Carnosol was purchased from Shanghai Shi Dande Services Ltd. and was dissolved in phosphate-buffered saline (PBS) solution as a stock solution. FBS, cell culture medium, penicillin and streptomycin were purchased from BioTNT (Shanghai, China).

Cell viability was assessed using an MTT kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Mouse bone marrow monocytes (BMMCs) and the mouse leukemia RAW264.7 cell line (Y-S Biotechnology) were seeded into a 96-well plate at a cell density of 1×10⁴ cells/well. Cells were treated with carnosol at doses of 0.1, 0.2, 0.5, 1, 2, 5 and 10 µM. BMMCs and RAW264.7 cells were cultured for 3 days at 37°C. Subsequently, 50 µg/µl MTT solution was added to each well and 0.04-0.1 N HCl in isopropanol was the solvent. Cellular activity was calculated by detecting the absorbance at 490 nm.

BMMCs were isolated from the femoral bone marrow of C57BL/6 mice (age: 4 weeks, 14.3-16.2g, n=3 per group). For the in vivo experiments another group of mice (age: 8 weeks, 21.5-23.1 g, n=3 per group) were used. BMMCs and RAW264.7 cells were cultured in low-glucose DMEM (Corning) at 37°C with 5% CO₂. Non-adherent cells were discarded by changing the medium every 72 h.

Third generation of cultured cells were seeded into 24-well plates and induced with 30 ng/m M-CSF (cat. no. 416-ML-050; R&D Systems, Inc.) and 100 ng/ml RANKL (cat. no. 462-TEC-010; R&D Systems, Inc.) at 37°C for 7 days. RANKL + M-CSF induction is named as RANKL group. Different concentrations of carnosol (0, 0.25, 0.5 and 1 µM) were added at 37°C for 7 days. Fixation was performed using 10% formalin for 5 min at room temperature. TRAP staining was performed according to the manufacturer's instructions (cat. no. PMC-AK04F-COS, Cosmo Bio) at 37°C for 30 min. TRAP-positive cells with more than three nuclei were classified as osteoclasts. RAW264.7 cells were plated at 4×10⁴ cells/ml in α-minimal essential media (HyClone; Cytiva) containing 10% FBS (BioTNT), penicillin and streptomycin, 20 ng/ml M-CSF and 10 ng/ml RANKL at 37°C. Following 7 days in culture, the cells were subjected to TRAP staining and imaged by light microscope (Olympus). Carnosol was also added on day 1, 3 and 5 of induction to explore its role in different stage.

To assess the formation of F-actin rings, BMMCs were fixed using 4% paraformaldehyde for 1 h at room temperature and then washed at least three times with PBS solution. BMMCs were subsequently incubated with fluorescein isothiocyanate (FITC)-phalloidin for 30 min followed by 4′6-diamidino-2-phenylindole for 10 min at room temperature (17). For assay of pit formation, BMMCs (1×10⁶/plate) were seeded into a bone biomimetic plate (Corning, Inc.). The resorbing area was visualized via optical light microscopy and quantified using ImageJ (v1.8.0, National Institutes of Health).

Total DNA was extracted from RAW264.7 cells using gDNA Extraction kits (Invitrogen; Thermo Fisher Scientific, Inc.) and PCR was performed as previously described (18). Takara Taq^™ (Takara) was used as DNA polymerase. The thermocycling conditions (30 cycles) were: 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. The primers used for PCR analysis were as follows: Recombinant nuclear factor of activated T cells (NFATc1) forward (F), 5′-ATGACGGGGCTGGAGCAGGA-3′ and reverse (R), 5′-TTAGGAGTGGGGGGATCGTGC−3′; β-actin F, 5′-GTGACGTTGACATCCGTAAAGA-3′ and R, 5′-GCCGGACTCATCGTACTCC-3′. The percentage of agarose gel was 0.9% and ethidium bromide was used for visualisation. β-actin was used as reference gene. Image Lab (V6.1, Bio-RAD) was used for densitometry. For RT-qPCR, total RNA was also isolated using TRIzol reagent according to the manufacturer's protocol. Total RNA (1 mg) was reverse transcribed using the ThermoScript RT-PCR System (Vazyme Biotech Co., Ltd.) to produce first-strand complementary (c)DNA. PrimeScript RT Master Mix (Takara, Japan) was used to synthesized cDNA. The RT kit was used according to the manufacturer's protocol. The thermocycling conditions were: 37°C for 15 min and 85°C for 15 sec. For qPCR, cDNA was amplified using the Real-Time PCR Detection System (Roche 480; Roche Applied Science). TaqMan probe (Thermo Fisher Scientific, Inc.) was used as fluorophore. The thermocycling conditions were: initial denaturation (95°C for 10 min); 40 cycles of denaturation (95°C for 10 sec), annealing (60°C for 10 sec) and elongation (72°C for 30 sec); and final extension (72°C for 5 min). β-actin was used as the internal reference gene. 2^−∆∆Cq method was used for quantification (18). The data represents three independent experiments. The following primers were used for qPCR: NFATc1 F, 5′-ACCACTCCACCCACTTCTG-3′ and R, 5′-GCTGCCTTCCGTCTCATAG-3′; and β-actin F, 5′-GTCCCTCACCCTCCCAAAAG-3′ and R, 5′-GCTGCCTCAACACCTCAACCC−3′.

In vivo experiments were performed at Zhoupu Hospital (Shanghai, China) and were approved by the Ethics Committee of Zhoupu Hospital. Briefly, 8-week-old female C57BL/6 mice (n=20) were obtained from Slac Laboratory Animal (Shanghai, China). Animal health and behavior were monitored every day. The housing conditions were: Humidity 50%, temperature 21°C, light/dark cycles 12/12 h, and accessible to food and water. The mice were randomly divided into three groups, with six mice per group. The three treatments groups were as follows: i) Control or sham treatment; ii) ovariectomy (OVX) and treatment with PBS; and iii) OVX and treatment with carnosol (10 mg/kg). The dose was determined using a preliminary study to test the toxicity of carnosol (data not shown). Using a 10 mg/kg dose, mice displayed no significant toxicity and abnormalities. Mice were injected intraperitoneally with PBS or carnosol daily. After 6 weeks of treatment when OVX-induced bone loss was significant, as previously described (18), all mice were euthanized using pentobarbital sodium (100 mg/kg via intraperitoneal injection). Failure to detect respiration and no heartbeat for a period of >5 min was used to confirm death. Mouse femurs were isolated and fixed with 4% paraformaldehyde for 24 h at room temperature. and 1 ml blood was collected via cardiac puncture for ELISA.

The effects of carnosol (1 µM) on nuclear translocation of p65 were examined in RAW264.7 cells. Immunofluorescence staining was conducted as described previously (19). Osteoclasts were fixed with 4% paraformaldehyde for 1 h at room temperature and washed at least three times with PBS solution. Subsequently, cells were incubated for 20 min with 0.2% Triton X-100 at room temperature and then blocked with 1% BSA (ThermoFisher Scientific, USA) for 30 min at room temperature. Cells were then incubated with a biotin-conjugated p65 IgG antibody (1:2,000; cat. no. 10745-1; ProteinTech Group, Inc.) for 60 min at 37°C and a fluorescein-conjugated streptavidin secondary antibody for 30 min at 37°C (1:2,000; cat. no. 00003-2; ProteinTech Group, Inc.). Cells were counterstained with propidium iodide for 15 min at 37°C. The inverted fluorescence microscope (Olympus) was used to image cells and ImageJ (v1.8.0, National Institutes of Health).

RAW264.7 cells were treated with RANKL or carnosol (1 µM) in 6-well plates for 0, 5, 10, 15, 30 and 60 min. The protein was extracted by RIPA Lysis Buffer (cat. no. BL504A; Biosharp) and assessed by the BCA protein assay kit (cat. no. BL521A; Biosharp). 100 µg protein was loaded per lane. The percentage of gel for Histone H3 was 15% and for others were 12%. PVDF membrane was used for transfer. The blots were probed with primary monoclonal antibodies against mouse TRAP (1:2,000; cat. no. ab191406; Abcam), cathepsin K (1:2,000; cat. no. ab187647; Abcam), MMP-9 (1:2,000; cat. no. ab76003; Abcam), p65 (1:2,000; cat. no. 10745-1; ProteinTech Group, Inc.), phosphorylated (p)-p65 (1:2,000; cat. no. ab76302; Abcam), IκBa (1:5,000; cat. no. ab32518; Abcam), β-actin (1:2,000; cat. no. GB11001; Servicebio, Inc.) and Histone H3 (1:1,000; cat. no. ab176842; Abcam). Following primary incubation (4°C, overnight), membranes were washed by TBST (0.1% Tween-20) and incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 h at 4°C (1:10,000; cat. no. ab205718; Abcam). After washing, the bands were detected via ECL Western Blotting substrate (cat. no. BL520A, Biosharp) and were imaged using the Bio-Rad ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.).

Mouse femurs were isolated and fixed using 4% paraformaldehyde for 3 days at room temperature and subsequently decalcified over 3 weeks at room temperature. The femurs were sliced into 4-µm-thick sections. Hematoxylin and eosin (H&E) staining was subsequently performed as previously described and was used to measure the area of the femoral trabecular bone (20). The light microscope (Olympus) was used to observe the results and ImageJ (v1.8.0, National Institutes of Health) was used for quantification.

Mouse femurs were isolated for 6 weeks following OVX and then analyzed via high solution X-ray micro-CT (Skyscan, Germany). For each femur, 200 sections (8 µm) below the growth plate were analyzed. The bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N) and bone surface area/total volume (BS/TV) were assessed by CTAn (V1.10, Skysan).

Blood samples were centrifuged at 4°C for 5 min at 1,509 × g and the serum were collected. IL-6 ELISA Kit (cat. no. BMS603-2, Invitrogen, USA), C-terminal telopeptide (CTX-1) ELISA Kit (cat. no. MBS458686, Mybiosource, USA), tartrate-resistant acid phosphatase type 5b (TRAcp5b) ELISA kit (cat. no. MBS163341, Mybiosource, USA) and osteocalcin (OCN) ELISA Kit (cat. no. MBS725134, Mybiosource, USA) were used for ELISA according to the manufacturer's protocol.

Data are presented as the mean ± SD (n=3). Three or more groups were statistically compared using one-way ANOVA analysis followed by Tukey's post hoc test. Data were analyzed using SPSS (V21.0, IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

The MTT assay was used to investigate the cytotoxicity of carnosol. Cells were treated with different concentrations of carnosol and the results demonstrated that below a concentration of 1 µM, carnosol was not cytotoxic in BMMCs or RAW264.7 cells, which represent established cellular models of osteoclastogenesis. The concentrations higher than 1 µM significantly inhibited cell viability in RAW264.7 cells and higher than 2 µM inhibited cell viability in BMMCs (Fig. 1A). The effects of carnosol on osteoclastogenesis were further examined. Cells were induced with RANKL and M-CSF and then treated with 0, 0.25, 0.5 and 1 µM carnosol. After 7 days, the number of TRAP-positive cells was calculated. The results demonstrated that TRAP-positive cells were abundant following induction with RANKL and M-CSF for 7 days (Fig. 1B). However, the number of TRAP-positive cells decreased following treatment with 0.25 µM carnosol. This decline was statistically significant following treatment with 0.5 and 1 µM carnosol compared with the control. These results indicated that carnosol suppressed osteoclastogenesis in a dose-dependent manner. The inhibitory effects of carnosol on osteoclastogenesis were also confirmed in RAW264.7 cells (Fig. 1C).

Bone resorption assays were performed to identify the role of carnosol in osteoclast functioning. The results demonstrated that after induction by RANKL and M-CSF, mature osteoclasts were able to form pits on a bone biomimetic synthetic plate. However, following the administration of carnosol, the resorbed area was reduced significantly compared with the control (Fig. 2A).

The effects of carnosol on the formation of the cytoskeleton was investigated by assessing the formation of F-actin rings during osteoclastogenesis, an essential process of osteoclast formation. F-actin ring formation is the prerequisite for osteoclast resorption. The ring forms a sealing zone with unique low pH microenvironment in which bone resorption occurs. Therefore, F-actin ring formation reflects the bone resorption function of osteoclasts (17,21). BMMCs were induced with RANKL and M-CSF and then treated with 0.25, 0.5 and 1 µM carnosol. FITC-phalloidin staining indicated that the formation of F-actin rings was significantly reduced in a dose-dependent manner compared with the control (Fig. 2B). This result confirmed that carnosol may inhibit the formation of F-actin rings during osteoclastogenesis.

Western blotting experiments demonstrated that expression levels of osteoclastogenesis-related proteins, including TRAP, cathepsin K and MMP-9, were markedly decreased following treatment with carnosol compared with RANKL group (Fig. 2C). In conclusion, these results indicated that carnosol may suppress osteoclast functioning in vitro.

To identify more precisely when osteoclastogenesis is influenced by carnosol, BMMCs and RAW264.7 cells were treated with carnosol at different stages of differentiation. Carnosol was added on day 1, 3 and 5 of induction. The purpose of this experiment was to determine the osteoclast differentiation stage where carnosol takes effect (18,21,22). Osteoclast differentiation was significantly inhibited after carnosol treatment compared with the control at days 1, 3 and 5, especially on the first day. Moreover, treatment was markedly less effective during the later stages of cell differentiation compared with the earlier stages, whereby inhibition was lowest on day 5 for RAW 264.7 cells BMMCs (Fig. 3A and B). NFATc1 is an essential transcription factor for osteoclast differentiation via its regulation of the expression of osteoclast-associated genes (23). PCR results of NFATc1 in RAW264.7 cells showed that addition of carnosol decreased the level of NFATc1 compared with the RANKL group (Fig. 3C). These results indicated that carnosol may inhibit osteoclast differentiation at an early stage rather than at a late stage.

The NF-κB signaling pathway is essential during osteoclastogenesis and p65 is one of the most important proteins in this pathway (23). Immunofluorescence staining of RAW264.7 cells was performed to investigate the effects of carnosol on the nuclear localization of p65. The results demonstrated that p65 was mainly distributed in the cytoplasm in the absence of RANKL (Fig. 4A). However, after cells were induced by RANKL, p65 was translocated to the nucleus and treatment with carnosol significantly inhibited nuclear translocation compared with the RANKL group. Western blotting was performed to detect the phosphorylation of p65. The results demonstrated that carnosol markedly inhibited the phosphorylation of p65 following RANKL treatment compared with the RANKL group (Fig. 4B). Subsequently, nuclear p-p65 and p65 protein expression levels were investigated. The results demonstrated that following RANKL induction, nuclear p-p65 and p65 protein expression levels were markedly increased, whereas carnosol treatment inhibited this increase compared with the RANKL group (Fig. 4C). The level of IκBα was also decreased by carnosol treatment compared with the RANKL group. These data indicated that carnosol may suppress the activation of the NF-κB signaling pathway during osteoclastogenesis.

To investigate the function of carnosol in vivo, an OVX mouse model was used to mimic PMOP in human clinical practice. As previously reported, carnosol treatment was administered just after the OVX (18,22). Mice were injected intraperitoneally with carnosol (10 mg/kg) daily for 6 weeks. The bone mass of the femurs was assessed, with H&E staining demonstrating that 6 weeks after OVX the femoral trabecular bone mass was significantly decreased compared with the sham group. However, carnosol treatment significantly attenuated bone loss in OVX mice compared with the OVX only group (Fig. 5A). The results of the micro-CT analysis were consistent with those of H&E staining. Following treatment with carnosol, femoral bone loss was significantly reduced compared with the OVX group, indicated by the increase of BMD, BV/TV, Tb.N and BS/TV (Fig. 5B and C). Serum TRAcp5b, CTX-1 and IL-6 levels were determined to assess osteoclast and osteoblast activity. Following carnosol treatment, TRAcp5b, CTX-1 and IL-6 levels decreased significantly compared with the OVX group (Fig. 5D), which indicated inhibition of osteoclastogenesis. Serum OCN levels were also investigated and the results demonstrated that carnosol displayed no significant impact on serum OCN levels in mice (Fig. 5E). These results indicated that carnosol may attenuate bone loss in OVX mice by suppressing osteoclast differentiation in vivo.