荜茇酰胺抑制破骨细胞分化的机制研究

Piperlongumine inhibits osteoclast differentiation and its mechanism

目的探讨荜茇酰胺对破骨细胞分化的影响及机制。方法通过CCK-8法评估荜茇酰胺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)染色评估其对BMMs破骨分化的影响,利用Osteo Assay Surface骨细胞表面培养板研究其对破骨细胞骨吸收能力的影响,并采用qRT-PCR法验证荜茇酰胺处理下BMMs破骨分化过程中核心转录因子活化T-细胞核因子1(NFATc1)和相关破骨特征基因在转录水平的变化。结果2μmol/L及以下浓度的荜茇酰胺对BMMs增殖无明显毒性反应,并且荜茇酰胺可浓度依赖性地抑制BMMs破骨分化过程,在1μmol/L荜茇酰胺干预下几乎无成熟破骨细胞生成。与此同时,荜茇酰胺可显著抑制破骨细胞的骨吸收功能。在机制上,荜茇酰胺可通过抑制NF-κB受体活化因子配体(RANKL)刺激BMMs破骨分化过程中核心转录因子NFATc1的表达,从而下调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)等破骨特征基因的转录,抑制破骨分化过程。结论荜茇酰胺可通过下调RANKL刺激BMMs破骨分化过程中NFATc1的表达,抑制BMMs破骨分化过程及破骨细胞的骨吸收能力。

Objective To investigate the effect of monomer piperlongumine on osteoclast differentiation and its mechanism.Methods Mouse bone marine-derived monocytes/macrophages(BMMs)were treated with piperlongumine at different concentrations.CCK-8 assay was used to determine the cell proliferation;tartrate resistant acid phosphatase(TRAP)staining was used to detect the osteoclast differentiation from BMMs;and Osteo Assay Surface plate was used to investigate the bone resorption capacity of osteoclasts;qRT-PCR was used to detect the mRNA expression of nuclear factor of activated T cells c1(NFATc1)and genes related to osteoclast differentiation.Results Piperlongumine showed no obvious toxic effect on the cell proliferation of BMMs under concentration 2μmol/L,and it inhibited osteoclast differentiation from BMMs in a concentration-dependent manner under concentration of 1μmol/L.Meanwhile,piperlongumine significantly inhibited the bone resorption function of osteoclasts.Piperlongumine hindered the osteoclastogenesis process by inhibiting the expression of NFATc1,a core transcription factor of osteoclast differentiation,down-regulating the transcription of osteoclast feature genes including ACP5,OSCAR,CTSK,MMP-9.Conclusion Piperlongumine inhibits osteoclastic differentiation of BMMs and bone resorption of osteoclast by down-regulating the expression of NFATc1 stimulated by RANKL.