三叶苷对破骨细胞分化影响的研究OA

Objective To evaluate the effects of trifolirhizin on osteoclast differentiation and functional formation by integrating network pharmacology prediction with in vitro cell experiments,and to explore the underlying mechanism of action.Methods Differentially expressed genes associated with osteoclast differentiation were screened from the GSE235773 dataset and intersected with predic-ted targets of trifolirhizin to construct protein-protein interaction(PPI)network;key targets were identified through pathway enrichment analysis and molecular docking.Bone marrow-derived macro-phages(BMMs)were isolated from mice and induced to differentiate into osteoclasts.The effects of trifolirhizin were assessed using Cell Counting Kit-8(CCK-8)assay,tartrate-resistant acid phospha-tase(TRAP)staining,F-actin ring staining and Western blot analysis.Functional validation was per-formed by exogenous supplementation of prostaglandin E2(PGE2)and cyclooxygenase-2(COX2)small interfering RNA(siRNA)-mediated knockdown.Results A total of 17 potential target genes were identi-fied,among which prostaglandin-endoperoxide synthase 2(Ptgs2,encoding COX2)occupied central posi-tion in the PPI network.Molecular docking revealed that trifolirhizin exhibited favorable binding affinity to COX2(binding energy:-7.4 kcal/mol).In vitro experiments demonstrated that trifolirhizin dose-de-pendently suppressed RANKL-induced osteoclast formation and F-actin ring structure within non-cytotoxic concentration range,and downregulated the expression of osteoclast-associated marker proteins,including matrix metalloproteinase 9(MMP9),tartrate-resistant acid phosphatase 5(ACP5)and COX2.Ex-ogenous PGE2 supplementation partially reversed the inhibitory effects of trifolirhizin.COX2 knock-down significantly suppressed the expression of osteoclast-related proteins,and trifolirhizin did not exert additionalinhibitory effects under COX2-silenced conditions.Conclusion Trifolirhizin inhibits osteoclast differentiation and functional formation in vitro,and this effect is at least partially media-ted through the COX2 signaling axis.