OBJECTIVE: This study aimed to determine the effectiveness of annatto-tocotrienol on the bone turnover markers and bone histomorphometry in a model of male osteoporosis induced by buserelin (a GnRH agonist).
METHODS: Forty-six three-months-old male Sprague-Dawley rats (three months old; 300-350 g) were randomly divided 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 daily 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 daily. The calcium control (n = 8) received 1% calcium in drinking water and subcutaneous buserelin injection 75 μg/kg/day. The remaining rats were treated with two different treatments, i.e., (1) oral annatto tocotrienol at 60 mg/kg/day plus subcutaneous buserelin injection 75 μg/kg/day (n = 8); (2) oral annatto tocotrienol at 100 mg/kg/day plus subcutaneous buserelin injection 75 μg/kg/day (n = 8). The rats were injected with calcein twice before being sacrificed to label the bones. The rats were euthanized, and their blood and right femur were harvested at the end of the treatment for bone turnover markers and bone histomorphometry examination.
RESULTS: Both serum osteocalcin and C-telopeptide of type 1 collagen were not significantly different between treated groups and buserelin control (P > 0.05). The buserelin control group had a significantly lower bone volume and higher eroded surface compared with the normal control group (P
METHODS: Murine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 104 cells/mL and treated with 4 concentrations of AnTT (0.001-1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.
RESULTS: The results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.
CONCLUSION: AnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.