Fisetin经c－Fos／NFATc1信号通路抑制小鼠巨噬细胞分化为破骨细胞的研究

摘要

Objective To explore the impacts of Fisetin on titanium particles -induced differentiation of mouse macrophages into osteoclasts via the c-Fos/NFATc1 signaling pathway.Methods Mouse RAW264.7 macrophages cul-tivated in vitro were divided into five groups according to their different processing conditions:Group A (blank control), Group B (titanium particles alone), Group C (1.25μmol/L Fisetin +titanium particles), Group D (2.5μmol/L Fi-setin +titanium particles), and Group E (5 μmol/L Fisetin +titanium particles).The proliferation of RAW264.7 cells in each group was examined using CCK-8 assay.After six days of treatment, the number of osteoclasts in each group was measured by tartrate resistant acid phosphatase ( TRAP) staining.The amounts of c-Fos/NFATcl mRNA and protein were detected by Western blotting and RT-PCR, respectively.Results According to CCK-8 results, no sta-tistical differences were seen as to the proliferation of RAW264.7 cells among the five groups (P>0.05).Although mul-tinucleated cells with positive TRAP staining formed in each group, Group B produced the largest number of the cells.As the concentrations of Fisetin rose, the number of multinucleated cells with positive TRAP staining was declined from Group C to Group E.The number of the cells in Group A was the least.Furthermore, according to RT-PCR and West-ern blotting analysis, Group B presented the quantities of c-Fos/NFATc1 mRNA and protein which were substantially higher than those of Groups C, D, and E (P<0.05).Conclusion Fisetin can suppress titanium particles-induced differentiation of mouse macrophages into osteoclasts in a dose-dependent manner, which may be associated with the c-Fos/NFATc1 signaling pathway.%目的：探讨Fisetin阻断c－Fos／NFATc1信号通路对钛颗粒诱导小鼠巨噬细胞向破骨细胞分化的影响。方法体外培养小鼠巨噬细胞RAW264．7，根据处理条件不同分为5组：A组（空白对照组）、B组（钛颗粒对照组）、C组（1．25μmol／L Fisetin＋钛颗粒）、D组（2．5μmol／L Fisetin＋钛颗粒）、E组（5μmol／L Fisetin＋钛颗粒）。采用CCK－8法检测各组RAW264．7细胞增殖活性。培养6天后，抗酒石酸酸性磷酸酶（ TRAP）染色检测各组细胞破骨细胞（细胞核≥3）数目，免疫印迹法检测各组细胞中c－Fos／NFATcl蛋白表达，RT－PCR法检测c－Fos／NFATc1的基因表达。结果 CCK－8法检测结果显示各组RAW264．7细胞增殖能力差异无统计学意义（P＞0．05）。 TRAP染色结果显示各组细胞均有TRAP阳性多核细胞生成，B组TRAP阳性多核细胞数目最高，C、D、E组呈现逐渐减少趋势，A组最少。 RT－PCR和Western blot检测表明，B组c－Fos／NFATc1表达最高，C、D、E组表达逐渐减少，与B组比较差异具有统计学意义（ P＜0．05）。结论 Fisetin可通过c－Fos／NFATc1信号通路抑制钛颗粒作用下小鼠巨噬细胞向破骨细胞的分化，其抑制程度与Fisetin药物浓度有关。