Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis

Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis | 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 Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis Xishuai Tong, Gengsheng Yu, Xiaohui Fu, Dehui Zhou, Yonggang Ma, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8702171/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract As a ubiquitous environmental pollutant, cadmium (Cd) has been strongly implicated in the development of osteoporosis. However, the cellular and molecular mechanisms mediating its skeletal toxicity remain incompletely understood. Here, we identify osteocytes as a highly vulnerable and previously underrecognized cellular target in Cd-induced osteoporosis. We demonstrate that Cd accumulates in cortical bone and induces pronounced osteocyte senescence in vivo and in vitro. Cd exposure provokes oxidative stress, DNA damage, and cell-cycle arrest in osteocytes, collectively disturbing bone metabolic homeostasis by amplifying senescence signals to neighboring osteoblasts, osteoclasts, and bone marrow stromal cells. Mechanistically, we reveal that brain and muscle ARNT-Like 1 (BMAL1) acts as a pivotal regulator of Cd-induced osteocyte senescence. BMAL1 deficiency exacerbated oxidative stress, senescence, and trabecular and cortical bone deterioration. Conversely, BMAL1 overexpression restored redox balance and markedly attenuated Cd-induced osteocyte senescence. Moreover, eliminating senescent cells using dasatinib plus quercetin markedly reduced osteocyte senescence and improved bone integrity, and co-treatment with melatonin further enhanced these protective effects. These findings reveal an unrecognized osteocyte-centered senescence axis in Cd-induced skeletal toxicity and position BMAL1 as a promising therapeutic target for mitigating pollutant-related bone damage. Biological sciences/Cell biology/Senescence Biological sciences/Cell biology/Circadian rhythms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Osteoporosis is a prevalent and progressive bone disease marked by decreased bone mass and disruption of bone microarchitecture, leading to a markedly elevated risk of fractures and a substantial global public health burden 1 . Cadmium (Cd), a pervasive environmental pollutant, exerts toxicity across multiple organ systems and has been closely associated with osteoporosis, compromised bone strength, and increased fracture risk 2 , 3 . Osteocytes, which derive from terminally differentiated osteoblasts, are now recognized as the principal regulators of bone remodeling and mineral metabolism 4 . Osteocytes sense mechanical signals and secrete key regulatory factors, including sclerostin (SOST) and receptor activator of nuclear factor κB ligand (RANKL), thereby coordinating bone formation and resorption to preserve skeletal homeostasis 5 . Osteocyte function is vulnerable to various intrinsic and extrinsic insults, including physiological ageing, osteoporosis, and chronic systemic inflammation, which can perturb the production of these regulatory molecules and thereby promote skeletal pathology 6 , 7 . Nonetheless, whether osteocytes are direct targets of Cd toxicity and the mechanisms by which they might contribute to Cd-induced osteoporosis remain incompletely defined. Given their pivotal functions in bone remodeling, elucidating how osteocytes respond to Cd is essential for understanding the development of Cd-induced osteoporosis and for informing preventive and therapeutic strategies. Cellular senescence, a hallmark of chronic stress, is defined by irreversible cell cycle arrest and the production of a proinflammatory senescence-associated secretory phenotype (SASP) 8 . Cd is a potent inducer of cellular senescence, Cd exposure promotes senescence in BMSCs via activation of NF-κB signaling, resulting in DNA damage and cell-cycle arrest 9 . Oxidative stress is a key driver of Cd-induced senescence, while senescent cells generate excessive reactive oxygen species (ROS) that further damage DNA and impair mitochondrial function, forming a self-amplifying loop between oxidative stress and cellular senescence that aggravates tissue injury 10 , 11 . Senescent cells impair tissue function by altering the local microenvironment, propagating damage signals, and compromising regenerative capacity [18, 19]. In bone, the accumulation of senescent osteocytes has been implicated as a critical driver of age-related bone loss. Brain and Muscle ARNT-Like 1 (BMAL1) serves as a core transcription factor within the molecular circadian clock 14 . Beyond its canonical role in circadian regulation, BMAL1 has emerged as an important regulator of skeletal biology and bone metabolism 15 . Studies show that BMAL1 regulates the differentiation program of mesenchymal stem cells into osteoblasts, a process that is essential for bone formation and structural integrity 16 . BMAL1 has also been implicated in the control of osteoclastogenesis and osteoclast activity, and disruption of BMAL1 expression or circadian rhythmicity promotes osteoclast differentiation and a low-bone-mass phenotype 17 . Moreover, BMAL1 functions as a transcriptional hub for cellular metabolism, redox homeostasis, and ageing, these roles raise the possibility that BMAL1 contributes to osteocyte resilience against Cd-induced damage 18 . In this study, we explored Cd-induced osteocyte senescence and its effects on bone metabolism. Using in vivo and in vitro models, we found that Cd promotes osteocyte senescence, which is worsened by BMAL1 deficiency. Notably, treatment with the senolytic cocktail dasatinib plus quercetin and the circadian modulator melatonin alleviated Cd-induced bone loss. These results uncover a BMAL1-senescence regulatory axis in osteocytes and highlight cellular senescence as a potential therapeutic target for heavy metal-related osteoporosis. MATERIAL AND METHODS Chemicals and reagents Comprehensive information regarding all chemicals and reagents utilized in this study is listed in Supplementary Table 1. Animals and experimental design All C57BL/6J mice were obtained from the Comparative Medicine Research Center of Yangzhou University. Animals were maintained under specific-pathogen-free conditions with a 12-hour light/dark cycle, 22 ± 2°C temperature, and 50 ± 10% relative humidity. All procedures were approved by the Institutional Animal Care and Use Committee of Yangzhou University (Protocol No. SYXK[Su]-2022-0044) For the Cd exposure experiment, six-week-old female C57BL/6J mice (n = 8) received 50 mg/L CdCl₂ in drinking water for 12 weeks. BMAL1 knockout ( Bmal1 −/− ) mice were generated by crossbreeding Bmal1 wt/− mice (kindly provided by Dr. Ying Xu, Soochow University, Jiangsu, China). Genotyping was confirmed by extracting genomic DNA from tail tissue. Six-week-old female WT and Bmal1 −/− mice were divided into four groups (n = 10): WT, Bmal1 −/− , WT + Cd, and Bmal1 −/− +Cd. Mice in the Cd-treated groups received 50 mg/L CdCl₂ in drinking water for 12 weeks. For the therapeutic assessment, six-week-old female C57BL/6J mice were randomly assigned to four groups (n = 8): Control, Cd, DQ + Cd, and DQ + MT+Cd. Mice in the Cd groups received 50 mg/L CdCl₂ in drinking water. Dasatinib (5 mg/kg) and quercetin (30 mg/kg) were dissolved in a DMSO:PEG-400:saline mixture (5:40:55) and administered by oral gavage three times per week, while melatonin (20 mg/kg) was given every other day. Cd exposure lasted 12 weeks, and DQ and/or melatonin treatments began at week 5 for the remaining 8 weeks. CdCl₂ dosage are based on e previous established bonetoxicity models 9 , 19 , and DQ/MT doses were selected according to previous efficacy studies 20 , 21 . At the end of the treatment, mice were euthanized at indicated time points according to institutional guidelines. Blood and tissue samples were then collected and stored at − 80°C for subsequent analyses. Cadmium content determination Rat and mouse bone samples were dried at 80°C for 24 hours. Cortical bone and bone marrow were separated from rat bones, while whole mouse bones were used for microwave-assisted acid digestion. Approximately 0.2 g of bone tissue was immersed in 4 mL guaranteed-grade nitric acid and pre-digested at room temperature for 24 hours. For cell culture medium and cell samples, digestion was performed directly without pre-drying. Microwave digestion was conducted using an Anton Paar system (Shanghai, China) with a temperature program: the temperature was ramped to 90°C, then to 120°C, and finally to 170°C, which was maintained for 25 minutes. Cadmium concentrations were measured using a PinAAcle 900F flame atomic absorption spectrometer (PerkinElmer Inc., Waltham, MA, USA). Micro-computed tomography (micro-CT) Femora were carefully dissected, and surrounding muscles were removed. Bones were fixed in 4% paraformaldehyde and scanned using a high-resolution micro-CT system (Suzhou Hiscan Information Technology Co., Ltd.) at 60 kV and 134 µA. Images were acquired at 10 µm resolution with a 0.5° rotation step over 360°, using a 500 ms exposure per step. Reconstruction and analysis were performed using Hiscan Reconstruct and Hiscan Analyzer software (Version 3.0). H&E and immunohistochemical analysis Femurs were fixed in 4% paraformaldehyde for 24 hours and decalcified in 10% EDTA at room temperature for 30 days. Decalcified bones were embedded in paraffin and sectioned at 5 µm using a rotary microtome (Leica, Wetzlar, Germany). Sections were stained with H&E (Servicebio Technology Co., Ltd., Wuhan, China) to evaluate osteocyte lacunae. For immunohistochemistry, sections were incubated overnight at 4°C with primary antibodies against SOST and p16, followed by HRP-conjugated secondary antibodies at 37°C for 30 minutes. Signals were developed using DAB and counterstained with hematoxylin. Images were captured with a Leica inverted microscope. Cell culture The murine osteocyte-like cell line MLO-Y4 was obtained from Pricella Life Science & Technology Co., Ltd. (Wuhan, Hubei, China). Cells were cultured in α-MEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (EallBio Biomedical Technology Co., Ltd., Beijing, China) at 37°C in a humidified atmosphere containing 5% CO₂. SA-β-Gal staining Cells were washed with PBS and fixed in 1 mL β-galactosidase fixative at room temperature for 15 minutes, followed by three PBS washes. Cells were then incubated with SA-β-gal staining solution at 37°C for 24 hours. Images were captured with a Leica inverted microscope, and SA-β-gal-positive areas were quantified using ImageJ software. ELISA Mouse serum and cell culture supernatants were analyzed. Blood samples were allowed to clot at 37°C for 30 minutes and centrifuged at 3000 × g for 15 minutes to obtain serum, which was diluted 1:5 with assay diluent. Diluted samples were added to antibody-coated wells and incubated at room temperature for 30 minutes, followed by HRP-conjugated detection antibodies at 37°C for 60 minutes. Wells were washed three times with PBS, and TMB substrate was added for 15 minutes at 37°C in the dark. The reaction was stopped, and absorbance at 450 nm was measured using a BioTek Synergy HTX reader. Blank-corrected values were calculated using standard curves. Estimation of antioxidant enzymes activities Samples analyzed included both mouse serum and cultured cells. For cell samples, protein concentrations were first quantified using a BCA protein assay kit and normalized to equal levels across samples. Antioxidant enzyme activities were then measured using colorimetric assay kits, following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China). OD values were detected using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Flow cytometry analysis Cell cycle analysis: Cells were fixed in 70% ethanol at 4°C for 12 hours and stained with propidium iodide (PI) at 37°C for 30 minutes in the dark. Cell cycle distribution was assessed by flow cytometry (BD Biosciences, San Jose, CA, USA). ROS measurement: Cells were incubated with 10 µM DCFH-DA in serum-free medium at 37°C for 20 minutes, washed three times, and analyzed by flow cytometry. Apoptosis analysis: Cells were stained with Annexin V-FITC and PI for 10 minutes at 25°C according to the manufacturer’s instructions, and apoptotic populations were quantified by flow cytometry. All flow cytometry data were analyzed using FlowJo software (version 10.8.1; BD, Franklin Lakes, NJ, USA). Immunofluorescence Cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were incubated with primary antibodies overnight at 4°C, followed by Alexa Fluor–conjugated secondary antibodies for 2 hours at room temperature. Nuclei were counterstained with DAPI for 10 minutes. Images were acquired using a Leica TCS SP8 STED super-resolution confocal microscope. Conditioned medium (CM) preparation For Cd-CM, MLO-Y4 cells were treated with 4 µM Cd for 24 hours, washed with PBS, and cultured in fresh medium for 12 hours. The supernatant was collected and centrifuged to obtain Cd-CM. For H₂O₂-CM, cells were treated with 200 µM H₂O₂ for 2 hours followed by a 48-hour incubation to induce cellular senescence, washed, and incubated in fresh medium for 12 hours before collecting and centrifuging the supernatant. Conditioned medium from untreated MLO-Y4 cells was used as a Con-CM. Isolation of BMSCs and trilineage differentiation BMSCs were isolated from the femora and tibiae of 4-week-old C57BL/6J mice. Bone marrow was flushed with α-MEM, passed through a 40-µm cell strainer, and centrifuged at 1000 × g for 5 minutes. The resulting cell pellet was resuspended in α-MEM containing 10% FBS and seeded into 100-mm culture dishes. After 24 hours, nonadherent cells were removed, and the medium was refreshed every 2 days. Cells were passaged with 0.25% trypsin at 80–90% confluence, and mycoplasma-free BMSCs at passages 2–3 were used for subsequent experiments. Osteogenic differentiation: BMSCs were cultured in CM supplemented with 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone, with medium changes every 2 days. Alkaline phosphatase (ALP) staining was performed on day 7 to evaluate osteogenic differentiation. Chondrogenic differentiation: BMSCs were cultured in CM supplemented with 1% ITS (insulin-transferrin-selenium), 50 µg/mL ascorbic acid, and 100 nM dexamethasone for 21 days, with medium changes every 2 days. Cartilage matrix production was assessed by Alcian Blue staining. Cells were rinsed twice with PBS, fixed in methanol for 5 minutes at room temperature, then stained with Alcian Blue for 1 hour. Adipogenic differentiation: BMSCs were induced with CM containing 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 10 µg/mL insulin, and 100 µM indomethacin for 3 days, followed by maintenance in insulin-containing medium for 14 days. Lipid accumulation was assessed by Oil Red O staining after fixation with 4% paraformaldehyde. Isolation of primary osteoblasts and Alizarin Red mineralization assay Primary osteoblasts were isolated from calvariae of neonatal C57BL/6J mice (postnatal days 1–3). After dissection and removal of soft tissues, calvariae were digested with 0.25% trypsin-EDTA for 10 minutes, followed by sequential digestion with 0.1% collagenase II. Fractions 2–5 were collected, centrifuged, and cultured in DMEM supplemented with 10% FBS at 37°C with 5% CO₂. Nonadherent cells were removed after 24 hours, and passage 2 cells were used for experiments. For mineralization assays, cells were seeded in 6-well plates and induced with osteogenic medium (CM containing 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) at ~ 60% confluence. Medium was replaced every 2 days. On day 12, cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S, followed by imaging under an inverted microscope. Isolation of bone marrow macrophages (BMMs) and osteoclast differentiation BMMs were isolated from the femora and tibiae of 3-week-old C57BL/6J mice. Bone marrow cells were flushed with α-MEM, filtered through a 40-µm strainer, and centrifuged at 1000 × g for 5 minutes. Cells were resuspended in α-MEM containing 10% FBS and plated for 12 hours to allow stromal cell adherence. Nonadherent cells were collected and cultured with 30 ng/mL M-CSF for 48 hours to obtain BMMs. For osteoclast differentiation, BMMs were seeded in 12-well plates and cultured in CM supplemented with 30 ng/mL M-CSF and 50 ng/mL RANKL, with medium changes every 2 days. After 4 days, TRAP-positive multinucleated cells (≥ 3 nuclei) were defined as mature osteoclasts. ALP staining For cultured cells, BMSCs were induced toward osteogenic differentiation as described above, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and rinsed with PBS. ALP activity was detected using a commercial staining kit. Cells were equilibrated in alkaline buffer for 5 minutes, incubated with substrate solution for 30 minutes in the dark, and washed with distilled water to terminate the reaction. For bone sections, decalcified, paraffin-embedded femora were deparaffinized in xylene and rehydrated through graded ethanol. After PBS washing, sections were equilibrated in alkaline buffer and incubated with ALP staining solution for 2 hours in the dark. Sections were counterstained with hematoxylin, dehydrated, mounted, and analyzed for ALP-positive areas using ImageJ software. Tartrate-resistant acid phosphatase (TRAP) staining For cultured osteoclasts, BMM-derived cells were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes at room temperature. After three PBS washes, TRAP staining was performed using a commercial kit according to the manufacturer’s instructions as previously reported 22 . For bone sections, paraffin-embedded sections were deparaffinized, rehydrated, and equilibrated in acetate buffer. Sections were incubated with TRAP working solution at 37°C for 30 minutes, rinsed, counterstained with hematoxylin, dehydrated, cleared, and mounted. Images were captured using an inverted microscope, and TRAP-positive areas were quantified with ImageJ using color thresholding. RNA-seq analysis MLO-Y4 cells were treated with 4 µM Cd for 24 h. Total RNA was extracted using Trizol reagent and transferred to RNase-free tubes. After quality assessment, RNA was reverse transcribed into cDNA. RNA sequencing and data analysis were performed by Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China). Circadian rhythm induction MLO-Y4 cells were treated with Cd (4 µmol/L) for 24 h, the medium was replaced with fresh medium supplemented with 1 µmol/L dexamethasone for 30 min to synchronize circadian rhythms, followed by replacement with fresh complete medium, which was designated as circadian time 0 (CT0). No further medium changes were performed until cell harvest. Cells were collected at CT0, 6, 12, 18, and 24 for total protein extraction. Lentivirus (LV) infection and siRNA transfection MLO-Y4 cells were seeded in 6-well plates at ~ 20% confluence. Lentiviral particles (Shanghai Genechem Co., Ltd.) were added according to the manufacturer’s instructions and incubated for 12 hours, followed by selection with 4 µg/mL puromycin for 48 hours to generate stable cell lines. For siRNA transfection, siRNAs were combined with Polyplus transfection reagent and applied to antibiotic-free medium for 24 hours. Knockdown efficiency was verified by Western blotting. siRNA sequences are provided in Supplementary Table 4. Western blot analysis Proteins were extracted from bone tissues or cultured cells using RIPA buffer containing protease and phosphatase inhibitors. For bone samples, femora were ground in liquid nitrogen prior to lysis. Protein concentrations were measured by BCA assay. Equal amounts of protein were separated by 10–12% SDS–PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 2 hours and incubated overnight at 4°C with primary antibodies. After washing, membranes were incubated with secondary antibodies for 2 hours at room temperature. Signals were developed using an ECL kit and visualized with a Tanon-5200 imaging system (Shanghai, China). Band intensities were quantified using ImageJ software. Antibody details are listed in Supplementary Table 2. Statistical analysis Data were analyzed using one-way ANOVA with SPSS software (version 26.0; IBM, Armonk, NY, USA). All experiments were performed in triplicate or more. Results are expressed as mean ± standard deviation (SD). Statistical significance was set at P < 0.05, and high significance at P < 0.01. RESULTS Cadmium exposure induces bone loss and osteocyte senescence in mice To examine the impact of Cd on osteocytes, mice were given drinking water containing 50 mg/L CdCl₂ for three months. Micro-CT analysis of the femur revealed significant bone loss in Cd-exposed mice, as evidenced by significant reductions in BV, BS, BMD, Tb.N, and Tb.Th, together with increased Tb.Sp (Fig. 1 a, b). Due to separating cortical bone from marrow is technically difficult in mice, femora from Sprague-Dawley rats exposed to 50 mg/L CdCl₂ for three months were analyzed for Cd content to assess cortical accumulation. Results showed that the majority of Cd was localized to the marrow compartment, while measurable Cd was also detected in cortical bone (Fig. 1 c). Micro-CT assessment of cortical bone in mice showed decreased Ct.Th and increased cortical porosity after Cd exposure (Fig. 1 d, e). H&E staining showed a significantly increased number of empty osteocyte lacunae in the cortical bone of Cd-treated mice (Fig. 1 f). At the protein level, Western blotting revealed that osteocyte related markers-DMP1, SOST, OPN, and OCN were markedly downregulated following Cd exposure (Fig. 1 g). These findings were corroborated by immunohistochemistry showing decreased SOST expression in osteocytes from Cd-exposed mice (Fig. 1 h). Concurrently, bone tissue from Cd-treated mice exhibited robust upregulation of senescence-associated proteins p53, p21, and p16 (Fig. 1 i), consistent with increased p16 immunostaining in osteocytes (Fig. 1 j). Collectively, these data indicate that chronic Cd exposure results Cd accumulation in cortical bone, promotes osteocyte senescence, and is associated with trabecular and cortical bone deterioration in vivo. Cadmium exposure induces osteocyte senescence through oxidative stress in vitro To determine whether Cd-induced osteocyte senescence in vitro, MLO-Y4 cells were exposed to increasing concentrations of CdCl₂ (0, 2, 4, and 6 µM) for 24 h. SA-β-gal staining revealed a dose-dependent increase in senescent MLO-Y4 cells after Cd exposure (Fig. 2 a). Western blotting revealed that Cd treatment significantly upregulated the expression of senescence markers p53, p21 and p16 (Fig. 2 b), which was further corroborated by an increase in the intensity of nuclear p16 immunofluorescence (Fig. 2 c). Flow cytometry showed an accumulation of cells in G1 phase following Cd treatment (Fig. 2 d), consistent with decreased protein levels of cell-cycle regulators cyclin B1, cyclin E1, CDK2 and CDK4 (Fig. 2 e). Increased DNA damage in Cd-treated cells was evident from elevated γ-H2AX staining and corresponding increases detected by Western blot (Fig. 2 f, g). Oxidative stress is a key trigger of senescence 23 , we measured intracellular ROS and antioxidant capacity. Cd exposure markedly increased intracellular ROS levels (Fig. S1 a, b) while concurrently reducing capacity of T-AOC and activities of CAT, GSH and SOD (Fig. S1 c–f). Consistent with the development of a senescence-associated secretory phenotype (SASP), Cd activated the NLRP3 inflammasome pathway and increased secretion of proinflammatory cytokines TNF-α, IL-6 and IL-1β (Fig. S1 g–j). Collectively, these data demonstrate that Cd exposure induces osteocyte senescence in a dose-dependent manner, characterized by cell-cycle arrest, DNA damage, oxidative stress, and activation of a proinflammatory SASP. Senescent osteocytes propagate senescence within the bone microenvironment To determine whether senescent osteocytes propagate aging signals via secreted factors, conditioned medium (CM) was collected from Cd- or H₂O₂-induced senescent MLO-Y4 cells and applied to BMSCs, osteoblasts, and osteoclasts (Fig. 3 a). Cd content analysis confirmed minimal residual Cd levels in the Cd-CM (Fig. S2a-c). To further verify that the biological effects of Cd-CM were mediated by SASP factors rather than direct Cd toxicity, H₂O₂-CM derived from oxidative stress-induced senescent osteocytes was used as a positive control for cellular senescence (Fig. S2d-g). ELISA assays showed that both Cd-CM and H₂O₂-CM contained markedly elevated levels of SASP factors, including TNF-α, IL-1β and IL-6 (Fig. 3 b–d). Following exposure to Cd-CM or H₂O₂-CM, SA-β-gal staining revealed a higher proportion of senescent BMSCs in the Cd-CM and H₂O₂-CM groups (Fig. 3 e), and Western blotting showed upregulation of p53, p21 and p16 (Fig. 3 f). Osteoblasts treated with Cd-CM or H₂O₂-CM exhibited similar phenotypes, elevated SA-β-gal activity, and increased p53, p21 and p16 expression (Fig. 3 g, h). Likewise, osteoclasts exposed to Cd-CM or H₂O₂-CM similarly exhibited increased SA-β-gal activity and elevated expression of p53, p21, and p16 (Fig. 3 i, l). Together, these data indicate that senescent osteocytes secrete SASP factors that act in an autocrine and paracrine manner to induce senescence in neighboring bone-resident cells, thereby facilitating the propagation of cellular aging throughout the bone microenvironment. Senescent osteocytes disrupt bone metabolic homeostasis To evaluate how senescent osteocytes affect bone remodeling, CM was applied to BMSCs, osteoblasts, and osteoclasts. In BMSC trilineage differentiation assays, both Cd-CM and H₂O₂-CM markedly suppressed osteogenic differentiation, as indicated by a reduced ALP-positive area (Fig. 4 a). Chondrogenic differentiation was similarly impaired, as evidenced by attenuated Alcian Blue staining following treatment with Cd-CM or H₂O₂-CM (Fig. 4 b). By contrast, adipogenic differentiation was significantly enhanced by Cd-CM and H₂O₂-CM, as demonstrated by increased Oil Red O-positive lipid accumulation (Fig. 4 c). Primary osteoblasts exposed to Cd-CM or H₂O₂-CM displayed impaired mineralization, evidenced by weaker Alizarin Red staining (Fig. 4 d). In osteoclastogenesis assays, senescent-osteocyte CM did not alter the apoptosis of osteoclast precursors (Fig. 4 e), but TRAP staining revealed a reduction in multinucleated osteoclast formation under Cd-CM or H₂O₂-CM treatment (Fig. 4 g). Mechanistically, Western blotting revealed downregulation of DCSTAMP-a membrane fusogen essential for precursor fusion (Fig. 4 f). Collectively, these findings indicate that senescent osteocytes skew BMSC lineage commitment away from osteogenesis, while impairing osteoclast precursor fusion and maturation, thereby disrupting bone remodeling homeostasis. BMAL1 is involved in cadmium-induced osteocyte senescence To investigate molecular mechanisms underlying Cd-induced osteocyte senescence, RNA sequencing was performed on MLO-Y4 cells exposed to 4 µM Cd for 24 h. Compared with controls, 631 genes were differentially expressed, including 469 upregulated and 162 downregulated genes (Fig. 5 a). KEGG and pathway enrichment analyses revealed significant overrepresentation of pathways related to reactive oxygen species and inflammatory responses, including Wnt and TNF signaling (Fig. 5 b), consistent with previous studies, and many of these genes have been previously validated for roles in cellular senescence 24 , 25 . Notably, several circadian rhythm-associated genes were altered in Cd-treated osteocytes, with BMAL1 exhibiting the most pronounced downregulation (Fig. 5 c). Consistent with the transcriptomic data, BMAL1 protein expression in osteocytes was decreased in a dose-dependent manner after Cd treatment in vitro (Fig. 5 d). In addition, the rhythmic amplitudes of multiple clock proteins were reduced following Cd exposure (Fig. S3), suggesting a potential involvement of the circadian clock in Cd-induced osteocyte senescence. Given BMAL1’s central role as a core circadian transcription factor, we focused on its potential involvement in Cd-elicited osteocyte aging. BMAL1 regulates cadmium-induced osteocyte senescence through the oxidative stress pathway RNA sequencing results indicated that the ROS signaling pathway is significantly altered in Cd-treated osteocytes, with BMAL1 implicated in the regulation of ROS generation 26 . To investigate BMAL1’s role in Cd-induced osteocyte senescence, Bmal1 overexpression and knockdown experiments were performed. Bmal1 was stably overexpressed using a lentiviral vector (LV-Bmal1) with puromycin selection (Fig. S4a, b), and overexpression was confirmed by Western blotting (Fig. 6 a). Compared with LV-vector + Cd group, LV-Bmal1 + Cd group exhibited a marked reduction in SA-β-gal positive cells (Fig. 6 b), accompanied by significantly decreased expression of senescence markers p53, p21, and p16 (Fig. 6 c). Moreover, Bmal1 overexpression attenuated Cd-induced oxidative stress, as evidenced by reduced intracellular ROS levels (Fig. 6 d) and increased capacity of T-AOC and CAT activity (Fig. 6 e, f). To further validate BMAL1’s function, BMAL1 was knocked down using si-Bmal1 (Fig. S4c), and its effects on osteocyte senescence were assessed. Compared with NC + Cd, si-Bmal1 + Cd exhibited a marked increase in the proportion of SA-β-gal positive cells and further upregulated the expression of p53, p21 and p16 (Fig. 6 g, h). In line with enhanced senescence, si-Bmal1 + Cd cells displayed significantly higher ROS levels (Fig. 6 i) and reduced T-AOC and CAT activities relative to NC + Cd cells (Fig. 6 j, k). Collectively, these results indicate that BMAL1 plays a crucial role in modulating Cd-induced osteocyte senescence by regulating oxidative stress. Bmal1 deficiency exacerbates cadmium-induced osteoporosis and osteocyte senescence in mice To interrogate the role of BMAL1 in Cd-induced osteoporosis and osteocyte senescence, Bmal1 ⁻/⁻ mice were generated by intercrossing heterozygotes ( Bmal1 ⁺/⁻ ) (Fig. S5a–c). WT and Bmal1 ⁻/⁻ mice then received 50 mg/L CdCl₂ in drinking water for 3 months (Fig. 7 a). Bmal1 deficiency attenuation the circadian rhythmicity of serum melatonin and cortisol, Cd exposure further exacerbated this disruption (Fig. S6a, b). Cd content analysis revealed pronounced Cd accumulation in bone, with significantly higher levels in Bmal1 ⁻/⁻ +Cd mice than in WT + Cd (Fig. 7 b). Micro-CT demonstrated substantial deterioration of both trabecular and cortical bone in Cd-exposed and Bmal1 ⁻/⁻ mice, with the most severe loss observed in the Bmal1 ⁻/⁻ +Cd group (Fig. 7 c, e). Quantitative analyses demonstrated reductions in BV, BS, BMD, Tb.N, Tb.Th, and Ct.Th, together with increased Tb.Sp and cortical porosity in Bmal1 ⁻/⁻ +Cd mice compared with WT + Cd mice (Fig. 7 d, f). H&E staining showed a higher proportion of empty osteocyte lacunae in Bmal1 ⁻/⁻ and Cd-treated mice, most prominently in Bmal1 ⁻/⁻ +Cd (Fig. 7 g, h). Consistently, osteocyte markers E11 and SOST were markedly reduced in Bmal1 ⁻/⁻ +Cd compared with WT + Cd, indicating aggravated osteocyte dysfunction (Fig. 7 i). At the molecular level, Bmal1 deficiency amplified Cd-induced oxidative stress and senescence. The capacity of T-AOC and CAT activity were decreased in Bmal1 ⁻/⁻ +Cd relative to WT + Cd (Fig. 7 j, k). Moreover, enhanced osteocyte senescence was confirmed by stronger p16 immunostaining and elevated p53, p21, and p16 protein levels on Western blot (Fig. 7 l–n). Collectively, these findings indicate that loss of BMAL1 exacerbates skeletal Cd accumulation, intensifies oxidative stress and osteocyte senescence, and accelerates both trabecular and cortical bone loss. BMAL1 deficiency enhances osteocyte-mediated bone resorption and aggravates bone metabolic imbalance Previous studies indicate that Cd disrupts bone metabolic balance 2 . To determine whether Bmal1 deficiency modifies this effect, we assessed systemic bone turnover markers and local indices of formation and resorption. Serum procollagen type I N-terminal propeptide (PINP) was significantly reduced in Bmal1 ⁻/⁻ and Cd-treated mice, with the lowest levels in the Bmal1 ⁻/⁻ +Cd group (Fig. S7a). Conversely, the resorption marker CTX-I was elevated in both Bmal1 ⁻/⁻ and Cd-exposed mice and increased further in Bmal1 ⁻/⁻ +Cd animals (Fig. S7b). Moreover, osteocyte-derived osteoclast regulators were altered: OPG decreased whereas RANKL increased, producing a higher RANKL/OPG ratio in Bmal1 ⁻/⁻ +Cd mice (Fig. S7c–e). Histological analyses revealed a shift in the balance between bone formation and resorption. ALP staining showed markedly reduced osteogenic activity, with the smallest ALP-positive area in Bmal1 ⁻/⁻ +Cd (Fig. S7f, g). In trabecular regions, TRAP staining demonstrated a significant reduction in TRAP-positive area, indicating impaired osteoclast formation and activity in that compartment (Fig. S7h, i). By contrast, cortical bone exhibited a striking increase in TRAP-positive osteocytes in Bmal1 ⁻/⁻ and Cd-exposed groups, most prominently in Bmal1 ⁻/⁻ +Cd mice (Fig. S7j, k). Together, these findings indicate that Bmal1 deficiency under Cd stress diverts bone resorption from canonical osteoclast-dependent mechanisms toward osteocyte-mediated pathways, thereby exacerbating Cd-induced remodeling imbalance. Senolytic treatment combined with melatonin alleviates cadmium-induced osteocyte senescence and bone loss To further assess the contributions of cellular senescence to Cd-induced osteoporosis, we tested senescent-cell clearance with dasatinib plus quercetin (DQ) and combined with melatonin (MT). Bone Cd measurements showed that DQ alone did not alter Cd accumulation, whereas DQ + MT reduced bone Cd content (Fig. 8 b). Micro-CT analysis of the femur revealed that both DQ and DQ + MT improved bone microarchitecture (Fig. 8 c, e), increasing BV, BS, BMD, Tb.N, Tb.Th, and Ct.Th and reducing Tb.Sp and cortical porosity (Fig. 8 d, f). Notably, DQ + MT produced greater recovery than DQ alone, suggesting a synergistic benefit of circadian support on senolytic therapy. H&E staining revealed fewer empty osteocyte lacunae in DQ + Cd and DQ + MT+Cd groups, indicating improved osteocyte viability (Fig. 8 g, h). Consistently, osteocyte markers E11 and SOST were partially restored by DQ and most strongly rescued by DQ + MT (Fig. 8 i). At the molecular level, both interventions attenuated oxidative stress and senescence: T-AOC and CAT activity were increased in the DQ + Cd and DQ + MT+Cd groups relative to Cd alone (Fig. 8 j, k). Moreover, p16 immunoreactivity and the protein levels of p16, p21, and p53 were markedly reduced after treatment, with the largest decreases observed in the DQ + MT group (Fig. 8 l–n). Collectively, these results indicate that senescent-cell clearance, particularly when combined with circadian rhythm restoration, mitigates Cd-induced osteocyte senescence, and bone loss, supporting DQ + MT as a promising therapeutic strategy for Cd-related skeletal toxicity. DISCUSSION Bone is a multifunctional organ that not only provides mechanical support and protection for vital tissues, but also functions as a dynamic endocrine organ and mineral reservoir essential for calcium-phosphate homeostasis 27 . Cd is a well-established skeletal toxicant and has long been implicated in the pathogenesis of osteoporosis 3 . In the present study, we show that Cd accumulates in bone, induces osteocyte senescence, and that osteocytes actively contribute to Cd-induced skeletal pathology. Mechanistically, Cd disrupts osteocyte redox homeostasis, triggering cellular senescence and a proinflammatory senescence-associated secretory phenotype that propagates ageing-related dysfunction to neighboring bone-resident cells. These results reposition osteocytes from passive bystanders to active mediators of cadmium-induced bone loss, providing a mechanistic link between environmental Cd burden, osteocyte dysfunction, and the collapse of bone-remodeling homeostasis. Cellular senescence is a hallmark of organismal ageing that contributes to physiological decline and is closely linked to the onset and progression of osteoporosis 28 . In a longitudinal comparison of female and male C57BL/6 mice, age-related loss of spinal and hindlimb strength was detectable nine months before measurable declines in bone mineral density 29 . Senescence can be triggered by diverse insults, including intrinsic factors such as oxidative stress and mitochondrial dysfunction as well as extrinsic exposures such as chemotherapeutic agents and environmental toxins 30 . These stimuli converge on DNA damage responses that activate the p53, p21 and p16 pathways, ultimately culminating in irreversible cell cycle arrest 31 . Although skeletal ageing is inevitable, environmental factors can accelerate its onset. Cd is a potent pro-senescent agent that induces cellular senescence through multiple routes, even at low doses. In BMSCs, Cd activates NF-κB signaling to drive senescence, as evidenced by upregulation of p53, p21 and p16, G0/G1 cell-cycle arrest, DNA damage, mitochondrial dysfunction, and disruption of the osteogenic-adipogenic balance 9 . Our study demonstrated that Cd-induced bone loss in vivo and in vitro is accompanied by osteocyte senescence: osteocytes exhibited strong SA-β-gal positivity, increased expression of p53, p21 and p16, elevated intracellular ROS, DNA damage markers, and G1 cell-cycle arrest. Collectively, these findings establish a mechanistic link between osteocyte senescence and Cd-induced osteoporosis. Senescent cells secrete a broad repertoire of bioactive molecules, including inflammatory cytokines, chemokines, matrix-degrading enzymes, and ROS-collectively known as the SASP 32 , 33 . In our experiments, conditioned medium from senescent osteocytes contained elevated levels of inflammatory cytokines. It is widely accepted that primary, damage-induced senescence can induce secondary senescence in neighboring cells via SASP factors 34 , 35 . This secondary spread occurs through two non-exclusive mechanisms: paracrine senescence driven by soluble SASP components, and juxtacrine senescence mediated by direct cell-cell contact; together, these processes allow a small initial pool of senescent cells to amplify tissue dysfunction and promote age-related disease 36 , 37 . For example, glucocorticoid therapy can trigger primary senescence in a subset of bone-marrow adipocyte lineage cells (BMAd) via the 15d-PGJ2–PPARγ–p16 axis; these BMAd then disseminate senescence to other bone and marrow populations through SASP, and suppression of BMAd senescence or blockade of SASP signaling attenuates glucocorticoid-induced bone loss 38 . Our data demonstrate that senescent osteocytes propagate senescence to other bone-resident cells by secreting SASP, impair the tri-lineage differentiation potential of BMSCs, reduce osteoblast mineralization, and impede osteoclast-precursor fusion, collectively disrupting bone remodeling. Thus, an osteocyte-centered model of senescence propagation reframes cadmium-induced osteoporosis as a network-level dysfunction driven by osteocyte-derived signaling rather than isolated effects on osteoblasts or osteoclasts. Circadian disruption is increasingly implicated in metabolic and age-related diseases, and the skeleton is no exception 39 . Epidemiological studies report a higher incidence of osteoporosis and fractures among shift workers 40 , 41 . Experimental models likewise show that continuous light exposure impairs musculoskeletal performance and remodels bone microarchitecture, producing features consistent with early osteoporosis 42 . BMAL1, a central component of the molecular clock, has emerged as a key regulator of skeletal health. In osteoblasts, BMAL1 knockdown reduces expression of osteogenic markers, alkaline phosphatase activity, and mineralization, while increasing apoptosis and inflammatory signaling 43 . In osteoclasts, BMAL1 modulates resorption by binding E-box elements in the NFATc1 promoter and cooperating with steroid receptor coactivators to promote NFATc1 transcription 44 . Beyond its timekeeping role, BMAL1 influences cellular metabolism, DNA-repair pathways, and oxidative-stress responses. For example, in aged human lens epithelial cells, downregulation of the NRF2/antioxidant-response-element (ARE) pathway coincides with reduced BMAL1 expression, leading to ROS accumulation and cell death, similarly, BMAL1 deficiency amplifies P. gingivalis-induced atherosclerosis via NF-κB activation and increased oxidative stress 26 , 45 . In the present study, Cd exposure markedly reduced BMAL1 levels in osteocytes, and genetic ablation of BMAL1 substantially exacerbated Cd-induced bone loss and osteocyte senescence, indicating that BMAL1 is essential for skeletal integrity under toxic stress. Mechanistically, BMAL1 likely attenuates oxidative stress by upregulating antioxidant enzymes and facilitating ROS clearance, consistent with its role as a core regulator of cellular redox homeostasis in other tissues. Effective treatments for skeletal diseases caused by environmental pollutants remain scarce, underscoring the need for innovative therapeutic strategies. Targeting senescent cells to ameliorate age-related disorders has recently emerged as a rapidly advancing approach 33 . In mouse models that recapitulate the severe bone loss of human senile osteoporosis, three classes of interventions-genetic depletion of senescent cells, pharmacologic senolysis, and pharmacologic suppression of the SASP, each significantly improved skeletal phenotypes 46 . Among senolytics, the dasatinib-quercetin combination (D + Q) is the most extensively studied and has shown promise in mitigating age-associated physiological dysfunction in both mice and humans 47 , 48 . Dasatinib is a clinically used tyrosine-kinase inhibitor employed in certain leukemias, while quercetin is a natural flavonoid that promotes apoptosis in senescent endothelial cells 49 . Recent preclinical data indicate that D + Q reduces age-related increases in SA-β-gal activity in periadrenal white adipose tissue and improves metabolic function in aged mice 20 . Building on this rationale, we evaluated D + Q in a Cd intoxication model: D + Q attenuated Cd-induced osteocyte senescence and improved femoral microarchitecture. Co-administration of melatonin further augmented these benefits, preserved BMAL1-centered circadian regulation, and reduced skeletal Cd burden. Mechanistically, this combination is rational and complementary: D + Q removes the primary source of SASP, whereas melatonin-acting as both a chronobiotic and an antioxidant-limits senescence propagation by restoring formation-resorption rhythmicity and strengthening redox homeostasis. In summary, our findings suggest that osteocytes play a central role in Cd-induced bone loss by undergoing senescence and impairing bone remodeling. BMAL1 serves as a protective factor by limiting oxidative stress and senescence in osteocytes. Importantly, interventions that restore or enhance BMAL1 function could help mitigate Cd-induced osteocyte dysfunction and bone loss, offering a potential therapeutic avenue for heavy metal-associated skeletal disorders. Declarations COMPETING INTERESTS The authors declare no competing interests. ACKNOWLEDGEMENTS This research was supported by the following sources: National Key R&D Program of China (2023YFD1801100), National Natural Science Foundation of China (32102732, 32072933, 32273086, 32072923), Jiangsu Provincial Natural Science Foundation of China (BK20210806), Graduate International Academic Exchange Special Fund Project of Yangzhou University (YZUF2024211) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Shen Y, Huang X, Wu J, Lin X, Zhou X, Zhu Z et al. The Global Burden of Osteoporosis, Low Bone Mass, and Its Related Fracture in 204 Countries and Territories, 1990–2019. Front Endocrinol 2022; 13: 882241. Tang C, Lv X, Zou L, Rong Y, Zhang L, Xu M et al. Cadmium exposure and osteoporosis: epidemiological evidence and mechanisms. Toxicol Sci 2025; 205: 1–10. Åkesson A, Bjellerup P, Lundh T, Lidfeldt J, Nerbrand C, Samsioe G et al. Cadmium-Induced Effects on Bone in a Population-Based Study of Women. 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Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInformation.docx Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 09 Mar, 2026 Review # 1 received at journal 08 Mar, 2026 Review # 2 received at journal 05 Mar, 2026 Reviewer # 2 agreed at journal 27 Feb, 2026 Reviewer # 1 agreed at journal 25 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Submission checks completed at journal 27 Jan, 2026 First submitted to journal 27 Jan, 2026 Unknown event 26 Jan, 2026 Editor assigned by journal 26 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8702171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":590562718,"identity":"e24d27b6-8b6e-4377-96ff-09dd7754c92a","order_by":0,"name":"Xishuai Tong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYPACGwjFQ5xqZhCRRrqWwyRoMTh//uDjgl/nE+fPSGB88LaNQd6ckBbJGcnMxjP7bic2zkhgNpzbxmC4s4GAFn4JZjZp3p7bic0SCUBGG0OCwQECWtj4D4O0nEtsk0hg/02UFn6GZDZpnh8HEnuAtjATpQXoF2Nj3oZk4xk8D5sl55yTMNxASIvB+YMPH/P8sZOd35588MObMht5graAAWMbg2MDA2MDkClBjHoQ+MNgT6zSUTAKRsEoGIEAAP+7OvrIvIYtAAAAAElFTkSuQmCC","orcid":"","institution":"Yangzhou University","correspondingAuthor":true,"prefix":"","firstName":"Xishuai","middleName":"","lastName":"Tong","suffix":""},{"id":590562719,"identity":"d35feb59-05b5-4a5c-b9eb-d52738647f2d","order_by":1,"name":"Gengsheng Yu","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Gengsheng","middleName":"","lastName":"Yu","suffix":""},{"id":590562720,"identity":"75d2b2fa-d7da-4192-8d6b-1c5eacefb764","order_by":2,"name":"Xiaohui Fu","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Fu","suffix":""},{"id":590562721,"identity":"629e6cf9-ae5b-463f-bb99-ed4a1025bcbc","order_by":3,"name":"Dehui Zhou","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Dehui","middleName":"","lastName":"Zhou","suffix":""},{"id":590562722,"identity":"63d5b4ba-a08e-4a36-9ef8-2cba115f7b24","order_by":4,"name":"Yonggang Ma","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yonggang","middleName":"","lastName":"Ma","suffix":""},{"id":590562723,"identity":"b2489619-5fc6-418b-b044-1ac29f64b813","order_by":5,"name":"Hui Zou","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zou","suffix":""},{"id":590562724,"identity":"56a35008-74b8-4f57-beee-9d066fe59456","order_by":6,"name":"Ruilong Song","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ruilong","middleName":"","lastName":"Song","suffix":""},{"id":590562725,"identity":"1e870d69-abc2-4356-84a6-18e1485e407b","order_by":7,"name":"Jianchun Bian","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jianchun","middleName":"","lastName":"Bian","suffix":""},{"id":590562726,"identity":"e2af59f8-3a9b-4b5c-91a0-a6da9c29f30d","order_by":8,"name":"Zongping Liu","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zongping","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-26 16:08:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8702171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8702171/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102918208,"identity":"be6b4c82-3329-44fc-a83f-75f4c056831a","added_by":"auto","created_at":"2026-02-18 11:53:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11588368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCadmium exposure induces bone loss and osteocyte senescence in vivo.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Representative micro-CT images of femoral bones from Con and Cd-exposed mice. \u003cstrong\u003eb \u003c/strong\u003eQuantitative analysis of trabecular bone parameters: bone volume (BV), bone surface (BS), bone density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) (n = 3). \u003cstrong\u003ec\u003c/strong\u003e Female SD rats were administered water containing 50 mg/L CdCl₂ for three months. Cadmium concentrations in the femoral bone marrow and cortical bone were determined using flame atomic absorption spectrometry (n = 3). \u003cstrong\u003ed\u003c/strong\u003e Representative micro-CT images of mice femoral cortical bone. \u003cstrong\u003ee \u003c/strong\u003eQuantitative analysis of cortical thickness (Ct.Th) and cortical bone porosity (n = 3). \u003cstrong\u003ef \u003c/strong\u003eRepresentative H\u0026amp;E staining of cortical bone, black arrows indicating the lacunae of osteocytes (scale bar = 50 µm). \u003cstrong\u003eg\u003c/strong\u003e Western blot analysis and quantification of osteocyte functional proteins DMP1, SOST, OPN and OCN in femoral tissue (n = 3). \u003cstrong\u003eh\u003c/strong\u003e Immunohistochemical staining for SOST in bone sections (black arrows indicate positive osteocytes) (scale bar = 50 µm). \u003cstrong\u003ei \u003c/strong\u003eWestern blot analysis and quantification of the senescence markers p53, p21, and p16 in femoral tissue (n = 3). \u003cstrong\u003ej\u003c/strong\u003e Immunohistochemical staining for p16 in bone sections (black arrows indicate positive osteocytes) (scale bar = 50 µm). Data are presented as mean ± SD. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Con group.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/f3985654920645e61d756d78.png"},{"id":102918212,"identity":"af2c7373-1502-4911-88db-eee254679e10","added_by":"auto","created_at":"2026-02-18 11:53:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8618487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCadmium induces cellular senescence in MLO-Y4 osteocytes. a\u003c/strong\u003e SA-β-gal staining and quantification of senescent cells following Cd exposure (n = 3). \u003cstrong\u003eb\u003c/strong\u003e Western blot analysis and quantification of senescence markers p53, p21, and p16 (n = 3). \u003cstrong\u003ec\u003c/strong\u003e Immunofluorescence staining of p16 (green) and DAPI (blue) after 4 μM Cd exposure (n = 3). \u003cstrong\u003ed\u003c/strong\u003e Flow cytometric analysis of cell cycle distribution (n = 3). \u003cstrong\u003ee\u003c/strong\u003e Western blot analysis and quantification of cell cycle regulatory proteins cyclinB1, cyclinE1, CDK2, and CDK4 (n = 3). \u003cstrong\u003ef\u003c/strong\u003e Immunofluorescence staining of γ-H2AX (red) and DAPI (blue) after 4 μM Cd exposure (n = 3). \u003cstrong\u003eg\u003c/strong\u003e Western blot analysis and quantification of DNA damage marker γ-H2AX expression (n = 3). Data are presented as mean ± SD; \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. 0 μM Cd; ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/f9508cf881d1d707c0bad341.png"},{"id":102918209,"identity":"8f115f22-50cf-4991-8d2c-58ce6c8a9165","added_by":"auto","created_at":"2026-02-18 11:53:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16744577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenescent osteocytes propagate cellular senescence within the bone microenvironment.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic of the experimental design: CM was collected from MLO-Y4 cells treated with cadmium (Cd-CM) or H₂O₂ (H₂O₂-CM), and applied to BMSCs, osteoblasts, and osteoclasts. \u003cstrong\u003eb-d\u003c/strong\u003e ELISA quantification of TNF-α, IL-1β, and IL-6 levels in different CM groups (n = 3). \u003cstrong\u003ee\u003c/strong\u003e SA-β-gal staining and quantification of senescent BMSCs (n = 3). \u003cstrong\u003ef\u003c/strong\u003e Western blot analysis and quantification of p53, p21, and p16 in BMSCs (n = 3). \u003cstrong\u003eg\u003c/strong\u003e SA-β-gal staining and quantification of senescent osteoblasts (n = 3). \u003cstrong\u003eh\u003c/strong\u003e Western blot analysis and quantification of p53, p21, and p16 in osteoblasts (n = 3). Osteoclasts were cultured in CM for 48 hours. \u003cstrong\u003ei\u003c/strong\u003e SA-β-gal staining and quantification of senescent osteoclasts (n = 3).\u003cstrong\u003e j\u003c/strong\u003e Western blot analysis and quantification of p53, p21, and p16 in osteoclasts (n = 3). Data are presented as mean ± SD; \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. Con-CM; ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/9b1f8639e86c1569b6d88e31.png"},{"id":102918218,"identity":"a3bc6b17-0bb2-4d14-900e-289c4b2e2063","added_by":"auto","created_at":"2026-02-18 11:53:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27500002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenescent osteocytes disrupt bone metabolic homeostasis.\u003c/strong\u003e BMSCs were cultured in CM for 7 days. \u003cstrong\u003ea\u003c/strong\u003e Representative images and quantification of ALP staining in BMSCs (n = 3). BMSCs were cultured in CM for 21 days. \u003cstrong\u003eb\u003c/strong\u003e Representative images and quantification of alcian blue staining in BMSCs (n = 3). BMSCs were cultured in CM for 14 days. \u003cstrong\u003ec\u003c/strong\u003e Representative images and quantification of Oil Red O staining in BMSCs (n = 3). Osteoblasts were cultured in CM for 12 days. \u003cstrong\u003ed\u003c/strong\u003e Representative images and quantification of alizarin red staining in osteoblasts (n = 3). BMMs were cultured in CM for 48 hours. \u003cstrong\u003ee\u003c/strong\u003e Flow cytometric analysis of apoptosis in BMMs (n = 3). \u003cstrong\u003ef\u003c/strong\u003e Western blot analysis and quantification of precursor fusion protein DCSTAMP in BMMs (n = 3). BMMs were cultured in CM with osteoclast differentiation inducers for 5 days. \u003cstrong\u003eg\u003c/strong\u003e Representative images and quantification of TRAP staining (n = 3). Data are presented as mean ± SD; \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. Con-CM; ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/f90f11c6ee0989ddccf07ea9.png"},{"id":102918221,"identity":"ccaaee9a-1b3a-4d7a-8f2d-1fb118834154","added_by":"auto","created_at":"2026-02-18 11:53:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1621736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMAL1 is involved in cadmium-induced osteocyte senescence.\u003c/strong\u003e MLO-Y4 cells were treated with 4 μM Cd for 24 h, followed by RNA-sequencing analysis (n = 3). \u003cstrong\u003ea\u003c/strong\u003e Volcano plot showing differentially expressed genes in Cd-treated cells relative to control group. Red points represent upregulated genes, blue points represent downregulated genes, and grey points indicate genes with no significant change. \u003cstrong\u003eb\u003c/strong\u003e Pathway enrichment analysis based on RNA-seq data. \u003cstrong\u003ec\u003c/strong\u003e Heatmap displaying differentially expressed genes in MLO-Y4 cells with or without Cd exposure. \u003cstrong\u003ed\u003c/strong\u003e Western blot analysis and quantification of BMAL1 protein expression in MLO-Y4 cells following Cd exposure (n = 3). Data are presented as mean ± SD; \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. 0 μM Cd.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/acc0f310ab8295d3a5a25c47.png"},{"id":102918215,"identity":"9c73f098-798e-409a-9607-3fc8d5caa350","added_by":"auto","created_at":"2026-02-18 11:53:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12478164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMAL1 regulates cadmium-induced osteocyte senescence through the oxidative stress pathway.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Bmal1 overexpression was verified by Western blot. After Bmal1 overexpression, MLO-Y4 cells were treated with 4 μM Cd for 24 hours. \u003cstrong\u003eb\u003c/strong\u003e SA-β-gal staining and quantification of senescent MLO-Y4 cells (n = 3). \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis and quantification of p53, p21, and p16 in MLO-Y4 cells (n = 3). \u003cstrong\u003ed\u003c/strong\u003e Flow cytometric analysis of intracellular ROS levels in MLO-Y4 cells (n = 3). \u003cstrong\u003ee, f\u003c/strong\u003e Measurement of T-AOC capacity and CAT activity using biochemical assay kits (n = 3). After transfecting 50 nM si-Bmal1 for 24 hours, MLO-Y4 cells were treated with 4 μM Cd for 24 hours. \u003cstrong\u003eg\u003c/strong\u003e SA-β-gal staining and quantification of senescent MLO-Y4 cells (n = 3). \u003cstrong\u003eh\u003c/strong\u003e Western blot analysis and quantification of p53, p21, and p16 in MLO-Y4 cells (n = 3). \u003cstrong\u003ei\u003c/strong\u003e Flow cytometric analysis of intracellular ROS levels in MLO-Y4 cells (n = 3). \u003cstrong\u003ej, k\u003c/strong\u003e Measurement of T-AOC capacity and CAT activity using biochemical assay kits (n = 3). Data are presented as mean ± SD. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. LV-vector/NC; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. LV-vector+Cd/NC+Cd; ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/7b40fda884cf3c479c176014.png"},{"id":103049781,"identity":"98b51dd9-a444-41f7-a2ff-590093f375c3","added_by":"auto","created_at":"2026-02-20 07:46:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":72353942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/c74021ee-e4b3-4389-872e-06be17ad0c12.pdf"},{"id":102918217,"identity":"03431349-af6d-45cb-9414-f2fd979618e7","added_by":"auto","created_at":"2026-02-18 11:53:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6531019,"visible":true,"origin":"","legend":"Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8702171/v1/35459751c0dbe80fd82e41ac.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOsteoporosis is a prevalent and progressive bone disease marked by decreased bone mass and disruption of bone microarchitecture, leading to a markedly elevated risk of fractures and a substantial global public health burden \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Cadmium (Cd), a pervasive environmental pollutant, exerts toxicity across multiple organ systems and has been closely associated with osteoporosis, compromised bone strength, and increased fracture risk \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Osteocytes, which derive from terminally differentiated osteoblasts, are now recognized as the principal regulators of bone remodeling and mineral metabolism \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Osteocytes sense mechanical signals and secrete key regulatory factors, including sclerostin (SOST) and receptor activator of nuclear factor κB ligand (RANKL), thereby coordinating bone formation and resorption to preserve skeletal homeostasis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Osteocyte function is vulnerable to various intrinsic and extrinsic insults, including physiological ageing, osteoporosis, and chronic systemic inflammation, which can perturb the production of these regulatory molecules and thereby promote skeletal pathology \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nonetheless, whether osteocytes are direct targets of Cd toxicity and the mechanisms by which they might contribute to Cd-induced osteoporosis remain incompletely defined. Given their pivotal functions in bone remodeling, elucidating how osteocytes respond to Cd is essential for understanding the development of Cd-induced osteoporosis and for informing preventive and therapeutic strategies.\u003c/p\u003e \u003cp\u003eCellular senescence, a hallmark of chronic stress, is defined by irreversible cell cycle arrest and the production of a proinflammatory senescence-associated secretory phenotype (SASP) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Cd is a potent inducer of cellular senescence, Cd exposure promotes senescence in BMSCs via activation of NF-κB signaling, resulting in DNA damage and cell-cycle arrest \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Oxidative stress is a key driver of Cd-induced senescence, while senescent cells generate excessive reactive oxygen species (ROS) that further damage DNA and impair mitochondrial function, forming a self-amplifying loop between oxidative stress and cellular senescence that aggravates tissue injury \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Senescent cells impair tissue function by altering the local microenvironment, propagating damage signals, and compromising regenerative capacity [18, 19]. In bone, the accumulation of senescent osteocytes has been implicated as a critical driver of age-related bone loss.\u003c/p\u003e \u003cp\u003eBrain and Muscle ARNT-Like 1 (BMAL1) serves as a core transcription factor within the molecular circadian clock \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Beyond its canonical role in circadian regulation, BMAL1 has emerged as an important regulator of skeletal biology and bone metabolism \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Studies show that BMAL1 regulates the differentiation program of mesenchymal stem cells into osteoblasts, a process that is essential for bone formation and structural integrity \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. BMAL1 has also been implicated in the control of osteoclastogenesis and osteoclast activity, and disruption of BMAL1 expression or circadian rhythmicity promotes osteoclast differentiation and a low-bone-mass phenotype \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, BMAL1 functions as a transcriptional hub for cellular metabolism, redox homeostasis, and ageing, these roles raise the possibility that BMAL1 contributes to osteocyte resilience against Cd-induced damage \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we explored Cd-induced osteocyte senescence and its effects on bone metabolism. Using in vivo and in vitro models, we found that Cd promotes osteocyte senescence, which is worsened by BMAL1 deficiency. Notably, treatment with the senolytic cocktail dasatinib plus quercetin and the circadian modulator melatonin alleviated Cd-induced bone loss. These results uncover a BMAL1-senescence regulatory axis in osteocytes and highlight cellular senescence as a potential therapeutic target for heavy metal-related osteoporosis.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eComprehensive information regarding all chemicals and reagents utilized in this study is listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals and experimental design\u003c/h3\u003e\n\u003cp\u003eAll C57BL/6J mice were obtained from the Comparative Medicine Research Center of Yangzhou University. Animals were maintained under specific-pathogen-free conditions with a 12-hour light/dark cycle, 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C temperature, and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity. All procedures were approved by the Institutional Animal Care and Use Committee of Yangzhou University (Protocol No. SYXK[Su]-2022-0044)\u003c/p\u003e \u003cp\u003eFor the Cd exposure experiment, six-week-old female C57BL/6J mice (n\u0026thinsp;=\u0026thinsp;8) received 50 mg/L CdCl₂ in drinking water for 12 weeks. BMAL1 knockout (\u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice were generated by crossbreeding \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (kindly provided by Dr. Ying Xu, Soochow University, Jiangsu, China). Genotyping was confirmed by extracting genomic DNA from tail tissue. Six-week-old female WT and \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were divided into four groups (n\u0026thinsp;=\u0026thinsp;10): WT, \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, WT\u0026thinsp;+\u0026thinsp;Cd, and \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e+Cd. Mice in the Cd-treated groups received 50 mg/L CdCl₂ in drinking water for 12 weeks. For the therapeutic assessment, six-week-old female C57BL/6J mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;8): Control, Cd, DQ\u0026thinsp;+\u0026thinsp;Cd, and DQ\u0026thinsp;+\u0026thinsp;MT+Cd. Mice in the Cd groups received 50 mg/L CdCl₂ in drinking water. Dasatinib (5 mg/kg) and quercetin (30 mg/kg) were dissolved in a DMSO:PEG-400:saline mixture (5:40:55) and administered by oral gavage three times per week, while melatonin (20 mg/kg) was given every other day. Cd exposure lasted 12 weeks, and DQ and/or melatonin treatments began at week 5 for the remaining 8 weeks. CdCl₂ dosage are based on e previous established bonetoxicity models \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and DQ/MT doses were selected according to previous efficacy studies \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. At the end of the treatment, mice were euthanized at indicated time points according to institutional guidelines. Blood and tissue samples were then collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analyses.\u003c/p\u003e\n\u003ch3\u003eCadmium content determination\u003c/h3\u003e\n\u003cp\u003eRat and mouse bone samples were dried at 80\u0026deg;C for 24 hours. Cortical bone and bone marrow were separated from rat bones, while whole mouse bones were used for microwave-assisted acid digestion. Approximately 0.2 g of bone tissue was immersed in 4 mL guaranteed-grade nitric acid and pre-digested at room temperature for 24 hours. For cell culture medium and cell samples, digestion was performed directly without pre-drying. Microwave digestion was conducted using an Anton Paar system (Shanghai, China) with a temperature program: the temperature was ramped to 90\u0026deg;C, then to 120\u0026deg;C, and finally to 170\u0026deg;C, which was maintained for 25 minutes. Cadmium concentrations were measured using a PinAAcle 900F flame atomic absorption spectrometer (PerkinElmer Inc., Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eMicro-computed tomography (micro-CT)\u003c/h3\u003e\n\u003cp\u003eFemora were carefully dissected, and surrounding muscles were removed. Bones were fixed in 4% paraformaldehyde and scanned using a high-resolution micro-CT system (Suzhou Hiscan Information Technology Co., Ltd.) at 60 kV and 134 \u0026micro;A. Images were acquired at 10 \u0026micro;m resolution with a 0.5\u0026deg; rotation step over 360\u0026deg;, using a 500 ms exposure per step. Reconstruction and analysis were performed using Hiscan Reconstruct and Hiscan Analyzer software (Version 3.0).\u003c/p\u003e\n\u003ch3\u003eH\u0026E and immunohistochemical analysis\u003c/h3\u003e\n\u003cp\u003eFemurs were fixed in 4% paraformaldehyde for 24 hours and decalcified in 10% EDTA at room temperature for 30 days. Decalcified bones were embedded in paraffin and sectioned at 5 \u0026micro;m using a rotary microtome (Leica, Wetzlar, Germany). Sections were stained with H\u0026amp;E (Servicebio Technology Co., Ltd., Wuhan, China) to evaluate osteocyte lacunae. For immunohistochemistry, sections were incubated overnight at 4\u0026deg;C with primary antibodies against SOST and p16, followed by HRP-conjugated secondary antibodies at 37\u0026deg;C for 30 minutes. Signals were developed using DAB and counterstained with hematoxylin. Images were captured with a Leica inverted microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe murine osteocyte-like cell line MLO-Y4 was obtained from Pricella Life Science \u0026amp; Technology Co., Ltd. (Wuhan, Hubei, China). Cells were cultured in α-MEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (EallBio Biomedical Technology Co., Ltd., Beijing, China) at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSA-β-Gal staining\u003c/h3\u003e\n\u003cp\u003eCells were washed with PBS and fixed in 1 mL β-galactosidase fixative at room temperature for 15 minutes, followed by three PBS washes. Cells were then incubated with SA-β-gal staining solution at 37\u0026deg;C for 24 hours. Images were captured with a Leica inverted microscope, and SA-β-gal-positive areas were quantified using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eMouse serum and cell culture supernatants were analyzed. Blood samples were allowed to clot at 37\u0026deg;C for 30 minutes and centrifuged at 3000 \u0026times; g for 15 minutes to obtain serum, which was diluted 1:5 with assay diluent. Diluted samples were added to antibody-coated wells and incubated at room temperature for 30 minutes, followed by HRP-conjugated detection antibodies at 37\u0026deg;C for 60 minutes. Wells were washed three times with PBS, and TMB substrate was added for 15 minutes at 37\u0026deg;C in the dark. The reaction was stopped, and absorbance at 450 nm was measured using a BioTek Synergy HTX reader. Blank-corrected values were calculated using standard curves.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of antioxidant enzymes activities\u003c/h2\u003e \u003cp\u003eSamples analyzed included both mouse serum and cultured cells. For cell samples, protein concentrations were first quantified using a BCA protein assay kit and normalized to equal levels across samples. Antioxidant enzyme activities were then measured using colorimetric assay kits, following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China). OD values were detected using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eCell cycle analysis: Cells were fixed in 70% ethanol at 4\u0026deg;C for 12 hours and stained with propidium iodide (PI) at 37\u0026deg;C for 30 minutes in the dark. Cell cycle distribution was assessed by flow cytometry (BD Biosciences, San Jose, CA, USA). ROS measurement: Cells were incubated with 10 \u0026micro;M DCFH-DA in serum-free medium at 37\u0026deg;C for 20 minutes, washed three times, and analyzed by flow cytometry. Apoptosis analysis: Cells were stained with Annexin V-FITC and PI for 10 minutes at 25\u0026deg;C according to the manufacturer\u0026rsquo;s instructions, and apoptotic populations were quantified by flow cytometry. All flow cytometry data were analyzed using FlowJo software (version 10.8.1; BD, Franklin Lakes, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eCells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were incubated with primary antibodies overnight at 4\u0026deg;C, followed by Alexa Fluor\u0026ndash;conjugated secondary antibodies for 2 hours at room temperature. Nuclei were counterstained with DAPI for 10 minutes. Images were acquired using a Leica TCS SP8 STED super-resolution confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConditioned medium (CM) preparation\u003c/h2\u003e \u003cp\u003eFor Cd-CM, MLO-Y4 cells were treated with 4 \u0026micro;M Cd for 24 hours, washed with PBS, and cultured in fresh medium for 12 hours. The supernatant was collected and centrifuged to obtain Cd-CM. For H₂O₂-CM, cells were treated with 200 \u0026micro;M H₂O₂ for 2 hours followed by a 48-hour incubation to induce cellular senescence, washed, and incubated in fresh medium for 12 hours before collecting and centrifuging the supernatant. Conditioned medium from untreated MLO-Y4 cells was used as a Con-CM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of BMSCs and trilineage differentiation\u003c/h2\u003e \u003cp\u003eBMSCs were isolated from the femora and tibiae of 4-week-old C57BL/6J mice. Bone marrow was flushed with α-MEM, passed through a 40-\u0026micro;m cell strainer, and centrifuged at 1000 \u0026times; g for 5 minutes. The resulting cell pellet was resuspended in α-MEM containing 10% FBS and seeded into 100-mm culture dishes. After 24 hours, nonadherent cells were removed, and the medium was refreshed every 2 days. Cells were passaged with 0.25% trypsin at 80\u0026ndash;90% confluence, and mycoplasma-free BMSCs at passages 2\u0026ndash;3 were used for subsequent experiments.\u003c/p\u003e \u003cp\u003eOsteogenic differentiation: BMSCs were cultured in CM supplemented with 50 \u0026micro;g/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone, with medium changes every 2 days. Alkaline phosphatase (ALP) staining was performed on day 7 to evaluate osteogenic differentiation. Chondrogenic differentiation: BMSCs were cultured in CM supplemented with 1% ITS (insulin-transferrin-selenium), 50 \u0026micro;g/mL ascorbic acid, and 100 nM dexamethasone for 21 days, with medium changes every 2 days. Cartilage matrix production was assessed by Alcian Blue staining. Cells were rinsed twice with PBS, fixed in methanol for 5 minutes at room temperature, then stained with Alcian Blue for 1 hour. Adipogenic differentiation: BMSCs were induced with CM containing 0.5 mM isobutylmethylxanthine, 1 \u0026micro;M dexamethasone, 10 \u0026micro;g/mL insulin, and 100 \u0026micro;M indomethacin for 3 days, followed by maintenance in insulin-containing medium for 14 days. Lipid accumulation was assessed by Oil Red O staining after fixation with 4% paraformaldehyde.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of primary osteoblasts and Alizarin Red mineralization assay\u003c/h2\u003e \u003cp\u003ePrimary osteoblasts were isolated from calvariae of neonatal C57BL/6J mice (postnatal days 1\u0026ndash;3). After dissection and removal of soft tissues, calvariae were digested with 0.25% trypsin-EDTA for 10 minutes, followed by sequential digestion with 0.1% collagenase II. Fractions 2\u0026ndash;5 were collected, centrifuged, and cultured in DMEM supplemented with 10% FBS at 37\u0026deg;C with 5% CO₂. Nonadherent cells were removed after 24 hours, and passage 2 cells were used for experiments. For mineralization assays, cells were seeded in 6-well plates and induced with osteogenic medium (CM containing 50 \u0026micro;g/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) at ~\u0026thinsp;60% confluence. Medium was replaced every 2 days. On day 12, cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S, followed by imaging under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of bone marrow macrophages (BMMs) and osteoclast differentiation\u003c/h2\u003e \u003cp\u003eBMMs were isolated from the femora and tibiae of 3-week-old C57BL/6J mice. Bone marrow cells were flushed with α-MEM, filtered through a 40-\u0026micro;m strainer, and centrifuged at 1000 \u0026times; g for 5 minutes. Cells were resuspended in α-MEM containing 10% FBS and plated for 12 hours to allow stromal cell adherence. Nonadherent cells were collected and cultured with 30 ng/mL M-CSF for 48 hours to obtain BMMs. For osteoclast differentiation, BMMs were seeded in 12-well plates and cultured in CM supplemented with 30 ng/mL M-CSF and 50 ng/mL RANKL, with medium changes every 2 days. After 4 days, TRAP-positive multinucleated cells (\u0026ge;\u0026thinsp;3 nuclei) were defined as mature osteoclasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eALP staining\u003c/h2\u003e \u003cp\u003eFor cultured cells, BMSCs were induced toward osteogenic differentiation as described above, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and rinsed with PBS. ALP activity was detected using a commercial staining kit. Cells were equilibrated in alkaline buffer for 5 minutes, incubated with substrate solution for 30 minutes in the dark, and washed with distilled water to terminate the reaction. For bone sections, decalcified, paraffin-embedded femora were deparaffinized in xylene and rehydrated through graded ethanol. After PBS washing, sections were equilibrated in alkaline buffer and incubated with ALP staining solution for 2 hours in the dark. Sections were counterstained with hematoxylin, dehydrated, mounted, and analyzed for ALP-positive areas using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTartrate-resistant acid phosphatase (TRAP) staining\u003c/h2\u003e \u003cp\u003eFor cultured osteoclasts, BMM-derived cells were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes at room temperature. After three PBS washes, TRAP staining was performed using a commercial kit according to the manufacturer\u0026rsquo;s instructions as previously reported \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For bone sections, paraffin-embedded sections were deparaffinized, rehydrated, and equilibrated in acetate buffer. Sections were incubated with TRAP working solution at 37\u0026deg;C for 30 minutes, rinsed, counterstained with hematoxylin, dehydrated, cleared, and mounted. Images were captured using an inverted microscope, and TRAP-positive areas were quantified with ImageJ using color thresholding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells were treated with 4 \u0026micro;M Cd for 24 h. Total RNA was extracted using Trizol reagent and transferred to RNase-free tubes. After quality assessment, RNA was reverse transcribed into cDNA. RNA sequencing and data analysis were performed by Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCircadian rhythm induction\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells were treated with Cd (4 \u0026micro;mol/L) for 24 h, the medium was replaced with fresh medium supplemented with 1 \u0026micro;mol/L dexamethasone for 30 min to synchronize circadian rhythms, followed by replacement with fresh complete medium, which was designated as circadian time 0 (CT0). No further medium changes were performed until cell harvest. Cells were collected at CT0, 6, 12, 18, and 24 for total protein extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus (LV) infection and siRNA transfection\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells were seeded in 6-well plates at ~\u0026thinsp;20% confluence. Lentiviral particles (Shanghai Genechem Co., Ltd.) were added according to the manufacturer\u0026rsquo;s instructions and incubated for 12 hours, followed by selection with 4 \u0026micro;g/mL puromycin for 48 hours to generate stable cell lines. For siRNA transfection, siRNAs were combined with Polyplus transfection reagent and applied to antibiotic-free medium for 24 hours. Knockdown efficiency was verified by Western blotting. siRNA sequences are provided in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from bone tissues or cultured cells using RIPA buffer containing protease and phosphatase inhibitors. For bone samples, femora were ground in liquid nitrogen prior to lysis. Protein concentrations were measured by BCA assay. Equal amounts of protein were separated by 10\u0026ndash;12% SDS\u0026ndash;PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 2 hours and incubated overnight at 4\u0026deg;C with primary antibodies. After washing, membranes were incubated with secondary antibodies for 2 hours at room temperature. Signals were developed using an ECL kit and visualized with a Tanon-5200 imaging system (Shanghai, China). Band intensities were quantified using ImageJ software. Antibody details are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using one-way ANOVA with SPSS software (version 26.0; IBM, Armonk, NY, USA). All experiments were performed in triplicate or more. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and high significance at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eCadmium exposure induces bone loss and osteocyte senescence in mice\u003c/h2\u003e \u003cp\u003eTo examine the impact of Cd on osteocytes, mice were given drinking water containing 50 mg/L CdCl₂ for three months. Micro-CT analysis of the femur revealed significant bone loss in Cd-exposed mice, as evidenced by significant reductions in BV, BS, BMD, Tb.N, and Tb.Th, together with increased Tb.Sp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Due to separating cortical bone from marrow is technically difficult in mice, femora from Sprague-Dawley rats exposed to 50 mg/L CdCl₂ for three months were analyzed for Cd content to assess cortical accumulation. Results showed that the majority of Cd was localized to the marrow compartment, while measurable Cd was also detected in cortical bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Micro-CT assessment of cortical bone in mice showed decreased Ct.Th and increased cortical porosity after Cd exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). H\u0026amp;E staining showed a significantly increased number of empty osteocyte lacunae in the cortical bone of Cd-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). At the protein level, Western blotting revealed that osteocyte related markers-DMP1, SOST, OPN, and OCN were markedly downregulated following Cd exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). These findings were corroborated by immunohistochemistry showing decreased SOST expression in osteocytes from Cd-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Concurrently, bone tissue from Cd-treated mice exhibited robust upregulation of senescence-associated proteins p53, p21, and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), consistent with increased p16 immunostaining in osteocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Collectively, these data indicate that chronic Cd exposure results Cd accumulation in cortical bone, promotes osteocyte senescence, and is associated with trabecular and cortical bone deterioration in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eCadmium exposure induces osteocyte senescence through oxidative stress in vitro\u003c/h2\u003e \u003cp\u003eTo determine whether Cd-induced osteocyte senescence in vitro, MLO-Y4 cells were exposed to increasing concentrations of CdCl₂ (0, 2, 4, and 6 \u0026micro;M) for 24 h. SA-β-gal staining revealed a dose-dependent increase in senescent MLO-Y4 cells after Cd exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Western blotting revealed that Cd treatment significantly upregulated the expression of senescence markers p53, p21 and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), which was further corroborated by an increase in the intensity of nuclear p16 immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Flow cytometry showed an accumulation of cells in G1 phase following Cd treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), consistent with decreased protein levels of cell-cycle regulators cyclin B1, cyclin E1, CDK2 and CDK4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Increased DNA damage in Cd-treated cells was evident from elevated γ-H2AX staining and corresponding increases detected by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). Oxidative stress is a key trigger of senescence \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we measured intracellular ROS and antioxidant capacity. Cd exposure markedly increased intracellular ROS levels (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b) while concurrently reducing capacity of T-AOC and activities of CAT, GSH and SOD (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec\u0026ndash;f). Consistent with the development of a senescence-associated secretory phenotype (SASP), Cd activated the NLRP3 inflammasome pathway and increased secretion of proinflammatory cytokines TNF-α, IL-6 and IL-1β (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg\u0026ndash;j). Collectively, these data demonstrate that Cd exposure induces osteocyte senescence in a dose-dependent manner, characterized by cell-cycle arrest, DNA damage, oxidative stress, and activation of a proinflammatory SASP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eSenescent osteocytes propagate senescence within the bone microenvironment\u003c/h2\u003e \u003cp\u003eTo determine whether senescent osteocytes propagate aging signals via secreted factors, conditioned medium (CM) was collected from Cd- or H₂O₂-induced senescent MLO-Y4 cells and applied to BMSCs, osteoblasts, and osteoclasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Cd content analysis confirmed minimal residual Cd levels in the Cd-CM (Fig. S2a-c). To further verify that the biological effects of Cd-CM were mediated by SASP factors rather than direct Cd toxicity, H₂O₂-CM derived from oxidative stress-induced senescent osteocytes was used as a positive control for cellular senescence (Fig. S2d-g). ELISA assays showed that both Cd-CM and H₂O₂-CM contained markedly elevated levels of SASP factors, including TNF-α, IL-1β and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;d). Following exposure to Cd-CM or H₂O₂-CM, SA-β-gal staining revealed a higher proportion of senescent BMSCs in the Cd-CM and H₂O₂-CM groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), and Western blotting showed upregulation of p53, p21 and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Osteoblasts treated with Cd-CM or H₂O₂-CM exhibited similar phenotypes, elevated SA-β-gal activity, and increased p53, p21 and p16 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h). Likewise, osteoclasts exposed to Cd-CM or H₂O₂-CM similarly exhibited increased SA-β-gal activity and elevated expression of p53, p21, and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, l). Together, these data indicate that senescent osteocytes secrete SASP factors that act in an autocrine and paracrine manner to induce senescence in neighboring bone-resident cells, thereby facilitating the propagation of cellular aging throughout the bone microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eSenescent osteocytes disrupt bone metabolic homeostasis\u003c/h2\u003e \u003cp\u003eTo evaluate how senescent osteocytes affect bone remodeling, CM was applied to BMSCs, osteoblasts, and osteoclasts. In BMSC trilineage differentiation assays, both Cd-CM and H₂O₂-CM markedly suppressed osteogenic differentiation, as indicated by a reduced ALP-positive area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Chondrogenic differentiation was similarly impaired, as evidenced by attenuated Alcian Blue staining following treatment with Cd-CM or H₂O₂-CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). By contrast, adipogenic differentiation was significantly enhanced by Cd-CM and H₂O₂-CM, as demonstrated by increased Oil Red O-positive lipid accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Primary osteoblasts exposed to Cd-CM or H₂O₂-CM displayed impaired mineralization, evidenced by weaker Alizarin Red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In osteoclastogenesis assays, senescent-osteocyte CM did not alter the apoptosis of osteoclast precursors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), but TRAP staining revealed a reduction in multinucleated osteoclast formation under Cd-CM or H₂O₂-CM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Mechanistically, Western blotting revealed downregulation of DCSTAMP-a membrane fusogen essential for precursor fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Collectively, these findings indicate that senescent osteocytes skew BMSC lineage commitment away from osteogenesis, while impairing osteoclast precursor fusion and maturation, thereby disrupting bone remodeling homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBMAL1 is involved in cadmium-induced osteocyte senescence\u003c/h3\u003e\n\u003cp\u003eTo investigate molecular mechanisms underlying Cd-induced osteocyte senescence, RNA sequencing was performed on MLO-Y4 cells exposed to 4 \u0026micro;M Cd for 24 h. Compared with controls, 631 genes were differentially expressed, including 469 upregulated and 162 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). KEGG and pathway enrichment analyses revealed significant overrepresentation of pathways related to reactive oxygen species and inflammatory responses, including Wnt and TNF signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), consistent with previous studies, and many of these genes have been previously validated for roles in cellular senescence \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Notably, several circadian rhythm-associated genes were altered in Cd-treated osteocytes, with BMAL1 exhibiting the most pronounced downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Consistent with the transcriptomic data, BMAL1 protein expression in osteocytes was decreased in a dose-dependent manner after Cd treatment in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In addition, the rhythmic amplitudes of multiple clock proteins were reduced following Cd exposure (Fig. S3), suggesting a potential involvement of the circadian clock in Cd-induced osteocyte senescence. Given BMAL1\u0026rsquo;s central role as a core circadian transcription factor, we focused on its potential involvement in Cd-elicited osteocyte aging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eBMAL1 regulates cadmium-induced osteocyte senescence through the oxidative stress pathway\u003c/h2\u003e \u003cp\u003eRNA sequencing results indicated that the ROS signaling pathway is significantly altered in Cd-treated osteocytes, with BMAL1 implicated in the regulation of ROS generation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To investigate BMAL1\u0026rsquo;s role in Cd-induced osteocyte senescence, Bmal1 overexpression and knockdown experiments were performed. Bmal1 was stably overexpressed using a lentiviral vector (LV-Bmal1) with puromycin selection (Fig. S4a, b), and overexpression was confirmed by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Compared with LV-vector\u0026thinsp;+\u0026thinsp;Cd group, LV-Bmal1\u0026thinsp;+\u0026thinsp;Cd group exhibited a marked reduction in SA-β-gal positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), accompanied by significantly decreased expression of senescence markers p53, p21, and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Moreover, Bmal1 overexpression attenuated Cd-induced oxidative stress, as evidenced by reduced intracellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) and increased capacity of T-AOC and CAT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). To further validate BMAL1\u0026rsquo;s function, BMAL1 was knocked down using si-Bmal1 (Fig. S4c), and its effects on osteocyte senescence were assessed. Compared with NC\u0026thinsp;+\u0026thinsp;Cd, si-Bmal1\u0026thinsp;+\u0026thinsp;Cd exhibited a marked increase in the proportion of SA-β-gal positive cells and further upregulated the expression of p53, p21 and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). In line with enhanced senescence, si-Bmal1\u0026thinsp;+\u0026thinsp;Cd cells displayed significantly higher ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei) and reduced T-AOC and CAT activities relative to NC\u0026thinsp;+\u0026thinsp;Cd cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej, k). Collectively, these results indicate that BMAL1 plays a crucial role in modulating Cd-induced osteocyte senescence by regulating oxidative stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eBmal1 deficiency exacerbates cadmium-induced osteoporosis and osteocyte senescence in mice\u003c/h2\u003e \u003cp\u003eTo interrogate the role of BMAL1 in Cd-induced osteoporosis and osteocyte senescence, \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e mice were generated by intercrossing heterozygotes (\u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁺/⁻\u003c/em\u003e\u003c/sup\u003e) (Fig. S5a\u0026ndash;c). WT and \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e mice then received 50 mg/L CdCl₂ in drinking water for 3 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Bmal1 deficiency attenuation the circadian rhythmicity of serum melatonin and cortisol, Cd exposure further exacerbated this disruption (Fig. S6a, b). Cd content analysis revealed pronounced Cd accumulation in bone, with significantly higher levels in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd mice than in WT\u0026thinsp;+\u0026thinsp;Cd (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Micro-CT demonstrated substantial deterioration of both trabecular and cortical bone in Cd-exposed and \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e mice, with the most severe loss observed in the \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, e). Quantitative analyses demonstrated reductions in BV, BS, BMD, Tb.N, Tb.Th, and Ct.Th, together with increased Tb.Sp and cortical porosity in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd mice compared with WT\u0026thinsp;+\u0026thinsp;Cd mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, f). H\u0026amp;E staining showed a higher proportion of empty osteocyte lacunae in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e and Cd-treated mice, most prominently in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, h). Consistently, osteocyte markers E11 and SOST were markedly reduced in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd compared with WT\u0026thinsp;+\u0026thinsp;Cd, indicating aggravated osteocyte dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). At the molecular level, Bmal1 deficiency amplified Cd-induced oxidative stress and senescence. The capacity of T-AOC and CAT activity were decreased in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd relative to WT\u0026thinsp;+\u0026thinsp;Cd (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej, k). Moreover, enhanced osteocyte senescence was confirmed by stronger p16 immunostaining and elevated p53, p21, and p16 protein levels on Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el\u0026ndash;n). Collectively, these findings indicate that loss of BMAL1 exacerbates skeletal Cd accumulation, intensifies oxidative stress and osteocyte senescence, and accelerates both trabecular and cortical bone loss.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eBMAL1 deficiency enhances osteocyte-mediated bone resorption and aggravates bone metabolic imbalance\u003c/h2\u003e \u003cp\u003ePrevious studies indicate that Cd disrupts bone metabolic balance \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To determine whether Bmal1 deficiency modifies this effect, we assessed systemic bone turnover markers and local indices of formation and resorption. Serum procollagen type I N-terminal propeptide (PINP) was significantly reduced in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e and Cd-treated mice, with the lowest levels in the \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd group (Fig. S7a). Conversely, the resorption marker CTX-I was elevated in both \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e and Cd-exposed mice and increased further in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd animals (Fig. S7b). Moreover, osteocyte-derived osteoclast regulators were altered: OPG decreased whereas RANKL increased, producing a higher RANKL/OPG ratio in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd mice (Fig. S7c\u0026ndash;e). Histological analyses revealed a shift in the balance between bone formation and resorption. ALP staining showed markedly reduced osteogenic activity, with the smallest ALP-positive area in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd (Fig. S7f, g). In trabecular regions, TRAP staining demonstrated a significant reduction in TRAP-positive area, indicating impaired osteoclast formation and activity in that compartment (Fig. S7h, i). By contrast, cortical bone exhibited a striking increase in TRAP-positive osteocytes in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e and Cd-exposed groups, most prominently in \u003cem\u003eBmal1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e+Cd mice (Fig. S7j, k). Together, these findings indicate that Bmal1 deficiency under Cd stress diverts bone resorption from canonical osteoclast-dependent mechanisms toward osteocyte-mediated pathways, thereby exacerbating Cd-induced remodeling imbalance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eSenolytic treatment combined with melatonin alleviates cadmium-induced osteocyte senescence and bone loss\u003c/h2\u003e \u003cp\u003eTo further assess the contributions of cellular senescence to Cd-induced osteoporosis, we tested senescent-cell clearance with dasatinib plus quercetin (DQ) and combined with melatonin (MT). Bone Cd measurements showed that DQ alone did not alter Cd accumulation, whereas DQ\u0026thinsp;+\u0026thinsp;MT reduced bone Cd content (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Micro-CT analysis of the femur revealed that both DQ and DQ\u0026thinsp;+\u0026thinsp;MT improved bone microarchitecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, e), increasing BV, BS, BMD, Tb.N, Tb.Th, and Ct.Th and reducing Tb.Sp and cortical porosity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, f). Notably, DQ\u0026thinsp;+\u0026thinsp;MT produced greater recovery than DQ alone, suggesting a synergistic benefit of circadian support on senolytic therapy. H\u0026amp;E staining revealed fewer empty osteocyte lacunae in DQ\u0026thinsp;+\u0026thinsp;Cd and DQ\u0026thinsp;+\u0026thinsp;MT+Cd groups, indicating improved osteocyte viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg, h). Consistently, osteocyte markers E11 and SOST were partially restored by DQ and most strongly rescued by DQ\u0026thinsp;+\u0026thinsp;MT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei). At the molecular level, both interventions attenuated oxidative stress and senescence: T-AOC and CAT activity were increased in the DQ\u0026thinsp;+\u0026thinsp;Cd and DQ\u0026thinsp;+\u0026thinsp;MT+Cd groups relative to Cd alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ej, k). Moreover, p16 immunoreactivity and the protein levels of p16, p21, and p53 were markedly reduced after treatment, with the largest decreases observed in the DQ\u0026thinsp;+\u0026thinsp;MT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003el\u0026ndash;n). Collectively, these results indicate that senescent-cell clearance, particularly when combined with circadian rhythm restoration, mitigates Cd-induced osteocyte senescence, and bone loss, supporting DQ\u0026thinsp;+\u0026thinsp;MT as a promising therapeutic strategy for Cd-related skeletal toxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBone is a multifunctional organ that not only provides mechanical support and protection for vital tissues, but also functions as a dynamic endocrine organ and mineral reservoir essential for calcium-phosphate homeostasis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Cd is a well-established skeletal toxicant and has long been implicated in the pathogenesis of osteoporosis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the present study, we show that Cd accumulates in bone, induces osteocyte senescence, and that osteocytes actively contribute to Cd-induced skeletal pathology. Mechanistically, Cd disrupts osteocyte redox homeostasis, triggering cellular senescence and a proinflammatory senescence-associated secretory phenotype that propagates ageing-related dysfunction to neighboring bone-resident cells. These results reposition osteocytes from passive bystanders to active mediators of cadmium-induced bone loss, providing a mechanistic link between environmental Cd burden, osteocyte dysfunction, and the collapse of bone-remodeling homeostasis.\u003c/p\u003e \u003cp\u003eCellular senescence is a hallmark of organismal ageing that contributes to physiological decline and is closely linked to the onset and progression of osteoporosis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In a longitudinal comparison of female and male C57BL/6 mice, age-related loss of spinal and hindlimb strength was detectable nine months before measurable declines in bone mineral density \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Senescence can be triggered by diverse insults, including intrinsic factors such as oxidative stress and mitochondrial dysfunction as well as extrinsic exposures such as chemotherapeutic agents and environmental toxins \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. These stimuli converge on DNA damage responses that activate the p53, p21 and p16 pathways, ultimately culminating in irreversible cell cycle arrest \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Although skeletal ageing is inevitable, environmental factors can accelerate its onset. Cd is a potent pro-senescent agent that induces cellular senescence through multiple routes, even at low doses. In BMSCs, Cd activates NF-κB signaling to drive senescence, as evidenced by upregulation of p53, p21 and p16, G0/G1 cell-cycle arrest, DNA damage, mitochondrial dysfunction, and disruption of the osteogenic-adipogenic balance \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our study demonstrated that Cd-induced bone loss in vivo and in vitro is accompanied by osteocyte senescence: osteocytes exhibited strong SA-β-gal positivity, increased expression of p53, p21 and p16, elevated intracellular ROS, DNA damage markers, and G1 cell-cycle arrest. Collectively, these findings establish a mechanistic link between osteocyte senescence and Cd-induced osteoporosis.\u003c/p\u003e \u003cp\u003eSenescent cells secrete a broad repertoire of bioactive molecules, including inflammatory cytokines, chemokines, matrix-degrading enzymes, and ROS-collectively known as the SASP \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In our experiments, conditioned medium from senescent osteocytes contained elevated levels of inflammatory cytokines. It is widely accepted that primary, damage-induced senescence can induce secondary senescence in neighboring cells via SASP factors \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This secondary spread occurs through two non-exclusive mechanisms: paracrine senescence driven by soluble SASP components, and juxtacrine senescence mediated by direct cell-cell contact; together, these processes allow a small initial pool of senescent cells to amplify tissue dysfunction and promote age-related disease \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. For example, glucocorticoid therapy can trigger primary senescence in a subset of bone-marrow adipocyte lineage cells (BMAd) via the 15d-PGJ2\u0026ndash;PPARγ\u0026ndash;p16 axis; these BMAd then disseminate senescence to other bone and marrow populations through SASP, and suppression of BMAd senescence or blockade of SASP signaling attenuates glucocorticoid-induced bone loss \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our data demonstrate that senescent osteocytes propagate senescence to other bone-resident cells by secreting SASP, impair the tri-lineage differentiation potential of BMSCs, reduce osteoblast mineralization, and impede osteoclast-precursor fusion, collectively disrupting bone remodeling. Thus, an osteocyte-centered model of senescence propagation reframes cadmium-induced osteoporosis as a network-level dysfunction driven by osteocyte-derived signaling rather than isolated effects on osteoblasts or osteoclasts.\u003c/p\u003e \u003cp\u003eCircadian disruption is increasingly implicated in metabolic and age-related diseases, and the skeleton is no exception \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Epidemiological studies report a higher incidence of osteoporosis and fractures among shift workers \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Experimental models likewise show that continuous light exposure impairs musculoskeletal performance and remodels bone microarchitecture, producing features consistent with early osteoporosis \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. BMAL1, a central component of the molecular clock, has emerged as a key regulator of skeletal health. In osteoblasts, BMAL1 knockdown reduces expression of osteogenic markers, alkaline phosphatase activity, and mineralization, while increasing apoptosis and inflammatory signaling \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In osteoclasts, BMAL1 modulates resorption by binding E-box elements in the NFATc1 promoter and cooperating with steroid receptor coactivators to promote NFATc1 transcription \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Beyond its timekeeping role, BMAL1 influences cellular metabolism, DNA-repair pathways, and oxidative-stress responses. For example, in aged human lens epithelial cells, downregulation of the NRF2/antioxidant-response-element (ARE) pathway coincides with reduced BMAL1 expression, leading to ROS accumulation and cell death, similarly, BMAL1 deficiency amplifies P. gingivalis-induced atherosclerosis via NF-κB activation and increased oxidative stress \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In the present study, Cd exposure markedly reduced BMAL1 levels in osteocytes, and genetic ablation of BMAL1 substantially exacerbated Cd-induced bone loss and osteocyte senescence, indicating that BMAL1 is essential for skeletal integrity under toxic stress. Mechanistically, BMAL1 likely attenuates oxidative stress by upregulating antioxidant enzymes and facilitating ROS clearance, consistent with its role as a core regulator of cellular redox homeostasis in other tissues.\u003c/p\u003e \u003cp\u003eEffective treatments for skeletal diseases caused by environmental pollutants remain scarce, underscoring the need for innovative therapeutic strategies. Targeting senescent cells to ameliorate age-related disorders has recently emerged as a rapidly advancing approach \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In mouse models that recapitulate the severe bone loss of human senile osteoporosis, three classes of interventions-genetic depletion of senescent cells, pharmacologic senolysis, and pharmacologic suppression of the SASP, each significantly improved skeletal phenotypes \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Among senolytics, the dasatinib-quercetin combination (D\u0026thinsp;+\u0026thinsp;Q) is the most extensively studied and has shown promise in mitigating age-associated physiological dysfunction in both mice and humans \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Dasatinib is a clinically used tyrosine-kinase inhibitor employed in certain leukemias, while quercetin is a natural flavonoid that promotes apoptosis in senescent endothelial cells \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Recent preclinical data indicate that D\u0026thinsp;+\u0026thinsp;Q reduces age-related increases in SA-β-gal activity in periadrenal white adipose tissue and improves metabolic function in aged mice \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Building on this rationale, we evaluated D\u0026thinsp;+\u0026thinsp;Q in a Cd intoxication model: D\u0026thinsp;+\u0026thinsp;Q attenuated Cd-induced osteocyte senescence and improved femoral microarchitecture. Co-administration of melatonin further augmented these benefits, preserved BMAL1-centered circadian regulation, and reduced skeletal Cd burden. Mechanistically, this combination is rational and complementary: D\u0026thinsp;+\u0026thinsp;Q removes the primary source of SASP, whereas melatonin-acting as both a chronobiotic and an antioxidant-limits senescence propagation by restoring formation-resorption rhythmicity and strengthening redox homeostasis.\u003c/p\u003e \u003cp\u003eIn summary, our findings suggest that osteocytes play a central role in Cd-induced bone loss by undergoing senescence and impairing bone remodeling. BMAL1 serves as a protective factor by limiting oxidative stress and senescence in osteocytes. Importantly, interventions that restore or enhance BMAL1 function could help mitigate Cd-induced osteocyte dysfunction and bone loss, offering a potential therapeutic avenue for heavy metal-associated skeletal disorders.\u003c/p\u003e"},{"header":"Declarations","content":" \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis research was supported by the following sources: National Key R\u0026amp;D Program of China (2023YFD1801100), National Natural Science Foundation of China (32102732, 32072933, 32273086, 32072923), Jiangsu Provincial Natural Science Foundation of China (BK20210806), Graduate International Academic Exchange Special Fund Project of Yangzhou University (YZUF2024211) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShen Y, Huang X, Wu J, Lin X, Zhou X, Zhu Z \u003cem\u003eet al.\u003c/em\u003e The Global Burden of Osteoporosis, Low Bone Mass, and Its Related Fracture in 204 Countries and Territories, 1990\u0026ndash;2019. \u003cem\u003eFront Endocrinol\u003c/em\u003e 2022; 13: 882241.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang C, Lv X, Zou L, Rong Y, Zhang L, Xu M \u003cem\u003eet al.\u003c/em\u003e Cadmium exposure and osteoporosis: epidemiological evidence and mechanisms. \u003cem\u003eToxicol Sci\u003c/em\u003e 2025; 205: 1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Aring;kesson A, Bjellerup P, Lundh T, Lidfeldt J, Nerbrand C, Samsioe G \u003cem\u003eet al.\u003c/em\u003e Cadmium-Induced Effects on Bone in a Population-Based Study of Women. \u003cem\u003eEnviron Health Perspect\u003c/em\u003e 2006; 114: 830\u0026ndash;834.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDallas SL, Prideaux M, Bonewald LF. 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Senolytic drugs: from discovery to translation. \u003cem\u003eJ Intern Med\u003c/em\u003e 2020; 288: 518\u0026ndash;536.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8702171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8702171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs a ubiquitous environmental pollutant, cadmium (Cd) has been strongly implicated in the development of osteoporosis. However, the cellular and molecular mechanisms mediating its skeletal toxicity remain incompletely understood. Here, we identify osteocytes as a highly vulnerable and previously underrecognized cellular target in Cd-induced osteoporosis. We demonstrate that Cd accumulates in cortical bone and induces pronounced osteocyte senescence in vivo and in vitro. Cd exposure provokes oxidative stress, DNA damage, and cell-cycle arrest in osteocytes, collectively disturbing bone metabolic homeostasis by amplifying senescence signals to neighboring osteoblasts, osteoclasts, and bone marrow stromal cells. Mechanistically, we reveal that brain and muscle ARNT-Like 1 (BMAL1) acts as a pivotal regulator of Cd-induced osteocyte senescence. BMAL1 deficiency exacerbated oxidative stress, senescence, and trabecular and cortical bone deterioration. Conversely, BMAL1 overexpression restored redox balance and markedly attenuated Cd-induced osteocyte senescence. Moreover, eliminating senescent cells using dasatinib plus quercetin markedly reduced osteocyte senescence and improved bone integrity, and co-treatment with melatonin further enhanced these protective effects. These findings reveal an unrecognized osteocyte-centered senescence axis in Cd-induced skeletal toxicity and position BMAL1 as a promising therapeutic target for mitigating pollutant-related bone damage.\u003c/p\u003e","manuscriptTitle":"Cadmium targeting the circadian transcription factor BMAL1 to induce osteocyte senescence contributes to osteoporosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 11:52:57","doi":"10.21203/rs.3.rs-8702171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-09T06:03:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-09T01:30:25+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-05T06:46:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-27T05:37:10+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-26T01:06:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-13T01:05:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T01:48:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2026-01-27T16:28:37+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-01-26T23:55:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T15:55:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53cfd9dc-7b6f-4ca2-a7bb-be8ad22e2bf7","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62844445,"name":"Biological sciences/Cell biology/Senescence"},{"id":62844446,"name":"Biological sciences/Cell biology/Circadian rhythms"}],"tags":[],"updatedAt":"2026-03-09T06:06:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 11:52:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8702171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8702171","identity":"rs-8702171","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}