Abstract

Background

   Cellular senescence is a key driver of decreased bone formation and
   osteoporosis. Leptin (LEP) has been implicated in cellular senescence
   and osteogenic differentiation. The aim of this study was to
   investigate the mechanisms by which LEP mediates cellular senescence
   and osteogenic differentiation.

Methods

   C3H10T1/2 cells were treated with etoposide to induce cellular
   senescence, which was assessed by β-galactosidase staining.
   Quantitative real-time PCR and western blotting were used to measure
   the levels of senescence markers p21 and p16, as well as osteogenic
   differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline
   phosphatase (ALP) staining and alizarin red S staining were performed
   to evaluate osteogenic differentiation. The NF-κB pathway and
   O-GlcNAcylation were assessed by western blotting.

Results

   Etoposide treatment increased the number of senescent cells and the
   levels of p21 and p16, along with elevated LEP expression. These
   effects were reversed by LEP knockdown. Additionally, LEP knockdown
   increased ALP staining density and osteoblast mineralization nodules,
   as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2,
   indicating that LEP knockdown promoted osteogenic differentiation in
   C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB
   pathway by inhibiting the nuclear translocation of p65. Furthermore,
   OGT was found to promote O-GlcNAcylation of LEP at the S50 site.

Conclusion

   Our findings demonstrated that O-GlcNAcylation of LEP inactivated the
   NF-κB pathway by reducing LEP protein levels, thereby inhibiting
   cellular senescence and promoting osteogenic differentiation in
   C3H10T1/2 cells. This study may provide a novel therapeutic target for
   the treatment of osteoporosis.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12860-024-00523-7.

   Keywords: Osteogenic differentiation, Cellular senescence, LEP, NF-κB
   pathway, O-GlcNAcylation, Osteoporosis

Introduction

   The global trend towards population aging has led to an increased
   incidence of age-related diseases. Cellular senescence, an intrinsic
   aging mechanism, plays a pivotal role in various age-related
   conditions, including osteoporosis, atherosclerosis, diabetes,
   cataracts, and osteoarthritis [[28]1–[29]3]. Numerous studies have
   explored the mechanisms by which cellular senescence contributes to
   osteoporosis. For instance, a prior study confirmed that bone loss in
   aging mice results from increased osteoblastic bone formation coupled
   with a reduction in bone marrow adipose tissue, leading to a shift in
   bone marrow mesenchymal stem cell (BMSC) differentiation from
   osteoblasts to adipocytes [[30]4]. Other research has suggested that an
   imbalance between osteoblast-mediated bone formation and
   osteoclast-mediated bone resorption underlies age-related bone loss
   [[31]5–[32]7]. Furthermore, the removal of senescent osteoblasts has
   been shown to improve bone microarchitecture and strength [[33]4].
   Consequently, targeting cellular senescence represents a promising
   strategy for mitigating bone loss in osteoporosis.

   Mesenchymal stem cells (MSCs) are pluripotent adult stem cells with the
   potential to differentiate into multiple cell types, including
   chondrocytes, myocytes, osteoblasts, and adipocytes [[34]8–[35]11].
   Osteoporosis arises from a decline in the osteogenic differentiation
   capacity of these cells. Therefore, enhancing osteogenic
   differentiation while inhibiting adipogenic differentiation of MSCs is
   crucial for treating osteoporosis [[36]12–[37]14]. However, senescent
   MSCs exhibit reduced osteogenic differentiation and enhanced adipogenic
   differentiation, leading to diminished bone formation and limiting
   their regenerative potential [[38]15–[39]17]. These findings underscore
   the importance of MSC differentiation potential in bone formation,
   suggesting that investigating MSC senescence and osteogenic
   differentiation may provide an effective therapeutic target for
   alleviating osteoporosis.+.

   Leptin (LEP) is a circulating adipokine produced by adipocytes that
   plays a critical role in the regulation of energy balance and body
   weight [[40]18–[41]20]. Additionally, LEP is involved in numerous
   physiological activities and disease processes, including diabetes,
   chronic kidney disease, tumor development, and apoptosis
   [[42]21–[43]24]. Several studies have elucidated the mechanisms by
   which LEP mediates cellular senescence. For example, high doses of
   leptin have been shown to induce cell cycle arrest and senescence in
   chondrogenic progenitor cells (CPCs) via the activation of the p53/p21
   pathway [[44]25]. Furthermore, LEP has been reported to enhance the
   osteogenic potential of CPCs [[45]25]. It has also been proposed that
   LEP serves as a biomarker for aging and age-related diseases [[46]26].
   LEP is a regulator of osteogenic differentiation; targeting LEP
   promotes the osteogenic differentiation of BMSCs [[47]27, [48]28].
   Additionally, the regulation of LEP has been shown to promote
   osteogenic differentiation while inhibiting adipocyte formation in
   high-fat-diet-induced models [[49]29]. These findings suggested that
   LEP plays a role in cellular senescence and osteogenic differentiation,
   although the specific mechanisms underlying LEP-mediated osteoporosis
   require further investigation.

   In this study, we aimed to investigate the effects and underlying
   mechanisms of LEP on cellular senescence and osteogenic differentiation
   in MSCs, which may provide a new therapeutic target for the treatment
   of osteoporosis.

Methods

Cell culture and treatment

   Mouse mesenchymal stem cells (MSCs), specifically C3H10T1/2 cells, were
   obtained from the American Type Culture Collection (ATCC, Manassas, VA,
   USA). The cells were maintained in Dulbecco’s Modified Eagle Medium
   (DMEM, Gibco, Grand Island, NY, USA) supplemented with 100 µg/mL
   penicillin, 100 U/mL streptomycin, and 10% fetal bovine serum (FBS,
   Gibco) in a humidified incubator at 37 °C with 5% CO2. To establish
   cellular senescence models, cells were treated with 20 µM etoposide for
   24 h. For inducing osteogenic differentiation in C3H10T1/2 cells, they
   were cultured in osteogenic induction medium containing DMEM, 10% FBS,
   10 mM β-glycerophosphate, 50 µg/L L-ascorbic acid, and 100 nM
   dexamethasone for 14 days.

Cell transfection

   Short hairpin RNA (shRNA) targeting leptin (shLEP), shRNA negative
   control (shNC), empty vector (pcDNA3.1), OGT-overexpressing plasmids
   (pcDNA3.1-OGT), and OGA-overexpressing plasmids (pcDNA3.1-OGA) were
   obtained from Genechem (Shanghai, China). C3H10T1/2 cells, either
   treated with etoposide or untreated, were seeded into 6-well plates the
   day before transfection to achieve approximately 70% confluence.
   Transfections were performed using Lipofectamine 2000 (Invitrogen,
   Carlsbad, CA, USA) according to the manufacturer’s instructions. After
   48 h, the transfection was complete, and the cells were harvested for
   further analysis.

Bioinformatics analysis

   The [50]GSE35956 dataset was obtained from the Gene Expression Omnibus
   (GEO) database ([51]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi).
   Differentially expressed genes (DEGs) were identified based on criteria
   of P < 0.05 and |log(fold change)| > 2. Data analysis was conducted
   using GEO2R, and visualization was performed using the Sangerbox
   platform ([52]http://vip.sangerbox.com/). The STRING database
   ([53]https://cn.string-db.org/) was utilized to construct a
   protein-protein interaction (PPI) network of the DEGs relevant to
   osteoporosis. DEGs were subjected to Kyoto Encyclopedia of Genes and
   Genomes (KEGG) enrichment analysis to identify enriched pathways. An
   enrichment threshold of P < 0.05 was applied. O-GlcNAcylation sites in
   LEP were predicted using the DictyOGlyc-1.1 database
   ([54]https://services.healthtech.dtu.dk/services/DictyOGlyc-1.1/).

β-galactosidase staining

   Cellular senescence was detected using a senescence β-galactosidase
   staining kit (Beyotime, Beijing, China). Cells were washed once with
   phosphate-buffered saline (PBS, Gibco), fixed with β-galactosidase
   fixation solution for 15 min at room temperature, and then washed three
   times with PBS (3 min per wash). Subsequently, cells were stained with
   the staining solution overnight at 37 °C. Senescent cells were
   visualized and quantified using a light microscope.

Quantitative real-time PCR (qPCR)

   Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and
   quantified using a NanoDrop spectrophotometer (ThermoFisher Scientific,
   Waltham, MA, USA). RNA was reverse-transcribed into cDNA using the
   PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) following the
   manufacturer’s protocol. The cDNA was diluted tenfold, and qPCR was
   performed using SYBR Green Master Mix (Yeasen, Shanghai, China). The
   reaction mixture consisted of 10 µL SYBR Green Master Mix, 9.2 µL
   diluted cDNA, and 0.8 µL of each primer. Amplification was carried out
   on a QuantStudio 6 (ThermoFisher Scientific). Relative expression was
   calculated using the ^2−ΔΔCt method and GAPDH was used as a endogenous
   reference. The qPCR primers for LEP were as follows: forward,
   5’-GTGGCTTTGGTCCTATCTGTC-3’; reverse, 5’-CGTGTGTGAAATGTCATTGATCC-3’.

Nuclear and cytoplasmic protein isolation

   Nuclear and cytoplasmic proteins were extracted from C3H10T1/2 cells
   using a nuclear and cytoplasmic protein extraction kit (Beyotime).
   Cells were washed with PBS, and the supernatant was removed by
   centrifugation to collect the cell pellet. Cytoplasmic protein
   extraction reagent A was added to the cell pellet, and the mixture was
   vortexed vigorously for 5 s, followed by incubation on ice for 10 min.
   Cytoplasmic protein extraction reagent B was then added, and the
   mixture was vortexed vigorously for 5 s and incubated on ice for 1 min.
   The mixture was centrifuged at 12,000 × g for 5 min at 4 °C. The
   supernatant, containing the cytoplasmic protein, was transferred to a
   pre-chilled tube. Nuclear protein extraction reagent was added to the
   remaining pellet, which was vortexed vigorously for 30 s and incubated
   on ice for 30 min with intermittent vortexing every 2 min for 30 s. The
   mixture was then centrifuged at 12,000 × g for 10 min at 4 °C. The
   supernatant, containing the nuclear protein, was collected.

Western blot

   Total protein was extracted using RIPA lysis buffer (Beyotime) and
   quantified using a BCA protein assay kit (Beyotime). Equal amounts of
   protein were loaded onto 10% SDS-PAGE gels and subsequently transferred
   to PVDF membranes. The membranes were blocked with 5% non-fat milk in
   TBS-T for 1 h at room temperature. After blocking, the membranes were
   washed and incubated with primary antibodies overnight at 4 °C. The
   following day, the membranes were washed again and incubated with
   horseradish peroxidase-conjugated secondary antibodies (1:10,000,
   ab205718, Abcam, Cambridge, UK) for 2 h at room temperature. Protein
   bands were visualized using an ECL chemiluminescence reagent (Yeasen)
   and quantified using ImageJ software. The primary antibodies used in
   this study were as follows: anti-p21 (1/1000, ab109520, Abcam),
   anti-p16 (1/1000, #80772, CellSignaling, Danvers, MA, USA), anti-ALP
   (1/1000, ab307726, Abcam), anti-COL1A1 (1/1000, ab34710, Abcam),
   anti-RUNX2 (1/1000, ab236639, Abcam), anti-p65 (1/1000, ab32536,
   Abcam), anti-p-p65 (1/1000, ab76302, Abcam), anti-OGT (1/1000,
   ab96718), anti-OGA (1/5000, ab124807), anti-LEP (1/1000, ab16227,
   Abcam), anti-GAPDH (1/10000, ab181602, Abcam), anti-Lamin B (1/10000,
   ab133741, Abcam) and anti-O-GlcNAc (1: 1000, MA1-072, Thermo
   Scientific, Waltham, MA, USA).

Identification of osteogenic differentiation

   To identify osteogenic differentiation of C3H10T1/2 cells after 14 days
   of osteogenic induction, alkaline phosphatase (ALP) staining and
   alizarin red S (ARS) staining were performed. ALP staining was
   conducted using a BCIP/NBT ALP Color Development Kit (Beyotime)
   following the manufacturer’s protocol. Briefly, cells were washed three
   times with PBS and stained with BCIP/NBT stain solution for 30 min in
   the dark. For ARS staining, an ARS staining kit (Beyotime) was used
   according to the manufacturer’s instructions. Cells were washed with
   PBS, fixed with the provided fixative for 20 min, and then stained with
   ARS solution for 30 min at room temperature. Images were captured using
   a microscope.

Protein stability assay

   To assess the stability of LEP protein, C3H10T1/2 cells transfected
   with pcDNA3.1 or pcDNA3.1-OGT were treated with 10 µM cycloheximide
   (MKBio, Shanghai, China). The stability of LEP protein was evaluated by
   Western blot analysis at baseline and 8, 16, and 24 h post-treatment.

Statistical analysis

   All statistical analyses were performed using GraphPad Prism 7.0. Data
   are presented as the mean ± standard deviation. Each experimental group
   included at least three replicates. Comparisons between two groups were
   made using Student’s t-test, while comparisons among multiple groups
   were performed using one-way analysis of variance (ANOVA). Statistical
   significance was set at P < 0.05.

Results

LEP expression is upregulated in elderly patients with osteoporosis

   Osteoporosis is a chronic condition with a high prevalence among older
   individuals. To further explore the mechanisms underlying osteoporosis,
   we performed microarray analysis to identify DEGs in human MSCs derived
   from elderly patients with osteoporosis compared to middle-aged
   non-osteoporotic donors. Our results indicated that the expression of
   LEP was significantly upregulated in elderly patients with osteoporosis
   (Fig. [55]1A, B). Protein-protein interaction (PPI) network analysis
   revealed several proteins that interact with LEP (Fig. [56]1C). These
   findings suggested that LEP was upregulated in elderly patients with
   osteoporosis, indicating its potential as a therapeutic target for
   osteoporosis.

Fig. 1.

   [57]Fig. 1
   [58]Open in a new tab

   LEP expression was upregulated in elderly patients with osteoporosis (A
   and B) Differentially expressed genes in elderly patients with
   osteoporosis and middle-aged non-osteoporotic donors were analyzed by
   microarray analysis. (C) PPI network analysis of differentially
   expressed genes

Etoposide increases LEP expression and promotes cellular senescence in
C3H10T1/2 cells

   To investigate the effects of cellular senescence, C3H10T1/2 cells were
   treated with etoposide to induce a senescence model. Our results showed
   that the expression of LEP was significantly elevated in
   etoposide-treated C3H10T1/2 cells (Fig. [59]2A). Furthermore, etoposide
   treatment increased the number of senescent cells (Fig. [60]2B, C). We
   also measured the expression of cellular senescence markers in
   C3H10T1/2 cells and confirmed that the expression of p21 and p16 was
   significantly upregulated by etoposide (Fig. [61]2D, E). Western blot
   analysis corroborated these findings, showing increased protein levels
   of p21 and p16 in etoposide-induced C3H10T1/2 cells (Fig. [62]2F).
   Collectively, these data demonstrated that etoposide increased LEP
   expression and promoted cellular senescence in C3H10T1/2 cells.

Fig. 2.

   [63]Fig. 2
   [64]Open in a new tab

   Etoposide increased LEP expression and promotes cellular senescence in
   C3H10T1/2 cells (A) The expression of LEP was measured using qPCR. (B
   and C) Cellular senescence was assessed by β-galactosidase staining. (D
   and E) qPCR was performed to measure the expression of aging markers
   p21 and p16. (F) The protein levels of p21 and p16 were evaluated by
   western blot

LEP knockdown inhibits etoposide-induced cellular senescence in C3H10T1/2
cells

   To further elucidate the role of LEP, C3H10T1/2 cells were transfected
   with shLEP, resulting in a significant decrease in LEP expression
   (Fig. [65]3A). Subsequently, the number of senescent cells induced by
   etoposide was notably reduced upon LEP knockdown (Fig. [66]3B, C).
   Additionally, LEP knockdown inhibited the etoposide-induced increase in
   the expression of p21 and p16 (Fig. [67]3D, E). Consistent with these
   observations, Western blot analysis revealed that the protein levels of
   p21 and p16, which were upregulated by etoposide, were restored to
   lower levels by LEP knockdown (Fig. [68]3F). Collectively, these
   results indicated that LEP knockdown inhibited etoposide-induced
   cellular senescence in C3H10T1/2 cells.

Fig. 3.

   [69]Fig. 3
   [70]Open in a new tab

   LEP knockdown inhibited etoposide-induced cellular senescence in
   C3H10T1/2 cells (A) The expression of LEP was measured using qPCR. (B
   and C) Cellular senescence was assessed by β-galactosidase staining. (D
   and E) qPCR was performed to measure the expression of aging markers
   p21 and p16. (F) The protein levels of p21 and p16 were evaluated by
   western blot

LEP knockdown promotes osteogenic differentiation in C3H10T1/2 cells

   To further explore the relationship between LEP and osteogenic
   differentiation, C3H10T1/2 cells were transfected with shLEP. Compared
   to the control group, LEP knockdown significantly increased the ALP
   staining density in C3H10T1/2 cells (Fig. [71]4A, B). ARS staining
   further confirmed that LEP knockdown enhanced the formation of
   osteoblastic mineralization nodules in C3H10T1/2 cells (Fig. [72]4C,
   D). Moreover, the expression levels of osteogenic
   differentiation-related genes, including ALP, COL1A1, and RUNX2, were
   markedly elevated by LEP knockdown (Fig. [73]4E). Western blot analysis
   corroborated these findings, demonstrating that the protein levels of
   ALP, COL1A1, and RUNX2 were also upregulated upon LEP knockdown
   (Fig. [74]4F). Moreover, LEP knockdown promoted osteogenic
   differentiation in vivo (Figure S5A-H). Conversely, we found that LEP
   overexpression inhibited osteogenic differentiation in C3H10T1/2 cells
   (Figure [75]S1A-G). In summary, these results demonstrated that LEP
   knockdown promoted osteogenic differentiation in C3H10T1/2 cells.

Fig. 4.

   [76]Fig. 4
   [77]Open in a new tab

   LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells (A
   and B) An ALP staining kit was applied to evaluate the ALP staining
   density. (C and D) ARS staining was performed to assess the osteoblast
   mineralization nodules of C3H10T1/2 cells. (E) The expression of
   osteogenic differentiation-related genes ALP, COL1A1 and RUNX2 was
   measured by qPCR. (F) The protein levels of ALP, COL1A1 and RUNX2 were
   evaluated by western blot

LEP promotes osteoporosis through the activation of NF-κB signaling pathway

   To identify DEGs affected by LEP knockdown in C3H10T1/2 cells, we
   performed transcriptomic analysis. The top ten upregulated and
   downregulated genes are displayed in a heatmap (Fig. [78]5A). KEGG
   pathway enrichment analysis of these DEGs revealed significant
   enrichment in the NF-κB signaling pathway (Fig. [79]5B). Given that the
   NF-κB pathway is a key regulatory pathway in osteoporosis [[80]30], we
   further investigated whether LEP modulates this pathway in C3H10T1/2
   cells. Our results showed that LEP knockdown inhibited the
   phosphorylation of p65 (Fig. [81]5C). Additionally, LEP knockdown
   suppressed the nuclear translocation of p65 (Fig. [82]5D). These
   findings suggested that LEP knockdown inactivated the NF-κB pathway in
   C3H10T1/2 cells. Moreover, activation of NF-κB pathway in C3H10T1/2
   cells inhibited osteogenic differentiation increased by LEP knockdown
   (Figure [83]S2A-F). In conclusion, our data confirmed that LEP promoted
   osteoporosis through the activation of the NF-κB signaling pathway.

Fig. 5.

   [84]Fig. 5
   [85]Open in a new tab

   LEP promoted osteoporosis through the activation of NF-κB signaling
   pathway (A) The DEGs in C3H10T1/2 cells with or without LEP knockdown
   were shown in a heatmap. (B) Bubble diagram of KEGG pathway enrichment
   analysis. (C) The protein levels of p65 were assessed by western blot.
   (D) Western blot was performed to measure the protein levels of p65 in
   cytoplasm and nucleus

OGT promotes O-GlcNAcylation of LEP at S50 site

   Previous studies have indicated that O-GlcNAcylation plays a role in
   osteogenic differentiation [[86]31]. To investigate whether
   O-GlcNAcylation is involved in LEP-mediated osteogenic differentiation
   in C3H10T1/2 cells, we first assessed the O-GlcNAcylation level in
   these cells. Western blot analysis revealed that etoposide treatment
   inhibited O-GlcNAcylation in C3H10T1/2 cells (Fig. [87]6A). Since
   O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and
   O-GlcNAcase (OGA), we measured the protein levels of both enzymes.
   Etoposide treatment specifically downregulated OGT levels without
   affecting OGA (Fig. [88]6A). We then overexpressed OGT to examine its
   impact on LEP. Overexpression of OGT reduced the protein level of LEP
   but increased its O-GlcNAcylation (Fig. [89]6B). Conversely,
   overexpression of OGA did not affect the protein level or
   O-GlcNAcylation status of LEP (Fig. [90]6C). To identify specific
   O-GlcNAcylation sites on LEP, we predicted several potential sites
   (Fig. [91]6D). The top three predicted sites were illustrated in
   Fig. [92]6E. To validate which site was indeed modified by
   O-GlcNAcylation, we mutated these sites to alanine (A) using
   site-directed mutagenesis. Western blot analysis showed that mutating
   S50 to alanine (S50A) increased the protein level of LEP and decreased
   its O-GlcNAcylation, whereas mutations at S91 and S164 did not alter
   the protein level or O-GlcNAcylation status of LEP (Fig. [93]6F).
   Finally, we found that OGT overexpression accelerated the degradation
   of LEP and decreased its protein stability (Fig. [94]6G). Collectively,
   these results indicated that OGT promoted O-GlcNAcylation of LEP at the
   S50 site. Moreover, OGT overexpression inhibited etoposide-induced
   cellular senescence and osteogenic differentiation in C3H10T1/2 cells
   (Figure [95]S3 A-F and [96]S4 A-F).

Fig. 6.

   [97]Fig. 6
   [98]Open in a new tab

   OGT promoted O-GlcNAcylation of LEP at S50 site. (A) The
   O-GlcNAcylation level and the protein levels of OGT and OGA were
   measured by western blot. (B and C) The protein levels of OGT, OGA and
   LEP and the O-GlcNAcylation levels of LEP in C3H10T1/2 cells with OGT
   or OGA overexpression were measured by western blot. (D and E) The
   potential O-GlcNAcylation sites in LEP was predicted using
   DictyOGlyc-1.1 database. (F) After mutating the potential
   O-GlcNAcylation sites in LEP, the protein and O-GlcNAcylation levels of
   LEP was measured by western blot. (G) After C3H10T1/2 cells were
   treated with 10 µM cycloheximide for 0, 8, 16, and 24 h, the stability
   of LEP protein was measured by western blot

Discussion

   Osteoporosis is a chronic metabolic bone disorder predominantly
   affecting the elderly population. Cellular senescence is a key driver
   of age-related osteoporosis, and inhibiting cellular senescence has
   been shown to be an effective strategy for improving osteoporotic
   conditions. Previous research has highlighted the protective effects of
   Sirt3 overexpression against bone marrow stromal cell (BMSC)
   senescence, thereby ameliorating senile osteoporosis [[99]32].
   Similarly, Liu et al. [[100]33] demonstrated that reducing bone loss
   through the inhibition of BMSC senescence can improve osteoporosis in
   mice.

   Studies have also reported a decline in osteogenic differentiation
   potential in MSCs derived from older individuals [[101]34].
   Additionally, Zhang et al. [[102]35] observed that aged BMSCs
   preferentially differentiate into adipocytes rather than osteoblasts,
   indicating an imbalance between bone formation and resorption in
   senescent MSCs. This imbalance is attributed to reduced osteogenic
   differentiation potential, and enhancing the osteogenic differentiation
   capacity of senescent cells can aid in restoring bone formation. For
   instance, Liu et al. [[103]36] found that UBE2E3 positively correlates
   with osteogenesis-related genes and that its overexpression attenuates
   cellular senescence in BMSCs while promoting osteogenic
   differentiation. Chen et al. [[104]37] also demonstrated that
   overexpression of YBX1 enhances bone formation and reduces fat
   accumulation in aged mice. Collectively, these findings underscore the
   interplay between cellular senescence and osteogenic differentiation,
   highlighting their critical roles in bone metabolism. In our study, we
   observed that LEP was highly expressed in etoposide-induced senescent
   cells, and knockdown of LEP inhibited cellular senescence while
   promoting osteogenic differentiation in senescent MSCs. LEP, known as a
   regulator of lipid metabolism, has been implicated in various diseases
   such as diabetes and cardiovascular disorders [[105]38, [106]39].
   Recent studies have begun to elucidate the role of LEP in bone
   metabolism, suggesting a multifaceted involvement in skeletal health.
   For instance, Lindenmaier et al. [[107]40] demonstrated that LEP
   treatment reduces marrow adipose tissue and enhances bone formation in
   leptin-deficient mice. Mei et al. [[108]27] confirmed that miR-874-3p
   promotes the proliferation and differentiation of human BMSCs by
   downregulating LEP expression, thereby inhibiting osteoporosis.
   Additionally, Zhao et al. [[109]25] suggested that LEP-mediated
   cellular senescence acts as a modulator of obesity-related
   osteoarthritis. These findings collectively suggested that LEP may
   serve as a regulator of osteogenic differentiation and osteoporosis.
   Compared to these previous studies, our results confirmed that LEP
   knockdown inhibited cellular senescence while promoting osteogenic
   differentiation, indicating that LEP played a regulatory role in the
   development of osteoporosis.

   Furthermore, we demonstrated that LEP knockdown inactivated the NF-κB
   signaling pathway and inhibitd the nuclear translocation of p65. NF-κB
   is a key mediator of inflammatory responses and plays a crucial role in
   immune regulation, being involved in numerous diseases [[110]41].
   Multiple studies have highlighted the role of the NF-κB pathway in
   osteoporosis. For example, Li et al. [[111]42] found that urolithin B
   attenuates bone loss in ovariectomized (OVX) mice by inhibiting
   osteoclast formation and activation via down-regulation of the
   ERK/NF-κB signaling pathway. Sun et al. [[112]30] revealed that
   inhibition of NF-κB signaling attenuates osteoclastogenesis and bone
   loss in OVX-induced mice. Moreover, certain drugs improve osteoporosis
   by inhibiting osteoclast differentiation and bone resorption through
   the suppression of the NF-κB signaling pathway [[113]43, [114]44].
   Additionally, NF-κB is considered a therapeutic target for
   aging-related osteoporosis, and its inhibition mitigates cellular
   senescence and rescues osteoporosis in premature aging mice [[115]45].
   Hu et al. [[116]17] confirmed that NAP1L2 inhibits etoposide-induced
   senescence in MSCs and improves impaired osteogenesis. Collectively,
   these findings demonstrate that NF-κB is a potential regulator of
   aging-related osteoporosis. In this study, we confirmed that LEP
   knockdown inactivated the NF-κB pathway, revealing for the first time
   the mechanism by which LEP regulated osteoporosis through the NF-κB
   pathway.

   Finally, we confirmed that OGT mediated O-GlcNAcylation in C3H10T1/2
   cells, and that OGT promotes LEP O-GlcNAcylation at the S50 site,
   leading to a reduction in the protein level of LEP. O-GlcNAcylation is
   a post-translational modification whose role in cancer, cardiovascular
   diseases, and immune system disorders has been extensively studied
   [[117]46]. This modification is regulated by two enzymes: OGT, which
   adds O-GlcNAc to proteins, and OGA, which removes O-GlcNAc [[118]47].
   Recent studies have highlighted the functions of O-GlcNAcylation in
   osteogenic differentiation. Nagel and Ball [[119]31] initially found
   that suppression of OGA enhances ALP activity in BMSCs. Zhang et al.
   [[120]48] also revealed that O-GlcNAcylation promotes osteogenic
   differentiation of BMSCs by modifying and activating RUNX2. Consistent
   with these findings, our study demonstrated that increased
   O-GlcNAcylation of LEP promoted osteogenic differentiation in mouse
   mesenchymal stem cells. However, contrasting results have been reported
   in periodontal ligament stem cells (PDLCs), where suppression of OGT or
   O-GlcNAcylation levels promoted osteogenic differentiation [[121]49,
   [122]50]. We hypothesize that these differences may be due to distinct
   regulatory mechanisms of O-GlcNAcylation on osteogenic differentiation
   in mesenchymal stem cells versus periodontal ligament stem cells,
   warranting further investigation.

   In conclusion, our study demonstrates that O-GlcNAcylation of LEP at
   the S50 site inactivates the NF-κB pathway by suppressing LEP protein
   levels, thereby inhibiting cellular senescence and promoting osteogenic
   differentiation in C3H10T1/2 cells. This research provides a novel
   theoretical foundation for understanding the mechanisms underlying
   osteoporosis.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [123]Supplementary Material 1^ (22KB, xls)
   [124]Supplementary Material 2^ (163.1MB, doc)
   [125]12860_2024_523_MOESM3_ESM.jpg^ (1.1MB, jpg)

   Supplementary Material 3: Figure S1 LEP overexpression inhibited
   osteogenic differentiation in C3H10T1/2 cells. (A) LEP expression in
   C3H10T1/2 cells was measured by qPCR. (B and C) An ALP staining kit was
   applied to evaluate the ALP staining density. (D and E) ARS staining
   was performed to assess the osteoblast mineralization nodules of
   C3H10T1/2 cells. (F) The expression of osteogenic
   differentiation-related genes ALP, COL1A1 and RUNX2 was measured by
   qPCR. (G) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by
   western blot.
   [126]12860_2024_523_MOESM4_ESM.jpg^ (1.4MB, jpg)

   Supplementary Material 4: Figure S2 Activation of NF-?B pathway
   inhibited osteogenic differentiation in C3H10T1/2 cells enhanced by LEP
   knockdown. (A and B) An ALP staining kit was applied to evaluate the
   ALP staining density. (C and D) ARS staining was performed to assess
   the osteoblast mineralization nodules of C3H10T1/2 cells. (E) The
   expression of osteogenic differentiation-related genes ALP, COL1A1 and
   RUNX2 was measured by qPCR. (F) The protein levels of ALP, COL1A1 and
   RUNX2 were evaluated by western blot.
   [127]12860_2024_523_MOESM5_ESM.jpg^ (2.4MB, jpg)

   Supplementary Material 5: Figure S3 OGT overexpression inhibited
   etoposide-induced cellular senescence in C3H10T1/2 cells.(A) The
   expression of OGT was measured using qPCR. (B and C) Cellular
   senescence was assessed by ?-galactosidase staining. (D and E) qPCR was
   performed to measure the expression of aging markers p21 and p16. (F)
   The protein levels of p21 and p16 were evaluated by western blot.
   [128]12860_2024_523_MOESM6_ESM.jpg^ (968.2KB, jpg)

   Supplementary Material 6: Figure S4 OGT overexpression promoted
   osteogenic differentiation in C3H10T1/2 cells. (A and B) An ALP
   staining kit was applied to evaluate the ALP staining density. (C and
   D) ARS staining was performed to assess the osteoblast mineralization
   nodules of C3H10T1/2 cells. (E) The expression of osteogenic
   differentiation-related genes ALP, COL1A1 and RUNX2 was measured by
   qPCR. (F) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by
   western blot.
   [129]12860_2024_523_MOESM7_ESM.jpg^ (1.8MB, jpg)

   Supplementary Material 7: Figure S5 LEP knockdown inhibited
   osteoporosis in vivo of mice. (A) Representative images from micro-CT
   analysis of 3D image reconstruction of the femoral midshaft corticoid
   bone of 6-month-old and 20-month-old mice transfected with shNC or
   shLEP. (B) The vBMD of trabecular quantified by micro-CT. (C)
   Trabecular BV/TV, (D) Tb.Th, (E) Tb.Sp were measured by micro-CT to
   analyze trabecular bone. (F) The vBMD of cortical was quantified by
   micro-CT. (G) cortical BV/TV and (H) Ct. Th of corticoid bone was
   analyzed by micro-CT.

Acknowledgements