Abstract: Objective To explore the mechanism by which Gubao Capsule treats osteoporosis by regulating the nuclear factor-kappa B(NF-κB)signaling pathway to inhibit osteocyte apoptosis and promote osteogenic differentiation. Methods Network pharmacology was used to retrieve targets related to the active ingredients of Gubao Capsule, osteogenic differentiation, and osteoporosis, and their overlapping targets were obtained. A protein-protein interaction(PPI)network was constructed using the STRING database, and topological analysis was performed with Cytoscape 3.8.0. Core targets(degree values above the median)were selected for gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses. In animal experiments, 36 SD rats were divided into three groups: control group(no treatment, n=12), model group(ovariectomy-induced osteoporosis, n=12), and treatment group(Gubao Capsule gavage based on the model group, n=12). After intervention, hematoxylin-eosin, Sirius red, toluidine blue, and safranin O-fast green staining were performed to observe bone pathological changes. TUNEL and immunofluorescence assays were used to detect osteocyte apoptosis. Panoramic pathological analysis evaluated NF-κB pathway activation. Immunohistochemistry detected the expression of osteogenic proteins: bone morphogenetic protein 2(BMP2), osteopontin(OPN), osterix, and runt-related transcription factor 2(RUNX2). Results A total of 643 targets of Gubao Capsule, 2,998 osteogenic differentiation-related targets, and 7,707 osteoporosis-related targets were retrieved, with 266 overlapping targets. Among them, 132 core targets with degree values above the median were identified. KEGG enrichment showed that the core targets were mainly enriched in osteogenic differentiation and NF-κB signaling pathways; GO analysis revealed that theenrichment in biological processes such as apoptosis and programmed cell death.Animal experiments showed that compared with the control group, the model group exhibited severe bone tissue destruction, trabecular fracture, and loss of fibrin and proteoglycans. Meanwhile, NF-κB p65 and Caspase3 expression were significantly upregulated(P<0.05), accompanied by a marked increase in osteocyte apoptosis rate(P<0.05). Panoramic pathological analysis confirmed abnormal activation of the NF-κB pathway and significantly decreased osteogenic protein expression in the model group(P<0.05). All these abnormalities were significantly reversed after Gubao Capsule intervention. Conclusion Gubao Capsule can treat osteoporosis by inhibiting abnormal activation of the NF-κB signaling pathway in trabecular bone tissue, reducing osteocyte apoptosis, upregulating osteogenic protein expression, and promoting osteogenic differentiation.

Key words: Gubao Capsule, Nuclear factor-kappa B, Signaling pathway, Osteocyte apoptosis, Osteogenic differentiation, Osteoporosis