(PTFS11-02-24) Bioinformatics Analysis of RNA-Seq Data Reveals Inhibitory Effects of Ent-7β-Hydroxy-15-Oxokaur-16-En-18-Yl Acetate on Osteoclastogenesis and Mitochondrial Function

Objectives: Given the adverse effects associated with anti-osteoclastic drugs in osteoporosis treatment, there is a growing need to explore alternative therapeutics utilizing natural compounds. Here, we investigated the effects of ent-7β-hydroxy-15-oxokaur-16-en-18-yl acetate (Ent7), a major diterpene isolated from the leaves of Croton tonkinensis, on osteoclast (OC) differentiation and underlying molecular mechanisms using a bioinformatic approach.

Methods: Mouse bone marrow-derived macrophages were differentiated into OC in the presence of Ent7 (0-250 ng/ml) for 72 h. Cells were subsequently analyzed for TRAP assay, F-actin ring staining, and evaluation of osteoclastogenic marker expression. RNA transcriptome was analyzed using bioinformatic tools including DAVID, GSEA, and STRING. MitoTracker, MitoSOX, and JC-1 were performed to measure mitochondrial mass, ROS, and membrane potential in mature OC.

Results: Ent7 significantly reduced RANKL-stimulated OC formation without cytotoxicity. Ent7 treatment suppressed the level of osteoclastogenic markers including Nfatc1, Dc-stamp, Calcitonin receptor, Atp6v0d2, Trap, Cathepsin k, and Mmp9 in a dose-dependent manner, which was associated with the inhibition of MAPK (JNK, ERK, and p38), NF-κB (IκBα and p65), and Akt pathways. Furthermore, functional enrichment analysis using 623 DEGs with |fold change| >2 indicated Ent7 affected downregulation of OC differentiation, positive regulation of OC differentiation, and mitochondrial energy metabolism. Pathway enrichment analysis revealed significant regulation of various pathways involving cell cycle, oxidative phosphorylation, TCA cycle, ROS, and OC differentiation in response to Ent7. PPI network analysis identified ATP6v0d2 involved in proton transport as one of the hub genes that play a pivotal role both in OC differentiation and mitochondrial energy metabolism. Notably, we confirmed Ent7 reduced mitochondrial mass, ROS level, membrane potential, and the level of markers related with mitochondrial biogenesis, fusion, and fission.

Conclusions: Ent7 effectively suppresses the MAPK, NF-κB, and Akt pathways and mitochondrial function, thereby restraining osteoclastogenesis. Our findings imply Ent7 holds promise as a potential candidate for an anti-osteoporotic supplement.

Funding Sources: National Research Foundation of Korea