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Ferrostatin-1 inhibits osteoclast differentiation and prevents osteoporosis by suppressing lipid peroxidation

Journal of Orthopaedic Surgery and Research volume 20, Article number: 117 (2025) Cite this article

Abstract

Background

Osteoporosis (OP) is a systemic disease characterized by low bone mass. New progress has been made in the study of OP, such as lipid peroxidation. However, the role of lipid peroxides in osteoclast differentiation is still unclear.

Methods

Bone marrow macrophages (BMMs) were extracted from C57BL/6J mice and induced to differentiate into osteoclasts, which were observed via TRAP staining, Phalloidin staining and bone pit assays. Related substances of lipid peroxidation were detected during osteoclastogenesis. The levels of osteoclastogenesis and lipid peroxides were measured by qRT-PCR, Western Blot and immunofluorescence. Activation of the p38/JNK/MAPK pathway was detected by Western Blot. The capacity for osteogenesis and angiogenesis of cells after treatment with supernatant of BMMs was evaluated. Furthermore, Ferrostatin-1 (Fer-1), from which femur and serum samples were comprehensively evaluated, was used in OVX mice.

Results

During osteoclastogenesis, the levels of ROS, MDA, ACSL4 and LPCAT3 increased with increasing duration of RANKL stimulation, while there were no significant changes in the levels of GSH or GPX4. Fer-1 inhibited osteoclast differentiation and decreased the level of lipid peroxides. In addition, Fer-1 inhibited osteoclast-related markers by inhibiting the p38/JNK/MAPK pathway. Furthermore, the supernatant of BMMs after Fer-1 treatment promoted osteogenesis and angiogenesis. Finally, Fer-1 successfully alleviated OP in OVX mice by reducing the level of lipid peroxidation in vivo.

Conclusion

Fer-1 suppresses osteoclast differentiation by reducing lipid peroxidation levels regulated by ACSL4, which is mediated through the p38/JNK/MAPK signaling pathway. Additionally, Fer-1 enhances the coupling between osteogenesis and angiogenesis and has an anti-OP effect in vivo.

Highlights

Lipid peroxide levels increase with osteoclast differentiation, with no significant difference in GSH or GPX4.

Fer-1 suppresses osteoclast differentiation in vitro by reducing the lipid peroxidation levels regulated by ACSL4. This effect appears to be mediated through the inhibition of the p38/JNK/MAPK signaling pathway.

Fer-1 enhances the coupling between osteogenesis and angiogenesis in vitro.

Fer-1 has an anti-osteoporosis effect by reducing the level of lipid peroxidation in vivo.

Introduction

Osteoporosis (OP) is a systemic disease characterized by low bone mass and increased bone fragility, which results in a heavy burden on society [1, 2]. According to epidemiological data from 2018, more than 200 million people worldwide suffer from OP, with regional variation [3,4,5]. Osteoclasts are giant multinucleated cells derived from mononuclear/ macrophage precursors. The functions of osteoclasts and osteoblasts in healthy people are in dynamic balance [6]. In addition to being the only bone-resorbing cells, osteoclasts can also secrete cytokines to regulate the functions of osteoblasts and endothelial cells [7]. Therefore, osteoclasts are among the core targets for the treatment of OP, which is a type of bone remodeling disease.

In recent years, new progress has been made in the study of OP [8, 9]. The accumulation of lipid peroxides has been shown to be involved in the occurrence and development of a variety of diseases [10,11,12]. Studies have shown that inhibiting the lipid peroxidation of osteoblasts and bone marrow mesenchymal stem cells (BMSCs) promotes their osteogenic capacity [13, 14]. However, there are few and conflicting reports on the lipid peroxidation of osteoclasts. In a study by Jin, artesunate promoted osteoclast lipid peroxidation and inhibited OP in mice [15]. However, a moderate number of lipid peroxides can also act as signaling molecules involved in cellular activities. Interestingly, Aconine was found to inhibit osteoclast differentiation as an antioxidant, which also decreased lipid peroxides [16]. Thus, the role of lipid peroxidation in osteoclast differentiation remains unclear. Moreover, several antiosteoclast drugs may also decrease the secretion of osteogenic and angiogenic factors during osteoclast differentiation. Therefore, inhibiting osteoclast differentiation while preserving their osteogenic and angiogenic functions is a key issue in the treatment of OP.

This study demonstrated the effects of Ferrostatin-1 (Fer-1), an anti-lipid peroxidation drug, on the inhibition of osteoclast differentiation in vitro and on the promotion of the secretion of osteogenic and angiogenic factors by osteoclast precursor cells, which could prevent OP in mice to a certain extent, providing a new theoretical basis and evidence for the treatment of OP by targeting lipid peroxidation.

Materials and methods

Isolation of mouse bone marrow macrophages

Bone Marrow Macrophages (BMMs) were extracted according to the methods of Song [17]. Briefly, C57BL/6J mice were sacrificed after decapitation and immersed in 75% ethanol for 10 min. Bilateral lower limbs were carefully separated, from which soft tissue and adipose tissue were removed. Both ends of the tibia and femur were cut off, and the bone marrow cavity was repeatedly rinsed with α-MEM basal medium (Cytiva, USA, SH30265.01). Cells were collected and treated with ACK lysis buffer (Beyotime, China, C3702) for 10 min. The cells were subsequently resuspended in α-MEM supplemented with 10% fetal bovine serum (ExCell Bio, China, FSD500), 1% penicillin and 1% streptomycin (Beyotime, China, C0222) and incubated in a cell incubator at 37℃ and 5% CO2 overnight.

Differentiation of osteoclasts

Unadhered cells were collected and reseeded. Macrophage Colony-stimulating Factor (M-CSF) (Amizona, China, AM10003-010) was added to the cells at a final concentration of 40 ng/ml. After 72 h, Receptor Activator of Nuclear Factor-κB Ligand (RANKL) (Amizona, China, AM10004-010) was subsequently added at a final concentration of 50 ng/ml. In accordance with the needs of subsequent experiments, Fer-1 (MCE, China, HY-100579) and Erastin (MCE, China, HY-15763) were added to the medium 48 h before the induction of RANKL after the appropriate concentration was determined. The complete medium was changed every 48 h, and typical multinucleated osteoclasts were observed after 5 days.

Identification of bone marrow macrophages via flow cytometry

The cells were digested and resuspended in phosphate-buffered saline (PBS). CD11b (Biolegend, USA, 982614) and F4/80 antibodies (Biolegend, USA, 123107) were subsequently added to the cells after blocking with FC. After incubation in the dark for 30 min, the cells were washed once and analyzed.

Cell viability assay

The cells were seeded in 96-well plates at a density of approximately 4000 cells per well. After adhesion, the cells in the different groups were treated with 10 μl of Cell Count Kit-8 (CCK-8) reagent (Beyotime, China, C0037) at 24 h and 48 h and then placed in an incubator for 30 min in the dark. The absorbance of each well was measured at 450 nm with a microplate reader. The cell viability was subsequently calculated according to the instructions.

Detection of intracellular GSH and MDA

The cells were disrupted with an ultrasonic cell crusher. After centrifugation at 3500 ×g for 10 min at 4℃, the mixture of dd-H2O, Glutathione (GSH) standard solution (Solarbio, China, BC1170), Malondialdehyde (MDA) standard solution (Solarbio, China, BC0025) and the supernatant to be tested with a chromogenic agent were boiled and centrifuged. The absorbance was measured at 412 nm for GSH detection and at 450 nm, 520 nm and 600 nm for MDA detection via a microplate reader. The concentration of each marker was subsequently calculated according to the instructions.

Detection of intracellular ROS and lipid peroxide

The cells were seeded in 24-well plates at a density of approximately 105 cells per well. After treatment, the medium was replaced with H2DCFDA (Biosharp, China, BL714A) for reactive oxygen species (ROS) or C11 BODIPY 581/591 (GLPBIO, USA, GC40165) for lipid peroxide working solution. The cells were then incubated at 37℃ for 2 h in the dark and washed twice with serum-free medium. The cells were then observed under a fluorescence microscope with a FITC filter.

qRT‒PCR

Total RNA was extracted from BMMs with TRIzol (Beyotime, China, R0016) according to the protocol. After the concentration was determined, 2 μg of total RNA was used to synthesize cDNA via a cDNA synthesis kit. SYBR Green (ES Science, China, QP002) was used to perform quantitative real-time PCR (qRT‒PCR). GAPDH served as an internal reference to normalize the expression level via the 2∆∆CT algorithm. The sequences of the primers used are listed in Table 1.

Table 1 Primers used for qRT-PCR
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Western blot

The cells were lysed in RIPA buffer (Beyotime, China, P0013B) supplemented with 1% PMSF (Beyotime, China, ST506) for 10 min. Protein concentrations were determined via a bicinchoninic acid assay (Beyotime, China, P0012S). The proteins were subsequently separated via 10% or 12.5% SDS‒PAGE (Epizyme, China, PG112, PG113) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (0.2 μm; Bio-Rad, USA). After blocking with 5% skim milk (Beyotime, China, P0216-300 g) for 120 min, the membranes were incubated overnight at 4 °C with the following primary antibodies: GPX4 (Affinity, USA, DF6701), ACSL4 (Affinity, USA, DF12141), LPCAT3 (Proteintech, China, 67882-1-Ig), P38 (Proteintech, China, 14064-1-AP), p-P38 (Proteintech, China, 28796-1-AP), JNK (Proteintech, China, 51151-1-AP), p-JNK (Proteintech, China, 80024-1-RR), GAPDH (Proteintech, China, 10494-1-AP) and β-actin (Wanleibio, China, WL01372). The membranes were subsequently incubated with secondary antibodies (Wanleibio, China, WLA023a) for 1 h. The blots were visualized via enhanced chemiluminescence reagents (Beyotime, China, P0018S). The density of each band was quantified via ImageJ version 1.51.

TRAP staining

Tartrate Resistant Acid Phosphatase (TRAP) staining was performed according to the instructions of the TRAP Kit (Amizona, China, AMK1002-005). Briefly, the cells were washed with PBS twice. For the paraffin sections, dewaxing and dehydration were performed. The cells or sections were fixed with fixation solution for 5 min. The working solutions were prepared according to the instructions and added to the cells. Images were obtained after the cells or sections were incubated at 37 °C for 1 h.

Immunofluorescence and phalloidin staining

The cells or sections were fixed with 4% paraformaldehyde (Beyotime, China, P0099-100 ml) at room temperature for 15 min. After being washed three times, the cells or sections were infiltrated with 0.1% Triton X-100 (Biosharp, China, BS084) for 30 min and blocked with goat serum (Boster, China, AR0009) for 1 h. The cells or sections were then incubated with different antibodies at 4℃ overnight. For Phalloidin staining, BMMs were treated with F-Actin Tracker (Beyotime, China, C2203S) for 1 h. The next day, the BMMs or slices were incubated with a Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime, China, BL033A) for 2 h in the dark and then with DAPI (Beyotime, China, C1005) for 10 min. Images were obtained under a fluorescence microscope.

Bone pit assay

BMMs were seeded at a concentration of 105/well on bone slices (Amizona, China, AMB1001–020), which were placed into 24-well plates in advance. Osteoclastogenesis was induced via the method described above, and the complete medium was changed every 48 h. After treatment with RANKL for 14 days, the bone slices were observed by scanning electron microscopy.

Treatment of BMSCs and ECs

A mouse BMSC (Princella, China, CP-M131) cell line and a mouse Umbilical Vein Endothelial Cells (UVECs, Princella, China, CP-M232) cell line were used for the evaluation of osteogenesis and angiogenesis. Briefly, BMMs were treated with Fer-1 for 2 days and then induced to differentiate into osteoclasts. After 5 days, the supernatant was collected, centrifuged, and used for BMSC and ECs culture.

ALP and alizarin red S staining

BMSCs were fixed with 4% paraformaldehyde at room temperature for 15 min. Then, the cells were treated with the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China, C3206) or Alizarin Red S (Beyotime, China, C0140) according to the protocol.

Tube formation assay

ECs were seeded on Matrigel-coated 24-well plates and cultured for 6 h. Then, the formation tubes were observed under an optical microscope. The number of meshes was used as an indicator for evaluating angiogenesis.

Establishment of the ovariectomy model

The study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (Ref: YJSDW2023-172). Six-week-old female C57BL/6J mice were anesthetized with afudine through intraperitoneal injection. The incision was made 0.5 cm beside the dorsal bilateral spine. Cauliflower-shaped ovaries surrounded by adipose tissue were carefully recognized. The uterine horn was ligated, and the ovaries were removed. After the operation, 2 × 104 units of penicillin were injected intraperitoneally into each mouse for 3 days. The mice were divided into three groups: the Sham group (mice that underwent only skin and peritoneal incisions without ovary removal), the OVX group and the OVX + Fer-1 group (Fer-1 was administered at a concentration of 1 mg/kg via intraperitoneal injection every other day after ovariectomy), with 5 mice in each group. After 8 weeks, the model was considered complete. Femur and serum samples were isolated for subsequent experiments.

Micro-CT

The femur samples from each mouse were fixed in 4% paraformaldehyde and subsequently scanned via a micro-CT system (SCANCO MEDICAL, Switzerland). The scan parameters were set as follows: 70 kVp, 114 μA, and 10 μm. The classification of each pixel was delineated with a threshold of 1,000 for bone and 184 for nonbone. The scanning range was determined from the middle section of the femoral shaft to the distal metaphysis of the femur, including trabecular bone beams measuring 1 mm long. After scanning, parameters such as the bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated via SCANCO MEDICAL software.

Hematoxylin and eosin (H&E) staining

The sections were dewaxed and stained with hematoxylin and eosin, and the reaction was terminated with ddH2O. After dehydration, the sections were sealed with neutral gum.

ELISA analysis

The cell supernatant or serum was collected and centrifuged for 20 min at 1000×g at 2–8℃ and then added to a 96-well plate covered by ELISA antibodies against TGF-β (Elabscience, USA, E-EL-M1191), SDF-1 (Elabscience, USA, E-EL-M3046), S1P (Meimian, China, MM-44778M2), PDGF-BB (Elabscience, USA, E-EL-M0632), Trap-5b (Elabscience, USA, E-EL-M1116), RANKL (Elabscience, USA, E-EL-M0644), CTX-1 (Elabscience, USA, E-EL-M3023) and PINP (Elabscience, USA, E-EL-M0233). 100 μl was added to each well. After incubation for 90 min at 37℃, Biotinylated Detection Ab was added to each well. After incubation for 60 min at 37 °C, 100 μl of HRP Conjugate was added, and the cells were incubated for 30 min at 37℃ away from light. 90 μl of Substrate Reagent and 50 μl of stop solution were added. The optical density (OD value) of each well was immediately determined via a microplate reader at 450 nm.

Statistical analysis

The quantification was conducted via at least three separate experiments, and the results are reported as the means ± standard deviation (SD). Statistical analyses were performed via GraphPad Prism version 9.5. Differences in numerical data between two groups were determined by Student’s t test, whereas differences among more than two groups were determined by one-way ANOVA followed by a Bonferroni post hoc correction. P < 0.05 was considered statistically significant.

Results

Induction of osteoclastogenesis of primary bone marrow macrophages in vitro

After treatment with M-CSF at a concentration of 40 ng/ml for 3 days, 95.3% of the cells were positive for CD11b, and 95.0% were positive for F4/80; these findings are consistent with the characteristics of BMMs (Fig. 1A). Subsequently, osteoclast induction was successfully induced by 50 ng/ml RANKL. The extracted BMMs were short shaped under light microscope after they were firmly adherent. While after 5 days of stimulation with RANKL, giant multinucleated cells were observed (Fig. 1B). TRAP staining revealed that BMMs without RANKL stimulation were TRAP-, whereas multinucleated cells were TRAP+ (Fig. 1C). By Phalloidin staining, the cytoskeleton was labeled in red, accompanied by blue nuclei clustered inside (Fig. 1D).

Fig. 1
figure 1

Induction of osteoclastogenesis of primary bone marrow macrophages in vitro. (A) Visualization of BMMs surface markers: CD11b and F4/80. (B) Observation during osteoclast differentiation under optical microscope, Scale bar = 200 μm. Yellow arrows represent BMMs, and red arrows represent osteoclasts. (C) TRAP staining during osteoclast differentiation, Scale bar = 200 μm. (D) Phalloidin staining during osteoclast differentiation, Scale bar = 200 μm

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Changes in markers of lipid peroxidation during osteoclast differentiation

Several key markers of lipid peroxidation were sequentially detected over time. The MDA levels increased with increasing duration of RANKL stimulation, peaking at the point of maximal osteoclast fusion (Fig. 2A), whereas there were no significant changes in the intracellular or supernatant GSH levels, although there was a decreasing trend over time (Fig. 2B). Moreover, fluorescence microscopy revealed that the levels of ROS and lipid peroxides also increased with increasing duration of RANKL stimulation (Fig. 2C-D). The results of PCR revealed that the levels of ACSL4 and LPCAT3, two key genes involved in lipid peroxidation, significantly increased after 3 days of RANKL treatment, whereas GPX4 levels remained stable throughout the entire process (Fig. 2E). The Western blot results provided similar evidence at the protein level, indicating that the levels of ACSL4 and LPCAT3 gradually increased, whereas GPX4 levels showed no significant differences across the various time points (Fig. 2F-G).

Fig. 2
figure 2

Changes in markers of lipid peroxidation during osteoclast differentiation. (A) Concentration of intracellular MDA during osteoclast differentiation. (B) Concentration of intracellular GSH during osteoclast differentiation. (C) The level of ROS during osteoclast differentiation evaluated by immunofluorescence staining, followed by mean density of ROS. Scale bar = 100 μm. (D) The level of lipid peroxide during osteoclast differentiation evaluated by immunofluorescence staining, followed by mean density of C11. Scale bar = 100 μm. (E) Relative mRNA expression of GPX4, ACSL4, LPCAT3 during osteoclast differentiation. (F) Protein expression of GPX4, ACSL4, LPCAT3 during osteoclast differentiation through Western Blot. (G) The band density ratio of GPX4, ACSL4, LPCAT3 to GAPDH in Western Blot quantified by densitometry. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Fer-1 inhibits osteoclast differentiation in vitro

Given the gradual increase in ROS, lipid peroxides, and ACSL4 levels during osteoclast differentiation, Fer-1 was aimed to inhibit osteoclastogenesis, which suppresses lipid peroxide levels. Concurrently, the lipid peroxidation agonist Erastin was used to reverse this process. Cytotoxicity assays indicated that treatment with Fer-1 at a concentration of 2 μM and Erastin at a concentration of 7.5 μM was safe for BMMs, and these concentrations were used in subsequent experiments (Fig. 3A-B). The results demonstrated that osteoclast differentiation was significantly inhibited in the RANKL + Fer-1 group, whereas Erastin partially reversed this effect. Similar results were observed through TRAP staining and Phalloidin staining (Fig. 3C-F). After 14 days of culture on bone slices, the bone resorption area in the RANKL + Fer-1 group was significantly reduced compared with that in the RANKL group, with slight recovery in the RANKL + Fer-1 + Erastin group (Fig. 3G-H).

Fig. 3
figure 3

Fer-1 inhibits osteoclast differentiation in vitro. (A) Viability of BMMs after treatment of different concentration of Fer-1 for 12 h and 24 h. (B) Viability of BMMs after treatment of different concentration of Erastin for 12 h and 24 h. (C) TRAP staining of each group. Scale bar = 200 μm. (D) Ratio of TRAP+ area. (E) Phalloidin staining of each group. Scale bar = 200 μm. (F) Ratio of F-actin Ring area. (G) Bone pit area of each group under scanning electron microscope. Scale bar = 400 μm. (H) Proportion of bone pit area. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Fer-1 reduces oxidative and lipid peroxide levels during osteoclast differentiation

The fluorescence microscopy results revealed that the ROS levels were significantly lower in the RANKL + Fer-1 group than in the RANKL group (Fig. 4A). C11 and MDA assays revealed that lipid peroxide levels were significantly lower in the RANKL + Fer-1 group than in the RANKL group (Fig. 4B-C). The results of PCR revealed that ACSL4 and LPCAT3 expression levels were significantly lower in the RANKL + Fer-1 group than in the RANKL group (Fig. 4D-E). These findings were further corroborated by Western Blot (Fig. 4F-H) and immunofluorescence (Fig. 4I-L), which were consistent with the results of PCR.

Fig. 4
figure 4

Fer-1 reduces oxidation and lipid peroxidation during osteoclast differentiation. (A) The level of ROS of each group evaluated by immunofluorescence staining. Scale bar = 100 μm. (B) The level of lipid peroxide of each group evaluated by immunofluorescence staining. Scale bar = 100 μm. (C) Concentration of intracellular MDA of each group. (D-E) Relative mRNA expression of ACSL4 and LPCAT3 of each group. (F) Protein expression of ACSL4 and LPCAT3 of each group through Western Blot. (G-H) The band density ratio of ACSL4 and LPCAT3 to β-actin in Western Blot quantified by densitometry. (I-J) Protein level of ACSL4 and LPCAT3 in cells of each group evaluated by immunofluorescence staining. Scale bar = 100 μm. (K-L) Relative fluorescent density of ACSL4 and LPCAT3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Fer-1 reduces the expression of osteoclast-related markers by inhibiting p38/JNK/MAPK pathway

c-Fos and NFATc1 are key genes that initiate osteoclast differentiation and are regulated by the p38/JNK/MAPK pathway. Like DC-STAMP, MMP-9, Trap, and ATP6V0D2, these genes serve as markers for evaluating the extent of osteoclast differentiation and bone resorption capacity. In this study, the mRNA expression levels of these genes were measured, and the results demonstrated that these genes were significantly lower in cells in the RANKL + Fer-1 group than in those in the RANKL group, with the exception of DC-STAMP (Fig. 5A-F). Furthermore, c-Fos protein levels were evaluated by immunofluorescence, and the results were consistent with the mRNA levels (Fig. 5G). Additionally, the protein phosphorylation levels of the p38/JNK/MAPK pathway were examined at 15 and 30 min posttreatment. The phosphorylation levels of p38 and JNK were markedly increased after RANKL stimulation but were significantly decreased in the RANKL + Fer-1 group (Fig. 5H-J). Collectively, these results suggest that Fer-1 may inhibit RANKL-induced osteoclast differentiation via suppression of the p38/JNK/MAPK pathway.

Fig. 5
figure 5

Fer-1 reduces the expression of osteoclast-related markers by inhibiting p38/JNK/MAPK pathway. (A-F) Relative mRNA expression of c-Fos, NFATc1, DC-STAMP, MMP-9, Trap and ATP6V0D2 of each group. (G) Protein level of c-Fos in cells of each group evaluated by immunofluorescence staining. Scale bar = 100 μm. (H) Protein expression of p-JNK, JNK, p-p38 and p38 of each group through Western Blot. (I-J) The band density ratio of p-JNK and p-p38 to JNK and p38 respectively in Western Blot quantified by densitometry. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Supernatant of BMM after Fer-1 treatment promotes osteogenesis and angiogenesis

During osteoclast differentiation, osteogenic and angiogenic factors are also secreted into the supernatant, and their levels are assessed via ELISA. The results indicated that the levels of TGF-β, SDF-1, S1P and PDGF-BB were significantly greater in the RANKL + Fer-1 group than in the RANKL group (Fig. 6A-D). The supernatants from each group of BMMs were co-cultured with BMSCs. The results of ALP staining and Alizarin Red staining demonstrated that the BMSCs cultured with the RANKL + Fer-1 group supernatants exhibited increased osteogenic potential (Fig. 6E-H). The supernatants from each group of BMMs were co-cultured with ECs, subsequently. The results of the angiogenesis assays indicated that ECs cultured with RANKL + Fer-1 group supernatants displayed greater angiogenic capacity (Fig. 6I-J).

Fig. 6
figure 6

Supernatant of osteoclast after Fer-1 treatment promotes osteogenesis and angiogenesis. (A-D) Concentration of TGF-β, SDF-1, S1P and PDGF-BB in the supernatant of osteoclast of each group. (E) ALP staining of BMSCs of each group after co-culture with supernatant of osteoclast for 14 days. Scale bar = 200 μm. (F) Positive area of ALP staining. (G) Alizarin Red staining of BMSCs of each group after co-culture with supernatant of osteoclast for 14 days. Scale bar = 200 μm. (H) Positive area of Alizarin Red staining. (I) Tube formation assay of ECs of each group after co-culture with supernatant of osteoclast for 6 h. (J) Numbers of Meshes of Tube formation assay. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Fer-1 inhibits osteoporosis of OVX mice

Ovariectomized C57BL/6J mice were used to validate the inhibitory effect of Fer-1 on osteoclast differentiation in vivo. After 8 weeks of treatment, the femoral micro-CT results were analyzed as shown in Fig. 7A. Compared with the OVX group, the Fer-1-treated group presented greater bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) and lower trabecular separation (Tb.Sp) (Fig. 7B-F). Histological examination via H&E staining revealed that, compared with the sham group, the OVX group presented a significantly lower trabecular number and sparser distribution, whereas Fer-1 treatment reversed these effects (Fig. 7G). Subsequently, ELISA analysis of blood collected from the ocular veins revealed that the levels of TRAP, RANKL and CTX-1 were significantly lower in the OVX + Fer-1 group than in the OVX group (Fig. 7H). TRAP staining revealed significantly lower TRAP levels in the metaphysis in the OVX + Fer-1 group than in the OVX group (Fig. 7I-J).

Fig. 7
figure 7

Fer-1 inhibits osteoporosis of OVX mice. (A) Reconstruction of bone trabecular of distal femurs by Micro-CT of each group. Scale bar = 200 μm. (B-F) Quantitative micro-CT analysis of bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). (G) HE staining of tissue sections, followed by the quantitative analysis of Tb.N and Tb.Sp of HE staining. Scale bar = 200 μm. (H) Relative concentration of TRAP, RANKL, CTX-1 and PINP in serum of each group. (I) TRAP staining of tissue sections. Scale bar = 200 μm. (J) Quantitative analysis of osteoclast number per trabecular bone surface (N. OC/BS). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Fer-1 inhibits lipid peroxidation level in OVX mice

To investigate whether Fer-1 reduces lipid peroxidation levels in vivo, serum MDA levels were measured. The results revealed that MDA levels were significantly greater in the OVX group than in the Sham group, whereas MDA levels were significantly lower in the OVX + Fer-1 group than in the OVX group (Fig. 8A). After the treatment of each group, protein was extracted from the bone tissue, and the mRNAs were extracted from the BMMs. The Western Blot results revealed that the protein levels of ACSL4 and LPCAT3 in the bone tissue were significantly elevated in the OVX group but markedly reduced in the OVX + Fer-1 group (Fig. 8B-D). PCR analysis of extracted BMMs revealed that the mRNA expression levels of ACSL4 and LPCAT3 were significantly lower in the OVX + Fer-1 group than in the OVX group (Fig. 8E-F). To explore the effect of Fer-1 on ACSL4 expression, double immunofluorescence staining for ACSL4 and CTSK (an osteoclast marker) was performed. The results revealed significant co-localization of ACSL4 and CTSK in the metaphysis of the OVX group, whereas the expression of both proteins was significantly reduced in the OVX + Fer-1 group (Fig. 8G).

Fig. 8
figure 8

Fer-1 inhibits lipid peroxidation in OVX mice. (A) Concentration of MDA in serum of each group. (B) Protein expression of ACSL4 and LPCAT3 in bone tissue of each group through Western Blot. (C-D) The band density ratio of ACSL4 and LPCAT3 to β-actin in Western Blot quantified by densitometry. (E-F) Relative mRNA expression of ACSL4 and LPCAT3 in BMMs of each group. (G) Immunofluorescence staining of bone tissue sections of each group. Scale bar = 100 μm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

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Discussion

Osteoporosis commonly affects seniors and postmenopausal women, leading to decreased bone mass and, in severe cases, often resulting in fractures [18]. Excessive osteoclast activation is one of the key pathological changes in osteoporosis; thus, significant efforts have been made to identify preventive or therapeutic strategies [19]. RANKL is a critical factor for the differentiation of BMMs into osteoclasts. However, drugs targeting RANKL, such as bisphosphonates and alendronate, have strong side effects and often cause rebound effects after discontinuation [20]. Additionally, some anti-osteoporosis medications, while inhibiting osteoclast activity, may also negatively impact the secretion of osteogenic or angiogenic factors [21]. Therefore, further investigations are needed to explore the mechanisms of osteoclast differentiation and to develop strategies that not only inhibit osteoclast formation but also promote or minimally affect interactions with osteogenesis and angiogenesis.

The lipid peroxidation has been shown to be activated in BMSCs and osteoblasts and to contribute to the progression of osteoporosis. Existing studies have utilized lipid peroxidation inhibitors for osteocytes, BMSCs, and osteoblasts in an attempt to promote osteogenic differentiation and enhance osteogenic capacity by downregulating lipid peroxidation [22, 23]. However, the relationship between lipid peroxidation and osteoclasts in osteoporosis has not been fully elucidated. In this study, osteoclast differentiation was induced via the use of 40 ng/ml M-CSF and 50 ng/ml RANKL, following previously described methods. In this study, we focused on changes in oxidation and lipid peroxidation. The levels of ROS and lipid peroxidation gradually increased over time, peaking in mature osteoclasts. Further analysis revealed that ACSL4 and LPCAT3, key regulators of lipid peroxidation, were significantly elevated [24]. Notably, the levels of GSH, a critical antioxidant, remained stable throughout the process of osteoclast differentiation, whereas the levels of GPX4 did not significantly change at the mRNA or protein level [25]. These findings suggest that the expression of these antioxidants was neither suppressed nor upregulated in response to the high oxidative levels observed during osteoclast differentiation [26].

Therefore, we focused on the relationship between lipid peroxidation and osteoclast differentiation and used Fer-1, which reduces lipid peroxide levels, to inhibit osteoclast differentiation [27]. The results showed that co-treatment with Fer-1 significantly suppressed the formation and fusion of TRAP + osteoclast precursor cells and markedly reduced the bone resorption capacity of osteoclasts. Furthermore, mice treated with Fer-1 for 8 weeks resulted in a significant increase in bone density and trabecular number compared with those of the OVX group, with higer BMD, BV/TV, Tb.Th and Tb.N, while with lower Tb.Sp. RANKL, Trap, and CTX-1 are markers of bone resorption in serum, and their expression is elevated in osteoporosis models [28]. PINP, a marker reflecting bone turnover, is elevated in the serum of women with OP and decreases after effective treatment for bone resorption. Our study demonstrated that Fer-1 significantly inhibited the secretion of these factors. These findings indicate that Fer-1 has a substantial inhibitory effect on osteoclast differentiation and bone destruction both in vitro and in vivo.

Lipid peroxidation refers to the oxidative deterioration of polyunsaturated fatty acids (PUFAs) and lipids, which can destroy the membrane of mitochondria and cells, leading to cell dysfunction, which is upregulated by ACSL4 and LPCAT3 [29,30,31]. We found that Fer-1 significantly reduced lipid peroxidation levels during osteoclast differentiation and decreased the expression of ACSL4 and LPCAT3 at both the mRNA and protein levels, which is consistent with its effects on neurons and chondrocytes [11, 32]. Moreover, this effect was reversed by Erastin, an agonist of lipid peroxidation that inhibits the production of antioxidants [33]. In in vivo experiments, we observed that serum lipid peroxide levels were lower in the Fer-1-treated mice than in control mice. Additionally, ACSL4 and LPCAT3 expression levels were reduced in the bone tissue and bone marrow cells of the Fer-1 group, and femoral sections from the Fer-1 group presented significantly lower levels of ACSL4 in osteoclasts. These findings suggest that Fer-1 inhibits osteoclast differentiation and mitigates osteoporosis by decreasing lipid peroxidation levels. The p38/JNK/MAPK signaling pathway plays crucial roles in the cascade amplification process during the RANKL-mediated activation of c-Fos and NFATc1 [34]. Inhibition of the MAPK signaling pathway prevents the fusion of osteoclast precursors into multinucleated osteoclasts. Our study demonstrated that Fer-1 significantly suppressed the phosphorylation of p38/JNK/MAPK pathway proteins in BMMs, which is consistent with findings reported by Sun M in hepatocytes [35].

The stability of bone metabolism relies on the balance between osteoblasts, osteoclasts, and ECs [36]. For example, OBs secrete RANKL, a key factor that promotes osteoclast differentiation by binding to RANK on the membrane of preosteoclasts. Concurrently, OBs also secrete osteoprotegerin (OPG), which competes with RANKL for binding to RANK, thereby inhibiting osteoclast differentiation [37]. Special ECs characterized by high expression of CD31 and Emcn form H-type vessels, which recruit a substantial number of osteoprogenitor cells [38]. Additionally, pOCs secrete osteogenic factors such as TGF-β, SDF-1, and S1P during their differentiation into osteoclasts [39, 40], as well as angiogenic factors such as PDGFBB [41]. Our study demonstrated that the use of Fer-1 to inhibit lipid peroxidation not only suppresses osteoclast differentiation but also promotes the secretion of osteogenic and angiogenic factors. Furthermore, the supernatant from Fer-1-treated osteoclasts enhances osteogenic and angiogenic capabilities. Thus, we propose that Fer-1, while inhibiting osteoclast differentiation, also augments the coupling between osteoclasts, osteoblasts, and ECs, thereby promoting bone formation.

This study has several limitations. While we discovered that Fer-1 inhibits osteoclast differentiation by reducing lipid peroxidation through the ACSL4/LPCAT3 axis and suppressing the p38/JNK/MAPK pathway, the relationship between ACSL4 and the p38/MAPK pathway has not been fully proven. In addition, specific molecular targets of Fer-1 and the critical targets through which lipid peroxidation affects osteoclast differentiation have not been thoroughly investigated. Additionally, osteoclast differentiation is a two-step process: the differentiation of BMMs into TRAP+ preosteoclasts and the fusion of TRAP + preosteoclasts into mature osteoclasts. Although our study explored the role of lipid peroxidation throughout this process, it did not pinpoint its effect on each intermediate step. Moreover, although our study demonstrated that Fer-1 significantly inhibits bone resorption in vivo, its efficacy in OVX mice is less pronounced than that of bisphosphonates, which have been reported to completely reverse bone loss and even increase bone density [42]. This may be due to the use of commonly used concentrations of Fer-1 for intraperitoneal injection due to concerns about toxicity without exploring dose-dependent effects or ensuring optimal absorption efficiency [43]. Therefore, future research needs to focus on determining the appropriate dosage and administration route for Fer-1 and developing effective drug delivery systems to enhance its therapeutic efficacy against osteoporosis.

Conclusion

In summary, we found that Fer-1 suppresses osteoclast differentiation by reducing lipid peroxidation levels regulated by ACSL4. This effect appears to be mediated through the inhibition of the p38/JNK/MAPK signaling pathway. Additionally, while inhibiting osteoclast differentiation, Fer-1 enhances the coupling between osteogenesis and angiogenesis and has an anti-osteoporosis effect in vivo. These findings suggest that inhibitors of lipid peroxidation, such as Fer-1, have potential as therapies for osteoporosis by targeting osteoclast differentiation.

Data availability

All data generated or analysed during this study are included in this published article.

Abbreviations

ACSL:

Acyl - Coenzyme A Synthetase Long - chain

BMD:

Bone Mineral Density

BMM:

Bone Marrow Macrophage

BMSC:

Bone Marrow Derived Mesenchymal stem cell

BP:

Biological Process

BV/TV:

Bone Nolume/Total Nolume ratio

CCK-8:

Cell Count Kit-8

c-Fos:

proto-oncogene protein c-Fos

Era:

Erastin

Fer-1:

Ferrostatin-1

GPX4:

Glutathione Peroxidase 4

GSH:

Glutathione

H&E:

Hematoxylin and eosin

LPCAT3:

Lysophosphatidylcholine Acyltransferase 3

MAPK:

Mitogen-Activated Protein Kinase

M-CSF:

Monocyte Colony- Stimulating Factor

MDA:

Malondialdehyde

NFATc1:

Nuclear Factor of Activated T cells cytoplasmic 1

OB:

Osteoblast

OC:

Osteoclast

OP:

Osteoporosis

OVX:

Ovariectomy

PDGF-BB:

Platelet-derived growth factor-BB

RANKL:

Receptor Activator of Nuclear Factor Kappa-B Ligand

ROS:

Reactive Oxygen Species

Tb.N:

Trabecular Number

Tb.Sp:

Trabecular Separation

Tb.Th:

Trabecular Thickness

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Funding

This research was funded by the National Natural Science Foundation of China (82072472 and U23A20412), Natural Science Foundation of Heilongjiang Province (LH2021H029), and Key Program of National Natural Science Foundation of Heilongjiang Province (ZD2020H003).

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Author notes

  1. Chunyang Xi and Jinglong Yan contributed equally to this work.

  2. Wenbo Xu and Shiyan Lv contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopedics, The Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, 150001, Heilongjiang Province, China

    Wenbo Xu, Shiyan Lv, Xiaoyan Wang, Chengchao Song, Chunyang Xi & Jinglong Yan

  2. Heilongjiang Provincial Key Laboratory of Hard Tissue Development and Regeneration, Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Harbin, 150001, China

    Wenbo Xu

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Contributions

Wenbo Xu : Data curation, Formal analysis, Writing – original draft. Shiyan Lv : Data curation, Project administration. Xiaoyan Wang : Validation. Chunyang Xi : Software. Chengchao Song : Writing – review and editing. Jinglong Yan : Conceptualization and Writing – review and editing.

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Xu, W., Lv, S., Wang, X. et al. Ferrostatin-1 inhibits osteoclast differentiation and prevents osteoporosis by suppressing lipid peroxidation. J Orthop Surg Res 20, 117 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13018-025-05544-2

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Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X