Total hip arthroplasty (THA), total knee arthroplasty (TKA), and total joint arthroplasty (TJA) are among the most common orthopedic surgeries used to treat severe joint injuries, rheumatoid arthritis, osteoarthritis, and other end-stage joint diseases [1]. TKA surgery is typically performed in elderly patients with conditions such as rheumatoid arthritis [2], while TJA is a common and effective surgery for older patients (> 65 years) with late-stage osteoarthritis [3, 4]. Despite the maturity of these surgeries, some patients still require further surgical revision, mainly due to aseptic loosening [5]. The main cause of aseptic loosening is the accumulation of wear particles around the implant, such as titanium (Ti) particles [6]. Wear particles are reported to induce osteolysis by disrupting the differentiation, survival, and function of osteoblasts and osteoclasts, resulting in an imbalance between bone formation and resorption [7-10]. Bone marrow-derived mesenchymal stem cells (BMSCs) are important for bone regeneration due to their multipotent differentiation ability, as they can differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, thereby aiding in bone tissue regeneration and repair [11-14]. In addition, BMSCs secrete growth factors and extracellular matrix molecules that promote angiogenesis, cell proliferation, and differentiation [15]. Therefore, it is important to study the differentiation mechanism of BMSCs to promote osteoblast differentiation and alleviate osteolysis around prostheses.
Carvacrol (2-methyl-5-[1-methylethyl] phenol) is a volatile oil component found primarily in herbaceous plants such as oregano, peppermint, and thyme [16]. It has anti-inflammatory, antioxidant, and antimicrobial properties [17, 18]. Research suggests that carvacrol can effectively reduce tissue damage and bone loss associated with ligature-induced periodontitis [19, 20]. Deepak et al. discovered that carvacrol inhibits osteoclastogenesis and negatively regulates the survival of mature osteoclasts [18]. In addition, carvacrol has a bacteriostatic effect on osteoblast proliferation without cytotoxic effects [21]. Based on the existing literature, we speculate that carvacrol may have a therapeutic effect in osteolysis. There is evidence that carvacrol is involved in the differentiation of mesenchymal stem cells. Specifically, it has been observed that carvacrol can reduce the differentiation of mesenchymal stem cells derived from umbilical cord tissue into adipocytes [22]. Furthermore, at low concentrations, carvacrol can promote the paracrine potential and endothelial differentiation of bone marrow mesenchymal stem cells in angiogenesis [23]. Therefore, we speculate that carvacrol induces osteogenic differentiation of BMSCs to alleviate osteolysis.
Numerous studies have demonstrated that silent information regulator type 1 (SIRT1) has a therapeutic effect on osteolysis [24-27]. Studies have shown that promoting osteogenic potential in BMSCs through SIRT1 can improve osteolysis [27]. Upregulation of SIRT1 was found to promote osteogenic differentiation in BMSCs [28, 29]. Runx2 and Osterix, which are the primary transcription factors involved in osteogenesis [30], are positively regulated by SIRT1. In addition, the expression of osteogenesis-related factors, including osteocalcin (OCN), osteopontin (OPN), and sialoprotein, is reduced in SIRT1 knockout cells [31-33]. Although research has shown that carvacrol upregulates SIRT1 levels [34], the specific regulatory mechanism is not yet understood.
Long non-coding RNAs (lncRNAs), as a type of noncoding RNA, have been recognized as key regulatory molecules involved in gene expression, epigenetic modifications, and protein activity [35]. Recently, lncRNAs have been found to be involved in the process of osteolysis. For example, lncRNA TSIX has been shown to promote osteoblast apoptosis in particle-induced osteolysis [36]. LncRNA DANCR contributes to osteolysis by inhibiting osteoblast differentiation [37]. Similarly, LncRNA KCNQ1OT1 improves particle-induced osteolysis by inducing macrophage polarization [38]. NEAT1 and XIST promote osteogenic differentiation of human bone marrow mesenchymal stem cells [39, 40], but there is no clear literature reporting their involvement in osteolysis.
Therefore, this study proposes to use a mouse model of osteolysis and Ti particle-induced BMSCs to investigate the potential therapeutic effects of carvacrol on osteolysis in aged mice. In addition, this study will investigate whether carvacrol induces the expression of SIRT1 through the mediation of NEAT1 to promote the osteogenic differentiation of BMSCs. The results of this study promise to provide new ideas and methods for the treatment of osteolysis.

Ti particle-induced osteolysis model

As previously described, a mouse model of osteolysis was established to determine the protective effect of carvacrol on osteolysis in aged mice [41]. SPF grade C57BL/6 mice (18 months old) were obtained from Changzhou Cavens Laboratory Animal Co., Ltd. The living conditions of the mice were as described previously [42]. The mice were housed in a controlled environment with a temperature of 22-24℃ and humidity of 50%. Prior to the start of the experiment, the mice underwent a 1-week period of ad libitum feeding and acclimatization.
Mice were anesthetized by intraperitoneal injection of ketamine and xylazine. After anesthesia, the skin at the calvarial area was gently scraped and disinfected with 10% povidone-iodine solution, and then cut along the midline with a sharp scalpel. Then, 30 mg titanium particles (30 μL) were evenly distributed on the surface of the bilateral parietal bones, and the surgical skin incision was closed. In the sham + PBS group, an equivalent volume of PBS (30 μL) was used instead of the titanium particle suspension. After establishment of the model, carvacrol (5 or 10 mg/kg) was administered intragastrically daily for 2 weeks, while PBS was administered daily to mice in the sham + PBS and Ti + PBS groups. For the Ti + Carvacrol + LV-shRNA and Ti + Carvacrol + LV-sh-NEAT1 groups, 70 µL of LV-sh-NEAT1 or LV-shRNA (titer: 8×10⁸ TU/mL) was injected locally into the calvarial area immediately after completion of surgery. The injection site was determined to be 2 mm from the midline and on the right lateral side of the surgical incision. Upon completion of the injection, the mice were administered carvacrol at a dose of 10 mg/kg. To euthanize the mice, an intraperitoneal injection of 480 mg/kg Avertin was administered. Calvarial bones were dissected and fixed in 4% paraformaldehyde for 2 days. After fixation, calvarial samples were washed three times with PBS and then stored in 75% ethanol at 4°C until further analysis.

Micro-computed tomography (Micro-CT) scan

Carefully remove Ti particles from each calvaria to avoid metal artifacts in the scan results. Then, fix the harvested mouse calvaria and use a micro-CT scanner (Skyscan 1272, Bruker, Germany). Set the scan resolution to 9 μm and the X-ray energy to 50 kV and 500 μA. Initiate the scanning process by rotating the scanning stage and exposing the specimens to the X-ray beam, taking a series of projection images of the calvarial region. Use the software provided by the manufacturer to perform three-dimensional image reconstruction. Following the methods established in previous studies [43], use the micro-CT analysis software (Skyscan) to evaluate different tissue morphometric measurements: bone density (g/cm³) and bone volume/total volume (BV/TV, %).

Cell culture of BMSCs and osteogenic differentiation

BMSCs were cultured and differentiated as previously described [44]. Briefly, BMSCs were isolated from the femur and tibia of 18-month-old C57BL/6 mice. Cells were cultured in MEM alpha medium (α-MEM, 11900024, Gibco, USA) supplemented with 2 mM glutamine, 20% fetal bovine serum (FBS, 12483020, Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (15140122, Invitrogen, USA). Cells were maintained in a cell culture incubator at 37℃ with 5% carbon dioxide. BMSCs were seeded in 12-well plates at a density of 1 × 10⁵ cells per well. When the cell density reached approximately 70-80%, osteogenic induction differentiation was initiated. For osteogenic differentiation, cells were incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 10% horse serum, 12 mM Lglutamine, 20 mM β-glycerol phosphate, 100 μg/mL streptomycin, 50 ng/mL thyroxine, 1 nM dexamethasone, 100 U/mL penicillin, and 0.5 μM ascorbate 2-phosphate. For Ti and carvacrol treatments, Ti (1.0 mg/mL) or carvacrol (12.5, 25, and 50 μM) was added simultaneously at the start of osteogenic induction.

Lentivirus infection

NEAT1 overexpression lentiviruses (LV-NEAT1), SIRT1 overexpression lentiviruses (LV-SIRT1), negative control (LV-NC), NEAT1 RNA interference lentiviruses (LV-shNEAT1), and negative control (LV-shRNA) were purchased from GenePharma (China). The lentiviral suspensions containing polybrene (5 mg/mL) were added to the cell proliferation medium. After 48 hours of incubation, cells were selected with puromycin (1 µg/mL).

Alkaline phosphatase (ALP) activity

After 7 days of cultivation in osteogenic differentiation medium, BMSCs were subjected to ALP activity assessment using an ALP detection kit (P0321S, Beyotime, China). Cells were lysed using RIPA lysis buffer (P0013B, Beyotime, China), and the appropriate reagents were added according to the manufacturer's instructions. The mixture was incubated at 37℃ for 10 minutes. After the reaction was stopped, the absorbance was measured at 405 nm to quantify the ALP activity in the samples.

Alizarin red S (ARS) staining

The mineralization ability of the cells was evaluated using an ARS staining kit (C0148S, Beyotime, China). After 21 days of induction and differentiation, the culture medium was removed and the cells were washed once with PBS. The cells were then fixed with a fixative solution for 20 min, followed by three washes with PBS. Next, 0.5% ARS working solution was added to detect calcium deposits according to the kit instructions. The samples were examined under a light microscope (Leica, Germany).

Western blot analysis

The experimental procedure for protein extraction from cryopreserved calvarial tissue samples was as follows: samples were washed 2-3 times with prechilled PBS. A mixture of 2% protease phosphatase inhibitor and 200 μL RIPA protein lysis buffer was then added to the samples, followed by thorough grinding. After sufficient grinding, 300 μL protein lysis buffer was added and mixed evenly. For BMSCs, lysis was performed on ice using RIPA protein lysis buffer containing phenylmethanesulfonyl fluoride (PMSF, 100 mM). After lysis was completed, the supernatant was collected by centrifugation. Then, 2 μL of the protein solution was collected for protein concentration measurement using a BCA assay kit (P0012, Beyotime, China). Next, the protein samples were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred to a polyvinylidene fluoride membrane (PVDF, 88518, ThermoFisher Scientific, USA). The membrane was blocked with 5% skim milk for one hour at room temperature. Specific primary antibodies were added and incubated with the PVDF membrane overnight at 4℃: anti-SIRT1 (ab263965, Abcam, USA), anti-RUNX2 (ab114133, Abcam, USA), anti-Osterix (ab209484, Abcam, USA), anti-OCN (ab93876, Abcam, USA), anti-OPN (ab8448, Abcam, USA), and anti-β-actin (ab8227, Abcam, USA). After three washes with TrisHCl buffer saline and Tween (TBST), the membrane was incubated with the corresponding secondary antibody (HRP-conjugated anti-mouse IgG or anti-rabbit IgG) for 1 hour. Specific protein signals were detected using a chemiluminescence assay (PE0010, Solarbio, China). Images were captured using an Amersham Imager 600 system (GE Healthcare, USA).

Reverse transcription-quantitative PCR (RT-qPCR)

After culturing BMSCs in osteogenic induction medium for seven days, Trizol reagent (15596026, ThermoFisher Scientific, USA) was added to completely lyse the cells for RNA extraction. After the RNA concentration was detected by spectrophotometer, the extracted total RNA was used as a template, and the reverse transcription reaction was performed according to the instructions of the M-MLV Reverse Transcription Kit manual (M1701, Promega, USA). The PCR reaction system was constructed according to the Fast SYBR Mixture reaction kit (CW0955M, CWBIO, China). Finally, qPCR was performed using the ABI 7000 real-time fluorescence quantitative PCR system (Applied Biosystems, USA). Relative gene expression levels were calculated using the 2^-ΔΔCt formula.

RNA pull-down

RNA probes were synthesized by GenePharma and labeled with biotin (Bio-NEAT1 or Bio-NC). The experiment was performed according to the instructions of the RNA-Protein Pull-Down Assay Kit (20164, ThermoFisher Scientific, USA). Briefly, BMSCs were lysed with lysis buffer, and then the synthesized RNA probes were incubated with the lysate at room temperature for 30 min. The biotinylated RNA probe specifically binds to streptavidin-coated magnetic beads to form an RNA probe-bead complex. Wash buffer is used several times to remove non-specifically bound proteins and RNA molecules. The specific binding proteins are eluted from the RNA probe-bead complex and the RNA-protein complex is detected by Western blotting.

RNA immunoprecipitation (RIP)

RIP experiments were performed using the EZ-Magna RIP Kit (17-701, Millipore, USA) to verify the binding between SIRT1 and NEAT1. The cross-linked cells were completely lysed with RIPA lysis buffer. SIRT1 antibody and magnetic beads were then added to the lysed samples for immunoprecipitation. Non-specifically bound proteins and impurities were removed by multiple washing steps. Finally, RNA was extracted from the immunoprecipitated samples and the RNA samples were analyzed by RT-qPCR.

Ubiquitination assay

BMSCs were transfected with HA-ubiquitin (Ub) and LV-NEAT1 (or LV-NC). After 24 hours of transfection, MG132 (10 μM) was added to the medium and the cells were incubated for 4 hours. The cells were washed twice with PBS and lysed with RIPA lysis buffer. After centrifugation, the supernatant was transferred to a new EP tube and 50 μL beads were added to the supernatant. The cell lysate was incubated with anti-SIRT1 antibody at 4℃ overnight for immunoprecipitation. Wash solution (500 μL sterile PBS buffer + 50 μL protease inhibitor) was prepared. Centrifuge at 7500 rpm for 5 min and discard the supernatant. Wash solution is added to the tube, quickly mixed and centrifuged. Discard the supernatant, leaving the beads in each tube. 50 μL of 6 × loading buffer was added to each tube, vortexed to mix thoroughly, and boiled in a 100℃-metal bath for 10 min. Immunoblotting (IB) was then performed to detect binding proteins using anti-HA (ab137838, Abcam, USA) and anti-SIRT1.

Immunoprecipitation (IP) and IB assay

BMSCs were transfected with LV-NEAT1 (or LV-NC). After 24 h of transfection, MG132 (10 μM) was added to the medium and the cells were incubated for 4 hours. Cells were lysed with RIPA lysis buffer containing PMSF. The supernatant of the lysed cells was mixed with protein A/G agarose, and the centrifuge tube was gently shaken at 4℃. Anti-SMURF2 (ab313470, Abcam, USA) was then added and incubated overnight at 4℃. Finally, the target protein and its interacting proteins were eluted with elution buffer. Immunoblotting and detection of eluted proteins were performed using anti-SIRT1 and anti-SMURF2.

Statistical analysis

All data analysis was performed using GraphPad Prism 6.0. All numerical values are presented as mean ± standard deviation (SD). Two-tailed unpaired Student's t-test was used to compare mean differences between two independent samples. Comparison between groups was performed using one-way ANOVA followed by Tukey's multiple comparison test or two-way ANOVA followed by Bonferroni's multiple comparison test. A P value < 0.05 was considered statistically significant.

Carvacrol alleviates osteolysis in aged mice in vivo

To investigate the role of carvacrol in osteolysis, we examined the calvarial condition of elderly mice in a mouse model of osteolysis induced by Ti particles treated with different doses of carvacrol. We evaluated the calvarial conditions of different groups using micro-CT scanning. Three-dimensional reconstructed images showed that, compared with the sham + PBS group, the calvarial bone of the Ti + PBS group was extensively eroded and the calvarial thickness was reduced. However, carvacrol treatment significantly improved the calvarial erosion (Figure 1A). Further analysis revealed that the Ti + PBS group had a significant reduction in bone density and BV/TV compared to the sham + PBS group (Figure 1B-C). Interestingly, treatment with 5 or 10 mg/kg carvacrol significantly increased the bone density reduction induced by Ti particles (Figure 1B), and only 10 mg/kg carvacrol treatment significantly upregulated the BV/TV level (Figure 1C). Taken together, these results indicate that carvacrol has a protective effect against Ti particle-induced osteolysis.
To further elucidate the role of carvacrol in osteoblast differentiation, we examined the expression levels of SIRT1 and osteoblast differentiation-related factors (RUNX2, Osterix, OCN, and OPN) in the calvaria of aged mice. Western blot analysis revealed that the expression of SIRT1, RUNX2, Osterix, OCN, and OPN was significantly decreased after Ti treatment, while carvacrol treatment effectively reversed this phenomenon (Figure 1D). These results suggest that carvacrol has the potential to promote osteoblast differentiation.

Carvacrol alleviates osteogenic differentiation of Tiinhibited BMSCs in vitro

To determine the appropriate concentration of carvacrol to promote the differentiation of BMSCs into osteoblasts, we performed ARS staining and ALP activity analysis on different concentrations of carvacrol-treated groups and control groups. The results showed that the mineralization ability of BMSCs was significantly enhanced when the concentration of carvacrol exceeded 25 μM (Figure S1A). When the concentration of carvacrol exceeded 12.5 μM, the ALP activity of BMSCs was observed to be promoted (Figure S1B). At the same time, we evaluated the expression of osteogenic differentiation-related factors and found that the expression of RUNX2 and OCN in BMSCs was promoted when the concentration of carvacrol exceeded 25 μM (Figure S1C&E). In addition, the expression of Osterix and OPN in BMSCs was also promoted when the concentration of carvacrol exceeded 12.5 μM (Figure S1D&F). Based on these results, we concluded that carvacrol at a concentration of 25 μM significantly promoted osteogenic differentiation. Therefore, we chose this concentration for the following experiments.
To further investigate whether the protective effect of carvacrol against osteolysis occurs by promoting osteogenic differentiation of BMSCs, we first measured the expression of osteogenic differentiation-related factors. The results showed that carvacrol treatment significantly promoted the expression of RUNX2, Osterix, OCN, and OPN in Ti-inhibited mouse BMSCs (Figure 2A-D). To further elucidate the role of carvacrol in the function of BMSCs, we next examined ALP activity. Ti inhibited the ALP activity of BMSCs, whereas carvacrol treatment reversed this result (Figure 2E). To evaluate whether carvacrol promotes the mineralization capacity of BMSCs, we performed ARS staining. Ti significantly reduced the osteogenic capacity of BMSCs, whereas carvacrol reversed these effects (Figure 2F). Taken together, these observations suggest that carvacrol effectively alleviates the Ti-inhibited osteogenic differentiation of BMSCs. Furthermore, compared with the control group, the expression of SIRT1 was significantly decreased in the Ti group, whereas the addition of carvacrol treatment significantly upregulated the expression level of SIRT1, which is consistent with the results in mice (Figure 2G). Therefore, we speculate that carvacrol may induce osteogenic differentiation of BMSCs and alleviate osteolysis by upregulating the expression of osteogenic differentiation-related factors and SIRT1. However, further validation is needed to confirm this hypothesis.

Carvacrol alleviates osteogenic differentiation of Ti-inhibited BMSCs by upregulating SIRT1

To further confirm the regulatory role of carvacrol on SIRT1 during osteogenic differentiation of BMSCs, we transfected BMSCs with LV-sh-SIRT1 or LV-shRNA (negative control). Compared with the Ti + Carvacrol + LV-shRNA group, the significant decrease of SIRT1 expression in BMSCs transfected with LV-sh-SIRT1 indicates the efficacy of transfection (Figure 3A). Carvacrol treatment significantly upregulated the expression of osteoblast differentiation-related factors (RUNX2, Osterix,OCN, OPN), and LV-sh-SIRT1 partially reversed the effect of carvacrol (Figure 3B-E). Further analysis of the osteogenic potential of BMSCs revealed that carvacrol promoted ALP activity and mineralization ability, which was partially reversed by LV-sh-SIRT1 (Figure 3F-G). Collectively, these results suggest that carvacrol alleviates titanium inhibition of osteogenic differentiation in BMSCs by upregulating SIRT1.

Carvacrol promotes SIRT1 expression through NEAT1

To further investigate the promoting effect of carvacrol on SIRT1 expression, we used software prediction and combined with existing literature to identify potential lncRNAs that may interact with SIRT1 and play a role in osteolysis or osteoblast differentiation processes. The selected lncRNAs are as follows: TSIX [36], DANCR [37], KCNQ1OT1 [38], NEAT1 [39], and XIST [40]. Therefore, we cultured BMSCs under the treatment of carvacrol at a concentration of 25 μM and evaluated the expression levels of lncRNAs by RT-qPCR after 48 hours of incubation. The results showed a significant upregulation of NEAT1 expression in response to carvacrol (Figure 4A). Therefore, we hypothesize that carvacrol may affect SIRT1 by regulating NEAT1.
To determine the interaction between NEAT1 and SIRT1, we performed an RNA pull-down assay using biotinlabeled lncRNA-NEAT1 (Bio-NEAT1) and detected SIRT1 in the pulled-down complexes by Western blotting. The results showed that SIRT1 was enriched in the BioNEAT1 pulled-down complexes compared to the BioNC (Figure 4B). In addition, we performed a RIP assay using specific antibodies to precipitate SIRT1, followed by RT-qPCR to determine the levels of NEAT1 in the precipitated material. The results showed a significant enrichment of NEAT1 in the immunoprecipitated complex of SIRT1 (Figure 4C). Furthermore, we used LV-sh-NEAT1 plasmids to transfect BMSCs. Remarkably, the downregulation of NEAT1 resulted in a significant decrease in the expression level of SIRT1 (Figure 4D), providing further evidence for the interaction between NEAT1 and SIRT1.
We then investigated whether carvacrol affects SIRT1 expression through NEAT1. BMSCs were divided into the following groups: control, Ti, Ti + carvacrol, Ti + carvacrol + LV-shRNA, and Ti + carvacrol + LV-sh-NEAT1. Western blot analysis revealed that carvacrol significantly promoted the expression of SIRT1 protein, but this promotion was altered in the presence of LV-sh-NEAT1 (Figure 4E). In addition, RT-qPCR analysis of the calvarial samples in Figure 1 showed that NEAT1 expression was significantly downregulated in the Ti + PBS group compared with the sham + PBS group. However, the introduction of carvacrol resulted in the upregulation of NEAT1 levels (Figure 4F). The data presented strongly support the notion that NEAT1 plays a critical role in facilitating the upregulation of SIRT1 expression induced by carvacrol.

NEAT1 stabilizes the expression of SIRT1 by inhibiting its ubiquitination levels

Previous studies have shown that lncRNAs can regulate the expression of SIRT1 through ubiquitination pathways [45-47]. Based on this evidence, we hypothesized that NEAT1 may modulate SIRT1 expression through ubiquitination. To investigate this hypothesis, we used cycloheximide (CHX) treatment to evaluate the effect of NEAT1 on SIRT1 protein stability. BMSCs were transfected with LV-NEAT1 plasmid followed by treatment with 10 μg/mL CHX for 0, 3, 6, and 9 hours. Our results showed that NEAT1 overexpression significantly inhibited the timedependent degradation of SIRT1 protein in BMSCs under CHX treatment (Figure 5A). Next, BMSCs were treated with (or without) 10 μM proteasome inhibitor MG132 for 4 hours. Western blot analysis showed that MG132 significantly increased the protein level of SIRT1, confirming the successful blockade of proteasome-mediated protein degradation (Figure 5B). In the absence of MG132 treatment, NEAT1 overexpression resulted in a significant increase in SIRT1 protein levels. However, when BMSCs were pretreated with MG132, the effect of LV-NEAT1 on SIRT1 protein levels was relatively modest. This observation supports the notion that the effect of NEAT1 on SIRT1 is dependent on proteasome-mediated degradation (Figure 5B). The ubiquitination assay further confirmed that NEAT1 overexpression inhibited the ubiquitination modification of SIRT1 (Figure 5C). According to the literature, the E3 ubiquitin ligase SMURF2 interacts with SIRT1 and facilitates its ubiquitination and degradation [48]. We hypothesized that NEAT1 regulates the ubiquitination levels of SIRT1 by modulating the interaction between SIRT1 and SMURF2. The IP and IB assay revealed a significant decrease in the enrichment of SIRT1 within the SMURF2 immunoprecipitation complex in BMSCs transfected with LV-NEAT1 (Figure 5D). Overall, these results suggest that NEAT1 plays a pivotal role in stabilizing SIRT1 and effectively inhibiting its ubiquitination modification levels.

Carvacrol promotes SIRT1 expression by upregulating NEAT1 and alleviates Ti-inhibited osteoblast differentiation

We first examined the expression levels of NEAT1, SIRT1, and osteogenic differentiation-related factors in BMSCs after different treatments. The results showed that knockdown of NEAT1 significantly inhibited the carvacrolinduced increase in NEAT1 expression, providing compelling evidence for the efficacy of transfection (Figure 6A). Furthermore, LV-sh-NEAT1 suppressed the carvacrolpromoted expression of SIRT1, RUNX2, Osterix, OCN, and OPN, whereas LV-SIRT1 reversed the effects of LVsh-NEAT1 (Figure 6B-F). Next, we performed osteogenic differentiation of BMSCs in different treatment groups. The ALP activity was significantly increased in the Ti + Carvacrol group, while the Ti + Carvacrol + LV-shNEAT1 group showed a significant decrease. As expected, ALP activity increased again in the Ti+Carvacrol+LV-shNEAT1+LV-SIRT1 group (Figure 6G). The ARS staining results showed enhanced mineralization ability in BMSCs of the Ti + Carvacrol group, whereas decreased mineralization ability was observed in the Ti + Carvacrol + LVsh-NEAT1 group. After transfection with LV-SIRT1, the mineralization ability was restored (Figure 6H). These results demonstrate that silencing of NEAT1 can inhibit the osteogenic effect of carvacrol, but this inhibition can be reversed by overexpression of SIRT1.

Carvacrol alleviates osteolysis in aged mice by upregulating NEAT1

We randomly divided 18-month-old mice into four groups: Ti + PBS, Ti + Carvacrol, Ti + Carvacrol + LVshRNA, and Ti + Carvacrol + LV-sh-NEAT1 groups. Two weeks after surgery, we collected calvarial tissue from the mice and performed micro-CT scanning, quantitative micro-CT analysis, and Western blotting. Analysis of the three-dimensional reconstructed images revealed extensive erosion and reduced thickness of the calvarial bone in the Ti + PBS group. However, application of carvacrol ameliorated calvarial erosion, and injection of LV-sh-NEAT1 partially reversed the effects of carvacrol (Figure 7A). Furthermore, carvacrol also upregulated bone density and BV/TV levels, which were partially counteracted by LV-sh-NEAT1 injection (Figure 7B-C). This suggests that NEAT1 may play a critical role in mediating the protective effects of carvacrol on calvarial bone erosion. Additionally, LV-sh-NEAT1 attenuated the carvacrol-induced increase in the protein levels of SIRT1, RUNX2, Osterix, OCN, and OPN (Figure 7D). These findings demonstrate that carvacrol alleviates osteolysis in aged mice by upregulating NEAT1.

This study demonstrated the potential bone-protective effects of carvacrol. Carvacrol enhances SIRT1 expression by upregulating NEAT1, thereby alleviating the inhibitory effect of titanium particles on osteoblast differentiation. In summary, carvacrol may have potential therapeutic effects on osteolysis in aged mice by promoting osteogenic differentiation of BMSCs. This finding provides a promising direction for the development of new treatment strategies and drugs (Figure 8).

Osteolysis refers to a pathological condition characterized by increased bone resorption and decreased bone formation in bone tissue, resulting in destruction and loss of bone mass [49]. This pathological process is typically triggered by wear particles following joint replacement surgery [6]. The probability of wear particle-induced osteolysis is significantly higher in elderly patients [50]. Therefore, inducing osteogenesis promotion may be one of the most promising therapeutic approaches to treat osteolysis. BMSCs have the potential for osteogenic differentiation, which can promote bone formation and repair by differentiating into osteoblasts and osteocytes [51, 52]. Therefore, the development of drugs that enhance the osteogenic differentiation capacity of BMSCs offers a new therapeutic approach for the treatment of osteolysis.
Previous studies have revealed the potential regulatory effects of carvacrol on osteoblasts and osteoclasts [18, 21]. Additionally, carvacrol has been suggested to reduce tissue damage and bone loss caused by ligature-induced periodontitis [53, 20]. However, the potential of carvacrol in regulating osteogenic differentiation for the treatment of particle-induced osteolysis has not been explored until now.
In this study, we demonstrated for the first time that carvacrol attenuated particle-induced osteolysis in an osteolysis model of aged mice. It is worth noting that the accumulation of wear particles (Ti particles) around implants can lead to local bone resorption and ultimately calvarial erosion [54, 55]. As expected, micro-CT imaging, bone density and BV/TV statistical analysis showed that carvacrol exhibited a protective effect against Ti-induced bone destruction. Furthermore, a recent study by Spalletta et al. reported the involvement of carvacrol in the differentiation of umbilical cord mesenchymal stem cells [22]. Therefore, in this study, we also evaluated the effect of carvacrol on the osteogenic differentiation and osteogenesis of titanium-inhibited BMSCs. The results showed that titanium treatment suppressed cell mineralization, activity of osteogenic differentiation marker ALP, and expression of osteogenic differentiation-related factors (RUNX2, Osterix, OCN, and OPN). However, carvacrol was able to reverse these effects induced by titanium.
The reduction of SIRT1 levels and activity is associated with the progression of osteolysis [24, 25, 56, 57]. Furthermore, SIRT1 is involved in the differentiation of BMSCs in aged mice and promotes bone formation [58, 59]. SIRT1 effectively improves osteolysis by enhancing the osteogenic potential of BMSCs [27]. Our research showed that carvacrol upregulates SIRT1 levels, which is consistent with previous findings [34]. A previous study showed that melatonin promotes the osteogenic potential of BMSCs by regulating SIRT1-mediated antioxidant properties [29]. Eldecalcitol inhibits BMSC senescence by modulating the SIRT1-Nrf2 pathway [60]. Based on these findings, we further investigate whether carvacrol regulates SIRT1 to influence the osteogenic differentiation of BMSCs. The inhibitory effect of sh-SIRT1 on carvacrolinduced osteogenic differentiation of BMSCs was confirmed by ALP activity assay, ARS staining, and RT-qPCR analysis.
Many studies have highlighted the critical impact of lncRNAs on various processes. Increasing evidence suggests that lncRNAs are involved in BMSC osteogenic differentiation [61], bone formation [62], and osteoblast matrix mineralization [63]. To gain a deeper understanding of the molecular mechanism by which carvacrol regulates osteolysis, we screened several lncRNAs potentially involved in SIRT1 interaction and osteolysis. Surprisingly, only NEAT1 expression was regulated by the addition of carvacrol to the culture medium. LncRNA NEAT1 was found to activate BTK and regulate the NF-κB pathway, thereby participating in the process of wear particleinduced osteolysis [64]. Our results also show that inhibition of NEAT1 attenuates the protective effect of carvacrol on calvarial bone and is involved in osteolysis. LncRNAs can be involved in the regulation of gene expression through various mechanisms [65, 66], including the regulation of SIRT1 through ubiquitination [67, 46, 47]. To further investigate the relationship between NEAT1 and SIRT1, we performed RNA pull-down and RIP experiments. We found that NEAT1 stabilizes SIRT1 expression by inhibiting its ubiquitination modification. In addition, NEAT1 has been shown to promote osteogenic differentiation of human bone marrow mesenchymal stem cells [39]. The results of our study indicate that in Ti-induced in vitro BMSCs, LV-sh-NEAT1 inhibits the osteogenic differentiation promoted by carvacrol, while LV-SIRT1 reverses the effect of sh-NEAT1.

In conclusion, carvacrol promotes SIRT1 expression by upregulating NEAT1 and induces the expression of osteogenesis-related transcription factors. This mechanism may contribute to the promotion of osteogenic differentiation of BMSCs and the alleviation of osteolysis. However, this study has only been discussed in animal models, and there is a lack of further clinical research evidence.