Inhibitory Effect of LGS and ODE Isolated from the Twigs of Syringa oblata subsp. dilatata on RANKL-Induced Osteoclastogenesis in Macrophage Cells

Abstract

Osteoblasts and osteoclasts play a pivotal role in maintaining bone homeostasis, of which excessive bone resorption by osteoclasts can cause osteoporosis and various bone diseases. However, current osteoporosis treatments have many side effects, and research on new treatments that can replace these treatments is ongoing. Therefore, in this study, the roles of ligustroside (LGS) and oleoside dimethylester (ODE), a natural product-derived compound isolated from Syringa oblata subsp. dilatata as a novel, natural product-derived osteoporosis treatments were investigated. In the results of this study, LGS and ODE inhibited the differentiation of receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced RAW264.7 cells into osteoclasts without cytotoxicity, and down-regulated the activity of TRAP, a specific biomarker of osteoclasts. In addition, it inhibited bone resorption and actin ring formation, which are important functions and features of osteoclasts. Also, the effects of LGS and ODE on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and phosphoinositide 3-kinases (PI3K)/ protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling pathways that play important roles in osteoclast differentiation were evaluated. In the results, LGS and ODE downregulated the phosphorylation of RANKL-induced MAPK and PI3K/Akt/mTOR proteins in a concentration-dependent manner, translocation of NF-κB into the nucleus was inhibited. As a result, the compounds LGS and ODE isolated from S. oblate subsp. dilatata effectively regulated the differentiation of RANKL-induced osteoclasts and inhibited the phosphorylation of signaling pathways that play a pivotal role in osteoclast differentiation. Therefore, these results suggest the possibility of LGS and ODE as new natural product treatments for bone diseases caused by excessive osteoclasts.

Keywords: Syringa oblata subsp. dilatata; ligustroside; oleoside dimethylester; osteoclastogenesis.

Figure 1

Effect of ligustroside (LGS) (A) and oleoside dimethylester (ODE) (B) on cytotoxicity in RAW 264.7 cells. The RAW 264.7 cells were treated with various concentrations of LGS (C) and ODE (D) (5, 10, 20, 40 μM) for 7 days and cell viability was determined by MTT assay. Mean value of three experiments ± S.D. was presented.

Figure 2

Inhibitory effect of LGS and ODE on osteoclast differentiation. RAW 264.7 cells were cultured for 7 days with or without RANKL (50 ng/mL) in the indicated concentration of LGS (A) and ODE (B). Cells were fixed in 4% formaldehyde and stained for TRAP and measured TRAP activity. Mean value of three experiments ± S.D. was presented. * p < 0.05 versus the RANKL-treated group.

Figure 3

Inhibitory effects of LGS and ODE on the actin ring structure formation during osteoclast formation. RAW 264.7 cells were cultured in 24-well plates (5 × 10³ cell/well) for 7 days in the presence or absence of LGS (A) and ODE (B) with RANKL (50 ng/mL). Cells were fixed in 0.1% Triton X-100, after then stained with Alexa 488 and 4′,6-diamidino-2-phenylindole (DAPI). The mean value of three experiments ± S.D. was presented. * p < 0.05 versus the RANKL-treated group.

Figure 4

LGS and ODE inhibit bone resorption by RANKL-induced osteoclasts. RAW 264.7 cells (2 × 10³ cell/well) were cultured during 7 days treated or untreated with RANKL, ODE (A) and LGS (B) in an osteo-surface assay plate. Cells were removed and resorption pits were identified by light microscopy. Mean value of three experiments ± S.D. was presented. * p < 0.05 versus the RANKL-treated group.

Figure 5

LGS and ODE inhibit RANKL-induced osteoclast formation master transcription factor and osteoclast-specific gene expression. RAW 264.7 cell were pretreated with or without LGS and ODE for 2 h and then treated with RANKL (50 ng/mL) for 24 h. Cellular proteins were extracted and identified c-Fos and NFATc1 dependent pathways through western blot analysis. Expression levels of c-Fos and NFATc1 were quantified by western blotting (A,B). LGS and ODE regulate RANKL-mediated osteoclast-specific gene expression. Osteoclast-specific gene expression was analyzed using real-time PCR and results were standardized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (C,D). The mean value of three experiments ± S.D. was presented. * p < 0.05 versus the RANKL-treated group.

Figure 6

LGS and ODE inhibit the pathways of MAPK in osteoclastogenesis induced by RANKL. RAW 264.7 cells were pretreated with or without LGS (A) and ODE (B) for 2 h and treated with RANKL (50 ng/mL) for 30 min. Then, the whole protein was extracted and western blotting was performed. Quantitated signals are plotted. The gel images are representative of three independent experiments. Mean value of three experiments ± S.D. were presented. * p < 0.05 versus the RANKL-treated group.

Figure 7

RAW 264.7 cells were pretreated with or without LGS (A) and ODE (B) for 2 h and then treated with RANKL (50 ng/mL) for 30 min. In order to confirm the expression of NF-κB, the cytoplasm and nucleus were separated according to the instructions to measure protein expression of IκBα, and NF-κB p65. Cell lysates were analyzed by Western blot. Quantitated signals are plotted. The gel images are representative of three independent experiments. The mean value of three experiments ± S.D. was presented.

Figure 8

LGS and ODE inhibit the pathways of PI3K/Akt/mTOR in osteoclastogenesis induced by RANKL. RAW264.7 cells were treated with RANKL (50 ng/mL) at the indicated concentrations of LGS (A) and ODE (B) (5–40 μM) for 30 min. Thereafter, total protein was extracted from each group and evaluated by western blot. Relative protein expression levels were measured using ImageJ software. Mean value of three experiments ± S.D. was presented. * p < 0.05 versus the RANKL-treated group.