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This study investigated whether nasunin, the major component of anthocyanin pigment of eggplant (Solanum melongena L.), known for its antioxidant effects, counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells. Pretreatment with purified nasunin (10−10–10−6 M) significantly increased viability and reduced apoptosis of MC3T3-E1 cells cultured with t-BHP (250 µM, 3 h). Furthermore, nasunin (10−9 M) prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organisation due to both an increase of intracellular glutathione and a decrease of intracellular reactive oxygen species. Moreover, t-BHP-induced reduction in osteoblast differentiation markers, such as alkaline phosphatase and collagen content, was recovered in nasunin-pretreated cells. Nasunin protects against t-BHP-induced osteoblastic dysfunction by activating the phosphoinositide 3-kinase/Akt signalling pathway (PI3K/Akt) since LY294002, a PI3K-specific inhibitor, worsened the cytotoxic effect of t-BHP and reversed the protective action of nasunin. Furthermore, nasunin prevented the decreased Akt phosphorylation observed in t-BHP-treated cells.

Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk. Ghrelin (10−10M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys3-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression.