Osteoporosis represents a major global health challenge. Cyclolinopeptides (CLPs), which are cyclic hydrophobic peptides derived from flaxseed oil, exhibit antioxidative, immunomodulatory, and antiosteoporotic activities; however, their therapeutic utility in osteoporosis remains insufficiently studied. This study aimed to explore the potential protective effects of CLP-A, CLP-E, and CLP-P in the context of osteoporosis using in vitro models.Potential overlapping targets of CLP-A, CLP-E, and CLP-P with osteoporosis were identified through network pharmacology using databases including GeneCards, DrugBank, DisGeNET, PharmMapper, and BindingDB. Core targets were highlighted via protein–protein interaction (PPI) network analysis in Cytoscape. Cytotoxicity of CLPs was evaluated on MC3T3-E1 and RAW264.7 cells using the CCK-8 assay, while osteogenic differentiation was assessed via alizarin red S staining and alkaline phosphatase (ALP) activity measurement.Network pharmacology analysis indicated that CLP-A, CLP-E, and CLP-P potentially regulate multiple biological processes implicated in osteoporosis, such as signal transduction and positive regulation of transcription from RNA polymerase II promoters, predominantly through pathways linked to cancer, PI3K-Akt signaling, and Ras signaling. In vitro experiments showed that CLPs exhibited no cytotoxicity toward MC3T3-E1 or RAW264.7 cells within 24–48 h. Furthermore, CLP-A, CLP-E, and CLP-P promoted osteogenic differentiation and increased ALP activity in MC3T3-E1 cells.Our findings suggest that CLP-A, CLP-E, and CLP-P enhance osteogenesis and ALP activity in MC3T3-E1 cells without cytotoxic effects, highlighting their promise as antiosteoporotic agents. Network pharmacology analysis further implicates their involvement in critical signaling pathways, including PI3K-Akt and Ras, that are fundamental to bone metabolism and osteoporosis, although further mechanistic studies are warranted.
