Forty 8-week-old, BALB/c female mice, weighing 20.84 ± 1.21 g,
were obtained from the Experimental Animal Center of The Fourth
Military Medical University (Xi'an, China). There was no significant
difference in the initial body weights of the mice among all 4 groups
in this experiment. The mice were allowed to adapt to the laboratory
environment (a well-ventilated controlled room at 20 °C on a 12-h
light/dark cycle; the animals were given free access to water and
food) for 1 week before the surgery. Subsequently, the mice experienced
sham-operation (n = 10) or were surgically ovariectomized
(OVX) (n = 30) under anesthesia using pentobarbital sodium
(50 mg/kg body weight, i.p.). The ovariectomy operation was performed
according to Steven K. Boyd's procedure [31]. A total of 30
BALB/c female mice were randomly divided into three groups: 1) OVX
group, administered intraperitoneally with distilled water (n = 10);
2) OVX group, administered intraperitoneally with Rb2 (body weight,
4.6 μmol/kg; n = 10) daily; and 3) OVX group, administered intraperitoneally
with Rb2 (body weight, 18.5 μmol/kg; n = 10) daily.
Rb2 was dissolved in distilled water. One week after the operation,
the treatments commenced and continued for 12 weeks. Blood
samples were obtained from the hearts in anesthetized mice and
serum samples were prepared by centrifugation. The left femurs
and 4th lumbar vertebrae (L4) of the mice were collected and the
adherent tissue was discarded. All of the experimental procedures
were officially approval by the Ethics in the Animal Research Committee
of the Fourth Military Medical University (permission code
2010C00843).