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.
METHODS: Thirty-two female Wistar rats were randomly divided into four groups: Sham-operated (Sham), ovariectomized control (OVXC), ovariectomized with Labisia pumila var. alata (LPva) and ovariectomized with ERT (Premarin) (ERT). The LPva and ERT were administered via daily oral gavages at doses of 17.5 mg/kg and 64.5 μg/kg, respectively. Following two months of treatment, the rats were euthanized and the gene expressions of BMP-2, OPG, RANKL and MCSF in the femoral bones were measured using a branch - DNA technique.
RESULTS: The RANKL gene expression was increased while the OPG and BMP-2 gene expressions were reduced in the OVXC group compared to the SHAM group. There were no significant changes in the MCSF gene expressions among the groups. Treatment with either LPva or ERT was able to prevent these ovariectomy-induced changes in the gene expressions in ovariectomized rats with similar efficacy.
CONCLUSION: LPva may protect bone against estrogen deficiency-induced changes by regulating the RANKL, OPG and BMP-2 gene expressions.
METHODS: Fifty-six female Sprague-Dawley rats were randomly allocated into eight groups (n = 7): SHAM (healthy sham control); OVX (ovarietomized) nontreated rats (negative control); OVX + Remifemin (100 mg/kg body weight), and 2% green tea extract (positive controls); OVX + OS 50% ethanolic and aqueous extracts, both at either 150 or 300 mg/kg. After 16 weeks, the rats' bones and blood were evaluated for osteoporosis indicators (protein and mRNA expressions), micro-computed tomography for bone histomorphometry, and three-point bending test for tibia mechanical strength.
RESULTS: The extracts dose-dependently and significantly (P bone strength and flexibility, bone mineral density, bone formation protein markers (P1NP), and bone histomorphometry. All extracts reduced the inflammation biomarker (interleukin-6). The extracts up-regulated osteoblastogenesis (bone morphogenetic protein-2) and collagen-1 synthesis (collagen type 1 alpha-1) mRNA expressions, and down-regulated bone resorption (TNFSF11 and nuclear factor-kappa B) mRNA expressions. Both the water and 50% ethanolic extract were effective. The effective dose is equivalent to 25 to 50 mg/kg extract for humans.
CONCLUSIONS: The extract showed bone-protective and antiosteoporotic effects (improving bone strength, flexibility, bone density, and bone morphometry) by reducing inflammation and the bone resorption biomarkers, while enhancing bone formation biomarkers and collagen synthesis.