Osteoblasts are derived from mesenchymal progenitor cells and are the main functional cells for bone formation [[1], [2], [3]]. They are responsible for the synthesis, secretion and mineralization of bone matrix and can secrete various bioactive substances for bone formation and reconstruction [[1], [2], [3]]. In the pathogenesis of osteoporosis, uncontrolled reactive oxygen species (ROS) production and excessive oxidative injury will lead to significant damage and death of osteoblasts [[4], [5], [6]], causing bone mass reduction and microstructure disruption [[4], [5], [6]]. In in vitro experiment system, hydrogen peroxide (H₂O₂) or other oxidative stimuli were added to cultured osteoblasts/osteoblastic cells [[7], [8], [9], [10], [11], [12], [13]]. H₂O₂ treatment in osteoblasts/osteoblastic cells was shown to cause oxidative stress, protein denaturation, lipid peroxidation as well as DNA damage, eventually cell death and apoptosis [[8], [9], [10], [11],13].

Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein, essential for synapse development and function [14,15]. Its mutation is associated with autism [14]. Interestingly, early studies have shown that neuronal activity could enable neurons to secrete NLGN3, thereby promoting surrounding glioma cell growth and proliferation [[16], [17], [18], [19]]. NLGN3 was shown to phosphorylate multiple receptor tyrosine kinases (RTKs) and other receptors in glioma cells, activating downstream signaling cascades, including PI3K-Akt-mTOR and Erk-MAPK [[16], [17], [18], [19]]. Zelano et al., have shown that NLGN3 is expressed in the bone [20] and its expression was downregulated in spinal motoneurons after axotomy [20]. We here explored its potential activity against H₂O₂-induced oxidative injury in cultured osteoblasts.

One of the best characterized anti-oxidant cascades is nuclear-factor-E2-related factor 2 (Nrf2) [[21], [22], [23]]. Under the unstimulated basal condition, Nrf2 protein in the cytosol associates with its suppressor protein Kelch-1ike ECH- associated protein l (Keap1). The latter, together with Cullin 3 (Cul3), initiates Nrf2 proteasomal degradation through the ubiquitin system [[21], [22], [23]]. Activated Nrf2 protein, i.e. through phosphorylation, separates from Keap1. This will strop Nrf2 ubiquitin process and cause its stabilization [[21], [22], [23]]. The stabilized Nrf2 protein then enters nucleus and binds to anti-oxidant response element (ARE) along with small Maf proteins, promoting transcription and expression of over 250 Nrf2 cascade genes [[21], [22], [23]]. Proteins encoded by Nrf2-cascade genes are mostly antioxidant and cytoprotective proteins or detoxification enzymes, including ?-glutamyl cysteine ligase catalytic subunit (GCLC), the modified subunit (GCLM), NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO1) [[21], [22], [23]]. Activation of Nrf2 cascade, through genetic means or various pharmacological agents, can protect osteoblasts/osteoblastic cells from H₂O₂ [7,10,13,[24], [25], [26], [27]] and other oxidative stimuli [[28], [29], [30]]. The results of this study will show that NLGN3 activates Nrf2 cascade to protect osteoblasts from H₂O₂.