Abstract: BACKGROUND: The differentiation of osteoclasts caused by wear particles is one of the main causes of periprosthesis osteolysis. Inhibiting the differentiation and absorption of osteoclasts is an important research direction in the treatment of periprosthesis osteolysis. Studies have shown that isoliquiritigenin can promote the maturation of osteoblasts and inhibit the bone resorption of osteoclasts.
OBJECTIVE: To investigate the effect of isoliquiritigenin on osteoclast formation and differentiation induced by wear particles.
METHODS: (1) Cell experiment: Passage 3 RAW264.7 cells were divided into five different groups. The control group was not treated with any treatment. The titanium particle group was added with titanium particles. The osteoclast induction group was added with nuclear factor κB receptor activating factor ligand to induce osteoclast differentiation. The osteoclast induction+titanium particle group was added with nuclear factor κB receptor activating factor ligand and titanium particles at the same time, and the isoliquiritigenin group was added with nuclear factor κB receptor activating factor ligand and titanium particles. On the 2nd day of treatment, 20 μmol/L isoliquiritigenin was added. After 5 days of induction with receptor activator of nuclear factor-κB ligand, tartrate-resistant acid phosphatase and F-actin staining were used to evaluate osteoclast formation and bone resorption. Western blot assay was used to detect the effect of isoliquiritigenin on the phosphorylation of IκBα and p-65 in the nuclear factor-κB pathway. Enzyme-linked immunosorbent assay was used to detect the levels of inflammatory factors (tumor necrosis factor-α, interleukin-6, and matrix metalloproteinase-9) in the cell supernatant. Reverse transcription polymerase chain reaction was used to detect the expressions of osteoclast-related genes (cathepsin K, nuclear factor of activated T cells 1, proto-oncogene protein, and tartrate-resistant acid phosphatase). (2) Animal experiment: Totally 32 BALB/c mice were used to construct dorsal airbag bone graft models and randomly divided into four intervention groups: the blank group (n=8) was injected with PBS into the airbag, and the model group (n=8), solvent group (n=8), and isoliquiritigenin group (n=8) were injected with titanium particle suspension into the airbag. On the second day after the titanium particle suspension was injected, the blank group, model group, solvent group, and isoliquiritigenin group were intraperitoneally injected with phosphate buffered saline, phosphate buffered saline, dimethyl sulfoxide+corn oil, and 40 mg/kg isoliquiritigenin, respectively, once every other day for 2 consecutive weeks. After the intervention, tartrate-resistant acid phosphatase staining was used to detect the number of osteoclasts in the airbag. Enzyme-linked immunosorbent assay was used to detect the level of inflammatory factors in the grinding fluid of the airbag. Reverse transcription polymerase chain reaction was used to detect the expression of osteoclast-related genes in the grinding fluid of the airbag.
RESULTS AND CONCLUSION: (1) Cell experiment: Compared with the control group, the number of osteoclasts formed in the other four groups increased, and the bone resorption capacity was enhanced. Among them, the osteoclast induction+titanium particle group had the largest number of osteoclasts formed and the strongest bone resorption capacity. Compared with the osteoclast induction+titanium particle group, the number of osteoclasts formed in the isoliquiritigenin group decreased, and the bone resorption capacity decreased. Compared with the control group, the phosphorylation of IκBα and p65 in the other four groups increased, indicating that the number of osteoclasts increased. Among them, the phosphorylation of IκBα and p65 in the osteoclast induction+titanium particle group was the highest, and it was significantly reduced after isoliquiritigenin treatment. Compared with the control group, the levels of inflammatory factors and the expression of osteoclast-related genes in the other four groups increased, among which the osteoclast induction+titanium particle group was the highest. Compared with the osteoclast induction+titanium particle group, the levels of inflammatory factors and the expression of osteoclast-related genes were reduced in the isoliquiritigenin group. (2) Animal experiment: Compared with the blank group, the number of osteoclasts, the level of inflammatory factors and the expression of osteoclast-related genes increased in the other three groups. Compared with the model group, the number of osteoclasts, the level of inflammatory factors and the expression of osteoclast-related genes decreased in the isoliquiritigenin group. These findings indicate that isoliquiritigenin could suppress the formation and differentiation of osteoclasts in vivo and in vitro induced by wear particles. 

Key words: isoliquiritigenin, wear particle, RAW264.7 cell, air bag implant model, aseptic loosening