Effects of cuttlebone powders and their structured calcium compounds against glucocorticoid-induced osteoporosis via MAPK signaling-mediated bone formation

Abstract

The effects of dried and calcined cuttlebone powders (DCB and CCB, respectively) and their structured calcium compounds (DCB-OH, DCB-Lac, DCB-CiMa, CCB-OH, CCB-Lac, and CCB-CiMa) on MC3T3-E1 osteoblasts were investigated in vitro . Cytotoxicity analysis using the MTT assay revealed that lower concentrations (10–100 μg/mL) of all compounds were non-toxic, whereas higher concentrations (250–1000 μg/mL) showed dose-dependent cytotoxic effects. Using dexamethasone-induced osteoporosis-like MC3T3-E1 cells, cuttlebone-derived compounds significantly enhanced matrix mineralization as well as alkaline phosphatase (ALP) mRNA expression and activity, indicating restoration of osteoblast function. Additionally, treatment with all cuttlebone compounds reduced dexamethasone-induced apoptosis, demonstrating cytoprotective effects. Gene expression analysis showed suppression of ER stress markers ( CHOP, GRP78, ATF4 ) by most compounds, suggesting attenuation of ER stress-related osteoblast dysfunction. Osteoblast-related genes linked to matrix remodeling and bone formation ( MMP2, MMP9, COL1A1 ) were modulated; treatments generally downregulated MMP2 and upregulated COL1A1 expression. Key osteogenic transcription factors and markers ( BMP4, OC, OSX, RUNX2 ) decreased by dexamethasone were significantly upregulated by cuttlebone compounds, supporting enhanced osteoblast differentiation. Protein phosphorylation studies revealed that these compounds decreased the expression of pERK and p-p38, pathways that are often associated with impaired osteogenesis under glucocorticoid stress. Collectively, these findings demonstrate that cuttlebone powders and their structured calcium compounds mitigate dexamethasone-induced osteoporotic changes in MC3T3-E1 cells by enhancing cell viability, promoting osteoblast differentiation, and reducing apoptosis and ER stress, effects that are associated with suppression of MAPK pathway activation. This finding suggests a possible role in alleviating glucocorticoid-induced dysfunction in MC3T3-E1 osteoblasts and indicates potential relevance for future development as functional food ingredients or nutraceuticals for bone health. • Cuttlebone powders and their calcium compounds enhance osteoblast function in osteoporosis model. • They reduce apoptosis and relieve ER stress in dexamethasone-treated osteoblasts. • The compounds inhibit MAPK signaling pathways linked to impaired bone formation. • These effects support their potential as therapeutics for glucocorticoid-induced osteoporosis.