Ceria nanoparticles guard against glucocorticoid induced bone homeostasis imbalance through the promotion of osteogenesis and inhibition of osteoclastogenesis

Abstract

Osteoporosis induced by the long-term administration of glucocorticoids is a common clinical problem. Current anti-osteoporosis therapies mainly focus on bone resorption inhibition or bone formation promotion unilaterally. However, the imbalance between bone formation and resorption often leads to suboptimal therapeutic outcomes. This study confirms the pathways through which dexamethasone (DEX), a typical glucocorticoid, triggers GIO, and explores both the therapeutic potential and underlying mechanisms of ceria nanoparticles (CNPs) in mitigating GIO. Our results demonstrate that DEX-induced bone homeostasis imbalance is the primary mechanism underlying GIO development in mice. Specifically, DEX inhibits pre-osteoblast proliferation and differentiation by inducing apoptosis and ferroptosis. Concurrently, DEX promotes osteoclast precursor proliferation and differentiation, significantly accelerating bone resorption. Administration of CNPs significantly mitigates DEX-induced disruption of bone homeostasis by ameliorating intracellular reactive oxygen species (ROS) accumulation via regulation of the Keap1/Nrf2 signaling pathway. Regarding osteogenesis, CNPs are capable of alleviating DEX-induced pre-osteoblast apoptosis and ferroptosis through the regulation of GPX4/ACSL4 signaling pathway. In terms of bone resorption, CNPs inhibit the RANKL-dependent osteoclast formation pathway. These findings validate the potential application of inorganic nanoparticles in GIO prevention.

Graphical abstract

1. Introduction

Glucocorticoids (GCs) are a class of steroid hormones that play a crucial role in suppressing inflammatory responses and regulating autoimmune disorders [1]. However, accumulating evidence suggests that excessive and prolonged exposure to GCs can lead to various adverse health effects, such as type 2 diabetes mellitus and osteoporosis [2]. Glucocorticoid-induced osteoporosis (GIO), the most common form of secondary osteoporosis, is characterized by decreased bone mass, deterioration of bone architecture, and increased fracture risk [3]. Epidemiological studies have indicated that the increased risk of fractures, especially vertebral fractures, is closely related to the cumulative dose and duration of GCs. Additionally, the risk of fractures can be partially reduced after discontinuing the medication [4]. Despite the known association between long-term GC use and bone damage, the understanding and treatment of GIO remain insufficient. Therefore, it is crucial to uncover the pathological mechanism of GIO and discover new interventions with both clinical and social value.

The bone homeostasis is maintained by the dynamic balance between osteogenesis (regulated mainly by osteoblasts) and bone resorption (regulated mainly by osteoclasts). Previous studies have demonstrated that GC-induced oxidative stress plays a significant role in disrupting the balance of bone homeostasis [[5], [6], [7]]. Specifically, GCs directly inhibit pre-osteoblasts proliferation and differentiation through reactive oxygen species (ROS)-mediated signaling pathways, leading to reduced bone formation [8,9]. While ROS negatively affect osteoblasts, they do not necessarily have detrimental effects on osteoclasts. In fact, ROS stimulate the expression of RANKL in osteoblasts, which in turn promotes osteoclastogenesis through the OPG/RANKL/RANK signaling pathway [10,11]. Interestingly, a growing body of researches have indicated that GIO is associated with ferroptosis, which is characterized by the iron accumulation and increased levels of lipid ROS [12,13]. A recent study has reported that iron accumulation increases NADPH oxidase 4, an enzyme that enhances ROS levels and intracellular lipid peroxides, driving ferroptosis in osteoblasts [14].

Currently, numerous studies have focused on exploring ways to alleviate oxidative stress in order to counteract the consequences of bone loss caused by GCs. Many molecules or agents with antioxidant properties, such as flavonoid polyphenols [15], vitamin E [16], and N-Acetylcysteine [17], have been used to improve the bone metabolism and represent potential and effective therapies for GIO. However, these antioxidants face challenges in targeting the bone microenvironment, and their inability to precisely regulate the bidirectional effects between osteoblasts and osteoclasts results in limited therapeutic efficacy. Recently, the application of ceria nanoparticles (CNPs) in biomedicine has drawn considerable attention due to their capability to induce cell proliferation and differentiation owing to their excellent ROS scavenging activity [18]. The reversible redox cycling between surface Ce³⁺ and Ce⁴⁺ enables CNPs to maintain sustained ROS scavenging activity, unlike conventional antioxidants that are irreversibly consumed [19]. We have reported that CNPs are beneficial for promoting bone regeneration through several pathways including inhibition of mature osteoclasts, induction of angiogenesis in regenerated bone tissue, facilitation of endochondral ossification [20,21]. Current studies primarily focus on the impact of ROS on bone homeostasis. However, emerging evidence indicates that ferroptosis can exacerbate GIO through ROS-independent metabolic pathways, such as lipid metabolism dysregulation and iron homeostasis imbalance. Notably, the specific mechanisms by which CNPs intervene in ferroptosis-related GIO progression remain to be elucidated.

Herein, we evaluated the effects of CNPs on the bone homeostasis under the GIO condition induced by dexamethasone (DEX). Our results demonstrated that DEX significantly disrupted both osteogenesis and bone resorption processes. On the osteogenesis side, DEX inhibited the proliferation and differentiation of pre-osteoblasts through the promotion of apoptosis and ferroptosis. On the bone resorption side, DEX enhanced the differentiation of pre-osteoclasts resulted in the accelerated bone resorption. Notably, the presence of CNPs could effectively inhibit DEX-induced pre-osteoblast apoptosis and ferroptosis through the regulation of GPX4/ACSL4 signaling pathway. Also, the differentiation of pre-osteoclasts could be effectively inhibited by CNPs.

2. Materials and methods

2.1. Reagents

Cerium nitrate hexahydrate (Ce(NO₃)₃·6H₂O) and 1-octadecene (ODE) were purchased from Acros. Trioctylphosphine oxide (TOPO), succinic acid (SA), oleylamine (OAm), riboflavin, nitrotetrazolium blue chloride (NBT), N-hydroxysuccinimide (NHS), dexamethasone (DEX), calcein, β-glycerophosphate, ascorbic acid, rabbit anti-PMP70 antibody and tartrate resistant acid phosphatase (TRAP) kit were purchased from Sigma-Aldrich. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Aladdin. Paraformaldehyde and hydrogen peroxide (H₂O₂) kit were purchased from Solarbio. Mouse PINP ELISA kit, mouse OC/BGP ELISA kit, TUNEL kit, mitochondrial membrane potential kit (JC-1) and ferrous iron colorimetric assay kit were purchased from Elabsciense. Mouse CTX-I ELISA kit was purchased from Henghuibio. Alfa-minimum essential medium (α-MEM) and fetal bovine serum (FBS) were obtained from Gibco Life Technologies. CCK-8 assay kit, 3-Methyladenine (3-MA), necrostatin-1 (Nec-1), Z-VAD (OH)-FMK (Z-VAD), ferrostatin-1 (Fer-1) and RSL3 were purchased from MedChemExpress. Dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), alkaline phosphatase (ALP) staining solution, alizarin red S (ARS) staining solution (0.2 %, pH 8.3), ALP activity kit, Annexin V-FITC apoptosis kit, reduced glutathione (GSH)/oxidized glutathione (GSSG) kit, total superoxide dismutase (SOD) kit, catalase (CAT) assay kit and malondialdehyde (MDA) kit were purchased from Beyotime Biotechnology. Annexin V-FITC/PI double staining apoptosis kit was purchased from BD Pharmingen. C11-BODIPY 581/591 probe was purchased from Invitrogen. TraKine™ F-actin staining kit was purchased from Abbkine. Rabbit anti-ACSL4, ALPL, RUNX2, osteopontin (OPN), collagen type I (COL-I), osteocalcin (OCN), cleaved caspase-3, catalase (CAT) and NADPH oxidase (NOX1) antibodies were purchased from Proteintech. Rabbit anti-15 lipoxygenase (ALOX15) antibody was purchased from Abmart. Mouse anti-GPX4, caspase-3, NFATc1, c-Fos, β-actin antibody, HRP-conjugated affinipure goat anti-rabbit or mouse IgG (H + L) were purchased from Proteintech. M-CSF and RANKL were purchased from R&D Systems.

2.2. Ethics statement

All animal experimental procedures were approved by the Animal Ethics Committee of Chongqing General Hospital, Chongqing University (KY S2023-021-01) and the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (AMUWEC20230271). Six-week-old male C57BL/6J mice were purchased from the Laboratory Animal Center of the Army Medical University, where maintained under pathogen-free environment at a 12 h light/dark cycle, 24 ± 2 °C with 55 % ± 10 % humidity. The experimental procedure complied with the NIH Guidelines for the Care and Use of Laboratory Animals.

2.3. CNPs synthesis and characterization

CNPs synthesis. The CNPs were synthesized by our previous method [22]. In brief, Ce(NO₃)₃·6H₂O (0.7 g) and TOPO (1 g) were dissolved in ethanol (3 mL) at 90 °C. After that, ODE (5 mL) was added to the mixture and the ethanol was removed through vacuum for 15 min. After cooling to room temperature, OAm (100 μL) was slowly added under stirring condition and the solution was heated at 190 °C for 15 min. After cooling to room temperature, the nanoparticles were washed by acetone for several times until the supernatant was colorless. The nanoparticles were then dispersed in tetrahydrofuran (10 mL). Subsequently, the nanoparticles were modified with SA ligands using our previous method [23]. Briefly, SA (109 mg), NHS (105 mg) and EDC (175 mg) were dissolved in dichloromethane (10 mL) and stirred at room temperature for 8 h. The solvent was removed by rotary evaporation. Then a mixture solution (20 mL) containing alendronate sodium (AL) (100 mg) and Na₂CO₃ (250 mg) was added to the activated ligand and stirred at room temperature overnight. The SA ligand solution (5 mL) was mixed with the synthesized nanoparticles (1 mL) and tetrahydrofuran (5 mL). The mixture was stirred at 80 °C for 4 h. After cooling to room temperature, the water layer containing the surface modified nanoparticles was separated and the nanoparticles were precipitated out by adding acetone. Finally, the nanoparticles were dialyzed in water for 24 h. Of particular note, alendronate sodium merely served as a bridging molecule between CNPs and SA, without utilizing its therapeutic effects for osteoporosis. Firstly, the amino group in alendronate reacted with the carboxyl group in SA through amidation, resulting in the AL-SA intermediate. Subsequently, due to the strong affinity between cerium ions and phosphate groups, AL-SA was conjugated onto the surface of CNPs.

CNPs characterization. The morphology of the CNPs was observed on transmission electron microscopy (TEM) (FEL Talos F200X) operated at 200 kV. The size of the nanoparticles was determined by Nano Measurer 1.2 software based on the TEM images. The Ce³⁺/Ce⁴⁺ ratio in the CNPs was measured by X-ray photoelectron spectroscopy (XPS) (Scientific Escalab 250), and the data was analyzed by XPS Peak Fit 4.0 software. The zeta potential of the CNPs was measured using a Malvern Zetasizer Nano ZS at 25 °C, with a sample concentration of 0.1 mg/mL. The ROS scavenging activity of the CNPs was assessed by our previous procedures [23]. In brief, a working solution containing EDTA-Na₂ (0.1 M, 800 μL), NBT (2 mM, 300 μL) and riboflavin (0.6 mM, 200 μL) and sodium phosphate buffer (10 mM, pH 7.8, 10.9 mL) was prepared. Different concentrations of CNPs were mixed with the working solution at volume ratio of 1: 2. The mixture was shaken for 60 s and exposed to white light for 90 s. The ROS scavenging activity was determined by detecting the absorbance at 560 nm.

2.4. Animal experiment

Establishment and treatment of GIO mice. The GIO mouse model was established by previous method [24]. The C57BL/6J mice were randomly divided into three groups including control group (n = 6), DEX group (n = 6) and DEX + CNPs group (n = 6) after one week of adaptive feeding. The three groups of mice were injected with saline (i.v.), DEX (s.c., 10 mg kg⁻¹) and DEX (s.c., 10 mg kg⁻¹) + CNPs (i.v., 1 mg kg⁻¹) respectively at administration frequency of 3 times per week for 4 weeks. The mice were sacrificed at week 4 for further analysis. The dosage of CNPs in vivo tests is based on previous research by the group. The dosage of CNPs used in the in vivo experiments was determined based on our group's previous research findings [25,26].

Micro-CT analysis. The bone trabecular structure of the distal femurs was evaluated using cone-beam micro-CT scanner (Skyscan 1272) with resolution of 5 μm. The scanning parameters were set as X-ray power of 40 kV and tube current of 0.25 mA. The average of three images taken at each angle (0.9°) was used to generate the final image. The results were processed by NRecon software. A total of 4 mm wide trabecular bone close to the distal growth plate of the femur was designated the ROI for each group (n = 6). Trabecular bone parameters including trabecular bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp) were analyzed to assess the therapeutic effects of the CNPs.

Histological analysis. The bone trabecular structure of the distal femurs was further evaluated by histological analysis. The collected femurs were fixed in paraformaldehyde (4 %) at 4 °C for 24 h and decalcified in EDTA (10 %) decalcification solution for 2 weeks at room temperature. Then the femurs were embedded in paraffin and cut into sections with thickness of 5 μm. The sections were stained by hematoxylin and eosin (HE), Masson's trichrome, TRAP. The ferroptosis marker proteins of GPX4, ACSL4 and the osteogenic marker protein of osteocalcin (OCN) were stained by immunohistochemistry method using anti-mouse/rabbit antibodies respectively. The histological images were collected on slide scanner (Olympus Slide VS200).

ELISA procedures. The osteogenesis and osteoclastogenesis markers in the serum for each group (n = 6) were determined by ELISA. The blood samples were collected from orbital vein and the serum samples were isolated by centrifugation (3000 r/min for 5 min). The marker proteins including PINP, OC/BGP and CTX-I were quantified using their corresponding ELISA kits.

Double-calcein labeling. Mice were injected intraperitoneally with 30 mg kg⁻¹ calcein (1 mg ml⁻¹ in 2 % NaHCO₃ solution) at 13 and 3 days before euthanasia. On day 0, the mice were euthanized, and the femurs were fixed and dehydrated and embedded. Samples were cut into 5 μm with a hard tissue cutter, and fluorescence-labelled images were captured using a microscope (Olympus Slide VS200). Quantified bone mineral apposition rate (MAR) was measured.

Pharmacokinetic and biodistribution assessment. Administered CNPs via tail vein injection (5 mg kg⁻¹) in C57BL/6 mice. Blood samples were collected via cardiac puncture under anesthesia at the designated time points. Quantified cerium (Ce) content in blood at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h post-injection using inductively coupled plasma mass spectrometry (ICP-MS). Additionally, major organs including the heart, liver, spleen, lungs, and kidneys, as well as the femurs, were harvested at 1, 2, 3 and 7 days post-injection for biodistribution analysis. These tissues were digested using a suitable acid mixture, and the cerium (Ce) content was determined using ICP-MS to evaluate the tissue distribution and clearance of CNPs.

2.5. Pre-osteoblasts culture and treatment

Cell culture. The mouse pre-osteoblast cell line MC3T3-E1 was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were cultured in α-MEM supplemented with FBS (10 %) and penicillin-streptomycin (1 %) in a humidified atmosphere containing 5 % CO₂ at 37 °C. To induce the osteogenic differentiation, the cells were cultured in medium additionally supplemented with DEX (10⁻⁷ M), β-glycerophosphate (10 mM) and ascorbic acid (50 mg L⁻¹).

Cell viability. The pre-osteoblasts were seeded on 96 well plate (5 × 10³ cells/well) for 12 h. The cells were then treated by different regents including DEX, DEX + CNPs, 3-MA, Nec-1, Z-VAD and Fer-1 for 24 h. After treatment, the cells were incubated with CCK-8 (10 %) for 1 h. The cell viability was determined by detecting the absorbance at 450 nm using microplate reader (Bio-Tek synergy 4).

ALP and ARS analysis. The pre-osteoblasts were seeded on 24 well plate (2 × 10⁴ cells/well) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs. For the ALP analysis, the cells were cultured in osteogenic differentiation medium for 14 days. The ALP activity was assessed by ALP activity assay kit using the procedures provided by the manufacturer. As for the ALP staining, the cells were washed with PBS and fixed with paraformaldehyde (4 %) for 30 min. Then the cells were stained with ALP staining solution. For the ARS analysis, the cells were cultured in osteogenic differentiation medium for 28 days. The cells were then fixed with ethanol (90 %) for 20 min and washed by PBS twice. The ARS staining solution was added to the cell and the calcium nodules were stained for 40 min.

Intracellular ROS measurement. The pre-osteoblasts were seeded on 60 mm cell culture dish (5 × 10⁵ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. After that, the cells were incubated with ROS probe DCFH-DA for 30 min. The intracellular ROS was quantified by flow cytometer (CytoFLEX LX, Beckman Coulter).

DHE staining. The pre-osteoblasts were seeded on 12 well plate (2 × 10⁴ cells/well) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. After that, the cells were incubated with DHE (5 μM in PBS) for 30 min in the dark at 37 °C. Then the nuclei were stained by Hoechst 33342 for 5 min in the dark at 37 °C. The samples were washed by PBS and immediately imaged on microscope (Olympus IX71).

Western blot (WB) analysis. The pre-osteoblasts were seeded on 100 mm dish (1 × 10⁶ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX, RSL3, DEX + CNPs or DEX + Fer-1 and the cells were cultured for 24 h. The cells were washed by cold PBS and lysed by cell lysis buffer containing phenylmethanesulfonyl fluoride (1.0 mM). The total protein was extracted by centrifugation at 12,000 g at 4 °C for 15 min. The protein concentration was measured by BCA method using BCA protein assay kit. The proteins were separated by electrophoresis and transferred onto 0.22 μm PVDF membrane. The membrane was blocked using 5 % nonfat milk for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. The membrane was washed by TBST for three times and incubated with secondary antibody for 1 h at room temperature. The protein bands were collected on imaging system (Bio-rad ChemiDoc XRS+). The optical densities of the bands were measured by Image J software.

Annexin V-FITC staining. For the collection of optical images, the pre-osteoblasts were seeded on 96 well plate (1 × 10⁴ cells/well) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. The cells were stained by Annexin V-FITC kit using procedures provided by the manufacturer. The nuclei were stained by Hoechst 33342. The optical images were collected on microscope. For the collection of flow cytometer data, the pre-osteoblasts were seeded on 60 mm dish (5 × 10⁵ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. The cells were stained by Annexin V-FITC kit using procedures provided by the manufacturer. The nuclei were stained by PI. The stained cells were subjected to flow cytometer for apoptosis analysis.

TUNEL assay. For the cell samples, the pre-osteoblasts were seeded on cell climbing films placed in the 24 well plate (5 × 10⁴ cells/well) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. The cell climbing films were fixed according to the procedures provided by the TUNEL assay kit manufacturer. The cells were then incubated with TUNEL working solution at 37 °C for 60 min. For the tissue samples, antigen repair was performed using proteinase K (20 μg mL⁻¹) at 37 °C for 20 min after deparaffinization and hydration. After that, the tissue sections were incubated with TUNEL working solution at 37 °C for 60 min. The nuclei of the cells were stained by DAPI solution for 5 min at room temperature. The samples were imaged on microscope and the pictures were analyzed by Image J software.

Mitochondrial membrane potential assessment. The pre-osteoblasts were seeded on 60 mm dish (5 × 10⁵ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. After that, the cells were collected and incubated with JC-1 at 37 °C for 20 min. Then the cells were re-suspended in fresh HBSS (500 μL) and analyzed by flow cytometer equipped with 488 nm laser for excitation.

Intracellular Fe²⁺ measurement. The pre-osteoblasts were seeded on 100 mm dish (1 × 10⁶ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX, RSL3, DEX + CNPs or DEX + Fer-1 and the cells were cultured for 24 h. The cells were harvested and lysed by iron assay buffer supplied with the Fe²⁺ assay kit. The lysate was centrifuged at 15,000 g for 10 min. The supernatant (80 μL) was mixed with the working solution (80 μL) at 37 °C for 10 min and the OD value (593 nm) of the samples was detected on microplate reader. The Fe²⁺ concentrations were determined according to the absorbance of the standard curve.

Lipid peroxidation assay. The pre-osteoblasts were seeded on 60 mm dish (5 × 10⁵ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX or DEX + CNPs and the cells were cultured for 24 h. After that, the cells were incubated with C11-BODIPY 581/591 (2 μM) in a serum-free medium for 20 min in the dark at 37 °C. The fluorescence intensity of the lipid peroxidation levels were detected by flow cytometer.

Transmission electron microscope (TEM) analysis. The pre-osteoblasts were seeded on 100 mm dish (1 × 10⁶ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX, RSL3, DEX + CNPs or DEX + Fer-1 and the cells were cultured for 24 h. The cells were collected and washed twice by cold PBS. The cells were then fixed with glutaraldehyde solution (2.5 %) at 4 °C for 24 h. After fixation, the cells were dehydrated, embedded, sectioned and stained. The morphology of the mitochondria was visualized on TEM (Hitachi HT-7700).

Immunofluorescence staining. The pre-osteoblasts were seeded on cell climbing films placed in the 24 well plate (2 × 10⁴ cells/well) and cultured overnight. The medium was replaced by new medium containing DEX, RSL3, DEX + CNPs or DEX + Fer-1 and the cells were cultured for 24 h. The cell climbing films were washed twice by cold PBS, fixed with paraformaldehyde (4 %) for 10 min at room temperature and incubated with Triton X-100 (0.3 %) for 10 min on ice. After that, the cells were blocked in goat serum (10 %) at room temperature for 60 min and incubated with primary antibody against PMP70 (dilution 1:100) at 4 °C overnight. The next day, the cell climbing films were washed twice by PBS and incubated with anti-rabbit IgG (dilution 1:100) secondary antibody at room temperature for 60 min in the dark. The nuclei were stained by DAPI solution for 5 min. The fluorescence was visualized on microscope.

GSH/GSSG, H₂O₂, SOD and MDA measurement. The pre-osteoblasts were seeded on 100 mm dish (1 × 10⁶ cells/dish) and cultured overnight. The medium was replaced by new medium containing DEX, RSL3, DEX + CNPs or DEX + Fer-1 and the cells were cultured for 24 h. The GSH/GSSG, H₂O₂, SOD and MDA were measured using procedures provided by the kits manufacturer.

Quantitative RT-PCR. Total RNA was extracted from cells using TRIzol reagent (AGbio, China). RNA concentration and purity were determined by measuring A260/A280 and A260/A230 ratios using NanoDrop ND-1000 Microplate Reader (Thermo Fisher Scientific). For cDNA synthesis, 2 μg of total RNA was reverse transcribed using the reverse transcriptase kit (AGbio, China). Real-time PCR was performed using SYBR PremixExTaq (AGbio, China) by CFX connect (Bio-rad, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control for normalization. The relative mRNA expression levels of target genes were calculated using the 2-ΔΔCT method. All primer sequences were designed and synthesized by Sangon Biotech (Shanghai, China), as listed in Supplementary Table S1.

2.6. Osteoclasts culture and treatment

Cell culture. The pre-osteoclasts were obtained through the isolation of mononuclear macrophages from mouse bone marrow (BMMs) under specific induction culture conditions. In briefly, the femurs were collected from euthanized animals (6 week old C57BL/6J) and the soft tissues were removed. Bone marrow was flushed with sterilized PBS into 100 mm dish. The cells were transferred into centrifugation tube and centrifuged at 1200 r/min for 3 min. The cells were added ACK lysis buffer to lyse the red blood cells. After washed twice by PBS, the cells were seeded on culture flask (25 cm²) and cultured in α-MEM medium containing FBS (10 %) and penicillin-streptomycin (1 %) for 24 h. After that, the non-adherent cells were collected and cultured in medium containing M-CSF (50 ng mL⁻¹). To induce the osteoclast differentiation, the cells were cultured in medium containing M-CSF (50 ng mL⁻¹) and RANKL (100 ng mL⁻¹) for 5 days.

TRAP and F-actin staining. The BMMs were seeded on 96 well plate (1 × 10³ cells/well) overnight. The procedures used to induce the differentiation of the pre-osteoclasts were mentioned above. The reagents including CNPs (50 μM), DEX (0.1 μM) or DEX + CNPs (0.1 μM + 50 μM) were added to the culture medium. For TRAP staining, the cells were fixed in paraformaldehyde (4 %) for 10 min and incubated in Triton X-100 (0.1 %) for 5 min. After that, the cells were stained using the protocol provided by the assay kit manufacturer. The TRAP-positive multinucleated cells with ≥3 nuclei were considered as mature osteoclast. The F-actin ring staining was performed using the protocol provided by the assay kit manufacturer.

GSH/GSSG, CAT, SOD and MDA measurement. The BMMs were seeded on 60 mm dish (1 × 10⁵ cells/dish) and cultured overnight. The procedures used to induce the differentiation of the pre-osteoclasts were mentioned above. The medium was added containing CNPs, DEX or DEX + CNPs and the cells were cultured for 3 day. The GSH/GSSG, CAT, SOD and MDA were measured using procedures provided by the kits manufacturer.

Osteoclast resorption activity (pit assay). For osteoclast resorption activity assay, BMMs were seeded on collagen I-coated plates (354407; Corning). The cells were treated with M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of the indicated doses of DEX and CNPs or indicated treatments for five days. Cells were detached from the plate by washing buffer and were gently washed away, and images of bone resorption pits were captured by a light microscope and the areas of the pits were measured by ImageJ software (USA).

2.7. Statistical analysis

Statistical significance was evaluated using one-way ANOVA (Fisher's LSD test) with a p < 0.05 indicating significance, and GraphPad PRISM software (La Jolla, CA, USA) was utilized for this purpose. Mean ± SD was used to present all of the data. The statistical significance was calculated, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns, no significant.

3. Results

3.1. CNPs maintained bone homeostasis under the GIO condition

The CNPs featured with a size of 4 nm were synthesized by the thermal decomposition method. The CNPs were coated with succinic acid conjugated alendronate to ensure their hydrophilicity (Fig. 1, Fig. 2A). The ROS scavenging activity of the surface coated CNPs was confirmed by an O₂^•− inhibition experiment (Fig. S1). The GIO mouse model was established by the subcutaneous injection (s.c.) of DEX. The CNPs were administered through tail vein injection (i.v.). The femurs and orbital blood were collected to evaluate the performance of CNPs in guarding against the bone homeostasis under the GIO condition by detecting bone formation and bone resorption.

Fig. 1.

Schematic illustration of experimental design.

Fig. 2.

Effects of CNPs on the bone homeostasis under the GIO condition. (A) Transmission electron microscope (TEM) image of CNPs coated by succinic acid conjugated alendronate. Scale bar: 10 nm. (B) Representative images of the distal femurs microarchitecture (upper panel, scale bar: 1 mm) and reconstructed trabecular structure (lower panel, scale bar: 500 μm) (n = 6). (C–G) Quantitative analysis of the micro-CT data, including BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp (n = 6). (H) Representative HE, Masson's trichrome and TRAP staining images of the distal femur trabecular (n = 6). Scale bar: 50 μm. Semi-quantitative analysis of collagen volume (I) and TRAP-positive cells (J) based on Masson's trichrome staining and TRAP staining respectively (n = 6). (K–L) Representative images of double-calcein labeling of femurs from Control, DEX, and DEX + CNPs group mice and quantitative parameters of MAR (n = 6). Scale bar: 50 μm. (M–N) Representative IHC images of OCN staining. Scale bar: 50 μm. Semi-quantitative analysis of OCN expression levels in the trabecular bone of the distal femur (n = 6). (O–Q) The serum levels of PINP, OC/BGP and CTX-I were measured by ELISA (n = 6). Data were presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. One-way ANOVA with Tukey's post-hoc test.

On the bone formation side, the micro-CT images revealed that the GIO mice exhibited trabecular deterioration and bone loss compared with the control group, which confirmed the occurrence of bone homeostasis imbalance (Fig. 2B). While, the bone homeostasis for the CNPs treated GIO mice was well maintained as their trabecular number and density were much higher than the GIO mice. This visual observation was confirmed by the quantitation of the micro-CT data. For the GIO mice, the BMD, BV/TV, Tb.N and Tb.Th were decreased and the Tb.Sp was increased compared with the control mice (Fig. 2C–G). However, for the CNPs treated GIO mice, the BMD, BV/TV, Tb.N and Tb.Th were increased and the Tb.Sp was decreased compared with the GIO mice. The micro-CT results were further verified by HE staining and Masson's trichrome staining, where the trabecular of the GIO mice was sparser and smaller than that of the control mice, while this pathological change was significantly inhibited for the CNPs treated GIO mice (Fig. 2H–J and S2A-B). As shown in Fig. 2K–N, double-calcein labeling and OCN immunohistochemical staining were performed to assess bone formation in vivo. The results demonstrated that the bone mineral apposition rate was decreased in the GIO mice compared with the control mice. Notably, treatment with CNPs significantly increased the bone mineral apposition rate in the GIO mice, indicating that CNPs had a positive effect on bone formation. The enhanced OCN-positive osteoblast surface observed in the CNP group (Fig. 2M − N) further corroborated the anabolic effects of CNPs on osteoblastic bone formation. Additionally, the ELISA results revealed that the bone formation markers including OC/BGP and PINP in the serum of CNPs treated GIO mice were higher than those of the GIO mice (Fig. 2O–P). Besides, TUNEL assay indicated that CNPs could effectively protect the osteocytes from DEX-induced cell death in the bone tissue (Fig. S3).

On the bone resorption side, we further probed if the CNPs could alleviate the bone resorption under the GIO condition. The TRAP staining for the bone tissue indicated that the active osteoclasts in the GIO mice were obviously more than those of the control mice as well as the CNPs treated GIO mice, which implied that CNPs could effectively inhibit the formation of osteoclasts (Fig. 2H and S2C). The bone resorption marker CTX-I in the serum of CNPs treated GIO mice was obviously lower than that of the GIO mice (Fig. 2Q). By the way, the HE staining and pharmacokinetic analysis results revealed that the CNPs had no effects on the tissue structure and morphology of the major organs implying good biocompatibility of the CNPs (Fig. S4 and S5). These results indicated that CNPs could effectively maintained bone homeostasis under the GIO condition.

3.2. CNPs preserved the proliferation and differentiation activities of pre-osteoblasts

To understand the mechanisms underlying the capability of CNPs to guard against the bone homeostasis under the GIO condition, the effects of CNPs on the proliferation and differentiation activities of pre-osteoblasts in vitro were explored. The effects of DEX on the proliferation activity of MC3T3-E1 osteoblasts was firstly analyzed. It could be seen in the cell viability assay results that the proliferation activity of the cells was affected by DEX in a dose-dependent manner. Low concentration of DEX (< 0.1 μM) did not inhibit the cell proliferation activity, while high concentration of DEX (> 1.0 μM) exhibited obvious inhibition effects on the cell proliferation activity (Fig. 3A). The capability of CNPs to rescue pre-osteoblasts from the suppression of DEX (100 μM) was evaluated by incubating the cells with different concentrations of CNPs. When the concentration of CNPs was higher than 50 μM, the rescue of the cells from DEX induced proliferation suppression was observed (Fig. 3B). This result was reasonable because the pre-osteoblasts cultured with CNPs in absence of DEX could promote the proliferation of the cells in concentrations ranging from 10 to 500 μM (Fig. S6). Furthermore, our investigation revealed that CNPs significantly reduced DEX-induced oxidative stress levels through intracellular ROS detection (Fig. 3C).

Fig. 3.

CNPs preserved the proliferation and differentiation activities of pre-osteoblasts. (A) Cell viabilities of pre-osteoblasts cultured with different concentrations of DEX for 24 h. (B) Cell viabilities of pre-osteoblasts cultured with 100 μM DEX and different concentrations of CNPs for 24 h. (C) Intracellular ROS quantified by DCFH-DA staining and flow cytometry detection. (D) Quantified ALP activities for the pre-osteoblasts treated with DEX or DEX + CNPs for 14 days. (E) Representative images of ALP and ARS staining for 14 days and 28 days respectively. Scale bar: 200 μm. (F) Expression of osteogenic differentiation indicators analyzed by WB technology. (G) Schematic diagram of different outcomes of pre-osteoblasts cultured with DEX or DEX + CNPs. Data were presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns no significant. One-way ANOVA with Tukey's post-hoc test.

Aside from the suppression of proliferation activity, glucocorticoids could also impair anabolic function as well as the subsequent matrix mineralization of osteoblasts [27,28]. To this end, the effects of CNPs on the differentiation and mineralization of pre-osteoblasts in the presence of DEX was evaluated by detecting the expression of typical osteogenic differentiation indicators, including ALP, RUNX2, OPN and COL I, and the formation of calcium nodules. It could be seen in the staining images and the quantified data that the ALP positive area of the cells treated with DEX was obviously decreased compared with the control group (Fig. 3D and E). However, the addition of CNPs could effectively reverse this declining tendency. The other osteogenic indicators quantified by WB technology also supported the capability of CNPs to preserve the differentiation activity of the pre-osteoblasts (Fig. 3F). The generation of calcium nodules stained by ARS confirmed this conclusion (Fig. 3E). Moreover, the mRNA expression levels of Runx2, Alpl, and Col1α1 further validated the osteogenic differentiation potential of the pre-osteoblasts treated with CNPs. As illustrated in Fig. S7, a significant upregulation of Runx2 mRNA was observed in the CNP-treated group compared to the DEX group. Similarly, the expression levels of Alpl and Col1α1 were also notably increased, indicating enhanced osteogenic differentiation and matrix mineralization. Taking together, it could be concluded that the suppression of proliferation and differentiation activities on the pre-osteoblasts induced by DEX could be effectively alleviated by the CNPs (Fig. 3G).

3.3. CNPs alleviated DEX induced pre-osteoblasts apoptosis and ferroptosis

Previous studies revealed that glucocorticoids participated in various programmed cell death [29]. To confirm these results and figure out how could the CNPs reverse the DEX induced pre-osteoblasts suppression, the cell death pathways including necroptosis, apoptosis, autophagy and ferroptosis of the osteoblasts were evaluated. The contribution of the four pathways to the inhibition of the pre-osteoblasts activity was screened using the specific inhibitors of these pathways. The concentrations of the inhibitors were referred to the literature [30,31]. At these concentrations, all of the inhibitors showed no inhibition effects on the cells cultured without DEX (Fig. S8A). The autophagy inhibitor (3-MA) and the necroptosis inhibitor (Nec-1) could not rescue the osteoblasts from DEX induced inhibition of cell viability (Fig. 4A and B). Besides, the Nec-1 at higher concentrations (≥50 μM) showed synergistic effect with DEX in inhibiting the cell viability of the pre-osteoblasts (Fig. 4B). In contrast, the apoptosis inhibitor (Z-VAD) and the ferroptosis inhibitor (Fer-1) could effectively reverse the DEX induced inhibition of cell viability (Fig. 4C and D). Based on these results, we believed that the induction of apoptosis and ferroptosis were the major reason for DEX in the inhibition of pre-osteoblasts viability. Also, it could be speculated from this conclusion that the alleviation of pre-osteoblasts apoptosis and ferroptosis might be an important pathway for CNPs to protect the pre-osteoblasts from DEX induced inhibition of cell viability. To confirm it, the effects of CNPs on the apoptosis and ferroptosis of the pre-osteoblasts in the presence of DEX were carefully evaluated.

Fig. 4.

CNPs alleviated DEX induced pre-osteoblasts apoptosis and ferroptosis. (A–D) Cell viabilities of the pre-osteoblasts cultured with DEX and different cell death pathway inhibitors including autophagy inhibitor 3-MA, necroptosis inhibitor Nec-1, apoptosis inhibitor Z-VAD and ferroptosis inhibitor Fer-1 for 24 h. (E) Microscope images of Annexin V-FITC labelled apoptotic cells. All of the images shared a same scale bar of 100 μm. (F) Annexin V-FITC labelled apoptotic cells quantified by flow cytometry. (G) Apoptosis-related caspase-3 and cleaved caspase-3 proteins quantified by WB. (H) JC-1 probed mitochondrial membrane potential quantified by flow cytometry. (I) Intracellular Fe²⁺ concentration tested by iron assay kit. (J) Ferroptosis-related GPX4 and ACSL4 proteins quantified by WB. (K) Representative mitochondrial ultrastructure TEM images of the pre-osteoblasts cultured with DEX, RSL3, DEX + CNPs and DEX + Fer-1. The normal mitochondria were labelled by red star and the abnormal mitochondria were labelled by green triangle. The upper panel images shared a same scale bar of 5 μm and the lower panel shared a same scale bar of 1 μm. The concentration for both the DEX and the CNPs was 100 μM. Data were presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control group or DEX group, ns no significant. One-way ANOVA with Tukey's post-hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The apoptosis was firstly determined by Annexin V-FITC staining. The microscope images revealed that a large amount of apoptotic cells was detected for the pre-osteoblasts cultured with DEX. As expected, the presence of CNPs resulted in an obvious reduction of apoptotic cells (Fig. 4E). This observation was further confirmed by the quantification of the apoptotic cell using flow cytometry. As shown in the results, the early and late apoptosis ratios for the control cells were 1.77 % and 6.77 %, while these two ratios increased to 16.96 % and 18.03 % for the cells cultured with DEX. In the presence of both DEX and CNPs, these two ratios decreased to 6.61 % and 11.56 % (Fig. 4F and S8B). The TUNEL assay similarly confirmed that DEX could lead to cell death, which could be inverted by CNPs treatment (Fig. S8C). Further, the apoptosis was evaluated by the quantification of caspase-3 which was generally considered as apoptotic effector [32]. As shown in the WB assay results, DEX could significantly downregulate caspase-3 and upregulate cleaved caspase-3. The addition of CNPs resulted in the decrease of cleaved caspase-3 indicating the inhibition of pre-osteoblasts apoptosis (Fig. 4G and S9A, B). The mitochondrial membrane potential determined by JC-1 dye further supported that the CNPs could effectively inhibit the DEX induced pre-osteoblasts apoptosis (Fig. 4H and S9C).

The ferroptosis was evaluated by the quantification of intracellular Fe²⁺ concentration and ferroptosis marker proteins including GPX4 and ACSL4. To better compare the results, a ferroptosis induction reagent RSL3 and a ferroptosis inhibition reagent Fer-1 were used as positive and negative controls respectively. The Fe²⁺ quantified by iron assay kit indicated that the culture of pre-osteoblasts with both of the DEX and RSL3 could obviously augment the intracellular Fe²⁺ accumulation, while the addition of CNPs and Fer-1 resulted in an obvious reduction of intracellular Fe²⁺ concentration (Fig. 4I). The ferroptosis marker proteins quantified by WB technology indicated that the presence of DEX resulted in the downregulation of GPX4 and upregulation of ACSL4 in the pre-osteoblasts. However, the addition of CNPs could effectively inhibit the downregulation of GPX4 and upregulation of ACSL4 (Fig. 4J and S10). The immunohistochemistry staining for the tissue samples collected from the trabecular bone of the distal femur supported this conclusion in vivo (Fig. S11). Mitochondria morphology change due to the excessive iron loading induced membrane peroxidation is a typical characteristic of ferroptosis [33]. Therefore, the TEM images of the pre-osteoblasts culture with DEX, RSL3, DEX + CNPs, and DEX + Fer-1 were collected. The results revealed that the mitochondrial ultrastructure of the pre-osteoblasts cultured with DEX and RSL3 exhibited typical ferroptosis characteristics including mitochondrial shrinkage, membrane rupture, and a decrease in tubular morphology, while the cells cultured in the presence of CNPs or Fer-1 showed similar mitochondrial ultrastructure as the control cells (Fig. 4K). It could be concluded based on these observations that the induction of apoptosis and ferroptosis were the major pathways for DEX to decrease the activity of pre-osteoblasts. Meanwhile, the CNPs showed impressive capability to guard against the pre-osteoblasts from DEX induced apoptosis and ferroptosis.

3.4. CNPs inhibited the apoptosis and ferroptosis through the modulation of redox homeostasis

Superoxide anion (O₂^•−) generated from mitochondrial electron transport chain is a typical ROS that can reflect the intracellular redox homeostasis [34]. Therefore, an O₂^•− specific probe DHE was used to evaluate the intracellular ROS level. As expected, the pre-osteoblasts cultured with DEX exhibited higher O₂^•− level than the control cells, while the addition of CNPs could effectively maintain the intracellular O₂^•− level (Fig. 5A). Under physiological conditions, the excessive O₂^•− can be eliminated by endogenous antioxidant system such as GSH and SOD [35]. However, the presence of DEX disrupted the antioxidant system as both of the GSH/GSSG ratio and the SOD activity were seriously reduced. The addition of CNPs could restore, at least partially, the GSH/GSSG ratio and the SOD activity (Fig. 5B and C). This conclusion was further supported by the quantification of lipid peroxidation products, where both of the MDA and C11-BODIPY staining quantification indicated that the CNPs could effectively inhibit the generation of lipid peroxidation products (Fig. 5D, E and S12A). Besides, the expression of NADPH oxidase (NOX1), a protein that critical in the generation of O₂^•−, was significantly inhibited by CNPs (Fig. S12B and C). Taking together, CNPs could not only directly eliminate the excessive O₂^•− but also play an important role in maintaining the endogenous antioxidant system.

Fig. 5.

CNPs inhibited the apoptosis and ferroptosis of pre-osteoblasts through the modulation of redox homeostasis. (A) Intracellular O₂^•− measured by DHE staining (upper panel) and the peroxisome membrane marker protein PMP70 detected by immunofluorescence staining (lower panel). All of the images shared a same scale bar of 100 μm. (B, C) Intracellular GSH/GSSG ratio and SOD activity. (D, E) Lipid peroxidation evaluated by MDA assay kit and C11-BODIPY 581/591 probe respectively. (F) The expression of PMP70, catalase, ALOX15, Keap1 and Nrf2 analyzed by WB technology. (G) Schematic illustration of CNPs regulated the redox homeostasis of pre-osteoblasts through scavenging ROS produced by the abnormal chondriosomes and peroxisomes. Data were presented as mean ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control group or DEX group. One-way ANOVA with Tukey's post-hoc test.

It should be noticed that the excessive O₂^•− can be transferred into H₂O₂ by SOD, thus the intracellular H₂O₂ concentration (∼10⁻⁸ M) is generally much higher than O₂^•− (∼10⁻¹¹ M) under physiological condition [36]. From this perspective, the regulation of H₂O₂ concentration is also of great importance in determining the intracellular redox homeostasis. As expected, the pre-osteoblasts cultured with DEX resulted in the over accumulation of intracellular H₂O₂, while the presence of CNPs could effectively rectify the H₂O₂ imbalance (Fig. S12D). The concentration of intracellular H₂O₂ is generally regulated by endogenous catalase. On the other hand, peroxisome is a critical source of intracellular catalase [37]. Based on these principles, we believed that the DEX and/or CNPs might modulate the intracellular H₂O₂ through the change of peroxisome density. To prove it, immunofluorescence staining was carried out to probe the peroxisome membrane marker protein PMP70. The results indicated that the treatment of osteoblasts with DEX resulted in the suppression of PMP70 expression. In accordance with expectation, the addition of CNPs could restore the PMP70 expression (Fig. 5A). This observation was confirmed by WB analysis, which revealed that CNPs could effectively restore the expression of PMP70 and catalase (Fig. 5F and S13). Besides, the expression of ALOX15, a peroxisome autolysis protein, was obviously inhibited by CNPs. The Keap1/Nrf2 pathway [38], known for its crucial role in antioxidant response, was also investigated in our study. Notably, the treatment with CNPs resulted in a significant downregulation of Keap1 expression. Concurrently, there was a marked upregulation of Nrf2 expression, suggesting the activation of the Nrf2 signaling pathway (Fig. 5F). Based on these results, we believed that the major reason for CNPs to inhibit DEX induced pre-osteoblasts apoptosis and ferroptosis was that they could modulate the intracellular redox homeostasis through directly eliminating ROS and maintaining the function of the endogenous antioxidant system (Fig. 5G).

3.5. CNPs inhibited the differentiation activity of osteoclasts under the GIO condition

As mentioned above, the bone homeostasis is maintained by the dynamic balance between bone formation and bone resorption regulated mainly by osteoblasts and osteoclasts respectively. Therefore, the modulation of osteoclasts activity under GIO condition could be another critical reason for CNPs in guarding against the bone homeostasis imbalance. To prove it, pre-osteoclasts were isolated from bone marrow and their differentiation activity in the presence of DEX and/or CNPs was evaluated (Fig. 6A). Before doing it, the effect of DEX on the cell viability of the pre-osteoclast was determined. The result revealed that low dosage of DEX (<0.1 μM) was beneficial to the proliferation of pre-osteoclasts (Fig. 6B and S14). Subsequently, the differentiation of the pre-osteoclasts was induced by M-CSF and RANKL. As shown in the TRAP staining and F-action staining results, DEX could obviously promote the differentiation of pre-osteoclasts, while the osteoclastogenesis was significantly inhibited in the presence of CNPs (Fig. 6C–G).

Fig. 6.

(A) Schematic illustration of CNPs inhibited DEX induced pre-osteoclasts differentiation. (B) Cell viabilities of the pre-osteoclasts cultured with different concentrations of DEX. (C) Effects of CNPs, DEX and DEX + CNPs on the differentiation of pre-osteoclasts. The TRAP staining (upper panel) and F-action ring staining (lower panel) were performed at day 5. In the TRAP staining panel, the images in the first row shared a same scale bar of 200 μm and images in the second row shared a same scale bar of 500 μm. In the F-actin staining panel, all images shared a same scale bar of 500 μm. (D–G) The areas and numbers of osteoclasts calculated based on the TRAP staining and F-actin ring staining respectively. (H–I) Pit assay showing resorption pit by RANKL-induced osteoclasts treated with CNPs and DEX. Scale bar, 500 μm. (J–K) WB analysis for the osteoclasts differentiation-related proteins NFATc1, c-Fos, CtsK and intracellular ROS regulation protein NOX1. Data were presented as mean ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001. One-way ANOVA with Tukey's post-hoc test.

To further explore the effect of CNPs on osteoclasts function, bone resorption assay was carried out on collagen plates. Quantification analysis for the pit area revealed that DEX could significantly enhance the bone resorption activity of osteoclasts, leading to an increased pit area on the collagen plates. In contrast, the presence of CNPs markedly suppressed the bone resorption capacity of osteoclasts, resulting in a reduced pit area compared to the DEX group (Fig. 6H–I). This conclusion was further supported by the quantification of the osteoclasts differentiation-related proteins NFATc1, c-Fos and CtsK (Fig. 6J–K). Furthermore, qPCR analyses of NFATc1 and CtsK showed an inhibitory effect of CNPs on osteoclasts function (Fig. S15). Interestingly, in the presence of both CNPs and DEX, inhibition of osteoclasts formation was observed, which implied that CNPs could effectively modulate the activity of osteoclasts under GIO condition.

4. Discussion

Currently, increasing evidence suggests that oxidative stress can inhibit osteoclasts differentiation and mineralization, leading to osteocytes necrosis and increasing the expression of various cytokines, thereby inducing osteoporosis [5]. Previous antioxidants have shown promising results in reducing oxidative stress and its subsequent anti-osteoporosis effects, such as the use of natural compounds like polyphenols and flavonoids [15,39]. However, the efficacy of these compounds in vivo remains undeveloped due to variable bioavailability and potential side effects. In this study, we selected ceria nanoparticles as an antioxidant to investigate their influence on bone homeostasis disruption caused by DEX. CNPs offer unique advantages for treating GIO due to their nanoscale size, which enables them to enter bone tissue and interact with bone cells. Serving as nanoenzymes, CNPs possess the capability to mimic the functions of natural enzymes, demonstrating superior stability and catalytic efficiency compared to natural enzymes. Additionally, CNPs have high biocompatibility, ensuring long-term presence in vivo with minimal side effects. This study demonstrates that the metabolism and distribution of cerium oxide in vivo exhibit distinct biphasic characteristics: an early rapid phase (0.25–4 h) and a late slow phase (>8 h). This phenomenon reveals the pharmacokinetic properties of cerium oxide in the body, particularly its accumulation and clearance processes in the liver. In most tissues (e.g., heart, spleen, lung, kidney), cerium concentrations remained relatively stable or changed slowly over time. The femur showed minor fluctuations but maintained overall stability. These observations suggest that cerium oxide distributes to varying degrees across different tissues, and its metabolic elimination rate correlates with tissue-specific characteristics. The above results demonstrate that CNPs hold great potential in counteracting the negative impacts of DEX on bone homeostasis, potentially serving as a novel therapeutic strategy for GIO.

Cellular programmed death is crucial for various physiological processes in multicellular organisms [40]. It is well known that there is a mutual promotion between ROS and apoptosis in the cells. High ROS level can induce apoptosis through the activation of apoptosis-related signaling pathways, such as mitochondrial and death receptor pathways [41]. While, the destruction of intracellular antioxidant system in the apoptotic cells can in turn promote the accumulation of ROS [42]. Similar relationship has been established between ROS and ferroptosis [43]. On the other hand, CNPs featured with excellent ROS scavenging activity due to the transient and reversible exchange between Ce³⁺ and Ce⁴⁺ in their crystals [19]. In our study, our research further substantiated that DEX can disrupt the cellular redox homeostasis, leading to decreased GSH/GSSG ratios and SOD activity, and elevated MDA levels. CNPs could effectively eliminate the surplus ROS generated by DEX, thereby restoring redox balance. Our study provides evidence that CNPs modulate the Keap1/Nrf2 signaling axis, a master regulator of cellular antioxidant defense mechanisms [37]. This coordinated response – Keap1 downregulation coupled with Nrf2 upregulation – strongly indicates the activation of the Nrf2 signaling pathway by CNPs. While the exact molecular mechanism by which CNPs induce Keap1 downregulation remains to be fully elucidated. Further studies, such as assessing Nrf2 nuclear translocation, ARE-luciferase reporter activity, and the expression of key Nrf2 target genes (e.g., HO-1, NQO1), are crucial to fully confirm the functional activation of the pathway and pinpoint the precise mechanism of Keap1 suppression by CNPs.

Peroxisomes are the main source of catalase within the cell and play a crucial role in maintaining cellular redox homeostasis [37]. When peroxisomal dysfunction occurs, cells produce excessive amounts of ROS. Our experiments revealed that exposure to high doses of DEX led to a reduction in the quantity of peroxisomes in MC3T3-E1 cells, along with a decrease in the expression levels of PMP70 and catalase. The peroxisome density could be regulated by several pathways, within which ALOX15 mediated peroxisome autolysis could be an important reason for DEX to decrease the peroxisome density. Previous research has established a strong correlation between ALOX15 and ferroptosis [44,45]. A study reported that selective 12/15-LOX inhibitors and pan-LOX inhibitor nordihydroguaiaretic acid protect acute lymphoblastic leukemia (ALL) cells from RSL3-induced ferroptosis, indicating that LOX regulates lipid peroxidation generation and ferroptosis occurrence induced by RLS3 [46]. Correspondingly, we found that the DEX and RSL3 could significantly increase the expression of ALOX15, while the presence of CNPs or Fer-1 could obviously reduce the DEX induced high expression of ALOX15. In other words, CNPs could reduce the protein level of ALOX15, increase the number of peroxisomes, increase the expression of catalase, and ultimately restore cellular redox homeostasis.

It is widely recognized that ROS act as powerful promoters of osteoclastogenesis [47,48]. In this study, we found that DEX enhanced the formation of TRAP(+) multinucleated osteoclasts by increasing the production of ROS and promoting the development of cytoskeletal F-actin ring. These findings are consistent with previous studies [49,50]. As demonstrated in our previous study, CNPs selectively promoting apoptosis specifically in mature osteoclasts (mOCs) without affecting precursor osteoclasts (pOCs) [51]. However, the previous research reveals a more nuanced mechanism: CNPs function by excessively activating reactive oxygen species (ROS) production, thereby shortening the osteoclast formation cycle. This excessive ROS elevation leads to premature apoptosis of mature osteoclasts, resulting in their clearance. Intriguingly, our findings indicate that CNPs alone do not significantly elevate ROS levels during the early stages of osteoclast differentiation (Fig. S16). This may be related to the unique oxidative stress vulnerability of mOCs in the late stage of differentiation and the positive feedback loop between Ca²⁺ oscillation and ROS production. Moreover, our findings reveal that the application of RANKL, with or without DEX co-treatment, significantly disrupts the antioxidant system in osteoclast precursor cells during their differentiation process, as evidenced by a marked reduction in the GSH/GSSG ratio, CAT activity, and SOD activity. Importantly, CNPs effectively counteract this oxidative stress by restoring these antioxidant parameters, thereby inhibiting osteoclast formation through the modulation of ROS generation, which is critical for osteoclastogenesis.

5. Conclusion

In summary, the potential of CNPs in guarding against the DEX-induced bone homeostasis imbalance was evaluated at tissue, cellular and molecular levels (Fig. 7). Our results indicated that DEX could seriously impair the bone homeostasis on both the bone formation and bone resorption sides. On the bone formation side, DEX inhibited the proliferation and differentiation activities of the pre-osteoblasts through the induction of apoptosis and ferroptosis through the regulation of GPX4/ACSL4 signaling pathway. On the bone resorption side, DEX promoted osteoclast formation via the RANKL-dependent differentiation pathway. The loss of bone homeostasis in the presence of DEX was highly related to excessive accumulation of ROS in both pre-osteoblasts and pre-osteoclasts. The administration of CNPs could effectively modulate redox homeostasis in the cells through their excellent ROS scavenging activity and ability to maintain the endogenous antioxidant system. Consequently, the apoptosis and ferroptosis of the pre-osteoblasts were obviously ameliorated, and the differentiation of the pre-osteoclasts was significantly intercepted. Our results suggested that CNPs were potential preventative agents for GIO.

Fig. 7.

Schematic illustration of CNPs guarded against DEX induced bone homeostasis imbalance through the promotion of osteogenesis and inhibition of osteoclastogenesis.

CRediT authorship contribution statement

Anwei Zhang: Writing – original draft, Project administration, Investigation, Funding acquisition, Data curation. Zhiwei Liu: Validation, Software, Project administration, Investigation, Funding acquisition, Data curation. Ling Zou: Project administration, Methodology, Investigation. Lu Yang: Project administration, Investigation. Yonghui Wu: Investigation, Data curation. Xinxing Wang: Investigation, Data curation. Daigui Cao: Supervision, Funding acquisition. Junli Liu: Supervision, Funding acquisition, Conceptualization. Xiaochao Yang: Writing – review & editing, Supervision, Conceptualization. Shengli Zhang: Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.