhenide (SPN), a key bioactive compound in FLL, is recognized as a phytochemical marker for assessing the herb\u0026rsquo;s quality, as described in the current edition of the Chinese Pharmacopoeia[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. SPN has demonstrated significant antioxidative properties, notably its ability to mitigate OS caused by free radicals[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but its potential to alleviate BPA-induced testicular cell death remains unclear. Previous studies have shown that SPN can attenuate CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury in mice by counteracting OS through activation of the NRF2 signaling pathway and suppressing apoptosis through downregulation of the BCL-2 pathway[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These mechanisms suggest a potential parallel in the ability of SPN to counteract BPA-induced testicular OS and apoptosis. Thus, we speculate that SPN has the potential to alleviate BPA-induced testicular injury.\u003c/p\u003e \u003cp\u003eIn this review, we investigate how SPN can ameliorate BPA-induced testicular damage, with a focus on elucidating its role in modulating key cell death pathways, such as apoptosis and ferroptosis. By integrating findings from in vivo studies, we aim to provide a comprehensive understanding of the underlying mechanisms through which SPN exerts its protective effects. This analysis not only highlights SPN\u0026rsquo;s therapeutic potential but also offers valuable scientific insights and references for strategies to safeguard and enhance reproductive health.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemical and Reagents\u003c/h2\u003e \u003cp\u003eBPA (purity\u0026thinsp;\u0026ge;\u0026thinsp;99%) was purchased from Sigma (USA) and stored at 4\u0026deg;C. Specnuezhenide (SPN, HPLC\u0026thinsp;\u0026gt;\u0026thinsp;98%) was sourced from Nanjing Jingzhu Biotechnology Co., Ltd. (Jiangsu, China), with its content and composition verified by HPLC; the corresponding results are provided in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Corn oil was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China) to use as a solvent. Biochemical kits for superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), nitric oxide (NO), nitric oxide synthase (NOS), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) were sourced from Nanjing Jiancheng Institute of Biotechnology Co., Ltd. (Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kits for hormones of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH), while increasing estradiol (E2), cytokines of interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) were supplied by Jingmei Biotechnology Co., Ltd. (Jiangsu, China). Additionally, cysteine- dependent aspartate-specific protease-3 (Caspase-3) and cysteine- dependent aspartate-specific protease-9 (Caspase-9) assay kits, immunofluorescence staining reagents (anti-rabbit Alexa Fluor 488), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals and treatments\u003c/h2\u003e \u003cp\u003eThe experiments were conducted with male ICR mice weighting 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g, supplied by the Changchun Yisi Laboratory Animal Technology Co. Ltd. (License number: SCXK(Ji)2018-0007, Jilin, China. No.01021685682051708). The animals were housed under controlled conditions, with a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, and water with 12-hour light-dark cycle. They were fed a laboratory diet (SCXK(liao) 2022-0001). Sixty mice were randomly divided into three groups, each receiving treatments detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Mice in BPA and BPA\u0026thinsp;+\u0026thinsp;SPN groups were intragastric administration with BPA for 28 days. Then, mice in BPA\u0026thinsp;+\u0026thinsp;SPN groups were intragastric administration with SPN for 21-, 28-, and 35-days, respectively. At each experimental endpoint (21, 28, and 35 days), mice were anesthetized and euthanized to collect testes and serum for subsequent analyses. The body weight and testis weight of each mouse were recorded, and the relative organ weight was determined by the ratio of testis weight to body weight. For consistency, samples from 15 mice in either the control or model group were pooled for experimental analysis and data calculation, as per the study design.\u003c/p\u003e \u003cp\u003e The experimental protocol was approved by the Ethics Committee of Jilin Medical University, Jilin, China (2023-LW015). All procedures were performed by qualified technicians with appropriate laboratory animal handling certifications, strictly adhering to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals regarding rodent disposal standards. The reporting complies with the ARRIVE guidelines. The entire experiment followed the \"3R principles\" (Reduction, Refinement, Replacement). After a 6-hour preoperative fast, the animals were deeply anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg), with the disappearance of corneal reflexes and absence of response to painful stimuli serving as the criteria for effective anesthesia. For euthanasia, cervical dislocation was performed on mice after confirming a state of anesthesia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Epididymal sperm analysis\u003c/h2\u003e \u003cp\u003eEpididymal tails from each group of mice were dissected into small pieces and subsequent incubated in the 0.9% NaCl solution (0.5 mL) that prewarmed to 37\u0026deg;C for 30 mins. The resulting suspension was used to isolate epididymal sperm. A 20 \u0026micro;L aliquot of the sperm suspension was loaded onto a Makler counting chamber and then using a computer-assisted sperm analysis system (SCA, Microptic, Spain) to analyze them. For each sample, ten fields of view were selected to assess sperm concentration and viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Analysis of serum hormone and inflammatory cytokine levels\u003c/h2\u003e \u003cp\u003eThe serum samples were analyzed quantitatively using ELISA kits to measure the hormones of T, LH, FSH, and E2, pro-inflammatory cytokines of IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokines of IL-4 and IL-10 levels in accordance with the manufacturer\u0026rsquo;s instructions (Jiangsu Jingmei Biotechnology Co., Ltd.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of ferritin contents in serum and Fe\u003csup\u003e2+\u003c/sup\u003e levels in testicular tissues\u003c/h2\u003e \u003cp\u003eSerum ferritin levels were quantified using a mouse ferritin ELISA kit (Jiangsu Jingmei Biotechnology Co., Ltd.), under the guidance of the manufacturer\u0026rsquo;s protocol. The absorbance was measured at the wavelength of 450 nm using an Epoch plate reader (Epoch, BioTek, America), and the ferritin concentration was subsequently calculated.\u003c/p\u003e \u003cp\u003eIron concentrations in testicular tissues were assessed by means of Iron Assay Kit (Nanjing Jiancheng Institute of Biotechnology Co., Ltd.). Testicular cell lysates were treated with an iron reducer to convert ferric iron (Fe\u0026sup3;⁺) into ferrous iron (Fe\u0026sup2;⁺), followed by incubation in a water bath at 100\u0026deg;C, with 5 mins, allowing the formation of a colored complex. The absorbance measurement was performed at the wavelength of 520 nm, and Fe\u0026sup2;⁺ levels were determined according to the provided protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Mitochondrial function analysis\u003c/h2\u003e \u003cp\u003eTesticular homogenates were collected at 3000 g, 4\u0026deg;C for 20 mins at to obtain supernatants for subsequent analyses. The electron transport chain (ETC) complexes (I, II, and V) activities in testicular homogenates were measured by means of ELISA kits (Beijing Solarbio Science \u0026amp; Technology Co., Ltd., China) in accordance with the protocols, with absorbance values recorded on an Epoch microplate reader (Epoch, BioTek, America). All experiments were performed in triplicate.\u003c/p\u003e \u003cp\u003eAdenosine triphosphate (ATP) levels in testicular homogenates were measured employing an ATP assay kit (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China). Tissue samples were lysed with an ATP-releasing reagent, and subsequently, lysates were mixed with ATP detection solution. ATP concentrations were normalized to protein content, which was determined using an enhanced BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China).\u003c/p\u003e \u003cp\u003eThe NAD⁺/NADH and NADPH/NADP⁺ ratios in testicular homogenates were measured by means of assay kits (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China). The absorbance measurement was performed at 570 nm with a microplate reader, and the ratios were determined according to the absorbance values as specified in the kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Oxidative stress assay\u003c/h2\u003e \u003cp\u003eROS levels in testicular homogenates of mice were measured using a ROS assay kit (Jingmei Biotechnology Co., Ltd., Jiangsu, China) under the guidance of the manufacturer\u0026rsquo;s instructions. Briefly, 50 \u0026micro;L of testicular homogenate was added to an ELISA plate in duplicate, incubated at 37\u0026deg;C for 30 mins, and subsequently washing. After a second incubation at same conditions with 50 \u0026micro;L of enzyme-labeled reagents, followed by washing, a colorimetric reaction was initiated. Then using an Epoch microplate reader (Epoch, BioTek, America) to measure the values of optical density (OD) at 450 nm.\u003c/p\u003e \u003cp\u003e Enzymatic activities of SOD, NOS, CAT, and GSH-Px, along with the concentrations of MDA, GSH, and NO, were assessed in serum and testicular supernatants by means of commercial kits (Nanjing Jiancheng Institute of Biotechnology, Jiangsu, China) in accordance with the manufacturer\u0026rsquo;s instructions. The supernatants protein concentrations were determined by means of Bradford Protein Assay Kits (BCA, Beyotime Biotechnology Co., Ltd., Shanghai, China). All measurements were conducted in triplicate to ensure accuracy.\u003c/p\u003e \u003cp\u003eFor further analysis the anti-oxidative effect of SPN, the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (\u003cb\u003e\u0026middot;\u003c/b\u003eOH) radical scavenging capacity and iron reducing power were also measured in vitro (Detailed procedures were shown in Fig. S2 and S8)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Histopathological analysis\u003c/h2\u003e \u003cp\u003eTesticular tissues were fixed and preserved in 4% paraformaldehyde, dehydrated, and subsequently embedded in paraffin. Sections, 5 \u0026micro;m thick, were prepared by means of a rotary microtome (RM2245, Leica, Wetzlar, Germany) and subsequently stained with hematoxylin and eosin (H\u0026amp;E). Light microscope (BX53, Olympus, Tokyo, Japan) was use to examine the histopathological changes. To ensure consistency, three mice were analyzed, with three sections evaluated per mouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Immunofluorescence analysis\u003c/h2\u003e \u003cp\u003eThe subcellular localization of target proteins was assessed using immunofluorescence, following previously established protocols[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Paraffin-embedded testicular sections were successively deparaffinized, rehydrated, permeabilized, and blocked. The slides were subsequently reacted with primary antibodies (details provided in Table S2) and then exposed to an Alexa Fluor 488-conjugated secondary antibody using an immunofluorescence staining kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Cellular nuclei were stained with DAPI as a counterstain, and the slides were mounted with anti-fluorescence quenching solution and sealed. Fluorescence signals were observed by means of a fluorescence microscope (BX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Caspase-3 and \u0026minus;\u0026thinsp;9 measurement\u003c/h2\u003e \u003cp\u003eCaspase-3 and \u0026minus;\u0026thinsp;9 activities in testicular homogenates were measured using commercial assay kits (Beyotime Biotechnology Co., Ltd., Shanghai, China). The absorbance measurement was determined at a wavelength of 405 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Western blotting\u003c/h2\u003e \u003cp\u003eTotal protein from testes were extracted, separated by means of 10\u0026ndash;12% SDS-PAGE, and then transferred onto the PVDF membranes (Millipore, Merck, Germany). Those samples were exposed to 5% BSA in TBST buffer (0.1% Triton X-100 in PBS), for 1 h with pH of 7.4, at room temperature. After subsequent blocking, they were exposed to the specified primary antibodies (Table S3) at 4\u0026deg;C overnight, followed by the corresponding secondary antibodies at 37\u0026deg;C for 1 h. After three washes with TBST, protein bands were detected using a chemiluminescence reagent (Beijing Solarbio Science \u0026amp; Technology Co., Ltd., China). The levels of relative protein were normalized to β-actin, which served as the loading control. Band intensities were quantified using a Bio-Rad GelDoc XR\u003csup\u003e+\u003c/sup\u003e system and analyzed with Image-Pro Plus software (Bio-Rad Laboratories, Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. qPCR amplification\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from testicular tissues by means of Trizol reagent (Invitrogen, Thermo Fisher Scientific, USA) in accordance with the manufacturer\u0026rsquo;s protocols. The first strand cDNAs were synthesized by means of the Start Script First-Strand cDNA Synthesis Kit (GenStar, Co., Ltd., China). Quantitative PCR was conducted employing the RealStar Green Fast Kit (GenStar, Co., Ltd., China) in an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA). Primer sequences for target genes (listed in Table S4) were synthesized and provided by Sangon Biotech Co., Ltd. (Shanghai, China). The qPCR protocol can separate in two main steps: an initial denaturation at 95\u0026deg;C for 2 mins, and a second step of successively 40 cycles at 95\u0026deg;C for 15 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s. Each experimental group included samples from five individual mice, with three technical replicates per sample. GAPDH served as the internal control, and the expression levels of relative mRNA were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences between groups were evaluated using one-way ANOVA, with a statistical significance of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Fig.s and visualizations were created using GraphPad Prism software to illustrate the results.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. SPN alleviates BPA-induced sperm quality reduction\u003c/h2\u003e \u003cp\u003eBody weight changes were monitored throughout the experimental period. After BPA treatment, the body weight was deceased compared to control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), whereas there was no remarkable difference between control and BPA groups at the beginning of the experiments (Table S5). After SPN treatment, the body weights of BPA + SPN groups were increased relative to the BPA group from day 0 to day 35 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Table S6). On day 21, the body weight of BPA + SPN (35) were higher compared with BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). On day 28, the body weights of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) were increased relative to BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). On day 35, the body weights of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) showed an increase as compared to BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). The body weight gain was dependent on SPN dose increase.\u003c/p\u003e \u003cp\u003eThe effects of BPA and SPN treatments on testicular and sperm parameters were further analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d). BPA exposure resulted in significant reductions in testicular weight, testicular organ coefficient, sperm concentration, and sperm motility relative to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). SPN supplementation effectively counteracted these adverse effects. The testicular weight, testicular organ coefficient, sperm concentration and sperm motility of BPA + SPN (21), BPA + SPN (28) and BPA + SPN (35) increased significantly compared with that of BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Notably, the testicular weight in the BPA + SPN (28) and BPA + SPN (35) groups was statistically higher than that in the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Similarly, sperm concentration in the BPA + SPN (28) group showed a prominent increase relative to the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. SPN alleviates BPA-induced hormonal disorder and testicular tissue damage\u003c/h2\u003e \u003cp\u003eHormonal analysis revealed significant alterations in plasma hormone levels following BPA exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). T, FSH and LH levels were dramatically reduced in the BPA group when compared with the results of control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), while E2 levels showed a considerable increase (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). SPN treatment significantly restored T, FSH, LH, and E2 levels in all BPA + SPN groups relative to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Notably, T, FSH, and E2 levels displayed statistically remarkable differences among the BPA + SPN treatment groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). LH levels were meaningfully higher in the BPA + SPN (35) group relative to the BPA + SPN (21) and BPA + SPN (28) groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). H\u0026amp;E staining showed well-ordered testicular cells with regular structure in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Seminiferous tubules were enlarged with disordered testicular cells in model group. Granular degeneration, nuclear condensation, interstitial proliferation and inflammatory cell infiltration were also pronounced in model group. BPA + SPN (35) group showed apparently alleviated pathological lesions in testis with slight enlargement of seminiferous tubules. SPN treatment effectively protected testes structures against BPA damage and reduced the loss of spermatophores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. SPN alleviates BPA-induced testicular mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003eThe BPA-treated mice exhibited a remarkable increment in the ROS level in comparison with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Concurrently, the relative ratios of NAD⁺/NADH and NADPH/NADP⁺ in testicular tissues were reduced significantly in the BPA group versus the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). SPN administration decreased the ROS levels and increased the testicular NAD\u003csup\u003e+\u003c/sup\u003e/NADH and NADPH/NADP\u003csup\u003e+\u003c/sup\u003e relative ratios (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This situation was developed time-dependently (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The effects of BPA and SPN on ATP levels and the activity of ETC complexes (I, II, and V) were also examined. BPA exposure resulted in significant reductions in ATP content and the activities of ETC complexes (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), while SPN administration markedly improved ATP levels and ETC complex activities in SPN-treated mice (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. SPN alleviates BPA-induced oxidative damage in mice\u003c/h2\u003e \u003cp\u003eBoth in vitro and in vivo experiments were performed to confirm the anti-oxidant activity of SPN. In vitro, anti-oxidant activity was calculated based on clearance rates, with lower IC-50 values indicating higher activity. The IC-50 values of the SPN (Fig. S2a and S2b), calculated using DPPH and hydroxyl radical scavenging capacity, demonstrated that the removal of different kinds of free radicals may be related to the different biological activities of SPN. In addition, the IC-50 of the SPN (DPPH IC-50 = 271.5 µg/mL, hydroxyl radical IC-50 = 572.98 µg/mL) is lower compared to the VC (DPPH IC-50 = 44.69 µg/mL, hydroxyl radical IC-50 = 375.80 µg/mL) (Fig. S2c).\u003c/p\u003e \u003cp\u003eIn vivo experiments, the antioxidant oxidase activity and the markers of oxidative damage were also determined by a mice model. Compared with control group, BPA exposure led to significant reductions in SOD and GSH-Px activities in testicular tissues and serum, accompanied by increased MDA, NOS, and NO levels, while CAT activity in testicular tissue also decreased significantly (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). However, SPN supplementation restored the SOD, GSH-Px, CAT and NOS activities, the MDA and NO contents in BPA + SPN (28) and BPA + SPN (35) groups when compared with BPA-treated group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Moreover, SPN supplementation significantly restored the SOD and CAT activities, MDA and NO contents of serum and testicular tissues, NOS activity of serum in BPA + SPN (21) group relative to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). SPN treatment for 28 and 35 days showed better resistance to BPA- induced oxidative damage than SPN treatment for 21 days. SPN treatment for 35 days restored the MDA content in serum and testicular tissues, SOD activity in testes, NOS activity and NO content in serum when compared to SPN treatment for 28 days (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). No-significant change in the CAT and GSH-Px activities of serum and testes was observed between BPA + SPN (28) group and BPA + SPN (35) group (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eMoreover, the NAD(P)H dehydrogenase, quinone 1 (NQO1) expression level in BPA group was higher compared with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), while SPN supplementation significantly reduced NQO1 expression levels in SPN-treated mice (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). In contrast, no significant differences in the NF-E2-related factor 2 (NRF2) expression were observed among five groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). The heme oxygenase-1 (HO-1) expression in the BPA + SPN (35) group was meaningfully lower than that of other groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eImmunofluorescence analysis confirmed that BPA exposure markedly increased neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression levels, and decreased CAT, superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) expression levels in comparison with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig. S3-S7), while it was reversed by SPN administration in a time-dependent manner (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e \u003cp\u003eGSH content in testicular tissues was notably lower in the BPA group when compared with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). SPN supplementation significantly enhanced GSH contents in all BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), with BPA + SPN (28) and BPA + SPN (35) groups showing higher GSH levels than the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Non-significant change was observed between BPA + SPN (35) group and BPA + SPN (28) group (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. SPN alleviates BPA-induced inflammation in testes\u003c/h2\u003e \u003cp\u003eThe mRNA and protein levels of IL-6, IL-1β, and TNF-α in BPA group have a significantly increase when compared to the control group due to the BPA exposure (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, SPN supplementation significantly reduced the mRNA and protein expressions of IL-6 and IL-1β in BPA + SPN groups relative to the BPA-treated group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), although no remarkable differences in IL-1β expressions were observed among the BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Changes in IL-6 mRNA and TNF-α protein levels were significant among BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), with TNF-α mRNA levels notably lower in BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e \u003cp\u003eThe levels of IL-4 and IL-10 mRNA and protein have a marked decrease in the BPA group when in comparison with the results of control group due to the BPA exposure (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). SPN supplementation significantly increased IL-4 and IL-10 expressions in BPA + SPN (28) and BPA + SPN (35) groups relative to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). IL-10 mRNA levels in BPA + SPN (28) and BPA + SPN (35) groups were higher than those in BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), while IL-4 mRNA levels in BPA + SPN (35) group were higher than in BPA + SPN (21) and BPA + SPN (28) groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Protein expressions of IL-4 and IL-10 in the BPA + SPN (35) group were also markedly elevated as compared to the BPA + SPN (21) group due to the SPN treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effects of SPN on iron metabolism pathways\u003c/h2\u003e \u003cp\u003eTo assess whether BPA exposure increased iron uptake in testicular cells, we investigated the mRNA and protein expressions of major extracellular ferric iron importers, including transferrin receptor1 (TFR1, CD71), solute carrier family 39 (zinc transporter), member 8 (ZIP8, SLC39A8) and member 14 (ZIP14, SLC39A14). RT-PCR examination shown that the levels of mRNA of Tfr1 and Zip14 have significant changes (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), whereas SPN treatment mitigated these effects, reducing iron absorption (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Notably, the expression of Tfr1 mRNA in the BPA + SPN (35) group was higher versus with BPA + SPN (21) and BPA + SPN (28) groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Conversely, Zip14 mRNA expression reduced in BPA + SPN (28) and BPA + SPN (35) groups relative to BPA + SPN (21) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). At the protein level, TFR1 expression was elevated in the BPA group compared to the control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), but SPN treatment significantly reduced TFR1 levels in BPA + SPN (21) and BPA + SPN (28) groups compared to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). TFR1 protein levels further declined in BPA + SPN (28) group compared to BPA + SPN (21) group (p \u0026lt; 0.05), with an increase in the BPA + SPN (35) group relative to BPA + SPN (28) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). For ZIP14, no remarkable differences were observed in protein expression across the groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Similarly, Zip8 mRNA and protein expressions did not show substantial differences between the BPA and control groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, Zip8 mRNA was lower in BPA + SPN (28) compared to BPA + SPN (21) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), and the level of Zip8 mRNA or ZIP8 protein displayed higher in BPA + SPN (35) compared to the BPA + SPN (28) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eSince increased intracellular ferrous iron may result from impaired export function, we examined the expression of the primary iron exporter, ferroportin1 (FPN1, SLC40A1). ANOVA analysis shown that BPA exposure substantially increased the expression of Fpn1 mRNA compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). SPN treatment considerably decreased Fpn1 mRNA expression in the BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). However, BPA and SPN had no considerable influence on FPN1 protein expression across all groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eBPA exposure also significantly upregulated mRNA levels of ferritin heavy chain (Fth) and ferritin light chain (Ftl), key components of iron storage (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). SPN supplementation caused significant differences in Ftl mRNA expression between the BPA and BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), but no considerable changes were observed in FTL protein expression between the control and other groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Similarly, no meaningful differences in FTH protein expression were observed between the BPA and BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Notably, Fth mRNA expression changes were significantly difference among BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eAfter entering the endosome, imported intracellular iron is reduced to ferrous iron by the metalloreductase six-transmembrane epithelial antigen of the prostate 3 (STEAP3) and subsequently released into the cytoplasmic labile iron pool via divalent metal transporter 1 (DMT1) for cellular utilization or storage in ferritin. ANOVA analysis revealed that BPA exposure markedly affected Steap3 mRNA expression, which was reversed by SPN treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Steap3 mRNA expression was lower in the BPA + SPN (28) and BPA + SPN (35) groups relative to the BPA + SPN (21) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). However, BPA exposure did not alter STEAP3 protein levels (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), and while STEAP3 protein levels tended to decrease in the BPA + SPN groups, the differences were not significant in comparison with the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Similarly, DMT1(SLC11A2) protein levels not displayed significantly differences between BPA, SPN, and control groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). However, BPA exposure significantly increased Dmt1 mRNA expression compared with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). SPN supplementation reduced Dmt1 mRNA levels in all BPA + SPN groups compared to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Furthermore, Dmt1 mRNA levels were lower in the BPA + SPN (28) and BPA + SPN (35) groups compared to the BPA + SPN (21) group, but it was reversed in protein levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eActing as an autophagic cargo receptor, NCOA4 facilitates the degradation of ferritin, resulting in elevated levels of free iron. To determine whether the observed iron accumulation in our model was associated with ferritinophagy, we analysed the protein and mRNA expression levels of NCOA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). The results indicated no noticeable differences in NCOA4 protein expression among the groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). However, the levels of Ncoa4 mRNA in the BPA group were higher when compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), and were lower in the BPA + SPN groups relative to the BPA group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Additionally, the levels of Ncoa4 mRNA in BPA + SPN (28) group were decreased in comparison with BPA + SPN (21) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), but increased in the BPA + SPN (35) group in comparison with BPA + SPN (28) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7. SPN alleviates BPA-induced testicular ferroptosis\u003c/h2\u003e \u003cp\u003eTo assess the role of iron in SPN-mediated protection against oxidative damage, the iron-reducing power of SPN was evaluated in vitro. As shown in Fig. S8, SPN demonstrated strong iron-reducing ability. In vivo results further indicated that BPA exposure increased serum ferritin levels and testicular ferrous iron content compared with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). These effects were reversed in BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Western blotting assay showed BPA dramatically decreased the expression of glutathione peroxidase 4 (GPX4) in testicular tissues (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) and these effects were reversed in BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The levels of solute carrier family 7 member 11 (SLC7A11) in BPA group were decreased in comparison with control group (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), and it was reversed by SPN (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Addition, the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) in BPA group were decreased in comparison with control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), and it was reversed by SPN (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). These results were further supported by immunofluorescence staining, which revealed enhanced expression of GPX4, SLC7A11, and ACSL4 after SPN treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003ed and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e-\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). Moreover, SPN upregulated the mRNA expression of Gpx4 and Acsl4 in testes exposed to BPA (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig. S12).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8. SPN alleviates BPA-induced testicular apoptosis\u003c/h2\u003e \u003cp\u003eNo significant differences in BCL2-associated X protein (BAX) and B-cell CLL/lymphoma 2 (BCL-2) protein expression were observed between the BPA and BPA + SPN groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and \u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Immunofluorescence staining showed apparent changes in BAX and BCL-2 antibodies between BPA + SPN(35) and BPA groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS13\u003c/span\u003e and \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003eS14\u003c/span\u003e). At the mRNA level, BPA exposure upregulated the expression of the pro-apoptotic marker Bax and downregulated the anti-apoptotic marker Bcl-2 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). SPN treatment reversed these changes, normalizing the mRNA levels of Bax and Bcl-2 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Furthermore, BPA exposure increased the protein and mRNA expression of Caspase-3 and Caspase-9 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ed and \u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). SPN treatment suppressed the expression of these apoptosis-related markers at both the protein and mRNA levels.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSome studies demonstrated that BPA exposure induces OS, iron deposition, and mitochondrial structural abnormalities, while also altering the expression of proteins related to ferritinophagy, ferroptosis, and apoptosis, suggesting that BPA triggers OS, promotes iron accumulation, and leads to cell death in mouse testes[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SPN exhibits diverse pharmacological activities, which may stem from its strong free radical scavenging ability, as observed in this study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Based on these properties, we hypothesized that SPN could mitigate testicular cell death-induced by BPA through modulating OS and iron accumulation pathways. To investigate the therapeutic effects of SPN on BPA-induced testicular damage, we conducted comprehensive analyses of dosage optimization for both compounds.\u003c/p\u003e\u003cp\u003e The establishment of an effective mouse model required careful determination of BPA dosage. Our BPA exposure regimen (50 mg/kg BW/day) was selected based on the previously reported NOAEL (No Observable Adverse Effect Level) for reproductive and developmental toxicity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Subsequent evaluation of SPN's therapeutic potential revealed dose-dependent effects on reproductive system toxicity (Fig. S15). Sperm motility improvements showed progressive enhancement across the 30–60 mg/kg SPN range. Hormonal analyses demonstrated concomitant dose-dependent increases in T, FSH, and LH levels, along with a reduction in E2 concentrations. Comparative analysis between 15 mg/kg and 30 mg/kg SPN doses revealed comparable therapeutic efficacy against BPA-induced testicular toxicity. We selected 30 mg/kg as the optimal therapeutic dose, balancing efficacy with safety considerations while avoiding potential toxicity associated with higher doses. Treatment duration analysis revealed temporal patterns in therapeutic response (Fig. S16). Biochemical parameters including serum hormone levels (T, FSH, LH, E2) and testicular oxidative stress markers (SOD, MDA) demonstrated significantly enhanced recovery in the 21–35 days treatment groups compared to shorter durations (7–14 days). Based on these findings, we focused subsequent investigations on 21-, 28-, and 35-day treatment periods to identify the optimal therapeutic window. To elucidate the molecular mechanisms underlying SPN's protective effects, we systematically investigated its capacity to mitigate BPA-induced iron accumulation and associated pathological processes in testicular tissue. This iron dysregulation, if unaddressed, could lead to chronic cellular toxicity and programmed cell death.\u003c/p\u003e\u003cp\u003eIn testicular tissue, BPA exhibits antiandrogenic and estrogen-like effects, causing further damage to testicular cells[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a potent endocrine disruptor, it impairs male gonads and disrupts the redox balance in testes and sperm[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Studies have shown that BPA negatively affects sperm parameters, including reduced testicular organ coefficients, sperm motility, and concentration[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consistent with these findings, the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e1\u003c/span\u003e support this hypothesis in this work. Furthermore, our results also demonstrate that SPN supplementation effectively reversed these changes. Prolonged SPN treatment led to increased testicular weight, organ coefficients, sperm concentration, and motility.\u003c/p\u003e\u003cp\u003eAs noted, BPA disrupted the endocrine system in treated mice. Recent epidemiological studies report that BPA exposure decreases LH, FSH, and T levels while increasing progesterone and E2 levels[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, prior studies have shown reduced LH, FSH and T levels in rats treated with BPA at 50 mg/kg/day for 30 days [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These findings align closely with our laboratory results. Moreover, in the present study, hormone levels in BPA + SPN groups were higher compared to the BPA group, suggesting that SPN facilitates the restoration and/or enhancement of antioxidant capacity, which supports spermatogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The protective role of SPN against BPA-induced reproductive toxicity was further confirmed through H\u0026amp;E staining results.\u003c/p\u003e\u003cp\u003eOur results provide evidence that BPA exposure significantly increased ROS in the testes, suggesting BPA heightened OS (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to our findings, SPN is capable of scavenge and neutralize free radicals in vitro and it can significantly mitigated BPA-induced ROS production, similarly to other researchers' findings[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. NADPH plays a vital role in converting oxidized glutathione (GSSG) into reduced GSH[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], with NADPH, GSH, and SOD serving as key endogenous antioxidants [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, BPA exposure markedly decreased the NADPH/NADP⁺ ratio, GSH content, and SOD activity in testicular tissues, indicating reduced antioxidant capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003e). SPN treatment not only restored the NADPH/NADP⁺ ratio but also increased GSH content and SOD activity after BPA treated, confirming its antioxidant properties.\u003c/p\u003e\u003cp\u003eIn the cytoplasm, lactate dehydrogenase reduces NAD⁺ to NADH [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] while the oxidative phosphorylation ETC complex I (NADH dehydrogenase) oxidizes NADH back to NAD⁺, facilitating the reduction of oxygen to water and ATP production[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Electron leakage from the ETC and reduced ATP synthesis can elevate mitochondrial ROS levels, triggering apoptosis[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, BPA exposure decreased the NAD⁺/NADH ratio, which was restored by SPN supplementation. Similarly, BPA reduced ATP content and inhibited ETC complex (I, II, and V) activities, leading to elevated ROS levels. Expectedly, SPN reverse the decrease of ATP synthesis and ETC complexes (I, Ⅱ, and V) levels in our results. It is implied that SPN can improve the function of mitochondria.\u003c/p\u003e\u003cp\u003eThe mitochondrial antioxidant enzyme defense system plays a crucial role in the normal process of spermatogenesis. Key antioxidant enzymes such as SOD, CAT and GSH-Px help eliminate ROS and maintain redox balance[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. BPA exposure has been shown to increase NO, nitric oxide synthase 2A (NOS2A), and ROS levels while reducing NADPH levels[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Notably, BPA-induced NO production is largely driven by inducible nitric oxide synthase (iNOS) in cells[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, SPN treatment effectively prevented testicular oxidative damage in BPA-exposed mice by enhancing antioxidant levels, including SOD, CAT, GSH-Px and GSH in serum and testicular tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e4\u003c/span\u003e). SPN also mitigated BPA-induced toxicity by reducing NOS activity, MDA, and NO levels in serum and testes. In line with the results of NOS, the increase of nNOS and iNOS, decrease of CAT, SOD1 and SOD2 expressions-treated by BPA were also confirmed by immunofluorescence and reversed by SPN treatment (Fig. S3-S7). Downregulation nNOS and iNOS (markers of RNS), upregulation of SOD1 and SOD2 (markers of ROS) levels would be an effective way to alleviate BPA-induced testicular destroy in mice.\u003c/p\u003e\u003cp\u003eOur study provides evidence that SPN alleviates oxidative damage induced by BPA. BPA also promotes the accumulation of proteins associated with OS signaling[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The NRF2/HO-1/NQO1 signaling pathway has garnered attention for its critical role in regulating cellular OS and offering protection against BPA-induced reproductive toxicity[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, the NRF2/HO-1 pathway exhibits anti-inflammatory effects by suppressing inflammatory proteins such as TNF-α and IL-6[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Contrary to the previous findings, our study observed increased NRF2, HO-1, and NQO1 protein levels following BPA exposure. This elevation is likely a compensatory response to BPA-induced testicular injury and was reversed by SPN treatment in BPA-exposed mice. Furthermore, SPN reduced the expression of IL-6, IL-1β and TNF-α significantly, while enhancing IL-4 and IL-10 at both protein and gene levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar effects of IL-4 and IL-10 have been reported in BPA-exposed mice in previous studies[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Overall, SPN appears to mitigate BPA-induced damage by activating antioxidant and anti-inflammatory pathways at both the expression of protein and gene.\u003c/p\u003e\u003cp\u003eResearch indicates that ferrous iron accumulation is mainly attributed to disturbances in iron homeostasis, which are intrinsically connected to iron metabolism. Under normal conditions, circulating ferric iron is taken up by cells through TFR1[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Extracellular iron can also be imported via metal transporters such as ZIP8 and ZIP14. Once inside the cell, ferric iron is reduced to ferrous iron by STEAP3 in the endosome and subsequently released into the cytoplasmic labile iron pool by DMT1[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Ferrous iron is then used for physiological reactions or stored in FTH, while excess iron is exported by FPN1[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Disruptions in these pathways can trigger ferroptosis. In this study, BPA exposure decreased DMT1, FPN1, FTH and FTL protein levels, while increasing the TFR1, ZIP8, ZIP14, and STEAP3 protein expressions. However, compared to the control group, BPA exposure did not significantly alter the protein expressions of DMT1, FPN1, ZIP8, ZIP14, FTH, or FTL, suggesting that iron import via ZIP8 and ZIP14, export by FPN1, and storage by FTH and FTL were not evidently affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e6\u003c/span\u003e). SPN treatment reversed these proteins expression changes, notably downregulating TFR1, indicating a decrease in intracellular iron transport demand. Additionally, ferritinophagy mediated by NCOA4 has been reported as a pathway for ferroptosis induction[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. While silencing NCOA4 has been shown to reverse BPA-induced FTH degradation and MDA accumulation[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], our results revealed no remarkable differences in NCOA4 protein expression between the BPA and control groups. However, NCOA4 expression was lower in the SPN-treated groups compared to the BPA group, although this difference lacked statistical significance. These results indicate that BPA-induced iron accumulation may involve ferritinophagy, and SPN could partially reverse this process. Overall, BPA-induced iron accumulation appears to result from enhanced iron absorption and transport combined with reduced storage and export. SPN alleviates these effects by regulating iron metabolism and inhibiting ferritinophagy, a pathway associated with autophagy-dependent ferroptosis. As a potential negative regulator of ferritinophagy in testicular cells, SPN may contribute to maintaining iron homeostasis. Notably, some mRNA results were inconsistent with protein findings, suggesting that SPN's regulation of iron homeostasis may extend beyond the pathways examined here, warranting further investigation into the underlying mechanisms.\u003c/p\u003e\u003cp\u003eIron metabolism in testicular tissues must be tightly regulated to prevent iron overload. Ferritin, a highly conserved protein composed of FTH and FTL polypeptide chains, plays a key role in maintaining iron homeostasis. FTH catalyzes Fe²⁺ oxidation, while FTL facilitates ferric iron (Fe³⁺) storage[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Ferroptosis regulation involves GPX4, an antioxidant enzyme in mitochondria, SLC7A11, a cysteine transporter crucial for GSH synthesis, and ACSL4, which influences lipid composition[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. GPX4 converts lipid hydroperoxides to lipid alcohols, preventing Fe²⁺-dependent toxic lipid ROS formation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. BPA has been reported to induce Fe²⁺ accumulation in testes, along with decreased GPX4 expression and increased ferritin levels, contributing to ferroptosis in testicular cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, BPA treatment significantly elevated Fe²⁺ levels in testes and ferritin levels in serum while reducing GPX4 and SLC7A11 expression in testicular tissue, consistent with previous findings, suggesting ferroptosis as a key mechanism in BPA-induced testicular injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Meanwhile, SPN-mediated ferroptosis resistance was achieved via decrease Fe\u003csup\u003e2+\u003c/sup\u003e and ferritin contents, upregulation of the GPX4 and SLC7A11 expression levels, suggesting that SPN can also inhibit BPA-induced ferroptosis in testes. Besides, expressions of GPX4 and SLC7A11 were also assessed by immunofluorescence. Immunofluorescence analysis further confirmed that SPN significantly suppressed BPA-induced NRF2 overexpression while restoring GPX4, GPX4, and SLC7A11 levels. These results suggest that SPN activates NRF2 downstream pathways involving GSH, SLC7A11, and GPX4, thereby protecting against BPA-induced testicular ferroptosis.\u003c/p\u003e\u003cp\u003eACSL4 is a key isozyme involved in the metabolism of polyunsaturated fatty acids (PUFAs), playing a crucial role in determining ferroptosis sensitivity[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Ferroptosis is a controlled cell death mechanism driven by iron-dependent lipid peroxidation reaching toxic levels[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. While the role of ACSL4 in BPA-induced lipid peroxidation and ferroptosis remains unclear, increased ACSL4 levels have been observed in BPA-induced lipid accumulation in the liver[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. To investigate the protective effects of ACSL4, western blotting and immunofluorescence analyses were conducted on testicular tissue undergoing BPA-induced ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e8\u003c/span\u003e). BPA significantly downregulated ACSL4 expression in the testes, which was notably ameliorated by SPN treatment. These findings suggest that ACSL4 is a critical determinant of ferroptosis sensitivity and that SPN may exert protective effects by modulating ACSL4 expression and maintaining cellular homeostasis.\u003c/p\u003e\u003cp\u003eBPA-induced testicular damage through OS can occur via both ferroptosis and apoptosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. To explore how SPN mitigates BPA-induced testicular apoptosis, we investigated the expression of apoptosis-related proteins. Previous studies have reported that BPA exposure increases the expression of Caspase-3, Caspase-9, and Bax, while reducing Bcl-2 expression[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Our mRNA and protein analyses corroborated these findings, showing similar changes in apoptosis-related markers. Notably, SPN treatment reversed these alterations, suggesting that SPN alleviates testicular cell death by modulating the mitochondria-dependent apoptotic pathway.\u003c/p\u003e\u003cp\u003eIt is intriguing that in this study, SPN rescued BPA-induced damage do not seem to be at a time-dependent manner, the results in SPN + BPA (28) group were apparently superior to the other two groups. This nonlinear relationship may be due to the fact that although SPN is an antioxidant, high dose SPN produced toxic side effects and enhanced the toxicity of BPA.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study is the first to demonstrate that SPN has an observable testicular protective effect on BPA exposure mice by increasing its ability to suppress hormonal imbalance, OS, iron accumulation, ferroptosis, and apoptosis. It is also proposed for the first time that the regulation of NAD\u003csup\u003e+\u003c/sup\u003e/NADH, NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratios and ACSL4 expression are involved in the protective effects of SPN against BPA-induced reproductive system damage. These findings provide a foundation for the potential prevention and treatment of BPA-induced testicular toxicity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"557\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eBPA \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOS \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSPN \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eFLL \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eH\u0026amp;E \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eT \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eLH \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eFSH \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eE2 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eGpx4,GPX4\u003c/p\u003e\n \u003cp\u003eSLC7A11\u003c/p\u003e\n \u003cp\u003eCaspase-3\u003c/p\u003e\n \u003cp\u003eCaspase-9\u003c/p\u003e\n \u003cp\u003eBax, BAX\u003c/p\u003e\n \u003cp\u003eBcl-2, BCL-2\u003c/p\u003e\n \u003cp\u003eCCF\u003c/p\u003e\n \u003cp\u003eCr\u003c/p\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003cp\u003eNOS\u003c/p\u003e\n \u003cp\u003eGSH-Px\u003c/p\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003cp\u003eIL-4\u003c/p\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003cp\u003eETC\u003c/p\u003e\n \u003cp\u003eATP\u003c/p\u003e\n \u003cp\u003eOD\u003c/p\u003e\n \u003cp\u003eBCA\u003c/p\u003e\n \u003cp\u003eDPPH\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003eOH\u003c/p\u003e\n \u003cp\u003eNQO1\u003c/p\u003e\n \u003cp\u003eHO-1\u003c/p\u003e\n \u003cp\u003eNRF2\u003c/p\u003e\n \u003cp\u003enNOS\u003c/p\u003e\n \u003cp\u003eiNOS\u003c/p\u003e\n \u003cp\u003eSOD1\u003c/p\u003e\n \u003cp\u003eSOD2\u003c/p\u003e\n \u003cp\u003eTFR1,CD71\u003c/p\u003e\n \u003cp\u003eZip8,ZIP8\u003c/p\u003e\n \u003cp\u003eZip14,ZIP14\u003c/p\u003e\n \u003cp\u003eFpn1,FPN1\u003c/p\u003e\n \u003cp\u003eFth,FTH\u003c/p\u003e\n \u003cp\u003eFtl,FTL\u003c/p\u003e\n \u003cp\u003eSteap3, STEAP3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eDmt1, DMT1\u003c/p\u003e\n \u003cp\u003eAcsl4, ACSL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 406px;\"\u003e\n \u003cp\u003eBisphenol A\u003c/p\u003e\n \u003cp\u003eoxidative stress\u003c/p\u003e\n \u003cp\u003especnuezhenide\u003c/p\u003e\n \u003cp\u003eFructus Ligustri Lucidi\u003c/p\u003e\n \u003cp\u003ehematoxylin-eosin\u003c/p\u003e\n \u003cp\u003etestosterone\u003c/p\u003e\n \u003cp\u003eluteinizing hormone\u003c/p\u003e\n \u003cp\u003efollicle stimulating hormone\u003c/p\u003e\n \u003cp\u003eestradiol\u003c/p\u003e\n \u003cp\u003eglutathione peroxidase 4\u003c/p\u003e\n \u003cp\u003esolute carrier family 7 member 11\u003c/p\u003e\n \u003cp\u003ecysteine- dependent aspartate-specific protease-3\u003c/p\u003e\n \u003cp\u003ecysteine- dependent aspartate-specific protease-9\u003c/p\u003e\n \u003cp\u003eBCL2-associated X protein\u003c/p\u003e\n \u003cp\u003eB-cell CLL/lymphoma 2\u003c/p\u003e\n \u003cp\u003eCuscuta chinensis flavonoids\u003c/p\u003e\n \u003cp\u003eCrocin\u003c/p\u003e\n \u003cp\u003esuperoxide dismutase\u003c/p\u003e\n \u003cp\u003emalondialdehyde\u003c/p\u003e\n \u003cp\u003ecatalase\u003c/p\u003e\n \u003cp\u003enitric oxide\u003c/p\u003e\n \u003cp\u003enitric oxide synthase\u003c/p\u003e\n \u003cp\u003eglutathione peroxidase\u003c/p\u003e\n \u003cp\u003eglutathione\u003c/p\u003e\n \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e\n \u003cp\u003einterleukin-1\u0026beta;\u003c/p\u003e\n \u003cp\u003einterleukin-4\u003c/p\u003e\n \u003cp\u003einterleukin-6\u003c/p\u003e\n \u003cp\u003einterleukin-10\u003c/p\u003e\n \u003cp\u003etumor necrosis factor-\u0026alpha;\u003c/p\u003e\n \u003cp\u003ereactive oxygen species\u003c/p\u003e\n \u003cp\u003e2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride\u003c/p\u003e\n \u003cp\u003eelectron transport chain\u003c/p\u003e\n \u003cp\u003eAdenosine triphosphate\u003c/p\u003e\n \u003cp\u003eoptical density\u003c/p\u003e\n \u003cp\u003eBradford Protein Assay\u003c/p\u003e\n \u003cp\u003e1,1-diphenyl-2-picrylhydrazyl\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHydroxyl\u003c/p\u003e\n \u003cp\u003eNAD(P)H dehydrogenase, quinone 1\u003c/p\u003e\n \u003cp\u003eheme oxygenase-1\u003c/p\u003e\n \u003cp\u003eNF-E2-related factor 2\u003c/p\u003e\n \u003cp\u003eneuronal nitric oxide synthase\u003c/p\u003e\n \u003cp\u003einducible nitric oxide synthase\u003c/p\u003e\n \u003cp\u003esuperoxide dismutase 1\u003c/p\u003e\n \u003cp\u003esuperoxide dismutase 2\u003c/p\u003e\n \u003cp\u003etransferrin receptor1\u003c/p\u003e\n \u003cp\u003esolute carrier family 39 (zinc transporter), member 8\u003c/p\u003e\n \u003cp\u003esolute carrier family 39 (zinc transporter), member14\u003c/p\u003e\n \u003cp\u003eferroportin1\u003c/p\u003e\n \u003cp\u003eferritin heavy chain\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eferritin light chain\u003c/p\u003e\n \u003cp\u003emetalloreductase six-transmembrane epithelial antigen of the prostate 3\u003c/p\u003e\n \u003cp\u003edivalent metal transporter 1\u003c/p\u003e\n \u003cp\u003eacyl-CoA synthetase long-chain family member 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003eConceptualization: X.B. and Y.X.; Data Curation: B.J., W.D., B.W., H.S., and S.Y.; Writing-review and editing: X.B. and Y.X.; Funding acquisition: Y.X.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eDeclaration of Competing Interest\u003c/p\u003e\n\u003cp\u003eThe authors declared no competing interest.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis research was funded by Key Research and Development project of Science and Technology Department of Jilin Province (NOS.20220204035 YY), Innovation and Entrepreneurship Training project for College Students (NOS.\u0026nbsp;2021013). The author(s) would like to thank the Research and Experiment Center of Chronic Disease Prevention, Jilin Medical University, Jilin, China.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The study was approved by the Animal Care Committee of Jilin Medical University, China (2023-LW015). There are no human subjects in this article and informed consent is not applicable.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSu, Y., Quan, C., Li, X., Shi, Y., Duan, P., Yang, K., 2018. Mutual promotion of apoptosis and autophagy in prepubertal rat testes induced by joint exposure of bisphenol A and nonylphenol. Environ. Pollut. (Pt A):693\u0026ndash;702.\u003c/li\u003e\n\u003cli\u003eSoto, A.M., Sonnenschein, C., 2010. Environmental causes of cancer: endocrine disruptors as carcinogens. 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Toxicol. 3(6):637\u0026ndash;644.\u003c/li\u003e\n\u003cli\u003eRatajczak-Wrona, W., Nowak, K., Garley, M., Tynecka, M., Jablonska, E., 2019. Sex-specific differences in the regulation of inducible nitric oxide synthase by bisphenol A in neutrophils. Hum. Exp. Toxicol. 38(2):239\u0026ndash;246. \u003c/li\u003e\n\u003cli\u003eCarb\u0026oacute;, M., Chaturvedi, P., \u0026Aacute;lvarez, A., Pineda-Cevallos, D., Ghatak, A., Gonz\u0026aacute;lez, P.R., Ca\u0026ntilde;al, M.J., Weckwerth, W., Valledor, L., 2023. Ferroptosis is the key cellular process mediating Bisphenol A responses in Chlamydomonas and a promising target for enhancing microalgae-based bioremediation. J. Hazard Mater. 448:130997.\u003c/li\u003e\n\u003cli\u003eDuan, C., Wang, H., Jiao, D., Geng, Y., Wu, Q., Yan, H., Li, C., 2022. Curcumin Restrains Oxidative Stress of After Intracerebral Hemorrhage in Rat by Activating the Nrf2/HO-1 Pathway. Front Pharmacol. 13:889226.\u003c/li\u003e\n\u003cli\u003eSun, Y.Y., Zhu, H.J., Zhao, R.Y., Zhou, S.Y., Wang, M.Q., Yang, Y., Guo, Z.N., 2023. Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice. Redox Biol. 66:102852.\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zaydın, T., \u0026Ouml;znurlu, Y., Sur, E., \u0026Ccedil;elik, İ., Uluışık, D., 2018. The effects of bisphenol A on some plasma cytokine levels and distribution of CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes in spleen, ileal Peyer\u0026apos;s patch and bronchus associated lymphoid tissue in rats. Acta Histochem. 120(8):728\u0026ndash;733. \u003c/li\u003e\n\u003cli\u003eXiong, L., Bin, Z., Young, J.L., Wintergerst, K., Cai, L., 2022. Exposure to low-dose cadmium induces testicular ferroptosis. Ecotoxicol. Environ. Saf. 234:113373.\u003c/li\u003e\n\u003cli\u003eWang, C.Y., Jenkitkasemwong, S., Duarte, S., Sparkman, B.K., Shawki, A., Mackenzie, B., Knutson, M.D., 2012. ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading. J. Biol. Chem. 287(41):34032\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eLei, P., Bai, T., Sun, Y., 2019. Mechanisms of ferroptosis and relations with regulated cell death: a review. Front Physiol. 10:139. \u003c/li\u003e\n\u003cli\u003eChen, X., Li, J., Kang, R., Klionsky, D.J., Tang, D., 2021. Ferroptosis: machinery and regulation. Autophagy. 17(9):2054-2081. \u003c/li\u003e\n\u003cli\u003eZhang, H., Gao, Y., Wang, C., Huang, X., Li, T., Li, K., Peng, R., Li, F., Li, L., Zhang, X., Yin, L., Zhang, S., Zhang, J., 2023. NCOA4-mediated ferritinophagy aggravate intestinal oxidative stress and ferroptosis after traumatic brain injury. Biochem. Biophys. Res. Commun. 688:149065.\u003c/li\u003e\n\u003cli\u003eSun, Y., Sha, M., Qin, Y., Xiao, J., Li, W., Li, S., Chen, S., 2024. Bisphenol A induces placental ferroptosis and fetal growth restriction via the YAP/TAZ-ferritinophagy axis. Free Radic. Biol. Med. 211:127\u0026ndash;144.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003e[\u003c/strong\u003e46] Yuan, Y., Zhai, Y., Chen, J., Xu, X., Wang, H., 2021. Kaempferol ameliorates oxygen-glucose deprivation/reoxygenation-induced neuronal ferroptosis by activating Nrf2/SLC7A11/GPX4 axis. Biomolecules. 11(7):923.\u003c/li\u003e\n\u003cli\u003eHe, W., Gao, Z., Liu, S., Tan, L., Wu, Y., Liu, J., Zheng, Z., Fan, W., Luo, Y., Chen, Z., Song, S., 2023. G protein-coupled estrogen receptor activation by bisphenol-A disrupts lipid metabolism and induces ferroptosis in the liver. Environ Pollut. 334:122211. \u003c/li\u003e\n\u003cli\u003eKaur, S., Saluja, M., Aniqa, A., Sadwal, S., 2021. Selenium attenuates bisphenol A incurred damage and apoptosis in mice testes by regulating mitogen-activated protein kinase signalling. Andrologia. 53(3):e13975.\u003c/li\u003e\n\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bisphenol A, Specnuezhenide, ferroptosis, testicular toxicity, spermatogenesis, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6338624/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6338624/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/em\u003eBisphenol A (BPA), a xenoestrogenic compound, disrupts spermatogenesis by inducing oxidative stress (OS) through iron ion-dependent mechanisms, ultimately contributing to male infertility. Specnuezhenide (SPN), a secoiridoid derived from \u003cem\u003eFructus Ligustri Lucidi (FLL)\u003c/em\u003e, exhibits notable antioxidant and anti-inflammatory properties. However, whether SPN can protect against BPA-induced OS and its detrimental effects on spermatogenesis remains unclear. Furthermore, the underlying mechanisms by which SPN alleviates BPA-induced male reproductive toxicity are poorly understood.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eTo demonstrate the efficacy of SPN in mitigating BPA-provoked testicular damage.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStudy Design\u003c/em\u003e: Specnuezhenide was verified to attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/em\u003eThe male ICR mice have been divided into five groups to investigate these questions, including: the control group, the BPA group (50 mg/kg [bw], orally for 28 days), and three SPN+BPA groups receiving BPA (50 mg/kg [bw], orally for 28 days) along with SPN (30 mg/kg [bw], orally for 21, 28, and 35 days, respectively). The extent od testicular damage was evaluated by basic parameters of body weight, sperm quality, hormonal levels and hematoxylin-eosin (H\u0026amp;E) staining. The mRNA and protein levels of ferroptosis and apoptosis pathways in testes were evaluated by qPCR amplification, western blotting and immunofluorescence analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eBPA exposure significantly impaired sperm quality, induced OS, caused iron accumulation, and led to mitochondrial damage; restored serum hormone levels, including testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH), while increasing estradiol (E2) levels; reduced the activities of antioxidant enzyme, such as superoxide dismutase, catalase, and glutathione peroxidase; and sharply elevates in the expressions of NCOA4 ( a marker of ferritinophagy), GPX4 and SLC7A11 (markers of ferroptosis), cysteine- dependent aspartate-specific protease-3 (Caspase-3), cysteine- dependent aspartate-specific protease-9 (Caspase-9) and BCL2-associated X protein (Bax) (markers of apoptosis). Conversely, SPN supplementation considerably mitigated BPA-induced testicular damage by inhibiting iron accumulation and OS, thereby downregulating ferroptosis and apoptosis pathways.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eThese findings underscore potential of SPN as a therapeutic agent and highlight the necessity for in-depth investigation into the detailed mechanisms underlying BPA-induced toxicity.\u003c/p\u003e","manuscriptTitle":"Specnuezhenide attenuates bisphenol A-induced testicular damage through inhibiting iron accumulation, ferroptosis and apoptosis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 12:27:05","doi":"10.21203/rs.3.rs-6338624/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-22T10:08:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T08:46:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245029381928616457847889782097103380928","date":"2025-05-09T07:33:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T05:13:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211468563988830333654987889443394329759","date":"2025-05-09T01:28:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T00:11:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T00:09:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-25T11:39:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T04:53:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-30T13:09:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11c4a887-6392-472d-9663-5e37fe27e25f","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48294358,"name":"Biological sciences/Molecular biology"},{"id":48294359,"name":"Earth and environmental sciences/Environmental sciences"},{"id":48294360,"name":"Health sciences/Endocrinology"},{"id":48294361,"name":"Health sciences/Pathogenesis"},{"id":48294362,"name":"Biological sciences/Physiology/Metabolism"},{"id":48294363,"name":"Biological sciences/Physiology/Reproductive biology"}],"tags":[],"updatedAt":"2025-09-26T14:38:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 12:27:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6338624","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6338624","identity":"rs-6338624","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}