刺槐素调控NF-κB/NFATc1信号轴抑制破骨细胞分化的机制研究

Robinin inhibits osteoclast differentiation by down-regulating NF-κB/NFATc1 signaling pathway

目的探讨刺槐素对破骨细胞分化的影响及机制。方法通过细胞计数试剂盒-8法评估刺槐素对小鼠骨髓来源单核巨噬细胞(BMMs)增殖活性的影响,采用抗酒石酸酸性磷酸酶(TRAP)染色评估刺槐素对BMMs破骨分化诱导的影响,采用破骨细胞伪足小体免疫荧光染色研究刺槐素对破骨分化过程中伪足小体形成的影响,并采用qRT-PCR法验证刺槐素处理下BMMs破骨分化过程中核心转录因子活化T-细胞核因子1(NFATc1)和相关特征基因在转录水平的变化。最后通过Western blot法检测刺槐素对NF-κB受体活化因子配体(RANKL)刺激下NF-κB活性及破骨分化过程中NFATc1表达的影响。结果2μmol/L及以下浓度刺槐素对BMMs无明显毒性。刺槐素可浓度依赖性地抑制BMMs在RANKL刺激下破骨分化过程,在2μmol/L刺槐素干预下几乎无成熟破骨细胞生成。与此同时,刺槐素可显著抑制破骨细胞分化过程中伪足小体的形成。刺槐素可抑制破骨细胞分化过程中下游耐酒石酸酸性磷酸酶(ACP5)、破骨细胞相关受体(OSCAR)、组织蛋白酶K(CTSK)、基质金属蛋白酶9(MMP-9)等特征基因的转录。在机制上,刺槐素可通过抑制RANKL刺激下BMMs内NF-κB信号通路p65磷酸化激活,进而下调NFATc1、MMP-9的表达,达到抑制破骨细胞分化的作用。结论刺槐素可通过下调NF-κB/NFATc1信号轴抑制BMMs破骨分化过程。

Objective To investigate the effect and mechanism of robinin on osteoclast differentiation.Methods The effect of robinin on the proliferation activity of mouse bone marrow-derived monocyte/macrophage(BMMs)was evaluated by CCK-8 method,the effect of robinin on osteoclastic differentiation induction of BMMs was evaluated by tartrate resistant acid phosphatase(TRAP)staining,and the effect of robinin on the formation of pseudopodosomes during osteoclastic differentiation was studied by immunofluorescence staining of osteoclastic pseudopodosomes.qRT-PCR was used to verify the transcriptional changes of core transcription factor nuclear factor of activated T cells c1(NFATc1)and related osteoclastic characteristic genes during osteoclastic differentiation of BMMs treated with robinin.Finally,the effects of robinin on NF-κB activity stimulated by receptor activator of NF-κB ligand(RANKL)and NFATc1 expression during osteoclast differentiation were detected by Western blot analysis.Results Robinin had no significant toxic effect on the cell proliferation of BMMs under concentration of 2μmol/L.Robinin inhibited osteoclast differentiation from BMMs in a concentration-dependent manner,and almost no mature osteoclasts were generated with robinin treatment under 2μmol/L.Meanwhile,robinin significantly inhibited podosome formation during osteoclastogenesis.Robinin hindered the transcription of osteoclast-related genes including ACP5,OSCAR,CTSK,MMP-9.Robinin inhibited osteoclast differentiation by inhibiting the p65 phosphorylation of NF-κB signaling pathway in BMMs cells stimulated by RANKL,and down-regulated the expressions of NFATc1 and MMP-9.Conclusion Robinin can inhibit osteoclast differentiation of BMMs by down-regulating NF-κB/NFATc1 signaling pathway.