Brevilin A, a novel BNIP3 inhibitor suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss via impairing mitophagy and mitochondrial metabolism

Abstract

Background

The mitochondrial dysfunction and overactive osteoclasts is involved in the progress of osteoporosis. Brevilin A (BA), a sesquiterpene lactone, is a compound extracted and purified from Centipeda minima. It exhibits a range of pharmacological activities, such as anti-inflammatory and antioxidant effects. However, its specific impact on osteoporosis remains unclear. The present study is designed to explore BA as a novel osteoclast inhibitor for the treatment of osteoporosis as well as its molecular mechanisms of action via BNIP3-mediated mitophagy.

Methods

The cytotoxicity of BA in vitro was evaluated using the CCK8 assay, while tartrate-resistant acid phosphatase (TRAcP) staining and bone resorption assays were conducted to examine its effects on osteoclastogenesis and osteoclast function. To elucidate the molecular mechanisms by which BA targets BNIP3 in osteoclasts, RNA-seq, molecular docking analysis, Surface plasmon resonance (SPR), qPCR, western blot, mitochondrial oxygen consumption rate (OCR), transmission electron microscopy (TEM), Single cell sequencing and immunofluorescence staining were employed. In addition, a specific BNIP3 agonist IOX5, was used to revalidate the inhibitory effect of BA on BNIP3. To investigate the effects and protective role of BA in modulating BNIP3 on bone loss in osteoporotic mice induced by ovariectomy (OVX), we employed in vivo micro-CT scanning and histological immunostaining techniques.

Results

Our study demonstrated that BA inhibited RANKL-induced osteoclastogenesis in a concentration-dependent manner without any cell cytotoxicity. Further, BA abrogated MAPK-related proteins and intracellular and mitochondrial ROS level, subsequently inhibiting NFATc1 activity. RNA-seq analysis revealed that the molecular mechanism by which BA inhibited osteoclasts is closely related to mitophagy and mitochondrial function. Here, we found that BA suppressed oxygen consumption rate and mitochondrial oxidative phosphorylation during osteoclastogenesis. This compound abolished expression of ATG5, SIRT3, Beclin1 and LC3B. RANKL-induced mitophagy associated protein (PINK1 and Parkin) were also suppressed by BA. BA interacted with BNIP3 and IOX5 treatment further verified the targeted inhibition effect of BA on BNIP3. In addition, we found that BNIP3 deficient inhibited osteoclast differentiation related with mitophagy and mitochondrial function. In vivo experiments confirmed that BA significantly prevent OVX-induced bone loss associated with BNIP3-mediated mitophagy.

Conclusions

Our study reveals for the first time that BA acts as a novel inhibitor of BNIP3, which ameliorates osteoclast activity and OVX-induced osteoporosis via limiting mitophagy and mitochondrial energy production, suggesting that it could be a novel therapeutic strategy for osteoporosis.