Twenty male Sprague Dawley (SD) rats (purchased from the Experiment Animal Center of Zhejiang University) were divided into two groups (n=10) at random. The rats in the diabetic group were fasted for 10 h and injected intraperitoneally with streptozotocin (STZ) (Alexis Corporation, Switzerland) 65 mg/kg to induce diabetes. The other 10 rats formed the control group and they were fasted for 10 h and injected with 0.9% saline. The rats with blood glucose >16 mmol/l 48 h after injection were considered diabetic. All procedures were approved by the Zhejiang University Institutional Animal Care and Use Committee.

Six weeks after diabetes was induced, all rats were anesthetized, their thoracic cavities opened and perfused intracardially with normal saline. Following saline perfusion, the animals were perfused with 300–400 ml fixative containing 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4). After perfusion, both femurs were extracted. The BMD of the left femurs was determined using a small animal radiophotography system (Faxitron model MX20; Tucson, AZ, USA). The right femurs were used for hematoxylin and eosin (H&E) staining and immunohistochemistry assay.

Primary osteoblasts were derived from newborn rat calvaria as previously described (24). Cells were seeded in 6-, 24- or 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml) and 15% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were used between the second and the fourth passages. The third passage cells were divided into a normal medium group and a high-glucose medium group (concentrations of glucose 100 mmol/l). The osteoblasts of both groups were cultured for 48 h and washed twice with phosphate-buffered saline (PBS). Ten wells of osteoblasts for each group were used for CHOP western blot analysis.

The cells were prepared in lysis buffer and centrifuged (12,000 × g for 10 min at 4°C) to remove cellular debris. The protein concentration was determined by the Lowry method using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates containing equal amounts of protein (50 μg) were resolved using 8–10% SDS-polyacrylamide gel electrophoresis and transferred onto Millipore (Billerica, MA, USA) nitrocellulose membranes. The reactions were stopped with a solution containing 5% skimmed milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature and treated with CHOP antibodies (1:2,000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in TBST overnight at 4°C, washed for 1 h with TBST and further probed with secondary horseradish peroxidase (HRP; 1:2,000) in TBST for 1 h at room temperature. The immune complexes were visualized using an enhanced chemiluminescence (ECL) detection system according to the manufacturer’s protocols. The densitometric analysis of the bands was assayed using Quantity One (Bio-Rad).

The remaining right femurs were fixed in the same fixative for 4 h and placed in 30% phosphate-buffered sucrose until the tissue sank. Sections 12 μm thick were cut on a freezing microtome through the coronary planes of the proximal femur for H&E staining and diaminobenzidine (DAB) immunohistochemical staining.

Femur sections were rinsed in 0.01 M PBS and mounted onto 0.02% poly-l-lysine-coated slides. The ABC system was used with DAB as the chromagen. Tissue sections were first washed in PBS and incubated in 1% bovine serum albumin (BSA) for 30 min. Tissues were incubated overnight at 4°C in the medium of PBS with CHOP antibody (1:100) plus 1% BSA. Control sections were incubated in PBS and 1% BSA. The following day, the sections were incubated in a biotinylated goat-anti-mouse secondary antibody (diluted 1:200 in PBS, Boster Biotechnology Company, Wuhan, China) and subsequently in an avidin-HRP solution. Immunolabeling was visualized with 0.05% DAB plus 0.3% H₂O₂ in PBS. The sections were dehydrated through ethanol and xylene before mounting.

Immunohistochemistry results were analyzed using CHOP-positive osteoblast in femur per mm² of two groups of rats under a Nikon microscope (Nikon E600, Nikon Company, Japan) at final magnifications of ×400.

CHOP-positive cells in each visual field under the microscope were counted at ×200 magnification. The data represent the means ± SD. The differences were evaluated by analysis of two-tailed t-tests. P<0.05 was considered to indicate a statistically significant result. All computations were performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA).

Initially, the body weight and blood glucose showed no significant differences (P>0.05) in both groups of rats before STZ injection. After 6 weeks, the diabetic rats had a significantly higher blood glucose and lower body weight when compared with the control group rats (P<0.01). Compared with non-diabetic control rats, diabetic rats showed a decrease in femoral BMD (−15.4±2.3% vs. control, P<0.05, Fig. 1).

Osteoblasts were identified by a central lucency, eccentric nucleus and basophilic cytoplasmic stain. Osteoclasts were identified as single or multi-nucleated cells demonstrating foamy cytoplasm. Six weeks after inducing diabetes, the proximal femur H&E staining demonstrated a greater number of osteoclasts that clumped together, reduced cortical bone and a deteriorated bone micro-architecture in diabetic rats. The overall growth plate architecture was dominated by hypertrophic chondrocytes, with fewer proliferative and chondroblastic cells as compared with control rats. The femur of control rats showed a normal shape with intact cartilage, qualitatively equivalent cortical bone and a well-organized bone matrix composed of trabecular bone compared with the diabetic rats (Fig. 2).

CHOP immunoreactivity was visualized in a granular immunostain pattern in the nucleus. Caspase-12 immunohistochemistry staining positive cells with DAB staining, showed buff-colored granules in the cytoplasm. Quantitative analysis for the number and optical density of CHOP-positive cells with DAB immunostaining showed significant increases in osteoblast cells in diabetic rats and under high-glucose medium in vitro (P<0.05, Fig. 2 and Table I).

The number of CHOP-positive cells and the optical density was significantly increased in the rats of the diabetic group (P<0.05). Similarly, the number of positive cells and the optical density was upregulated in the osteoblasts of high-glucose medium compared with that of the normal medium group (P<0.05).

CHOP proteins in osteoblast cells were detected as single bands migrating ∼27 kDa. The densitometric analysis of the bands for CHOP revealed a significant increase in relative protein content (451.4±32.6%) in high-glucose medium osteoblasts when compared with those from the normal medium group (100.00%; P<0.05, Fig. 3). This suggested that CHOP was activated in osteoblasts under high-glucose medium.