Melatonin is a promising drug for improving bone mass in postmenopausal women. This study investigated the mechanism behind the regulation of melatonin on osteoblast differentiation. Mouse embryonic osteoblast precursor MC3T3-E1 cells were treated with melatonin or transfected with cathepsin D (CTSD) vectors. A cellular model was established by H2O2 treatment. Alkaline phosphatase (ALP) staining and Alizarin Red S staining were performed. An osteoporosis mouse model was established by ovariectomy (OVX) and treated with melatonin or transfected with a CTSD knockdown vector. Bone biomechanical testing was performed to assess bone strength. Histological bone damage was assessed, osteoclast differentiation was visualized using tartrate resistant acid phosphatase staining, osteocalcin (OCN) and type I collagen α1 chain (COL1A1) expression was visualized using immunohistochemistry, and serum bone turnover markers procollagen type I N-propeptide (PINP) and C-terminal telopeptide of type I collagen (CTX-1) levels were measured using enzyme-linked immunosorbent assay. Receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), Wnt3a, and β-catenin protein levels were determined using western blotting. Melatonin treatment or CTSD overexpression promoted ALP activity and mineralization in MC3T3-E1 cells. Melatonin upregulated CTSD expression. Melatonin treatment enhanced bone strength, inhibited osteoclast differentiation, increased OCN and COL1A1 expression, elevated PINP levels, and reduced CTX-1 in OVX mice. Moreover, melatonin suppressed RANKL expression and promoted OPG, Wnt3a, and β-catenin expression. CTSD knockdown abolished the regulatory effects of melatonin on MC3T3-E1 cells and OVX mice. In conclusion, melatonin increases CTSD expression to promote osteoblast differentiation and regulate the RANKL/OPG/Wnt signaling pathway, thereby slowing down the progression of osteoporosis.
Guo et al. (Wed,) studied this question.