To investigate the effect and mechanism of kurarinone on bone marrow-derived macrophages (BMDM) cells through regulating toll-like receptor 9 (TLR9) signaling pathway via cathepsin K (Ctsk).

BMDM cells were divided into control group, model group [receptor activator of nuclear factor-κB ligand (RANKL) 50 ng·mL^-1], kurarinone group (RANKL 50 ng·mL^-1, kurarinone 10 μmol·L^-1), kurarinone+oe-NC group (infected oe-NC, RANKL 50 ng·mL^-1, kurarinone 10 μmol·L^-1), and kurarinone+oe-Ctsk group (infected oe-Ctsk, RANKL 50 ng·mL^-1, kurarinone 10 μmol·L^-1). Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of Ctsk mRNA; Western blot was used to detect the expression levels of tartrate-resistant acid phosphatase (TRAP) and TLR9 signaling pathway-related proteins; immunofluorescence was used to detect the expression levels of osteoclast differentiation-related proteins; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors; in vitro bone resorption assay was used to detect bone resorption.

The relative expression levels of Ctsk mRNA in the control group, model group, kurarinone group, kurarinone+oe-NC group, and kurarinone+oe-Ctsk group were 1.00±0.15, 2.07±0.28, 1.31±0.18, 1.34±0.14 and 1.79±0.27, respectively; the relative expression levels of TRAP protein were 1.00±0.16, 2.31±0.44, 1.43±0.17, 1.38±0.22 and 1.91±0.30, respectively; the relative fluorescence levels of matrix metalloproteinase-9 (MMP-9) protein were 1.00±0.15, 2.18±0.37, 1.33±0.19, 1.28±0.17 and 1.62±0.31, respectively; the relative fluorescence levels of nuclear factor of activated T-cells 1 (NFATc1) protein were 1.00±0.13, 1.87±0.39, 1.13±0.21, 1.18±0.19 and 1.38±0.22, respectively; the relative fluorescence levels of integrin beta-1 (Itgb1) protein were 1.00±0.19, 1.49±0.25, 1.19±0.22, 1.14±0.17 and 1.32±0.18, respectively; the levels of interleukin-1β (IL-1β) were (15.28±2.05), (44.68±7.93), (31.40±6.08), (33.75±5.76) and (39.62±5.52) pg·mL^-1, respectively; the levels of interleukin-18 (IL-18) were (122.45±22.39), (317.56±51.51), (191.72±33.34), (185.93±32.19) and (217.89±33.58) pg·mL^-1, respectively; the levels of tumor necrosis factor-α (TNF-α) were (81.36±12.27), (216.93±34.39), (130.89±16.18), (125.76±15.15) and (162.94±26.59) pg·mL^-1, respectively; the number of bone resorption pits were (35.26±6.35), (84.72±16.10), (50.38±9.07), (47.85±9.09) and (61.63±10.89) per piece, respectively; the relative area of bone resorption pits were (0.26±0.05)%, (1.38±0.24)%, (1.04±0.18)%, (1.06±0.16)% and (1.27±0.19)%, respectively; the relative expression levels of TLR9 protein were 1.00±0.15, 2.05±0.31, 1.32±0.16, 1.27±0.15 and 1.53±0.24, respectively; the relative expression levels of myeloid differentiation factor 88 (MyD88) protein were 1.00±0.13, 2.18±0.33, 1.42±0.18, 1.36±0.17 and 1.62±0.26, respectively; p-p65/t-p65 were 1.00±0.11, 2.22±0.34, 1.40±0.19, 1.39±0.19, 1.76±0.27, respectively. Compared model group with control group, compared kurarinone group with the model group, compared the kurarinone+oe-Ctsk group with the kurarinone group, the differences of above indicators were all statistically significant (P<0.05, P<0.01, P<0.001).

Kurarinone can inhibit RANKL-induced osteoclast differentiation, inflammatory factor secretion, and bone resorption function by suppressing Ctsk, which may be achieved through inhibiting the TLR9 pathway.