Anti-osteoclastogenic Activity Of Isoliquiritigenin Via Inhibition Of NF-?B-dependent Autophagic Pathway

Recently, global research interest has showed increasing in the use of naturally bioactive constituents extracted from traditional plant medicine to treat human diseases. Isoliquiritigenin (ISL; 2',4',4-trihydroxychalcone) is a natural flavonoid and is the bioactive constituent of licorice. It has been proven its pharmacological safety and biological properties, including its anti-inflammatory, anti-cancer, and anti-oxidant activities. It has also been a promising agent for bone destructive diseases.Osteolytic bone diseases have abnormal increased osteoclasts which are exclusive bone-resorbing multinucleated cells in common derived from hematopoietic cells of monocyte/macrophage lineage. Receptor activator of nuclear factor-KB ligand (RANKL) is the essential cytokine for osteoclastogenesis initiation. Lipopolysaccharide (LPS) stimulation triggers an inflammatory state to induce osteoclastogenesis and subsequent bone resorption. Previous studies, including those from our laboratory, have demonstrated that the natural flavonoid ISL could inhibit RANKL-induced osteoclastogenesis in vitro and inflammatory bone loss in vivo. However, the mechanisms underlying its anti-osteoclastogenic effects are still far from clear.Autophagy is thought of as a key mechanism in various physiopathological processes and maintains cellular homeostasis. The autophagy-related proteins (ATGs) are known to participate and regulate autophagy process. Microtubule-associated protein light chain 3 (LC3) and Beclin 1 proteins are the key markers of autophagy. Autophagy is shown to play a crucial role in osteoclast differentiation and is concerned in some biological activities of flavonoids. However, whether the anti-osteoclastogenesis exerted by ISL is associated with autophagy remains unknown.In the present study, we investigate the potential role of autophagy during anti-osteoclastogenic effects by ISL in vitro and in vivo. We further delineate the nuclear factor-kappa B (NF-?B) signaling functions during ISL-mediated autophagy in osteoclasts. The present study aims to provide probable mechanisms underlying anti-osteoclastogenic effects of ISL and to design new treatment strategies for osteoclast-related bone diseases.Part ? Effects of ISL on NF-kB signaling-mediated autophagy during anti-osteoclastogenesis in vitro.Objective:This study aimed to evaluate the potential alterations of autophagy during anti-osteoclastogenic effects by ISL in vitro, as well as to delineate the regulation of NF-?B pathway on autophagy.Methods:RAW 264.7 cells were treated with RANKL plus varying concentrations of ISL. RANKL-induced osteoclast differentiation and function were assessed with TRAP staining, F-actin staining, and SEM. Cytotoxicity of material extracts was evaluated with Cell Counting Kit-8 assay. MDC staining, immunofluorescence. TEM, western blot, and quantitative real-time PCR (Real Time-PCR) were used to detect the specific features for autophagy. Then RAW 264.7 cells were preincubation with rapamycin, and then treated with RANKL and ISL. TRAP and F-actin staining was used to assess osteoclastogenesis. The mRNA expressions associated with osteoclast differentiation were detected with Real Time-PCR. And we also employed MDC staining, immunofluorescence, western blot and Real Time-PCR to observe the effects of Bay 11-7082 on autophagy.Results:We observed that ISL inhibited RANKL-induced osteoclastogenesis and suppressed autophagic LC3-II and Beclin 1 accumulation. ISL treatment resulted in the interruption of several specific features for autophagy in osteoclast precursors, including acidic vesicular organelle formation, LC3-II accumulation, and appearance of autophagic vacuoles. The RANKL-stimulated expression levels of autophagy-related genes and proteins also diminished in ISL-treated osteoclast precursors. The reactivation of autophagy by rapamycin almost reversed the ISL-elicited anti-osteoclastogenic effects. Interestingly, ISL inhibited the RANKL-stimulated NF-?B expression and nuclear translocation, whereas the NF-?B inhibitor Bay 11-7082 markedly suppressed the RANKL-induced autophagic activation.Conclusion:ISL inhibits osteoclast differentiation in an autophagic way by suppressing NF-?B signaling pathway in vitro.Part ? Effects of ISL on autophagy and NF-?B during inhibition of osteoclastogenesis and inflammatory bone resorption in vivo.Objective:To investigate the effects of ISL on LPS-induced bone destruction in vivo, and to explore the roles of autophagy and NF-?B signaling.Methods:For in vivo experiments, purchased BALB/c mice were subcutaneously injected LPS or PBS vehicle over the calvarias under light anesthesia, daily for 7 days. ISL was intraperitoneal injection 30 min earlier LPS injection. At end, mice were sacrificed and calvarias were removed for fixation. The bone microarchitecture of calvarias was scanned with microcomputed tomography and X-ray. Calvarias were decalcified, embedded in paraffin, and then sectioned into serial 5 ?m paraffin sections. Sections were stained by hematoxylin-eosin (HE) and TRAP for bone destruction or osteoclasts. At last, immunohistochemistry and immunofluorescence were used to examine the expression of LC3-? and NF-?B p65 in calvaria sections.Results:Consistent with the in vitro results, the administration of ISL could significantly inhibit LPS-induced osteoclast differentiation and inflammatory calvarial bone erosion in vivo. And ISL markedly attenuated osteoclastogenic cathespin K, autophagic LC3, and NF-?B expression.Conclusion:ISL could suppress LPS-mediated calvaria bone erosion in vivo and the inhibition of NF-?B and autophagy was involved.