OP0074 Ebselen Is A Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In Vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In Vivo

Background

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer.

Objectives

We investigated the effects of ebselen on RANKL-induced differentiation of osteoclasts and their functions and the underlying molecular mechanisms. Furthermore, we determined the effects of ebselen on LPS-induced bone erosion in vivo.

Methods

To generate osteoclasts from BMMs, we cultured BMMs for 4 days in the condition of M-CSF and RANKL pretreated with ebselen. The cells were then stained with TRAP solution, rhodamine-conjugated phalloidin for F-actin ring labeling and DAPI solution to detect apoptotic body formation. The change of F-actin ring on mature osteoclasts induced by ebselen was quantified by calculating the ratio of actin ring positive (AR+) osteoclasts versus actin ring negative (AR-) osteoclasts. to detect the formation of apoptotic osteoclasts, we performed TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay. Primary calvaria osteoblasts and BMCs were co-cultured and were re-seeded in hydroxyapatite-coated plates or dentin slices with or without ebselen. ICR mice were divided into 4 experimental groups comprising 5 mice each: phosphate-buffered saline-treated (control) group, ebselen only-treated group, LPS only-treated group, and LPS- and ebselen-treated group. Ebselen (10 mg/kg) or PBS was administered orally every 8 days, and LPS was injected intraperitoneally on days 1 and 4. Micro-computed tomography (μ-CT) data containing 3D images and bone parameters and histological data were acquired

Results

Ebselen suppressed the formation of TRAP-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of RANKL/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its μ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate receptor.

Conclusions

Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor.

Disclosure of Interest

None declared