Exploration of the potential mechanism of cyclolinopeptides-mediated antiosteoporosis effects via network pharmacology analysis

Background: Osteoporosis poses a significant global health burden. Cyclolinopeptides (CLPs), cyclic hydrophobic peptides derived from flaxseed oil, exhibit antiosteoporotic, antioxidant, and immunomodulatory properties, but their therapeutic potential for osteoporosis remains underexplored. This study was aimed to investigate the potential protective effects of CLP-A, CLP-E, and CLP-P on the treatment of osteoporosis in vitro.  Methods: Network pharmacology identified overlapping targets of CLP-A/E/P and osteoporosis (sourced from GeneCards, DrugBank, DisGeNET, PharmMapper, and BindingDB). Core targets were prioritized via PPI network analysis (Cytoscape). In vitro assays were used to assess CLP cytotoxicity (CCK-8 on MC3T3-E1 and RAW264.7 cells), osteogenic differentiation (alizarin red S staining), and alkaline phosphatase (ALP) activity.  Results: Network pharmacology analysis revealed that the compounds CLP-A/ E/P were involved in the regulation of diverse biological functions associated with osteoporosis, including signal transduction and positive regulation of transcription from the RNA polymerase II promoter. These signaling pathways are involved primarily in the pathways related to cancer, the PI3K-Akt signaling pathway and the Ras signaling pathway. The experimental results demonstrated that CLP-A/ E/P had no direct toxic effects on MC3T3-E1 Cells or RAW264.7 Cells for 24-48 h, respectively. CLP-A/E/P were shown to enhance MC3T3-E1 Cell osteogenesis and ALP activity.  Conclusions: This study demonstrated that CLP-A, CLP-E, and CLP-P enhance osteogenesis and ALP activity in MC3T3-E1 cells without causing cytotoxicity, suggesting their potential as therapeutic agents for osteoporosis. Network pharmacology further highlights their involvement in key signaling pathways, such as the PI3K-Akt and Ras signaling pathways, which are critical in bone metabolism and osteoporosis, however, further investigation is needed to elucidate the underlying mechanism involved.