Purpose Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. of AnTT treatment. Results The results showed that HMGR was up-regulated in the lovastatin group on day time 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day time 9 and 15) and improved BMP-2 protein (day time 15). On the other hand, AnTT at 0.001 g/mL (day time 3) and 0.1 g/mL (day time 21) significantly down-regulated HMGR gene manifestation compared to the control. On day time 21, HMGR gene manifestation was significantly reduced in all organizations compared to day time 15. AnTT at 0.1 g/mL significantly decreased RhoA activation on day time 9 compared to the control. AnTT at 1 g/mL significantly increased BMP-2 protein on day time 15 compared to the control (P 0.05). Mineralized calcium nodules were more abundant in AnTT treated organizations compared to the control on day time 21. Summary AnTT suppresses the mevalonate pathway by downregulating HMGR Semaxinib cell signaling gene manifestation and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This clarifies the stimulating effects of AnTT on osteoblast mineralization. strong class=”kwd-title” Keywords: bone, osteogenic, osteoporosis, tocotrienol, vitamin E Introduction Bone remodelling is controlled by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The imbalance of these two processes causes abnormalities in bone remodelling, which can produce a variety of bone disorders including osteoporosis.1 Osteoporosis is a silent bone degenerative disorder characterized by low bone mass and deterioration of skeletal microarchitecture, leading to bone Rabbit Polyclonal to PPP1R2 Semaxinib cell signaling fragility.2 Osteoporosis mainly affects postmenopausal ladies but it can also occur in males.3,4 It is probably one of the most under-recognised non-communicable health conditions influencing developing countries with increasing seniors population.5 The current therapies for osteoporosis include anti-resorptive agents (bisphosphonates, calcitonin, denosumab, estrogen + progesterone) and anabolic agents (teriparatide).6,7 These agents are effective against osteoporosis but they come with adverse side effects.8,9 The preventive agents for osteoporosis are limited to calcium with or without vitamin D. This shows a significant space for pharmacological prevention of osteoporosis. The mevalonate pathway, responsible for cholesterol synthesis, has been the prospective of drug treatment for osteoporosis.10 Statins, traditionally known as cholesterol-lowering agents, have been found to promote bone formation in vitro and in vivo.11C13 Statins regulate the mevalonate pathway by inhibiting the 3-hydroxy-3-methyl-glutaryl coenzyme-A (HMG-CoA) reductase (HMGR) enzyme, the rate-limiting enzyme for the mevalonate pathway, from transforming HMG-CoA into mevalonate. These will consequently suppress the synthesis of isoprenoids, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). FPP and GGPP are involved in the protein prenylation process, whereby the isoprenoids are bound covalently to the C-terminal of small G-proteins or GTPase.14 G-proteins consist of 5 family members; the Rho/Rac/Cdc42, Ras, Rab, Sar1/Arf, and Ran family members.15 Rho is involved in statin-induced osteogenesis. A study by Harmey et al showed that Rho-Rho kinase inhibition stimulated differentiation and calcium nodule formation of mouse calvariae cells.16 Inhibition of Rho kinase was shown to increase bone morphogenetic protein-2 (BMP-2) gene expression by pitavastatin in human osteoblasts.17 However, this stimulatory effect was abolished by mevalonate or GGPP, indicating that these effects originated from inhibition of the mevalonate pathway.17 BMP-2 takes on a crucial part in the differentiation of human being embryonic stem cells.18 BMP signalling regulates transcription factors including runt-related factor 2 (Runx2) and osterix (OSX) involved in the formation of osteoblasts and expression of downstream genes involved in bone formation.19 Tocotrienol is a member of the vitamin E family, along with tocopherol. It can be found in palm oil, wheat germ, grain bran, barley, and annatto bean. Both tocopherol and tocotrienol include 4 isomers, i.e. alpha (), beta (), delta () and gamma (), with regards to the relative aspect stores over the chromanol band. Tocotrienol exerts effective neuroprotective, antioxidant, lipid-lowering and anti-cancer properties, which differentiate it Semaxinib cell signaling from tocopherol.20 Furthermore, -tocotrienol preserved regular body structure and calcium mineral articles more in comparison to -tocopherol in dexamethasone-induced rats effectively. 21 Both hand annatto and tocotrienol derived-tocotrienol have already been reported to safeguard bone tissue in a variety of pet types of osteoporosis.22 A previous research suggested the participation of mevalonate pathway in the bone-sparing ramifications of -tocotrienol in ovariectomized mice.23 Annatto derived-tocotrienol (AnTT) from seed products of achiote tree native to tropical America contains 90% – and 10% -tocotrienol.24,25 In animal types of osteoporosis due to testosterone deficiency, AnTT was shown to prevent bone loss by increasing osteoblast number, osteoid volume and osteoid surface.26 Combination of AnTT and lovastatin Semaxinib cell signaling also increased bone formation, improved bone structure and bone strength in ovariectomized rats.27,28.