The molecular mechanism of melatonin in regulating osteoporosis based on the RANKL/OPG signaling axis

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.