Female 5-month-old Sprague-Dawley rats were obtained from the Experimental Animal Centre of China Medical University (Shenyang, China). The animals were housed under standard laboratory conditions at a stable temperature (22–24°C) and a 12/12-h light/dark cycle. This study was approved by the Ethics Committee of China Medical University (Shenyang, China).

The rats were randomly divided into 3 groups (n=6 per group) as follows: the control group, the DXM group and the DXM + curcumin group. The rats in the DXM and DXM + curcumin groups received subcutaneous injections of dexamethasone (0.1 mg/kg/day, Tianjin Pharmaceutical Group Xinzheng Co., Ltd., Zhengzhou, China) for 60 days. The rats in the control group were injected subcutaneously with the vehicle only. Bone mineral density (BMD) was measured at the proximal tibia to confirm that the model had been successfully established. The rats in the DXM + curcumin group received an intragastric administration of curcumin (100 mg/kg/day, Dalian Meilun Biology Technology Co., Ltd., Dalian, China) for a further 60 days. The rats in the control and DXM groups received the vehicle only [0.5% sodium carboxymethyl cellulose (CMC-Na)]. Following treatment, BMD was measured again, and the rats were euthanatized by an overdose of pentobarbital. Blood was collected from the inferior vena cava. The muscle around the femurs was removed using surgical scissors and the femurs were collected.

BMD was measured at the femurs by dual-energy X-ray absorptiometry bone densitometry using a Hologic QDR 4500 machine (Hologic, Bedford, MA, USA) and the accompanying experimental animal assessment software. BMD was represented as g/cm². All samples were measured 3 times, and the mean values were calculated. The coefficient of variation for the BMD measurements was 1.02%.

The activities of bone-specific ALP and TRAP were determined using commercial kits for ALP and TRAP activity assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. Briefly, the serum samples were mixed with the test solutions and at 37°C for the indicated periods of time. Finally, the absorbance at 520 nm (ALP) or 530 nm (TRAP) was read using a microplate reader (ELX-800; Bio-Tek Instruments, Winooski, VT, USA).

The serum levels of osteocalcin and CTX were determined using commercial ELISA kits specific to osteocalcin and CTX (USCN Life Science, Wuhan, China) according to the manufacturer's instructions. Briefly, the serum samples were diluted according to the manufacturer's instructions and incubated with antibody solutions specific for osteocalcin and CTX at 37°C for 1 h. After washing, streptavidin-HRP was added followed by incubation at 37°C for 30 min. The absorbance was measured following the addition of stop solution by a microplate reader (ELX-800; Bio-Tek Instruments) at 450 nm.

The femoral samples were fixed in 4% paraformaldehyde for 24 h, dehydrated using a series of ethanol and embedded in paraffin. Paraffin blocks were cut into 5-µm-thick slices, deparaffinized in xylene and hydrated using a series of ethanol. The femoral tissue sections were then stained with hematoxylin and eosin (H&E) solution (Solarbio Science & Technology Co., Ltd., Beijing, China) and observed under a light microscope (DP73; Olympus, Tokyo, Japan).

Calvarial bones from 40 24-h-old neonatal Sprague Dawley rats (Experimental Animal Centre of China Medical University, Shenyang, China) were used for primary osteoblast cell culture. Following euthanasia, the calvarial bones were removed and cut into 1–3-mm³ sections and digested in 1% trypsin and 0.2% collagenase type II at 37°C for 30 min. The digestion was then terminated, and the mixture was filtered and centrifuged at 300 × g for 10 min. The supernatant was discarded and the cells were washed twice using Tris-buffered saline (PBS) and resuspended in Dulbecco's modified essential medium (DMEM, Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 2 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were plated into the indicated culture plates and maintained in a humidified atmosphere of 5% CO₂–95% air at 37°C.

The cells were seeded into a 96-well plate at 4×10³ cells/well. The cells were exposed to DXM (0, 1, 10, 100 and 1,000 nM) for 24 h. Subsequently, the cells were seeded in a 96-well plate at 4×10³ cells/well and treated with curcumin (0, 0.5, 1 and 2 µM) for 24 h. The cells were then seeded in a 96-well plate at 4×10³ cells/well and pre-treated with curcumin (0.5, 1 and 2 µM) for 2 h, and then exposed to DXM (100 nM) for 24 h. Finally, the cells were seeded in a 96-well plate at 4×10³ cells/well and treated with curcumin (0.5, 1 and 2 µM and DXM 100 nM simultaneously for 24 h.

Following treatment, MTT (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to each well followed by incubation at 37°C for 4 h. The supernatant was then removed, and 200 µl dimethyl sulfoxide (DMSO) were added to solve the precipitate, and the absorbance at 570 nm was read using a microplate reader (ELX-800, Bio-Tek Instruments).

Total mRNA from the femoral tissues or primary rat osteoblasts was isolated using an RNA Simple Total RNA kit (Tiangen Biotech, Co., Ltd., Beijing, China) according to the manufacturer's instructions. cDNA was reverse transcribed with oligonucleotide primer using super Moloney Murine Leukemia Virus (M-MLV) (BioTeke, Beijing, China) in a 20-µl system. The qPCR reactions were performed in a 20-µl system containing 10 µl SYBR-Green Master Mix (Tiangen Biotech Co., Ltd.), 0.5 µM of forward and reverse primers, and 1 µl template cDNA on an Exicycler™ 96 real-time quantitative thermal block (Bioneer, Daejeon, Korea). The sequences of the primers are listed in Table I. Data were normalized to β-actin and analyzed using the comparative threshold cycle (CT) method.

The bone tissues or cells were homogenized in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). Following centrifugation at 12,000 × g at 4°C for 10 min, the supernatant was collected and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Proteins were then separated on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using the wet transfer method. The membranes with target proteins were blocked with 5% non-fat milk at room temperature for 1 h, and then incubated with the primary antibodies at 4°C overnight. After a washing stage, the membranes were subsequently incubated with horseradish peroxidase-labeled goat anti-rabbit or donkey anti-goat IgG (1:5,000, Beyotime Institute of Biotechnology) at 37°C for 45 min. The protein blots were visualized using enhanced chemiluminescence (7 Sea Pharmtech, Shanghai, China) and exposed to Fuji Rx 100 X-ray film (Fuji Photo Film, Tokyo, Japan). The gray values of the blots were analyzed using Gel-Pro-Analyzer (Media Cybernetics, Bethesda, MD, USA). β-actin was used as a loading control. The primary antibodies used in this study were as follows: phosphorylated glycogen synthase kinase (p-GSK-3β antibody (1:200, sc-11757; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GSK-3β antibody (1:200, sc-9166; Santa Cruz), OPG antibody (1:500, bs-0431R; Bioss, Beijing, China) and receptor activator for nuclear factor-kappa B ligand (RANKL) antibody (1:500, bs-0747R; Bioss, Beijing, China).

The cells were cultured on glass coverslips and fixed in 4% paraformaldehyde for 15 min. Slips were washed in PBS 3 times and incubated with 0.1% Triton X-100 for 30 min at room temperature. After a washing stage, the slips were blocked in goat serum for 15 min at room temperature. The cells were then incubated with anti-β-catenin antibody (1:200, BA0426; Boster, Wuhan, China) at 4°C overnight. Subsequently, the cells were washed with PBS and incubated with cy3-labeled goat anti-rabbit secondary antibody (1:200, Beyotime Institute of Biotechnology) at room temperature for 1 h. The cells were then co-stained with DAPI and then observed under a fluorescence microscope (BX53; Olympus).

Statistical analyses were conducted with SPSS19 software (IBM, New York, NY, USA). Data are expressed as the means ± standard deviation (SD). Differences between groups were evaluated by one-way ANOVA followed by Fisher's least significant difference (LSD) test. A P-value <0.05 was considered to indicate a statistically significant difference.

We measured the BMD of rats before and after treatment with curcumin. As illustrated in Fig. 1A, after 60 days of DXM administration, the BMD of the rats was significantly decreased compared with the rats in the control group (P<0.01). After a further 60 days of treatment with curcumin, the BMD of the femurs increased significantly (P<0.01 vs. DXM group and DXM + curcumin group post-treatment), and the value was similar to that of the control group (P>0.05).

Histological changes were examined by H&E staining. In the control group, the femurs exhibited a complete trabeculae structure and ordered arrangement of the trabeculae. In the DXM group, significantly reduced and thinning trabeculae and small numbers of empty bone lacunae were observed. Treatment with curcumin markedly reversed these changes (Fig. 1B).

The osteocalcin content was examined as a bone formation biomarker, and the CTX content was examined as a bone resorption biomarker. As shown in Fig. 2, the osteocalcin levels were markedly decreased and the CTX levels were markedly increased in the serum following the administration of DXM. However, treatment with curcumin significantly reversed these changes.

The mRNA expression levels of key proteins in the Wnt signaling pathway were measured by RT-qPCR. Following exposure to DXM, the mRNA expression levels of Wnt, β-catenin and LRP5 were significantly downregulated, whereas the mRNA expression levels of the two receptor inhibitors, sclerostin (SOST) and Dickkopf-1 were upregulated (Fig. 3A). In addition, the phosphorylation of GSK-3β, a cytosolic Wnt signaling inhibitor, was inhibited by DXM (Fig. 3B). However, treatment with curcumin effectively reversed these changes. These results indicated that DXM induced the inhibition of Wnt signaling in the femurs of rats, and that curcumin, at least partly, reversed this inhibition.

The effects of DXM and curcumin on the viability of primary osteoblasts were evaluated by MTT assay. As shown in Fig. 4A and B, exposure to DXM at 100 and 1,000 nM markedly decreased the viability of the osteoblasts, whereas at all concentrations (0.5, 1 or 2 µM) curcumin caused no toxicity to osteoblasts. Subsequently, the effects of curcumin on the viability of DXM-stimulated osteoblasts were evaluated. In the first experiment, the cells were pre-treated using the indicated concentrations of curcumin for 2 h and then exposed to 100 nM DXM for 24 h. In this experiment, curcumin at 1 and 2 µM significantly increased cell viability in a dose-dependent manner (Fig. 4C). In the second experiment, the cells were treated with the indicated concentrations of curcumin and 100 nM DXM simultaneously. In this experiment, curcumin also inhibited the toxic effects of DXM on primary osteoblasts in a dose-dependent manner, although the values were slightly lower than those in the first experiment (Fig. 4D).

Based on the results of MTT assay, we treated the cells with curcumin and DXM simultaneously in order to perform the following experiments. As expected, DXM at 100 nM induced a marked decrease in ALP activity in the primary osteoblasts. Curcumin at 2 µM significantly reversed this decrease, but exerted little effect on normal cells (cells not stimulated with DXM; Fig. 5A). In addition, the mRNA expression levels of osteoblast differentiation-associated proteins were measured. As illustrated in Fig. 5B, the mRNA expression levels of osteoclacin, collagen, type 1, alpha 1 (Col1A1), osteonectin, runt-related transcription factor-2 (Runx2) and osterix were markedly downregulated following stimulation with DXM. We also examined the expression of two crucial factors related to osteogenesis, OPG and RANKL. DXM induced the marked downregulation of OPG and the upregulation of RANKL mRNA and protein expression (Fig. 5C and D). Curcumin at 2 µM partly, but still significantly, reversed the effects of DXM on the expression of these genes.

The effects of curcumin on the Wnt signaling pathway were examined in vitro. In line with our in vivo experiments, DXM downregulated the mRNA expression of Wnt, β-catenin and LRP5, and upregulated the mRNA expression of Dickkopf-1 and SOST (Fig. 6A). In addition, GSK phosphorylation was inhibited by stimulation with DXM (Fig. 6B). These changes were partly reversed by treatment with curcumin. In addition, we used immunofluorescence staining in order to examine β-catenin trafficking in osteoblasts (Fig. 6C). In the control primary osteoblasts (no treatment), we observed a strong intranuclear staining of β-catenin, reflecting activated Wnt/β-catenin signaling. Exposure to DXM reduced the intranuclear staining of β-catenin, representing the significant inhibition of Wnt/β-catenin signaling. Treatment with curcumin markedly, although not completely, restored the intranuclear staining of β-catenin. Curcumin did not affect β-catenin nuclear staining in the normal osteoblasts.