Tetrapeptides derived from black bean enhance osteogenesis and alleviate ovariectomy-induced bone loss via activation of BMP-2/Smad signaling

INTRODUCTION: Osteoporosis, a "silent killer" among the elderly, is marked by progressive bone loss and microstructural deterioration. Oligopeptides derived from black bean, particularly tetrapeptides, have shown notable osteogenic potential, yet their therapeutic roles in regulating bone metabolism and preventing osteoporosis remain unclear. OBJECTIVES: This study explored the osteoanabolic effects of black bean-derived tetrapeptides and elucidated their underlying mechanisms. METHODS: In vitro assays were conducted using MC3T3-E1 pre-osteoblasts derived from mouse calvaria to assess osteogenic differentiation upon tetrapeptide treatment. Six black bean tetrapeptides were evaluated, including KIGT, KGVG, KTGV, SIKL, KLGT, and SLKL. In vivo efficacy was evaluated in an ovariectomized mouse model of osteoporosis via intragastric administration. Based on molecular docking, the mechanisms were explored using Western blotting and immunofluorescence and validated through pathway inhibitor and siRNA knockdown experiments. RESULTS: All six tetrapeptides significantly stimulated the osteogenic differentiation of MC3T3-E1 pre-osteoblasts, particularly during early osteogenesis. Notably, the peptides all exhibited osteogenic effects at low doses (0.1 μM), although their optimal concentrations varied. Mechanistic studies revealed a shared anabolic pathway among the tetrapeptides, in which lysine residues may play a pivotal role in mediating their similar interactions with bone morphogenetic protein-2 (BMP-2), thereby activating the BMP-2/Smad signaling. Furthermore, this potent osteoanabolic efficacy was confirmed in osteoporotic mice, where a 10-week treatment with black bean oligopeptides (<1 kD), KIGT, and KGVG (100 mg/kg) markedly attenuated bone loss. CONCLUSION: Lysine-containing tetrapeptides derived from black bean exert direct osteoanabolic activity by targeting BMP-2/Smad signaling in osteoblasts. These findings support their potential as novel anabolic candidates against osteoporosis, paving the way for the development of oligopeptide-based therapies targeting bone health.