Active fraction of Polyrhachis vicina (Rogers) inhibits osteoclastogenesis by targeting Trim38 mediated proteasomal degradation of TRAF6.

Reactive Oxygen Species (ROS) is a key factor in the pathogenesis of osteoporosis (OP) primarily characterized by excessive osteoclast activity. Active fraction of Polyrhachis vicina Rogers (AFPR) exerts antioxidant effects and possesses extensive promising therapeutic effects in various conditions, however, its function in osteoclastogenesis and OP is unknown. The aim of this study is to elucidate the cellular and molecular mechanisms of AFPR in OP. CCK8 assay was used to evaluate the cell viability under AFPR treatment. TRAcP staining, podosome belts staining and bone resorption were used to test the effect of AFPR on osteoclastogenesis. Immunofluorescence staining was used to observe the effect of AFPR on ROS production. si-RNA transfection, coimmunoprecipitation and Western-blot were used to clarify the underlying mechanisms. Further, an ovariectomy (OVX) -induced OP mice model was used to identify the effect of AFPR on bone loss using Micro-CT scanning and histological examination. In the present study, AFPR inhibited osteoclast differentiation and bone resorption induced by nuclear factor-κB receptor activator (NF-κB) ligand (RANKL) in dose-/ time-dependent with no cytotoxicity. Meanwhile, AFPR decreased RANKL-mediated ROS levels and enhanced ROS scavenging enzymes. Mechanistically, AFPR promoted proteasomal degradation of TRAF6 by significantly upregulating its K48-linked ubiquitination, subsequently inhibiting NFATc1 activity. We further observed that tripartite motif protein 38 (TRIM38) could mediate the ubiquitination of TRAF6 in response to RANKL. Moreover, TRIM38 could negatively regulate the RANKL pathway by binding to TRAF6 and promoting K48-linked polyubiquitination. In addition, TRIM38 deficiency rescued the inhibition of AFPR on ROS and NFATc1 activity and osteoclastogenesis. In line with these results, AFPR reduced OP caused by OVX through ameliorating osteoclastogenesis. AFPR alleviates ovariectomized-induced bone loss via suppressing ROS and NFATc1 by targeting Trim38 mediated proteasomal degradation of TRAF6. The research offers innovative perspectives on AFPR's suppressive impact in vivo OVX mouse model and in vitro , and clarifies the fundamental mechanism. Polyrhachis vicina is a species of ant insects (also called black ant) (Wei et al., 2023 ; Zhang et al., 2022). AFPR is the components of active fraction of Polyrchachis vicina Rogers. As shown in the figure, AFPR alleviates ovariectomized-induced bone loss via suppressing ROS and NFATc1 by targeting Trim38 mediated proteasomal degradation of TRAF6. AFPR reduced RANKL-mediated ROS level by down-regulating TRAF6/NOX1 signaling cascade and enhancing Nrf2-mediated ROS scavenging enzymes (HO-1, Nqo1), followed by inhibiting NF-κB and MAPK pathways, leading to the suppression of NFATc1 activity and the expression of osteoclast-related genes, such as Ctsk and Atp6v0d2. [Display omitted] [ABSTRACT FROM AUTHOR]