体外研究橙皮苷抑制钛颗粒介导的破骨细胞分化

目的研究橙皮苷对钛颗粒介导前破骨细胞分化及成熟的影响。 方法骨髓巨噬细胞在巨噬细胞集落刺激因子(M-CSF)30 ng/ml及核因子κB受体活化因子配体(Rankl)50 ng/ml刺激下，诱导分化为破骨细胞。扫描电镜观察钛磨损颗粒形貌结构。对不同浓度下橙皮苷对巨噬细胞增殖的影响进行t检验分析得出最低有效浓度；对抗酒石酸酸性磷酸酶(TRAP)阳性细胞数及骨吸收陷窝面积判断橙皮苷对破骨细胞分化及成熟的影响。最后通过实时定量PCR(RT-PCR)验证橙皮苷对钛颗粒介导的破骨基因，包括活化T细胞核因子(NFATc1)、组织蛋白酶K (CTSK)、TRAP的影响。TRAP阳性细胞数及骨吸收陷窝面积、RT-PCR结果数据均采用t检验分析。 结果扫描电镜显示钛磨损颗粒大小在1～3 μm。CCK-8实验结果显示橙皮苷对巨噬细胞增殖有促进作用，浓度超过40 μmol/L后，会对巨噬细胞产生抑制作用(F＝40.1, P<0.01)，所以选择40 μmol/L作为对巨噬细胞分化的影响。在抗酒石酸酸性磷酸酶染色中发现40 μmol/L的橙皮苷会明显抑制前破骨细胞的分化，TRAP阳性细胞数目及破骨细胞的面积明显减少(t＝5.5，P<0.05)。与对照组相比，扫描电镜观察钛颗粒介导骨吸收陷窝面积明显增多，但加入40 μmol/L的橙皮苷后，这种吸收效果或明显减少(t＝6.1，P<0.05)。最后，通过RT-PCR实验得出，40 μmol/L的橙皮苷会明显抑制破骨细胞分化相关NFATc1, CTSK，TRAP基因(t＝7.1、4.8、9.1，均为P<0.05)。 结论橙皮苷抑制钛颗粒介导的破骨细胞分化及成熟。

Hesperidin inhibits osteoclast differentiation mediated by titanium particles in vitro

ObjectiveTo investigate the effects of hesperidin on titanium particles-mediated osteoclast differentiation and maturation. MethodsBone marrow macrophages were induced to differentiate into osteoclasts under the stimulation of M-CSF (30ng / ml) and Rankl (50ng / ml). SEM was used to observe the morphology and structure of titanium particles. Cell counting kit 8(CCK8) was used to screen the lowest effective concentration of hesperidin. The effects of hesperidin on osteoclast differentiation and maturation was determined by observing the number of tanrate resistaIlt acid phosphatase (TRAP) positive cells and observing the area of bone resorption pits by SEM. The effects of hesperidin on titanium particle-mediated osteoclast marker genes including nuclear factor of activated T-cells cytoplasmic one(NFATc1), cathepsin K(CK), TRAP was detected by real-time PCR(RT-PCR). ResultsThe size of titanium particles were 1-3 μm.CCK-8 showed that hesperidin had a significant inhibitory effect on macrophages when the concentration of hesperidin exceeded 40 μmol/L (F=40.1, P<0.01). TRAP staining revealed that hesperidin significantly inhibited the differentiation of pre-osteoclasts. SEM showed that the area of the titanium particles mediated bone resorption was significantly increased, However, after the addition of 40 μmol/L hesperidin, the absorption effect was significantly reduced (t=6.1, P<0.05). Hesperidin significantly inhibited the expression of osteoclast-related genes, including NFATc1, CTSK, and TRAP(t=7.1, 4.8, 9.1, all P<0.05). ConclusionHesperidin may inhibite titanium particles-mediated osteoclast differentiation and maturation.