Effects of annatto tocotrienol on bone in a rat model of osteoporosis induced by buserelin

Abstract

Long-term administration of buserelin, a gonadotropin-releasing hormone (GnRH) agonist, is associated with osteoporosis. It reduces the skeletal strength and increases the risk for fragility fracture. Annatto tocotrienol (AnTT) has been shown to improve bone health in castrated osteoporotic rats. This study aimed to determine the antiosteoporotic effects of AnTT in a male osteoporotic rat model induced by buserelin. Forty-six male Sprague-Dawley rats aged three months old were randomly assigned into six groups. The baseline control group (n=6) was sacrificed at the onset of the study. The normal control group (n=8) received corn oil (the vehicle of tocotrienol) orally and normal saline (the vehicle of buserelin) subcutaneously daily. The buserelin control (n=8) received corn oil orally daily and subcutaneous buserelin injection 75 µg/kg/day. The calcium control (n=8) was given 1% calcium in drinking water and subcutaneous buserelin injection 75 µg/kg/day. The remaining two groups (n=8/group) were treated with AnTT orally at two different doses (60 or 100 mg/kg/day) and receiving subcutaneous buserelin injection 75 µg/kg/day. The rats were injected with calcein (20 mg/kg, i.p.) nine days and two days before they were sacrificed. At the end of treatment period, the rats were euthanized, and their blood, tibia and femur were harvested. Structural changes in the trabecular and cortical bone at the proximal tibia were examined using X-ray micro computed tomography. Structural, dynamic and static changes at the distal femur were examined using histomorphometric methods. Bone calcium content and biomechanical strength at the distal femur also were examined. Serum osteocalcin and C-terminal of type 1 collagen crosslinks (CTX-1) levels were measured. The groups treated with AnTT and 100 mg/kg demonstrated significant improvements in bone microstructure (bone volume, trabecular number and trabecular separation) and cortical thickness compared with the buserelin-treated osteoporotic rats (P<0.05). There were also significant improvements in structural (bone volume and trabecular thickness) and static histomorphometric indices (number of osteoblast) for both the AnTT-treated groups, as well as in dynamic histomorphometric indices (double-labeled surface) for the group treated with AnTT at 60 mg/kg (P<0.05). However, no significant changes in osteocalcin and CTX-1 levels were observed across the groups (P>0.05). AnTT at 60 mg/kg and 100 mg/kg increased femoral biomechanical strength (load, stress and elastic modulus) and bone calcium content significantly compared to the osteoporotic rats (P<0.05).The effects of calcium supplementation on the bone were similar with AnTT at 60 mg/kg. In conclusion, supplementation of AnTT at 60 mg/kg was shown to be more effective than 100 mg/kg in preventing the degeneration of the bone caused by buserelin. Effects of AnTT in preventing osteoporosis were comparable with calcium supplementation. Therefore, it has the potential to be developed as an antiosteoporotic agent for male patients receiving GnRH agonist therapy.