Characterization of Vitamin k2 FUNTIONS IN Saos-2 osteoblasts

Abstract

MK-7 as a potential treatment against osteoporosis, favoring bone formation, was explored in this thesis. Today most treatments for bone loss target suppression of osteoclastogenesis.

Biological activity and cytotoxicity of vitamin K2, MK-7, in osteoblasts were investigated to give a better understanding of MK-7’s effect on bone formation. In order to determine these characteristics, rezasurin assays, qPCR assays and arachidonic acid release assays of Saos-2 cells were used throughout this study. Vitamin K2, MK-7, found fermented in natto is a structural analogue to vitamin K2, MK-4, found in meat. These vitamin K2 derivatives were purified and synthesized by Kappa Bioscience. Prior to this study, in vivo vitamin K2 serum half-life assays indicated that MK-7 had longer half-life and higher potency compared to MK-4. Doses of 10 μM MK-7 were recommended. This master thesis evaluated MK-7 effects on osteoblast differentiation factors, bone remodeling factors RANKL and OPG, and factors involved in inflammation. Lastly vitamin K2 derivatives, MK-7 and MK-4, effects on IL-1β and TNFα cytokines role in arachidonic acid release in osteosarcoma cells Saos-2 were studied.

Of the two vitamin K2 derivatives, only vitamin MK-4 demonstrated cytotoxicity. Vitamin MK-7 displayed greater stability compared to MK-4. MK-7 was evaluated to favor osteoblast differentiation through suppression of PTHLH and Runx2 expression and elevated expression of SOST and SP7. RANKL and OPG were found to be down-regulated in early osteoblast cells in response to MK-7 whereas the expression of OPG was higher than RANKL indicating the cells might have suppressed ability to activate osteoclasts. Determining the role MK-7 on inflammatory factors has been evaluated by two methods. qPCR results indicate the MK-7 suppress the pro-inflammatory factor COX2 whereas the anti-inflammatory factor NFkBIA were up-regulated. Results from arachidonic acid release assays indicate that MK-7 in response to cytokine TNFα and IL-1β decrease AA release. These results propose that MK-7 might have ability to suppress inflammation in osteoblasts.

Summary of the results from this project strengthens findings that MK-7 has proven to be promising in effecting osteoblast activity. These results indicate that MK-7 displayed stability in osteoblasts, no cytotoxicity, affect differentiation, might favor bone formation through presumed suppressed ability to activate osteoclasts and has anti-inflammatory effect. For this reason, this study supports MK-7 as a future candidate for osteoporosis treatment.