br Materials and methods br Results br

Materials and methods
Results
Discussion Natural compounds and their derivatives have been investigated as candidates for the treatment of bone diseases and can also be the sources of nutraceutical agents and therapeutic agents [28]. Using peptides as therapeutic effects on bone-related diseases, such as osteoporosis and osteopenia, has been reported to enhance bone formation. Among them, mg115 is a potential prospect for its role in promoting osteogenic differentiation [19,29,30]. However, so far, the available data on the effects of the calcitonin-like peptide from C. intestinalis on the MC3T3-E1 cells including proliferation, differentiation, and mineralization is very limited. Thus, we evaluated in the present study by the efficacy of CCLP in osteogenesis in mouse pre-osteoblast MC3T3-E1 cells. The promising findings of this study support the hypothesis that CCLP increases the effect of osteogenic in MC3T3-E1 cells by increased activity of enzyme ALP and osteoblast proliferation. ALP is typically the most widely recognized biochemical marker for osteoblastic activity [31,32]. Wang et al. [33] researched that during osteoblast growth and differentiation, the cell proliferation after 3–5 day cultures then ALP synthesis and activity increase in matrix maturation phase after 5-15 days. Osteoblast differentiation takes place specifically in three stages: cell proliferation, matrix maturation, and matrix mineralization. In this study, CCLP was shown to stimulate cell proliferation in the concentration range of 7.5–30 μM during the early osteoblast proliferation phase (2 and 5 days) (Fig. 1). Calcitonin secreted by the thyroid and thymus glands can inhibit bone resorption through interaction with osteoclasts. In addition, calcitonin has been shown to delay the progress of early-stage osteoarthritis lesions induced by mechanical instability in a rabbit experimental model [34]. Human and salmon calcitonin induced proliferation and enhanced ALP-specific activity in osteoblast-line cells derived from normal human bone [35]. C. intestinalis, the representative of marine invertebrates, grows rapidly and has an early maturation period of approximately 2–3 months [3]. Thus, C. intestinalis is listed as a source organism for material supply. Sekiguchi et al. [20] deduced that the amino acid sequence of calcitonin was made by Cys1 and Cys7 with a disulfide bond and similar in essential sequence characteristics of vertebrate calcitonin. The disulfide bridge forms a ring structure at the amino-terminus and then they can oligosaccharides in complex glycopeptides play an important role in substrate-receptor recognition, cell–cell interaction, and stability. In addition, calcitonin gene in vertebrates displays six exons and five introns in its structure [36], whereas the C. intestinalis calcitonin gene is composed of four exons and three introns. The effects of salmon calcitonin treatment are mediated through binding to a G-protein-coupled receptor [37,38]. In one previous report, calcitonin was found to specifically inhibit osteoclasts by suppressing tartrate-resistant acid phosphatase activity [20]. We investigated that CCLP from C. intestinalis greatly enhanced on high the proliferation and differentiation of the MC3T3-E1 mouse osteoblastic cells. A previous study has shown that calcitonin from salmon increases the ALP content in osteoblast cell cultures and promotes matrix mineralization [35]. Enhanced ALP activity and calcium deposition were observed after CCLP treatment, compared to that in the control. We also revealed that CCLP could significantly increase OSC and OPN activity in MC3T3-E1 cells compared to the control. In this study, CCLP treatment increased ALP activity at days 2, 5, and 7 (Fig. 2). CCLP treatment compared to the control increased ALP mRNA and protein expression at day 7; ALP mRNA and protein expression decreased after 14 days of culture, which are the typical proliferation and maturation periods (Fig. 4, Fig. 5). ALP plays an important key factor in mineralization process during osteoblastic differentiation. In the bone tissue, it is known that ALP is expressed early stage in mineralization process, and is observed on the cell surface in matrix vesicles. Later in the mineralization process, while other factors are increased, ALP expression declines [39].