Six-week-old male C57BL/6J mice were obtained from the Shanghai Laboratory Animals Center (SLAC; Shanghai, China) and were allowed to acclimate to the environment for 1 week. All experimental procedures were performed in accordance with the Guidelines of the Department of Orthopaedics at Gongli Hospital of Pudong New Area (Shanghai, China) on Animal Care. This study was approved by the Ethics Committee of the Department of Orthopaedics at Gongli Hospital of Pudong New Area. All chemicals and reagents were purchased from Sigma-Aldrich Canada Co., (Oakville, Ontario, Canada), except where noted otherwise.

The mice were randomly divided into four groups: i) vehicle group mice (n=8) were injected intramuscularly with saline instead of DXM as controls; ii) the mice in the DXM group were injected intramuscularly with 5 mg/kg body weight DXM three times a week for 12 weeks (n=8); iii) the mice in the ALN group received alendronate orally at a dose of 0.1 mg/kg/day in combination with DXM for 12 weeks (n=8); and iv) the mice in the PS group received daily drinks of aqueous extract of pomegranate seed (AE-PS) in combination with DXM for 12 weeks (n=8). Pomegranate seeds (100 g) were chopped in a mortar and extracted in water (1,000 ml) by means of agitation for 10 min at room temperature. Supernatants were filtered out and the filtrates were given to the mice daily as drinking water. The pomegranates were purchased from the Chinese herbal medicine market in Nanjing (Jiangsu, China).

The concentrations of calcium (Ca) and testosterone in the serum as well as creatinine (Cr) in the urine were measured by ELISA using a microplate reader (BioTek, Winooski, VT, USA). Serum was collected by cardiac exsanguination under light ether anesthesia. Urine (24 h) was collected from the mice using metabolic cages. The level of urine Ca was corrected by the concentration of urine Cr. The serum levels of bone-specific alkaline phosphatase (ALP-b), testosterone, tartrate-resistant acid phosphatase (TRAP)-5b, C-terminal telopeptide of type I collagen (CTX) and osteocalcin were detected using a rat bioactive parathyroid hormone (PTH) enzyme-linked immunosorbent assay (ELISA; Immutopics, Inc., San Clemente, CA, USA) with an ELISA reader (MD SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA).

The tibias were incinerated at 800°C for 6 h and the ash weighed. Ten milligrams of bone ash was then dissolved in 1 ml of 37% HCl and diluted with Millin-Q water. The Ca content was determined using the same kits as those used for the serum and urine calcium assays (Biosino, Beijing, China).

Mice were sacrificed by cardiac exsanguination under light ether anesthesia. After cardiac exsanguination, the tibias were collected with all soft tissue removed and stored at −80°C. A total of 8 tibias were used for histomorphological analysis. The right tibia was used for histomorphological analysis, and the left tibia was used for the RT-PCR and western blot analysis (n=8 in each group). The tibias were decalcified in 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0), followed by dehydration through a graded series of ethanol solutions and then embedded in paraffin according to standard histological procedures (22). Sections, 5 µm in thickness, were cut and stained with hematoxylin and eosin (H&E; Xinfan, Shanghai, China), and visualized under a microscope (Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany).

TRAP staining was used for the identification of osteoclasts according to the manufacturer's instructions (387-A; Sigma-Aldrich, St. Louis, MO, USA). The number of TRAP-positive osteoclasts was counted to quantify the number of osteoclasts below the growth plates in the distal metaphysis of the femur. The assays were scored by averaging the number of osteoclasts per low-power field in at least four fields per slide, and visualized under a microscope (Leica DM 2500; Leica Microsystems GmbH).

After cardiac exsanguination, the kidneys were collected with all soft tissue removed and stored at −80°C. The kidneys of each animal were crushed under liquid nitrogen conditions and RNA extraction was performed using TRIzol according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The synthesis of cDNAs was performed by reverse transcription reactions with 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) with oligo(dT) 15 primers (Fermentas, Vilnius, Lithuania) as described by the manufacturer's instructions. The first strand cDNAs served as the template for the regular PCR performed using a Real-Time PCR System (ABI 7300; Applied Biosystems Life Technologies, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the data to determine the relative expression of the target genes. The reaction conditions were set according to the kit instructions. PCR was performed with the following primers: calcium sensing receptor (CaSR), forward, 5′-TAGGCATTCTGTAATAGCT-3′ and reverse, 5′-CTGGGCTGCTCAGTCGG-3′. The PCR conditions were as follows: denaturation of cDNA at 95°C for 3 min, denaturation at 95°C for 15 sec; annealing 56°C for 20 sec; extension at 72°C for 20 sec. The amplification cycle number was 40 for the target gene.

The tibias, kidney and duodenum were homogenized and extracted in NP-40 buffer, followed by 5–10 min of boiling and centrifugation (10,000 × g, at 4°C) in order to obtain the supernatant. The samples containing 50 µg of protein were separated on 10% SDS-PAGE gel, transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following antibodies: transient receptor potential vanilloid (TRPV)5 (sc-398345; 1:500), TRPV6 (sc-28763; 1:500), osteoprotegerin (OPG; sc-8468; 1:1,000) and receptor activator of nuclear factor-κB ligand (RANKL; sc-7628; 1:2,000), CaSR (sc-32181; 1:500), calbindin-D9k (CaBP-9k; sc-367381; 1:500), plasma membrane Ca²⁺-ATPase1 (PMCA1; sc-16488; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), overnight at 4°C. After three washes with TBST, the membranes were incubated with secondary immunoglobulins (Igs) conjugated to IRDye 800CW Infrared Dye (LI-COR Biosciences, Lincoln, NE, USA), including donkey anti-goat IgG and donkey anti-mouse IgG, at a dilution of 1:10,000–1:20,000. After a 1-h incubation at 37°C, the membranes were washed three times with TBST. The blots were visualized using the Odyssey Infrared Imaging system (LI-COR Biosciences). Signals were densitometrically assessed (Odyssey Application Software version 3.0; LI-COR Biosciences) and normalized to the GAPDH signals to correct for unequal loading using the mouse monoclonal anti-GAPDH antibody (AP0063; 1:20,000; Bioworld Technology, Inc., St. Louis Park, MN, USA).

All data are expressed as the means ± standard error of mean (SEM). The statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to perform comparisons among the different groups, and P<0.05 was considered to indicate a statistically significant difference.

Following DXM administration for 12 weeks, the GC-treated animals exhibited significant decreases in serum calcium (9.02±0.44 mg/dl), bone calcium content (5.38±0.43 mg) and calcium/bone ash (0.38±0.04) (Fig. 1A and C). Urine calcium levels were comparable among the four experimental groups. At 12 weeks, when comparing the urine calcium levels between the vehicle and the DXM groups, we observed that calcium secretion in the urine continually increased as the duration of DXM treatment increased (Fig. 1B). At the end of the treatment period, the AE-PS and alendronate increased serum calcium levels, bone calcium content and calcium/bone ash, and hypercalciuria was markedly inhibited in the mice with GIOP (Fig. 1A–C). There was no significant difference observed between the two treatment groups. Additionally, the results showed that the serum testosterone level in the DXM group was significantly decreased as compared with that of the control group; however, AE-PS treatment alone, not alendronate, was capable of reversing DXM-induced testosterone deficiency (Fig. 1D).

The serum concentrations of the bone turnover markers, TRAP-5b and CTX as a bone resorption markers and ALP-b and osteocalcin as a bone formation markers, were determined. The results showed that the serum levels of TRAP-5b and CTX in the DXM group were significantly increased, and the serum levels of ALP-b and osteocalcin were significantly decreased when compared with those of the control group (Fig. 2A–D). The AE-PS and alendronate significantly decreased the serum levels of TRAP-5b and CTX, and increased ALP-b and osteocalcin serum levels. However, ANOVA analysis revealed that the AE-PS significantly reduced TRAP-5b levels and raised ALP-b levels when compared with alendronate. There was no significant difference in the serum CTX levels between the two treatment groups (Fig. 2A–D).

To examine the mechanism responsible for the decreased serum calcium levels and the increased urine calcium concentrations in the DXM-treated mice, we analyzed the expression levels of calcium transport proteins in the kidney and in the duodenum. The expression of two calcium influx channels (TRPV5 and TRPV6), a reflux channel (PMCA1), and an intracellular calcium buffering protein (CaBP-9k) in the duodenum and in the kidney was examined. The protein levels of TRPV5, TRPV6, PMCA1 and CaBP-9k in the kidney were decreased by DXM treatment; however, the decreases in TRPV5, TRPV6, PMCA1 and CaBP-9k protein expression in the kidneys of mice with GIOP were reversed by the AE-PS or alendronate supplementation (Fig. 3A and B). Moreover, the protein levels of TRPV6 and CaBP-9k were significantly decreased by DXM in the duodenum. The AE-PS and alendronate upregulated the protein expression of TRPV6 and CaBP-9k in the duodenum as compared with that in the DXM-treated groups (Fig. 4A and B). There was no significant difference observed between the two treatment groups. The mRNA and protein expression of renal CaSR was increased in the mice with GIOP as compared with the expression levels in the untreated mice. Notably, treatment with the AE-PS significantly downregulated the mRNA expression of CaSR as well as the protein expression of CaSR in the kidneys of mice with GIOP (Fig. 5A–C). The results of western blot analyses revealed that the AE-PS reversed GIOP more effectively than alendronate, although both treatments exerted marked beneficial effects.

TRAP staining revealed that very few osteoclasts were identified in the trabecular bone area below the growth plate in the vehicle group, whereas the number of osteoclasts were significantly increased in this area in the DXM group (Fig. 6A). The treatment of the mice with GIOP with the AE-PS or alendronate significantly decreased the number of osteoclasts (Fig. 6A). As the maturation and formation of osteoclasts are mainly regulated by the balance between extracellular OPG and RANKL levels, the ratio of OPG/RANKL determines the number of matured osteoclasts. Consistent with the TRAP staining results, the results of western blot analysis revealed that the expression of OPG was significantly decreased whereas the expression of RANKL was significantly increased in the mice treated with DXM as compared with the control groups (Fig. 6B and C). The treatment of the DXM group with the AE-PS or alendronate significantly elevated the expression of OPG and suppressed the expression of RANKL (Fig. 6B and C). There was no marked difference between the two treatment groups.

Histological analysis of trabecular bone in the proximal metaphysis of the mice was performed using the H&E stain. The histology of trabecular bone near to the growth plate was markedly different in the four experimental groups. H&E staining revealed the increased disconnections and separation between the growth plate and the trabecular bone network as well as the reduction in trabecular bone mass of primary and secondary spongiosa throughout the proximal metaphysis of tibia in the DXM group. Notably, the AE-PS and alendronate reversed the deleterious effects on the trabecular bone induced by DXM and stimulated bone remodeling (Fig. 7A). Histological staining showed that the AE-PS exerted greater effects on bone structure compared with alendronate. Notably, the protein expression of CaSR was increased in the tibias of mice with GIOP compared with those of the untreated mice; however, AE-PS treatment significantly downregulated the protein expression of CaSR in the tibias of the mice with GIOP (Fig. 7B and C). By contrast, the protein expression of CaBP-9k in the tibias of mice with GIOP was decreased compared with those of the untreated mice. The administration of the AE-PS to the mice with GIOP significantly upregulated the protein expression of CaBP-9k in the tibias (Fig. 7B and C).