Establishment of ovariectomized rat model of osteoporosis

A total of 20 3-month-old healthy female Sprague-Dawley (SD) rats (weighing 280–300 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., and fed freely in a quiet and well-ventilated clean animal room. The rats were classified into two groups of 10 rats each in a random manner: OVX group and sham operated group (Sham). Ovariectomized (OVX) rat model of osteoporosis was constructed, and rats in OVX group were anaesthetized with 10% ketamine (100 mg/kg, Alfasan, the Netherlands) and 2% cilazin (10 mg/kg, Alfasan, the Netherlands) and fixed in the prone position. The ovaries were located near the inferior pole of the kidney through a dorsal median incision under sterile condition. The bilateral ovaries were ligated and removed with line 4, and the incisions were sutured layer by layer. In Sham group, only the same mass of adipose tissues around ovary was removed. After 12 weeks, rats were anaesthetized intraperitoneally by the combined use of ketamine and xylazine before being sacrificed. The right femora of rats were soaked in physiological saline and preserved at −20 °C for microstructure scanning with a micro-computerized tomography (micro-CT) and the detection of bone mineral density (BMD). Meanwhile, the right femora of rats were cryopreserved in liquid nitrogen at −80 °C for subsequent protein and RNA analysis.