Ent-7β-hydroxy-15-oxokaur-16-en-18-yl acetate suppresses RANKL-induced and LPS-enhanced osteoclastogenesis through NF-κB/MAPK signaling with involvement of the ACE–AT1R axis

Abstract

Osteoporosis is a metabolic bone disorder characterized by excessive osteoclast-mediated bone resorption, which is exacerbated by inflammatory and oxidative stress-associated signaling pathways. Although anti-resorptive drugs are used to prevent excessive bone resorption, their long-term use may cause adverse effects, highlighting the need for alternative pharmacological interventions targeting osteoclast differentiation and inflammation-associated signaling. Here, we investigated the anti-osteoclastogenic effects of ent-7β-hydroxy-15-oxokaur-16-en-18-yl acetate (Ent7), an ent-kaurane diterpenoid isolated from Croton tonkinensis, under RANKL-induced and inflammatory conditions using an integrated strategy combining transcriptomic profiling, network pharmacology, and molecular docking. Ent7 inhibited RANKL-induced osteoclast differentiation and bone resorption, accompanied by reduced osteoclastogenic marker expression and NF-κB, p38 MAPK, and Akt signaling. Transcriptomic profiling identified 384 differentially expressed genes, with enrichment of oxidative phosphorylation, phagosome, cell cycle, and osteoclast differentiation pathways. Functional validation further demonstrated alterations in cell-cycle progression, mitochondrial characteristics, and respiratory activity following Ent7 treatment. Ent7 also attenuated LPS-enhanced osteoclastogenesis and bone resorption, suppressing LPS-induced MAPK/c-Fos signaling. Integrated in silico analyses identified ACE as a candidate regulatory molecule associated with Ent7 activity, with a predicted binding energy of -9.3 kcal/mol. Consistently, Ent7 reduced the expression of ACE and its downstream receptor AT1R. Pharmacological inhibition of ACE or AT1R attenuated osteoclastogenesis, supporting the potential involvement of the ACE-AT1R axis in the anti-osteoclastogenic effects of Ent7. These findings provide a mechanistic basis for further evaluation of Ent7 in osteoclast-mediated pathological bone loss by demonstrating that Ent7 suppresses osteoclast differentiation under both RANKL-induced and inflammatory conditions through modulation of osteoclastogenic signaling and cellular processes.