Background: To investigate the effect and underlying mechanism of Bushenhuoxue formula (BSHXF) on steroid-related osteonecrosis of the femoral head (SONFH). Methods:Seventy-five male New Zealand white rabbits were divided into three groups: control group, model group and BSHXF group. Rabbit SONFH was induced by methylprednisolone (MPS) combined with lipopolysaccharide (LPS). At week 2 and 6 post induction, the corresponding number of rabbits were sacrificed, and the femoral heads were harvested

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... for tissue analyses, including histopathology, mechanical test of femoral heads, micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry for p-P65 and co-staining of TRAP and alkaline phosphatase (ALP). Additionally, the serum TRACP5b level was measured using enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also evaluated the effect of BSHXF treatment on osteoclastogenesis in vitro. The protein level of cathepsin K (CTSK), P65, p-P65 and NFATc1 in rabbit femoral heads and RAW264.7 cells were detected, respectively.Results: At weeks 2 and 6 post induction, the elevated TRAP, p-P65 expression and reduced ALP expression were observed in the model group, with decreases in weight-bearing capacity of femoral heads and bone mass. After BSHXF treatment, the ratio of empty lacuna and the incidence of osteonecrosis in BSHXF group were markedly lower than that in model group. Micro-CT evaluation indicated that BSHXF has a preventive effect on bone loss in rabbit SONFH. In addition, BSHXF treatment increased weight-bearing capacity of femoral heads and reduced TRAP+ osteoclasts and serum TRACP5b level. Interestingly, CTSK, p-P65 and NFATc1 upregulation in necrotic femoral head could be reversed by BSHXF treatment, which also effectively inhibited RANKL-induced osteoclast differentiation and down-regulated CTSK, p-P65 and NFATc1 expression in vitro.Conclusions: BSHXF could inhibit osteoclastogenesis and bone resorption on rabbit steroid-related osteonecrosis of the femoral head by suppressing NF-κB/NFATc1 pathway.