FIGURE 4

FIGURE 4

Gene expression analysis of RAW264.7 cells and zebrafish scales treated with 10-gingerol. (A,B) qPCR analysis of osteoclast marker genes in RAW264.7 cells with 10-gingerol. Oscar: Osteoclast-associated immunoglobulin-like receptor; Dc-stamp: Dendrocyte expressed seven transmembrane protein; Traf6: Tumor necrosis factor receptor-associated factor 6; Ctsk: Cathepsin k; Trap: Tartrate-resistant acid phosphatase; Mmp9: matrix metalloproteinases-9; Nfatc1: nuclear factor of activated T cells cytoplasmic 1. n = 3, error bars indicate SE; *p < 0.05, **p < 0.01. The data for GHE treatment were referred from our previous study (Ito et al., 2016). nd indicates no data. (C) qPCR analysis of osteoclast marker genes in osteoporotic zebrafish scales with 10-gingerol, GHE, or AL. ctsk: cathepsin k; mmp2: matrix metalloproteinases-2. *p < 0.05, **p < 0.01. n = 5, error bars indicate SE. (D) Fluorescent immunohistochemistry for Nfatc1 in RAW264.7 cells treated with sRANKL for 3 days. Red indicates NFATc1, and blue indicates nuclei (DAPI). (E) qPCR analysis of Nf-kb1 and Nf-kb2 in RAW264.7 cells with 10-gingerol. Nf-kb1: Nuclear factor kappa B subunit 1; Nf-kb2: Nuclear factor kappa B subunit 2. *p < 0.05. n = 5, error bars indicate SE. (F,G) qPCR analysis of nf-kb1, nf-kb2, (F) and rankl(G) in osteoporotic zebrafish scales (PN) with 10-gingerol, GHE, or AL. rankl: receptor activator of nuclear factor kappa B ligand. #p < 0.1, *p < 0.05 vs. PN group. n = 5, error bars indicate SE. (H) Cell-free CTSK inhibitor assay of 10-gingerol (100 μM) and GHE (100 μg/mL). FF-FMK (10 μM) was used as a positive control (98.3% inhibition to the negative control). **p < 0.01 vs. negative control. n = 3, error bars indicate SE.